Patent application title: SUCCINIC ACID PRODUCTION IN A EUKARYOTIC CELL
Inventors:
René Verwaal (Nootdorp, NL)
Liang Wu (Delft, NL)
Liang Wu (Delft, NL)
Robbertus Antonius Damveld (Berkel En Rodenrijs, NL)
Robbertus Antonius Damveld (Berkel En Rodenrijs, NL)
Cornelis Maria Jacobus Sagt (Utrecht, NL)
Cornelis Maria Jacobus Sagt (Utrecht, NL)
IPC8 Class: AC07C5510FI
USPC Class:
562590
Class name: Carboxylic acids and salts thereof acyclic polycarboxylic
Publication date: 2012-06-28
Patent application number: 20120165569
Abstract:
The present invention relates to a recombinant eukaryotic cell selected
from a yeast of a filamentous fungus comprising a nucleotide sequence
encoding a NAD(H)-dependent fumarate reductase that catalyses the
conversion of fumaric acid to succinic acid. The invention further
relates to a process for the production of succinic acid wherein the
eukaryotic cell according to the present invention is used.Claims:
1. A recombinant eukaryotic cell selected from the group consisting of a
yeast and a filamentous fungus comprising a nucleotide sequence encoding
a NAD(H)-dependent fumarate reductase that catalyses the conversion of
fumaric acid to succinic acid.
2. A cell according to claim 1, wherein the cell expresses a nucleotide sequence encoding an enzyme that catalyses the formation of succinic acid, wherein the nucleotide sequence encodes a NAD(H)-dependent fumarate reductase, comprising an amino acid sequence that has at least 40% sequence identity with the amino acid sequence of SEQ ID NO:1, and/or SEQ ID NO: 3, and/or SEQ ID NO:4, and/or SEQ ID NO: 6;
3. A cell according to claim 1, wherein the NAD(H)-dependent fumarate reductase is derived from a Trypanosoma sp.
4. A cell according to claim 1, wherein the NAD(H)-dependent fumarate reductase is active in the cytosol upon expression of the nucleotide sequence encoding NAD(H)-dependent fumarate reductase.
5. A cell according to claim 1, wherein the cell overexpresses a nucleotide sequence encoding a pyruvate carboxylase.
6. A cell according to claim 1, further comprising a nucleotide sequence encoding a heterologous phosphoenolpyruvate carboxykinase.
7. A cell according to claim 1, further comprising a nucleotide sequence encoding a malate dehydrogenase active in the cytosol upon expression of the nucleotide sequence encoding malate dehydrogenase.
8. A cell according to claim 1, further comprising a nucleotide sequence encoding an enzyme that catalyses the conversion of malic acid to fumaric acid in the cytosol, upon expression of the nucleotide sequence encoding enzyme that catalyses the conversion of malic acid to fumaric acid.
9. A cell according to claim 1, further comprising a nucleotide sequence encoding a dicarboxylic acid transporter.
10. A cell according to claim 1, wherein at least one gene encoding alcohol dehydrogenase is not functional.
11. A cell according to claim 1, wherein at least one gene encoding glycerol-3-phosphate dehydrogenase is not functional.
12. A cell according to claim 1, wherein at least one gene encoding succinate dehydrogenase is not functional.
13. A cell according to claim 1, which is an Aspergillus, preferably an Aspergillus niger.
14. A cell according to claim 1, which is a Saccharomyces cerevisiae
15. A process for the preparation of succinic acid, comprising fermenting a eukaryotic cell according to claim 1, in a suitable fermentation medium, wherein succinic acid is prepared.
16. A process according to claim 15, wherein the succinic acid prepared is used for the production of a pharmaceutical, cosmetic, food, feed or chemical product
17. A fermentation broth comprising succinic acid obtainable by the process according to claim 16.
Description:
[0001] The present invention relates to a recombinant eukaryotic cell
comprising a nucleotide sequence encoding a fumarate reductase and a
process for the production of succinic acid wherein the recombinant
eukaryotic cell is used.
[0002] Succinic acid is a potential precursor for numerous chemicals. For example, succinic acid can be converted into 1,4-butanediol (BDO), tetrahydrofuran, and gamma-butyrolactone. Another product derived from succinic acid is a polyester polymer which is made by linking succinic acid and BDO.
[0003] Succinic acid is predominantly produced through petrochemical processes by hydrogenation of butane. These processes are considered harmful for the environment and costly. The fermentative production of succinic acid may be an attractive alternative process for the production of succinic acid, wherein renewable feedstock as a carbon source may be used.
[0004] A number of different bacteria such as Escherichia coli, and the rumen bacteria Actinobacillus, Anaerobiospirillum, Bacteroides, Mannheimia, or Succinimonas, sp. are known to produce succinic acid. Metabolic engineering of these bacterial strains have improved the succinic acid yield and/or productivity, or reduced the by-product formation.
[0005] WO2007/061590 discloses a pyruvate decarboxylase negative yeast for the production of malic acid and/or succinic acid which is transformed with a pyruvate carboxylase enzyme or a phosphoenolpyruvate carboxylase, a malate dehydrogenase enzyme, and a malic acid transporter protein (MAE).
[0006] Despite the improvements that have been made in the fermentative production of succinic acid, there remains a need for improved microorganisms for the fermentative production of succinic acid.
[0007] The aim of the present invention is an alternative microorganism for the production of succinic acid.
[0008] The aim is achieved according to the invention with a recombinant eukaryotic cell selected from the group consisting of a yeast and a filamentous fungus comprising a nucleotide sequence encoding NAD(H)-dependent fumarate reductase that catalyses the conversion of fumaric acid to succinic acid.
[0009] Surprisingly it was found that the recombinant eukaryotic cell according to the present invention produces an increased amount of succinic acid compared to the amount of succinic acid produced by a wild-type eukaryotic cell. Preferably, a eukaryotic cell according to the present invention produces at least 1.2, preferably at least 1.5, preferably at least 2 times more succinic acid than a wild-type eukaryotic cell which does not comprise the nucleotide sequence encoding NAD(H)-dependent fumarate reductase.
[0010] As used herein, a recombinant eukaryotic cell according to the present invention is defined as a cell which contains, or is transformed or genetically modified with a nucleotide sequence or polypeptide that does not naturally occur in the eukaryotic cell, or it contains additional copy or copies of an endogenous nucleic acid sequence. A wild-type eukaryotic cell is herein defined as the parental cell of the recombinant cell.
[0011] The nucleotide sequence encoding a NAD(H)-dependent fumarate reductase that catalyses the conversion of fumaric acid to succinic acid may be a heterologous or homologous nucleotide sequence, or encodes a heterologous or homologous NAD(H)-dependent fumarate reductase, which may have been further genetically modified by mutation, disruption or deletion. Recombinant DNA techniques are well known in the art such as in Sambrook and Russel (2001) "Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory Press.
[0012] The term "homologous" when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain.
[0013] The term "heterologous" when used with respect to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous nucleic acids or proteins are not endogenous to the cell into which it is introduced, but have been obtained from another cell or synthetically or recombinantly produced.
[0014] A NAD(H)-dependent fumarate reductase according to the present invention uses NAD(H) as a cofactor, whereas most eukaryotic cells comprise a FADH2-dependent fumarate reductase, wherein FADH2 is the cofactor. It was found advantageous that the eukaryotic cell comprises a nucleotide sequence encoding a NAD(H)-dependent fumarate reductase, since the NAD(H)-dependent fumarate reductase provides the cell with further options to oxidise NAD(H) to NAD.sup.+ and influence the redox balance in the cell.
[0015] Preferably, the cell expresses a nucleotide sequence encoding an enzyme that catalyses the formation of succinic acid, wherein the nucleotide sequence preferably encodes a NAD(H)-dependent fumarate reductase, comprising an amino acid sequence that has at least 40%, preferably at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, 99% sequence identity with the amino acid sequence of SEQ ID NO: 1, and/or SEQ ID NO: 3, and/or SEQ ID NO: 4, and/or SEQ ID NO: 6. Preferably, the nucleotide sequence encodes a NAD(H)-dependent fumarate reductase comprising the amino acid sequence of SEQ ID NO: 1, and/or SEQ ID NO: 3, and/or SEQ ID NO: 4, and/or SEQ ID NO: 6.
[0016] Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. Usually, sequence identities or similarities are compared over the whole length of the sequences compared. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences.
[0017] Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include BLASTP and BLASTN, publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894). Preferred parameters for amino acid sequences comparison using BLASTP are gap open 11.0, gap extend 1, Blosum 62 matrix.
[0018] Nucleotide sequences encoding the enzymes expressed in the cell of the invention may also be defined by their capability to hybridise with the nucleotide sequences encoding a NAD(H) dependent fumarate reductase of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, and/or SEQ ID NO: 6, under moderate, or preferably under stringent hybridisation conditions. Stringent hybridisation conditions are herein defined as conditions that allow a nucleic acid sequence of at least about 25, preferably about 50 nucleotides, 75 or 100 and most preferably of about 200 or more nucleotides, to hybridise at a temperature of about 65° C. in a solution comprising about 1 M salt, preferably 6×SSC (sodium chloride, sodium citrate) or any other solution having a comparable ionic strength, and washing at 65° C. in a solution comprising about 0.1 M salt, or less, preferably 0.2×SSC or any other solution having a comparable ionic strength. Preferably, the hybridisation is performed overnight, i.e. at least for 10 hours and preferably washing is performed for at least one hour with at least two changes of the washing solution. These conditions will usually allow the specific hybridisation of sequences having about 90% or more sequence identity.
[0019] Moderate conditions are herein defined as conditions that allow a nucleic acid sequences of at least 50 nucleotides, preferably of about 200 or more nucleotides, to hybridise at a temperature of about 45° C. in a solution comprising about 1 M salt, preferably 6×SSC or any other solution having a comparable ionic strength, and washing at room temperature in a solution comprising about 1 M salt, preferably 6×SSC or any other solution having a comparable ionic strength. Preferably, the hybridisation is performed overnight, i.e. at least for 10 hours, and preferably washing is performed for at least one hour with at least two changes of the washing solution. These conditions will usually allow the specific hybridisation of sequences having up to 50% sequence identity. The person skilled in the art will be able to modify these hybridisation conditions in order to specifically identify sequences varying in identity between 50% and 90%.
[0020] To increase the likelihood that an introduced enzyme(s) is/are expressed in active form in a eukaryotic cell of the invention, the corresponding encoding nucleotide sequence may be adapted to optimise its codon usage to that of the chosen eukaryote host cell. Several methods for codon optimisation are known in the art. A preferred method to optimise codon usage of the nucleotide sequences to that of the eukaryotic cell is a codon pair optimization technology as disclosed in WO2008/000632. Codon-pair optimization is a method for producing a polypeptide in a host cell, wherein the nucleotide sequences encoding the polypeptide have been modified with respect to their codon-usage, in particular the codon-pairs that are used, to obtain improved expression of the nucleotide sequence encoding the polypeptide and/or improved production of the polypeptide. Codon pairs are defined as a set of two subsequent triplets (codons) in a coding sequence.
[0021] The term "gene", as used herein, refers to a nucleic acid sequence containing a template for a nucleic acid polymerase, in eukaryotes, RNA polymerase II. Genes are transcribed into mRNAs that are then translated into protein.
[0022] The term "nucleic acid" as used herein, includes reference to a deoxyribonucleotide or ribonucleotide polymer, i.e. a polynucleotide, in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof.
[0023] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids. The terms "polypeptide", "peptide" and "protein" are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.
[0024] The term "enzyme" as used herein is defined as a protein which catalyses a (bio)chemical reaction in a cell.
[0025] Usually, the nucleotide sequence encoding an enzyme is operably linked to a promoter that causes sufficient expression of the corresponding nucleotide sequence in the eukaryotic cell according to the present invention to confer to the cell the ability to produce succinic acid.
[0026] As used herein, the term "operably linked" refers to a linkage of polynucleotide elements (or coding sequences or nucleic acid sequence) in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
[0027] As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences known to one of skilled in the art. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation.
[0028] A promoter that could be used to achieve the expression of a nucleotide sequence coding for an enzyme such as NAD(H)-dependent fumarate reductase or any other enzyme introduced in the eukaryotic cell of the invention, may be not native to a nucleotide sequence coding for the enzyme to be expressed, i.e. a promoter that is heterologous to the nucleotide sequence (coding sequence) to which it is operably linked. Preferably, the promoter is homologous, i.e. endogenous to the host cell.
[0029] Suitable promoters in this context include both constitutive and inducible natural promoters as well as engineered promoters, which are well known to the person skilled in the art. Suitable promoters in eukaryotic host cells may be GAL7, GAL10, or GAL 1, CYC1, HIS3, ADH1, PGL, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, and AOX1. Other suitable promoters include PDC, GPD1, PGK1, TEF1, and TDH.
[0030] Usually a nucleotide sequence encoding an enzyme comprises a terminator. Any terminator, which is functional in the eukaryotic cell, may be used in the present invention. Preferred terminators are obtained from natural genes of the host cell. Suitable terminator sequences are well known in the art. Preferably, such terminators are combined with mutations that prevent nonsense mediated mRNA decay in the host cell of the invention (see for example: Shirley et al., 2002, Genetics 161:1465-1482).
[0031] In a preferred embodiment, a nucleotide sequence encoding a NAD(H)-dependent fumarate reductase may be overexpressed to achieve a sufficient production of succinic acid by the cell.
[0032] There are various means available in the art for overexpression of nucleotide sequences encoding enzymes in a eukaryotic cell of the invention. In particular, a nucleotide sequence encoding an enzyme may be overexpressed by increasing the copy number of the gene coding for the enzyme in the cell, e.g. by integrating additional copies of the gene in the cell's genome, by expressing the gene from a centromeric vector, from an episomal multicopy expression vector or by introducing an (episomal) expression vector that comprises multiple copies of the gene. Preferably, overexpression of the enzyme according to the invention is achieved with a (strong) constitutive promoter.
[0033] The invention also relates to a nucleotide construct comprising one or more nucleotide sequence(s) selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.
[0034] The nucleic acid construct may be a plasmid, for instance a low copy plasmid or a high copy plasmid. The eukaryotic cell according to the present invention may comprise a single, but preferably comprises multiple copies of the nucleotide sequence encoding a NAD(H) dependent fumarate reductase, for instance by multiple copies of a nucleotide construct.
[0035] The nucleic acid construct may be maintained episomally and thus comprise a sequence for autonomous replication, such as an autosomal replication sequence. If the eukaryotic cell is of fungal origin, a suitable episomal nucleic acid construct may e.g. be based on the yeast 2μ or pKD1 plasmids (Gleer et al., 1991, Biotechnology 9: 968-975), or the AMA plasmids (Fierro et al., 1995, Curr Genet. 29:482-489). Alternatively, each nucleic acid construct may be integrated in one or more copies into the genome of the eukaryotic cell. Integration into the cell's genome may occur at random by non-homologous recombination but preferably, the nucleic acid construct may be integrated into the cell's genome by homologous recombination as is well known in the art.
[0036] The nucleotide sequence encoding a NAD(H)-dependent fumarate reductase, may be a heterologous or a homologous nucleotide sequence. Preferably, the NADH-dependent fumarate reductase is a heterologous enzyme, which may be derived from any suitable origin, for instance bacteria, fungi, protozoa or plants. Preferably, the cell according to the invention comprises hetereologous a NAD(H)-dependent fumarate reductase, preferably derived from a Trypanosoma sp, for instance a Trypanosoma brucei.
[0037] In a preferred embodiment the nucleotide sequence encoding a NAD(H)-dependent fumarate reductase is expressed in the cytosol. Surprisingly, cytosolic activity of the enzyme resulted in an increased productivity of succinic acid by the eukaryotic cell.
[0038] In the event that the nucleotide sequence encoding a NAD(H)-dependent fumarate reductase comprises a peroxisomal or mitochondrial targeting signal, it may be essential to modify or delete a number of amino acids (and corresponding nucleotide sequences in the encoding nucleotide sequence) in order to prevent peroxisomal or mitochondrial targeting of the enzyme. The presence of a peroxisomal targeting signal may for instance be determined by the method disclosed by Schluter et al, Nucleic acid Research 2007, 35, D815-D822.
[0039] Preferably, the NAD(H)-dependent fumarate reductase lacks a peroxisomal or mitochondrial targeting signal for cytosolic activity of the enzyme upon expression of the encoding nucleotide sequence.
[0040] Preferably, the cell expresses a nucleotide sequence encoding an enzyme that catalyses the formation of succinic acid, wherein the nucleotide sequence preferably encodes a NAD(H)-dependent fumarate reductase, preferably a fumarate reductase comprising an amino acid sequence that has at least 40%, preferably at least 45, 50, 55, 60, 65 70, 75, 80, 85, 90, 95, 97, 98, 99% sequence identity with the amino acid sequence of SEQ ID NO: 3, and/or SEQ ID NO: 6. Preferably the nucleotide sequence encodes a NAD(H)-dependent fumarate reductase comprising the amino acid sequence of SEQ ID NO: 3, and/or SEQ ID NO: 6.
[0041] The eukaryotic cell selected from the group consisting of a yeast and a filamentous fungus, preferably belongs to one of the genera Saccharomyces, Aspergillus, Penicillium, Pichia, Kluyveromyces, Yarrowia, Candida, Hansenula, Humicola, Rhizopus, Torulaspora, Trichosporon, Brettanomyces, Zygosaccharomyces, Pachysolen or Yamadazyma. More preferably, the eukaryotic cell is a Saccharomyces cervisiae, Saccharomyces uvarum, Saccharomyces bayanus, Aspergillus niger, Penicillium chrysogenum, Pichia stipidis, Kluyveromyces marxianus, K. lactis, K. thermotolerans, Yarrowia lipolytica, Candida sonorensis, C. glabrata, Hansenula polymorpha, Torulaspora delbrueckii, Brettanomyces bruxellensis, Rhizopus orizae or Zygosaccharomyces bailiff.
[0042] In addition to a nucleotide sequence encoding a NAD(H)-dependent fumarate reductase that catalyses the conversion of fumaric acid to succinic acid, recombinant eukaryotic cell according to the present invention may comprise further genetic modifications, for instance mutations, deletions or disruptions, in homologous nucleotide sequences and/or transformation with nucleotide sequences that encode homologous or heterologous enzymes that catalyse a reaction in the cell resulting in an increased flux towards succinic acid. It may for example be favourable to introduce, genetically modify and/or overexpress heterologous and/or homologous nucleotide sequences encoding i) an enzyme that catalyses the conversion of phosphoenolpyruvate or pyruvate to oxaloacetate; ii) a malate dehydrogenase which catalyses the conversion from OAA to malic acid; or iii) a fumarase, which catalyses the conversion of malic acid to fumaric acid.
[0043] A eukaryotic cell may be transformed or genetically modified with any suitable nucleotide sequence catalyzing the reaction from a C3 to C4 carbon molecule, such as phosphoenolpyruvate (PEP, C3) to oxaloacetate (OAA, C4) and pyruvate (C3) to OAA or malic acid (C3). Suitable enzymes are PEP carboxykinase (EC 4.1.1.49, EC 4.1.1.38) and PEP carboxylase (EC 4.1.1.31) which catalyse the conversion of PEP to OAA; pyruvate carboxylase (EC 6.4.1.1.), that catalyses the reaction from pyruvate to OAA; or malic enzyme (EC 1.1.1.38), that catalyses the reaction from pyruvate to malic acid.
[0044] Preferably a eukaryotic cell according to the present invention overexpresses a nucleotide sequence encoding a pyruvate carboxylase (PYC), preferably a pyruvate carboxylase that is active in the cytosol upon expression of the nucleotide sequence encoding a PYC, for instance a PYC comprising an amino acid sequence according to SEQ ID NO: 41. Preferably, an endogenous or homologous pyruvate carboxylase is overexpressed. Surprisingly, it was found that overexpressing an endogenous pyruvate carboxylase resulted in increased succinic acid production levels by the eukaryotic cell according to the present invention.
[0045] In another preferred embodiment, a eukaryotic cell according to the present invention further comprises a nucleotide sequence encoding a heterologous PEP carboxykinase (EC 4.1.1.49) catalysing the reaction from phosphoenolpyruvate to oxaloacetate. Surprisingly it was found that a eukaryotic cell according to the present invention which further comprises a heterologous PEP carboxykinase produced an increased amount of succinic acid as compared to a eukaryotic cell that does not comprise the heterologous PEP carboxykinase. Preferably, a PEP carboxykinase that is derived from bacteria, more preferably the enzyme having PEP carboxykinase activity is derived from Escherichia coli, Mannheimia sp., Actinobacillus sp., or Anaerobiospirillum sp., more preferably Mannheimia succiniciproducens, Actinobacillus succinogenes, or Anaerobiospirillum succiniciproducens. Preferably, the PEP carboxykinase is active in the cytosol upon expression of the nucleotide sequence encoding PEP carboxykinase since it was found that this resulted in an increase succinic acid production. In one embodiment the PEP carboxykinase of Actinobacillus succinogenes (PCKa) has been modified to replace EGY at position 120-122 with a DAF amino acid sequence. Preferably, a eukaryotic cell according to the present invention comprises a PEP carboxykinase which has at least 80, 85, 90, 95 or 99% sequence identity with SEQ ID NO: 14 or SEQ ID NO: 17, preferably a PEP carboxykinase comprising SEQ ID NO: 14 or SEQ ID NO: 17. Surprisingly it was found that the concomitant (over)expression of a PYC and a PEP carboxykinase as described herein resulted in at least 1.5 increase in succinic acid production.
[0046] In another preferred embodiment a cell according to the present invention further comprises a nucleotide sequence encoding a malate dehydrogenase (MDH) which is active in the cytosol upon expression of the nucleotide sequence. A cytosolic MDH may be any suitable homologous or heterologous malate dehydrogenase. The MDH may be a S. cerevisiae MDH3 or S. cerevisiae MDH1. Preferably, the MDH lacks a peroxisomal or mitochondrial targeting signal in order to localize the enzyme in the cytosol. Alternatively, the MDH is S. cerevisiae MDH2 which has been modified such that it is not inactivated in the presence of glucose and is active in the cytosol. It is known that the transcription of MDH2 is repressed and Mdh2p is degraded upon addition of glucose to glucose-starved cells. Mdh2p deleted for the first 12 amino-terminal amino acids is less-susceptible for glucose-induced degradation (Minard and McAlister-Henn, J. Biol Chem. 1992 Aug. 25; 267(24):17458-64). Preferably, a eukaryotic cell according to the present invention comprises a nucleotide sequence encoding a malate dehydrogenase that has at least 70%, preferably at least 75, 80, 85, 90, 92, 94, 95, 96, 97, 98, 99% sequence identity with the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 21. Preferaby the malate dehydrogenase comprises SEQ ID NO: 19 or SEQ ID NO: 21. Preferably, the activity of malate dehydrogenase is increased by overexpressing the encoding nucleotide sequence by known methods in the art.
[0047] Preferably, a eukaryotic cell according to the present invention further comprises a nucleotide sequence encoding an enzyme that catalyses the conversion of malic acid to fumaric acid, which may be a heterologous or homologous enzyme, for instance a fumarase (FUM). A nucleotide sequence encoding an heterologous enzyme that catalyses the conversion of malic acid to fumaric acid, may be derived from any suitable origin, preferably from microbial origin, preferably from a yeast, for instance Saccharomyces cerevisiae or a filamentous fungus, for instance Rhizopus oryzae. Preferably, a eukaryotic cell according to the present invention comprises a nucleotide sequence encoding a fumarase that has at least 70%, preferably at least 75, 80, 85, 90, 92, 94, 95, 96, 97, 98, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 23. Preferably, the fumarase comprises SEQ ID NO: 23. Preferably the enzyme having fumarase activity is active in the cytosol upon expression of the nucleotide sequence encoding the enzyme having fumarase activity. Surprisingly, it was found that a eukaryotic cell further comprising an enzyme having fumarase activity as described herein produced an increased amount of succinic acid.
[0048] In another embodiment, a eukaryotic cell according to the present invention comprises a nucleotide sequence encoding a dicarboxylic acid transporter protein, preferably a malic acid transporter protein (MAE). A dicarboxylic acid transporter protein may be a homologous or heterologous protein. Preferably the dicarboxylic acid transporter protein is a heterologous protein. A dicarboxylic acid transporter protein may be derived from any suitable organism, preferably from Schizosaccharomyces pombe. Preferably, a dicarboxylic acid transporter protein is a malic acid transporter protein (MAE) which has at least 80, 85, 90, 95 or 99% sequence identity with SEQ ID NO: 36. Preferably the MAE comprises SEQ ID NO: 36. Surprisingly, it was found that a eukaryotic cell according to the present invention further comprising a dicarboxylic acid transporter, such as a malic acid transporter as described herein produced an increased amount of succinic acid as compared to a eukaryote cell not comprising a dicarboxylic acid transporter protein.
[0049] The present invention also relates to the use of a dicarboxylic acid transporter, preferably a malic acid transporter protein, in a eukaryotic cell to increase succinic acid production. Preferably, the malic acid transporter is derived from Schizosaccharomyces pombe.
[0050] In a preferred embodiment a eukaryotic cell according to the present invention is a yeast comprising nucleotide sequences encoding a NAD(H)-dependent fumarate reductase, a malate dehydrogenase, a heterologous fumarase, a heterologous PEP carboxykinase and a heterologous dicarboxylic acid transporter and overexpresses a pyruvate carboxylase (PYC), as described, including the preferred embodiments, herein above. Surprisingly, it found that a yeast of the invention comprising the nucleotide sequences encoding the enzymes as described herein produced an increased amount of succinic acid as compared to a yeast comprising either of the nucleotide sequences alone.
[0051] In another preferred embodiment a eukaryotic cell according to the present invention comprises reduced activity of enzymes that convert NAD(H) to NAD.sup.+ compared to the activity of these enzymes in a wild-type cell.
[0052] Preferably, the cell according to the present invention is a cell wherein at least one gene encoding alcohol dehydrogenase is not functional. An alcohol dehydrogenase gene that is not functional is used herein to describe a eukaryotic cell which comprises a reduced alcohol dehydrogenase activity compared to a cell wherein all genes encoding an alcohol dehydrogenase are functional. A gene may become not functional by known methods in the art, for instance by mutation, disruption, or deletion, for instance by the method disclosed by Gueldener et. al. 2002, Nucleic Acids Research, Vol. 30, No. 6, e23. Preferably, a eukaryotic cell is a yeast cell such as Saccharomyces cerevisiae, wherein one or more genes adh1 and/or adh2, encoding alcohol dehydrogenase are inactivated.
[0053] Preferably, the cell according to the present invention further comprises at least one gene encoding glycerol-3-phosphate dehydrogenase which is not functional. A glycerol-3-phosphate dehydrogenase gene that is not functional is used herein to describe a eukaryotic cell, which comprises a reduced glycerol-3-phosphate dehydrogenase activity, for instance by mutation, disruption, or deletion of the gene encoding glycerol-3-phosphate dehydrogenase, resulting in a decreased formation of glycerol as compared to the wild-type cell. Surprisingly, it was found that the eukaryotic cell comprising reduced alcohol dehydrogenase activity and/or glycerol-3-phosphate dehydrogenase activity and a NAD(H)-dependent fumarase resulted in an increased production of succinic acid as compared to a cell wherein one or more gene(s) encoding alcohol dehydrogenase and/or glycerol-3-phosphate dehydrogenase are not inactivated.
[0054] The present invention also relates to a process for the production of succinic acid comprising fermenting a eukaryotic cell comprising at least one gene encoding alcohol dehydrogenase is not functional and / or at least one gene encoding glycerol-3-phosphate dehydrogenase which is not functional.
[0055] In another preferred embodiment the recombinant eukaryotic cell according to the present invention comprises at least one gene encoding succinate dehydrogenase that is not functional. A succinate dehydrogenase that is not functional is used herein to describe a eukaryotic cell, which comprises a reduced succinate dehydrogenase activity by mutation, disruption, or deletion, of at least one gene encoding succinate dehydrogenase resulting in a increased formation of succinic acid as compared to the wild-type cell. A eukaryotic cell comprising a gene encoding succinate dehydrogenase that is not functional may for instance be Aspergillus niger, preferably an Aspergillus niger, wherein one or more genes encoding succinate dehydrogenase, such as sdhA and sdhB is/are not functional, for instance by deletion of these genes.
[0056] Preferably, a eukaryotic cell according to the invention is a yeast, preferably Saccharomyces cerevisiae, preferably a Saccharomyces cerevisiae comprising one or more of the nucleotide sequences selected from SEQ ID NO: 9 and SEQ ID NO: 10. A eukaryotic cell according to the present invention may also be a filamentous fungus, preferably A. niger, preferably A. niger comprising one or more nucleotide sequences selected from SEQ ID NO: 7 and SEQ ID NO: 8.
[0057] Preferably, a eukaryotic cell according to the present invention comprising any one of the genetic modifications described herein is capable of producing at least 0.3, 0.5, 0.7, g/L succinic acid, preferably at least 1 g/L succinic acid, preferably at least 1.5 preferably at least 2, or 2.5, 4.5 preferably at least 8, 10, 15, or 20 g / L succinic acid but usually below 200 or below 150 g / L.
[0058] A preferred eukaryotic cell according to the present invention may be able to grow on any suitable carbon source known in the art and convert it to succinic acid. The eukaryotic cell may be able to convert directly plant biomass, celluloses, hemicelluloses, pectines, rhamnose, galactose, fucose, maltose, maltodextrines, ribose, ribulose, or starch, starch derivatives, sucrose, lactose and glycerol. Hence, a preferred host organism expresses enzymes such as cellulases (endocellulases and exocellulases) and hemicellulases (e.g. endo- and exo-xylanases, arabinases) necessary for the conversion of cellulose into glucose monomers and hemicellulose into xylose and arabinose monomers, pectinases able to convert pectines into glucuronic acid and galacturonic acid or amylases to convert starch into glucose monomers. Preferably, the cell is able to convert a carbon source selected from the group consisting of glucose, fructose, galactose, xylose, arabinose, sucrose, raffinose, lactose and glycerol.
[0059] In another aspect, the present invention relates to a process for the preparation of succinic acid, comprising fermenting the eukaryotic cell according to the present invention, wherein succinic acid is prepared.
[0060] It was found advantageous to use a eukaryotic cell according to the invention in the process for the production of succinic acid, because most eukaryotic cells do not require sterile conditions for propagation and are insensitive to bacteriophage infections.
[0061] Preferably, the succinic acid that is prepared in the process according to the present invention is further converted into a desirable product. A desirable product may for instance be a polymer, such as polybutylene succinic acid (PBS), a deicing agent, or a surfactant.
[0062] The process according to the present invention may be run under aerobic and anaerobic conditions. Preferably, the process is carried out under anaerobic conditions or under micro-aerophilic or oxygen limited conditions. An anaerobic fermentation process is herein defined as a fermentation process run in the absence of oxygen or in which substantially no oxygen is consumed, preferably less than 5, 2.5 or 1 mmol/L/h, and wherein organic molecules serve as both electron donor and electron acceptors.
[0063] An oxygen-limited fermentation process is a process in which the oxygen consumption is limited by the oxygen transfer from the gas to the liquid. The degree of oxygen limitation is determined by the amount and composition of the ingoing gasflow as well as the actual mixing/mass transfer properties of the fermentation equipment used.
[0064] Preferably, in a process under oxygen-limited conditions, the rate of oxygen consumption is at least 5.5, more preferably at least 6 and even more preferably at least 7 mmol/L/h.
[0065] The process for the production of succinic acid according to the present invention may be carried out at any suitable pH between 1 and 9. Preferably, the pH in the fermentation broth is between 2 and 7, preferably between 3 and 5. It was found advantageous to be able to carry out the process according to the present invention at a low pH, since this prevents bacterial contamination. In addition, since the pH drops during succinic acid production, a lower amount of titrant may be needed to keep the pH at a desired level.
[0066] A suitable temperature at which the process according to the present invention may be carried out is between 5 and 60° C., preferably between 10 and 50° C., more preferably between 15 and 35° C., more preferably between 18° C. and 30° C. The skilled man in the art knows which optimal temperatures are suitable for fermenting a specific eukaryotic cell.
[0067] Preferably, succinic acid is recovered from the fermentation broth by a suitable method known in the art, for instance by crystallisation and ammonium precipitation.
[0068] Preferably, the succinic acid that is prepared in the process according to the present invention is further converted into a pharmaceutical, cosmetic, food, feed, or chemical product. Succinic acid may be further converted into a polymer, such as polybutylene succinate (PBS) or other suitable polymers derived therefrom.
[0069] The present invention also relates to a fermentation broth comprising a succinic acid obtainable by a process according to the present invention.
[0070] The invention relates to a process for the production of succinic acid by a yeast or a filamentous fungus as succinic acid producer, whereby fumarate reductase from Trypanosoma brucei is used to increase succinic acid production, wherein preferably the fumarate reductase is active in the cytosol.
[0071] Genetic Modifications
[0072] Standard genetic techniques, such as overexpression of enzymes in the host cells, genetic modification of host cells, or hybridisation techniques, are known methods in the art, such as described in Sambrook and Russel (2001) "Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987). Methods for transformation, genetic modification etc of fungal host cells are known from e.g. EP-A-0 635 574, WO 98/46772, WO 99/60102 and WO 00/37671, W090/14423, EP-A-0481008, EP-A-0635 574 and U.S. Pat. No. 6,265,186.
[0073] The following examples are for illustrative purposes only and are not to be construed as limiting the invention.
DESCRIPTION OF THE FIGURES
[0074] FIG. 1. Map of the pGBTOP-11 vector used for expression of fumarate reductase in A. niger
[0075] FIG. 2: Plasmid map of pGBS414SUS-07, encoding mitochondrial fumarate reductase m1 (FRDm1) from Trypanosoma brucei for expression in Saccharomyces cerevisiae. CPO denotes codon pair optimized.
[0076] FIG. 3: Plasmid map of pGBS414SUS-08, encoding glycosomal fumarate reductase (FRDg) from Trypanosoma brucei for expression in Saccharomyces cerevisiae. CPO denotes codon pair optimized.
[0077] FIG. 4: Plasmid map of pDEL-SDHA
[0078] FIG. 5: Map of plasmid pGBTPAn1, for overexpression FRDm1 in A. niger.
[0079] FIG. 6: Replacement scheme of sdhA
[0080] FIG. 7: Plasmid map of pGBS416FRD-1, encoding mitochondrial fumarate reductase m1 (FRDm1) from Trypanosoma brucei for expression in Saccharomyces cerevisiae. CPO denotes codon pair optimized.
[0081] FIG. 8: Plasmid map of pGBS416FRE-1, encoding glycosomal fumarate reductase (FRDg) from Trypanosoma brucei for expression in Saccharomyces cerevisiae. CPO denotes codon pair optimized.
[0082] FIG. 9: Plasmid map of pGBS414PPK-1, containing PEP carboxykinase from Actinobacillus succinogenes (PCKa) for expression in Saccharomyces cerevisiae. The synthetic gene construct TDH1 promoter-PCKa-TDH1 terminator was cloned into expression vector pRS414. CPO denotes codon pair optimized.
[0083] FIG. 10: Plasmid map of pGBS414PPK-2, containing PEP carboxykinase from Actinobacillus succinogenes (PCKa) and mitochondrial fumarate reductase m1 from Trypanosoma brucei (FRDm1) for expression in Saccharomyces cerevisiae. The synthetic gene constructs TDH1 promoter-PCKa-TDH1 terminator and TDH3 promoter-FRDm1-TDH3 terminator were cloned into expression vector pRS414. CPO denotes codon pair optimized.
[0084] FIG. 11: Plasmid map of pGBS414PPK-3, containing PEP carboxykinase from Actinobacillus succinogenes (PCKa) and glycosomal fumarate reductase from Trypanosoma brucei (FRDg) for expression in Saccharomyces cerevisiae. The synthetic gene constructs TDH1 promoter-PCKa-TDH1 terminator and TDH3 promoter-FRDg-TDH3 terminator were cloned into expression vector pRS414. CPO denotes codon pair optimized.
[0085] FIG. 12: Plasmid map of pGBS414PEK-1, containing PEP carboxykinase from Mannheimia succiniciproducens (PCKm) for expression in Saccharomyces cerevisiae. The synthetic gene construct TDH1 promoter-PCKm-TDH1 terminator was cloned into expression vector pRS414. CPO denotes codon pair optimized.
[0086] FIG. 13: Plasmid map of pGBS414PEK-2, containing PEP carboxykinase from Mannheimia succiniciproducens (PCKm) and mitochondrial fumarate reductase m1 from Trypanosoma brucei (FRDm1) for expression in Saccharomyces cerevisiae. The synthetic gene constructs TDH1 promoter-PCKm-TDH1 terminator and TDH3 promoter-FRDm1-TDH3 terminator were cloned into expression vector pRS414. CPO denotes codon pair optimized.
[0087] FIG. 14: Plasmid map of pGBS414PEK-3, containing PEP carboxykinase from Mannheimia succiniciproducens (PCKm) and glycosomal fumarate reductase from Trypanosoma brucei (FRDg) for expression in Saccharomyces cerevisiae. The synthetic gene constructs TDH1 promoter-PCKm-TDH1 terminator and TDH3 promoter-FRDg-TDH3 terminator were cloned into expression vector pRS414. CPO denotes codon pair optimized.
[0088] FIG. 15: Plasmid map of pGBS415FUM-2, containing fumarase from Rhizopus oryzae (FUMR) and cytoplasmic malate dehydrogenase from Saccharomyces cerevisiae truncated for the first 12 amino acids (delta12N MDH2) for expression in Saccharomyces cerevisiae. The synthetic gene constructs TDH1 promoter-FUMR-TDH1 terminator and DH3 promoter-MDH3-TDH3 terminator were cloned into expression vector pRS415. CPO denotes codon pair optimized.
[0089] FIG. 16: Plasmid map of pGBS415FUM-3, containing fumarase from Rhizopus oryzae (FUMR) and peroxisomal malate dehydrogenase from Saccharomyces cerevisiae (MDH3) for expression in Saccharomyces cerevisiae. The synthetic gene constructs TDH1 promoter-FUMR-TDH1 terminator and TDH3 promoter-MDH3-TDH3 terminator were cloned into expression vector pRS415. CPO denotes codon pair optimized.
[0090] FIG. 17: Succinic acid levels in strains SUC-101 (◯, empty vectors control), SUC-148 (.box-solid., overexpression of PCKa, MDH3, FUMR, FRDm1), SUC-149 (quadrature, PCKa, MDH3, FUMR, FRDg), SUC-150 (.diamond-solid., PCKm, MDH3, FUMR, FRDm1), SUC-151 (⋄, PCKm, MDH3, FUMR, FRDg), SUC-152 ( , PCKa, MDH3, FUMR), SUC-154 (×, PCKm, MDH3, FUMR) and SUC-169 (.tangle-solidup., PCKm, delta12NMDH2, FUMR, FRDm1). All overexpressed genes were codon pair optimized for expression in S. cerevisiae. All data represent averages of 3 independent growth experiments of SUC-148, 149, 150, 151, 152, 154 and SUC-169 and averages of 6 independent growth experiments of SUC-101.
[0091] FIG. 18: Plasmid map of pGBS416MAE-1, containing malate permease from Schizosaccharomyces pombe (SpMAE1) for expression in Saccharomyces cerevisiae. The synthetic gene construct EnoI promoter-MAE1-Eno1 terminator was cloned into expression vector pRS416. CPO denotes codon pair optimized.
[0092] FIG. 19: Succinic acid levels in strains SUC-101 (◯, empty vectors control), SUC-169 (.tangle-solidup., PCKm, delta12NMDH2, FUMR, FRDm1) and SUC-194 (.box-solid., PCKm, delta12NMDH2, FUMR, FRDm1, SpMAE1). All overexpressed genes were codon pair optimized for expression in S. cerevisiae. All data represent averages of 3 independent growth experiments of SUC-169 and SUC-194 and averages of 6 independent growth experiments of SUC-101.
[0093] FIG. 20: Succinic acid levels in strains SUC-103 (◯, adh1/2 and gpd1 deletion mutant; empty vectors control), SUC-201 (quadrature, adh1/2 and gpd1 deletion mutant; PCKa, MDH3, FUMR, FRDg) and SUC-200 (.box-solid., adh1/2 and gpd1 deletion mutant; PCKa, MDH3, FUMR, FRDg, SpMAE1). All overexpressed genes were codon pair optimized for expression in S. cerevisiae.
[0094] FIG. 21: Plasmid map of pGBS426PYC-2, containing pyruvate carboxylase from Saccharomyces cerevisiae for expression in Saccharomyces cerevisiae. The PYC2 coding nucleotide sequence was obtained by PCR using genomic DNA from strain CEN.PK113-5D as template and the PCR product was cloned into expression vector p426GPD.
[0095] FIG. 22: Plasmid map of pGBS414FRE-1, encoding glycosomal fumarate reductase (FRDg) from Trypanosoma brucei for expression in Saccharomyces cerevisiae. The synthetic gene construct TDH3 promoter-FRDg-TDH3 terminator was cloned into expression vector pRS414.
[0096] FIG. 23: Succinic acid levels in strains SUC-226 (quadrature, PCKa, MDH3, FUMR, FRDg), -227 (.tangle-solidup., PYC2, PCKa, MDH3, FUMR, FRDg), SUC-228 (.box-solid., PYC2, MDH3, FUMR, FRDg) and SUC-230 (◯, MDH3, FUMR, FRDg). Data represents the average of 3 independent growth experiments.
EXAMPLES
Example 1
Cloning of Fumarate Reductases from Trypanosoma Brucei in Aspergillus Niger
1.1. Expression Constructs
[0097] Mitochondrial fumarate reductase ml (FRDm1) [E.C. 1.3.1.6], GenBank accession number 60460035, from Trypanosoma brucei was analysed for the presence of signal sequences using SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/) Bendtsen, J. et al. (2004) Mol. Biol., 340:783-795 and TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP/) Emanuelsson, O. et al. (2007) Nature Protocols 2, 953-971. A putative mitochondrial targeting sequence in the N-terminal half of the protein was identified, including a possible cleavage site between pos. 25 and 26 (D-S).
[0098] It was shown that FRDm1 recombinant protein lacking the 68 N-terminal residues, relocalized to the cytosol of the procyclic trypanosomes (Coustou et al., J Biol Chem. 2005 Apr. 29; 280(17):16559-70). These results indicate that the predicted N-terminal signal motif of FRDm1 is required for targeting to the mitochondrion. The first 68 amino acids were removed from SEQ ID NO: 1 (corresponding to nucleotide sequence SEQ ID NO: 2) and a new methionine amino acid was reintroduced, which resulted in SEQ ID NO: 3. SEQ ID NO: 3 was subjected to the codon-pair method as disclosed in WO2008/000632 for A. niger. The resulting sequence SEQ ID NO: 7 was put behind the constitutive GPDA promoter sequence SEQ ID NO: 11, wherein the last 10 nucleotide sequences were replaced with optimal Kozak sequence CACCGTAAA. Convenient restriction sites were added. The stop codon TAA in SEQ: ID NO: 7 was modified to TAAA. The resulting sequence was synthesised at Sloning (Puchheim, Germany). The fragment was SnaBI, SfiI cloned in the A. niger expression vector pGBTOP11 (FIG. 1) using appropriate restriction sites. The resulting plasmid comprising FRDm1 was named pGBTOPAn1 (FIG. 5).
[0099] Likewise, glycosomal fumarate reductase (FRDg) [E.C. 1.3.1.6], GenBank accession number 23928422, from Trypanosoma brucei was analysed for peroxisomal targeting in filamentous fungi using the PTS1 predictor http://mendel.imp.ac.at/mendeljsp/sat/pts1/PTS1predictor.jsp with the fungi-specific prediction function. The C-terminal amino acids at position 1140-1142 (SKI) were removed from the protein SEQ ID NO: 4 (corresponding to nucleotide sequence SEQ ID NO: 5), resulting in SEQ ID NO: 6. SEQ ID NO: 6, was subjected to the codon-pair method as disclosed in PCT/EP2007/05594 for A. niger. The stop codon TAA in SEQ ID NO: 8 was modified to TAAA. The resulting sequence SEQ ID NO: 8 was put behind the constitutive GPDA promoter sequence SEQ ID NO: 11, and convenient restriction sites were added. The resulting sequence was synthesised at Sloning (Puchheim, Germany). The fragment was SnaBI, SfiI cloned in the A. niger expression vector pGBTOP11 (FIG. 1) using appropriate restriction sites.
1.2. Transformation of A. Niger
[0100] A. niger WT-1: This A. niger strain is CBS513.88 comprising deletions of the genes encoding glucoamylase (glaA), fungal amylase and acid amylase. A. niger WT 1 was constructed by using the "MARKER-GENE FREE" approach as described in EP 0 635 574 B1.
[0101] The expression constructs are co-transformed to strain A. niger WT-1 according to the method described by Tilburn, J. et al. (1983) Gene 26, 205-221 and Kelly, J. & Hynes, M. (1985) EMBO J., 4, 475-479 with the following modifications:
[0102] Spores are germinated and cultivated for 16 hours at 30 degrees Celsius in a shake flask placed in a rotary shaker at 300 rpm in Aspergillus minimal medium (100 ml). Aspergillus minimal medium contains per litre: 6 g NaNO3, 0.52 g KCl, 1.52 g KH2PO4, 1.12 ml 4 M KOH, 0.52 g MgSO4.7H2O, 10 g glucose, 1 g casaminoacids, 22 mg ZnSO4.7H2O, 11 mg H3BO3, 5 mg FeSO4.7H2O, 1.7 mg CoCl2.6H2O, 1.6 mg CuSO4.5H2O, 5 mg MnCl2.2H2O, 1.5 mg Na2MoO4.2H2O, 50 mg EDTA, 2 mg riboflavin, 2 mg thiamine-HCl, 2 mg nicotinamide, 1 mg pyridoxine-HCL, 0.2 mg panthotenic acid, 4 g biotin, 10 ml Penicillin (5000 IU/ml) Streptomycin (5000 UG/ml) solution (Gibco).
[0103] Novozym 234® (Novo Industries) instead of helicase is used for the preparation of protoplasts;
[0104] After protoplast formation (60-90 minutes), KC buffer (0.8 M KCl, 9.5 mM citric acid, pH 6.2) is added to a final volume of 45 ml, the protoplast suspension is centrifuged for 10 minutes at 3000 rpm at 4 degrees Celsius in a swinging-bucket rotor. The protoplasts are resuspended in 20 ml KC buffer and subsequently 25 ml of STC buffer (1.2 M sorbitol, 10 mM Tris-HCl pH 7.5, 50 mM CaCl2) is added. The protoplast suspension is centrifuged for 10 minutes at 3000 rpm at 4 degrees Celsius in a swinging-bucket rotor, washed in STC-buffer and resuspended in STC-buffer at a concentration of 10E8 protoplasts/ml;
[0105] To 200 microliter of the protoplast suspension, the DNA fragment, dissolved in 10 microliter TE buffer (10 mM Tris-HCl pH 7.5, 0.1 mM EDTA) and 100 microliter of PEG solution (20% PEG 4000 (Merck), 0.8 M sorbitol, 10 mM Tris-HCl pH 7.5, 50 mM CaCl2) is added;
[0106] After incubation of the DNA-protoplast suspension for 10 minutes at room temperature, 1.5 ml PEG solution (60% PEG 4000 (Merck), 10 mM Tris-HCl pH 7.5, 50 mM CaCl2) is added slowly, with repeated mixing of the tubes. After incubation for 20 minutes at room temperature, suspensions are diluted with 5 ml 1.2 M sorbitol, mixed by inversion and centrifuged for 10 minutes at 4000 rpm at room temperature. The protoplasts are resuspended gently in 1 ml 1.2 M sorbitol and plated onto solid selective regeneration medium consisting of either Aspergillus minimal medium without riboflavin, thiamine.HCL, nicotinamide, pyridoxine, panthotenic acid, biotin, casaminoacids and glucose. In case of acetamide selection the medium contains 10 mM acetamide as the sole nitrogen source and 1 M sucrose as osmoticum and C-source. Alternatively, protoplasts are plated onto PDA (Potato Dextrose Agar, Oxoid) supplemented with 1-50 microgram/ml phleomycin and 1M sucrose as osmosticum. Regeneration plates are solidified using 2% agar (agar No. 1, Oxoid L11). After incubation for 6-10 days at 30 degrees Celsius, conidiospores of transformants are transferred to plates consisting of Aspergillus selective medium (minimal medium containing acetamide as sole nitrogen source in the case of acetamide selection or PDA supplemented with 1-50 microgram/ml phleomycin in the case of phleomycin selection) with 2% glucose and 1.5% agarose (Invitrogen) and incubated for 5-10 days at 30 degrees Celsius. Single transformants are isolated and this selective purification step is repeated once upon which purified transformants are stored.
1.3. Shake Flask Growth of A. Niger
[0107] In total 10 transformants are selected for each construct and the presence of the construct is confirmed by PCR using primers specific for the constructs. Subsequently spores are inoculated in 100 ml Aspergillus minimal enriched medium comprising 100 g/l glucose. Strains are grown in an incubator at 250 rotations per minute for four days at 34 degrees Celsius. The supernatant of the culture medium is analysed for oxalic acid, malic acid, fumaric acid and succinic acid formation by HPLC and compared to a non transformed strain.
1.4 HPLC Analysis
[0108] HPLC is performed for the determination of organic acids and sugars in different kinds of samples. The principle of the separation on a Phenomenex Rezex-RHM-Monosaccharide column is based on size exclusion, ion-exclusion and ion-exchange using reversed phase mechanisms. Detection takes place by differential refractive index and ultra violet detectors.
Example 2A
Cloning of Fumarate Reductases from Trypanosoma Brucei in Saccharomyces Cerevisiae
2A.1. Expression Constructs
[0109] Mitochondrial fumarate reductase m1 (FRDm1) [E.C. 1.3.1.6], GenBank accession number 60460035, from Trypanosoma brucei was analysed for the presence of signal sequences and codon optimized as described in section 1.1 for expression in S. cerevisiae. The resulting sequence SEQ ID NO: 9 was put behind the constitutive TDH3 promoter sequence SEQ ID NO: 12 and before the TDH3 terminator sequence SEQ ID NO: 13, and convenient restriction sites were added. The stop codon TGA in SEQ ID NO: 9 was modified to TAAG. The resulting sequence was synthesised at Sloning (Puchheim, Germany). The expression construct pGBS414SUS-07 was created after a BamHI/NotI restriction of the S. cerevisiae expression vector pRS414 (Sirkoski R. S. and Hieter P, Genetics, 1989, 122(1):19-27) and subsequently ligating in this vector a BamHI/NotI restriction fragment consisting of the fumarate reductase synthetic gene construct (FIG. 2). The ligation mix is used for transformation of E. coli DH10B (Invitrogen) resulting in the yeast expression construct pGBS414SUS-07 (FIG. 2).
[0110] Likewise, glycosomal fumarate reductase (FRDg) [E.C. 1.3.1.6], GenBank accession number 23928422, from Trypanosoma brucei was analysed for peroxisomal targeting and codon optimisation was applied as described in section 1.1 for expression in S. cerevisiae. The resulting sequence SEQ ID NO: 10 was put behind the constitutive TDH3 promoter sequence SEQ ID NO: 12 and before the TDH3 terminator sequence SEQ ID NO: 13, and convenient restriction sites were added. The stop codon TGA in SEQ ID NO: 10 was modified to TAAG. The resulting sequence was synthesised at Sloning (Puchheim, Germany). The expression construct pGBS414SUS-08 was created after a BamHI/NotI restriction of the S. cerevisiae expression vector pRS414 (Sirkoski R. S. and Hieter P, Genetics, 1989, 122(1):19-27) and subsequently ligating in this vector a BamHI/NotI restriction fragment consisting of the fumarate reductase synthetic gene construct (FIG. 3). The ligation mix is used for transformation of E. coli DH10B (Invitrogen) resulting in the yeast expression construct pGBS414SUS-08 (FIG. 3).
[0111] The constructs pGBS414SUS-07 and pGBS414SUS-08 are independently transformed into S. cerevisiae strains CEN.PK113-6B (MATA ura3-52 leu2-112 trp1-289), RWB066 (MATA ura3-52 leu2-112 trp1-289 adh1::lox adh2::Kanlox) and RWB064 (MATA ura3-52 leu2-112 trp1-289 adh1::lox adh2::lox gpd1::Kanlox). Transformation mixtures are plated on Yeast Nitrogen Base (YNB) w/o AA (Difco)+2% glucose supplemented with appropriate amino acids. Transformants are inoculated in Verduyn medium comprising glucose supplemented with appropriate amino acids (Verduyn et al., 1992, Yeast. July; 8(7):501-17) and grown under aerobic, anaerobic and oxygen-limited conditions in shake flasks. The medium for anaerobic cultivation is supplemented with 0.01 g/l ergosterol and 0.42 g/l Tween 80 dissolved in ethanol (Andreasen and Stier, 1953, J. cell. Physiol, 41, 23-36; Andreasen and Stier, 1954, J. Cell. Physiol, 43: 271-281). All yeast cultures are grown at 30° C. in a shaking incubator at 250-280 rpm. At different incubation times, aliquots of the cultures are removed, centrifuged and the medium is analysed by HPLC for formation of oxalic acid, malic acid, fumaric acid and succinic acid as described in section 1.4.
Example 2B
Cloning of Fumarate Reductases from Trypanosoma Brucei in Saccharomyces Cerevisiae
28.1. Expression Constructs
[0112] In a similar way as disclosed in Example 2A.1. mitochondrial fumarate reductase from Trypanosoma brucei (FRDm, SEQ ID NO: 9) was ligated in a S. cerevisiae expression vector pRS416 (Sirkoski R. S. and Hieter P, Genetics, 1989, 122(1):19-27). The ligation mix was used for transformation of E. coli TOP10 cells (Invitrogen) resulting in the yeast expression constructs and pGBS416FRD-1 (FIG. 7).
[0113] Likewise, glycosomal fumarate reductase (FRDg, SEQ ID NO: 10) from Trypanosoma brucei was ligated in an S. cerevisiae expression vector pRS416. The ligation mix was used for transformation of E. coli TOP10 cells (Invitrogen) resulting in the yeast expression construct pGBS416FRE-1 (FIG. 8).
28.2. Transformation and Microtiterplates (MTP's) Growth Experiments
[0114] The constructs pGBS416FRD-1 and pGBS416FRE-1 were independently transformed into S. cerevisiae strain CEN.PK113-5D (MATA ura3-52). As negative control, empty vector pRS416 was transformed into strain CEN.PK 113-5D. Transformation mixtures were plated on Yeast Nitrogen Base (YNB) w/o AA (Difco)+2% glucose. The following numbers of individual transformants were inoculated in duplo in 250 microlitres Verduyn medium comprising 2% glucose in 96 deep-well MTP's and pre-cultured at 30 degrees Celsius, 550 rpm, and a humidity of 80% in an Infors Microplate shaking incubator: 12 pGBS416FRD-1 (FRDm1), 12 pGBS416FRE-1 (FRDg) and 24 pRS416 empty vector control transformants. After 3 days, 25 microlitres of the pre-culture present in the wells of the MTP plates was transferred to new 96 deep-well MTP's containing Verduyn medium containing glucose and CaCO3 (end-concentrations: glucose 10%, CaCO3 1% w/v in a total volume of 250 microlitres). After 3 and 7 days of growth at 30° C., 550 rpm, and a humidity of 80% in an Infors Microplate shaking incubator, the MTP's were centrifuged for 2 minutes at 2000 rpm, and 200 microliters of supernatant was harvested using the Multimek 96 (Beckman). The supernatant was analyzed by HPLC as described in Example 1.4 for the presence succinic acid. The results are shown in Table 1.
TABLE-US-00001 TABLE 1 Effect of introduction of mitochondrial (FRDm1) and glycosomal fumarate reductase (FRDg) from T. brucei in S. cerevisiae on the succinic acid production levels after 3 and 7 days of incubation S. cerevisiae comprising Succinic acid (mg/l) Succinic acid (mg/l) plasmid: after 3 days after 7 days Empty vector pRS416 138 ± 18 (n = 48) 203 ± 48 (n = 48) pGBS416FRD-1 (FRDm1) 340 ± 65 (n = 24) 399 ± 72 (n = 24) pGBS416FRE-1 (FRDg) 489 ± 30 (n = 24) 516 ± 57 (n = 24)
[0115] The results in Table 1 show that introduction and overexpression of mitochondrial fumarate reductase (FRDm1) from T. brucei resulted in increased succinic acid production levels (2.47 fold, p=6.96E-14, Student's t-test, after 3 days incubation and 1.97 fold, p=8.63E-14, Student's t-test after 7 days incubation).
[0116] Likewise, introduction and overexpression of glycosomal fumarate reductase (FRDg) from T. brucei resulted in increased succinic acid production levels (3.55 fold, p=5.08E-32, Student's t-test, after 3 days incubation and a 2.55 fold increase, p=8.63E-25, Student's t-test after 7 days incubation).
Example 2C
Expression of PEP Carboxykinase from Actinobacillus Succinogenes or Mannheimia Succiniciproducens and Malate Dehydrogenase from Saccharomyces Cerevisiae and Fumarase from Rhizopus Oryzae and Fumarate Reductase from Trypanosoma Brucei in Saccharomyces Cerevisiae
2C.1 Gene Sequences
Phosphoenolpyruvate Carboxykinase:
[0117] Phosphoenolpyruvate carboxykinase [E.C. 4.1.1.49], Gen Bank accession number 152977907, from Actinobacillus succinogenes was analysed for the presence of signal sequences using SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/) Bendtsen, J. et al. (2004) Mol. Biol., 340:783-795 and TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP/) Emanuelsson, O. et al. (2007) Nature Protocols 2, 953-971. Analysis as described by Schluter et al., (2007) NAR, 35, D815-D822 revealed a putative PTS2 signal sequence at position 115-123. The A. succinogenes sequence was modified to resemble the Mannheimia succiniciproducens protein sequence by replacing the amino acids EGY at position 120-122 with DAF resulting in amino acid sequence SEQ ID NO: 14 (nucleotide sequence SEQ ID NO: 15). SEQ ID NO: 14 was subjected to the codon-pair method as disclosed in WO2008/000632 for S. cerevisiae. The stop codon TAA in the resulting nucleotide sequence SEQ ID NO: 16 was modified to TAAG. This SEQ ID NO: 16 containing stop codon TAAG was put behind the constitutive TDH1 promoter sequence SEQ ID NO: 25 and before the TDH1 terminator sequence SEQ ID NO: 26, and convenient restriction sites were added. The resulting sequence SEQ ID NO: 29 was synthesised at Sloning (Puchheim, Germany).
[0118] Likewise phosphoenolpyruvate carboxykinase [E.C. 4.1.1.49], GenBank accession number 52426348, from Mannheimia succiniciproducens was analysed for the presence of signal sequences as described in Schluter et al., (2007) NAR, 35, D815-D822. The sequence as shown in SEQ ID NO: 17 required no modifications. SEQ ID NO: 17 was subjected to the codon-pair method as disclosed in WO2008/000632 for S. cerevisiae. The stop codon TAA in the resulting sequence SEQ ID NO: 18 was modified to TAAG. SEQ ID NO: 18 containing stop codon TAAG was put behind the constitutive TDH1 promoter sequence SEQ ID NO: 25 and before the TDH1 terminator sequence SEQ ID NO: 26. Convenient restriction sites were added. The resulting synthetic construct (SEQ ID NO: 30) was synthesised at Sloning (Puchheim, Germany).
Malate Dehydrogenase
[0119] Cytoplasmic malate dehydrogenase (Mdh2p) [E.C. 1.1.1.37], GenBank accession number 171915, is regulated by carbon catabolite repression: transcription of MDH2 is repressed and Mdh2p is degraded upon addition of glucose to glucose-starved cells. Mdh2p deleted for the 12 amino-terminal amino acids is less-susceptible for glucose-induced degradation (Minard and McAlister-Henn, J Biol Chem. 1992 Aug. 25; 267(24):17458-64). To avoid glucose-induced degradation of Mdh2, the nucleotides encoding the first 12 amino acids were removed, and a new methionine amino acid was introduced (SEQ ID NO: 19) for overexpression of Mdh2 in S. cerevisiae. SEQ ID NO: 19 was subjected to the codon-pair method as disclosed in WO2008/000632 for S. cerevisiae. The stop codon TAA in the resulting in SEQ ID NO: 20, was modified to TAAG. SEQ ID NO: 20 containing a modified stop codon TAAG, encoding delta12NMDH2, was put behind the constitutive TDH3 promoter sequence SEQ ID NO: 12 and before the TDH3 terminator sequence SEQ ID NO: 13, and convenient restriction sites were added. The resulting synthetic construct (SEQ ID NO: 31) was synthesised at Sloning (Puchheim, Germany).
[0120] Peroxisomal malate dehydrogenase (Mdh3p) [E.C. 1.1.1.37], GenBank accession number 1431095, was analysed for peroxisomal targeting in filamentous fungi using the PTS1 predictor http://mendel.imp.ac.at/mendeljsp/sat/pts1/PTS1predictor.jsp with the fungi-specific prediction function. The C-terminal amino acids at position 341-343 (SKL) were removed from protein MDH3 resulting in SEQ ID NO: 21. SEQ ID NO: 21 was subjected to the codon-pair method as disclosed in WO2008/000632 for S. cerevisiae. The stop codon TGA in the resulting sequence SEQ ID NO: 22 was modified to TAAG. SEQ ID NO: 22 containing TAAG as stop codon was synthesized behind the constitutive TDH3 promoter sequence SEQ ID NO: 27 (600 by upstream of start codon) and before the TDH3 terminator sequence SEQ ID NO: 28 (300 by downstram of stop codon), and convenient restriction sites were added. The resulting sequence SEQ ID NO: 32 was synthesised at Sloning (Puchheim, Germany).
Fumarase:
[0121] Fumarase [E.C. 4.2.1.2], GenBank accession number 469103, from Rhizopus oryzae (FumR) was analysed for the presence of signal sequences using SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/) Bendtsen, J. et al. (2004) Mol. Biol., 340:783-795 and TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP/) Emanuelsson, O. et al. (2007) Nature Protocols 2, 953-971. A putative mitochondrial targeting sequence in the first 23 amino acid of the protein was identified. To avoid potential targeting to mitochondria in S. cerevisiae, the first 23 amino acids were removed from FumR and a methionine amino acid was reintroduced resulting in SEQ ID NO: 23. SEQ ID NO: 23 was subjected to the codon-pair method as disclosed in WO2008/000632 for S. cerevisiae resulting in SEQ ID NO: 24. The stop codon TAA in SEQ ID NO: 24 was modified to TAAG. SEQ ID NO: 24 containing TAAG as stop codon was synthesized behind the constitutive TDH1 promoter sequence SEQ ID NO: 25 and before the TDH1 terminator sequence SEQ ID NO: 26 and convenient restriction sites were added. The resulting synthetic construct SEQ ID NO: 33 was synthesised at Sloning (Puchheim, Germany).
Fumarate Reductase:
[0122] Gene sequences of mitochondrial fumarate reductase (FRDm1) and glycosomal fumarate reductase (FRDg) from T. brucei were designed and synthesized as described under 2A.1.
2C.2. Construction of Expression Constructs
[0123] The expression constructs pGBS414PPK-1 (FIG. 9), pGBS414PPK-2 (FIG. 10) and pGBS414PPK-3 (FIG. 11) were created after a BamHI/NotI restriction of the S. cerevisiae expression vector pRS414 (Sirkoski R. S. and Hieter P, Genetics, 1989, 122(1):19-27) and subsequently ligating in this vector a BamHI/NotI restriction fragment consisting of the phosphoenolpyruvate carboxykinase (origin Actinobacillus succinogenes) synthetic gene construct (SEQ ID NO: 29). The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS414PPK-1. Subsequently, pGBK414PPK-1 was restricted with AscI and NotI. To create pGBS414PPK-2, an AscI/NotI restriction fragment consisting of mitochondrial fumarate reductase from T. brucei (FRDm1) synthetic gene construct (SEQ ID NO: 34) was ligated into the restricted pGBS414PPK-1 vector. The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS414PPK-2 (FIG. 10). To create pGBS414PPK-3, an AscI/NotI restriction fragment consisting of glycosomal fumarate reductase from T. brucei (FRDg) synthetic gene construct (SEQ ID NO: 35) was ligated into the restricted pGBS414PPK-1 vector. The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS414PPK-3 (FIG. 11).
[0124] The expression constructs pGBS414PEK-1 (FIG. 12), pGBS414PEK-2 (FIG. 13) and pGBS414PEK-3 (FIG. 14) were created after a BamHI/NotI restriction of the S. cerevisiae expression vector pRS414 (Sirkoski R. S. and Hieter P, Genetics, 1989, 122(1):19-27) and subsequently ligating in this vector a BamHI/NotI restriction fragment consisting of the phosphoenolpyruvate carboxykinase (origin Mannheimia succiniciproducens) synthetic gene construct (SEQ ID NO: 30). The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS414PEK-1. Subsequently, pGBK414PEK-1 was restricted with AscI and NotI. To create pGBS414PEK-2, an AscI/NotI restriction fragment consisting of mitochondrial fumarate reductase from T. brucei (FRDm1) synthetic gene construct (SEQ ID NO: 34) was ligated into the restricted pGBS414PEK-1 vector. The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS414PEK-2 (FIG. 13). To create pGBS414PEK-3, an AscI/NotI restriction fragment consisting of glycosomal fumarate reductase from T. brucei (FRDg) synthetic gene construct (SEQ ID NO: 35) was ligated into the restricted pGBS414PEK-1 vector. The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS414PEK-3 (FIG. 14).
[0125] The expression constructs pGBS415FUM-2 (FIG. 15) and pGBS415FUM-3 (FIG. 16) were created after a BamHI/NotI restriction of the S. cerevisiae expression vector pRS415 (Sirkoski R. S. and Hieter P, Genetics, 1989, 122(1):19-27) and subsequently ligating in this vector a BamHI/NotI restriction fragment consisting of the fumarase (origin Rhizopus oryzae) synthetic gene construct (SEQ ID NO: 33). The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS415FUM-1. Subsequently, pGBK415FUM-1 was restricted with AscI and NotI. To create pGBS415FUM-2, an AscI/NotI restriction fragment consisting of cytoplasmic malate dehydrogenase from S. cerevisiae (delta12N MDH2) synthetic gene construct (SEQ ID NO: 31) was ligated into the restricted pGBS415FUM-1 vector. The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS415FUM-2 (FIG. 15). To create pGBS415FUM-3, an AscI/NotI restriction fragment consisting of peroxisomal malate dehydrogenase from S. cerevisiae (MDH3) synthetic gene construct (SEQ ID NO: 32) was ligated into the restricted pGBS415FUM-1 vector. The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS415FUM-3 (FIG. 16).
2C.3. S. Cerevisiae Strains
[0126] Different combinations of plasmids pGBS414PPK-1, pGBS414 PPK-2, pGBS414PPK-3, pGBS414PEK-1, pGBS414PEK-2, pGBS414PEK-3, pGBS415FUM-2, pGBS415-FUM-3 were transformed into S. cerevisiae strain CEN.PK113-6B (MATA ura3-52 leu2-112 trp1-289), resulting in the yeast strains depicted in Table 2. In addition to the mentioned plasmids, pRS416 (empty vector) was transformed to create prototrophic yeast strains. The expression vectors were transformed into yeast by electroporation. The transformation mixtures were plated on Yeast Nitrogen Base (YNB) w/o AA (Difco)+2% glucose.
TABLE-US-00002 TABLE 2 Yeast strains constructed for Example 2C. Name Background Plasmids Genes SUC-148 CEN.PK113-6B pGBS414PPK-2 PCKa, FRDm1 pGBS415FUM-3 FUMR, MDH3 pRS416 (empty vector) SUC-149 CEN.PK113-6B pGBS414PPK-3 PCKa, FRDg pGBS415FUM-3 FUMR, MDH3 pRS416 (empty vector) SUC-150 CEN.PK113-6B pGBS414PEK-2 PCKm, FRDm1 pGBS415FUM-3 FUMR, MDH3 pRS416 (empty vector) SUC-151 CEN.PK113-6B pGBS414PEK-3 PCKm, FRDg pGBS415FUM-3 FUMR, MDH3 pRS416 (empty vector) SUC-152 CEN.PK113-6B pGBS414PPK-1 PCKa pGBS415FUM-3 FUMR, MDH3 pRS416 (empty vector) SUC-154 CEN.PK113-6B pGBS414PEK-1 PCKm pGBS415FUM-3 FUMR, MDH3 pRS416 (empty vector) SUC-169 CEN.PK113-6B pGBS414PEK-2 PCKm, FRDm1 pGBS415FUM-2 FUMR, Δ12NMDH2 pRS416 (empty vector) SUC-101 CEN.PK113-6B pRS414 (empty vector) pRS415 (empty vector) pRS415 (empty vector)
2C.4. Growth Experiments and Succinic Acid Production
[0127] Transformants were inoculated in 20 ml pre-culture medium consisting of Verduyn medium (Verduyn et al., 1992, Yeast. July; 8(7):501-17) comprising 2% galactose (w/v) and grown under aerobic conditions in 100 ml shake flasks in a shaking incubator at 30° C. at 250 rpm. After 72 hours, the culture was centrifuged for 5 minutes at 4750 rpm. 1 ml supernatant was used to measure succinic acid levels by HPLC as described in section 1.4. The remaining supernatant was decanted and the pellet (cells) was resuspended in 1 ml production medium. The production medium consisted of Verduyn medium with 10% galactose (w/v) and 1% CaCO3 (w/v). The resuspended cells were inoculated in 50 ml production medium in 100 ml shake flasks and grown in a shaking incubator at 30° C. at 100 rpm. At various time points, 1 ml sample was taken from the culture succinic acid levels were measured by HPLC as described in section 1.4 (FIG. 17).
[0128] Strains transformed with empty vectors (control strain) produced up to 0.3 g/L succinic acid. Overexpression of PEP carboxykinase from M. succiniciproducens (PCKm), peroxisomal malate dehydrogenase (MDH3) from S. cerevisiae and fumarase from R. oryzae (FUMR) resulted in production of 0.9 g/L succinic acid production. Overexpression of PEP carboxykinase from A. succinogenes (PCKa), MDH3 and FUMR resulted in a slight increase in succinic acid production to 1.0 g/L.
[0129] These results show that in S. cerevisiae as decribed increased succinic acid production about 3 times.
[0130] Additional overexpression of mitochondrial fumarate reductase (FRDm1) from T. brucei further increased succinic acid production levels; overexpression of PCKa, MDH3, FUMR, FRDm1 resulted in production of 2.6 g/L succinic acid, and overexpression of PCKm, MDH3, FUMR and FRDm1 resulted in production of 2.7 g/L succinic acid. Overexpression of delta12NMDH2 in combination with PCKm, FUMR and FRDm1 resulted in production of 2.7 g/L succinic acid, indicating that similar levels of succinic acid were produced using either truncated MDH2 or MDH3. Additional overexpression of glycosomal fumarate reductase (FRDg) from T. brucei resulted in an even higher increase in succinic acid production levels; overexpression of PCKa, MDH3, FUMR and FRDg resulted in production of 3.9 g/L succinic acid, whereas overexexpression of PCKm, MDH3, FUMR and FRDg resulted in slightly lower production of 3.6 g/L succinic acid.
[0131] The results show addition of NAD(H) dependent fumarate reductase as disclosed herein in a S. cerevisiae comprising a genetic modification of PCKa/m, MDH3 and FUMR significantly increased succinic acid production levels.
[0132] Overexpression of FRDg had a more positive effect on succinic acid production levels in S. cerevisiae compared to overexpression of FRDm1 in S. cerevisiae.
Example 2D
Effect of Overexpression of a Dicarboxylic Acid Transporter on Succinic Acid Production in Succinic Acid Producing S. Cerevisiae Cells
2D.1. Gene Sequences
[0133] Malate permease, GenBank accession number 119368831, from Schizosaccharomyces pombe (SEQ ID NO: 36) was subjected to the codon-pair method as disclosed in WO2008/000632 for S. cerevisiae resulting in SEQ ID NO: 37. The stop codon TAA in SEQ ID NO: 37 was modified to TAAG. SEQ ID NO: 37 containing TAAG as stop codon was put behind the constitutive ENO1 promoter sequence SEQ ID NO: 38 and before the ENO1 terminator sequence SEQ ID NO: 39, and convenient restriction sites were added. In the ENO1 promotor, T at position 596 (-5) was changed to A in order to obtain a better Kozak sequence. The resulting sequence SEQ ID NO: 40 was synthesised at Sloning (Puchheim, Germany).
2D.2. Construction of Expression Constructs
[0134] The expression constructs pGBS416MAE-1 (FIG. 18) was created after a BamHI/NotI restriction of the S. cerevisiae expression vector pRS416 (Sirkoski R. S. and Hieter P, Genetics, 1989, 122(1):19-27) and subsequently ligating in this vector a BamHI/NotI restriction fragment consisting of the Schizosaccharomyces pombe malate transporter synthetic gene construct (SEQ ID NO: 40). The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS416MAE-1.
2D.3. S. Cerevisiae Strains
[0135] Plasmids pGBS414PEK-2, pGBS415FUM-2 and pGBS416MAE-1 (described under 2C.2.) were transformed into S. cerevisiae strain CEN.PK113-6B (MATA ura3-52 leu2-112 trp1-289) to create strain SUC-194, overexpressing PCKm, delta12NMDH2, FUMR, FRDm1 and SpMAE1. All genes were codon pair optimized for expression in S. cerevisiae.
[0136] The expression vectors were transformed into yeast by electroporation. The transformation mixtures were plated on Yeast Nitrogen Base (YNB) w/o AA (Difco)+2% glucose. Strains SUC-101 is described in Table 2.
TABLE-US-00003 TABLE 3 Yeast strains constructed for Example 2D. Name Background Plasmids Genes SUC-132 CEN.PK113-6B pGBS414PEK-2 PCKm, FRDm1 pGBS415FUM-2 FUMR, Δ12NMDH2 pRS416 (empty vector) SUC-194 CEN.PK113-6B pGBS414PEK-2 PCKm, FRDm1 pGBS415FUM-2 FUMR, Δ12NMDH2 pRS416MAE-1 SpMAE1
2D.4. Growth Experiments and Succinic Acid Production in Wildtype CEN.PK Strains
[0137] Growth parameters and sample analysis were performed as described under example 2C.4 with the following modifications: pre-culturing was performed using 2% glucose (w/v) as carbon source. In the production medium 10% glucose (w/v) was used as carbon source.
[0138] Strains transformed with empty vectors (control strain) produced up to 0.3 g/L succinic acid. Additional overexpression of SpMAE1 in strain SUC-194, overexpressing PCKm, delta12NMDH2, FUMR and FRDm1 resulted in increased succinic acid production levels to 4.6 g/L, whereas strain SUC-132, overexpressing PCKm, delta12NMDH2, FUMR and FRDm1 resulted in production of 2.7 g/L succinic acid.
[0139] The results show that insertion of a malate transporter in a S. cerevisiae comprising the genetic modifications as described herein further increased succinic acid production at least 1.5 times.
Example 2E
Effect of a Dicarboxylic Acid Transporter in S. Cerevisiae Comprising a Deletion of the Genes Alcohol Dehydrogenase 1 and 2 (adh1, adh2) and the gene glycerol-3-phosphate dehydrogenase 1 (gpd1) on Succinic Acid Production Levels
2E.1. Gene Sequences
[0140] Described under 2D.1.
2E.2. Construction of Expression Constructs
[0141] Described under 2D.2.
2E.3. S. Cerevisiae Strains
[0142] Plasmids pGBS414PPK-3, pGBS415FUM-3 and pGBS416MAE-1 (described under 2C.2.) were transformed into S. cerevisiae strain RWB064 (MA TA ura3-52 leu2-112 trp1-289 adh1::lox adh2::lox gpd1::Kanlox) to create strain SUC-201, overexpressing PCKa, MDH3, FUMR, FRDg and SpMAE1. All genes were codon pair optimized for expression in S. cerevisiae.
TABLE-US-00004 TABLE 4 Yeast strains constructed for Example 2E. Name Background Plasmids Genes SUC-200 CEN.PK113-6B pGBS414PPK-3 PCKa, FRDg adh1::lox adh2::lox pGBS415FUM-3 FUMR, MDH3 gpd1::Kanlox pGBS416MAE-1 SpMAE1 SUC-201 CEN.PK113-6B pGBS414PPK-3 PCKa, FRDg adh1::lox adh2::lox pGBS415FUM-3 FUMR, MDH3 gpd1::Kanlox pRS416 (empty vector) SUC-103 CEN.PK113-6B pRS414 (empty vector) adh1::lox adh2::lox pRS415 (empty vector) gpd1::Kanlox pRS415 (empty vector)
2E.4. Growth Experiments and Succinic Acid Production in CEN.PK Strains Deleted for the Genes Alcohol Dehydrogenase 1 and 2 (Adh1, Adh2) and the Gene glycerol-3-phosphate dehydrogenase 1 (gpd1)
[0143] Growth parameters and sample analysis were performed as described under example 2C.4 with the following modifications: pre-culturing was performed using 2% galactose (w/v) as carbon source. 5% galactose (w/v) was added to the production medium at t=0, 3 and 7 days.
[0144] Strain SUC-103 transformed with empty vectors (control strain) produced 0.9 g/L succinic acid after growth for 10 days in production medium (FIG. 20). Overexpression of PCKa, MDH3, FUMR and FRDg in strain RWB064 resulted in increased succinic acid production levels to 2.5 g/L (strain SUC-201, FIG. 20). Additional overexpression of SpMAE1 besides PCKa, MDH3, FUMR and FRDg in strain RWB064 resulted in a further increase of succinic acid production levels to 11.9 g/L (strain SUC-200, FIG. 20).
[0145] The results show that overexpression of a malate transporter in S. cerevisiea comprising a deletion of alcohol dehydrogenase and glycerol-3-phosphate dehydrogenase genes resulted in a significant increase in succinic acid production levels. In addition it was shown that deletion of the gene adh1, adh2 and gpd1 (SUC 103) resulted in increased succinic acid production levels as compare to a wild type strain (SUC 101, Table 2).
Example 2F
Cloning of Phosphoenolpyruvate Carboxykinase from Actinobacillus Succinogenes, Pyruvate Carboxylase from Saccharomyces Cerevisiae, Malate Dehydrogenase from Saccharomyces Cerevisiae, Fumarase from Rhizopus Oryzae in Saccharomyces Cerevisiae and Fumarate Reductase from Trypanosoma Brucei
2F.1. Gene Sequences
[0146] Gene sequences of PEP carboxykinase from A. succinogenes, malate dehydrogenase from S. cerevisiae, fumarase from R. oryzae and fumarate reductase from T. brucei are described under 2F.1. Cytoplasmic pyruvate carboxylase from Saccharomyces cerevisiae (Pyc2p) [E.C. 6.4.1.1.], GenBank accession number 1041734, SEQ ID NO: 41, is encoded by the nucleotide sequence SEQ ID NO: 42. Genomic DNA from S. cerevisiae strain CEN.PK113-5D (MATA ura3-52) was used as template to amplify the PYC2 coding sequence (SEQ ID NO: 42), using primers P1 (SEQ ID NO: 43) and P2 (SEQ ID NO: 44), and the Phusion DNA polymerase (Finnzymes, Finland) according to manufacturer's instructions. Convenient restriction sites were included in the primers for further cloning purposes.
2F.2. Construction of Expression Constructs
[0147] The expression construct pGBS426PYC-2 (FIG. 21) was created after a SpeI/XhoI restriction of the S. cerevisiae expression vector p426GPD (Mumberg et al., Gene. 1995 Apr. 14; 156(1):119-22) and subsequently ligating in this vector a SpeI/XhoI restriction fragment consisting of the amplified PYC2 nucleotide sequence (SEQ ID NO: 42). The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS426PYC-2 (FIG. 21). Construction of expression vectors pGBS414PPK-3 and pGBS415FUM-3 is described under 2C.2. Expression construct pGBS414FRE-1 was created after a BamHI/NotI restriction of the S. cerevisiae expression vector pRS414 (Sirkoski R. S. and Hieter P, Genetics, 1989, 122(1):19-27) and subsequently ligating in this vector a BamHI/NotI restriction fragment consisting of the glycosomal fumarate reductase (origin Trypanosoma brucei) synthetic gene construct (SEQ ID NO: 35). The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS414FRE-1 (FIG. 22).
2F.3. S. Cerevisiae Strains
[0148] Strains SUC-226, SUC-227, SUC-228 and SUC-230 were obtained by transformation of different combinations of the plasmids pGBS414FRE-1, pGBS414PPK-3, pGBS415FUM-1, pGBS426PYC-2 and p426GPD into strain CEN.PK113-6B (MATA ura3-52 leu2-112 trp1-289), as depicted in Table 5.
TABLE-US-00005 TABLE 5 Yeast strains constructed for Example 2F. Name Background Plasmids Genes SUC-226 CEN.PK113-6B pGBS414PPK-3 PCKa, FRDg pGBS415FUM-3 FUMR, MDH3 p426GPD (empty vector) SUC-227 CEN.PK113-6B pGBS414PPK-3 PCKa, FRDg pGBS415FUM-3 FUMR, MDH3 pGBS426PYC-2 PYC2 SUC-228 CEN.PK113-6B pGBS414FRE-1 FRDg pGBS415FUM-3 FUMR, MDH3 pGBS426PYC-2 PYC2 SUC-230 CEN.PK113-6B pGBS414FRE-1 FRDg pGBS415FUM-3 FUMR, MDH3 p426GPD (empty vector)
2F.4. Growth Experiments and Succinic Acid Production
[0149] Growth parameters and sample analysis were performed as described under example 2C.4 with the following modifications: pre-culturing was performed using 2% glucose (w/v) as carbon source. In the production medium 10% glucose (w/v) was used as carbon source.
[0150] As depicted in FIG. 23 strain SUC-230, overexpressing MDH3, FUMR and FRDg, produced up to 3.0 g/L succinic acid. Additional overexpression of PCKa increased succinic acid production up to 3.4 g/L (strain SUC-226), and additional overexpression of PYC2 increased succinic acid production up to 3.7 g/L (strain SUC-228). Surprisingly, overexpression of both PCKa and PYC2 (SUC-227) resulted in 1.5 increase of succinic acid production levels up to 5.0 g/L, as compared to the effect of PCK and PYC alone. These results show a synergistic effect of combined overexpression of both PEP carboxykinase from A. succinogenes (PCKa) and pyruvate carboxylase from S. cerevisiae (PYC2) on succinic acid production levels in S. cerevisiae.
Example 3
Inactivation of Succinate Dehydrogenase Encoding Genes in Aspergillus Niger
3.1. Identification
[0151] Genomic DNA of Aspergillus niger strain CBS513.88 was sequenced and analyzed. Two genes with translated proteins annotated as homologues to succinate dehydrogenase proteins were identified and named sdhA and sdhB respectively. Sequences of the sdhA (An16g07150) and sdhB (An02g12770) loci are available on genbank with accession numbers 145253004 and 145234071 respectively. Gene replacement vectors for sdhA and sdhB were designed according to known principles and constructed according to routine cloning procedures (see FIG. 6). The vectors comprise approximately 1000 by flanking regions of the sdh ORFs for homologous recombination at the predestined genomic loci. In addition, they contain the A. nidulans bi-directional amdS selection marker driven by the gpdA promoter, in-between direct repeats. The general design of these deletion vectors were previously described in EP635574B and WO 98/46772.
3.2. Inactivation of the SdhA Gene in Aspergillus Niger.
[0152] Linear DNA of deletion vector pDEL-SDHA (FIG. 4) was isolated and used to transform Aspergillus niger CBS513.88 as described in: Biotechnology of Filamentous fungi: Technology and Products. (1992) Reed Publishing (USA); Chapter 6: Transformation p. 113 to 156. This linear DNA can integrate into the genome at the sdhA locus, thus substituting the sdhA gene by the amdS gene as depicted in FIG. 6. Transformants were selected on acetamide media and colony purified according to standard procedures as described in EP635574B. Spores were plated on fluoro-acetamide media to select strains, which lost the amdS marker. Growing colonies were diagnosed by PCR for integration at the sdhA locus and candidate strains tested by Southern analyses for deletion of the sdhA gene. Deletion of the sdhA gene was detectable by the ˜2.2 kb size reduction of DNA fragments (4.6 kb wild-type fragment versus 2.4 kb for a successful deletion of SDHA) covering the entire locus and hybridized to appropriate probes. Approximately 9 strains showed a removal of the genomic sdhA gene from a pool of approximately 96 initial transformants.
[0153] Strain dSDHA was selected as a representative strain with the sdhA gene inactivated. The succinic acid production of dSDHA was determined in microtiter plates as described in Example 4.
Example 4
Cloning of FRDm from Trypanosoma Brucei in Aspergillus Niger dSDHA
[0154] A. niger strain dSDHA of example 3.2. was transformed with the expression construct pGBTOPAn1 (FIG. 5) comprising truncated mitochondrial fumarate reductase m1 (FRDm1, SEQ ID NO:7) as described in Example 1.1. E. coli DNA was removed by NotI digestion. A. niger transformants were picked using Qpix and transferred onto MTP's containing Aspergillus selective media. After 7 days incubation at 30 degrees Celsius the biomass was transferred to microtiter plates (MTP's) containing PDA by hand or colony picker. After 7 days incubation at 30 degrees Celsius, the biomass was sporulated. These spores were resuspended using the Multimek 96 (Beckman) in 100 microlitres minimal enriched Aspergillus medium containing 10% glucose. Subsequently 2 MTP with 170 micolitres minimal enriched Aspergillus medium containing 10% glucose and 1% CaCO3 were inoculated with 30 microlitres of the spore suspension. Likewise, A. niger strains dSDHA and CBS513.88 were inoculated in the MTP's. These MTP's were incubated for 5 days at 34 degrees Celsius80% humidity. After 5 days 160 microlitres were harvested using the Multimek 96 (Beckman) and succinic acid was determined by HPLC as described in Example 1.4. The results are shown in Table 6.
TABLE-US-00006 TABLE 6 Effect of deletion of succinate dehydrogenase (SDHA) and insertion of mitochondrial fumarate reductase (FRDm1) from T. brucei in A. niger on succinic acid production levels. A. niger strain Succinic acid mg/l CBS513.88 38 dSDHA 50 dSDHA + gGBTOPAn1 583 (FRDm1)
[0155] Table 6 clearly shows an increased production of succinic acid by A. niger that comprises mitochondrial fumarate reductase from T. brucei
Sequence CWU
1
4411232PRTTrypanosoma brucei 1Met Leu Ser Thr Lys Gln Leu Leu Leu Arg Ala
Thr Ser Ala Leu Val1 5 10
15Ala Gly Ser Ser Gly Val Ala Arg Asp Ser Pro Ser Leu Val Gly Asp
20 25 30Pro Cys Asp Ser Val Ser Pro
Thr Arg Val Val Trp Gly Arg Phe Phe 35 40
45Lys Ser Leu Ala Pro Pro Ala Pro Ser Val Val Ser Cys Gln Lys
Arg 50 55 60Phe Thr Ser His Gly Ala
Asp Gly Ile Ser Ser Ala Ser Ile Val Val65 70
75 80Thr Asp Pro Glu Ala Ala Ala Lys Lys Arg Asp
Arg Met Ala Arg Glu 85 90
95Leu Leu Ser Ser Asn Ser Gly Leu Cys Gln Glu Asp Glu Pro Thr Ile
100 105 110Ile Asn Leu Lys Gly Leu
Glu His Thr Ile Pro Tyr Arg Leu Ala Val 115 120
125Val Leu Cys Asn Ser Arg Ser Thr Gly Glu Phe Glu Ala Lys
Ala Ala 130 135 140Glu Ile Leu Arg Lys
Ala Phe His Met Val Asp Tyr Ser Leu Asn Cys145 150
155 160Phe Asn Pro Glu Ser Glu Leu Ser Arg Val
Asn Ser Leu Pro Val Gly 165 170
175Glu Lys His Gln Met Ser Glu Asp Leu Arg His Val Met Glu Cys Thr
180 185 190Ile Ser Val His His
Ser Ser Gly Met Gly Phe Asp Pro Ala Ala Gly 195
200 205Pro Ile Ile Ser Arg Leu Arg Gly Ala Met Arg Asp
His Asn Asp Met 210 215 220Ser Asp Ile
Ser Val Thr Glu Ala Glu Val Glu Leu Phe Ser Leu Ala225
230 235 240Gln Ser Phe Asp Val Asp Leu
Glu Glu Gly Thr Ile Ala Arg Lys His 245
250 255Ser Glu Ala Arg Leu Asp Leu Gly Gly Val Asn Lys
Gly Tyr Thr Val 260 265 270Asp
Tyr Val Val Asp His Leu Arg Ala Ala Gly Met Pro Asn Val Leu 275
280 285Phe Glu Trp Gly Gly Asp Ile Arg Ala
Ser Gly Arg Asn Ile Lys Gly 290 295
300Asn Leu Trp Ala Val Ala Ile Lys Arg Pro Pro Ser Val Glu Glu Val305
310 315 320Ile Arg Arg Ala
Lys Gly Lys Met Leu Lys Met Gly Glu Glu Glu Gln 325
330 335Glu Glu Lys Asp Asp Asp Ser Pro Ser Leu
Leu His Val Val Glu Leu 340 345
350Asp Asp Glu Ala Leu Cys Thr Ser Gly Asp Tyr Glu Asn Val Leu Tyr
355 360 365His Pro Lys His Gly Val Ala
Gly Ser Ile Phe Asp Trp Gln Arg Arg 370 375
380Gly Leu Leu Ser Pro Glu Glu Gly Ala Leu Ala Gln Val Ser Val
Lys385 390 395 400Cys Tyr
Ser Ala Met Tyr Ala Asp Ala Leu Ala Thr Val Cys Leu Val
405 410 415Lys Arg Asp Ala Val Arg Ile
Arg Tyr Leu Leu Glu Gly Trp Arg Tyr 420 425
430Val Arg Ser Arg Val Thr Asn Tyr Phe Ala Tyr Thr Arg Gln
Gly Glu 435 440 445Arg Leu Ala His
Met His Glu Ile Ala Gln Glu Thr Arg Glu Leu Arg 450
455 460Glu Ile Arg Ile Ala Gly Ser Leu Pro Ser Arg Ile
Val Ile Val Gly465 470 475
480Gly Gly Leu Ala Gly Leu Ser Ala Ala Ile Glu Ala Ala Ser Cys Gly
485 490 495Ala Gln Val Ile Leu
Met Glu Lys Glu Gly Arg Ile Gly Gly Asn Ser 500
505 510Ala Lys Ala Thr Ser Gly Ile Asn Gly Trp Gly Thr
Arg Thr Gln Ala 515 520 525Lys Ser
Asp Ile Leu Asp Gly Gly Lys Tyr Phe Glu Arg Asp Thr Phe 530
535 540Leu Ser Gly Val Gly Gly Thr Thr Asp Pro Ala
Leu Val Lys Val Leu545 550 555
560Ser Val Lys Ser Gly Asp Ala Ile Gly Trp Leu Thr Ser Leu Gly Val
565 570 575Pro Leu Ser Val
Leu Ser Gln Leu Gly Gly His Ser Phe Lys Arg Thr 580
585 590His Arg Ala Pro Asp Lys Thr Asp Gly Thr Pro
Leu Pro Ile Gly His 595 600 605Thr
Ile Met Arg Thr Leu Glu Asp His Ile Arg Asn Asn Leu Ser Glu 610
615 620Arg Val Thr Ile Met Thr His Val Ser Val
Thr Glu Leu Leu His Glu625 630 635
640Thr Asp Thr Thr Pro Asp Gly Ala Ser Glu Val Arg Val Thr Gly
Val 645 650 655Arg Tyr Arg
Asp Leu Ser Asp Val Asp Gly Gln Pro Ser Lys Leu Leu 660
665 670Ala Asp Ala Val Val Leu Ala Thr Gly Gly
Phe Ser Asn Asp Arg Glu 675 680
685Glu Asn Ser Leu Leu Cys Lys Tyr Ala Pro His Leu Ala Ser Phe Pro 690
695 700Thr Thr Asn Gly Pro Trp Ala Thr
Gly Asp Gly Val Lys Leu Ala Thr705 710
715 720Ser Val Gly Ala Lys Leu Val Asp Met Asp Lys Val
Gln Leu His Pro 725 730
735Thr Gly Leu Ile Asp Pro Lys Asp Pro Ala Asn Thr Thr Lys Ile Leu
740 745 750Gly Pro Glu Ala Leu Arg
Gly Ser Gly Gly Ile Leu Leu Asn Lys Gln 755 760
765Gly Lys Arg Phe Val Asn Glu Leu Asp Leu Arg Ser Val Val
Ser Lys 770 775 780Ala Ile Asn Thr Gln
Gly Asn Glu Tyr Pro Gly Ser Gly Gly Cys Tyr785 790
795 800Phe Ala Tyr Cys Val Leu Asn Glu Asp Ala
Thr Asn Leu Phe Cys Gly 805 810
815Gly Ala Leu Gly Phe Tyr Gly Lys Lys Leu Gly Leu Phe Gln Arg Ala
820 825 830Glu Thr Val Glu Glu
Leu Ala Lys Leu Ile Gly Cys Asp Glu Gly Glu 835
840 845Leu Arg Asp Thr Leu Glu Lys Tyr Glu Thr Cys Ser
Lys Ala Lys Val 850 855 860Ala Cys Pro
Val Thr Gly Lys Val Val Phe Pro Cys Val Val Gly Thr865
870 875 880Arg Gly Pro Tyr Asn Val Ala
Phe Val Thr Pro Ser Ile His Tyr Thr 885
890 895Met Gly Gly Cys Leu Ile Ser Pro Ala Ala Glu Val
Leu Gln Glu Tyr 900 905 910Lys
Gly Leu Asn Ile Leu Glu Asn His Arg Pro Ile Arg Cys Leu Phe 915
920 925Gly Ala Gly Glu Val Thr Gly Gly Val
His Gly Gly Asn Arg Leu Gly 930 935
940Gly Asn Ser Leu Leu Glu Cys Val Val Phe Gly Lys Ile Ala Gly Asp945
950 955 960Arg Ala Ala Thr
Ile Leu Gln Lys Arg Glu Ile Ala Leu Ser Lys Thr 965
970 975Ser Trp Thr Ser Val Val Val Arg Glu Ser
Arg Ser Gly Glu Gln Phe 980 985
990Gly Thr Gly Ser Arg Val Leu Arg Phe Asn Leu Pro Gly Ala Leu Gln
995 1000 1005Arg Thr Gly Leu Asn Leu
Gly Glu Phe Val Ala Ile Arg Gly Glu 1010 1015
1020Trp Asp Gly Gln Gln Leu Val Gly Tyr Phe Ser Pro Ile Thr
Leu 1025 1030 1035Pro Glu Asp Leu Gly
Thr Ile Ser Leu Leu Val Arg Ala Asp Lys 1040 1045
1050Gly Thr Leu Lys Glu Trp Ile Cys Ala Leu Arg Pro Gly
Asp Ser 1055 1060 1065Val Glu Ile Lys
Ala Cys Gly Gly Leu Arg Ile Asp Gln Asp Pro 1070
1075 1080Val Lys Lys Cys Leu Leu Phe Arg Asn Arg Pro
Ile Thr Arg Phe 1085 1090 1095Ala Leu
Val Ala Ala Gly Thr Gly Val Ala Pro Met Leu Gln Val 1100
1105 1110Ile Arg Ala Ala Leu Lys Lys Pro Tyr Val
Asp Thr Leu Glu Ser 1115 1120 1125Ile
Arg Leu Ile Tyr Ala Ala Glu Glu Tyr Asp Thr Leu Thr Tyr 1130
1135 1140Arg Ser Ile Leu Gln Arg Phe Ala Glu
Glu Phe Pro Asp Lys Phe 1145 1150
1155Val Cys Asn Phe Val Leu Asn Asn Pro Pro Glu Gly Trp Thr Gly
1160 1165 1170Gly Val Gly Phe Val Asn
Lys Lys Ser Leu Gln Lys Val Leu Gln 1175 1180
1185Pro Pro Ser Ser Glu Pro Leu Ile Val Val Cys Gly Pro Pro
Val 1190 1195 1200Met Gln Arg Asp Val
Lys Asn Glu Leu Leu Ser Met Gly Tyr Asp 1205 1210
1215Lys Glu Leu Val His Thr Val Asp Gly Glu Ser Gly Thr
Leu 1220 1225 123023698DNATrypanosoma
brucei 2atgctctcaa cgaagcaact tctccttcga gccacatctg cattagtggc gggaagctct
60ggagttgcgc gagacagccc ttcgcttgtc ggcgaccctt gcgactcggt ttcaccaacg
120cgggtcgtat gggggcgctt cttcaaatcc ctagcgccac ccgctccctc ggttgtttca
180tgtcaaaagc gttttacgtc ccatggcgcc gatggtattt cctcggcttc gattgttgtc
240actgacccgg aggcggcagc aaagaagcgt gaccgcatgg cgcgcgagtt gctctcaagt
300aatagtggtc tttgtcaaga agatgaaccc actatcatta acttaaaggg gttggagcac
360acgattccgt acaggctcgc cgtggttctt tgtaactcgc gctctacagg tgaattcgaa
420gcaaaggcag ctgagatttt gcgaaaggca tttcacatgg tggactactc cctcaattgt
480ttcaatcctg aaagcgagtt gtcgcgtgtc aactctctgc cggtgggtga gaagcatcaa
540atgtcggagg atctccggca cgtgatggag tgcaccatca gtgtacatca ctccagcgga
600atgggcttcg acccggcggc aggtccaatt atcagccgac ttcggggggc aatgagggac
660cacaacgaca tgtccgacat ttccgtaacg gaagccgagg tagagctctt ctccttagcg
720caaagttttg acgtggacct cgaggaggga acaatagctc gcaagcactc tgaagcgagg
780cttgatcttg gtggtgtgaa caaaggctac acagttgatt atgtagtgga tcatcttcgt
840gcggccggta tgccaaacgt gctctttgag tggggcgggg atattcgagc gtcgggtagg
900aacatcaaag gaaacctatg ggcagttgct atcaaacgac cgccatctgt ggaggaggtg
960attcggcgcg ccaaagggaa aatgttaaaa atgggggagg aggagcagga agagaaggac
1020gatgattctc catccctgct tcatgtggtg gagcttgatg atgaagccct ttgcaccagt
1080ggtgactacg aaaacgtttt gtatcatcca aagcatggag tggcggggag catttttgac
1140tggcagcgaa gggggctact atctcctgag gaaggggcac tcgctcaagt gtctgtgaaa
1200tgttatagcg caatgtacgc tgatgctctg gcaacagtgt gccttgtgaa gcgtgatgct
1260gtgaggattc gctacttatt agagggctgg cgttacgttc gaagtcgtgt gacgaattac
1320tttgcctata cccgtcaggg cgagcggtta gcacatatgc acgagatagc gcaagaaaca
1380cgggagctac gtgaaatacg gattgccggg agtttgccct ccagaattgt tattgtgggt
1440ggaggtctag cgggcctttc agcggccatc gaagccgcaa gttgtggtgc acaagtcata
1500ctcatggaaa aggaaggaag aatcgggggg aacagcgcaa aggctacatc aggtattaat
1560gggtggggga cgcgtacgca ggcaaagtca gatattctcg acggtggaaa gtattttgag
1620cgtgacactt ttctctctgg cgttggcggt actaccgatc ctgccctcgt caaagtgctc
1680tcagttaaga gtggggacgc aattggttgg cttacttctc ttggtgtgcc actcagtgtc
1740ctctcgcaac ttggtggcca cagtttcaag cgaacccacc gtgccccgga caaaacggac
1800gggacacccc taccaattgg tcatacgatc atgagaaccc tcgaggatca catccgtaac
1860aacctctctg agcgagtaac gattatgaca catgtgtccg tgaccgagtt attgcacgaa
1920accgatacaa cacctgatgg cgcctccgaa gtgcgtgtta cgggtgtaag atacagggac
1980ctctccgatg tggatggcca gccatcaaaa ttgcttgcgg atgccgtcgt tcttgcaact
2040ggtggtttct ccaatgaccg tgaagaaaat tcactgctct gcaagtatgc gcctcacctg
2100gccagttttc caacgacaaa tggcccctgg gcgaccggtg acggggttaa actcgcaaca
2160tcggttggtg caaagcttgt ggatatggat aaggttcagc tacaccccac agggcttatc
2220gatccaaagg atcccgcgaa cacaacgaag attctcggcc cggaggcact ccgaggttca
2280ggtgggatat tactcaacaa gcaaggaaag cgcttcgtga atgaacttga cctccgctct
2340gttgtatcca aggcaattaa tacgcagggt aatgaatacc ctggatccgg tggatgttac
2400tttgcgtact gcgtgctcaa cgaagatgca acaaacctct tctgtggcgg tgcactgggg
2460ttctacggaa agaagcttgg tttgttccag cgtgctgaga ctgtggaaga gttggccaaa
2520ctgattggct gtgacgaagg tgaattacgg gatacgcttg aaaagtatga aacttgcagc
2580aaggccaaag ttgcgtgccc tgtgacgggg aaggtagtat tcccttgtgt ggtgggtaca
2640agggggccgt acaatgttgc ttttgtcacg ccttccattc attacacaat gggtggctgc
2700ctcatttcac cggctgctga agttcttcag gagtacaaag gtttaaatat tctggaaaac
2760catagaccga ttcgatgctt gtttggtgcc ggtgaagtga cgggtggtgt gcacggtggt
2820aaccgccttg gtggtaattc gctcttggaa tgtgtggtat tcgggaaaat tgcgggtgac
2880cgtgccgcaa caatacttca aaaacgtgag atagccctct ccaagacgag ttggacttcc
2940gttgttgtac gtgagtcccg ctccggcgaa cagttcggga ccggctctcg tgttcttcgt
3000tttaacctac ctggggcgct gcagcgcaca ggtctcaatc tgggcgaatt tgtggccatc
3060cgtggcgagt gggacggcca acaacttgtt ggttacttca gtccaattac actaccagag
3120gaccttggca ctatctccct tctggttcgt gccgacaagg gcacattgaa ggaatggatc
3180tgcgccttgc gaccgggcga ctccgtcgaa atcaaagcgt gtggaggtct tcgtattgat
3240caagacccgg taaagaagtg tctgctgttt cgtaaccggc ctattacgcg gtttgctctt
3300gtcgcggcag ggactggtgt cgcgcccatg ttgcaggtta ttcgtgcggc actcaagaag
3360ccttacgtgg acacgttgga aagcatccgt cttatatacg ccgcagaaga gtacgacaca
3420ttgacgtatc gctcaatttt gcagcggttt gcggaagagt tccccgacaa gttcgtctgc
3480aacttcgttc ttaacaaccc acccgaaggg tggacaggtg gagtggggtt tgtcaacaaa
3540aaatccctgc agaaggtgct gcaaccgcca tcgagtgagc cgctgattgt tgtgtgtgga
3600ccgcccgtga tgcagcgcga cgtgaagaat gagttactga gcatgggtta tgacaaagag
3660ctcgttcata cggttgacgg cgagtcggga acgctgta
369831164PRTArtificial sequenceFRDm Trypanosoma lacking 68 aa targeting
signal 3Met Ala Asp Gly Ile Ser Ser Ala Ser Ile Val Val Thr Asp Pro Glu1
5 10 15Ala Ala Ala Lys
Lys Arg Asp Arg Met Ala Arg Glu Leu Leu Ser Ser 20
25 30Asn Ser Gly Leu Cys Gln Glu Asp Glu Pro Thr
Ile Ile Asn Leu Lys 35 40 45Gly
Leu Glu His Thr Ile Pro Tyr Arg Leu Ala Val Val Leu Cys Asn 50
55 60Ser Arg Ser Thr Gly Glu Phe Glu Ala Lys
Ala Ala Glu Ile Leu Arg65 70 75
80Lys Ala Phe His Met Val Asp Tyr Ser Leu Asn Cys Phe Asn Pro
Glu 85 90 95Ser Glu Leu
Ser Arg Val Asn Ser Leu Pro Val Gly Glu Lys His Gln 100
105 110Met Ser Glu Asp Leu Arg His Val Met Glu
Cys Thr Ile Ser Val His 115 120
125His Ser Ser Gly Met Gly Phe Asp Pro Ala Ala Gly Pro Ile Ile Ser 130
135 140Arg Leu Arg Gly Ala Met Arg Asp
His Asn Asp Met Ser Asp Ile Ser145 150
155 160Val Thr Glu Ala Glu Val Glu Leu Phe Ser Leu Ala
Gln Ser Phe Asp 165 170
175Val Asp Leu Glu Glu Gly Thr Ile Ala Arg Lys His Ser Glu Ala Arg
180 185 190Leu Asp Leu Gly Gly Val
Asn Lys Gly Tyr Thr Val Asp Tyr Val Val 195 200
205Asp His Leu Arg Ala Ala Gly Met Pro Asn Val Leu Phe Glu
Trp Gly 210 215 220Gly Asp Ile Arg Ala
Ser Gly Arg Asn Ile Lys Gly Asn Leu Trp Ala225 230
235 240Val Ala Ile Lys Arg Pro Pro Ser Val Glu
Glu Val Ile Arg Arg Ala 245 250
255Lys Gly Lys Met Leu Lys Met Gly Glu Glu Glu Gln Glu Glu Lys Asp
260 265 270Asp Asp Ser Pro Ser
Leu Leu His Val Val Glu Leu Asp Asp Glu Ala 275
280 285Leu Cys Thr Ser Gly Asp Tyr Glu Asn Val Leu Tyr
His Pro Lys His 290 295 300Gly Val Ala
Gly Ser Ile Phe Asp Trp Gln Arg Arg Gly Leu Leu Ser305
310 315 320Pro Glu Glu Gly Ala Leu Ala
Gln Val Ser Val Lys Cys Tyr Ser Ala 325
330 335Met Tyr Ala Asp Ala Leu Ala Thr Val Cys Leu Val
Lys Arg Asp Ala 340 345 350Val
Arg Ile Arg Tyr Leu Leu Glu Gly Trp Arg Tyr Val Arg Ser Arg 355
360 365Val Thr Asn Tyr Phe Ala Tyr Thr Arg
Gln Gly Glu Arg Leu Ala His 370 375
380Met His Glu Ile Ala Gln Glu Thr Arg Glu Leu Arg Glu Ile Arg Ile385
390 395 400Ala Gly Ser Leu
Pro Ser Arg Ile Val Ile Val Gly Gly Gly Leu Ala 405
410 415Gly Leu Ser Ala Ala Ile Glu Ala Ala Ser
Cys Gly Ala Gln Val Ile 420 425
430Leu Met Glu Lys Glu Gly Arg Ile Gly Gly Asn Ser Ala Lys Ala Thr
435 440 445Ser Gly Ile Asn Gly Trp Gly
Thr Arg Thr Gln Ala Lys Ser Asp Ile 450 455
460Leu Asp Gly Gly Lys Tyr Phe Glu Arg Asp Thr Phe Leu Ser Gly
Val465 470 475 480Gly Gly
Thr Thr Asp Pro Ala Leu Val Lys Val Leu Ser Val Lys Ser
485 490 495Gly Asp Ala Ile Gly Trp Leu
Thr Ser Leu Gly Val Pro Leu Ser Val 500 505
510Leu Ser Gln Leu Gly Gly His Ser Phe Lys Arg Thr His Arg
Ala Pro 515 520 525Asp Lys Thr Asp
Gly Thr Pro Leu Pro Ile Gly His Thr Ile Met Arg 530
535 540Thr Leu Glu Asp His Ile Arg Asn Asn Leu Ser Glu
Arg Val Thr Ile545 550 555
560Met Thr His Val Ser Val Thr Glu Leu Leu His Glu Thr Asp Thr Thr
565 570 575Pro Asp Gly Ala Ser
Glu Val Arg Val Thr Gly Val Arg Tyr Arg Asp 580
585 590Leu Ser Asp Val Asp Gly Gln Pro Ser Lys Leu Leu
Ala Asp Ala Val 595 600 605Val Leu
Ala Thr Gly Gly Phe Ser Asn Asp Arg Glu Glu Asn Ser Leu 610
615 620Leu Cys Lys Tyr Ala Pro His Leu Ala Ser Phe
Pro Thr Thr Asn Gly625 630 635
640Pro Trp Ala Thr Gly Asp Gly Val Lys Leu Ala Thr Ser Val Gly Ala
645 650 655Lys Leu Val Asp
Met Asp Lys Val Gln Leu His Pro Thr Gly Leu Ile 660
665 670Asp Pro Lys Asp Pro Ala Asn Thr Thr Lys Ile
Leu Gly Pro Glu Ala 675 680 685Leu
Arg Gly Ser Gly Gly Ile Leu Leu Asn Lys Gln Gly Lys Arg Phe 690
695 700Val Asn Glu Leu Asp Leu Arg Ser Val Val
Ser Lys Ala Ile Asn Thr705 710 715
720Gln Gly Asn Glu Tyr Pro Gly Ser Gly Gly Cys Tyr Phe Ala Tyr
Cys 725 730 735Val Leu Asn
Glu Asp Ala Thr Asn Leu Phe Cys Gly Gly Ala Leu Gly 740
745 750Phe Tyr Gly Lys Lys Leu Gly Leu Phe Gln
Arg Ala Glu Thr Val Glu 755 760
765Glu Leu Ala Lys Leu Ile Gly Cys Asp Glu Gly Glu Leu Arg Asp Thr 770
775 780Leu Glu Lys Tyr Glu Thr Cys Ser
Lys Ala Lys Val Ala Cys Pro Val785 790
795 800Thr Gly Lys Val Val Phe Pro Cys Val Val Gly Thr
Arg Gly Pro Tyr 805 810
815Asn Val Ala Phe Val Thr Pro Ser Ile His Tyr Thr Met Gly Gly Cys
820 825 830Leu Ile Ser Pro Ala Ala
Glu Val Leu Gln Glu Tyr Lys Gly Leu Asn 835 840
845Ile Leu Glu Asn His Arg Pro Ile Arg Cys Leu Phe Gly Ala
Gly Glu 850 855 860Val Thr Gly Gly Val
His Gly Gly Asn Arg Leu Gly Gly Asn Ser Leu865 870
875 880Leu Glu Cys Val Val Phe Gly Lys Ile Ala
Gly Asp Arg Ala Ala Thr 885 890
895Ile Leu Gln Lys Arg Glu Ile Ala Leu Ser Lys Thr Ser Trp Thr Ser
900 905 910Val Val Val Arg Glu
Ser Arg Ser Gly Glu Gln Phe Gly Thr Gly Ser 915
920 925Arg Val Leu Arg Phe Asn Leu Pro Gly Ala Leu Gln
Arg Thr Gly Leu 930 935 940Asn Leu Gly
Glu Phe Val Ala Ile Arg Gly Glu Trp Asp Gly Gln Gln945
950 955 960Leu Val Gly Tyr Phe Ser Pro
Ile Thr Leu Pro Glu Asp Leu Gly Thr 965
970 975Ile Ser Leu Leu Val Arg Ala Asp Lys Gly Thr Leu
Lys Glu Trp Ile 980 985 990Cys
Ala Leu Arg Pro Gly Asp Ser Val Glu Ile Lys Ala Cys Gly Gly 995
1000 1005Leu Arg Ile Asp Gln Asp Pro Val
Lys Lys Cys Leu Leu Phe Arg 1010 1015
1020Asn Arg Pro Ile Thr Arg Phe Ala Leu Val Ala Ala Gly Thr Gly
1025 1030 1035Val Ala Pro Met Leu Gln
Val Ile Arg Ala Ala Leu Lys Lys Pro 1040 1045
1050Tyr Val Asp Thr Leu Glu Ser Ile Arg Leu Ile Tyr Ala Ala
Glu 1055 1060 1065Glu Tyr Asp Thr Leu
Thr Tyr Arg Ser Ile Leu Gln Arg Phe Ala 1070 1075
1080Glu Glu Phe Pro Asp Lys Phe Val Cys Asn Phe Val Leu
Asn Asn 1085 1090 1095Pro Pro Glu Gly
Trp Thr Gly Gly Val Gly Phe Val Asn Lys Lys 1100
1105 1110Ser Leu Gln Lys Val Leu Gln Pro Pro Ser Ser
Glu Pro Leu Ile 1115 1120 1125Val Val
Cys Gly Pro Pro Val Met Gln Arg Asp Val Lys Asn Glu 1130
1135 1140Leu Leu Ser Met Gly Tyr Asp Lys Glu Leu
Val His Thr Val Asp 1145 1150 1155Gly
Glu Ser Gly Thr Leu 116041142PRTTrypanosoma brucei 4Met Val Asp Gly
Arg Ser Ser Ala Ser Ile Val Ala Val Asp Pro Glu1 5
10 15Arg Ala Ala Arg Glu Arg Asp Ala Ala Ala
Arg Ala Leu Leu Gln Asp 20 25
30Ser Pro Leu His Thr Thr Met Gln Tyr Ala Thr Ser Gly Leu Glu Leu
35 40 45Thr Val Pro Tyr Ala Leu Lys Val
Val Ala Ser Ala Asp Thr Phe Asp 50 55
60Arg Ala Lys Glu Val Ala Asp Glu Val Leu Arg Cys Ala Trp Gln Leu65
70 75 80Ala Asp Thr Val Leu
Asn Ser Phe Asn Pro Asn Ser Glu Val Ser Leu 85
90 95Val Gly Arg Leu Pro Val Gly Gln Lys His Gln
Met Ser Ala Pro Leu 100 105
110Lys Arg Val Met Ala Cys Cys Gln Arg Val Tyr Asn Ser Ser Ala Gly
115 120 125Cys Phe Asp Pro Ser Thr Ala
Pro Val Ala Lys Ala Leu Arg Glu Ile 130 135
140Ala Leu Gly Lys Glu Arg Asn Asn Ala Cys Leu Glu Ala Leu Thr
Gln145 150 155 160Ala Cys
Thr Leu Pro Asn Ser Phe Val Ile Asp Phe Glu Ala Gly Thr
165 170 175Ile Ser Arg Lys His Glu His
Ala Ser Leu Asp Leu Gly Gly Val Ser 180 185
190Lys Gly Tyr Ile Val Asp Tyr Val Ile Asp Asn Ile Asn Ala
Ala Gly 195 200 205Phe Gln Asn Val
Phe Phe Asp Trp Gly Gly Asp Cys Arg Ala Ser Gly 210
215 220Met Asn Ala Arg Asn Thr Pro Trp Val Val Gly Ile
Thr Arg Pro Pro225 230 235
240Ser Leu Asp Met Leu Pro Asn Pro Pro Lys Glu Ala Ser Tyr Ile Ser
245 250 255Val Ile Ser Leu Asp
Asn Glu Ala Leu Ala Thr Ser Gly Asp Tyr Glu 260
265 270Asn Leu Ile Tyr Thr Ala Asp Asp Lys Pro Leu Thr
Cys Thr Tyr Asp 275 280 285Trp Lys
Gly Lys Glu Leu Met Lys Pro Ser Gln Ser Asn Ile Ala Gln 290
295 300Val Ser Val Lys Cys Tyr Ser Ala Met Tyr Ala
Asp Ala Leu Ala Thr305 310 315
320Ala Cys Phe Ile Lys Arg Asp Pro Ala Lys Val Arg Gln Leu Leu Asp
325 330 335Gly Trp Arg Tyr
Val Arg Asp Thr Val Arg Asp Tyr Arg Val Tyr Val 340
345 350Arg Glu Asn Glu Arg Val Ala Lys Met Phe Glu
Ile Ala Thr Glu Asp 355 360 365Ala
Glu Met Arg Lys Arg Arg Ile Ser Asn Thr Leu Pro Ala Arg Val 370
375 380Ile Val Val Gly Gly Gly Leu Ala Gly Leu
Ser Ala Ala Ile Glu Ala385 390 395
400Ala Gly Cys Gly Ala Gln Val Val Leu Met Glu Lys Glu Ala Lys
Leu 405 410 415Gly Gly Asn
Ser Ala Lys Ala Thr Ser Gly Ile Asn Gly Trp Gly Thr 420
425 430Arg Ala Gln Ala Lys Ala Ser Ile Val Asp
Gly Gly Lys Tyr Phe Glu 435 440
445Arg Asp Thr Tyr Lys Ser Gly Ile Gly Gly Asn Thr Asp Pro Ala Leu 450
455 460Val Lys Thr Leu Ser Met Lys Ser
Ala Asp Ala Ile Gly Trp Leu Thr465 470
475 480Ser Leu Gly Val Pro Leu Thr Val Leu Ser Gln Leu
Gly Gly His Ser 485 490
495Arg Lys Arg Thr His Arg Ala Pro Asp Lys Lys Asp Gly Thr Pro Leu
500 505 510Pro Ile Gly Phe Thr Ile
Met Lys Thr Leu Glu Asp His Val Arg Gly 515 520
525Asn Leu Ser Gly Arg Ile Thr Ile Met Glu Asn Cys Ser Val
Thr Ser 530 535 540Leu Leu Ser Glu Thr
Lys Glu Arg Pro Asp Gly Thr Lys Gln Ile Arg545 550
555 560Val Thr Gly Val Glu Phe Thr Gln Ala Gly
Ser Gly Lys Thr Thr Ile 565 570
575Leu Ala Asp Ala Val Ile Leu Ala Thr Gly Gly Phe Ser Asn Asp Lys
580 585 590Thr Ala Asp Ser Leu
Leu Arg Glu His Ala Pro His Leu Val Asn Phe 595
600 605Pro Thr Thr Asn Gly Pro Trp Ala Thr Gly Asp Gly
Val Lys Leu Ala 610 615 620Gln Arg Leu
Gly Ala Gln Leu Val Asp Met Asp Lys Val Gln Leu His625
630 635 640Pro Thr Gly Leu Ile Asn Pro
Lys Asp Pro Ala Asn Pro Thr Lys Phe 645
650 655Leu Gly Pro Glu Ala Leu Arg Gly Ser Gly Gly Val
Leu Leu Asn Lys 660 665 670Gln
Gly Lys Arg Phe Val Asn Glu Leu Asp Leu Arg Ser Val Val Ser 675
680 685Lys Ala Ile Met Glu Gln Gly Ala Glu
Tyr Pro Gly Ser Gly Gly Ser 690 695
700Met Phe Ala Tyr Cys Val Leu Asn Ala Ala Ala Gln Lys Leu Phe Gly705
710 715 720Val Ser Ser His
Glu Phe Tyr Trp Lys Lys Met Gly Leu Phe Val Lys 725
730 735Ala Asp Thr Met Arg Asp Leu Ala Ala Leu
Ile Gly Cys Pro Val Glu 740 745
750Ser Val Gln Gln Thr Leu Glu Glu Tyr Glu Arg Leu Ser Ile Ser Gln
755 760 765Arg Ser Cys Pro Ile Thr Arg
Lys Ser Val Tyr Pro Cys Val Leu Gly 770 775
780Thr Lys Gly Pro Tyr Tyr Val Ala Phe Val Thr Pro Ser Ile His
Tyr785 790 795 800Thr Met
Gly Gly Cys Leu Ile Ser Pro Ser Ala Glu Ile Gln Met Lys
805 810 815Asn Thr Ser Ser Arg Ala Pro
Leu Ser His Ser Asn Pro Ile Leu Gly 820 825
830Leu Phe Gly Ala Gly Glu Val Thr Gly Gly Val His Gly Gly
Asn Arg 835 840 845Leu Gly Gly Asn
Ser Leu Leu Glu Cys Val Val Phe Gly Arg Ile Ala 850
855 860Gly Asp Arg Ala Ser Thr Ile Leu Gln Arg Lys Ser
Ser Ala Leu Ser865 870 875
880Phe Lys Val Trp Thr Thr Val Val Leu Arg Glu Val Arg Glu Gly Gly
885 890 895Val Tyr Gly Ala Gly
Ser Arg Val Leu Arg Phe Asn Leu Pro Gly Ala 900
905 910Leu Gln Arg Ser Gly Leu Ser Leu Gly Gln Phe Ile
Ala Ile Arg Gly 915 920 925Asp Trp
Asp Gly Gln Gln Leu Ile Gly Tyr Tyr Ser Pro Ile Thr Leu 930
935 940Pro Asp Asp Leu Gly Met Ile Asp Ile Leu Ala
Arg Ser Asp Lys Gly945 950 955
960Thr Leu Arg Glu Trp Ile Ser Ala Leu Glu Pro Gly Asp Ala Val Glu
965 970 975Met Lys Ala Cys
Gly Gly Leu Val Ile Glu Arg Arg Leu Ser Asp Lys 980
985 990His Phe Val Phe Met Gly His Ile Ile Asn Lys
Leu Cys Leu Ile Ala 995 1000
1005Gly Gly Thr Gly Val Ala Pro Met Leu Gln Ile Ile Lys Ala Ala
1010 1015 1020Phe Met Lys Pro Phe Ile
Asp Thr Leu Glu Ser Val His Leu Ile 1025 1030
1035Tyr Ala Ala Glu Asp Val Thr Glu Leu Thr Tyr Arg Glu Val
Leu 1040 1045 1050Glu Glu Arg Arg Arg
Glu Ser Arg Gly Lys Phe Lys Lys Thr Phe 1055 1060
1065Val Leu Asn Arg Pro Pro Pro Leu Trp Thr Asp Gly Val
Gly Phe 1070 1075 1080Ile Asp Arg Gly
Ile Leu Thr Asn His Val Gln Pro Pro Ser Asp 1085
1090 1095Asn Leu Leu Val Ala Ile Cys Gly Pro Pro Val
Met Gln Arg Ile 1100 1105 1110Val Lys
Ala Thr Leu Lys Thr Leu Gly Tyr Asn Met Asn Leu Val 1115
1120 1125Arg Thr Val Asp Glu Thr Glu Pro Ser Gly
Ser Ser Lys Ile 1130 1135
114053429DNATrypanosoma brucei 5atggtagacg ggcgatcttc tgcatcaatt
gttgccgttg atcccgaaag ggctgcgcgt 60gagcgcgacg cagcagcgcg tgcccttctt
caagacagtc cgctacacac gaccatgcaa 120tatgcaacgt ctggtcttga gcttaccgtt
ccctatgcac ttaaggtggt tgccagtgct 180gacaccttcg atcgcgctaa ggaggttgcc
gatgaggtgc tacgctgcgc atggcaactc 240gccgacaccg tgttgaacag tttcaacccg
aacagtgagg tttcactcgt gggtcgcctg 300cctgtggggc agaagcacca aatgtctgct
ccactcaagc gtgtgatggc atgctgccag 360cgtgtgtata actcatcggc tggatgtttt
gatccctcca cagcacccgt cgcaaaggcg 420ctgcgtgaga ttgcactggg gaaggagcgg
aacaatgctt gtctggaggc acttactcaa 480gcgtgtacgc ttcccaacag ttttgtgatc
gatttcgaag ctggaactat cagccgtaag 540cacgagcatg cgtctctgga cctaggtggg
gttagcaaag gttatatcgt tgattatgtc 600attgataata tcaatgctgc tggatttcaa
aacgtttttt ttgactgggg tggagactgc 660cgtgcgagtg gtatgaatgc gcgcaatacc
ccgtgggttg ttggtataac tcgccctccg 720tcccttgata tgctccctaa cccgccaaag
gaggcgtcgt atatcagcgt tatctctctc 780gacaacgagg cccttgccac gagtggcgat
tatgaaaact taatatacac cgctgatgat 840aaacccctta cctgcactta tgactggaag
gggaaggaac tgatgaaacc ttctcagtcc 900aatatcgcgc aggtatcggt taaatgttat
agcgccatgt acgctgacgc gcttgcgact 960gcgtgtttca taaagcggga tcccgcgaag
gttcgacagc tgctggacgg ttggcgttac 1020gtgcgtgata cagtgagaga ttacagggtc
tacgttcgtg aaaatgagcg agtagcgaag 1080atgtttgaga tcgccacaga ggatgcggaa
atgaggaaga ggcggatcag caacacactt 1140cccgctcgtg tcattgtggt gggcggtggt
cttgcgggtt tgtccgcggc catcgaagct 1200gcaggatgcg gtgctcaggt tgtgcttatg
gagaaggagg cgaagctcgg aggcaacagc 1260gccaaggcga catctggtat caacggatgg
ggcacacgtg ctcaggcgaa ggcaagcatt 1320gtggatggtg ggaaatactt cgagcgtgac
acatacaagt ctggtatcgg gggtaacacc 1380gatcctgccc ttgtgaagac actttctatg
aaaagtgctg acgctattgg gtggctgacc 1440tcgttgggtg taccgctgac ggtattgtca
cagcttgggg gtcacagccg caagcgcaca 1500catcgggcac cggataagaa agatggtaca
cctctaccta tcggatttac aatcatgaaa 1560accctcgagg atcacgtgcg tggtaacctt
tctggccgca tcaccataat ggaaaactgc 1620agtgtaacgt cgttgctcag tgagacgaag
gaacggccag atggcactaa acagatacga 1680gttactggtg tggagttcac gcaggctggc
agtgggaaga cgaccatact tgcagatgct 1740gtcatccttg ccactggtgg attttctaac
gacaaaactg cagactccct gcttcgtgag 1800cacgccccgc acttggtcaa cttccctacg
acgaatggcc cgtgggcgac aggtgatggc 1860gtgaaacttg cacagcgact tggcgctcaa
ctggtggata tggacaaggt ccagttgcat 1920ccgacaggcc tcatcaaccc gaaggatcca
gcgaacccta caaagttcct tggacctgag 1980gcgctacgtg gatccggtgg cgttttgttg
aacaagcaag gcaagcgctt cgttaatgaa 2040cttgacctcc gttctgtggt atcgaaagcc
atcatggaac agggtgcgga atatcctgga 2100tcgggtggta gcatgttcgc ctactgtgtg
ttgaatgctg cggcgcagaa gctctttggt 2160gtcagctcac acgagttcta ctggaagaag
atgggtctct tcgtgaaggc tgacaccatg 2220agggacctcg ctgcactcat tgggtgccca
gtggaatctg tgcagcagac gctggaggag 2280tacgagcggc tctccatatc acagcgttcc
tgccccatca cgcgcaaaag cgtctatccg 2340tgcgtgctcg gcactaaggg cccctactac
gtcgccttcg tgacaccttc gattcactac 2400acaatgggtg gatgtctcat ctcgccttct
gctgaaatac aaatgaagaa cacatcatca 2460cgcgctccac tgagtcacag caacccaatc
ctcgggttat ttggtgccgg tgaggtaacg 2520ggtggtgtgc acggtgggaa ccggttgggc
ggcaattcgc tgcttgagtg cgtcgtgttt 2580gggagaattg cgggtgatcg ggcctcgacc
atccttcaga ggaagtcctc agcactttcc 2640ttcaaggtgt ggacgaccgt ggtgctgcgt
gaagtacgcg aaggtggtgt gtacggtgct 2700gggtcccgcg tgcttcgctt taatttaccc
ggggcgctgc aacggtctgg tctgagcctc 2760ggccaattta tcgcaattcg tggtgattgg
gacggtcagc agttgatcgg ttattacagt 2820cccatcacgc tgccagatga tcttggcatg
atcgatatac tcgcccgcag tgataagggg 2880acgctgaggg agtggatttc cgctctggag
ccgggtgacg ctgtggagat gaaggcatgc 2940ggtggtctgg tgattgagcg ccgcttaagc
gataagcact ttgtgttcat gggacacatt 3000atcaacaagc tttgtctaat tgctggtgga
acgggtgtgg caccgatgct gcaaataatc 3060aaagcagcct ttatgaaacc cttcattgac
acattggaga gcgttcatct catctatgcc 3120gcggaggacg tgacggagtt gacgtatcgc
gaggtgctgg aggagcgccg tcgtgagtca 3180cgtggaaagt tcaagaaaac gtttgtcctc
aaccggcccc cgcccctatg gactgatggt 3240gttggcttca tcgaccgggg catcctcaca
aatcatgtgc agccgccatc tgacaacctg 3300ctggtggcca tatgcggacc accggtaatg
cagcgcattg taaaggcgac cctgaagact 3360ttgggctaca acatgaacct tgtgaggact
gtggatgaaa cggagccgag cggctcatcc 3420aaaatttga
342961139PRTArtificial sequenceFRDg
lacking 3 aa C-terminal targeting signal 6Met Val Asp Gly Arg Ser Ser Ala
Ser Ile Val Ala Val Asp Pro Glu1 5 10
15Arg Ala Ala Arg Glu Arg Asp Ala Ala Ala Arg Ala Leu Leu
Gln Asp 20 25 30Ser Pro Leu
His Thr Thr Met Gln Tyr Ala Thr Ser Gly Leu Glu Leu 35
40 45Thr Val Pro Tyr Ala Leu Lys Val Val Ala Ser
Ala Asp Thr Phe Asp 50 55 60Arg Ala
Lys Glu Val Ala Asp Glu Val Leu Arg Cys Ala Trp Gln Leu65
70 75 80Ala Asp Thr Val Leu Asn Ser
Phe Asn Pro Asn Ser Glu Val Ser Leu 85 90
95Val Gly Arg Leu Pro Val Gly Gln Lys His Gln Met Ser
Ala Pro Leu 100 105 110Lys Arg
Val Met Ala Cys Cys Gln Arg Val Tyr Asn Ser Ser Ala Gly 115
120 125Cys Phe Asp Pro Ser Thr Ala Pro Val Ala
Lys Ala Leu Arg Glu Ile 130 135 140Ala
Leu Gly Lys Glu Arg Asn Asn Ala Cys Leu Glu Ala Leu Thr Gln145
150 155 160Ala Cys Thr Leu Pro Asn
Ser Phe Val Ile Asp Phe Glu Ala Gly Thr 165
170 175Ile Ser Arg Lys His Glu His Ala Ser Leu Asp Leu
Gly Gly Val Ser 180 185 190Lys
Gly Tyr Ile Val Asp Tyr Val Ile Asp Asn Ile Asn Ala Ala Gly 195
200 205Phe Gln Asn Val Phe Phe Asp Trp Gly
Gly Asp Cys Arg Ala Ser Gly 210 215
220Met Asn Ala Arg Asn Thr Pro Trp Val Val Gly Ile Thr Arg Pro Pro225
230 235 240Ser Leu Asp Met
Leu Pro Asn Pro Pro Lys Glu Ala Ser Tyr Ile Ser 245
250 255Val Ile Ser Leu Asp Asn Glu Ala Leu Ala
Thr Ser Gly Asp Tyr Glu 260 265
270Asn Leu Ile Tyr Thr Ala Asp Asp Lys Pro Leu Thr Cys Thr Tyr Asp
275 280 285Trp Lys Gly Lys Glu Leu Met
Lys Pro Ser Gln Ser Asn Ile Ala Gln 290 295
300Val Ser Val Lys Cys Tyr Ser Ala Met Tyr Ala Asp Ala Leu Ala
Thr305 310 315 320Ala Cys
Phe Ile Lys Arg Asp Pro Ala Lys Val Arg Gln Leu Leu Asp
325 330 335Gly Trp Arg Tyr Val Arg Asp
Thr Val Arg Asp Tyr Arg Val Tyr Val 340 345
350Arg Glu Asn Glu Arg Val Ala Lys Met Phe Glu Ile Ala Thr
Glu Asp 355 360 365Ala Glu Met Arg
Lys Arg Arg Ile Ser Asn Thr Leu Pro Ala Arg Val 370
375 380Ile Val Val Gly Gly Gly Leu Ala Gly Leu Ser Ala
Ala Ile Glu Ala385 390 395
400Ala Gly Cys Gly Ala Gln Val Val Leu Met Glu Lys Glu Ala Lys Leu
405 410 415Gly Gly Asn Ser Ala
Lys Ala Thr Ser Gly Ile Asn Gly Trp Gly Thr 420
425 430Arg Ala Gln Ala Lys Ala Ser Ile Val Asp Gly Gly
Lys Tyr Phe Glu 435 440 445Arg Asp
Thr Tyr Lys Ser Gly Ile Gly Gly Asn Thr Asp Pro Ala Leu 450
455 460Val Lys Thr Leu Ser Met Lys Ser Ala Asp Ala
Ile Gly Trp Leu Thr465 470 475
480Ser Leu Gly Val Pro Leu Thr Val Leu Ser Gln Leu Gly Gly His Ser
485 490 495Arg Lys Arg Thr
His Arg Ala Pro Asp Lys Lys Asp Gly Thr Pro Leu 500
505 510Pro Ile Gly Phe Thr Ile Met Lys Thr Leu Glu
Asp His Val Arg Gly 515 520 525Asn
Leu Ser Gly Arg Ile Thr Ile Met Glu Asn Cys Ser Val Thr Ser 530
535 540Leu Leu Ser Glu Thr Lys Glu Arg Pro Asp
Gly Thr Lys Gln Ile Arg545 550 555
560Val Thr Gly Val Glu Phe Thr Gln Ala Gly Ser Gly Lys Thr Thr
Ile 565 570 575Leu Ala Asp
Ala Val Ile Leu Ala Thr Gly Gly Phe Ser Asn Asp Lys 580
585 590Thr Ala Asp Ser Leu Leu Arg Glu His Ala
Pro His Leu Val Asn Phe 595 600
605Pro Thr Thr Asn Gly Pro Trp Ala Thr Gly Asp Gly Val Lys Leu Ala 610
615 620Gln Arg Leu Gly Ala Gln Leu Val
Asp Met Asp Lys Val Gln Leu His625 630
635 640Pro Thr Gly Leu Ile Asn Pro Lys Asp Pro Ala Asn
Pro Thr Lys Phe 645 650
655Leu Gly Pro Glu Ala Leu Arg Gly Ser Gly Gly Val Leu Leu Asn Lys
660 665 670Gln Gly Lys Arg Phe Val
Asn Glu Leu Asp Leu Arg Ser Val Val Ser 675 680
685Lys Ala Ile Met Glu Gln Gly Ala Glu Tyr Pro Gly Ser Gly
Gly Ser 690 695 700Met Phe Ala Tyr Cys
Val Leu Asn Ala Ala Ala Gln Lys Leu Phe Gly705 710
715 720Val Ser Ser His Glu Phe Tyr Trp Lys Lys
Met Gly Leu Phe Val Lys 725 730
735Ala Asp Thr Met Arg Asp Leu Ala Ala Leu Ile Gly Cys Pro Val Glu
740 745 750Ser Val Gln Gln Thr
Leu Glu Glu Tyr Glu Arg Leu Ser Ile Ser Gln 755
760 765Arg Ser Cys Pro Ile Thr Arg Lys Ser Val Tyr Pro
Cys Val Leu Gly 770 775 780Thr Lys Gly
Pro Tyr Tyr Val Ala Phe Val Thr Pro Ser Ile His Tyr785
790 795 800Thr Met Gly Gly Cys Leu Ile
Ser Pro Ser Ala Glu Ile Gln Met Lys 805
810 815Asn Thr Ser Ser Arg Ala Pro Leu Ser His Ser Asn
Pro Ile Leu Gly 820 825 830Leu
Phe Gly Ala Gly Glu Val Thr Gly Gly Val His Gly Gly Asn Arg 835
840 845Leu Gly Gly Asn Ser Leu Leu Glu Cys
Val Val Phe Gly Arg Ile Ala 850 855
860Gly Asp Arg Ala Ser Thr Ile Leu Gln Arg Lys Ser Ser Ala Leu Ser865
870 875 880Phe Lys Val Trp
Thr Thr Val Val Leu Arg Glu Val Arg Glu Gly Gly 885
890 895Val Tyr Gly Ala Gly Ser Arg Val Leu Arg
Phe Asn Leu Pro Gly Ala 900 905
910Leu Gln Arg Ser Gly Leu Ser Leu Gly Gln Phe Ile Ala Ile Arg Gly
915 920 925Asp Trp Asp Gly Gln Gln Leu
Ile Gly Tyr Tyr Ser Pro Ile Thr Leu 930 935
940Pro Asp Asp Leu Gly Met Ile Asp Ile Leu Ala Arg Ser Asp Lys
Gly945 950 955 960Thr Leu
Arg Glu Trp Ile Ser Ala Leu Glu Pro Gly Asp Ala Val Glu
965 970 975Met Lys Ala Cys Gly Gly Leu
Val Ile Glu Arg Arg Leu Ser Asp Lys 980 985
990His Phe Val Phe Met Gly His Ile Ile Asn Lys Leu Cys Leu
Ile Ala 995 1000 1005Gly Gly Thr
Gly Val Ala Pro Met Leu Gln Ile Ile Lys Ala Ala 1010
1015 1020Phe Met Lys Pro Phe Ile Asp Thr Leu Glu Ser
Val His Leu Ile 1025 1030 1035Tyr Ala
Ala Glu Asp Val Thr Glu Leu Thr Tyr Arg Glu Val Leu 1040
1045 1050Glu Glu Arg Arg Arg Glu Ser Arg Gly Lys
Phe Lys Lys Thr Phe 1055 1060 1065Val
Leu Asn Arg Pro Pro Pro Leu Trp Thr Asp Gly Val Gly Phe 1070
1075 1080Ile Asp Arg Gly Ile Leu Thr Asn His
Val Gln Pro Pro Ser Asp 1085 1090
1095Asn Leu Leu Val Ala Ile Cys Gly Pro Pro Val Met Gln Arg Ile
1100 1105 1110Val Lys Ala Thr Leu Lys
Thr Leu Gly Tyr Asn Met Asn Leu Val 1115 1120
1125Arg Thr Val Asp Glu Thr Glu Pro Ser Gly Ser 1130
113573498DNAArtificial sequenceFRDm1 codon optimised for A.
niger 7atgggtgccg atggtatctc ctctgcctcc attgtcgtca ccgaccccga ggctgctgcc
60aagaagcgtg accgcatggc ccgtgagctc ctctcctcca actccggtct ttgccaggag
120gatgagccca ccatcatcaa cctgaagggt ctggaacaca ccatccccta ccgtcttgct
180gttgtccttt gcaactctcg cagcactggt gaattcgagg ccaaggctgc tgagatcctc
240cgcaaggctt tccacatggt tgactactct ctgaactgct tcaaccccga gtccgagctc
300tcccgtgtca acagcttgcc tgtcggtgag aagcaccaga tgagcgaaga tctgcgccac
360gtcatggagt gcaccatctc cgtccaccac tcctctggca tgggtttcga ccctgctgct
420ggtcccatca tctcccgtct gcgtggtgcc atgcgcgacc acaacgacat gtccgacatc
480tccgtcaccg aggctgaggt tgagctgttc tcgctagcgc agtcgttcga tgttgacctc
540gaggagggca ccattgctcg caagcactcc gaggctcgcc tcgaccttgg tggtgtcaac
600aagggctaca ctgttgacta cgtggtggac cacctccgcg ctgctggcat gcccaacgtc
660ctgttcgaat ggggtggtga catccgtgcc tccggccgca acatcaaggg caacctctgg
720gctgttgcca tcaagcgccc tccctccgtt gaggaggtca tccgccgtgc caagggcaag
780atgctcaaga tgggtgaaga agaacaggag gagaaggatg atgactctcc cagccttctg
840cacgttgttg agctcgatga tgaggccctc tgcacctccg gtgactacga gaacgtcctc
900taccacccca agcacggtgt tgctggcagc atcttcgact ggcagcgccg tggtctgctg
960tctcctgagg agggtgctct tgctcaggtt tccgtcaagt gctactctgc catgtacgcc
1020gatgcccttg ccaccgtctg cctggtcaag cgtgatgccg tccgtatccg ctacctcctg
1080gaaggctggc gctacgtgcg ctctcgtgtc accaactact tcgcctacac ccgccagggt
1140gagcgtcttg ctcacatgca cgaaattgcc caggagactc gtgagctccg tgagatccgc
1200attgctggct ccctcccctc ccgtatcgtc atcgtcggtg gtggtctggc cggtctgtct
1260gctgccattg aggctgcctc ctgcggtgct caggtcatcc tgatggagaa ggagggtcgt
1320attggtggca actctgccaa ggccacctcc ggtatcaacg gctggggtac tcgcactcag
1380gccaagtccg acatcctgga tggcggcaag tacttcgagc gtgacacctt cctgagcggt
1440gttggtggta ccactgaccc tgctctggtc aaggtcctct ccgtcaagtc cggtgatgcc
1500attggctggt tgaccagcct tggtgttcct ctttctgttc tctcccagct gggtggtcac
1560tctttcaagc gtacccaccg tgctcctgac aagactgatg gcactcctct ccccatcggt
1620cacaccatca tgcgcaccct cgaggaccac atccgcaaca acctgagcga acgtgtcacc
1680atcatgaccc acgtttccgt cactgagctc ctccacgaga ctgacaccac tcccgatggt
1740gcctccgagg tccgtgtcac cggtgtccgc taccgtgacc tctccgatgt tgacggccag
1800cccagcaagc tccttgccga tgccgttgtc cttgccactg gtggtttctc caacgaccgc
1860gaggagaaca gcttgctttg caagtacgcc ccccacctgg cctccttccc caccaccaac
1920ggcccttggg ccactggtga tggtgtcaag ctggccacct ccgtcggtgc caagctcgtc
1980gacatggaca aggtccagct gcaccccact ggcttgattg accccaagga ccccgccaac
2040accaccaaga tcctgggccc cgaggctctc cgtggcagcg gtggtatcct gctcaacaag
2100cagggcaagc gcttcgtcaa cgagcttgac ctccgcagcg ttgtctccaa ggccatcaac
2160actcagggca acgaataccc cggcagcggt ggctgctact tcgcctactg cgtgttgaac
2220gaagatgcca ccaacctgtt ctgcggtggt gctcttggat tctacggcaa gaagcttggt
2280ctgttccagc gtgctgagac tgttgaggag cttgccaagt tgattggctg cgatgagggc
2340gagctccgtg acaccctcga gaagtacgag acttgctcga aggccaaggt tgcctgcccc
2400gtgaccggca aggtcgtgtt cccctgcgtt gttggtaccc gtggtcccta caacgtcgct
2460ttcgtcaccc cctccatcca ctacaccatg ggtggctgct tgatttctcc tgctgctgag
2520gtcctccagg aatacaaggg tctgaacatc ctggagaacc accgtcccat tcgctgcttg
2580ttcggtgctg gtgaagtcac cggtggtgtc cacggtggca accgcctggg tggcaactcc
2640ctcctcgagt gcgttgtgtt cggcaagatc gctggtgacc gtgctgccac cattctccag
2700aagcgcgaaa ttgccctctc caagaccagc tggacctccg tcgtcgtccg cgagtcccgc
2760tctggcgagc agttcggtac cggctctcgt gtcctccgct tcaacctgcc cggtgctctc
2820cagcgcactg gtctgaacct gggtgagttc gtcgccatcc gtggtgaatg ggatggccag
2880cagctggtcg gctacttctc ccccatcacc ctccccgaag atcttggtac catctccctc
2940ctggtccgtg ccgacaaggg caccctcaag gaatggatat gtgccctccg ccccggtgac
3000agcgttgaga tcaaggcctg cggtggtctg cgtatcgacc aggaccctgt caagaagtgc
3060ttgctattcc gcaaccgccc catcacccgc ttcgctcttg ttgctgctgg tactggtgtt
3120gctcccatgc tccaggtcat ccgtgctgct ctcaagaagc cctacgtgga tacattggag
3180tccatccgtc tgatctacgc tgctgaagaa tacgacaccc tgacctaccg ctccatcctc
3240cagcgcttcg ctgaggagtt ccccgacaag ttcgtctgca acttcgtcct caacaaccct
3300cctgaaggct ggactggtgg tgttggtttc gtcaacaaga agtccctcca gaaggtcctc
3360cagcctccta gctctgagcc tctgattgtc gtctgcggtc ctcctgtcat gcagcgtgat
3420gtcaagaacg agctcctcag catgggctac gacaaggagc ttgtccacac cgttgacggc
3480gagtctggca ccctataa
349883420DNAArtificial sequenceFRDg gene optimised for A. niger
8atggtcgatg gccgctcctc cgcctccatt gttgctgttg accccgagcg tgctgctcgt
60gagcgtgatg ctgctgctcg tgccctcctc caggactctc ccttgcacac caccatgcag
120tacgccacct ccggtctgga attgactgtt ccctacgccc tcaaggttgt tgcctccgcc
180gacaccttcg accgtgccaa ggaggttgcc gatgaggtcc tccgctgcgc ctggcagctg
240gccgacaccg tcctcaactc tttcaacccc aacagcgaag tctctctggt cggccgcctc
300cccgtcggtc agaagcacca gatgagcgct cctctcaagc gtgtcatggc ctgctgccag
360cgtgtctaca acagctctgc tggctgcttc gaccccagca ctgctcctgt tgccaaggcc
420ctccgtgaga tcgctcttgg caaggagcgc aacaacgcct gcttggaggc tcttactcag
480gcctgcaccc tccccaactc gttcgtcatt gacttcgagg ctggcaccat ctcccgcaag
540cacgaacacg cctccctcga tcttggtggt gtcagcaagg gctacatcgt cgactacgtc
600attgacaaca tcaacgctgc tggtttccag aacgttttct tcgactgggg tggtgactgc
660cgtgcctccg gcatgaacgc ccgcaacacc ccctgggttg ttggtatcac ccgccccccg
720tcattggaca tgcttcccaa ccctcccaag gaggccagct acatctccgt catctccctc
780gacaacgagg ctcttgccac cagcggtgac tacgagaacc tgatctacac tgccgatgac
840aagcctctga cctgcaccta cgactggaag ggcaaggagc tcatgaagcc cagccagtcc
900aacattgccc aggtcagcgt caagtgctac tctgccatgt acgccgatgc ccttgccact
960gcttgcttca tcaagcgtga ccccgccaag gtccgccagc tgttggatgg ctggcgctac
1020gtgcgcgaca ccgtccgtga ctaccgtgtc tacgtgcgcg agaacgagcg tgttgccaag
1080atgttcgaaa ttgccactga ggatgccgag atgcgcaagc gccgtatctc caacaccctc
1140cctgctcgtg tcattgttgt tggtggtggt ctggctggtc tttctgctgc cattgaggct
1200gctggctgcg gtgctcaggt tgtcctgatg gagaaggagg ccaagctcgg tggcaactcc
1260gccaaggcca cctccggtat caacggctgg ggtactcgtg ctcaggccaa ggcctccatc
1320gtcgatggcg gcaagtactt cgagcgtgac acctacaagt ccggtatcgg tggcaacacc
1380gaccctgctc tggtcaagac cctgagcatg aagtccgccg atgccattgg ctggttgacc
1440agccttggtg ttcctcttac tgtcctttct cagctgggtg gccactctcg caagcgcacc
1500caccgtgctc ctgacaagaa ggacggcacc cccctcccca tcggtttcac catcatgaaa
1560actctcgagg accacgtccg tggcaacctg tctggccgta tcaccatcat ggagaactgc
1620tcggtgacct cgctactctc cgagactaag gagcgccccg atggcaccaa gcagatccgt
1680gtcaccggtg ttgagttcac ccaggctggc tctggcaaga ccaccatcct ggccgatgcc
1740gtcatcctgg ccactggtgg tttctccaac gacaagactg ccgactcgct actccgcgaa
1800cacgctcccc acctggtcaa cttccccacc accaacggcc cctgggcgac tggtgatggt
1860gtcaagctgg cccagcgtct gggtgctcag ctcgtcgaca tggacaaggt ccagctccac
1920cccactggtc tgatcaaccc caaggaccct gccaacccca ccaagttcct tggacctgag
1980gctctccgtg gctccggtgg tgtccttctg aacaagcagg gcaagcgctt cgtcaacgag
2040ctcgatctcc gcagcgttgt ctccaaggcc atcatggagc agggtgctga ataccccggc
2100agcggtggca gcatgttcgc ctactgcgtt ctcaacgctg ctgctcagaa gctgttcggt
2160gtctcctccc acgaattcta ctggaagaag atgggtctgt tcgtcaaggc cgacaccatg
2220cgtgatcttg ctgctctgat cggttgcccc gttgagagcg tgcagcagac cctggaagaa
2280tacgagcgcc tctccatctc ccagcgctct tgccccatca cccgcaagtc ggtgtaccct
2340tgcgtgcttg gcaccaaggg tccctactac gtggctttcg tcaccccctc catccactac
2400accatgggtg gctgcttgat ctctccttct gctgagatcc agatgaagaa cacctcctcc
2460cgtgctcctc tctcccactc caaccccatc ctcggtctgt tcggtgctgg tgaagtcact
2520ggtggtgtcc acggtggcaa ccgtcttggt ggcaactccc tcctcgagtg cgttgtgttc
2580ggccgtatcg ctggtgaccg tgccagcacc atcctccagc gcaagagctc tgctctctcc
2640ttcaaggtct ggaccactgt tgtcctccgc gaagtccgcg agggtggtgt ctacggtgct
2700ggctctcgtg tcctccgctt caacctcccc ggtgctctcc agcgctccgg tctgtctctt
2760ggccagttca ttgccatccg tggtgactgg gatggccagc agctcattgg ctactactct
2820cccatcaccc tccccgatga tcttggaatg atcgacatcc tggctcgctc cgacaagggt
2880accctccgcg aatggatctc cgctctggag cccggtgatg ccgttgagat gaaggcctgc
2940ggtggtctgg tcattgagcg tcgtctgtcc gacaagcact tcgtgttcat gggtcacatc
3000atcaacaagc tctgcttgat tgccggtggt actggtgttg ctcccatgct tcagatcatc
3060aaggctgctt tcatgaagcc cttcattgac accctcgagt ccgtccacct gatctacgct
3120gctgaggatg tcactgagct gacctaccgt gaggtccttg aggagcgccg ccgcgagtcc
3180cgtggcaagt tcaagaaaac cttcgtcctg aaccgccctc ctcctctctg gactgatggt
3240gttggtttca ttgaccgtgg tatcctgacc aaccacgtcc agcctccctc cgacaaccta
3300ttagtggcca tctgcggtcc tcctgtcatg cagcgcattg tcaaggccac tctcaagacc
3360ctaggataca acatgaacct ggtccgcact gttgatgaga ctgagccctc cggatcataa
342093498DNAArtificial sequenceFRDm1 gene optimsied for S. cerevisiae
9atgggtgctg atggtatttc ttctgcttcc attgttgtta ctgacccaga agctgctgcc
60aagaagcgtg acagaatggc cagagaattg ttgtcctcca actctggtct atgtcaagaa
120gatgaaccaa ccatcatcaa cttaaagggt ttggaacaca ccattccata cagattggcc
180gttgttttgt gtaactccag atccactggt gaattcgaag ccaaggctgc tgaaatcttg
240agaaaggctt tccacatggt tgactactct ttgaattgtt tcaacccaga atctgaattg
300tcccgtgtca actctttacc agtcggtgaa aagcaccaaa tgtccgaaga tctaagacat
360gtcatggaat gtaccatttc tgtccaccac tcctctggta tgggtttcga cccagctgct
420ggtccaatca tctccagatt gagaggtgcc atgagagatc acaacgacat gtccgatatc
480tccgtcactg aagctgaagt tgaattattc tctttggctc aatctttcga tgtcgacttg
540gaagaaggta ctattgccag aaagcactct gaagccagat tggatttggg tggtgtcaac
600aagggttaca ctgttgacta cgttgttgac catttgagag ctgctggtat gccaaacgtc
660ttgttcgaat ggggtggtga tatcagagct tctggtagaa acatcaaggg taacttgtgg
720gctgttgcca tcaagcgtcc accatctgtt gaagaagtta tccgtcgtgc caagggtaag
780atgttaaaga tgggtgaaga agaacaagaa gaaaaggacg atgactctcc atctttgttg
840cacgttgttg aattggatga cgaagctttg tgtacctctg gtgactacga aaacgtctta
900taccatccaa agcacggtgt tgctggttcc attttcgact ggcaacgtcg tggtttattg
960tctccagaag aaggtgcttt agctcaagtt tccgtcaaat gttactctgc catgtacgct
1020gatgctttgg ccactgtttg tttggtcaag agagatgctg tcagaatcag atacttgttg
1080gaaggttgga gatacgtcag atctcgtgtc accaactact tcgcttacac cagacaaggt
1140gaaagattgg ctcacatgca cgaaattgct caagaaacca gagaattaag agaaatcaga
1200attgctggtt ctttgccatc cagaattgtt atcgtcggtg gtggtttggc tggtctatcc
1260gctgccattg aagctgcttc ttgtggtgct caagtcattt tgatggaaaa ggaaggtaga
1320attggtggta actctgccaa ggctacctct ggtatcaacg gttggggtac cagaacccaa
1380gccaagtctg atatcttgga tggtggtaag tactttgaaa gagacacttt cttgtccggt
1440gtcggtggta ccactgaccc agctttggtc aaggtcttgt ccgtcaaatc tggtgacgct
1500atcggttggt taacttcttt gggtgtccca ttgtccgttt tgtctcaatt gggtggtcac
1560tctttcaaga gaactcacag agctccagac aagactgatg gtactccatt accaattggt
1620cacaccatca tgagaacttt ggaagatcat atcagaaaca acttgtctga aagagttacc
1680atcatgaccc acgtttctgt tactgaattg ttgcacgaaa ctgacaccac tccagatggt
1740gcttctgaag ttcgtgtcac cggtgtccgt tacagagact tgtctgatgt cgatggtcaa
1800ccttccaaac tattggctga cgctgttgtt ttggccactg gtggtttctc caacgacaga
1860gaagaaaact ctttgttgtg taaatacgct cctcatttgg cttctttccc aactaccaac
1920ggtccatggg ctactggtga cggtgtcaaa ttggccacct ccgttggtgc caagttggtt
1980gacatggaca aggttcaatt gcacccaact ggtttgattg acccaaagga cccagctaac
2040accactaaga tcttgggtcc agaagctttg agaggttctg gtggtatttt gttgaacaag
2100caaggtaaga gattcgtcaa cgaattggac ttgagatccg ttgtttccaa ggccattaac
2160actcaaggta acgaataccc aggttctggt ggttgttact ttgcttactg tgtcttaaac
2220gaagatgcta ccaacttatt ctgtggtggt gctttgggtt tctacggtaa gaaattaggt
2280ttgttccaaa gagctgaaac tgttgaagaa ttggccaaat tgattggttg tgacgaaggt
2340gaattgagag acactttgga aaaatacgaa acctgttcca aggccaaggt tgcttgtcca
2400gtcactggta aggttgtttt cccatgtgtt gtcggtacca gaggtccata caatgttgct
2460ttcgtcactc catccatcca ctacaccatg ggtggttgtt tgatctctcc agctgctgaa
2520gtcttgcaag aatacaaggg tttgaatatc ttggaaaacc acagaccaat cagatgtttg
2580ttcggtgctg gtgaagtcac tggtggtgtc cacggtggta acagattagg tggtaactct
2640ctattggaat gtgttgtctt tggtaagatt gctggtgaca gagctgccac tatcttgcaa
2700aagagagaaa ttgctttgtc caagacctcc tggacctctg ttgttgtcag agaatccaga
2760tctggtgaac aattcggtac cggttccaga gttttgagat tcaacttgcc aggtgcttta
2820caaagaaccg gtttgaactt gggtgaattc gttgccatca gaggtgaatg ggatggtcaa
2880caattagtcg gttacttctc tccaatcact ttgccagaag atttgggtac catctctttg
2940ttggtcagag ctgacaaggg tactttgaag gaatggatct gtgctttgcg tccaggtgac
3000tccgttgaaa tcaaggcttg tggtggtcta agaattgacc aagatccagt caagaaatgt
3060ttgttgttca gaaacagacc aattaccaga tttgctttgg ttgctgctgg taccggtgtt
3120gctccaatgt tgcaagttat cagagctgct ttgaagaagc catacgtcga cactttggaa
3180tccatcagat tgatctacgc tgctgaagaa tatgacactt taacctacag atctatcttg
3240caaagatttg ctgaagaatt cccagacaaa ttcgtttgta acttcgtctt aaacaaccct
3300ccagaaggtt ggaccggtgg tgttggtttc gtcaacaaga aatctttgca aaaggttttg
3360caaccacctt cttctgaacc attgattgtt gtttgtggtc cacctgttat gcaaagagat
3420gtcaaaaatg aattgttgtc catgggttac gacaaggaat tggttcacac tgtcgatggt
3480gaatctggta ccttgtaa
3498103420DNAArtificial sequenceFRDg gene optimised for S. cerevisiae
10atggttgatg gtagatcttc tgcttccatt gttgccgttg acccagaaag agctgccaga
60gaaagagatg ctgctgccag agctttgttg caagactctc cattgcacac caccatgcaa
120tacgctacct ctggtttgga attgactgtt ccatacgctt tgaaggttgt tgcttctgct
180gacactttcg acagagccaa ggaagttgct gatgaagtct tgagatgtgc ctggcaattg
240gctgacaccg ttttgaactc tttcaaccca aactctgaag tctctttagt cggtagatta
300ccagtcggtc aaaagcatca aatgtctgct ccattgaaac gtgtcatggc ttgttgtcaa
360agagtctaca actcctctgc tggttgtttc gacccatcca ctgctccagt tgccaaggct
420ttgagagaaa ttgctttggg taaggaaaga aacaatgctt gtttggaagc tttgactcaa
480gcttgtacct tgccaaactc tttcgtcatt gatttcgaag ctggtactat ctccagaaag
540cacgaacacg cttctttgga tttgggtggt gtttccaagg gttacatcgt cgattacgtc
600attgacaaca tcaatgctgc tggtttccaa aacgttttct ttgactgggg tggtgactgt
660cgtgcctccg gtatgaacgc cagaaacact ccatgggttg tcggtatcac tagacctcct
720tccttggaca tgttgccaaa ccctccaaag gaagcttctt acatctccgt catctctttg
780gacaatgaag ctttggctac ctctggtgat tacgaaaact tgatctacac tgctgacgat
840aaaccattga cctgtaccta cgattggaaa ggtaaggaat tgatgaagcc atctcaatcc
900aatatcgctc aagtttccgt caagtgttac tctgccatgt acgctgacgc tttggctacc
960gcttgtttca tcaagcgtga cccagccaag gtcagacaat tgttggatgg ttggagatac
1020gttagagaca ccgtcagaga ttaccgtgtc tacgtcagag aaaacgaaag agttgccaag
1080atgttcgaaa ttgccactga agatgctgaa atgagaaaga gaagaatttc caacacttta
1140ccagctcgtg tcattgttgt tggtggtggt ttggctggtt tgtccgctgc cattgaagct
1200gctggttgtg gtgctcaagt tgttttgatg gaaaaggaag ccaagttggg tggtaactct
1260gccaaggcta cctctggtat caacggttgg ggtactagag ctcaagctaa ggcttccatt
1320gtcgatggtg gtaagtactt cgaaagagat acctacaagt ctggtatcgg tggtaacacc
1380gatccagctt tggttaagac tttgtccatg aaatctgctg acgctatcgg ttggttgact
1440tctctaggtg ttccattgac tgttttgtcc caattaggtg gtcactccag aaagagaact
1500cacagagctc cagacaagaa ggatggtact ccattgccaa ttggtttcac catcatgaaa
1560actttagaag atcatgttag aggtaacttg tccggtagaa tcaccatcat ggaaaactgt
1620tccgttacct ctttgttgtc tgaaaccaag gaaagaccag acggtaccaa gcaaatcaga
1680gttaccggtg tcgaattcac tcaagctggt tctggtaaga ccaccatttt ggctgatgct
1740gttatcttgg ccaccggtgg tttctccaac gacaagactg ctgattcttt gttgagagaa
1800catgccccac acttggttaa cttcccaacc accaacggtc catgggctac tggtgatggt
1860gtcaagttgg ctcaaagatt aggtgctcaa ttggtcgata tggacaaggt tcaattgcac
1920ccaactggtt tgatcaaccc aaaggaccca gccaacccaa ccaaattctt gggtccagaa
1980gctctaagag gttctggtgg tgttttgttg aacaaacaag gtaagagatt tgtcaacgaa
2040ttggatttga gatctgttgt ttccaaggcc atcatggaac aaggtgctga atacccaggt
2100tctggtggtt ccatgtttgc ttactgtgtc ttgaacgctg ctgctcaaaa attgtttggt
2160gtttcctctc acgaattcta ctggaagaag atgggtttgt tcgtcaaggc tgacaccatg
2220agagacttgg ctgctttgat tggttgtcca gttgaatccg ttcaacaaac tttagaagaa
2280tacgaaagat tatccatctc tcaaagatct tgtccaatta ccagaaaatc tgtttaccca
2340tgtgttttgg gtaccaaagg tccatactat gtcgcctttg tcactccatc tatccactac
2400accatgggtg gttgtttgat ttctccatct gctgaaatcc aaatgaagaa cacttcttcc
2460agagctccat tgtcccactc caacccaatc ttgggtttat tcggtgctgg tgaagtcacc
2520ggtggtgtcc acggtggtaa cagattaggt ggtaactctt tgttggaatg tgttgttttc
2580ggtagaattg ccggtgacag agcttctacc attttgcaaa gaaagtcctc tgctttgtct
2640ttcaaggtct ggaccactgt tgttttgaga gaagtcagag aaggtggtgt ctacggtgct
2700ggttcccgtg tcttgagatt caacttacca ggtgctctac aaagatctgg tctatccttg
2760ggtcaattca ttgccatcag aggtgactgg gacggtcaac aattgattgg ttactactct
2820ccaatcactt tgccagacga tttgggtatg attgacattt tggccagatc tgacaagggt
2880actttacgtg aatggatctc tgctttggaa ccaggtgacg ctgtcgaaat gaaggcttgt
2940ggtggtttgg tcatcgaaag aagattatct gacaagcact tcgttttcat gggtcacatt
3000atcaacaagc tatgtttgat tgctggtggt accggtgttg ctccaatgtt gcaaatcatc
3060aaggccgctt tcatgaagcc attcatcgac actttggaat ccgtccactt gatctacgct
3120gctgaagatg tcactgaatt gacttacaga gaagttttgg aagaacgtcg tcgtgaatcc
3180agaggtaaat tcaagaaaac tttcgttttg aacagacctc ctccattatg gactgacggt
3240gtcggtttca tcgaccgtgg tatcttgacc aaccacgttc aaccaccatc tgacaactta
3300ttggttgcca tctgtggtcc accagttatg caaagaattg tcaaggccac tttaaagact
3360ttaggttaca acatgaactt ggtcagaacc gttgacgaaa ctgaaccatc tggaagttaa
342011898DNAArtificial sequenceGPDA promotor 11tcagcgtcca attcgagctc
tgtacagtga ccggtgactc tttctggcat gcggagacac 60ggacggtcgc agagaggagg
gctgagtaat aagcgcactc atgtcagctc tggcgctctg 120aggtgcagtg gatgattatt
aatccgggac cggccgcccc tccgccccga agtggaaagg 180ctggtgtgcc cctcgttgac
caagaatcta ttgcatcatc ggagaatatg gagcttcatc 240gaatcaccgg cagtaagcga
aggagaatgt gaagccaggg gtgtatagcc gtcggcgaaa 300tagcatgcca ttaacctagg
tacagaagtc caattgcttc cgatctggta aaagattcac 360gagatagtac cttctccgaa
gtaggtagag cgagtacccg gcgcgtaagc tccctaattg 420gcccatccgg catctgtagg
gcgtccaaat atcgtgcctc tcctgctttg cccggtgtat 480gaaaccggaa aggccgctca
ggagctggcc agcggcgcag accgggaaca caagctggca 540gtcgacccat ccggtgctct
gcactcgacc tgctgaggtc cctcagtccc tggtaggcag 600ctttgccccg tctgtccgcc
cggtgtgtcg gcggggttga caaggtcgtt gcgtcagtcc 660aacatttgtt gccatatttt
cctgctctcc ccaccagctg ctcttttctt ttctctttct 720tttcccatct tcagtatatt
catcttccca tccaagaacc tttatttccc ctaagtaagt 780actttgctac atccatactc
catccttccc atcccttatt cctttgaacc tttcagttcg 840agctttccca cttcatcgca
gcttgactaa cagctacccc gcttgagcca ccgtcaaa 898121000DNAArtificial
sequenceTDH3 promotor 12ctattttcga ggaccttgtc accttgagcc caagagagcc
aagatttaaa ttttcctatg 60acttgatgca aattcccaaa gctaataaca tgcaagacac
gtacggtcaa gaagacatat 120ttgacctctt aacaggttca gacgcgactg cctcatcagt
aagacccgtt gaaaagaact 180tacctgaaaa aaacgaatat atactagcgt tgaatgttag
cgtcaacaac aagaagttta 240atgacgcgga ggccaaggca aaaagattcc ttgattacgt
aagggagtta gaatcatttt 300gaataaaaaa cacgcttttt cagttcgagt ttatcattat
caatactgcc atttcaaaga 360atacgtaaat aattaatagt agtgattttc ctaactttat
ttagtcaaaa aattagcctt 420ttaattctgc tgtaacccgt acatgcccaa aatagggggc
gggttacaca gaatatataa 480catcgtaggt gtctgggtga acagtttatt cctggcatcc
actaaatata atggagcccg 540ctttttaagc tggcatccag aaaaaaaaag aatcccagca
ccaaaatatt gttttcttca 600ccaaccatca gttcataggt ccattctctt agcgcaacta
cagagaacag gggcacaaac 660aggcaaaaaa cgggcacaac ctcaatggag tgatgcaacc
tgcctggagt aaatgatgac 720acaaggcaat tgacccacgc atgtatctat ctcattttct
tacaccttct attaccttct 780gctctctctg atttggaaaa agctgaaaaa aaaggttgaa
accagttccc tgaaattatt 840cccctacttg actaataagt atataaagac ggtaggtatt
gattgtaatt ctgtaaatct 900atttcttaaa cttcttaaat tctactttta tagttagtct
tttttttagt tttaaaacac 960caagaactta gtttcgaata aacacacata aacaaacaaa
100013500DNAArtificial sequenceTDH3 terminator
13gtgaatttac tttaaatctt gcatttaaat aaattttctt tttatagctt tatgacttag
60tttcaattta tatactattt taatgacatt ttcgattcat tgattgaaag ctttgtgttt
120tttcttgatg cgctattgca ttgttcttgt ctttttcgcc acatgtaata tctgtagtag
180atacctgata cattgtggat gctgagtgaa attttagtta ataatggagg cgctcttaat
240aattttgggg atattggctt ttttttttaa agtttacaaa tgaatttttt ccgccaggat
300aacgattctg aagttactct tagcgttcct atcggtacag ccatcaaatc atgcctataa
360atcatgccta tatttgcgtg cagtcagtat catctacatg aaaaaaactc ccgcaatttc
420ttatagaata cgttgaaaat taaatgtacg cgccaagata agataacata tatctagatg
480cagtaatata cacagattcc
50014538PRTArtificial sequenceA. succinogenes PEP carboxykinase wherein
EGY at position 120-122 is replaced by DAF 14Met Thr Asp Leu Asn Lys
Leu Val Lys Glu Leu Asn Asp Leu Gly Leu1 5
10 15Thr Asp Val Lys Glu Ile Val Tyr Asn Pro Ser Tyr
Glu Gln Leu Phe 20 25 30Glu
Glu Glu Thr Lys Pro Gly Leu Glu Gly Phe Asp Lys Gly Thr Leu 35
40 45Thr Thr Leu Gly Ala Val Ala Val Asp
Thr Gly Ile Phe Thr Gly Arg 50 55
60Ser Pro Lys Asp Lys Tyr Ile Val Cys Asp Glu Thr Thr Lys Asp Thr65
70 75 80Val Trp Trp Asn Ser
Glu Ala Ala Lys Asn Asp Asn Lys Pro Met Thr 85
90 95Gln Glu Thr Trp Lys Ser Leu Arg Glu Leu Val
Ala Lys Gln Leu Ser 100 105
110Gly Lys Arg Leu Phe Val Val Asp Ala Phe Cys Gly Ala Ser Glu Lys
115 120 125His Arg Ile Gly Val Arg Met
Val Thr Glu Val Ala Trp Gln Ala His 130 135
140Phe Val Lys Asn Met Phe Ile Arg Pro Thr Asp Glu Glu Leu Lys
Asn145 150 155 160Phe Lys
Ala Asp Phe Thr Val Leu Asn Gly Ala Lys Cys Thr Asn Pro
165 170 175Asn Trp Lys Glu Gln Gly Leu
Asn Ser Glu Asn Phe Val Ala Phe Asn 180 185
190Ile Thr Glu Gly Ile Gln Leu Ile Gly Gly Thr Trp Tyr Gly
Gly Glu 195 200 205Met Lys Lys Gly
Met Phe Ser Met Met Asn Tyr Phe Leu Pro Leu Lys 210
215 220Gly Val Ala Ser Met His Cys Ser Ala Asn Val Gly
Lys Asp Gly Asp225 230 235
240Val Ala Ile Phe Phe Gly Leu Ser Gly Thr Gly Lys Thr Thr Leu Ser
245 250 255Thr Asp Pro Lys Arg
Gln Leu Ile Gly Asp Asp Glu His Gly Trp Asp 260
265 270Glu Ser Gly Val Phe Asn Phe Glu Gly Gly Cys Tyr
Ala Lys Thr Ile 275 280 285Asn Leu
Ser Gln Glu Asn Glu Pro Asp Ile Tyr Gly Ala Ile Arg Arg 290
295 300Asp Ala Leu Leu Glu Asn Val Val Val Arg Ala
Asp Gly Ser Val Asp305 310 315
320Phe Asp Asp Gly Ser Lys Thr Glu Asn Thr Arg Val Ser Tyr Pro Ile
325 330 335Tyr His Ile Asp
Asn Ile Val Arg Pro Val Ser Lys Ala Gly His Ala 340
345 350Thr Lys Val Ile Phe Leu Thr Ala Asp Ala Phe
Gly Val Leu Pro Pro 355 360 365Val
Ser Lys Leu Thr Pro Glu Gln Thr Glu Tyr Tyr Phe Leu Ser Gly 370
375 380Phe Thr Ala Lys Leu Ala Gly Thr Glu Arg
Gly Val Thr Glu Pro Thr385 390 395
400Pro Thr Phe Ser Ala Cys Phe Gly Ala Ala Phe Leu Ser Leu His
Pro 405 410 415Ile Gln Tyr
Ala Asp Val Leu Val Glu Arg Met Lys Ala Ser Gly Ala 420
425 430Glu Ala Tyr Leu Val Asn Thr Gly Trp Asn
Gly Thr Gly Lys Arg Ile 435 440
445Ser Ile Lys Asp Thr Arg Gly Ile Ile Asp Ala Ile Leu Asp Gly Ser 450
455 460Ile Glu Lys Ala Glu Met Gly Glu
Leu Pro Ile Phe Asn Leu Ala Ile465 470
475 480Pro Lys Ala Leu Pro Gly Val Asp Pro Ala Ile Leu
Asp Pro Arg Asp 485 490
495Thr Tyr Ala Asp Lys Ala Gln Trp Gln Val Lys Ala Glu Asp Leu Ala
500 505 510Asn Arg Phe Val Lys Asn
Phe Val Lys Tyr Thr Ala Asn Pro Glu Ala 515 520
525Ala Lys Leu Val Gly Ala Gly Pro Lys Ala 530
535151617DNAArtificial sequencent. A. succinogenes PEP carboxykinase
encoding DAF instead of EGY 15atgactgact taaacaaact cgttaaagaa
cttaatgact tagggcttac cgatgttaag 60gaaattgtgt ataacccgag ttatgaacaa
cttttcgagg aagaaaccaa accgggtttg 120gagggtttcg ataaagggac gttaaccacg
cttggcgcgg ttgccgtcga tacggggatt 180tttaccggtc gttcaccgaa agataaatat
atcgtttgcg atgaaactac gaaagacacc 240gtttggtgga acagcgaagc ggcgaaaaac
gataacaaac cgatgacgca agaaacttgg 300aaaagtttga gagaattagt ggcgaaacaa
ctttccggta aacgtttatt cgtggtagac 360gcattctgcg gcgccagtga aaaacaccgt
atcggtgtgc gtatggttac tgaagtggca 420tggcaggcgc attttgtgaa aaacatgttt
atccgaccga ccgatgaaga gttgaaaaat 480ttcaaagcgg attttaccgt gttaaacggt
gctaaatgta ctaatccgaa ctggaaagaa 540caaggtttga acagtgaaaa ctttgtcgct
ttcaatatta ccgaaggtat tcagcttatc 600ggcggtactt ggtacggcgg tgaaatgaaa
aaaggtatgt tctcaatgat gaactacttc 660ctgccgttaa aaggtgtggc ttccatgcac
tgttccgcca acgtaggtaa agacggtgac 720gtggctattt tcttcggttt atccggtacg
ggtaaaacaa cgctttcgac cgatcctaaa 780cgccaattaa tcggtgatga cgaacacggt
tgggatgaat ccggcgtatt taactttgaa 840ggcggttgtt acgcgaaaac cattaactta
tctcaagaaa acgaaccgga tatttacggc 900gcaatccgtc gtgacgcatt attagaaaac
gtcgtggttc gtgcagacgg ttccgttgac 960tttgacgacg gttcaaaaac agaaaatacc
cgtgtttcat atccgattta ccacatcgac 1020aacatcgttc gtccggtatc gaaagccggt
catgcaacca aagtgatttt cttaaccgcg 1080gacgcattcg gcgtattgcc gccggtttca
aaactgactc cggaacaaac cgaatactac 1140ttcttatccg gctttactgc aaaattagcg
ggtacggaac gcggcgtaac cgaaccgact 1200ccgacattct cggcctgttt cggtgcggca
ttcttaagcc tgcatccgat tcaatatgcg 1260gacgtgttgg tcgaacgcat gaaagcctcc
ggtgcggaag cttatttggt gaacaccggt 1320tggaacggca cgggtaaacg tatttcaatc
aaagataccc gcggtattat cgatgcgatt 1380ttggacggtt caatcgaaaa agcggaaatg
ggcgaattgc caatctttaa tttagcgatt 1440cctaaagcat taccgggtgt tgatcctgct
attttggatc cgcgcgatac ttacgcagac 1500aaagcgcaat ggcaagttaa agcggaagat
ttggcaaacc gtttcgtgaa aaactttgtg 1560aaatatacgg cgaatccgga agcggctaaa
ttagttggcg ccggtccaaa agcataa 1617161617DNAArtificial sequenceCodon
pair optimised A. succinogenes PEPCK for S. cerevisiae 16atgactgatt
tgaacaaatt ggtcaaggaa ttgaatgatt tgggtttgac tgacgtcaag 60gaaattgtct
acaacccatc ttacgaacaa ttattcgaag aagaaaccaa gccaggtttg 120gaaggtttcg
acaagggtac tttgaccact ttaggtgctg ttgctgttga caccggtatt 180ttcaccggtc
gttctccaaa ggacaaatac attgtttgtg atgaaaccac caaggacacc 240gtctggtgga
actctgaagc tgccaagaac gataacaagc caatgactca agaaacctgg 300aaatctttga
gagaattggt tgccaagcaa ttgtctggta agagattatt cgttgttgac 360gctttctgtg
gtgcttctga aaagcacaga attggtgtca gaatggtcac tgaagttgct 420tggcaagctc
atttcgtcaa gaacatgttc atcagaccaa ctgacgaaga attgaagaac 480ttcaaggctg
acttcaccgt tttgaatggt gccaagtgta ccaacccaaa ctggaaggaa 540caaggtttga
actctgaaaa ctttgttgct ttcaacatca ctgaaggtat ccaattgatt 600ggtggtacct
ggtacggtgg tgaaatgaag aagggtatgt tctccatgat gaactatttc 660ttgccattga
aaggtgttgc ttccatgcac tgttctgcca atgtcggtaa ggatggtgac 720gttgccatct
tcttcggtct atccggtact ggtaagacca ctctatccac tgacccaaag 780agacaattga
ttggtgatga cgaacacggt tgggacgaat ctggtgtctt taactttgaa 840ggtggttgtt
acgccaagac catcaactta tctcaagaaa acgaaccaga tatctacggt 900gccatccgtc
gtgatgcttt gttggaaaac gttgttgtca gagctgacgg ttctgttgac 960ttcgacgacg
gttccaagac tgaaaacacc agagtttctt acccaatcta ccacattgac 1020aacattgtca
gacctgtttc caaggctggt cacgctacca aggttatctt cttgactgct 1080gatgctttcg
gtgtcttgcc acctgtttcc aaattgactc cagaacaaac cgaatactac 1140ttcttgtccg
gtttcactgc caaattggct ggtactgaaa gaggtgtcac tgaaccaact 1200ccaactttct
ctgcttgttt cggtgctgct ttcttatctt tgcacccaat ccaatacgct 1260gatgtcttgg
ttgaaagaat gaaggcttct ggtgctgaag cttacttggt caacaccggt 1320tggaacggta
ccggtaagag aatctccatc aaggatacca gaggtatcat tgatgctatc 1380ttggacggtt
ccattgaaaa ggctgaaatg ggtgaattgc caatcttcaa cttggccatt 1440ccaaaggctt
tgccaggtgt tgacccagcc atcttagatc caagagacac ctacgctgac 1500aaggctcaat
ggcaagtcaa ggctgaagat ttggctaaca gattcgtcaa gaactttgtc 1560aaatacactg
ctaacccaga agctgccaaa ttggttggtg ctggtccaaa ggcttaa
161717538PRTMannheimia succinicipoducens 17Met Thr Asp Leu Asn Gln Leu
Thr Gln Glu Leu Gly Ala Leu Gly Ile1 5 10
15His Asp Val Gln Glu Val Val Tyr Asn Pro Ser Tyr Glu
Leu Leu Phe 20 25 30Ala Glu
Glu Thr Lys Pro Gly Leu Glu Gly Tyr Glu Lys Gly Thr Val 35
40 45Thr Asn Gln Gly Ala Val Ala Val Asn Thr
Gly Ile Phe Thr Gly Arg 50 55 60Ser
Pro Lys Asp Lys Tyr Ile Val Leu Asp Asp Lys Thr Lys Asp Thr65
70 75 80Val Trp Trp Thr Ser Glu
Lys Val Lys Asn Asp Asn Lys Pro Met Ser 85
90 95Gln Asp Thr Trp Asn Ser Leu Lys Gly Leu Val Ala
Asp Gln Leu Ser 100 105 110Gly
Lys Arg Leu Phe Val Val Asp Ala Phe Cys Gly Ala Asn Lys Asp 115
120 125Thr Arg Leu Ala Val Arg Val Val Thr
Glu Val Ala Trp Gln Ala His 130 135
140Phe Val Thr Asn Met Phe Ile Arg Pro Ser Ala Glu Glu Leu Lys Gly145
150 155 160Phe Lys Pro Asp
Phe Val Val Met Asn Gly Ala Lys Cys Thr Asn Pro 165
170 175Asn Trp Lys Glu Gln Gly Leu Asn Ser Glu
Asn Phe Val Ala Phe Asn 180 185
190Ile Thr Glu Gly Val Gln Leu Ile Gly Gly Thr Trp Tyr Gly Gly Glu
195 200 205Met Lys Lys Gly Met Phe Ser
Met Met Asn Tyr Phe Leu Pro Leu Arg 210 215
220Gly Ile Ala Ser Met His Cys Ser Ala Asn Val Gly Lys Asp Gly
Asp225 230 235 240Thr Ala
Ile Phe Phe Gly Leu Ser Gly Thr Gly Lys Thr Thr Leu Ser
245 250 255Thr Asp Pro Lys Arg Gln Leu
Ile Gly Asp Asp Glu His Gly Trp Asp 260 265
270Asp Glu Gly Val Phe Asn Phe Glu Gly Gly Cys Tyr Ala Lys
Thr Ile 275 280 285Asn Leu Ser Ala
Glu Asn Glu Pro Asp Ile Tyr Gly Ala Ile Lys Arg 290
295 300Asp Ala Leu Leu Glu Asn Val Val Val Leu Asp Asn
Gly Asp Val Asp305 310 315
320Tyr Ala Asp Gly Ser Lys Thr Glu Asn Thr Arg Val Ser Tyr Pro Ile
325 330 335Tyr His Ile Gln Asn
Ile Val Lys Pro Val Ser Lys Ala Gly Pro Ala 340
345 350Thr Lys Val Ile Phe Leu Ser Ala Asp Ala Phe Gly
Val Leu Pro Pro 355 360 365Val Ser
Lys Leu Thr Pro Glu Gln Thr Lys Tyr Tyr Phe Leu Ser Gly 370
375 380Phe Thr Ala Lys Leu Ala Gly Thr Glu Arg Gly
Ile Thr Glu Pro Thr385 390 395
400Pro Thr Phe Ser Ala Cys Phe Gly Ala Ala Phe Leu Ser Leu His Pro
405 410 415Thr Gln Tyr Ala
Glu Val Leu Val Lys Arg Met Gln Glu Ser Gly Ala 420
425 430Glu Ala Tyr Leu Val Asn Thr Gly Trp Asn Gly
Thr Gly Lys Arg Ile 435 440 445Ser
Ile Lys Asp Thr Arg Gly Ile Ile Asp Ala Ile Leu Asp Gly Ser 450
455 460Ile Asp Lys Ala Glu Met Gly Ser Leu Pro
Ile Phe Asp Phe Ser Ile465 470 475
480Pro Lys Ala Leu Pro Gly Val Asn Pro Ala Ile Leu Asp Pro Arg
Asp 485 490 495Thr Tyr Ala
Asp Lys Ala Gln Trp Glu Glu Lys Ala Gln Asp Leu Ala 500
505 510Gly Arg Phe Val Lys Asn Phe Glu Lys Tyr
Thr Gly Thr Ala Glu Gly 515 520
525Gln Ala Leu Val Ala Ala Gly Pro Lys Ala 530
535181617DNAArtificial sequencePEPcarboxykinase M. succiniciproducens cpo
for S. cerevisiae 18atgaccgatt tgaaccaatt gactcaagaa ttgggtgctt
tgggtattca cgatgtccaa 60gaagttgtct acaacccatc ttacgaattg ttgtttgctg
aagaaaccaa gccaggtttg 120gaaggttacg aaaagggtac tgttaccaac caaggtgctg
ttgctgtcaa caccggtatc 180ttcaccggtc gttctccaaa ggacaaatac attgtcttgg
atgacaagac caaggacact 240gtctggtgga cttctgaaaa ggtcaagaac gacaacaaac
caatgtccca agacacttgg 300aactctttaa agggtttagt cgctgaccaa ttgtctggta
agagattatt cgttgtcgat 360gctttctgtg gtgccaacaa ggacaccaga ttagctgtca
gagttgtcac tgaagttgct 420tggcaagctc acttcgttac caacatgttc atcagaccat
ctgctgaaga attgaaaggt 480ttcaagccag atttcgttgt catgaacggt gccaaatgta
ccaacccaaa ctggaaggaa 540caaggtttga actctgaaaa ctttgttgct ttcaacatca
ctgaaggtgt tcaattgatt 600ggtggtacct ggtacggtgg tgaaatgaag aagggtatgt
tctccatgat gaactacttc 660ttgccattga gaggtattgc ttccatgcac tgttctgcca
atgtcggtaa ggacggtgac 720actgccatct tcttcggtct atccggtacc ggtaagacca
ctttgtccac tgacccaaag 780agacaattga ttggtgatga cgaacacggt tgggatgacg
aaggtgtttt caactttgaa 840ggtggttgtt acgccaagac catcaactta tctgctgaaa
atgaaccaga tatctacggt 900gccatcaagc gtgacgctct attggaaaac gttgttgttt
tggacaatgg tgacgtcgat 960tatgctgacg gttccaagac tgaaaacacc agagtttctt
acccaatcta ccatattcaa 1020aacattgtca agccagtttc caaggctggt ccagctacca
aagttatctt cttgtctgct 1080gatgctttcg gtgttttgcc tcctgtttcc aagttgactc
cagaacaaac caagtactac 1140ttcttgtctg gtttcaccgc caagttggct ggtactgaaa
gaggtatcac tgaaccaact 1200ccaactttct ctgcttgttt cggtgctgcc tttttgtctt
tgcacccaac tcaatacgct 1260gaagttttgg tcaagagaat gcaagaatct ggtgctgaag
cttacttggt caacactggt 1320tggaacggta ccggtaagag aatctccatc aaagatacca
gaggtatcat cgatgccatc 1380ttggatggtt ccattgacaa ggctgaaatg ggttctttgc
caattttcga tttctccatt 1440ccaaaggctt tgccaggtgt caacccagcc atcttagacc
caagagacac ctacgctgac 1500aaagctcaat gggaagaaaa ggctcaagac ttggctggta
gattcgtcaa gaacttcgaa 1560aaatacactg gtactgctga aggtcaagct ttggttgctg
ctggtccaaa ggcctaa 161719365PRTArtificial sequenceMDH2 S. cerevisiae
lacking first 12 a.a. 19Met Leu Lys Ile Ala Ile Leu Gly Ala Ala Gly Gly
Ile Gly Gln Ser1 5 10
15Leu Ser Leu Leu Leu Lys Ala Gln Leu Gln Tyr Gln Leu Lys Glu Ser
20 25 30Asn Arg Ser Val Thr His Ile
His Leu Ala Leu Tyr Asp Val Asn Gln 35 40
45Glu Ala Ile Asn Gly Val Thr Ala Asp Leu Ser His Ile Asp Thr
Pro 50 55 60Ile Ser Val Ser Ser His
Ser Pro Ala Gly Gly Ile Glu Asn Cys Leu65 70
75 80His Asn Ala Ser Ile Val Val Ile Pro Ala Gly
Val Pro Arg Lys Pro 85 90
95Gly Met Thr Arg Asp Asp Leu Phe Asn Val Asn Ala Gly Ile Ile Ser
100 105 110Gln Leu Gly Asp Ser Ile
Ala Glu Cys Cys Asp Leu Ser Lys Val Phe 115 120
125Val Leu Val Ile Ser Asn Pro Val Asn Ser Leu Val Pro Val
Met Val 130 135 140Ser Asn Ile Leu Lys
Asn His Pro Gln Ser Arg Asn Ser Gly Ile Glu145 150
155 160Arg Arg Ile Met Gly Val Thr Lys Leu Asp
Ile Val Arg Ala Ser Thr 165 170
175Phe Leu Arg Glu Ile Asn Ile Glu Ser Gly Leu Thr Pro Arg Val Asn
180 185 190Ser Met Pro Asp Val
Pro Val Ile Gly Gly His Ser Gly Glu Thr Ile 195
200 205Ile Pro Leu Phe Ser Gln Ser Asn Phe Leu Ser Arg
Leu Asn Glu Asp 210 215 220Gln Leu Lys
Tyr Leu Ile His Arg Val Gln Tyr Gly Gly Asp Glu Val225
230 235 240Val Lys Ala Lys Asn Gly Lys
Gly Ser Ala Thr Leu Ser Met Ala His 245
250 255Ala Gly Tyr Lys Cys Val Val Gln Phe Val Ser Leu
Leu Leu Gly Asn 260 265 270Ile
Glu Gln Ile His Gly Thr Tyr Tyr Val Pro Leu Lys Asp Ala Asn 275
280 285Asn Phe Pro Ile Ala Pro Gly Ala Asp
Gln Leu Leu Pro Leu Val Asp 290 295
300Gly Ala Asp Tyr Phe Ala Ile Pro Leu Thr Ile Thr Thr Lys Gly Val305
310 315 320Ser Tyr Val Asp
Tyr Asp Ile Val Asn Arg Met Asn Asp Met Glu Arg 325
330 335Asn Gln Met Leu Pro Ile Cys Val Ser Gln
Leu Lys Lys Asn Ile Asp 340 345
350Lys Gly Leu Glu Phe Val Ala Ser Arg Ser Ala Ser Ser 355
360 365201099DNAArtificial sequencecpo MDH2 S.
cerevisiae lacking fisrt 12 a.a. 20atgttgaaga ttgccatctt gggtgctgct
ggtggtatcg gtcaatcttt gtctttgttg 60ttgaaggctc aattgcaata ccaattgaag
gaatccaaca gatctgttac ccacattcat 120ttggctttgt acgatgtcaa ccaagaagct
atcaacggtg tcactgctga cttgtctcac 180atcgataccc caatctctgt ttcctctcac
tctccagctg gtggtattga aaactgtttg 240cacaacgctt ccattgttgt cattccagcc
ggtgttccaa gaaagccagg tatgacccgt 300gacgatttgt tcaacgtcaa tgccggtatc
atctctcaat taggtgattc cattgctgaa 360tgttgtgact tgtccaaggt tttcgtcttg
gttatctcca acccagtcaa ctctttggtt 420cctgttatgg tttccaacat cttgaagaac
cacccacaat ccagaaactc tggtattgaa 480agaagaatca tgggtgtcac caaattggac
attgtcagag cttccacttt cttgagagaa 540atcaacattg aatctggttt gactccaaga
gtcaactcca tgccagatgt tccagttatc 600ggtggtcact ctggtgaaac tatcatccca
ttattctctc aatctaactt cttgtccaga 660ttgaatgaag atcaattgaa atacttgatt
caccgtgtcc aatacggtgg tgacgaagtt 720gtcaaggcca agaacggtaa gggttctgct
actctatcca tggctcatgc cggttacaag 780tgtgttgtcc aattcgtttc tctattatta
ggtaacattg aacaaatcca cggtacctac 840tacgttccat tgaaagatgc taacaacttc
ccaattgctc caggtgctga ccaattattg 900ccattagtcg acggtgctga ctactttgcc
atcccattga ccatcactac caagggtgtt 960tcttacgttg actacgatat cgtcaacaga
atgaacgaca tggaaagaaa ccaaatgttg 1020cctatctgtg tttctcaatt gaagaagaac
attgacaagg gtttggaatt cgttgcttcc 1080agatctgctt ccagttaag
109921340PRTArtificial sequenceMDH3 S.
cerevisiae lacking C-terminal SKL 21Met Val Lys Val Ala Ile Leu Gly Ala
Ser Gly Gly Val Gly Gln Pro1 5 10
15Leu Ser Leu Leu Leu Lys Leu Ser Pro Tyr Val Ser Glu Leu Ala
Leu 20 25 30Tyr Asp Ile Arg
Ala Ala Glu Gly Ile Gly Lys Asp Leu Ser His Ile 35
40 45Asn Thr Asn Ser Ser Cys Val Gly Tyr Asp Lys Asp
Ser Ile Glu Asn 50 55 60Thr Leu Ser
Asn Ala Gln Val Val Leu Ile Pro Ala Gly Val Pro Arg65 70
75 80Lys Pro Gly Leu Thr Arg Asp Asp
Leu Phe Lys Met Asn Ala Gly Ile 85 90
95Val Lys Ser Leu Val Thr Ala Val Gly Lys Phe Ala Pro Asn
Ala Arg 100 105 110Ile Leu Val
Ile Ser Asn Pro Val Asn Ser Leu Val Pro Ile Ala Val 115
120 125Glu Thr Leu Lys Lys Met Gly Lys Phe Lys Pro
Gly Asn Val Met Gly 130 135 140Val Thr
Asn Leu Asp Leu Val Arg Ala Glu Thr Phe Leu Val Asp Tyr145
150 155 160Leu Met Leu Lys Asn Pro Lys
Ile Gly Gln Glu Gln Asp Lys Thr Thr 165
170 175Met His Arg Lys Val Thr Val Ile Gly Gly His Ser
Gly Glu Thr Ile 180 185 190Ile
Pro Ile Ile Thr Asp Lys Ser Leu Val Phe Gln Leu Asp Lys Gln 195
200 205Tyr Glu His Phe Ile His Arg Val Gln
Phe Gly Gly Asp Glu Ile Val 210 215
220Lys Ala Lys Gln Gly Ala Gly Ser Ala Thr Leu Ser Met Ala Phe Ala225
230 235 240Gly Ala Lys Phe
Ala Glu Glu Val Leu Arg Ser Phe His Asn Glu Lys 245
250 255Pro Glu Thr Glu Ser Leu Ser Ala Phe Val
Tyr Leu Pro Gly Leu Lys 260 265
270Asn Gly Lys Lys Ala Gln Gln Leu Val Gly Asp Asn Ser Ile Glu Tyr
275 280 285Phe Ser Leu Pro Ile Val Leu
Arg Asn Gly Ser Val Val Ser Ile Asp 290 295
300Thr Ser Val Leu Glu Lys Leu Ser Pro Arg Glu Glu Gln Leu Val
Asn305 310 315 320Thr Ala
Val Lys Glu Leu Arg Lys Asn Ile Glu Lys Gly Lys Ser Phe
325 330 335Ile Leu Asp Ser
340221024DNAArtificial sequenceMDH3 S. cerevisiae lacking SKL encoding
nt, cpo 22atggttaagg ttgccatctt aggtgcttct ggtggtgtcg gtcaaccatt
atctctatta 60ttgaaattgt ctccatacgt ttctgaattg gctttgtacg atatcagagc
tgctgaaggt 120attggtaagg atttgtccca catcaacacc aactcctctt gtgttggtta
cgacaaggat 180tccatcgaaa acactttgtc caatgctcaa gttgtcttga ttccagctgg
tgttccaaga 240aagccaggtt tgaccagaga tgatttgttc aagatgaacg ctggtatcgt
taagtctttg 300gttactgctg tcggtaaatt tgccccaaac gctcgtatct tagtcatctc
caaccctgtt 360aactctttgg ttccaattgc cgttgaaact ttgaagaaga tgggtaagtt
caagccaggt 420aacgttatgg gtgtcaccaa cttggatttg gtcagagctg aaactttctt
ggttgactac 480ttgatgttga agaacccaaa gatcggtcaa gaacaagaca agaccaccat
gcacagaaag 540gtcaccgtca tcggtggtca ctctggtgaa accatcattc caatcatcac
tgacaaatcc 600ttggttttcc aattggacaa gcaatacgaa catttcatcc acagagtcca
attcggtggt 660gacgaaattg tcaaggccaa gcaaggtgcc ggttctgcta ccttgtccat
ggctttcgct 720ggtgccaaat ttgctgaaga agtcttacgt tctttccaca acgaaaagcc
agaaactgaa 780tctttgtctg ctttcgtcta cttgccaggt ttgaagaacg gtaagaaggc
tcaacaatta 840gtcggtgaca actccattga atacttctct ttgccaattg ttttgagaaa
cggttccgtt 900gtttccattg acacttctgt tttggaaaaa ttgtctccaa gagaagaaca
attggtcaac 960actgctgtca aggaattgag aaagaacatt gaaaagggta agtctttcat
cttggacagt 1020taag
102423472PRTArtificial sequenceFumarase R. oryzae lacking
first 23 aa+ new M 23Met Ser Ser Ala Ser Ala Ala Leu Gln Lys Phe Arg Ala
Glu Arg Asp1 5 10 15Thr
Phe Gly Asp Leu Gln Val Pro Ala Asp Arg Tyr Trp Gly Ala Gln 20
25 30Thr Gln Arg Ser Leu Gln Asn Phe
Asp Ile Gly Gly Pro Thr Glu Arg 35 40
45Met Pro Glu Pro Leu Ile Arg Ala Phe Gly Val Leu Lys Lys Ala Ala
50 55 60Ala Thr Val Asn Met Thr Tyr Gly
Leu Asp Pro Lys Val Gly Glu Ala65 70 75
80Ile Gln Lys Ala Ala Asp Glu Val Ile Asp Gly Ser Leu
Ile Asp His 85 90 95Phe
Pro Leu Val Val Trp Gln Thr Gly Ser Gly Thr Gln Thr Lys Met
100 105 110Asn Val Asn Glu Val Ile Ser
Asn Arg Ala Ile Glu Leu Leu Gly Gly 115 120
125Glu Leu Gly Ser Lys Ala Pro Val His Pro Asn Asp His Val Asn
Met 130 135 140Ser Gln Ser Ser Asn Asp
Thr Phe Pro Thr Ala Met His Val Ala Ala145 150
155 160Val Val Glu Ile His Gly Arg Leu Ile Pro Ala
Leu Thr Thr Leu Arg 165 170
175Asp Ala Leu Gln Ala Lys Ser Ala Glu Phe Glu His Ile Ile Lys Ile
180 185 190Gly Arg Thr His Leu Gln
Asp Ala Thr Pro Leu Thr Leu Gly Gln Glu 195 200
205Phe Ser Gly Tyr Thr Gln Gln Leu Thr Tyr Gly Ile Ala Arg
Val Gln 210 215 220Gly Thr Leu Glu Arg
Leu Tyr Asn Leu Ala Gln Gly Gly Thr Ala Val225 230
235 240Gly Thr Gly Leu Asn Thr Arg Lys Gly Phe
Asp Ala Lys Val Ala Glu 245 250
255Ala Ile Ala Ser Ile Thr Gly Leu Pro Phe Lys Thr Ala Pro Asn Lys
260 265 270Phe Glu Ala Leu Ala
Ala His Asp Ala Leu Val Glu Ala His Gly Ala 275
280 285Leu Asn Thr Val Ala Cys Ser Leu Met Lys Ile Ala
Asn Asp Ile Arg 290 295 300Tyr Leu Gly
Ser Gly Pro Arg Cys Gly Leu Gly Glu Leu Ser Leu Pro305
310 315 320Glu Asn Glu Pro Gly Ser Ser
Ile Met Pro Gly Lys Val Asn Pro Thr 325
330 335Gln Cys Glu Ala Met Thr Met Val Cys Ala Gln Val
Met Gly Asn Asn 340 345 350Thr
Ala Ile Ser Val Ala Gly Ser Asn Gly Gln Phe Glu Leu Asn Val 355
360 365Phe Lys Pro Val Met Ile Lys Asn Leu
Ile Gln Ser Ile Arg Leu Ile 370 375
380Ser Asp Ala Ser Ile Ser Phe Thr Lys Asn Cys Val Val Gly Ile Glu385
390 395 400Ala Asn Glu Lys
Lys Ile Ser Ser Ile Met Asn Glu Ser Leu Met Leu 405
410 415Val Thr Ala Leu Asn Pro His Ile Gly Tyr
Asp Lys Ala Ala Lys Cys 420 425
430Ala Lys Lys Ala His Lys Glu Gly Thr Thr Leu Lys Glu Ala Ala Leu
435 440 445Ser Leu Gly Tyr Leu Thr Ser
Glu Glu Phe Asp Gln Trp Val Arg Pro 450 455
460Glu Asp Met Ile Ser Ala Lys Asp465
470241419DNAArtificial sequenceFumarase R. oryzae lacking nt encoding
first aa + M 24atgtcctctg cttctgctgc tttgcaaaaa ttcagagctg
aaagagatac cttcggtgac 60ttgcaagttc cagctgaccg ttactggggt gctcaaactc
aaagatcttt gcaaaacttt 120gacattggtg gtccaactga aagaatgcca gaaccattaa
tcagagcttt cggtgttttg 180aagaaggctg ctgccaccgt caacatgacc tacggtttgg
acccaaaggt tggtgaagcc 240atccaaaagg ctgctgacga agttatcgat ggttctttga
ttgaccattt cccattggtt 300gtctggcaaa ccggttctgg tactcaaacc aagatgaacg
tcaatgaagt catctccaac 360agagccattg aattgttggg tggtgaatta ggttccaagg
ctccagtcca cccaaacgat 420catgtcaaca tgtctcaatc ttccaacgac actttcccaa
ctgccatgca cgttgctgcc 480gttgttgaaa ttcacggtag attgattcca gctttgacca
ctttgagaga tgctttgcaa 540gccaaatctg ctgaattcga acacatcatc aagattggta
gaacccactt gcaagatgct 600accccattga ctttaggtca agaattctcc ggttacactc
aacaattgac ctacggtatt 660gctcgtgttc aaggtacttt ggaaagatta tacaacttgg
ctcaaggtgg tactgctgtc 720ggtactggtt tgaacaccag aaagggtttc gatgccaagg
ttgctgaagc cattgcttcc 780atcactggtt taccattcaa gaccgctcca aacaaattcg
aagctttggc tgctcacgac 840gctttggttg aagctcacgg tgctttgaac accgttgctt
gttctttgat gaagattgcc 900aacgatatcc gttacttggg ttctggtcca agatgtggtt
taggtgaatt gtctctacca 960gaaaacgaac caggttcttc catcatgcca ggtaaggtca
acccaactca atgtgaagct 1020atgaccatgg tttgtgctca agtcatgggt aacaacactg
ccatctctgt tgctggttcc 1080aacggtcaat tcgaattgaa tgtctttaaa ccagtcatga
tcaagaactt gatccaatcc 1140atcagattaa tctctgacgc ttccatctct ttcaccaaga
actgtgttgt cggtattgaa 1200gctaacgaaa agaagatctc ctccatcatg aacgaatctt
tgatgttggt cactgctttg 1260aaccctcaca ttggttacga caaggctgcc aagtgtgcca
agaaggctca caaggaaggt 1320accactttga aagaagctgc tctatctttg ggttacttga
cctctgaaga attcgaccaa 1380tgggttagac ctgaggacat gatttctgcc aaggattaa
1419251000DNAArtificial sequenceTDH1 promotor
25cttccctttt acagtgcttc ggaaaagcac agcgttgtcc aagggaacaa tttttcttca
60agttaatgca taagaaatat ctttttttat gtttagctaa gtaaaagcag cttggagtaa
120aaaaaaaaat gagtaaattt ctcgatggat tagtttctca caggtaacat aacaaaaacc
180aagaaaagcc cgcttctgaa aactacagtt gacttgtatg ctaaagggcc agactaatgg
240gaggagaaaa agaaacgaat gtatatgctc atttacactc tatatcacca tatggaggat
300aagttgggct gagcttctga tccaatttat tctatccatt agttgctgat atgtcccacc
360agccaacact tgatagtatc tactcgccat tcacttccag cagcgccagt agggttgttg
420agcttagtaa aaatgtgcgc accacaagcc tacatgactc cacgtcacat gaaaccacac
480cgtggggcct tgttgcgcta ggaataggat atgcgacgaa gacgcttctg cttagtaacc
540acaccacatt ttcagggggt cgatctgctt gcttccttta ctgtcacgag cggcccataa
600tcgcgctttt tttttaaaag gcgcgagaca gcaaacagga agctcgggtt tcaaccttcg
660gagtggtcgc agatctggag actggatctt tacaatacag taaggcaagc caccatctgc
720ttcttaggtg catgcgacgg tatccacgtg cagaacaaca tagtctgaag aaggggggga
780ggagcatgtt cattctctgt agcagtaaga gcttggtgat aatgaccaaa actggagtct
840cgaaatcata taaatagaca atatattttc acacaatgag atttgtagta cagttctatt
900ctctctcttg cataaataag aaattcatca agaacttggt ttgatatttc accaacacac
960acaaaaaaca gtacttcact aaatttacac acaaaacaaa
100026500DNAArtificial sequenceTDH1 terminator 26ataaagcaat cttgatgagg
ataatgattt ttttttgaat atacataaat actaccgttt 60ttctgctaga ttttgtgaag
acgtaaataa gtacatatta ctttttaagc caagacaaga 120ttaagcatta actttaccct
tttctcttct aagtttcaat actagttatc actgtttaaa 180agttatggcg agaacgtcgg
cggttaaaat atattaccct gaacgtggtg aattgaagtt 240ctaggatggt ttaaagattt
ttcctttttg ggaaataagt aaacaatata ttgctgcctt 300tgcaaaacgc acatacccac
aatatgtgac tattggcaaa gaacgcatta tcctttgaag 360aggtggatac tgatactaag
agagtctcta ttccggctcc acttttagtc cagagattac 420ttgtcttctt acgtatcaga
acaagaaagc atttccaaag taattgcatt tgcccttgag 480cagtatatat atactaagaa
50027600DNAArtificial
sequencesecond TDH3 promotor 27ttagtcaaaa aattagcctt ttaattctgc
tgtaacccgt acatgcccaa aatagggggc 60gggttacaca gaatatataa catcgtaggt
gtctgggtga acagtttatt cctggcatcc 120actaaatata atggagcccg ctttttaagc
tggcatccag aaaaaaaaag aatcccagca 180ccaaaatatt gttttcttca ccaaccatca
gttcataggt ccattctctt agcgcaacta 240cagagaacag gggcacaaac aggcaaaaaa
cgggcacaac ctcaatggag tgatgcaacc 300tgcctggagt aaatgatgac acaaggcaat
tgacccacgc atgtatctat ctcattttct 360tacaccttct attaccttct gctctctctg
atttggaaaa agctgaaaaa aaaggttgaa 420accagttccc tgaaattatt cccctacttg
actaataagt atataaagac ggtaggtatt 480gattgtaatt ctgtaaatct atttcttaaa
cttcttaaat tctactttta tagttagtct 540tttttttagt tttaaaacac caagaactta
gtttcgaata aacacacata aacaaacaaa 60028300DNAArtificial sequencesecond
TDH3 terminator 28gtgaatttac tttaaatctt gcatttaaat aaattttctt tttatagctt
tatgacttag 60tttcaattta tatactattt taatgacatt ttcgattcat tgattgaaag
ctttgtgttt 120tttcttgatg cgctattgca ttgttcttgt ctttttcgcc acatgtaata
tctgtagtag 180atacctgata cattgtggat gctgagtgaa attttagtta ataatggagg
cgctcttaat 240aattttgggg atattggctt ttttttttaa agtttacaaa tgaatttttt
ccgccaggat 300293148DNAArtificial sequenceTDH1p-PCKm-TDH1t synthetic
construct 29ggatcccttc ccttttacag tgcttcggaa aagcacagcg ttgtccaagg
gaacaatttt 60tcttcaagtt aatgcataag aaatatcttt ttttatgttt agctaagtaa
aagcagcttg 120gagtaaaaaa aaaaatgagt aaatttctcg atggattagt ttctcacagg
taacataaca 180aaaaccaaga aaagcccgct tctgaaaact acagttgact tgtatgctaa
agggccagac 240taatgggagg agaaaaagaa acgaatgtat atgctcattt acactctata
tcaccatatg 300gaggataagt tgggctgagc ttctgatcca atttattcta tccattagtt
gctgatatgt 360cccaccagcc aacacttgat agtatctact cgccattcac ttccagcagc
gccagtaggg 420ttgttgagct tagtaaaaat gtgcgcacca caagcctaca tgactccacg
tcacatgaaa 480ccacaccgtg gggccttgtt gcgctaggaa taggatatgc gacgaagacg
cttctgctta 540gtaaccacac cacattttca gggggtcgat ctgcttgctt cctttactgt
cacgagcggc 600ccataatcgc gctttttttt taaaaggcgc gagacagcaa acaggaagct
cgggtttcaa 660ccttcggagt ggtcgcagat ctggagactg gatctttaca atacagtaag
gcaagccacc 720atctgcttct taggtgcatg cgacggtatc cacgtgcaga acaacatagt
ctgaagaagg 780gggggaggag catgttcatt ctctgtagca gtaagagctt ggtgataatg
accaaaactg 840gagtctcgaa atcatataaa tagacaatat attttcacac aatgagattt
gtagtacagt 900tctattctct ctcttgcata aataagaaat tcatcaagaa cttggtttga
tatttcacca 960acacacacaa aaaacagtac ttcactaaat ttacacacaa aacaaaatga
ctgatttgaa 1020caaattggtc aaggaattga atgatttggg tttgactgac gtcaaggaaa
ttgtctacaa 1080cccatcttac gaacaattat tcgaagaaga aaccaagcca ggtttggaag
gtttcgacaa 1140gggtactttg accactttag gtgctgttgc tgttgacacc ggtattttca
ccggtcgttc 1200tccaaaggac aaatacattg tttgtgatga aaccaccaag gacaccgtct
ggtggaactc 1260tgaagctgcc aagaacgata acaagccaat gactcaagaa acctggaaat
ctttgagaga 1320attggttgcc aagcaattgt ctggtaagag attattcgtt gttgacgctt
tctgtggtgc 1380ttctgaaaag cacagaattg gtgtcagaat ggtcactgaa gttgcttggc
aagctcattt 1440cgtcaagaac atgttcatca gaccaactga cgaagaattg aagaacttca
aggctgactt 1500caccgttttg aatggtgcca agtgtaccaa cccaaactgg aaggaacaag
gtttgaactc 1560tgaaaacttt gttgctttca acatcactga aggtatccaa ttgattggtg
gtacctggta 1620cggtggtgaa atgaagaagg gtatgttctc catgatgaac tatttcttgc
cattgaaagg 1680tgttgcttcc atgcactgtt ctgccaatgt cggtaaggat ggtgacgttg
ccatcttctt 1740cggtctatcc ggtactggta agaccactct atccactgac ccaaagagac
aattgattgg 1800tgatgacgaa cacggttggg acgaatctgg tgtctttaac tttgaaggtg
gttgttacgc 1860caagaccatc aacttatctc aagaaaacga accagatatc tacggtgcca
tccgtcgtga 1920tgctttgttg gaaaacgttg ttgtcagagc tgacggttct gttgacttcg
acgacggttc 1980caagactgaa aacaccagag tttcttaccc aatctaccac attgacaaca
ttgtcagacc 2040tgtttccaag gctggtcacg ctaccaaggt tatcttcttg actgctgatg
ctttcggtgt 2100cttgccacct gtttccaaat tgactccaga acaaaccgaa tactacttct
tgtccggttt 2160cactgccaaa ttggctggta ctgaaagagg tgtcactgaa ccaactccaa
ctttctctgc 2220ttgtttcggt gctgctttct tatctttgca cccaatccaa tacgctgatg
tcttggttga 2280aagaatgaag gcttctggtg ctgaagctta cttggtcaac accggttgga
acggtaccgg 2340taagagaatc tccatcaagg ataccagagg tatcattgat gctatcttgg
acggttccat 2400tgaaaaggct gaaatgggtg aattgccaat cttcaacttg gccattccaa
aggctttgcc 2460aggtgttgac ccagccatct tagatccaag agacacctac gctgacaagg
ctcaatggca 2520agtcaaggct gaagatttgg ctaacagatt cgtcaagaac tttgtcaaat
acactgctaa 2580cccagaagct gccaaattgg ttggtgctgg tccaaaggct taaggcccgg
gcataaagca 2640atcttgatga ggataatgat ttttttttga atatacataa atactaccgt
ttttctgcta 2700gattttgtga agacgtaaat aagtacatat tactttttaa gccaagacaa
gattaagcat 2760taactttacc cttttctctt ctaagtttca atactagtta tcactgttta
aaagttatgg 2820cgagaacgtc ggcggttaaa atatattacc ctgaacgtgg tgaattgaag
ttctaggatg 2880gtttaaagat ttttcctttt tgggaaataa gtaaacaata tattgctgcc
tttgcaaaac 2940gcacataccc acaatatgtg actattggca aagaacgcat tatcctttga
agaggtggat 3000actgatacta agagagtctc tattccggct ccacttttag tccagagatt
acttgtcttc 3060ttacgtatca gaacaagaaa gcatttccaa agtaattgca tttgcccttg
agcagtatat 3120atatactaag aaggcgcgcc gcggccgc
3148303148DNAArtificial sequenceTDH1p-PCK1-TDH1t synthetic
construct 30ggatcccttc ccttttacag tgcttcggaa aagcacagcg ttgtccaagg
gaacaatttt 60tcttcaagtt aatgcataag aaatatcttt ttttatgttt agctaagtaa
aagcagcttg 120gagtaaaaaa aaaaatgagt aaatttctcg atggattagt ttctcacagg
taacataaca 180aaaaccaaga aaagcccgct tctgaaaact acagttgact tgtatgctaa
agggccagac 240taatgggagg agaaaaagaa acgaatgtat atgctcattt acactctata
tcaccatatg 300gaggataagt tgggctgagc ttctgatcca atttattcta tccattagtt
gctgatatgt 360cccaccagcc aacacttgat agtatctact cgccattcac ttccagcagc
gccagtaggg 420ttgttgagct tagtaaaaat gtgcgcacca caagcctaca tgactccacg
tcacatgaaa 480ccacaccgtg gggccttgtt gcgctaggaa taggatatgc gacgaagacg
cttctgctta 540gtaaccacac cacattttca gggggtcgat ctgcttgctt cctttactgt
cacgagcggc 600ccataatcgc gctttttttt taaaaggcgc gagacagcaa acaggaagct
cgggtttcaa 660ccttcggagt ggtcgcagat ctggagactg gatctttaca atacagtaag
gcaagccacc 720atctgcttct taggtgcatg cgacggtatc cacgtgcaga acaacatagt
ctgaagaagg 780gggggaggag catgttcatt ctctgtagca gtaagagctt ggtgataatg
accaaaactg 840gagtctcgaa atcatataaa tagacaatat attttcacac aatgagattt
gtagtacagt 900tctattctct ctcttgcata aataagaaat tcatcaagaa cttggtttga
tatttcacca 960acacacacaa aaaacagtac ttcactaaat ttacacacaa aacaaaatga
ccgatttgaa 1020ccaattgact caagaattgg gtgctttggg tattcacgat gtccaagaag
ttgtctacaa 1080cccatcttac gaattgttgt ttgctgaaga aaccaagcca ggtttggaag
gttacgaaaa 1140gggtactgtt accaaccaag gtgctgttgc tgtcaacacc ggtatcttca
ccggtcgttc 1200tccaaaggac aaatacattg tcttggatga caagaccaag gacactgtct
ggtggacttc 1260tgaaaaggtc aagaacgaca acaaaccaat gtcccaagac acttggaact
ctttaaaggg 1320tttagtcgct gaccaattgt ctggtaagag attattcgtt gtcgatgctt
tctgtggtgc 1380caacaaggac accagattag ctgtcagagt tgtcactgaa gttgcttggc
aagctcactt 1440cgttaccaac atgttcatca gaccatctgc tgaagaattg aaaggtttca
agccagattt 1500cgttgtcatg aacggtgcca aatgtaccaa cccaaactgg aaggaacaag
gtttgaactc 1560tgaaaacttt gttgctttca acatcactga aggtgttcaa ttgattggtg
gtacctggta 1620cggtggtgaa atgaagaagg gtatgttctc catgatgaac tacttcttgc
cattgagagg 1680tattgcttcc atgcactgtt ctgccaatgt cggtaaggac ggtgacactg
ccatcttctt 1740cggtctatcc ggtaccggta agaccacttt gtccactgac ccaaagagac
aattgattgg 1800tgatgacgaa cacggttggg atgacgaagg tgttttcaac tttgaaggtg
gttgttacgc 1860caagaccatc aacttatctg ctgaaaatga accagatatc tacggtgcca
tcaagcgtga 1920cgctctattg gaaaacgttg ttgttttgga caatggtgac gtcgattatg
ctgacggttc 1980caagactgaa aacaccagag tttcttaccc aatctaccat attcaaaaca
ttgtcaagcc 2040agtttccaag gctggtccag ctaccaaagt tatcttcttg tctgctgatg
ctttcggtgt 2100tttgcctcct gtttccaagt tgactccaga acaaaccaag tactacttct
tgtctggttt 2160caccgccaag ttggctggta ctgaaagagg tatcactgaa ccaactccaa
ctttctctgc 2220ttgtttcggt gctgcctttt tgtctttgca cccaactcaa tacgctgaag
ttttggtcaa 2280gagaatgcaa gaatctggtg ctgaagctta cttggtcaac actggttgga
acggtaccgg 2340taagagaatc tccatcaaag ataccagagg tatcatcgat gccatcttgg
atggttccat 2400tgacaaggct gaaatgggtt ctttgccaat tttcgatttc tccattccaa
aggctttgcc 2460aggtgtcaac ccagccatct tagacccaag agacacctac gctgacaaag
ctcaatggga 2520agaaaaggct caagacttgg ctggtagatt cgtcaagaac ttcgaaaaat
acactggtac 2580tgctgaaggt caagctttgg ttgctgctgg tccaaaggcc taaggcccgg
gcataaagca 2640atcttgatga ggataatgat ttttttttga atatacataa atactaccgt
ttttctgcta 2700gattttgtga agacgtaaat aagtacatat tactttttaa gccaagacaa
gattaagcat 2760taactttacc cttttctctt ctaagtttca atactagtta tcactgttta
aaagttatgg 2820cgagaacgtc ggcggttaaa atatattacc ctgaacgtgg tgaattgaag
ttctaggatg 2880gtttaaagat ttttcctttt tgggaaataa gtaaacaata tattgctgcc
tttgcaaaac 2940gcacataccc acaatatgtg actattggca aagaacgcat tatcctttga
agaggtggat 3000actgatacta agagagtctc tattccggct ccacttttag tccagagatt
acttgtcttc 3060ttacgtatca gaacaagaaa gcatttccaa agtaattgca tttgcccttg
agcagtatat 3120atatactaag aaggcgcgcc gcggccgc
3148312637DNAArtificial sequenceTDH3p-delta 12N MDH2-TDH3t
synthetic construct 31ggatccggcg cgccctattt tcgaggacct tgtcaccttg
agcccaagag agccaagatt 60taaattttcc tatgacttga tgcaaattcc caaagctaat
aacatgcaag acacgtacgg 120tcaagaagac atatttgacc tcttaacagg ttcagacgcg
actgcctcat cagtaagacc 180cgttgaaaag aacttacctg aaaaaaacga atatatacta
gcgttgaatg ttagcgtcaa 240caacaagaag tttaatgacg cggaggccaa ggcaaaaaga
ttccttgatt acgtaaggga 300gttagaatca ttttgaataa aaaacacgct ttttcagttc
gagtttatca ttatcaatac 360tgccatttca aagaatacgt aaataattaa tagtagtgat
tttcctaact ttatttagtc 420aaaaaattag ccttttaatt ctgctgtaac ccgtacatgc
ccaaaatagg gggcgggtta 480cacagaatat ataacatcgt aggtgtctgg gtgaacagtt
tattcctggc atccactaaa 540tataatggag cccgcttttt aagctggcat ccagaaaaaa
aaagaatccc agcaccaaaa 600tattgttttc ttcaccaacc atcagttcat aggtccattc
tcttagcgca actacagaga 660acaggggcac aaacaggcaa aaaacgggca caacctcaat
ggagtgatgc aacctgcctg 720gagtaaatga tgacacaagg caattgaccc acgcatgtat
ctatctcatt ttcttacacc 780ttctattacc ttctgctctc tctgatttgg aaaaagctga
aaaaaaaggt tgaaaccagt 840tccctgaaat tattccccta cttgactaat aagtatataa
agacggtagg tattgattgt 900aattctgtaa atctatttct taaacttctt aaattctact
tttatagtta gtcttttttt 960tagttttaaa acaccaagaa cttagtttcg aataaacaca
cataaacaaa caaaatgttg 1020aagattgcca tcttgggtgc tgctggtggt atcggtcaat
ctttgtcttt gttgttgaag 1080gctcaattgc aataccaatt gaaggaatcc aacagatctg
ttacccacat tcatttggct 1140ttgtacgatg tcaaccaaga agctatcaac ggtgtcactg
ctgacttgtc tcacatcgat 1200accccaatct ctgtttcctc tcactctcca gctggtggta
ttgaaaactg tttgcacaac 1260gcttccattg ttgtcattcc agccggtgtt ccaagaaagc
caggtatgac ccgtgacgat 1320ttgttcaacg tcaatgccgg tatcatctct caattaggtg
attccattgc tgaatgttgt 1380gacttgtcca aggttttcgt cttggttatc tccaacccag
tcaactcttt ggttcctgtt 1440atggtttcca acatcttgaa gaaccaccca caatccagaa
actctggtat tgaaagaaga 1500atcatgggtg tcaccaaatt ggacattgtc agagcttcca
ctttcttgag agaaatcaac 1560attgaatctg gtttgactcc aagagtcaac tccatgccag
atgttccagt tatcggtggt 1620cactctggtg aaactatcat cccattattc tctcaatcta
acttcttgtc cagattgaat 1680gaagatcaat tgaaatactt gattcaccgt gtccaatacg
gtggtgacga agttgtcaag 1740gccaagaacg gtaagggttc tgctactcta tccatggctc
atgccggtta caagtgtgtt 1800gtccaattcg tttctctatt attaggtaac attgaacaaa
tccacggtac ctactacgtt 1860ccattgaaag atgctaacaa cttcccaatt gctccaggtg
ctgaccaatt attgccatta 1920gtcgacggtg ctgactactt tgccatccca ttgaccatca
ctaccaaggg tgtttcttac 1980gttgactacg atatcgtcaa cagaatgaac gacatggaaa
gaaaccaaat gttgcctatc 2040tgtgtttctc aattgaagaa gaacattgac aagggtttgg
aattcgttgc ttccagatct 2100gcttccagtt aaggcccggg cgtgaattta ctttaaatct
tgcatttaaa taaattttct 2160ttttatagct ttatgactta gtttcaattt atatactatt
ttaatgacat tttcgattca 2220ttgattgaaa gctttgtgtt ttttcttgat gcgctattgc
attgttcttg tctttttcgc 2280cacatgtaat atctgtagta gatacctgat acattgtgga
tgctgagtga aattttagtt 2340aataatggag gcgctcttaa taattttggg gatattggct
ttttttttta aagtttacaa 2400atgaattttt tccgccagga taacgattct gaagttactc
ttagcgttcc tatcggtaca 2460gccatcaaat catgcctata aatcatgcct atatttgcgt
gcagtcagta tcatctacat 2520gaaaaaaact cccgcaattt cttatagaat acgttgaaaa
ttaaatgtac gcgccaagat 2580aagataacat atatctagat gcagtaatat acacagattc
cggccggccg cggccgc 2637321966DNAArtificial sequenceTDH3p-MDH3-TDH3t
synthetic construct 32ggatccggcg cgccacgcgt ggccggcctt agtcaaaaaa
ttagcctttt aattctgctg 60taacccgtac atgcccaaaa tagggggcgg gttacacaga
atatataaca tcgtaggtgt 120ctgggtgaac agtttattcc tggcatccac taaatataat
ggagcccgct ttttaagctg 180gcatccagaa aaaaaaagaa tcccagcacc aaaatattgt
tttcttcacc aaccatcagt 240tcataggtcc attctcttag cgcaactaca gagaacaggg
gcacaaacag gcaaaaaacg 300ggcacaacct caatggagtg atgcaacctg cctggagtaa
atgatgacac aaggcaattg 360acccacgcat gtatctatct cattttctta caccttctat
taccttctgc tctctctgat 420ttggaaaaag ctgaaaaaaa aggttgaaac cagttccctg
aaattattcc cctacttgac 480taataagtat ataaagacgg taggtattga ttgtaattct
gtaaatctat ttcttaaact 540tcttaaattc tacttttata gttagtcttt tttttagttt
taaaacacca agaacttagt 600ttcgaataaa cacacataaa caaacaaaat ggttaaggtt
gccatcttag gtgcttctgg 660tggtgtcggt caaccattat ctctattatt gaaattgtct
ccatacgttt ctgaattggc 720tttgtacgat atcagagctg ctgaaggtat tggtaaggat
ttgtcccaca tcaacaccaa 780ctcctcttgt gttggttacg acaaggattc catcgaaaac
actttgtcca atgctcaagt 840tgtcttgatt ccagctggtg ttccaagaaa gccaggtttg
accagagatg atttgttcaa 900gatgaacgct ggtatcgtta agtctttggt tactgctgtc
ggtaaatttg ccccaaacgc 960tcgtatctta gtcatctcca accctgttaa ctctttggtt
ccaattgccg ttgaaacttt 1020gaagaagatg ggtaagttca agccaggtaa cgttatgggt
gtcaccaact tggatttggt 1080cagagctgaa actttcttgg ttgactactt gatgttgaag
aacccaaaga tcggtcaaga 1140acaagacaag accaccatgc acagaaaggt caccgtcatc
ggtggtcact ctggtgaaac 1200catcattcca atcatcactg acaaatcctt ggttttccaa
ttggacaagc aatacgaaca 1260tttcatccac agagtccaat tcggtggtga cgaaattgtc
aaggccaagc aaggtgccgg 1320ttctgctacc ttgtccatgg ctttcgctgg tgccaaattt
gctgaagaag tcttacgttc 1380tttccacaac gaaaagccag aaactgaatc tttgtctgct
ttcgtctact tgccaggttt 1440gaagaacggt aagaaggctc aacaattagt cggtgacaac
tccattgaat acttctcttt 1500gccaattgtt ttgagaaacg gttccgttgt ttccattgac
acttctgttt tggaaaaatt 1560gtctccaaga gaagaacaat tggtcaacac tgctgtcaag
gaattgagaa agaacattga 1620aaagggtaag tctttcatct tggacagtta aggtgaattt
actttaaatc ttgcatttaa 1680ataaattttc tttttatagc tttatgactt agtttcaatt
tatatactat tttaatgaca 1740ttttcgattc attgattgaa agctttgtgt tttttcttga
tgcgctattg cattgttctt 1800gtctttttcg ccacatgtaa tatctgtagt agatacctga
tacattgtgg atgctgagtg 1860aaattttagt taataatgga ggcgctctta ataattttgg
ggatattggc tttttttttt 1920aaagtttaca aatgaatttt ttccgccagg atgggcccgc
ggccgc 1966332950DNAArtificial sequenceTDH1-FUMR-TDH1t
synthetic construct 33ggatcccttc ccttttacag tgcttcggaa aagcacagcg
ttgtccaagg gaacaatttt 60tcttcaagtt aatgcataag aaatatcttt ttttatgttt
agctaagtaa aagcagcttg 120gagtaaaaaa aaaaatgagt aaatttctcg atggattagt
ttctcacagg taacataaca 180aaaaccaaga aaagcccgct tctgaaaact acagttgact
tgtatgctaa agggccagac 240taatgggagg agaaaaagaa acgaatgtat atgctcattt
acactctata tcaccatatg 300gaggataagt tgggctgagc ttctgatcca atttattcta
tccattagtt gctgatatgt 360cccaccagcc aacacttgat agtatctact cgccattcac
ttccagcagc gccagtaggg 420ttgttgagct tagtaaaaat gtgcgcacca caagcctaca
tgactccacg tcacatgaaa 480ccacaccgtg gggccttgtt gcgctaggaa taggatatgc
gacgaagacg cttctgctta 540gtaaccacac cacattttca gggggtcgat ctgcttgctt
cctttactgt cacgagcggc 600ccataatcgc gctttttttt taaaaggcgc gagacagcaa
acaggaagct cgggtttcaa 660ccttcggagt ggtcgcagat ctggagactg gatctttaca
atacagtaag gcaagccacc 720atctgcttct taggtgcatg cgacggtatc cacgtgcaga
acaacatagt ctgaagaagg 780gggggaggag catgttcatt ctctgtagca gtaagagctt
ggtgataatg accaaaactg 840gagtctcgaa atcatataaa tagacaatat attttcacac
aatgagattt gtagtacagt 900tctattctct ctcttgcata aataagaaat tcatcaagaa
cttggtttga tatttcacca 960acacacacaa aaaacagtac ttcactaaat ttacacacaa
aacaaaatgt cctctgcttc 1020tgctgctttg caaaaattca gagctgaaag agataccttc
ggtgacttgc aagttccagc 1080tgaccgttac tggggtgctc aaactcaaag atctttgcaa
aactttgaca ttggtggtcc 1140aactgaaaga atgccagaac cattaatcag agctttcggt
gttttgaaga aggctgctgc 1200caccgtcaac atgacctacg gtttggaccc aaaggttggt
gaagccatcc aaaaggctgc 1260tgacgaagtt atcgatggtt ctttgattga ccatttccca
ttggttgtct ggcaaaccgg 1320ttctggtact caaaccaaga tgaacgtcaa tgaagtcatc
tccaacagag ccattgaatt 1380gttgggtggt gaattaggtt ccaaggctcc agtccaccca
aacgatcatg tcaacatgtc 1440tcaatcttcc aacgacactt tcccaactgc catgcacgtt
gctgccgttg ttgaaattca 1500cggtagattg attccagctt tgaccacttt gagagatgct
ttgcaagcca aatctgctga 1560attcgaacac atcatcaaga ttggtagaac ccacttgcaa
gatgctaccc cattgacttt 1620aggtcaagaa ttctccggtt acactcaaca attgacctac
ggtattgctc gtgttcaagg 1680tactttggaa agattataca acttggctca aggtggtact
gctgtcggta ctggtttgaa 1740caccagaaag ggtttcgatg ccaaggttgc tgaagccatt
gcttccatca ctggtttacc 1800attcaagacc gctccaaaca aattcgaagc tttggctgct
cacgacgctt tggttgaagc 1860tcacggtgct ttgaacaccg ttgcttgttc tttgatgaag
attgccaacg atatccgtta 1920cttgggttct ggtccaagat gtggtttagg tgaattgtct
ctaccagaaa acgaaccagg 1980ttcttccatc atgccaggta aggtcaaccc aactcaatgt
gaagctatga ccatggtttg 2040tgctcaagtc atgggtaaca acactgccat ctctgttgct
ggttccaacg gtcaattcga 2100attgaatgtc tttaaaccag tcatgatcaa gaacttgatc
caatccatca gattaatctc 2160tgacgcttcc atctctttca ccaagaactg tgttgtcggt
attgaagcta acgaaaagaa 2220gatctcctcc atcatgaacg aatctttgat gttggtcact
gctttgaacc ctcacattgg 2280ttacgacaag gctgccaagt gtgccaagaa ggctcacaag
gaaggtacca ctttgaaaga 2340agctgctcta tctttgggtt acttgacctc tgaagaattc
gaccaatggg ttagacctga 2400ggacatgatt tctgccaagg attaaggccc gggcataaag
caatcttgat gaggataatg 2460attttttttt gaatatacat aaatactacc gtttttctgc
tagattttgt gaagacgtaa 2520ataagtacat attacttttt aagccaagac aagattaagc
attaacttta cccttttctc 2580ttctaagttt caatactagt tatcactgtt taaaagttat
ggcgagaacg tcggcggtta 2640aaatatatta ccctgaacgt ggtgaattga agttctagga
tggtttaaag atttttcctt 2700tttgggaaat aagtaaacaa tatattgctg cctttgcaaa
acgcacatac ccacaatatg 2760tgactattgg caaagaacgc attatccttt gaagaggtgg
atactgatac taagagagtc 2820tctattccgg ctccactttt agtccagaga ttacttgtct
tcttacgtat cagaacaaga 2880aagcatttcc aaagtaattg catttgccct tgagcagtat
atatatacta agaaggcgcg 2940ccgcggccgc
2950345037DNAArtificial sequenceTDH3p-FRDm1-TDH3t
synthetic construct 34ggatccggcg cgccctattt tcgaggacct tgtcaccttg
agcccaagag agccaagatt 60taaattttcc tatgacttga tgcaaattcc caaagctaat
aacatgcaag acacgtacgg 120tcaagaagac atatttgacc tcttaacagg ttcagacgcg
actgcctcat cagtaagacc 180cgttgaaaag aacttacctg aaaaaaacga atatatacta
gcgttgaatg ttagcgtcaa 240caacaagaag tttaatgacg cggaggccaa ggcaaaaaga
ttccttgatt acgtaaggga 300gttagaatca ttttgaataa aaaacacgct ttttcagttc
gagtttatca ttatcaatac 360tgccatttca aagaatacgt aaataattaa tagtagtgat
tttcctaact ttatttagtc 420aaaaaattag ccttttaatt ctgctgtaac ccgtacatgc
ccaaaatagg gggcgggtta 480cacagaatat ataacatcgt aggtgtctgg gtgaacagtt
tattcctggc atccactaaa 540tataatggag cccgcttttt aagctggcat ccagaaaaaa
aaagaatccc agcaccaaaa 600tattgttttc ttcaccaacc atcagttcat aggtccattc
tcttagcgca actacagaga 660acaggggcac aaacaggcaa aaaacgggca caacctcaat
ggagtgatgc aacctgcctg 720gagtaaatga tgacacaagg caattgaccc acgcatgtat
ctatctcatt ttcttacacc 780ttctattacc ttctgctctc tctgatttgg aaaaagctga
aaaaaaaggt tgaaaccagt 840tccctgaaat tattccccta cttgactaat aagtatataa
agacggtagg tattgattgt 900aattctgtaa atctatttct taaacttctt aaattctact
tttatagtta gtcttttttt 960tagttttaaa acaccaagaa cttagtttcg aataaacaca
cataaacaaa caaaatgggt 1020gctgatggta tttcttctgc ttccattgtt gttactgacc
cagaagctgc tgccaagaag 1080cgtgacagaa tggccagaga attgttgtcc tccaactctg
gtctatgtca agaagatgaa 1140ccaaccatca tcaacttaaa gggtttggaa cacaccattc
catacagatt ggccgttgtt 1200ttgtgtaact ccagatccac tggtgaattc gaagccaagg
ctgctgaaat cttgagaaag 1260gctttccaca tggttgacta ctctttgaat tgtttcaacc
cagaatctga attgtcccgt 1320gtcaactctt taccagtcgg tgaaaagcac caaatgtccg
aagatctaag acatgtcatg 1380gaatgtacca tttctgtcca ccactcctct ggtatgggtt
tcgacccagc tgctggtcca 1440atcatctcca gattgagagg tgccatgaga gatcacaacg
acatgtccga tatctccgtc 1500actgaagctg aagttgaatt attctctttg gctcaatctt
tcgatgtcga cttggaagaa 1560ggtactattg ccagaaagca ctctgaagcc agattggatt
tgggtggtgt caacaagggt 1620tacactgttg actacgttgt tgaccatttg agagctgctg
gtatgccaaa cgtcttgttc 1680gaatggggtg gtgatatcag agcttctggt agaaacatca
agggtaactt gtgggctgtt 1740gccatcaagc gtccaccatc tgttgaagaa gttatccgtc
gtgccaaggg taagatgtta 1800aagatgggtg aagaagaaca agaagaaaag gacgatgact
ctccatcttt gttgcacgtt 1860gttgaattgg atgacgaagc tttgtgtacc tctggtgact
acgaaaacgt cttataccat 1920ccaaagcacg gtgttgctgg ttccattttc gactggcaac
gtcgtggttt attgtctcca 1980gaagaaggtg ctttagctca agtttccgtc aaatgttact
ctgccatgta cgctgatgct 2040ttggccactg tttgtttggt caagagagat gctgtcagaa
tcagatactt gttggaaggt 2100tggagatacg tcagatctcg tgtcaccaac tacttcgctt
acaccagaca aggtgaaaga 2160ttggctcaca tgcacgaaat tgctcaagaa accagagaat
taagagaaat cagaattgct 2220ggttctttgc catccagaat tgttatcgtc ggtggtggtt
tggctggtct atccgctgcc 2280attgaagctg cttcttgtgg tgctcaagtc attttgatgg
aaaaggaagg tagaattggt 2340ggtaactctg ccaaggctac ctctggtatc aacggttggg
gtaccagaac ccaagccaag 2400tctgatatct tggatggtgg taagtacttt gaaagagaca
ctttcttgtc cggtgtcggt 2460ggtaccactg acccagcttt ggtcaaggtc ttgtccgtca
aatctggtga cgctatcggt 2520tggttaactt ctttgggtgt cccattgtcc gttttgtctc
aattgggtgg tcactctttc 2580aagagaactc acagagctcc agacaagact gatggtactc
cattaccaat tggtcacacc 2640atcatgagaa ctttggaaga tcatatcaga aacaacttgt
ctgaaagagt taccatcatg 2700acccacgttt ctgttactga attgttgcac gaaactgaca
ccactccaga tggtgcttct 2760gaagttcgtg tcaccggtgt ccgttacaga gacttgtctg
atgtcgatgg tcaaccttcc 2820aaactattgg ctgacgctgt tgttttggcc actggtggtt
tctccaacga cagagaagaa 2880aactctttgt tgtgtaaata cgctcctcat ttggcttctt
tcccaactac caacggtcca 2940tgggctactg gtgacggtgt caaattggcc acctccgttg
gtgccaagtt ggttgacatg 3000gacaaggttc aattgcaccc aactggtttg attgacccaa
aggacccagc taacaccact 3060aagatcttgg gtccagaagc tttgagaggt tctggtggta
ttttgttgaa caagcaaggt 3120aagagattcg tcaacgaatt ggacttgaga tccgttgttt
ccaaggccat taacactcaa 3180ggtaacgaat acccaggttc tggtggttgt tactttgctt
actgtgtctt aaacgaagat 3240gctaccaact tattctgtgg tggtgctttg ggtttctacg
gtaagaaatt aggtttgttc 3300caaagagctg aaactgttga agaattggcc aaattgattg
gttgtgacga aggtgaattg 3360agagacactt tggaaaaata cgaaacctgt tccaaggcca
aggttgcttg tccagtcact 3420ggtaaggttg ttttcccatg tgttgtcggt accagaggtc
catacaatgt tgctttcgtc 3480actccatcca tccactacac catgggtggt tgtttgatct
ctccagctgc tgaagtcttg 3540caagaataca agggtttgaa tatcttggaa aaccacagac
caatcagatg tttgttcggt 3600gctggtgaag tcactggtgg tgtccacggt ggtaacagat
taggtggtaa ctctctattg 3660gaatgtgttg tctttggtaa gattgctggt gacagagctg
ccactatctt gcaaaagaga 3720gaaattgctt tgtccaagac ctcctggacc tctgttgttg
tcagagaatc cagatctggt 3780gaacaattcg gtaccggttc cagagttttg agattcaact
tgccaggtgc tttacaaaga 3840accggtttga acttgggtga attcgttgcc atcagaggtg
aatgggatgg tcaacaatta 3900gtcggttact tctctccaat cactttgcca gaagatttgg
gtaccatctc tttgttggtc 3960agagctgaca agggtacttt gaaggaatgg atctgtgctt
tgcgtccagg tgactccgtt 4020gaaatcaagg cttgtggtgg tctaagaatt gaccaagatc
cagtcaagaa atgtttgttg 4080ttcagaaaca gaccaattac cagatttgct ttggttgctg
ctggtaccgg tgttgctcca 4140atgttgcaag ttatcagagc tgctttgaag aagccatacg
tcgacacttt ggaatccatc 4200agattgatct acgctgctga agaatatgac actttaacct
acagatctat cttgcaaaga 4260tttgctgaag aattcccaga caaattcgtt tgtaacttcg
tcttaaacaa ccctccagaa 4320ggttggaccg gtggtgttgg tttcgtcaac aagaaatctt
tgcaaaaggt tttgcaacca 4380ccttcttctg aaccattgat tgttgtttgt ggtccacctg
ttatgcaaag agatgtcaaa 4440aatgaattgt tgtccatggg ttacgacaag gaattggttc
acactgtcga tggtgaatct 4500ggtaccttgt aaggcccggg cgtgaattta ctttaaatct
tgcatttaaa taaattttct 4560ttttatagct ttatgactta gtttcaattt atatactatt
ttaatgacat tttcgattca 4620ttgattgaaa gctttgtgtt ttttcttgat gcgctattgc
attgttcttg tctttttcgc 4680cacatgtaat atctgtagta gatacctgat acattgtgga
tgctgagtga aattttagtt 4740aataatggag gcgctcttaa taattttggg gatattggct
ttttttttta aagtttacaa 4800atgaattttt tccgccagga taacgattct gaagttactc
ttagcgttcc tatcggtaca 4860gccatcaaat catgcctata aatcatgcct atatttgcgt
gcagtcagta tcatctacat 4920gaaaaaaact cccgcaattt cttatagaat acgttgaaaa
ttaaatgtac gcgccaagat 4980aagataacat atatctagat gcagtaatat acacagattc
cggccggccg cggccgc 5037354959DNAArtificial SequenceTDH3p-FRDg-TDH3t
artificial sequence 35ggatccggcg cgccctattt tcgaggacct tgtcaccttg
agcccaagag agccaagatt 60taaattttcc tatgacttga tgcaaattcc caaagctaat
aacatgcaag acacgtacgg 120tcaagaagac atatttgacc tcttaacagg ttcagacgcg
actgcctcat cagtaagacc 180cgttgaaaag aacttacctg aaaaaaacga atatatacta
gcgttgaatg ttagcgtcaa 240caacaagaag tttaatgacg cggaggccaa ggcaaaaaga
ttccttgatt acgtaaggga 300gttagaatca ttttgaataa aaaacacgct ttttcagttc
gagtttatca ttatcaatac 360tgccatttca aagaatacgt aaataattaa tagtagtgat
tttcctaact ttatttagtc 420aaaaaattag ccttttaatt ctgctgtaac ccgtacatgc
ccaaaatagg gggcgggtta 480cacagaatat ataacatcgt aggtgtctgg gtgaacagtt
tattcctggc atccactaaa 540tataatggag cccgcttttt aagctggcat ccagaaaaaa
aaagaatccc agcaccaaaa 600tattgttttc ttcaccaacc atcagttcat aggtccattc
tcttagcgca actacagaga 660acaggggcac aaacaggcaa aaaacgggca caacctcaat
ggagtgatgc aacctgcctg 720gagtaaatga tgacacaagg caattgaccc acgcatgtat
ctatctcatt ttcttacacc 780ttctattacc ttctgctctc tctgatttgg aaaaagctga
aaaaaaaggt tgaaaccagt 840tccctgaaat tattccccta cttgactaat aagtatataa
agacggtagg tattgattgt 900aattctgtaa atctatttct taaacttctt aaattctact
tttatagtta gtcttttttt 960tagttttaaa acaccaagaa cttagtttcg aataaacaca
cataaacaaa caaaatggtt 1020gatggtagat cttctgcttc cattgttgcc gttgacccag
aaagagctgc cagagaaaga 1080gatgctgctg ccagagcttt gttgcaagac tctccattgc
acaccaccat gcaatacgct 1140acctctggtt tggaattgac tgttccatac gctttgaagg
ttgttgcttc tgctgacact 1200ttcgacagag ccaaggaagt tgctgatgaa gtcttgagat
gtgcctggca attggctgac 1260accgttttga actctttcaa cccaaactct gaagtctctt
tagtcggtag attaccagtc 1320ggtcaaaagc atcaaatgtc tgctccattg aaacgtgtca
tggcttgttg tcaaagagtc 1380tacaactcct ctgctggttg tttcgaccca tccactgctc
cagttgccaa ggctttgaga 1440gaaattgctt tgggtaagga aagaaacaat gcttgtttgg
aagctttgac tcaagcttgt 1500accttgccaa actctttcgt cattgatttc gaagctggta
ctatctccag aaagcacgaa 1560cacgcttctt tggatttggg tggtgtttcc aagggttaca
tcgtcgatta cgtcattgac 1620aacatcaatg ctgctggttt ccaaaacgtt ttctttgact
ggggtggtga ctgtcgtgcc 1680tccggtatga acgccagaaa cactccatgg gttgtcggta
tcactagacc tccttccttg 1740gacatgttgc caaaccctcc aaaggaagct tcttacatct
ccgtcatctc tttggacaat 1800gaagctttgg ctacctctgg tgattacgaa aacttgatct
acactgctga cgataaacca 1860ttgacctgta cctacgattg gaaaggtaag gaattgatga
agccatctca atccaatatc 1920gctcaagttt ccgtcaagtg ttactctgcc atgtacgctg
acgctttggc taccgcttgt 1980ttcatcaagc gtgacccagc caaggtcaga caattgttgg
atggttggag atacgttaga 2040gacaccgtca gagattaccg tgtctacgtc agagaaaacg
aaagagttgc caagatgttc 2100gaaattgcca ctgaagatgc tgaaatgaga aagagaagaa
tttccaacac tttaccagct 2160cgtgtcattg ttgttggtgg tggtttggct ggtttgtccg
ctgccattga agctgctggt 2220tgtggtgctc aagttgtttt gatggaaaag gaagccaagt
tgggtggtaa ctctgccaag 2280gctacctctg gtatcaacgg ttggggtact agagctcaag
ctaaggcttc cattgtcgat 2340ggtggtaagt acttcgaaag agatacctac aagtctggta
tcggtggtaa caccgatcca 2400gctttggtta agactttgtc catgaaatct gctgacgcta
tcggttggtt gacttctcta 2460ggtgttccat tgactgtttt gtcccaatta ggtggtcact
ccagaaagag aactcacaga 2520gctccagaca agaaggatgg tactccattg ccaattggtt
tcaccatcat gaaaacttta 2580gaagatcatg ttagaggtaa cttgtccggt agaatcacca
tcatggaaaa ctgttccgtt 2640acctctttgt tgtctgaaac caaggaaaga ccagacggta
ccaagcaaat cagagttacc 2700ggtgtcgaat tcactcaagc tggttctggt aagaccacca
ttttggctga tgctgttatc 2760ttggccaccg gtggtttctc caacgacaag actgctgatt
ctttgttgag agaacatgcc 2820ccacacttgg ttaacttccc aaccaccaac ggtccatggg
ctactggtga tggtgtcaag 2880ttggctcaaa gattaggtgc tcaattggtc gatatggaca
aggttcaatt gcacccaact 2940ggtttgatca acccaaagga cccagccaac ccaaccaaat
tcttgggtcc agaagctcta 3000agaggttctg gtggtgtttt gttgaacaaa caaggtaaga
gatttgtcaa cgaattggat 3060ttgagatctg ttgtttccaa ggccatcatg gaacaaggtg
ctgaataccc aggttctggt 3120ggttccatgt ttgcttactg tgtcttgaac gctgctgctc
aaaaattgtt tggtgtttcc 3180tctcacgaat tctactggaa gaagatgggt ttgttcgtca
aggctgacac catgagagac 3240ttggctgctt tgattggttg tccagttgaa tccgttcaac
aaactttaga agaatacgaa 3300agattatcca tctctcaaag atcttgtcca attaccagaa
aatctgttta cccatgtgtt 3360ttgggtacca aaggtccata ctatgtcgcc tttgtcactc
catctatcca ctacaccatg 3420ggtggttgtt tgatttctcc atctgctgaa atccaaatga
agaacacttc ttccagagct 3480ccattgtccc actccaaccc aatcttgggt ttattcggtg
ctggtgaagt caccggtggt 3540gtccacggtg gtaacagatt aggtggtaac tctttgttgg
aatgtgttgt tttcggtaga 3600attgccggtg acagagcttc taccattttg caaagaaagt
cctctgcttt gtctttcaag 3660gtctggacca ctgttgtttt gagagaagtc agagaaggtg
gtgtctacgg tgctggttcc 3720cgtgtcttga gattcaactt accaggtgct ctacaaagat
ctggtctatc cttgggtcaa 3780ttcattgcca tcagaggtga ctgggacggt caacaattga
ttggttacta ctctccaatc 3840actttgccag acgatttggg tatgattgac attttggcca
gatctgacaa gggtacttta 3900cgtgaatgga tctctgcttt ggaaccaggt gacgctgtcg
aaatgaaggc ttgtggtggt 3960ttggtcatcg aaagaagatt atctgacaag cacttcgttt
tcatgggtca cattatcaac 4020aagctatgtt tgattgctgg tggtaccggt gttgctccaa
tgttgcaaat catcaaggcc 4080gctttcatga agccattcat cgacactttg gaatccgtcc
acttgatcta cgctgctgaa 4140gatgtcactg aattgactta cagagaagtt ttggaagaac
gtcgtcgtga atccagaggt 4200aaattcaaga aaactttcgt tttgaacaga cctcctccat
tatggactga cggtgtcggt 4260ttcatcgacc gtggtatctt gaccaaccac gttcaaccac
catctgacaa cttattggtt 4320gccatctgtg gtccaccagt tatgcaaaga attgtcaagg
ccactttaaa gactttaggt 4380tacaacatga acttggtcag aaccgttgac gaaactgaac
catctggaag ttaaggcccg 4440ggcgtgaatt tactttaaat cttgcattta aataaatttt
ctttttatag ctttatgact 4500tagtttcaat ttatatacta ttttaatgac attttcgatt
cattgattga aagctttgtg 4560ttttttcttg atgcgctatt gcattgttct tgtctttttc
gccacatgta atatctgtag 4620tagatacctg atacattgtg gatgctgagt gaaattttag
ttaataatgg aggcgctctt 4680aataattttg gggatattgg cttttttttt taaagtttac
aaatgaattt tttccgccag 4740gataacgatt ctgaagttac tcttagcgtt cctatcggta
cagccatcaa atcatgccta 4800taaatcatgc ctatatttgc gtgcagtcag tatcatctac
atgaaaaaaa ctcccgcaat 4860ttcttataga atacgttgaa aattaaatgt acgcgccaag
ataagataac atatatctag 4920atgcagtaat atacacagat tccggccggc cgcggccgc
495936438PRTSchizosaccharomyces pombe 36Met Gly Glu
Leu Lys Glu Ile Leu Lys Gln Arg Tyr His Glu Leu Leu1 5
10 15Asp Trp Asn Val Lys Ala Pro His Val
Pro Leu Ser Gln Arg Leu Lys 20 25
30His Phe Thr Trp Ser Trp Phe Ala Cys Thr Met Ala Thr Gly Gly Val
35 40 45Gly Leu Ile Ile Gly Ser Phe
Pro Phe Arg Phe Tyr Gly Leu Asn Thr 50 55
60Ile Gly Lys Ile Val Tyr Ile Leu Gln Ile Phe Leu Phe Ser Leu Phe65
70 75 80Gly Ser Cys Met
Leu Phe Arg Phe Ile Lys Tyr Pro Ser Thr Ile Lys 85
90 95Asp Ser Trp Asn His His Leu Glu Lys Leu
Phe Ile Ala Thr Cys Leu 100 105
110Leu Ser Ile Ser Thr Phe Ile Asp Met Leu Ala Ile Tyr Ala Tyr Pro
115 120 125Asp Thr Gly Glu Trp Met Val
Trp Val Ile Arg Ile Leu Tyr Tyr Ile 130 135
140Tyr Val Ala Val Ser Phe Ile Tyr Cys Val Met Ala Phe Phe Thr
Ile145 150 155 160Phe Asn
Asn His Val Tyr Thr Ile Glu Thr Ala Ser Pro Ala Trp Ile
165 170 175Leu Pro Ile Phe Pro Pro Met
Ile Cys Gly Val Ile Ala Gly Ala Val 180 185
190Asn Ser Thr Gln Pro Ala His Gln Leu Lys Asn Met Val Ile
Phe Gly 195 200 205Ile Leu Phe Gln
Gly Leu Gly Phe Trp Val Tyr Leu Leu Leu Phe Ala 210
215 220Val Asn Val Leu Arg Phe Phe Thr Val Gly Leu Ala
Lys Pro Gln Asp225 230 235
240Arg Pro Gly Met Phe Met Phe Val Gly Pro Pro Ala Phe Ser Gly Leu
245 250 255Ala Leu Ile Asn Ile
Ala Arg Gly Ala Met Gly Ser Arg Pro Tyr Ile 260
265 270Phe Val Gly Ala Asn Ser Ser Glu Tyr Leu Gly Phe
Val Ser Thr Phe 275 280 285Met Ala
Ile Phe Ile Trp Gly Leu Ala Ala Trp Cys Tyr Cys Leu Ala 290
295 300Met Val Ser Phe Leu Ala Gly Phe Phe Thr Arg
Ala Pro Leu Lys Phe305 310 315
320Ala Cys Gly Trp Phe Ala Phe Ile Phe Pro Asn Val Gly Phe Val Asn
325 330 335Cys Thr Ile Glu
Ile Gly Lys Met Ile Asp Ser Lys Ala Phe Gln Met 340
345 350Phe Gly His Ile Ile Gly Val Ile Leu Cys Ile
Gln Trp Ile Leu Leu 355 360 365Met
Tyr Leu Met Val Arg Ala Phe Leu Val Asn Asp Leu Cys Tyr Pro 370
375 380Gly Lys Asp Glu Asp Ala His Pro Pro Pro
Lys Pro Asn Thr Gly Val385 390 395
400Leu Asn Pro Thr Phe Pro Pro Glu Lys Ala Pro Ala Ser Leu Glu
Lys 405 410 415Val Asp Thr
His Val Thr Ser Thr Gly Gly Glu Ser Asp Pro Pro Ser 420
425 430Ser Glu His Glu Ser Val
435371317DNAArtificial sequenceS. pombe malae permease cpo for S.
cerevisiae 37atgggtgaat tgaaggaaat cttgaagcaa cgttaccatg aattgttgga
ctggaacgtc 60aaggctccac acgttccatt gtctcaaaga ttgaagcatt tcacctggtc
ctggtttgct 120tgtaccatgg ccactggtgg tgtcggtttg atcattggtt ctttcccatt
cagattctac 180ggtttgaaca ccattggtaa gattgtctac atcttacaaa tcttcttatt
ctctttgttt 240ggttcttgta tgttgttcag attcatcaaa tacccatcta ccatcaagga
ctcctggaac 300caccacttgg aaaaattatt cattgctacc tgtttgctat ccatctccac
tttcattgac 360atgttggcca tctacgctta cccagacact ggtgaatgga tggtctgggt
tatcagaatc 420ttatactaca tctacgttgc tgtctctttc atctactgtg tcatggcttt
cttcaccatt 480ttcaacaacc acgtttacac cattgaaact gcttctccag cttggatctt
accaattttc 540ccaccaatga tctgtggtgt cattgctggt gctgtcaact ccactcaacc
agctcaccaa 600ttgaagaaca tggttatctt cggtatctta ttccaaggtt tgggtttctg
ggtttacttg 660ttgttgtttg ctgtcaacgt tttgagattc ttcaccgttg gtttggccaa
gcctcaagac 720agaccaggta tgttcatgtt tgttggtcca ccagctttct ccggtttggc
tttgatcaac 780attgcccgtg gtgctatggg ttccagacca tacattttcg tcggtgccaa
ttcttctgaa 840tacttgggtt tcgtttccac tttcatggcc attttcatct ggggtttggc
tgcttggtgt 900tactgtttgg ccatggtttc tttcttggct ggtttcttca ccagagctcc
attgaaattt 960gcttgtggtt ggtttgcttt catcttccca aacgtcggtt tcgttaactg
taccattgaa 1020attggtaaga tgattgactc caaggccttc caaatgttcg gtcacatcat
cggtgtcatc 1080ctatgtatcc aatggatctt gttgatgtac ttgatggtca gagctttctt
ggtcaacgat 1140ttgtgttacc caggtaagga tgaagatgct cacccacctc caaagccaaa
cactggtgtt 1200ttgaacccaa ctttcccacc agaaaaggct ccagcttctt tggaaaaggt
tgacacccac 1260gttacttcca ctggtggtga atctgatcct ccatcttctg aacacgaaag
cgtttaa 131738600DNAArtificial sequenceENO1 promotor T at position
-5 was changed to A in order to obtain a better Kozak sequence
38ccgcggaacc gccagatatt cattacttga cgcaaaagcg tttgaaataa tgacgaaaaa
60gaaggaagaa aaaaaaagaa aaataccgct tctaggcggg ttatctactg atccgagctt
120ccactaggat agcacccaaa cacctgcata tttggacgac ctttacttac accaccaaaa
180accactttcg cctctcccgc ccctgataac gtccactaat tgagcgatta cctgagcggt
240cctcttttgt ttgcagcatg agacttgcat actgcaaatc gtaagtagca acgtctcaag
300gtcaaaactg tatggaaacc ttgtcacctc acttaattct agctagccta ccctgcaagt
360caagaggtct ccgtgattcc tagccacctc aaggtatgcc tctccccgga aactgtggcc
420ttttctggca cacatgatct ccacgatttc aacatataaa tagcttttga taatggcaat
480attaatcaaa tttattttac ttctttcttg taacatctct cttgtaatcc cttattcctt
540ctagctattt ttcataaaaa accaagcaac tgcttatcaa cacacaaaca ctaaaacaaa
60039300DNAArtificial sequenceENO1 terminator 39agcttttgat taagccttct
agtccaaaaa acacgttttt ttgtcattta tttcattttc 60ttagaatagt ttagtttatt
cattttatag tcacgaatgt tttatgattc tatatagggt 120tgcaaacaag catttttcat
tttatgttaa aacaatttca ggtttacctt ttattctgct 180tgtggtgacg cgggtatccg
cccgctcttt tggtcaccca tgtatttaat tgcataaata 240attcttaaaa gtggagctag
tctatttcta tttacatacc tctcatttct catttcctcc 300402240DNAArtificial
sequenceENO1p-SpMAE-ENO1t synthetic construct 40ggatccggcg cgccccgcgg
aaccgccaga tattcattac ttgacgcaaa agcgtttgaa 60ataatgacga aaaagaagga
agaaaaaaaa agaaaaatac cgcttctagg cgggttatct 120actgatccga gcttccacta
ggatagcacc caaacacctg catatttgga cgacctttac 180ttacaccacc aaaaaccact
ttcgcctctc ccgcccctga taacgtccac taattgagcg 240attacctgag cggtcctctt
ttgtttgcag catgagactt gcatactgca aatcgtaagt 300agcaacgtct caaggtcaaa
actgtatgga aaccttgtca cctcacttaa ttctagctag 360cctaccctgc aagtcaagag
gtctccgtga ttcctagcca cctcaaggta tgcctctccc 420cggaaactgt ggccttttct
ggcacacatg atctccacga tttcaacata taaatagctt 480ttgataatgg caatattaat
caaatttatt ttacttcttt cttgtaacat ctctcttgta 540atcccttatt ccttctagct
atttttcata aaaaaccaag caactgctta tcaacacaca 600aacactaaaa caaaatgggt
gaattgaagg aaatcttgaa gcaacgttac catgaattgt 660tggactggaa cgtcaaggct
ccacacgttc cattgtctca aagattgaag catttcacct 720ggtcctggtt tgcttgtacc
atggccactg gtggtgtcgg tttgatcatt ggttctttcc 780cattcagatt ctacggtttg
aacaccattg gtaagattgt ctacatctta caaatcttct 840tattctcttt gtttggttct
tgtatgttgt tcagattcat caaataccca tctaccatca 900aggactcctg gaaccaccac
ttggaaaaat tattcattgc tacctgtttg ctatccatct 960ccactttcat tgacatgttg
gccatctacg cttacccaga cactggtgaa tggatggtct 1020gggttatcag aatcttatac
tacatctacg ttgctgtctc tttcatctac tgtgtcatgg 1080ctttcttcac cattttcaac
aaccacgttt acaccattga aactgcttct ccagcttgga 1140tcttaccaat tttcccacca
atgatctgtg gtgtcattgc tggtgctgtc aactccactc 1200aaccagctca ccaattgaag
aacatggtta tcttcggtat cttattccaa ggtttgggtt 1260tctgggttta cttgttgttg
tttgctgtca acgttttgag attcttcacc gttggtttgg 1320ccaagcctca agacagacca
ggtatgttca tgtttgttgg tccaccagct ttctccggtt 1380tggctttgat caacattgcc
cgtggtgcta tgggttccag accatacatt ttcgtcggtg 1440ccaattcttc tgaatacttg
ggtttcgttt ccactttcat ggccattttc atctggggtt 1500tggctgcttg gtgttactgt
ttggccatgg tttctttctt ggctggtttc ttcaccagag 1560ctccattgaa atttgcttgt
ggttggtttg ctttcatctt cccaaacgtc ggtttcgtta 1620actgtaccat tgaaattggt
aagatgattg actccaaggc cttccaaatg ttcggtcaca 1680tcatcggtgt catcctatgt
atccaatgga tcttgttgat gtacttgatg gtcagagctt 1740tcttggtcaa cgatttgtgt
tacccaggta aggatgaaga tgctcaccca cctccaaagc 1800caaacactgg tgttttgaac
ccaactttcc caccagaaaa ggctccagct tctttggaaa 1860aggttgacac ccacgttact
tccactggtg gtgaatctga tcctccatct tctgaacacg 1920aaagcgttta agagcttttg
attaagcctt ctagtccaaa aaacacgttt ttttgtcatt 1980tatttcattt tcttagaata
gtttagttta ttcattttat agtcacgaat gttttatgat 2040tctatatagg gttgcaaaca
agcatttttc attttatgtt aaaacaattt caggtttacc 2100ttttattctg cttgtggtga
cgcgggtatc cgcccgctct tttggtcacc catgtattta 2160attgcataaa taattcttaa
aagtggagct agtctatttc tatttacata cctctcattt 2220ctcatttcct ccgcggccgc
2240411180PRTSaccharomyces
cerevisiae 41Met Ser Ser Ser Lys Lys Leu Ala Gly Leu Arg Asp Asn Phe Ser
Leu1 5 10 15Leu Gly Glu
Lys Asn Lys Ile Leu Val Ala Asn Arg Gly Glu Ile Pro 20
25 30Ile Arg Ile Phe Arg Ser Ala His Glu Leu
Ser Met Arg Thr Ile Ala 35 40
45Ile Tyr Ser His Glu Asp Arg Leu Ser Met His Arg Leu Lys Ala Asp 50
55 60Glu Ala Tyr Val Ile Gly Glu Glu Gly
Gln Tyr Thr Pro Val Gly Ala65 70 75
80Tyr Leu Ala Met Asp Glu Ile Ile Glu Ile Ala Lys Lys His
Lys Val 85 90 95Asp Phe
Ile His Pro Gly Tyr Gly Phe Leu Ser Glu Asn Ser Glu Phe 100
105 110Ala Asp Lys Val Val Lys Ala Gly Ile
Thr Trp Ile Gly Pro Pro Ala 115 120
125Glu Val Ile Asp Ser Val Gly Asp Lys Val Ser Ala Arg His Leu Ala
130 135 140Ala Arg Ala Asn Val Pro Thr
Val Pro Gly Thr Pro Gly Pro Ile Glu145 150
155 160Thr Val Gln Glu Ala Leu Asp Phe Val Asn Glu Tyr
Gly Tyr Pro Val 165 170
175Ile Ile Lys Ala Ala Phe Gly Gly Gly Gly Arg Gly Met Arg Val Val
180 185 190Arg Glu Gly Asp Asp Val
Ala Asp Ala Phe Gln Arg Ala Thr Ser Glu 195 200
205Ala Arg Thr Ala Phe Gly Asn Gly Thr Cys Phe Val Glu Arg
Phe Leu 210 215 220Asp Lys Pro Lys His
Ile Glu Val Gln Leu Leu Ala Asp Asn His Gly225 230
235 240Asn Val Val His Leu Phe Glu Arg Asp Cys
Ser Val Gln Arg Arg His 245 250
255Gln Lys Val Val Glu Val Ala Pro Ala Lys Thr Leu Pro Arg Glu Val
260 265 270Arg Asp Ala Ile Leu
Thr Asp Ala Val Lys Leu Ala Lys Val Cys Gly 275
280 285Tyr Arg Asn Ala Gly Thr Ala Glu Phe Leu Val Asp
Asn Gln Asn Arg 290 295 300His Tyr Phe
Ile Glu Ile Asn Pro Arg Ile Gln Val Glu His Thr Ile305
310 315 320Thr Glu Glu Ile Thr Gly Ile
Asp Ile Val Ser Ala Gln Ile Gln Ile 325
330 335Ala Ala Gly Ala Thr Leu Thr Gln Leu Gly Leu Leu
Gln Asp Lys Ile 340 345 350Thr
Thr Arg Gly Phe Ser Ile Gln Cys Arg Ile Thr Thr Glu Asp Pro 355
360 365Ser Lys Asn Phe Gln Pro Asp Thr Gly
Arg Leu Glu Val Tyr Arg Ser 370 375
380Ala Gly Gly Asn Gly Val Arg Leu Asp Gly Gly Asn Ala Tyr Ala Gly385
390 395 400Ala Thr Ile Ser
Pro His Tyr Asp Ser Met Leu Val Lys Cys Ser Cys 405
410 415Ser Gly Ser Thr Tyr Glu Ile Val Arg Arg
Lys Met Ile Arg Ala Leu 420 425
430Ile Glu Phe Arg Ile Arg Gly Val Lys Thr Asn Ile Pro Phe Leu Leu
435 440 445Thr Leu Leu Thr Asn Pro Val
Phe Ile Glu Gly Thr Tyr Trp Thr Thr 450 455
460Phe Ile Asp Asp Thr Pro Gln Leu Phe Gln Met Val Ser Ser Gln
Asn465 470 475 480Arg Ala
Gln Lys Leu Leu His Tyr Leu Ala Asp Leu Ala Val Asn Gly
485 490 495Ser Ser Ile Lys Gly Gln Ile
Gly Leu Pro Lys Leu Lys Ser Asn Pro 500 505
510Ser Val Pro His Leu His Asp Ala Gln Gly Asn Val Ile Asn
Val Thr 515 520 525Lys Ser Ala Pro
Pro Ser Gly Trp Arg Gln Val Leu Leu Glu Lys Gly 530
535 540Pro Ser Glu Phe Ala Lys Gln Val Arg Gln Phe Asn
Gly Thr Leu Leu545 550 555
560Met Asp Thr Thr Trp Arg Asp Ala His Gln Ser Leu Leu Ala Thr Arg
565 570 575Val Arg Thr His Asp
Leu Ala Thr Ile Ala Pro Thr Thr Ala His Ala 580
585 590Leu Ala Gly Ala Phe Ala Leu Glu Cys Trp Gly Gly
Ala Thr Phe Asp 595 600 605Val Ala
Met Arg Phe Leu His Glu Asp Pro Trp Glu Arg Leu Arg Lys 610
615 620Leu Arg Ser Leu Val Pro Asn Ile Pro Phe Gln
Met Leu Leu Arg Gly625 630 635
640Ala Asn Gly Val Ala Tyr Ser Ser Leu Pro Asp Asn Ala Ile Asp His
645 650 655Phe Val Lys Gln
Ala Lys Asp Asn Gly Val Asp Ile Phe Arg Val Phe 660
665 670Asp Ala Leu Asn Asp Leu Glu Gln Leu Lys Val
Gly Val Asn Ala Val 675 680 685Lys
Lys Ala Gly Gly Val Val Glu Ala Thr Val Cys Tyr Ser Gly Asp 690
695 700Met Leu Gln Pro Gly Lys Lys Tyr Asn Leu
Asp Tyr Tyr Leu Glu Val705 710 715
720Val Glu Lys Ile Val Gln Met Gly Thr His Ile Leu Gly Ile Lys
Asp 725 730 735Met Ala Gly
Thr Met Lys Pro Ala Ala Ala Lys Leu Leu Ile Gly Ser 740
745 750Leu Arg Thr Arg Tyr Pro Asp Leu Pro Ile
His Val His Ser His Asp 755 760
765Ser Ala Gly Thr Ala Val Ala Ser Met Thr Ala Cys Ala Leu Ala Gly 770
775 780Ala Asp Val Val Asp Val Ala Ile
Asn Ser Met Ser Gly Leu Thr Ser785 790
795 800Gln Pro Ser Ile Asn Ala Leu Leu Ala Ser Leu Glu
Gly Asn Ile Asp 805 810
815Thr Gly Ile Asn Val Glu His Val Arg Glu Leu Asp Ala Tyr Trp Ala
820 825 830Glu Met Arg Leu Leu Tyr
Ser Cys Phe Glu Ala Asp Leu Lys Gly Pro 835 840
845Asp Pro Glu Val Tyr Gln His Glu Ile Pro Gly Gly Gln Leu
Thr Asn 850 855 860Leu Leu Phe Gln Ala
Gln Gln Leu Gly Leu Gly Glu Gln Trp Ala Glu865 870
875 880Thr Lys Arg Ala Tyr Arg Glu Ala Asn Tyr
Leu Leu Gly Asp Ile Val 885 890
895Lys Val Thr Pro Thr Ser Lys Val Val Gly Asp Leu Ala Gln Phe Met
900 905 910Val Ser Asn Lys Leu
Thr Ser Asp Asp Ile Arg Arg Leu Ala Asn Ser 915
920 925Leu Asp Phe Pro Asp Ser Val Met Asp Phe Phe Glu
Gly Leu Ile Gly 930 935 940Gln Pro Tyr
Gly Gly Phe Pro Glu Pro Leu Arg Ser Asp Val Leu Arg945
950 955 960Asn Lys Arg Arg Lys Leu Thr
Cys Arg Pro Gly Leu Glu Leu Glu Pro 965
970 975Phe Asp Leu Glu Lys Ile Arg Glu Asp Leu Gln Asn
Arg Phe Gly Asp 980 985 990Ile
Asp Glu Cys Asp Val Ala Ser Tyr Asn Met Tyr Pro Arg Val Tyr 995
1000 1005Glu Asp Phe Gln Lys Ile Arg Glu
Thr Tyr Gly Asp Leu Ser Val 1010 1015
1020Leu Pro Thr Lys Asn Phe Leu Ala Pro Ala Glu Pro Asp Glu Glu
1025 1030 1035Ile Glu Val Thr Ile Glu
Gln Gly Lys Thr Leu Ile Ile Lys Leu 1040 1045
1050Gln Ala Val Gly Asp Leu Asn Lys Lys Thr Gly Gln Arg Glu
Val 1055 1060 1065Tyr Phe Glu Leu Asn
Gly Glu Leu Arg Lys Ile Arg Val Ala Asp 1070 1075
1080Lys Ser Gln Asn Ile Gln Ser Val Ala Lys Pro Lys Ala
Asp Val 1085 1090 1095His Asp Thr His
Gln Ile Gly Ala Pro Met Ala Gly Val Ile Ile 1100
1105 1110Glu Val Lys Val His Lys Gly Ser Leu Val Lys
Lys Gly Glu Ser 1115 1120 1125Ile Ala
Val Leu Ser Ala Met Lys Met Glu Met Val Val Ser Ser 1130
1135 1140Pro Ala Asp Gly Gln Val Lys Asp Val Phe
Ile Lys Asp Gly Glu 1145 1150 1155Ser
Val Asp Ala Ser Asp Leu Leu Val Val Leu Glu Glu Glu Thr 1160
1165 1170Leu Pro Pro Ser Gln Lys Lys 1175
1180423543DNASaccharomyces cerevisiae 42atgagcagta
gcaagaaatt ggccggtctt agggacaatt tcagtttgct cggcgaaaag 60aataagatct
tggtcgccaa tagaggtgaa attccgatta gaatttttag atctgctcat 120gagctgtcta
tgagaaccat cgccatatac tcccatgagg accgtctttc aatgcacagg 180ttgaaggcgg
acgaagcgta tgttatcggg gaggagggcc agtatacacc tgtgggtgct 240tacttggcaa
tggacgagat catcgaaatt gcaaagaagc ataaggtgga tttcatccat 300ccaggttatg
ggttcttgtc tgaaaattcg gaatttgccg acaaagtagt gaaggccggt 360atcacttgga
tcggccctcc agctgaagtt attgactctg tgggtgacaa agtctctgcc 420agacacttgg
cagcaagagc taacgttcct accgttcccg gtactccagg acctatcgaa 480actgtgcaag
aggcacttga cttcgttaat gaatacggct acccggtgat cattaaggcc 540gcctttggtg
gtggtggtag aggtatgaga gtcgttagag aaggtgacga cgtggcagat 600gcctttcaac
gtgctacctc cgaagcccgt actgccttcg gtaatggtac ctgctttgtg 660gaaagattct
tggacaagcc aaagcatatt gaagttcaat tgttggctga taaccacgga 720aacgtggttc
atcttttcga aagagactgt tctgtgcaaa gaagacacca aaaagttgtc 780gaagtcgctc
cagcaaagac tttgccccgt gaagttcgtg acgctatttt gacagatgct 840gttaaattag
ctaaggtatg tggttacaga aacgcaggta ccgccgaatt cttggttgac 900aaccaaaaca
gacactattt cattgaaatt aatccaagaa ttcaagtgga gcataccatc 960actgaagaaa
tcaccggtat tgacattgtt tctgcccaaa tccagattgc cgcaggtgcc 1020actttgactc
aactaggtct attacaggat aaaatcacca cccgtgggtt ttccatccaa 1080tgtcgtatta
ccactgaaga tccctctaag aatttccaac cggataccgg tcgcctggag 1140gtctatcgtt
ctgccggtgg taatggtgtg agattggacg gtggtaacgc ttatgcaggt 1200gctactatct
cgcctcacta cgactcaatg ctggtcaaat gttcatgctc tggttctact 1260tatgaaatcg
tccgtaggaa gatgattcgt gccctgatcg aattcagaat cagaggtgtt 1320aagaccaaca
ttcccttcct attgactctt ttgaccaatc cagtttttat tgagggtaca 1380tactggacga
cttttattga cgacacccca caactgttcc aaatggtatc gtcacaaaac 1440agagcgcaaa
aactgttaca ctatttggca gacttggcag ttaacggttc ttctattaag 1500ggtcaaattg
gcttgccaaa actaaaatca aatccaagtg tcccccattt gcacgatgct 1560cagggcaatg
tcatcaacgt tacaaagtct gcaccaccat ccggatggag acaagtgcta 1620ctggaaaagg
gaccatctga atttgccaag caagtcagac agttcaatgg tactctactg 1680atggacacca
cctggagaga cgctcatcaa tctctacttg caacaagagt cagaacccac 1740gatttggcta
caatcgctcc aacaaccgca catgcccttg caggtgcttt cgctttagaa 1800tgttggggtg
gtgctacatt cgacgttgca atgagattct tgcatgagga tccatgggaa 1860cgtctgagaa
aattaagatc tctggtgcct aatattccat tccaaatgtt attacgtggt 1920gccaacggtg
tggcttactc ttcattacct gacaatgcta ttgaccattt tgtcaagcaa 1980gccaaggata
atggtgttga tatatttaga gtttttgatg ccttgaatga tttagaacaa 2040ttaaaagttg
gtgtgaatgc tgtcaagaag gccggtggtg ttgtcgaagc tactgtttgt 2100tactctggtg
acatgcttca gccaggtaag aaatacaact tagactacta cctagaagtt 2160gttgaaaaaa
tagttcaaat gggtacacat atcttgggta ttaaggatat ggcaggtact 2220atgaaaccgg
ccgctgccaa attattaatt ggctccctaa gaaccagata tccggattta 2280ccaattcatg
ttcacagtca tgactccgca ggtactgctg ttgcgtctat gactgcatgt 2340gccctagcag
gtgctgatgt tgtcgatgta gctatcaatt caatgtcggg cttaacttcc 2400caaccatcaa
ttaatgcact gttggcttca ttagaaggta acattgatac tgggattaac 2460gttgagcatg
ttcgtgaatt agatgcatac tgggccgaaa tgagactgtt gtattcttgt 2520ttcgaggccg
acttgaaggg accagatcca gaagtttacc aacatgaaat cccaggtggt 2580caattgacta
acttgttatt ccaagctcaa caactgggtc ttggtgaaca atgggctgaa 2640actaaaagag
cttacagaga agccaattac ctactgggag atattgttaa agttacccca 2700acttctaagg
ttgtcggtga tttagctcaa ttcatggttt ctaacaaact gacttccgac 2760gatattagac
gtttagctaa ttctttggac tttcctgact ctgttatgga cttttttgaa 2820ggtttaattg
gtcaaccata cggtgggttc ccagaaccat taagatctga tgtattgaga 2880aacaagagaa
gaaagttgac gtgccgtcca ggtttagaat tagaaccatt tgatctcgaa 2940aaaattagag
aagacttgca gaacagattc ggtgatattg atgaatgcga tgttgcttct 3000tacaatatgt
atccaagggt ctatgaagat ttccaaaaga tcagagaaac atacggtgat 3060ttatcagttc
taccaaccaa aaatttccta gcaccagcag aacctgatga agaaatcgaa 3120gtcaccatcg
aacaaggtaa gactttgatt atcaaattgc aagctgttgg tgacttaaat 3180aagaaaactg
ggcaaagaga agtgtatttt gaattgaacg gtgaattaag aaagatcaga 3240gttgcagaca
agtcacaaaa catacaatct gttgctaaac caaaggctga tgtccacgat 3300actcaccaaa
tcggtgcacc aatggctggt gttatcatag aagttaaagt acataaaggg 3360tctttggtga
aaaagggcga atcgattgct gttttgagtg ccatgaaaat ggaaatggtt 3420gtctcttcac
cagcagatgg tcaagttaaa gacgttttca ttaaggatgg tgaaagtgtt 3480gacgcatcag
atttgttggt tgtcctagaa gaagaaaccc tacccccatc ccaaaaaaag 3540taa
35434330DNAArtificial sequenceP1 primer 43ggactagtat gagcagtagc
aagaaattgg 304431DNAArtificial
sequenceP2 primer 44ccgctcgagt tacttttttt gggatggggg t
31
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