Patent application title: Isolated Polynucleotides and Methods of Promoting a Morphology in a Fungus
Inventors:
Linda L. Lasure (Fall City, WA, US)
Ziyu Dai (Richland, WA, US)
Assignees:
BATTELLE MEMORIAL INSTITUTE
IPC8 Class: AC12N1574FI
USPC Class:
435471
Class name: Chemistry: molecular biology and microbiology process of mutation, cell fusion, or genetic modification introduction of a polynucleotide molecule into or rearrangement of nucleic acid within a microorganism (e.g., bacteria, protozoa, bacteriophage, etc.)
Publication date: 2009-02-12
Patent application number: 20090042302
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Patent application title: Isolated Polynucleotides and Methods of Promoting a Morphology in a Fungus
Inventors:
Linda L. Lasure
Ziyu Dai
Agents:
WELLS ST. JOHN P.S.
Assignees:
BATTELLE MEMORIAL INSTITUTE
Origin: SPOKANE, WA US
IPC8 Class: AC12N1574FI
USPC Class:
435471
Abstract:
The invention includes isolated polynucleotide molecules that are
differentially expressed in a native fungus exhibiting a first morphology
relative to the native fungus exhibiting a second morphology. The
invention includes a method of enhancing a bioprocess utilizing a fungus.
A transformed fungus is produced by transforming a fungus with a
recombinant polynucleotide molecule. The recombinant polynucleotide
molecule contains an isolated polynucleotide sequence linked operably to
a promoter. The polynucleotide sequence is expressed to promote a first
morphology. The first morphology of the transformed fungus enhances a
bioprocess relative to the bioprocess utilizing a second morphology.Claims:
1. A method of promoting a morphology in a fungus comprising:providing a
recombinant polynucleotide comprising an antisense orientated sequence
that is complementary to a gene coding region that is differentially
expressed in a native fungus exhibiting a pellet morphology relative to
said native fungus exhibiting a filament morphology wherein the
complementary sequence is complementary to an entirety of any one of SEQ
ID NOs.:1, 6, 8, 12, 16, 18, 22, 24, 26, 28, 33, 36 and 37;transforming
Aspergillus niger, transcribing the antisense oriented sequence to
produce a transcription product of sufficient length to hybridize to a
gene coding sequence transcription product to block translation;
andsuppressing expression of the gene coding region utilizing
transcription products produced by expression of the recombinant
polynucleotide, the suppression promoting a pellet or filament morphology
capable of being assumed by the fungi in its native form.
2. A method of enhancing a bioprocess utilizing a fungus, comprising:producing a transformed fungus by transforming Aspergillus niger with a recombinant polynucleotide molecule comprising a polynucleotide sequence complementary to the entirety any one of SEQ ID NOs.: 1, 6, 8, 12, 16, 18, 22, 24, 26, 28, 33, 36 and 37, linked operably to a promoter, the polynucleotide sequence being in antisense orientation;transcribing the polynucleotide sequence to produce polynucleotide transcripts; andhybridizing the transcripts to mRNA to suppress gene expression and promote pellet or filament morphology, the promoted morphology enhancing a bioprocess relative to the bioprocess utilizing an opposing filament or pellet morphology of the transformed fungus.
3. The method of claim 2 wherein the promoted morphology is filament morphology.
4. The method of claim 2 wherein the promoted morphology is pellet morphology.
Description:
RELATED PATENT DATA
[0001]This patent is a divisional of U.S. patent application Ser. No. 10/442,017 that was filed May 19, 2003 and which claims benefit of priority under 35 U.S.C. §119 to U.S. Provisional Patent Ser. No. 60/382,132, which was filed May 20, 2002.
TECHNICAL FIELD
[0003]The invention pertains to isolated polynucleotide molecules, recombinant polynucleotide constructs, and methods of promoting a morphology in a fungus.
BACKGROUND OF THE INVENTION
[0004]Fungi are becoming increasingly utilized for production of numerous commercially useful products. A type of fungi known as "filamentous" fungi are currently used for the industrial scale production of metabolites such as antibiotics (penicillins and cephalosporins, for example) and organic acids (citric and fumaric acids for example). Filamentous fungi are additionally useful for the industrial production of enzymes such as, for example, proteases and lipases.
[0005]Utilization of a filamentous fungus species for production of desired compounds often involves growing submerged cultures of the fungus. Filamentous fungi can exhibit numerous morphologies in submerged cultures, one of which is the filamentous morphology. When fungi in culture exhibit a filamentous morphology, the filamentous growth can increase the viscosity of the culture medium. The increased viscosity can affect the mass transfer and aeration properties of the culture, can cause mixing problems in a bioreactor, and can typically be accompanied by decreased productivity.
[0006]Alternatively, "filamentous" fungi can exhibit a pellet morphology. In contrast to cultures of fungi exhibiting a filamentous morphology, the viscosity of cultures of fungi exhibiting a pellet morphology can be relatively low and can utilize less power for mixing and aeration of the culture. For many products, for example citric acid, itaconic acid, statins, penicillins, and various enzymes, productivity can be enhanced utilizing fungus exhibiting a pellet morphology relative to fungus exhibiting a filamentous morphology. However, at least in certain fungal species, production of peptic enzyme or fumaric acid, for example, can be enhanced by utilizing a fungus exhibiting a filamentous morphology.
[0007]It would be desirable to develop methods to promote a desired morphology in a fungus and to develop methods for influencing or controlling morphologies exhibited by a fungus in a culture to optimize productivity.
SUMMARY OF THE INVENTION
[0008]In one aspect, the invention encompasses an isolated polynucleotide molecule that is differentially expressed in a native fungus exhibiting a pellet morphology relative to the native fungus exhibiting a filamentous morphology.
[0009]In one aspect, the invention encompasses a method of enhancing a bioprocess utilizing a fungus. A transformed fungus is produced by transforming a fungus with a recombinant polynucleotide molecule. The recombinant polynucleotide molecule contains an isolated polynucleotide sequence linked operably to a promoter. A polypeptide encoded by the polynucleotide sequence is expressed to promote a pellet morphology. The pellet morphology of the transformed fungus enhances a bioprocess relative to the bioprocess utilizing a filamentous morphology of the transformed fungus.
[0010]In one aspect, the invention encompasses a method of promoting a morphology of a fungus and enhancing productivity of a bioprocess. A fungus is transformed with an antisense oriented polynucleotide sequence complimentary to a gene sequence. A transcription product of the polynucleotide sequence hybridizes to an mRNA and thereby suppresses expression of the gene. The gene suppression promotes a morphology and enhances a bioprocess relative to the bioprocess in an alternative fungal morphology.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011]Preferred embodiments of the invention are described below with reference to the following accompanying drawings.
[0012]FIG. 1 shows the results of Northern blot analysis of the transcriptional level of the native A. niger gene corresponding to the Balu-4 cDNA sequence set fourth in SEQ ID NO.:1. Lanes 1, 2 and 3 reflect transcription levels in the pellet morphology. Transcription levels in the filamentous morphology are shown at 20 minutes (lane 4), 40 minutes (lane 5) and 120 minutes (lane 6) after inducing the filamentous morphology.
[0013]FIG. 2 shows the alignment and comparison of the predicted amino acid sequence of A. niger Balu-4, SEQ ID NO.:2 (top sequence) and the amino acid sequence of Emericella nidulans G-protein beta subunit, SEQ ID NO.:3 (bottom sequence).
[0014]FIG. 3 shows the results of Northern blot analysis of transcription levels of the native A. niger gene corresponding to the Balu-42 cDNA sequence set forth in SEQ ID NO.:4. Lanes 1, 2 and 3 reflect transcription levels in the pellet morphology. Transcription levels in the filamentous morphology are shown at 20 minutes (lane 4), 40 minutes (lane 5) and 120 minutes (lane 6) after inducing the filamentous morphology.
[0015]FIG. 4 shows the results of Northern blot analysis of transcription levels of the native A. niger gene corresponding to the Brsa-25 cDNA sequence set forth in SEQ ID NO.:6. Lanes 1, 2 and 3 reflect transcription levels in the pellet morphology. Transcription levels in the filamentous morphology are shown at 20 minutes (lane 4), 40 minutes (lane 5) and 120 minutes (lane 6) after inducing the filamentous morphology.
[0016]FIG. 5 shows the results of Northern blot analysis of transcription levels of the native A. niger gene corresponding to the Brsa-43 cDNA sequence set forth in SEQ ID NO.:8. Lanes 1, 2 and 3 reflect transcription levels in the pellet morphology. Transcription levels in the filamentous morphology are shown at 20 minutes (lane 4), 40 minutes (lane 5) and 120 minutes (lane 6) after inducing the filamentous morphology.
[0017]FIG. 6 shows the alignment and comparison of the predicted amino acid sequence of A. niger Brsa-43, SEQ ID NO.:10 (top sequence), and the amino acid sequence of the Homo sapiens lysosomal pepstatin insensitive protease, SEQ ID NO. :11 (bottom sequence).
[0018]FIG. 7 shows the results of Northern blot analysis of transcription levels of the native A. niger gene corresponding to the Brsa-47 cDNA sequence set forth in SEQ ID NO.:12. Lanes 1, 2 and 3 reflect transcription levels in the pellet morphology. Transcription levels in the filamentous morphology are shown at 20 minutes (lane 4), 40 minutes (lane 5) and 120 minutes (lane 6) after inducing the filamentous morphology.
[0019]FIG. 8 shows the alignment and comparison of the predicted amino acid sequence of A. niger Brsa-47, SEQ ID NO.:14 (top sequence), and the amino acid sequence of Sesamum indicum Myo-inositol 1-phosphate synthase, SEQ ID NO.:15 (bottom sequence).
[0020]FIG. 9 shows the results of Northern blot analysis of transcription levels of the native A. niger gene corresponding to the Brsa-109 cDNA sequence set forth in SEQ ID NO.:16. Lanes 1, 2 and 3 reflect transcription levels in the pellet morphology. Transcription levels in the filamentous morphology are shown at 20 minutes (lane 4), 40 minutes (lane 5) and 120 minutes (lane 6) after inducing the filamentous morphology.
[0021]FIG. 10 shows the results of Northern blot analysis of transcription levels of the native A. niger gene corresponding to the Brsa-118 cDNA sequence set forth in SEQ ID NO.:18. Lanes 1, 2 and 3 reflect transcription levels in the pellet morphology. Transcription levels in the filamentous morphology are shown at 20 minutes (lane 4), 40 minutes (lane 5) and 120 minutes (lane 6) after inducing the filamentous morphology.
[0022]FIG. 11 shows the alignment and comparison of the predicted amino acid sequence of A. niger Brsa-118, SEQ ID NO.:20 (top sequence), and the Neurospora crassa probable hydroxymethylglutaryl-CoA synthase, SEQ ID NO.:21 (bottom sequence).
[0023]FIG. 12 shows the results of Northern blot analysis of transcription levels of the native A. niger gene corresponding to the Arsa-7 cDNA sequence set forth in SEQ ID NO.:22. Lanes 1, 2 and 3 reflect transcription levels in the pellet morphology. Transcription levels in the filamentous morphology are shown at 20 minutes (lane 4), 40 minutes (lane 5) and 120 minutes (lane 6) after inducing the filamentous morphology.
[0024]FIG. 13 shows the results of Northern blot analysis of transcription levels of the native A. niger gene corresponding to the Arsa-48 cDNA sequence set forth in SEQ ID NO.:24. Lanes 1, 2 and 3 reflect transcription levels in the pellet morphology. Transcription levels in the filamentous morphology are shown at 20 minutes (lane 4), 40 minutes (lane 5) and 120 minutes (lane 6) after inducing the filamentous morphology.
[0025]FIG. 14 shows the results of Northern blot analysis of transcription levels of the native A. niger gene corresponding to the A-37 cDNA sequence set forth in SEQ ID NO.:26. Lanes 1, 2 and 3 reflect transcription levels in the pellet morphology. Transcription levels in the filamentous morphology are shown at 20 minutes (lane 4), 40 minutes (lane 5) and 120 minutes (lane 6) after inducing the filamentous morphology.
[0026]FIG. 1 5 shows the results of Northern blot analysis of transcription levels of the native A. niger gene corresponding to the A-90 cDNA sequence set forth in SEQ ID NO.:28. Lanes 1, 2 and 3 reflect transcription levels in the pellet morphology. Transcription levels in the filamentous morphology are shown at 20 minutes (lane 4), 40 minutes (lane 5) and 120 minutes (lane 6) after inducing the filamentous morphology.
[0027]FIG. 16 shows the results of Northern blot analysis of transcription levels of the native A. niger gene corresponding to the Arsa-43 cDNA sequence set forth in SEQ ID NO.:33. Lanes 1, 2 and 3 reflect transcription levels in the pellet morphology. Transcription levels in the filamentous morphology are shown at 20 minutes (lane 4), 40 minutes (lane 5) and 120 minutes (lane 6) after inducing the filamentous morphology.
[0028]FIG. 17 shows the alignment and comparison of the predicted amino acid sequence of A. niger Arsa-43, SEQ ID NO.:34 (top sequence), and the Aspergillus nidulans polyubiquitin protein, SEQ ID NO.:35 (bottom sequence).
[0029]FIG. 18 shows the results of Northern blot analysis of transcription levels of the native A. niger gene corresponding to the Arsa-10 cDNA partial sequence set forth in SEQ ID NO.:36. Lanes 1, 2 and 3 reflect transcription levels in the pellet morphology. Transcription levels in the filamentous morphology are shown at 20 minutes (lane 4), 40 minutes (lane 5) and 120 minutes (lane 6) after inducing the filamentous morphology.
[0030]FIG. 19 shows the results of Northern blot analysis of transcription levels of the native A. niger gene corresponding to the Arsa-27 cDNA partial sequence set forth in SEQ ID NO.:37. Lanes 1, 2 and 3 reflect transcription levels in the pellet morphology. Transcription levels in the filamentous morphology are shown at 20 minutes (lane 4), 40 minutes (lane 5) and 120 minutes (lane 6) after inducing the filamentous morphology.
[0031]FIG. 20 shows a comparison of enhanced expression levels in filamentous morphology (right) relative to the pellet morphology (left) of native A. niger for each of the Balu-4, Brsa-25, Brsa-43, Brsa-47, Brsa-1 09, and Brsa-118 genes.
[0032]FIG. 21 shows a comparison of enhanced expression levels in the pellet morphology (left) relative to filamentous morphology (right) of native A. niger for each of the Arsa-7, Arsa-10, Arsa-27, A-27, Arsa-43 and A-90 genes.
[0033]FIG. 22 shows the results of Northern blot analysis of transcription levels of the native A. niger genes corresponding to the Balu-4, Balu-42, Brsa-25, Brsa-47, Brsa-109, and Brsa-118 cDNA sequences set forth in SEQ ID NOs.:1, 4, 6, 12, 16 and 18, respectively. Panel (A) shows transcription levels in native A. niger grown in 10 ppb Mn2+ (pellet morphology) for 14 hr (lane 1), 24 hr (lane 2), 48 hr (lane 3), 72 hr (lane 4), 96 hr (lane 5) and 120 hr (lane 6). Panel (B) shows transcription levels in native A. niger grown in 1 000 ppb Mn2+ (filamentous morphology) for 1 hr (lane 1), 2 hr (lane 2), 24 hr (lane 3), 36 hr (lane 4), 72 hr (lane 5) and 108 hr (lane 6).
[0034]FIG. 23 shows the results of Northern blot analysis of transcription levels of the native A. niger genes corresponding to the Arsa-7, A-37, Arsa-48, and A-90 cDNA sequences set forth in SEQ ID NOs.:22, 24, 26 and 28, respectively. Panel (A) shows transcription levels in native A. niger grown in 10 ppb Mn2+ (pellet morphology) for 14 hr (lane 1), 24 hr (lane 2), 48 hr (lane 3), 72 hr (lane 4), 96 hr (lane 5) and 120 hr (lane 6). Panel (B) shows transcription levels in native A. niger grown in 1000 ppb Mn2+ (filamentous morphology) for 1 hr (lane 1), 2 hr (lane 2), 24 hr (lane 3), 36 hr (lane 4), 72 hr (lane 5) and 108 hr (lane 6).
[0035]FIG. 24 is a flowchart diagram illustrating a particular aspect of the present invention.
[0036]FIG. 25 shows suppression results for A. niger transformed with antisense oriented polynucleotide sequences complimentary to Balu-42 (Panel A), Brsa-25 (Panel B) and Brsa-118 (Panel C). Each panel compares morphologies of control A. niger (left) and transformed A. niger (right) containing the corresponding antisense DNA construct grown in 15 ppb Mn2+medium.
[0037]FIG. 26 shows suppression results for A. niger transformed with antisense oriented polynucleotide sequences complimentary to cDNAs corresponding to Arsa-7 (Panel A), A-37 (Panel B) and A-90 (Panel C). Each panel compares morphologies of control A. niger (left) and transformed A. niger (right) grown in 12 ppb Mn2+ medium.
[0038]FIG. 27 shows the citric acid production of control A. niger and transformed A. niger containing antisense polynucleotide sequence complimentary to Balu-42 (strain 2805) or complimentary to Brsa-118 (strain 2808). Panel (A) shows measured citric acid production for individual transformation experiments. Panel (B) shows averaged values of the data depicted in Panel (A).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0039]The invention encompasses polynucleotides that can have differential expression in a native fungus. For purposes of the present description the term "expression" of a polynucleotide sequence can refer to the combined processes of transcription and translation, or can refer to a portion of the combined transcription and translation process. The term "differential expression" can refer to two or more differing levels of expression, or can refer to an absence in expression in a first instance relative to a presence of expression in a second instance.
[0040]The invention includes isolated polynucleotide molecules that can include a polynucleotide sequence that is differentially expressed in different morphologies exhibited by a native fungus. For purposes of the present description, the term "native" can refer to an organism that has not been genetically manipulated. The term "isolated" can refer to a naturally occurring molecule such as, for example, a polynucleotide or a polypeptide that has been recovered from the organism which produced it, or alternatively can refer to a synthetic molecule.
[0041]An isolated polynucleotide molecule according to the present invention can comprise a polynucleotide sequence that has an increased expression in a fungus exhibiting a pellet morphology relative to a lower level or an absence of expression in the filamentous morphology of the fungus. Alternatively, a polynucleotide molecule according to the present invention can comprise polynucleotide sequence having an increased expression level in a filamentous morphology of a native fungus relative to a lower level or absence of expression in the pellet morphology.
[0042]Isolated polynucleotides encompassed by the present invention can be isolated from any source fungus that is capable of exhibiting a filamentous morphology and a pellet morphology. A source fungus is not limited to a specific group of fungi and can be a member any of the three major fungi groups. An exemplary member of the Basidiomycetes group is Phanerochaete chrysosporium. Exemplary members of the group of Ascomycetes and Imperfect Fungus include Aspergillus niger, Aspergillus oryzae, Aspergillus terreus, Emericella nidulans, Neurospora crassa, Fusarium oxysporum, Penicillium chrysogenum, and Trichoderma reesei. Exemplary members of the Zygomycetes group include Rhizomucor miehei and Rhizopus oryzae.
[0043]An exemplary isolated polynucleotide molecule encompassed by the present invention can comprise a polynucleotide sequence isolated from A. niger that is differentially expressed in the filamentous morphology of native A. niger relative to the pellet morphology of native A. niger. The differentially expressed polynucleotide sequence can comprise, for example, a sequence as set forth in any of SEQ ID NOs.:1, 4, 6, 8, 12, 16, 18, 22, 24, 26, 28, 33, 36 and 37, or can comprise a sequence complimentary to any of those sequences. Each of the polynucleotide sequences set forth in SEQ ID NOs.:1, 4, 6, 8,12,16, 18, 22, 24, 26, 28, 33, 36 and 37, corresponds to the sequence determined from a full-length cDNA molecule prepared according to methods discussed below, with SEQ ID NOs.:36 and 37 being partial sequences determined from full length cDNA. It is to be understood that the isolation methods and techniques discussed herein are exemplary and that numerous conventional techniques can be utilized for producing the isolated polynucleotide molecules of the present invention.
[0044]Full-length cDNA molecules comprising the polynucleotide sequences set forth in SEQ ID NOs.:1, 4, 6, 8, 12, 16, 18, 22, 24, 26, 28, 33, 36 and 37, are obtained from A. niger strain ATCC11414 utilizing suppression subtractive hybridization techniques (Diatchenko et al., Proceedings National Academy of Science U.S.A. Vol. 93, pp. 6025-6030, 1996), in conjunction with PCR-SELECT® cDNA subtraction kit (CLONTECH, Palo Alto, Calif.). Two suppression subtractive cDNA libraries are constructed. A first cDNA library is constructed utilizing cDNA obtained from A. niger exhibiting the pellet type morphology as tester and cDNA obtained from A. niger exhibiting the filamentous morphology as a driver. The driver/tester ratio is increased threefold over the ratio suggested by the subtraction kit manual.
[0045]A second suppression subtractive cDNA library is created utilizing cDNA obtained from A. niger exhibiting the filamentous morphology as tester and utilizing cDNA obtained from A. niger exhibiting pellet morphology as driver. A first cDNA pool is generated from the first library and a second cDNA pool is generated from the second library. Differentially expressed cDNAs that are specifically present or enhanced in the pellet morphology are isolated from the first cDNA library by hybridization utilizing the first cDNA pool as probes and independently hybridizing utilizing the second cDNA pool as probes. Isolation of cDNA that is enhanced or specific to the filamentous morphology of A. niger is achieved by independently hybridizing the second cDNA library utilizing the first cDNA pool and the second cDNA pool as probes.
[0046]The segments of differentially expressed cDNAs that are isolated by suppression subtractive hybridization are selected for DNA sequencing. Sequencing of the segments is performed utilizing single pass sequencing with the T7-2 primer. The DNA fragments isolated by the suppression subtractive hybridization are used to design pairs of gene specific primers for utilization in isolating full-length cDNAs.
[0047]Full-length cDNA isolation is achieved utilizing the marathon cDNA amplification kit and the ADVANTAGE® cDNA polymerase (CLONTECH, Palo Alto, Calif.). The gene specific primers designed from the suppression subtractive hybridization clones are utilized for performing rapid amplification of cDNA ends PCR (RACE-PCR). The sequence of full-length cDNAs is determined using conventional automated DNA sequencing methods.
[0048]Twelve full-length cDNA clones and two partial-length cDNA clones are produced and sequenced according to the methods discussed above. The resulting sequences are presented as follows. The sequence of the Balu-4 cDNA is set forth in SEQ ID NO.:1; the sequence of the Balu-42 cDNA is set forth in SEQ ID NO.:4; the sequence of the Brsa-25 cDNA is set forth in SEQ ID NO.:6; the sequence of the Brsa-43 cDNA is set forth in SEQ ID NO.:8; the sequence of the Brsa-47 cDNA is set forth in SEQ ID NO.:12; the sequence of the Brsa-109 cDNA is set forth in SEQ ID NO.:16; the sequence of the Brsa-118 cDNA is set forth in SEQ ID NO.:18; the sequence of the Arsa-7 cDNA is set forth in SEQ ID NO.:22; the sequence of the Arsa-48 cDNA is set forth in SEQ ID NO.:24; the sequence of the A-37 cDNA is set forth in SEQ ID NO.:26; the sequence of the A-90 cDNA is set forth in SEQ ID NO.:28; the sequence of the Arsa-43 cDNA is set forth in SEQ ID NO.:33; the partial sequence of the Arsa-10 cDNA is set forth in SEQ ID NO.:36; and the partial sequence of the Arsa-27 cDNA is set forth in SEQ ID NO.:37.
[0049]The amino acid sequence of each of the fourteen determined polynucleotide sequences is predicted utilizing the known genetic code. Homology searches are performed utilizing BLASTP to investigate homology between a predicted amino acid sequence and the sequences in the NCBI non-redundant GenBank CDS. All homology searches are conducted utilizing a threshold E value of E=0.005. Accordingly, the results of each BLAST homology search (discussed below) are based upon this initial threshold value.
[0050]Northern blot analysis is utilized to analyze the expression levels of the genes in native A. niger corresponding to each of the fourteen cDNA clones. The expression of each gene by A. niger exhibiting filamentous morphology is compared to the expression of the same gene in A. niger exhibiting the pellet morphology. For expression analysis, A. niger is initially grown in a culture medium containing less than or equal to about 12 parts per billion (ppb) Mn2+ for 12 hours. After the initial 12 hours of growth the culture is divided into two halves, the first half is maintained at low Mn2+ concentration (less than or equal to about 12 ppb) and the other half is brought to a final concentration of approximately 1000 ppb Mn2+ (or in some instances to a final concentration of greater than or equal to about 15 ppb Mn2+). A. niger can be extremely sensitive to Mn2+ concentration. At Mn2+ concentrations at or below about 12 ppb, native A. niger exhibits the pelleted morphology, while at Mn2+ concentrations higher than about 12 ppb, native A. niger exhibits filamentous morphology. To simplify the present description, the point at which the culture is divided into two halves (after 12 hours of initial growth) can be referred to as time zero (t=0). Additionally, since the addition of Mn2+ to a final concentration of above 12 ppb promotes the filamentous morphology, the addition of Mn2+ can be referred to as filament induction.
[0051]Culture samples are collected at 20, 40, 60 and 120 minutes after time zero from both the non-induced culture (pellet morphology) and the induced culture (filamentous morphology). The samples are centrifuged to form culture pellets which are frozen with liquid nitrogen and stored at -80° C. for future total RNA extraction.
[0052]Total RNA can be isolated from the frozen culture pellets utilizing conventional methods. After size fractionation of the total RNA sample by conventional gel electrophoresis techniques and subsequent transfer to a blotting membrane, the total RNA samples collected at each time point are analyzed using hybridization of probes that are synthesized by randomly priming the isolated suppression subtractive hybridization cDNA fragments or by randomly priming fragments of full-length cDNA digested with restricting endonuclease. Probe synthesis includes incorporation of [32P]-a-dCTP. Hybridization results of the Northern blots can be visualized by exposing the blots to x-ray film.
[0053]FIG. 1 shows the x-ray film exposure of a Northern blot analysis of the expression of the A. niger gene corresponding to Balu-4 SEQ ID NO.:1. Increased hybridization is apparent in mRNA samples taken from filamentous cultures (lanes 4, 5 and 6) relative to mRNA produced in pellet morphology (lanes 1-3). Fifteen micrograms (μg) of total RNA is used for each lane. The RNA samples utilized are obtained from post t=0 pellet cultures at t=20 minutes (lane 1), t=40 minutes (lane 2) and t=120 minutes (lane 3); and from post-induction filamentous cultures at t=20 minutes (lane 4), t=40 minutes (lane 5) and t=1 20 minutes (lane 6). The total RNA used for each lane and the lane identification for each of the Northern blots discussed below is the same as that set forth for FIG. 1. The results shown in FIG. 1 indicate that Balu-4 is differentially expressed in native A. niger, with an increased level of expression detected in the filamentous morphology.
[0054]The predicted amino acid sequence of Balu-4 is set forth in SEQ ID NO.:2. The Balu-4 amino acid sequence is predicted from the Balu-4 cDNA sequence (SEQ ID NO.:1). As shown in FIG. 2, an amino acid sequence homology search utilizing BLASTP indicates that SEQ ID NO.:2 (top sequence) has a 97% identity with the amino acid sequence of a G-protein beta subunit of Emericella nidulans, SEQ ID NO.:3 (bottom sequence). Positions of sequence identity are indicated by the placement of the corresponding identical amino acid symbol between SEQ ID NO.:2 (top) and SEQ ID NO.:3 (bottom). The symbol "+" shown intermediate SEQ ID NO.:2 and SEQ ID NO.:3 indicates a conservative amino acid difference. For purposes of the present invention a conservative amino acid difference or a conservative amino acid substitution can refer to a substitution of one amino acid by another amino acid with similar chemical properties. Additionally, the term "homology" can, in some instances, refer to an identical or a conservative amino acid.
[0055]The appearance of an open space between corresponding positions in SEQ ID NO.:2 and SEQ ID NO.:3 in FIG. 2 indicates a non-conservative amino acid difference between the two aligned sequences. Three sections of SEQ ID NO.:2 having relatively minimal identity with SEQ ID NO.:3 are set forth as SEQ ID NOs.:30, 31 and 32. SEQ ID NO.:30 corresponds to amino acids 28-49 of SEQ ID NO.:2. SEQ ID NO.:31 corresponds to amino acids 194-209 of SEQ ID NO.:2. SEQ ID NO.:32 corresponds to amino acids 260-288 of SEQ ID NO.:2.
[0056]FIG. 3 shows the results of Northern blot analysis of the expression of the native gene corresponding to Balu-42, SEQ ID NO.:4. The increased detection of mRNA corresponding to Balu-42 in the filamentous morphology indicates that Balu-42 is differentially expressed with increased expression in filaments relative to the pellet morphology of native A. niger.
[0057]SEQ ID NO.:5 corresponds to the Balu-42 amino acid sequence predicted from SEQ ID NO.:4. A BLASTP homology search is unable to identify homology between SEQ ID NO.:5 and any sequence in the searched database.
[0058]FIG. 4 shows the results of Northern blot analysis of the expression of the native gene corresponding to the Brsa-25 cDNA sequence set forth in SEQ ID NO.:6. The results indicate that Brsa-25 is differentially expressed with increased expression in the filamentous morphology of native A. niger relative to the pellet morphology.
[0059]The predicted amino acid sequence of Brsa-25 SEQ ID NO.:6 is set forth in SEQ ID NO.:7. A BLASTP homology search was unable to identify homology between SEQ ID NO.:7 and any sequence in the searched database.
[0060]FIG. 5 shows results of the Northern blot analysis of the expression of the native gene corresponding to the Brsa-43 cDNA set forth in SEQ ID NO.:8. The Northern blot results indicate that Brsa-43 is differentially expressed with increased expression in the filamentous morphology of native A. niger relative to the pellet morphology.
[0061]The Brsa-43 amino acid sequence predicted from SEQ ID NO.: 8 is set forth in SEQ ID NO.: 9. SEQ ID NO.:10 corresponds to amino acids 29-594 of SEQ ID NO.:9. FIG. 6 shows the BLASTP alignment and comparison of Brsa-43 SEQ ID NO.:10 (top sequence) which has 31% identity to the amino acid sequence of human tripeptidyl-peptidase I precursor (lysosomal pepstatin insensitive protease), SEQ ID NO.:11 (bottom sequence). Indication of identity and homology between sequences is as discussed above with respect to FIG. 2.
[0062]FIG. 7 shows the results of Northern blot analysis of the expression of the native Brsa-47 gene corresponding to the cDNA sequence set forth in SEQ ID NO.:12. The results indicate that Brsa-47 is differentially expressed; with increased expression levels apparent in the filamentous morphology relative to the pellet morphology of native A. niger.
[0063]The amino acid sequence of Brsa-47 as predicted from SEQ ID NO.:12 is set forth in SEQ ID NO.:13. FIG. 8 shows the BLASTP homology search results for SEQ ID NO.:14 (top sequence) which corresponds to amino acids 26-530 of SEQ ID NO.:13. The BLASTP results indicate that SEQ ID NO.:14 has a 56% identity with the amino acid sequence of Myo-inositol 1-phosphate synthase from Sesamum indicum, SEQ ID NO.:15 (bottom sequence).
[0064]The results of Northern blot analysis of the expression of the Brsa-109 gene in native A. niger corresponding to the cDNA sequence set forth in SEQ ID NO. :16 is shown in FIG. 9. The results indicate that the Brsa-109 gene is differentially expressed, with increased expression detected in the filamentous morphology relative to the pellet morphology.
[0065]The Brsa-109 amino acid sequence predicted from SEQ ID NO.:16, is set forth in SEQ ID NO.:17. A BLASTP homology search is unable to identify homology between SEQ ID NO.:19 and any sequence in the database.
[0066]FIG. 10 shows the results of Northern blot analysis of the expression of the Brsa-118 gene in native A. niger corresponding to the cDNA sequence set forth in SEQ ID NO.:18. The results indicate that the Brsa-118 gene is differentially expressed, with increased expression in the filamentous morphology relative to the pellet morphology.
[0067]The amino acid sequence of Brsa-118 predicted from SEQ ID NO.:18 is set forth in SEQ ID NO.:19. FIG. 11 shows the BLASTP homology search results for Brsa-118. The results show that the predicted amino acid sequence of Brsa-118, SEQ ID NO.:20 (top sequence), has 66% identity with the amino acid sequence of probable hydroxymethylglutaryl-CoA synthase from Neurospora crassa, SEQ ID NO.:21 (bottom sequence).
[0068]FIG. 12 shows the results of Northern blot analysis of the expression of the Arsa-7 gene in native A. niger corresponding to the cDNA sequence set forth in SEQ ID NO.:22. The results indicate that the Arsa-7 gene is differentially expressed, with increased expression levels in the pellet morphology relative to expression levels in the filamentous morphology.
[0069]The amino acid sequence of Arsa-7 as predicted from SEQ ID NO.: 22 is set forth in SEQ ID NO.:23. BLAST homology search results were unable to identify any sequences with homology to the predicted amino acid sequence of Arsa-7.
[0070]FIG. 13 shows the results of Northern blot analysis and the expression of the Arsa-48 gene in native A. niger corresponding to the cDNA sequence set forth in SEQ ID NO.:24. The results indicate the Arsa-48 gene is differentially expressed, with increased expression levels occurring in the pellet morphology relative to the filamentous morphology.
[0071]The Arsa-48 amino acid sequence as predicted from SEQ ID NO.:24, is set forth in SEQ ID NO.:25. A BLASTP homology search was unable to identify homology between the Arsa-48 amino acid sequence and any other amino acid sequence in the searched database.
[0072]FIG. 14 shows the results of a Northern blot analysis of the expression of the A-37 gene in native A. niger corresponding to the cDNA sequence set forth in SEQ ID NO.:26. The results indicate that the A-37 gene is differentially expressed with increased expression occurring in the pellet morphology relative to the expression level detected in the filamentous morphology.
[0073]The A-37 amino acid sequence as predicted from SEQ ID NO.:26, is set forth in SEQ ID NO.:27. The BLASTP homology search was unable to detect any homology between the predicted A-37 amino acid sequence and other amino acid sequences in the searched database.
[0074]FIG. 15 shows the result of Northern blot analysis of the expression of the A-90 gene in native A. niger corresponding to the cDNA sequence set forth in SEQ ID NO.:28. The results indicate that A-90 is differentially expressed with an increased expression level occurring in the pellet morphology relative to the expression level detected in the filamentous morphology.
[0075]The amino acid sequence of A-90 as predicted from SEQ ID NO.:28, is set forth in SEQ ID NO.:29. A BLASTP homology search performed on SEQ ID NO.:29, is unable to detect any homology with any other amino acid sequence in the database.
[0076]FIG. 16 shows the results of Northern blot analysis of the expression of the Arsa-43 gene in native A. niger corresponding to the cDNA sequence set forth in SEQ ID NO.:33. The results indicate that the Arsa-43 gene is differentially expressed, with increased expression in the pellet morphology relative to the filamentous morphology.
[0077]The amino acid sequence of Arsa-43 predicted from SEQ ID NO.:33, is set forth in SEQ ID NO.:34. FIG. 17 shows the BLASTP homology search results for Arsa-43. The results show that the predicted amino acid sequence of Arsa-43, SEQ ID NO.:34 (top sequence), has 96% identity with the amino acid sequence of the polyubiquitin protein from Aspergillus nidulans, SEQ ID NO.:35 (bottom sequence).
[0078]FIG. 18 shows the results of Northern blot analysis of the expression of the Arsa-10 gene in native A. niger corresponding to the cDNA partial sequence set forth in SEQ ID NO.:36. The results indicate that the Arsa-43 gene is differentially expressed, with increased expression in the pellet morphology relative to the filamentous morphology. Homology searching is unable to detect any homology between SEQ ID NO.:36 and other polynucleotide sequences in the searched database
[0079]FIG. 19 shows the results of Northern blot analysis of the expression of the Arsa-27 gene in native A. niger corresponding to the cDNA sequence set forth in SEQ ID NO.:37. The results indicate that the Arsa-43 gene is differentially expressed, with increased expression in the pellet morphology relative to the filamentous morphology. Homology searching is unable to detect any homology between SEQ ID NO.:37 and other polynucleotide sequences in the searched database.
[0080]Referring to FIGS. 20 and 21, such show bar-chart comparison of differential expression of various A. niger genes. FIG. 20 shows transcript levels for genes Balu-4. Brsa-25, Brsa-43, Brsa-47, Brsa-109 and Brsa-118, which show increased expression in filamentous A. niger. FIG. 21 shows transcript levels for genes Arsa-7, Arsa-10, Arsa-27, A-37, Arsa-43, and A-90, which show increased expression in the pellet morphology of A. niger.
[0081]Additional expression analysis is conducted utilizing cultures grown for up to 5 days post t=0 (as defined above). Referring to FIG. 22, such shows the increased transcript levels for genes Balu-4, Balu-42, Brsa-25, Brsa-47, Brsa-109, and Brsa-118 in native A. niger grown in filamentous conditions (Panel B) as compared to transcript levels in A. niger grown in pellet conditions (Panel A). Referring to FIG. 23, such shows the increased transcript levels for genes Arsa-7, A-37, Arsa-48 and A-90 in native A. niger grown in pellet conditions (Panel A), as compared to levels of the corresponding transcript in filamentous cultures (Panel B).
[0082]In particular embodiments, the present invention encompasses isolated polypeptide molecules comprising an amino acid sequence set forth in any of SEQ ID NOs.:2, 5, 7, 9, 13, 17, 19, 23, 25, 27, 29 and 34, and functional equivalents thereof. For purposes of the present description, the term functional equivalent can refer to a truncated version or a conservatively substituted version of an amino acid sequence having substantially equivalent functional properties and/or biological activity relative to the non-truncated, non-substituted polypeptide. As will be understood by those skilled in the art, conventional methods can be utilized for truncating or introducing conservative amino acid substitutions into the amino acid sequences set forth in the sequence listing. Conventional methods are available that can be utilized for producing of the isolated polypeptides of the present invention.
[0083]In addition to the isolated polynucleotide molecules discussed above, the present invention encompasses polynucleotides comprising alternative polynucleotide sequences that encode the amino acid sequences set forth in SEQ ID NOs.:2, 5, 7, 9, 13, 17, 19, 23, 25, 27, 29 and 34, or that encode functional equivalents of those amino acid sequences. The invention also encompasses amino acid sequences encoded by SEQ ID NOs.:36 and 37, and functional equivalents, and alternate polynucleotide sequences encoding the amino acid sequences encoded by SEQ ID NOs.:36 and 37. As will be under stood by those skilled in the art, various modifications can be introduced into a polynucleotide sequence without affecting the resulting amino acid sequence due to the degenerative nature of the genetic code.
[0084]Various recombinant polynucleotide constructs are encompassed by the present invention. In particular embodiments, a recombinant polynucleotide construct according to the present invention can comprise any of the isolated polynucleotide sequences discussed above. All or part of any of the polynucleotide sequences discussed herein can be linked to a promoter, preferably operably linked to a promoter. Operable linkage of a polynucleotide to a promoter to form a recombinant polynucleotide construct can allow expression of the polynucleotide sequence to be controlled by the promoter. Alternatively, a sequence complimentary to at least a part of a sequence set forth in any one of SEQ ID NO.:1, 4, 6, 8, 12, 16, 18, 22, 24, 26, 28, 33, 36 and 37, can be utilized to form a recombinant polynucleotide, and can be incorporated in antisense orientation.
[0085]In particular aspects, the complementary sequence can comprise a portion of complementary sequence of sufficient length to enable suppression hybridization (discussed below). Although utilization of polynucleotide sequences of fewer than 30 nucleotides is contemplated, suppression hybridization can typically involve utilization of one or more polynucleotides having a length of greater than or equal to 30 nucleotides. Accordingly, the invention encompasses polynucleotide sequences comprising a fragment of any of the sequences set forth in any one of SEQ ID NO.:1, 4, 6, 8, 12, 16, 18, 22, 24, 26, 28, 33, 36 and 37, and complimentary fragments. Such fragments can preferably comprise a length of at least 30 nucleotides of the corresponding sequence, or complimentary sequence.
[0086]The invention also encompasses a vector comprising any of the isolated polynucleotide sequences discussed above. Vectors encompassed by the present invention are not limited to a particular type of vector and can be, for example, a plasmid, a cosmid or a viral vector. Vectors according to the present invention can be utilized for introducing into a host cell one or more of the isolated polynucleotide molecules discussed. The host cell is not limited to a particular cell type and can be, for example, a bacterium, a fungus, or a higher-eukaryotic cell. Additionally, vectors encompassed by the present invention can be cloning vectors, expression vectors and/or integration vectors.
[0087]The invention also encompasses a transformed host cell and cell cultures which have been transformed to comprise any of the isolated polynucleotide molecules discussed above. Conventional cell transformation techniques can be utilized for introduction of the isolated polynucleotide into a desired host cell.
[0088]The present invention encompasses methods for promoting a morphology in a fungus. A process for promoting a morphology in a fungus is described with reference to a flowchart in FIG. 24. At initial step 100, an isolated polynucleotide is provided. The isolated polynucleotide from step 100 can comprise any of the isolated polynucleotides discussed above.
[0089]The isolated polynucleotide from step 100 can be used to form a recombinant polynucleotide in step 110. As discussed above, formation of the recombinant polynucleotide can comprise operably linking a promoter and the isolated polynucleotide sequence. Additionally, formation of a recombinant nucleotide step 110 can comprise formation of a vector which can be utilized to transform a fungus in step 120. Numerous fungi are available for utilization in transformation step 120. Preferably the fungus to be transformed is capable of exhibiting a filamentous morphology and is additionally capable of exhibiting a pellet morphology. Exemplary fungi for purposes of step 120 can be, for example, any of the fungi discussed above with respect to source fungi.
[0090]After transformation step 120, a polypeptide encoded by the recombinant polynucleotide can be expressed from the transformed fungus in step 130. The expression in step 130 can promote a particular morphology of the fungus. The particular morphology promoted by the expression can be determined by the sequence of the isolated polynucleotide provided in step 100. For example, a filamentous morphology can be promoted by providing an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs.: 2, 5, 7, 9, 13, 17, and 19, and functional equivalents thereof. Alternatively, a pellet morphology can be promoted by providing an isolated polynucleotide in step 100 that encodes a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs.:23, 25, 27 29, and 34, or a functional equivalent thereof; or that encodes an amino acid sequence encoded by polynucleotide SEQ ID NO.: 36 or 37, or a functional equivalent thereof.
[0091]In an alternate embodiment of the present invention, a recombinant polynucleotide comprising an antisense oriented complimentary sequence (discussed above) can be utilized for transformation step 120. In a suppression step 140, the RNA produced from transcription of the antisense DNA can form an RNA duplex (dsRNA) with the native mRNA and thereby promote RNA degradation and/or inhibit or block translation of the mRNA. Accordingly, recombinant antisense constructs introduced in step 120 can suppress or block expression of the complimentary gene to promote a desired morphology. For example, a polynucleotide construct comprising, a sequence complimentary to a fragment or an entirety of any of SEQ ID NOs.:1, 4, 6, 8, 12, 16 or 18 can be introduced in step 120. In step 140, the transcript produced from the antisense complimentary sequence can hybridize to mRNA transcribed from genes Balu-4, Balu-42, Brsa-25, Brsa-43, Brsa-47, Brsa-109 or Brsa-118, respectively, and inhibit or block production of the corresponding protein product. Suppression of one or more of Balu-4, Balu-42, Brsa-25, Brsa-43, Brsa-47, Brsa-109 or Brsa-118 by methods in accordance with the present invention can promote pellet morphology in the transformed host. Similarly, polynucleotides having one or more sequences complimentary to a fragment or an entirety of any of SEQ ID NOs.: 22, 24, 26, 28, 33, 36, and 37, can be introduced in step 120, can inhibit or block expression of corresponding gene Arsa-7, Arsa-48, A-37, A-90, Arsa-43, Arsa-10 and Arsa-27. Suppression of one or more of Arsa-7, Arsa-48, A-37, A-90, Arsa-43, Arsa-10 and Arsa-27 in step 140 by methods in accordance with the present invention can promote filamentous morphology in the transformed host.
[0092]Although the process shown in FIG. 24 was discussed in terms of providing a single isolated polynucleotide in step 100, it is to be understood that the invention encompasses providing two or more of the isolated polynucleotide sequences discussed above. Additionally, it is to be understood that isolated polynucleotide sequences can be provided in step 100 wherein at least one of the isolated polynucleotides provided can promote pellet morphology when expressed and at least one other provided isolated polynucleotide can promote filamentous morphology when expressed. By operably linking differing isolated polynucleotides to differing inducible promoters in step 110, and using multiple recombinant polynucleotides for transformation step 120, it can be possible to selectively promote either the filamentous morphology or the pellet morphology by inducing expression in step 130 or 140.
[0093]It can be advantageous to promote a particular morphology in a fungus since utilization of a particular fungus morphology can enhance a bioprocess in a fungus culture. For example, utilization of a pellet form of a fungus can enhance various bioprocesses such as, for example, expressing hemicellulase, expressing cellulase, expressing lignase, converting biomass to alcohol, producing organic acids, producing glucoamylase, producing penicillin and producing lovastatin. Alternatively, utilization of filamentous fungal cultures can enhance bioprocesses such as fumaric acid production or peptic enzyme production.
[0094]The process shown in FIG. 24 can be utilized to produce a transformed fungus and to promote a pellet morphology in the transformed fungus which can be utilized to enhance production of a desired product in a culture containing the transformed fungus relative to non-transformed fungus cultures under otherwise identical conditions. Alternatively, the process can be utilized to produce a transformed fungus and to promote a filament morphology in the transformed fungus. The promoted filament morphology can enhance production of a desired product in a culture containing the transformed fungus relative to non-transformed fungus culture under otherwise substantially identical conditions.
[0095]The invention also contemplates co-introduction of one or more polynucleotides encoding one or more proteins of interest along with the morphology promoting constructs discussed above. The protein of interest can be native to the host or can be from a different fungal or non-fungal species. Where the protein(s) of interest have increased expression and/or activity in a first morphology relative to a second morphology, the morphology promoting construct co-introduced can preferably promote the first morphology. A protein of interest may be one that can be collected from the culture or can be one that is involved in a bioprocess that produces a desired product or compound.
EXAMPLES
Example 1
General Methods for DNA Isolation and Functional Analysis.
[0096]Escherichia coli (E. coli) strains DH5α and JM109 are used as hosts for cloning experiments. Agrobacterium tumefaciens strain AGL0 is utilized as host for binary vectors and transformation of A. niger.
[0097]For isolation of morphology associated genes by suppression subtractive hybridization (SSH), total RNA is isolated from A. niger according to the modified acid phenol-guanidinium isothiocyanate-chloroform extraction method described by Chomczynski and Sacch (Anal. Biochem. 162:156-159 (1987)). The SSH is performed utilizing the PCR-SELECT® cDNA subtraction kit (CLONTECH, Palo Alto Calif.) as described by the manufacturer, with the exception that the amount of amount of driver cDNA relative to tester utilized was tripled for each of the first and the second hybridizations.
[0098]Morphology associated clones are identified by differential screening of SSH cDNA libraries. Two oligonucleotides are designed against each newly isolated clone sequence. Rapid amplification of cDNA and PCR (RACE-PCR) is performed to isolate the 5'-end and the 3'-end of each cDNA clone.
[0099]Fungal transformation is achieved utilizing the Bgl Il/Xba I pGpdA-hph-TtrpC fragment in pAN7-1 (Punt and van der Hondel, Methods Enzymol. 216: 447-57 (1992)), inserted into binary vector pGA482 (An et al., Binary Vectors" in Plant Molecular Biology Manual, Gelvin and Schilperolands (1988), at pp A3/1-19). Introduction of constructs based on pGA482 into Agrobacterium tumefaciens strain AGL0 is conducted utilizing the freeze-and-thaw method (Ebert et al., Proc. Natl. Acad. Sci., USA 84: 5745-5749 (1987)). Plasmids are isolated from the transformed A. tumefaciens, are digested with various restriction enzymes, and are analyzed utilizing agarose gel electrophoresis to confirm transformation. Fungal transformations are performed as described by Groot et al. (Nat. Biotechnol. 18: 839-42 (1998). At least fifteen independently transformed fungi are selected and grown on agar minimum media containing 250 μg/ml of hygromycin, and 250 μg/ml cefotaxin for each transgenic event.
Example 2
Promoting a Morphology using Antisense Expression.
[0100]Individual transgene expression vectors are constructed to comprise polynucleotide sequence complimentary to one the following: Balu-42 (SEQ ID No.: 4); Brsa-25 (SEQ ID No.:6); Brsa-118 (SEQ ID No.:18); Arsa-7 (SEQ ID No.:22); A-37 (SEQ ID No.:26); and A-90 (SEQ ID No.:28). The complimentary sequences are incorporated into the vectors in antisense orientation under the control of A. nidulans phosphoglyceral dehydrogenase (gpdA) promoter and A. nidulans trpC terminator. The constructed vectors are independently introduced into A. niger utilizing Agrobacterium tumefaciens mediated transformation. Control A. niger is prepared by transformation with binary vector without incorporated antisense sequence.
[0101]Referring to FIG. 25, such shows the promotion of the pellet morphology in transgenic A. niger expressing antisense Balu-42, Brsa-25 and Brsa-118 (right), as compared to control A. niger cultured under identical conditions. FIG. 26 shows the promotion of filamentous morphology in transgenic A. niger expressing antisense Arsa-7, A-37 and A-90 (right), as compared to control A. niger cultured under identical conditions.
Example 3
Morphology Enhanced Bio-production.
[0102]Transgenic A. niger comprising antisense complimentary Balu-42 (strain 2805) or Brsa-118 (strain 2808) is prepared as described in Example 1. Multiple independently transformed cultures of each strain and multiple control cultures (prepared as described above) were grown at 30° C. for about 50 hours. Referring to FIG. 26, Panel A shows the citric acid production for individual cultures of transformed strains 2805 (Balu-42) and 2808 (Brsa-118), and for control A. niger. Panel B shows the average citric acid production for cultures of strains 2805 and 2808 relative to control cultures.
[0103]The results indicate that the methods and sequences of the invention can be utilized to promote morphology in fungi. The promotion of a morphology by methodology of the invention can be used for enhancing production of protein and/or enhancing a bioprocess utilizing transgenic fungi.
[0104]In compliance with the statute, the invention has been described in language more or less specific as to structural and methodical features. It is to be understood, however, that the invention is not limited to the specific features shown and described, since the means herein disclosed comprise preferred forms of putting the invention into effect. The invention is, therefore, claimed in any of its forms or modifications within the proper scope of the appended claims appropriately interpreted in accordance with the doctrine of equivalents.
Sequence CWU
1
3712208DNAAspergillus niger 1accgtcaact caatttctcc ccctcaggcc cgtcctccgt
ttcacaattg acatcttccc 60tctccagggg cttgttccgt caagatggcc gacatgtccg
gcgaacagat gcaggctaag 120attaccgcgg ctaggcgcga agccgaaggc ctgaaggaca
agatcaagcg cagaaaggat 180gagttggccg atacgactct ccgtcaagtc gcgcagaacc
aaactgaaac cttgcctcgt 240attggtatga agccccggcg gacgctcaag ggacatttgg
ccaagatcta cgccatgcat 300tggtcgaccg accgccgaca tctcgtctca gcctctcagg
acggaaagct catcatctgg 360gacgcctaca ccacgaacaa ggtccatgcg atcccgctga
ggtcatcatg ggtcatgacc 420tgtgcctatg ccccgagtgg aaactacgtc gcctgcggtg
gtctcgacaa catttgctcg 480atctacaacc tctcctctcg cgagggtccg acccgtgtcg
cgcgtgagct ctccggacac 540tctggctacc tctcttgctg ccggttcatc aacgatcgca
gaatcatcac gtcttccggc 600gacatgactt gcatgctgtg ggatatcgaa tcgggctcga
aagttactga attcgctgat 660caccttggcg acgtgatgtc aatcagcatc aacccgacaa
accagaacgt tttcgtttcg 720ggcgcctgtg atgccttcgc caagctgtgg gacattcgta
ccggaaaggc ggtgcaaact 780ttcgctggac acgaatccga catcaacgcc atccagttct
tccccgacgg aaacgctttc 840ggaacgggtt ccgacgacac ctcctgccgt ctgtttgaca
tccgtgcgga tcgcgaactc 900aacacctacc agagcgacca aatactgtgc ggtatcacct
ccgttgcctt ctccgtctct 960ggcagattgc tttttgctgg ttacgatgac ttcgagtgca
aggtctggga tgttctgcgc 1020ggagacaagg ttggatccct gagtggtcac gagaaccgcg
taagctgcct gggagtcagc 1080aacgatggca tcagcttgtg cactggatcc tgggattctc
tgctcaaggt ctgggcttgg 1140taaaaaagca aaacgaacaa aaacagcaaa gataccctgt
ctcagtcttt tgcgacgtcc 1200tcattccaag tttctctttt tttccttttt ctgcgccact
aggctaaatg tccgccattg 1260tacgataatc tttttcaccg ggagcaaatc ttgtcgccct
tgctccataa tgtactatct 1320cggagtaccg gcaaagttac cacgaaacga aaaaatcacg
gggcagtcag ggtgcctaga 1380catgtcgggg ttggggattc tgccgccttt cccagctggg
tgtgacgaga aagaataagc 1440caagaaaaga gcagaatgcc aacaagaacc gacaatgctc
gaatactggt gcggtgggtt 1500gtaataatgc tattgatgtt tgagacctcg ggatcgttgc
acggatatca gtgcgttgct 1560gggcaagagg cagcgcatct cacatgtcat cttttgagct
tcgaatattt gcaggcccct 1620gttcttatgt attcgcgggc ttgactttct acttatgttc
ctttttcttt cgatctcgct 1680ttacccttca cccttactaa ccccatcccc cccctccttc
gatgtcttgt tcttttcttc 1740aatttcttac ctcgattact accatgatcg agtattcttt
tgttcacttt tcattgtttc 1800cttctcttgc cccctctttt cttctctgac ctttcttact
cactatcttc tgtacttttt 1860tgcgggtgat ggatggaaag ggagggaatg tttccggata
ggccatgacg tttttctttc 1920gactcttact gcgatcccct tctgttacta atcatcagcc
tacgtcttga aagtgtcggt 1980tgtgtcattt gagtgttttc aagcgggctt ttttttcttt
tatatccggt gttgaaatcg 2040accatgtttc cagcaaatct ttcctttatc cctcgggggt
ttcgccccac gatgtcatgt 2100tccgcgacac tcttatgtcc cgacctggtg gtcagccaag
gtgtggcaga gagttgttag 2160gcagccacta gtacaaatga caggcaatac tttttggtaa
aaaaaaaa 22082352PRTAspergillus niger 2Met Ala Asp Met Ser
Gly Glu Gln Met Gln Ala Lys Ile Thr Ala Ala1 5
10 15Arg Arg Glu Ala Glu Gly Leu Lys Asp Lys Ile
Lys Arg Arg Lys Asp 20 25
30Glu Leu Ala Asp Thr Thr Leu Arg Gln Val Ala Gln Asn Gln Thr Glu
35 40 45Thr Leu Pro Arg Ile Gly Met Lys
Pro Arg Arg Thr Leu Lys Gly His 50 55
60Leu Ala Lys Ile Tyr Ala Met His Trp Ser Thr Asp Arg Arg His Leu65
70 75 80Val Ser Ala Ser Gln
Asp Gly Lys Leu Ile Ile Trp Asp Ala Tyr Thr 85
90 95Thr Asn Lys Val His Ala Ile Pro Leu Arg Ser
Ser Trp Val Met Thr 100 105
110Cys Ala Tyr Ala Pro Ser Gly Asn Tyr Val Ala Cys Gly Gly Leu Asp
115 120 125Asn Ile Cys Ser Ile Tyr Asn
Leu Ser Ser Arg Glu Gly Pro Thr Arg 130 135
140Val Ala Arg Glu Leu Ser Gly His Ser Gly Tyr Leu Ser Cys Cys
Arg145 150 155 160Phe Ile
Asn Asp Arg Arg Ile Ile Thr Ser Ser Gly Asp Met Thr Cys
165 170 175Met Leu Trp Asp Ile Glu Ser
Gly Ser Lys Val Thr Glu Phe Ala Asp 180 185
190His Leu Gly Asp Val Met Ser Ile Ser Ile Asn Pro Thr Asn
Gln Asn 195 200 205Val Phe Val Ser
Gly Ala Cys Asp Ala Phe Ala Lys Leu Trp Asp Ile 210
215 220Arg Thr Gly Lys Ala Val Gln Thr Phe Ala Gly His
Glu Ser Asp Ile225 230 235
240Asn Ala Ile Gln Phe Phe Pro Asp Gly Asn Ala Phe Gly Thr Gly Ser
245 250 255Asp Asp Thr Ser Cys
Arg Leu Phe Asp Ile Arg Ala Asp Arg Glu Leu 260
265 270Asn Thr Tyr Gln Ser Asp Gln Ile Leu Cys Gly Ile
Thr Ser Val Ala 275 280 285Phe Ser
Val Ser Gly Arg Leu Leu Phe Ala Gly Tyr Asp Asp Phe Glu 290
295 300Cys Lys Val Trp Asp Val Leu Arg Gly Asp Lys
Val Gly Ser Leu Ser305 310 315
320Gly His Glu Asn Arg Val Ser Cys Leu Gly Val Ser Asn Asp Gly Ile
325 330 335Ser Leu Cys Thr
Gly Ser Trp Asp Ser Leu Leu Lys Val Trp Ala Trp 340
345 3503352PRTEmericella nidulans 3Met Ala Asp Met
Ser Gly Glu Gln Met Gln Ala Lys Ile Thr Ala Ala1 5
10 15Arg Arg Glu Ala Glu Gly Leu Lys Asp Lys
Ile Arg Arg Arg Lys Asp 20 25
30Asp Leu Ala Asp Thr Thr Leu Arg Asp Val Ala Gln Asn Gln Thr Asp
35 40 45Ala Leu Pro Arg Ile Gly Met Lys
Pro Arg Arg Thr Leu Lys Gly His 50 55
60Leu Ala Lys Ile Tyr Ala Met His Trp Ser Thr Asp Arg Arg His Leu65
70 75 80Val Ser Ala Ser Gln
Asp Gly Lys Leu Ile Ile Trp Asp Ala Tyr Thr 85
90 95Thr Asn Lys Val His Ala Ile Pro Leu Arg Ser
Ser Trp Val Met Thr 100 105
110Cys Ala Tyr Ala Pro Ser Gly Asn Tyr Val Ala Cys Gly Gly Leu Asp
115 120 125Asn Ile Cys Ser Ile Tyr Asn
Leu Ser Ser Arg Glu Gly Pro Thr Arg 130 135
140Val Ala Arg Glu Leu Ser Gly His Ser Gly Tyr Leu Ser Cys Cys
Arg145 150 155 160Phe Ile
Asn Asp Arg Arg Ile Ile Thr Ser Ser Gly Asp Met Thr Cys
165 170 175Met Leu Trp Asp Ile Glu Ser
Gly Ser Lys Val Thr Glu Phe Ala Asp 180 185
190His Phe Gly Asp Val Met Ser Ile Ser Ile Asn Pro Thr Asn
Gln Asn 195 200 205Ile Phe Val Ser
Gly Ala Cys Asp Ala Phe Ala Lys Leu Trp Asp Ile 210
215 220Arg Thr Gly Lys Ala Val Gln Thr Phe Ala Gly His
Glu Ser Asp Ile225 230 235
240Asn Ala Ile Gln Phe Phe Pro Asp Gly Asn Ala Phe Gly Thr Gly Ser
245 250 255Asp Asp Thr Thr Cys
Arg Leu Phe Asp Ile Arg Ala Asp Arg Ser Leu 260
265 270Asn Thr Tyr Gln Ser Asp Gln Ile Leu Cys Gly Ile
Thr Ser Val Gly 275 280 285Phe Ser
Val Ser Gly Arg Leu Leu Phe Ala Gly Tyr Asp Asp Phe Glu 290
295 300Cys Lys Val Trp Asp Val Leu Arg Gly Asp Lys
Val Gly Ser Leu Ser305 310 315
320Gly His Glu Asn Arg Val Ser Cys Leu Gly Val Ser Asn Asp Gly Ile
325 330 335Ser Leu Cys Thr
Gly Ser Trp Asp Ser Leu Leu Lys Val Trp Ala Trp 340
345 35041149DNAAspergillus niger 4acgatttgac
gtccctcgcg ttttcgccct ctcccacggt agtcactcct ttgcactaca 60tacacgaagt
cttacttcca gtcactcttt gaaaccactt ctcaatatcc ctacctctta 120tcattcttta
cttcacgcac aagacacgaa agtgaacctg taaaaatgcg tttcttcacc 180accgcccttg
tctctgccct tgcggccctg gcctctgcct acactcagcc cgactactct 240cagaacccca
ccggcaatgc catcctcacc cccgaactga accaggttgt tcctgctggc 300aagcccttcg
agatcacctg ggaccccact acctcgggca ctgtgtctct tgtccttctg 360cgcggcccca
gcaccaacgt cgtccccatc cagaccattg tcgaagacat cgacaactct 420ggcagttact
cttggactcc cagcaccacc ctcgagcctg acaccaccca ctacggtatc 480ctccttgttg
tcgagggcac tggccagtac cagtactccg tccagttcgg catctccaac 540ccttactact
cttcttcttc ctctgttgcc gctgctacta gcaccactgc cgccgccgct 600gtgagctctg
atgcttccga gactagcgtt atcatcagca agatcaccag cactatctgc 660cccgagactg
ccactgccac tgccgacgtc aagcccacct ccgtccctgt ggtcggtggc 720aacaagccca
ccagcttcgt cgttgctccc tctgcctccg gctctgccag ccttatccgc 780agctctgcca
ctccctccgg cactcctgct gccagcagct cctccgtctc tcccgttttc 840accggtgctg
ctgaccgcaa cgccatcagc ctcggcgccg tcgccgtcgg tgtcgctgcc 900gtccttgctt
tctaaatggg gcagccatcc ggcattctta ggaatttgta aagtgatgga 960ggttgtacct
agctgagaca gttgtatgta caagaacgcg caagcgcgag agagtgtgtt 1020gagattattc
atgtttgcag cgattcgatt cgattcgtcg actctttact tatgacaata 1080tacccccata
gttaatgagc aagggtaata ataagagcat tgtattatcc caaaaaaaaa 1140aaaaaaaaa
11495249PRTAspergillus niger 5Met Arg Phe Phe Thr Thr Ala Leu Val Ser Ala
Leu Ala Ala Leu Ala1 5 10
15Ser Ala Tyr Thr Gln Pro Asp Tyr Ser Gln Asn Pro Thr Gly Asn Ala
20 25 30Ile Leu Thr Pro Glu Leu Asn
Gln Val Val Pro Ala Gly Lys Pro Phe 35 40
45Glu Ile Thr Trp Asp Pro Thr Thr Ser Gly Thr Val Ser Leu Val
Leu 50 55 60Leu Arg Gly Pro Ser Thr
Asn Val Val Pro Ile Gln Thr Ile Val Glu65 70
75 80Asp Ile Asp Asn Ser Gly Ser Tyr Ser Trp Thr
Pro Ser Thr Thr Leu 85 90
95Glu Pro Asp Thr Thr His Tyr Gly Ile Leu Leu Val Val Glu Gly Thr
100 105 110Gly Gln Tyr Gln Tyr Ser
Val Gln Phe Gly Ile Ser Asn Pro Tyr Tyr 115 120
125Ser Ser Ser Ser Ser Val Ala Ala Ala Thr Ser Thr Thr Ala
Ala Ala 130 135 140Ala Val Ser Ser Asp
Ala Ser Glu Thr Ser Val Ile Ile Ser Lys Ile145 150
155 160Thr Ser Thr Ile Cys Pro Glu Thr Ala Thr
Ala Thr Ala Asp Val Lys 165 170
175Pro Thr Ser Val Pro Val Val Gly Gly Asn Lys Pro Thr Ser Phe Val
180 185 190Val Ala Pro Ser Ala
Ser Gly Ser Ala Ser Leu Ile Arg Ser Ser Ala 195
200 205Thr Pro Ser Gly Thr Pro Ala Ala Ser Ser Ser Ser
Val Ser Pro Val 210 215 220Phe Thr Gly
Ala Ala Asp Arg Asn Ala Ile Ser Leu Gly Ala Val Ala225
230 235 240Val Gly Val Ala Ala Val Leu
Ala Phe 24562083DNAAspergillus niger 6aaagttcgtc
ctctattctg tctcccttcg gcgattgtct tcgtcattcg ccttgcttta 60ccatggccac
agaaatcgag ccggccgaga tccctcccgt gctgggagtc ctcccagcat 120acggacagga
taaagaaacc cccctgagga tggtgccacg tgccgatggt gttcgcgctc 180gcttagatcc
gaacgtcacc ctcgaggagt acatgtactg ggccaagatc gagcgtcagc 240tggaagagga
agagaaccgc cagtacgtgc tggagcgcgg gcctctgacc gtcggcaagg 300tcatccagaa
ccgtttctcc aagggcgtcc accatgagaa ggagaagaaa ggcgcccaga 360atagcccgca
gatcgaaggt gaaaagggca tggtcgcatc cactccctca gattcgtccc 420tagctgttac
cgatgaggaa tggagaactg cagctcgcgc cctccgaaca gccagttggg 480gtaccgtctt
ctacttgatt accaccgacg tgctaggctg ggcaaacgca ccgttcgtct 540ttgcaagtgt
gggatacggt cctgccgtgg ctttgttcat tgtttttggt tgcttcgccg 600gcttcagtgg
ctggattctg tggaaggtgt ttctagaact cgactcaacg cgctacccct 660tgatcaactt
tggtgacacc tactatcgtg tttttggagc ttggagtcgt catttggtca 720acatcggaca
gtcgctgcag ctgctgatgt cggtgtccgt gcttgttctg ggtaacggcc 780agatcctgtc
gcagctgtcc aatgaaagta tctgtttcgt ggcgtgcatg attatccatg 840atggtcatcg
gcatggtact gtggaagcat tcggtccctt gcagcgtctc ggatggctga 900ccaacgctgc
cgtctggttg aacatcgcgg acttcatcat gatcatggtc gctgctggtg 960gccactttgg
tatcgactat caggccgtca tctcatccac cttgatccag gtcgtcgagc 1020ccgtcaaggt
cttcgctggg ccaccgcccg acaagtatca gattcaggcg acagggttct 1080cgggacaatt
cactggtgtc gaccagatgg tttacagcta cggtggtgct attttatttg 1140ttgccttcct
ggctgaaatg cgccatccgt gggacttctg gaagggattg ttgtgcgccc 1200agatgttcat
ttgttttgtc tacatcttct tcggtgcctt tgtctacagc ttctacggcc 1260aatactccat
ctccaacctt tacaacgtgg ttgagccgaa aggtctacaa atggcagtaa 1320atatagtcta
ctttttaaca tccattattg cctgcattct ctacttcaat atcggcatga 1380agtccatcta
ccaacaggtc tttatggagc ttctcaactt cccagatatc tccaccaccc 1440gcggccgcat
gctctggtac ggtctcggcc cgatctactg ggtgattgcg ttcgtcatcg 1500ccgctgccgt
gccaaacttt agtggaattt ccagcatggt cggcgcggcc ctgatcctca 1560acttcaccta
cacgctcccg ggtattctat acgttggttt ccgatgccag aaggatgctg 1620cgctacccgg
agaggggtat gatcctgcga ccggggagac ggtgcgccat gattccggca 1680tgcagcggta
tatccgtggg ttcaagaagc actgggtgct gaatcttttc tgtatcttct 1740acttctgtgg
tggattggcg tgttctggta tgggcatgtg ggctgccatt gagagtttga 1800ttgaggtgtt
tgggcccggg ggtacggtgg ctacgtcttt tgggtgtgct gcacctgttt 1860agaggggaga
ttaaaggaga gtgcactgtg gagtagatgg gcactcttga tgagactgtc 1920tataatatta
ttgttagtag atggtgatga tggtatatat gctctcatct ctgtatatgt 1980ctgtgatggt
gtcattatca tgtatggtac gacatggatg tgattttaat gttaatgcta 2040tgatcttcta
tccccaaaaa aaaaaaaaaa aaaaaaaaaa aaa
20837599PRTAspergillus niger 7Met Ala Thr Glu Ile Glu Pro Ala Glu Ile Pro
Pro Val Leu Gly Val1 5 10
15Leu Pro Ala Tyr Gly Gln Asp Lys Glu Thr Pro Leu Arg Met Val Pro
20 25 30Arg Ala Asp Gly Val Arg Ala
Arg Leu Asp Pro Asn Val Thr Leu Glu 35 40
45Glu Tyr Met Tyr Trp Ala Lys Ile Glu Arg Gln Leu Glu Glu Glu
Glu 50 55 60Asn Arg Gln Tyr Val Leu
Glu Arg Gly Pro Leu Thr Val Gly Lys Val65 70
75 80Ile Gln Asn Arg Phe Ser Lys Gly Val His His
Glu Lys Glu Lys Lys 85 90
95Gly Ala Gln Asn Ser Pro Gln Ile Glu Gly Glu Lys Gly Met Val Ala
100 105 110Ser Thr Pro Ser Asp Ser
Ser Leu Ala Val Thr Asp Glu Glu Trp Arg 115 120
125Thr Ala Ala Arg Ala Leu Arg Thr Ala Ser Trp Gly Thr Val
Phe Tyr 130 135 140Leu Ile Thr Thr Asp
Val Leu Gly Trp Ala Asn Ala Pro Phe Val Phe145 150
155 160Ala Ser Val Gly Tyr Gly Pro Ala Val Ala
Leu Phe Ile Val Phe Gly 165 170
175Cys Phe Ala Gly Phe Ser Gly Trp Ile Leu Trp Lys Val Phe Leu Glu
180 185 190Leu Asp Ser Thr Arg
Tyr Pro Leu Ile Asn Phe Gly Asp Thr Tyr Tyr 195
200 205Arg Val Phe Gly Ala Trp Ser Arg His Leu Val Asn
Ile Gly Gln Ser 210 215 220Leu Gln Leu
Leu Met Ser Val Ser Val Leu Val Leu Gly Asn Gly Gln225
230 235 240Ile Leu Ser Gln Leu Ser Asn
Glu Ser Ile Cys Phe Val Ala Cys Met 245
250 255Ile Ile His Asp Gly His Arg His Gly Thr Val Glu
Ala Phe Gly Pro 260 265 270Leu
Gln Arg Leu Gly Trp Leu Thr Asn Ala Ala Val Trp Leu Asn Ile 275
280 285Ala Asp Phe Ile Met Ile Met Val Ala
Ala Gly Gly His Phe Gly Ile 290 295
300Asp Tyr Gln Ala Val Ile Ser Ser Thr Leu Ile Gln Val Val Glu Pro305
310 315 320Val Lys Val Phe
Ala Gly Pro Pro Pro Asp Lys Tyr Gln Ile Gln Ala 325
330 335Thr Gly Phe Ser Gly Gln Phe Thr Gly Val
Asp Gln Met Val Tyr Ser 340 345
350Tyr Gly Gly Ala Ile Leu Phe Val Ala Phe Leu Ala Glu Met Arg His
355 360 365Pro Trp Asp Phe Trp Lys Gly
Leu Leu Cys Ala Gln Met Phe Ile Cys 370 375
380Phe Val Tyr Ile Phe Phe Gly Ala Phe Val Tyr Ser Phe Tyr Gly
Gln385 390 395 400Tyr Ser
Ile Ser Asn Leu Tyr Asn Val Val Glu Pro Lys Gly Leu Gln
405 410 415Met Ala Val Asn Ile Val Tyr
Phe Leu Thr Ser Ile Ile Ala Cys Ile 420 425
430Leu Tyr Phe Asn Ile Gly Met Lys Ser Ile Tyr Gln Gln Val
Phe Met 435 440 445Glu Leu Leu Asn
Phe Pro Asp Ile Ser Thr Thr Arg Gly Arg Met Leu 450
455 460Trp Tyr Gly Leu Gly Pro Ile Tyr Trp Val Ile Ala
Phe Val Ile Ala465 470 475
480Ala Ala Val Pro Asn Phe Ser Gly Ile Ser Ser Met Val Gly Ala Ala
485 490 495Leu Ile Leu Asn Phe
Thr Tyr Thr Leu Pro Gly Ile Leu Tyr Val Gly 500
505 510Phe Arg Cys Gln Lys Asp Ala Ala Leu Pro Gly Glu
Gly Tyr Asp Pro 515 520 525Ala Thr
Gly Glu Thr Val Arg His Asp Ser Gly Met Gln Arg Tyr Ile 530
535 540Arg Gly Phe Lys Lys His Trp Val Leu Asn Leu
Phe Cys Ile Phe Tyr545 550 555
560Phe Cys Gly Gly Leu Ala Cys Ser Gly Met Gly Met Trp Ala Ala Ile
565 570 575Glu Ser Leu Ile
Glu Val Phe Gly Pro Gly Gly Thr Val Ala Thr Ser 580
585 590Phe Gly Cys Ala Ala Pro Val
59582006DNAAspergillus niger 8gtgtgttgtc cctctcaagg tctccagcat gcgttcttcc
ggtctctaca cagcactcct 60gtgctccctg gccgcctcga ccaacgcgat tgtccatgaa
aagctcgccg cggtcccctc 120cggctggcat cacgtcgaag atgctggctc cgaccaccag
atcagcctgt cgatcgcgct 180ggcacgcaag aacctcgatc agcttgaatc caagctgaaa
gacttgtcaa cacctggcga 240atcgcaatac ggccagtggc tggaccagga ggatgtcgac
acgctgttcc cagtggccag 300cgacaaggct gtgattaact ggctgcgcag cgccaatatc
acccatattt cccgccaggg 360cagcttggtg aactttgcga ccacggtcga taaggtgaac
aagcttctca acgccacctt 420tgcctactac caaagcggct cttcccagag attgcgcaca
acagagtact ccatcccgga 480tgatttggtc gactcaatcg acctcatctc cccaacgacg
ttcttcggca aggaaaagac 540tactgctggt ttgaaccagc gggcgcaaaa gattgacaac
catgtggcca aacgttccaa 600cagctcgtcc tgtgccgatc tcattacgct gtcctgcctg
aaggagatgt acaactttgg 660caactacact cccagcgctt cgtcgggcag caagctgggc
ttcggcagct tcctgaacga 720atccgcctcg tattctgacc ttgccaagtt cgagaagctg
tttaacctgc cctctcagag 780cttttccgtg gagttggtca acggcggcgt caatgatcag
aatcaatcga cggcttcctt 840gaccgaggcg gacctcgatg tggaattgct cgtcggagtt
gctcatcccc tccctgtgac 900tgagttcatc acttctggcg aacctccttt cattcccgac
cccgatgagc cgagtgccgc 960cgacaacgag aacgagcctt acctccagta ctatgagtac
ctcctctcca agcccaactc 1020ggctctgccc caagtgattt ccaactccta tggtgacgac
gaacagaccg ttcccgagta 1080ctacgccaag cgagtctgca acctgaccgg acttgttggc
ctgcgcggca tcagtgtcct 1140cgagtcgtcc ggtgacgaag gtattggatc cggctgccga
accaccgacg gcaccaaccg 1200aacccaattc aaccccatct tcccggccac ctgtccctac
gtgaccgccg tgggagggac 1260aatgtcctat gcccccgaga tcgcctggga agccagttcc
ggcggattca gcaactactt 1320cgagcgggcg tggttccaga aggaagctgt gcagaactac
ctggcgcacc acatcaccaa 1380cgagacgaag cagtactact cgcaattcgc caactttagc
ggtcgcggat tccctgacgt 1440tgctgcccat agcttcgagc cttcatatga ggtgatcttc
tacggcgccc gctacggctc 1500cggcggtacc tcagccgcgt gtcccctttt ctctgcgcta
gtgggcatgt tgaacgatgc 1560tcgtctgcgg gcgggcaagt ccacgctggg tttcttgaac
cccctgctct acagcaaggg 1620gtacagagcg ttgactgatg tgacgggggg ccagtcgatc
ggatgcaatg gcattgatcc 1680gcagaatgat gagactgttg ccggcgcggg cattatcccg
tgggcgcact ggaacgccac 1740ggtcggatgg gatccggtga ctggattggg acttcctgac
tttgagaagt tgaggcagtt 1800ggtgctgtcg ttgtagatgt atactatgta tatggtatga
gattttgtgt gtgatgtgtg 1860atcttatatg agagagaatg gtttagactg tgcgtgatat
acatggacag ttcattttct 1920catttaatga gacccttcat acggtagggg tcttagaggt
cctcccattg ttatgcaaaa 1980aaaaaaaaaa aaaaaaaaaa aaaaaa
20069595PRTAspergillus niger 9Met Arg Ser Ser Gly
Leu Tyr Thr Ala Leu Leu Cys Ser Leu Ala Ala1 5
10 15Ser Thr Asn Ala Ile Val His Glu Lys Leu Ala
Ala Val Pro Ser Gly 20 25
30Trp His His Val Glu Asp Ala Gly Ser Asp His Gln Ile Ser Leu Ser
35 40 45Ile Ala Leu Ala Arg Lys Asn Leu
Asp Gln Leu Glu Ser Lys Leu Lys 50 55
60Asp Leu Ser Thr Pro Gly Glu Ser Gln Tyr Gly Gln Trp Leu Asp Gln65
70 75 80Glu Asp Val Asp Thr
Leu Phe Pro Val Ala Ser Asp Lys Ala Val Ile 85
90 95Asn Trp Leu Arg Ser Ala Asn Ile Thr His Ile
Ser Arg Gln Gly Ser 100 105
110Leu Val Asn Phe Ala Thr Thr Val Asp Lys Val Asn Lys Leu Leu Asn
115 120 125Ala Thr Phe Ala Tyr Tyr Gln
Ser Gly Ser Ser Gln Arg Leu Arg Thr 130 135
140Thr Glu Tyr Ser Ile Pro Asp Asp Leu Val Asp Ser Ile Asp Leu
Ile145 150 155 160Ser Pro
Thr Thr Phe Phe Gly Lys Glu Lys Thr Thr Ala Gly Leu Asn
165 170 175Gln Arg Ala Gln Lys Ile Asp
Asn His Val Ala Lys Arg Ser Asn Ser 180 185
190Ser Ser Cys Ala Asp Leu Ile Thr Leu Ser Cys Leu Lys Glu
Met Tyr 195 200 205Asn Phe Gly Asn
Tyr Thr Pro Ser Ala Ser Ser Gly Ser Lys Leu Gly 210
215 220Phe Gly Ser Phe Leu Asn Glu Ser Ala Ser Tyr Ser
Asp Leu Ala Lys225 230 235
240Phe Glu Lys Leu Phe Asn Leu Pro Ser Gln Ser Phe Ser Val Glu Leu
245 250 255Val Asn Gly Gly Val
Asn Asp Gln Asn Gln Ser Thr Ala Ser Leu Thr 260
265 270Glu Ala Asp Leu Asp Val Glu Leu Leu Val Gly Val
Ala His Pro Leu 275 280 285Pro Val
Thr Glu Phe Ile Thr Ser Gly Glu Pro Pro Phe Ile Pro Asp 290
295 300Pro Asp Glu Pro Ser Ala Ala Asp Asn Glu Asn
Glu Pro Tyr Leu Gln305 310 315
320Tyr Tyr Glu Tyr Leu Leu Ser Lys Pro Asn Ser Ala Leu Pro Gln Val
325 330 335Ile Ser Asn Ser
Tyr Gly Asp Asp Glu Gln Thr Val Pro Glu Tyr Tyr 340
345 350Ala Lys Arg Val Cys Asn Leu Thr Gly Leu Val
Gly Leu Arg Gly Ile 355 360 365Ser
Val Leu Glu Ser Ser Gly Asp Glu Gly Ile Gly Ser Gly Cys Arg 370
375 380Thr Thr Asp Gly Thr Asn Arg Thr Gln Phe
Asn Pro Ile Phe Pro Ala385 390 395
400Thr Cys Pro Tyr Val Thr Ala Val Gly Gly Thr Met Ser Tyr Ala
Pro 405 410 415Glu Ile Ala
Trp Glu Ala Ser Ser Gly Gly Phe Ser Asn Tyr Phe Glu 420
425 430Arg Ala Trp Phe Gln Lys Glu Ala Val Gln
Asn Tyr Leu Ala His His 435 440
445Ile Thr Asn Glu Thr Lys Gln Tyr Tyr Ser Gln Phe Ala Asn Phe Ser 450
455 460Gly Arg Gly Phe Pro Asp Val Ala
Ala His Ser Phe Glu Pro Ser Tyr465 470
475 480Glu Val Ile Phe Tyr Gly Ala Arg Tyr Gly Ser Gly
Gly Thr Ser Ala 485 490
495Ala Cys Pro Leu Phe Ser Ala Leu Val Gly Met Leu Asn Asp Ala Arg
500 505 510Leu Arg Ala Gly Lys Ser
Thr Leu Gly Phe Leu Asn Pro Leu Leu Tyr 515 520
525Ser Lys Gly Tyr Arg Ala Leu Thr Asp Val Thr Gly Gly Gln
Ser Ile 530 535 540Gly Cys Asn Gly Ile
Asp Pro Gln Asn Asp Glu Thr Val Ala Gly Ala545 550
555 560Gly Ile Ile Pro Trp Ala His Trp Asn Ala
Thr Val Gly Trp Asp Pro 565 570
575Val Thr Gly Leu Gly Leu Pro Asp Phe Glu Lys Leu Arg Gln Leu Val
580 585 590Leu Ser Leu
59510566PRTAspergillus niger 10Val Pro Ser Gly Trp His His Val Glu Asp
Ala Gly Ser Asp His Gln1 5 10
15Ile Ser Leu Ser Ile Ala Leu Ala Arg Lys Asn Leu Asp Gln Leu Glu
20 25 30Ser Lys Leu Lys Asp Leu
Ser Thr Pro Gly Glu Ser Gln Tyr Gly Gln 35 40
45Trp Leu Asp Gln Glu Asp Val Asp Thr Leu Phe Pro Val Ala
Ser Asp 50 55 60Lys Ala Val Ile Asn
Trp Leu Arg Ser Ala Asn Ile Thr His Ile Ser65 70
75 80Arg Gln Gly Ser Leu Val Asn Phe Ala Thr
Thr Val Asp Lys Val Asn 85 90
95Lys Leu Leu Asn Ala Thr Phe Ala Tyr Tyr Gln Ser Gly Ser Ser Gln
100 105 110Arg Leu Arg Thr Thr
Glu Tyr Ser Ile Pro Asp Asp Leu Val Asp Ser 115
120 125Ile Asp Leu Ile Ser Pro Thr Thr Phe Phe Gly Lys
Glu Lys Thr Thr 130 135 140Ala Gly Leu
Asn Gln Arg Ala Gln Lys Ile Asp Asn His Val Ala Lys145
150 155 160Arg Ser Asn Ser Ser Ser Cys
Ala Asp Leu Ile Thr Leu Ser Cys Leu 165
170 175Lys Glu Met Tyr Asn Phe Gly Asn Tyr Thr Pro Ser
Ala Ser Ser Gly 180 185 190Ser
Lys Leu Gly Phe Gly Ser Phe Leu Asn Glu Ser Ala Ser Tyr Ser 195
200 205Asp Leu Ala Lys Phe Glu Lys Leu Phe
Asn Leu Pro Ser Gln Ser Phe 210 215
220Ser Val Glu Leu Val Asn Gly Gly Val Asn Asp Gln Asn Gln Ser Thr225
230 235 240Ala Ser Leu Thr
Glu Ala Asp Leu Asp Val Glu Leu Leu Val Gly Val 245
250 255Ala His Pro Leu Pro Val Thr Glu Phe Ile
Thr Ser Gly Glu Pro Pro 260 265
270Phe Ile Pro Asp Pro Asp Glu Pro Ser Ala Ala Asp Asn Glu Asn Glu
275 280 285Pro Tyr Leu Gln Tyr Tyr Glu
Tyr Leu Leu Ser Lys Pro Asn Ser Ala 290 295
300Leu Pro Gln Val Ile Ser Asn Ser Tyr Gly Asp Asp Glu Gln Thr
Val305 310 315 320Pro Glu
Tyr Tyr Ala Lys Arg Val Cys Asn Leu Thr Gly Leu Val Gly
325 330 335Leu Arg Gly Ile Ser Val Leu
Glu Ser Ser Gly Asp Glu Gly Ile Gly 340 345
350Ser Gly Cys Arg Thr Thr Asp Gly Thr Asn Arg Thr Gln Phe
Asn Pro 355 360 365Ile Phe Pro Ala
Thr Cys Pro Tyr Val Thr Ala Val Gly Gly Thr Met 370
375 380Ser Tyr Ala Pro Glu Ile Ala Trp Glu Ala Ser Ser
Gly Gly Phe Ser385 390 395
400Asn Tyr Phe Glu Arg Ala Trp Phe Gln Lys Glu Ala Val Gln Asn Tyr
405 410 415Leu Ala His His Ile
Thr Asn Glu Thr Lys Gln Tyr Tyr Ser Gln Phe 420
425 430Ala Asn Phe Ser Gly Arg Gly Phe Pro Asp Val Ala
Ala His Ser Phe 435 440 445Glu Pro
Ser Tyr Glu Val Ile Phe Tyr Gly Ala Arg Tyr Gly Ser Gly 450
455 460Gly Thr Ser Ala Ala Cys Pro Leu Phe Ser Ala
Leu Val Gly Met Leu465 470 475
480Asn Asp Ala Arg Leu Arg Ala Gly Lys Ser Thr Leu Gly Phe Leu Asn
485 490 495Pro Leu Leu Tyr
Ser Lys Gly Tyr Arg Ala Leu Thr Asp Val Thr Gly 500
505 510Gly Gln Ser Ile Gly Cys Asn Gly Ile Asp Pro
Gln Asn Asp Glu Thr 515 520 525Val
Ala Gly Ala Gly Ile Ile Pro Trp Ala His Trp Asn Ala Thr Val 530
535 540Gly Trp Asp Pro Val Thr Gly Leu Gly Leu
Pro Asp Phe Glu Lys Leu545 550 555
560Arg Gln Leu Val Leu Ser 56511532PRTHomo
sapiens 11Leu Pro Pro Gly Trp Val Ser Leu Gly Arg Ala Asp Pro Glu Glu
Glu1 5 10 15Leu Ser Leu
Thr Phe Ala Leu Arg Gln Gln Asn Val Glu Arg Leu Ser 20
25 30Glu Leu Val Gln Ala Val Ser Asp Pro Ser
Ser Pro Gln Tyr Gly Lys 35 40
45Tyr Leu Thr Leu Glu Asn Val Ala Asp Leu Val Arg Pro Ser Pro Leu 50
55 60Thr Leu His Thr Val Gln Lys Trp Leu
Leu Ala Ala Gly Ala Gln Lys65 70 75
80Cys His Ser Val Ile Thr Gln Asp Phe Leu Thr Cys Trp Leu
Ser Ile 85 90 95Arg Gln
Ala Glu Leu Leu Leu Pro Gly Ala Glu Phe His His Tyr Val 100
105 110Gly Gly Pro Thr Glu Thr His Val Val
Arg Ser Pro His Pro Tyr Gln 115 120
125Leu Pro Gln Ala Leu Ala Pro His Val Asp Phe Val Gly Gly Leu His
130 135 140His Phe Pro Pro Thr Ser Ser
Leu Arg Gln Arg Pro Glu Pro Gln Val145 150
155 160Thr Gly Thr Val Gly Leu His Leu Gly Val Thr Pro
Ser Val Ile Arg 165 170
175Lys Arg Tyr Asn Leu Thr Ser Gln Asp Val Gly Ser Gly Thr Ser Asn
180 185 190Asn Ser Gln Ala Cys Ala
Gln Phe Leu Glu Gln Tyr Phe His Asp Ser 195 200
205Asp Leu Ala Gln Phe Met Arg Leu Phe Gly Gly Asn Phe Ala
His Gln 210 215 220Ala Ser Val Ala Arg
Val Val Gly Gln Gln Gly Arg Gly Arg Ala Gly225 230
235 240Ile Glu Ala Ser Leu Asp Val Gln Tyr Leu
Met Ser Ala Gly Ala Asn 245 250
255Ile Ser Thr Trp Val Tyr Ser Ser Pro Gly Arg His Glu Gly Gln Glu
260 265 270Pro Phe Leu Gln Trp
Leu Met Leu Leu Ser Asn Glu Ser Ala Leu Pro 275
280 285His Val His Thr Val Ser Tyr Gly Asp Asp Glu Asp
Ser Leu Ser Ser 290 295 300Ala Tyr Ile
Gln Arg Val Asn Thr Glu Leu Met Lys Ala Ala Ala Arg305
310 315 320Gly Leu Thr Leu Leu Phe Ala
Ser Gly Asp Ser Gly Ala Gly Cys Trp 325
330 335Ser Val Ser Gly Arg His Gln Phe Arg Pro Thr Phe
Pro Ala Ser Ser 340 345 350Pro
Tyr Val Thr Thr Val Gly Gly Thr Ser Phe Gln Glu Pro Phe Leu 355
360 365Ile Thr Asn Glu Ile Val Asp Tyr Ile
Ser Gly Gly Gly Phe Ser Asn 370 375
380Val Phe Pro Arg Pro Ser Tyr Gln Glu Glu Ala Val Thr Lys Phe Leu385
390 395 400Ser Ser Ser Pro
His Leu Pro Pro Ser Ser Tyr Phe Asn Ala Ser Gly 405
410 415Arg Ala Tyr Pro Asp Val Ala Ala Leu Ser
Asp Gly Tyr Trp Val Val 420 425
430Ser Asn Arg Val Pro Ile Pro Trp Val Ser Gly Thr Ser Ala Ser Thr
435 440 445Pro Val Phe Gly Gly Ile Leu
Ser Leu Ile Asn Glu His Arg Ile Leu 450 455
460Ser Gly Arg Pro Pro Leu Gly Phe Leu Asn Pro Arg Leu Tyr Gln
Gln465 470 475 480His Gly
Ala Gly Leu Phe Asp Val Thr Arg Gly Cys His Glu Ser Cys
485 490 495Leu Asp Glu Glu Val Glu Gly
Gln Gly Phe Cys Ser Gly Pro Gly Trp 500 505
510Asp Pro Val Thr Gly Trp Gly Thr Pro Asn Phe Pro Ala Leu
Leu Lys 515 520 525Thr Leu Leu Asn
530121991DNAAspergillus niger 12acccatcaat accttcagtt cgttagcaat
cgtcttcccg tcgttcaatt caacttctga 60tcacactctc tgaggcgtgg tcgaatataa
accgtcaaaa ttttcgccac acttcttaac 120tcgcggcacc acccgttcaa cggccggcgc
tcatccaacc gtggtggggc accggactac 180gcattatacg tccagtaaac aactcgcagt
ctgaacactc gtattatctg tctcgcaccc 240caatctgtca actgtgaaca atggctcccc
acgcaagctc ggatgttgct gccaatggcg 300ccgtgaacgg gtccgctcgt gccaacgctc
ctttgtttac cgtcaactcg cccaacgtcg 360tgtacaccga caatgaaatc agaagccagt
atgcttatca taccactgat atcacccgca 420ctgccgacaa caagctcgtt gccactccca
aggccaccaa ctaccacttc aaggtcgacc 480gcaaggtggg caaggtgggc gtcatgatgg
tcggctgggg tggtaacaat ggttccaccg 540tgacgcaggt atccttgcca accgccgtgg
tctcgaatgg gagaccgcga gccatgcgcg 600cctccaacta ctacggctct gtggtcatgg
gttccaccat caagctcggt actgacgcca 660agaccggtga ggagattaac attcctttcc
acgacatgct gcccatggtc caccccaatg 720atctcgctat tggtggctgg gacattagca
gcctgaacct tgccgattcc atggaccgtg 780cccaggtcct ggagcctacc ctcaagcagc
aggttcgtaa ggagatggcc gagatgaagc 840ccctgcctag tatctactac ccggacttta
tcgctgccaa ccaggaggac cgggccgaca 900atgtgctcga gggctccaag gcatgctggg
ctcatgttga gaagatccag caggacattc 960gcgacttcaa ggctcagaac ggcctcgaca
aggtcatcgt gatgtggact gccaacaccg 1020agcgttacgc cgacatcctg cctggcgtca
atgacacggc cgacaacctc ctcaacgcta 1080tcaagaccgg ccacctggag gtgtccccgt
ccactgtctt tgctgtggcc tgtatcctgg 1140acaacgttcc cttcatcaac ggctctcccc
agaacacctt tgttcccggt gccatccagt 1200tggctgagca gcacaaggcc ttcattggcg
gagacgactt caagtctggc cagaccaaaa 1260tgaagtcggc tctggttgac ttcttgatca
acgccggtat caagctcacc tcgattgcca 1320gctacaacca cctgggcaac aacgacggca
agaacttgag ctcccagaag cagttccggt 1380ccaaggagat ctccaagtcc aacgtggtgg
acgacatggt cgcggctaac aagatcctct 1440acgccgagga cgagcacccc gaccacaccg
tggtgatcaa gtacatgcct gcggtgggcg 1500acaacaagcg cgcgctcgac gagtactacg
cggagatctt catgggtggc caccagacca 1560tcagtctgtt caacatctgc gaggactccc
tgctggcgtc tcccttgatc attgatctgg 1620tgctgattgc ggagatgatg acccgcatca
gctggaagtc ggacgaggcg gccgagtaca 1680agggcttcca cagcgtgctc agcgtgctca
gctacatgct caaggcgcct ctgactcccc 1740ctggcactcc tgtggtcaac tcgctgacca
agcagcgcag tgccttgacc aacatcttcc 1800gggcgtgcgt tggactgcag cctgaatccg
agatgactct ggagcacaag ctgttctaga 1860cacccaccta gtaatgctta gccatcatgc
taggcgttga tcacacttta cccattgtca 1920gccaactaca gccactcttt gaatatcagt
gactaccttc gaaaaaaaaa aaaaaaaaaa 1980aaaaaaaaaa a
199113532PRTAspergillus niger 13Met Ala
Pro His Ala Ser Ser Asp Val Ala Ala Asn Gly Ala Val Asn1 5
10 15Gly Ser Ala Arg Ala Asn Ala Pro
Leu Phe Thr Val Asn Ser Pro Asn 20 25
30Val Val Tyr Thr Asp Asn Glu Ile Arg Ser Gln Tyr Ala Tyr His
Thr 35 40 45Thr Asp Ile Thr Arg
Thr Ala Asp Asn Lys Leu Val Ala Thr Pro Lys 50 55
60Ala Thr Asn Tyr His Phe Lys Val Asp Arg Lys Val Gly Lys
Val Gly65 70 75 80Val
Met Met Val Gly Trp Gly Gly Asn Asn Gly Ser Thr Val Thr Gln
85 90 95Val Ser Leu Pro Thr Ala Val
Val Ser Asn Gly Arg Pro Arg Ala Met 100 105
110Arg Ala Ser Asn Tyr Tyr Gly Ser Val Val Met Gly Ser Thr
Ile Lys 115 120 125Leu Gly Thr Asp
Ala Lys Thr Gly Glu Glu Ile Asn Ile Pro Phe His 130
135 140Asp Met Leu Pro Met Val His Pro Asn Asp Leu Ala
Ile Gly Gly Trp145 150 155
160Asp Ile Ser Ser Leu Asn Leu Ala Asp Ser Met Asp Arg Ala Gln Val
165 170 175Leu Glu Pro Thr Leu
Lys Gln Gln Val Arg Lys Glu Met Ala Glu Met 180
185 190Lys Pro Leu Pro Ser Ile Tyr Tyr Pro Asp Phe Ile
Ala Ala Asn Gln 195 200 205Glu Asp
Arg Ala Asp Asn Val Leu Glu Gly Ser Lys Ala Cys Trp Ala 210
215 220His Val Glu Lys Ile Gln Gln Asp Ile Arg Asp
Phe Lys Ala Gln Asn225 230 235
240Gly Leu Asp Lys Val Ile Val Met Trp Thr Ala Asn Thr Glu Arg Tyr
245 250 255Ala Asp Ile Leu
Pro Gly Val Asn Asp Thr Ala Asp Asn Leu Leu Asn 260
265 270Ala Ile Lys Thr Gly His Leu Glu Val Ser Pro
Ser Thr Val Phe Ala 275 280 285Val
Ala Cys Ile Leu Asp Asn Val Pro Phe Ile Asn Gly Ser Pro Gln 290
295 300Asn Thr Phe Val Pro Gly Ala Ile Gln Leu
Ala Glu Gln His Lys Ala305 310 315
320Phe Ile Gly Gly Asp Asp Phe Lys Ser Gly Gln Thr Lys Met Lys
Ser 325 330 335Ala Leu Val
Asp Phe Leu Ile Asn Ala Gly Ile Lys Leu Thr Ser Ile 340
345 350Ala Ser Tyr Asn His Leu Gly Asn Asn Asp
Gly Lys Asn Leu Ser Ser 355 360
365Gln Lys Gln Phe Arg Ser Lys Glu Ile Ser Lys Ser Asn Val Val Asp 370
375 380Asp Met Val Ala Ala Asn Lys Ile
Leu Tyr Ala Glu Asp Glu His Pro385 390
395 400Asp His Thr Val Val Ile Lys Tyr Met Pro Ala Val
Gly Asp Asn Lys 405 410
415Arg Ala Leu Asp Glu Tyr Tyr Ala Glu Ile Phe Met Gly Gly His Gln
420 425 430Thr Ile Ser Leu Phe Asn
Ile Cys Glu Asp Ser Leu Leu Ala Ser Pro 435 440
445Leu Ile Ile Asp Leu Val Leu Ile Ala Glu Met Met Thr Arg
Ile Ser 450 455 460Trp Lys Ser Asp Glu
Ala Ala Glu Tyr Lys Gly Phe His Ser Val Leu465 470
475 480Ser Val Leu Ser Tyr Met Leu Lys Ala Pro
Leu Thr Pro Pro Gly Thr 485 490
495Pro Val Val Asn Ser Leu Thr Lys Gln Arg Ser Ala Leu Thr Asn Ile
500 505 510Phe Arg Ala Cys Val
Gly Leu Gln Pro Glu Ser Glu Met Thr Leu Glu 515
520 525His Lys Leu Phe 53014505PRTAspergillus niger
14Phe Thr Val Asn Ser Pro Asn Val Val Tyr Thr Asp Asn Glu Ile Arg1
5 10 15Ser Gln Tyr Ala Tyr His
Thr Thr Asp Ile Thr Arg Thr Ala Asp Asn 20 25
30Lys Leu Val Ala Thr Pro Lys Ala Thr Asn Tyr His Phe
Lys Val Asp 35 40 45Arg Lys Val
Gly Lys Val Gly Val Met Met Val Gly Trp Gly Gly Asn 50
55 60Asn Gly Ser Thr Val Thr Gln Val Ser Leu Pro Thr
Ala Val Val Ser65 70 75
80Asn Gly Arg Pro Arg Ala Met Arg Ala Ser Asn Tyr Tyr Gly Ser Val
85 90 95Val Met Gly Ser Thr Ile
Lys Leu Gly Thr Asp Ala Lys Thr Gly Glu 100
105 110Glu Ile Asn Ile Pro Phe His Asp Met Leu Pro Met
Val His Pro Asn 115 120 125Asp Leu
Ala Ile Gly Gly Trp Asp Ile Ser Ser Leu Asn Leu Ala Asp 130
135 140Ser Met Asp Arg Ala Gln Val Leu Glu Pro Thr
Leu Lys Gln Gln Val145 150 155
160Arg Lys Glu Met Ala Glu Met Lys Pro Leu Pro Ser Ile Tyr Tyr Pro
165 170 175Asp Phe Ile Ala
Ala Asn Gln Glu Asp Arg Ala Asp Asn Val Leu Glu 180
185 190Gly Ser Lys Ala Cys Trp Ala His Val Glu Lys
Ile Gln Gln Asp Ile 195 200 205Arg
Asp Phe Lys Ala Gln Asn Gly Leu Asp Lys Val Ile Val Met Trp 210
215 220Thr Ala Asn Thr Glu Arg Tyr Ala Asp Ile
Leu Pro Gly Val Asn Asp225 230 235
240Thr Ala Asp Asn Leu Leu Asn Ala Ile Lys Thr Gly His Leu Glu
Val 245 250 255Ser Pro Ser
Thr Val Phe Ala Val Ala Cys Ile Leu Asp Asn Val Pro 260
265 270Phe Ile Asn Gly Ser Pro Gln Asn Thr Phe
Val Pro Gly Ala Ile Gln 275 280
285Leu Ala Glu Gln His Lys Ala Phe Ile Gly Gly Asp Asp Phe Lys Ser 290
295 300Gly Gln Thr Lys Met Lys Ser Ala
Leu Val Asp Phe Leu Ile Asn Ala305 310
315 320Gly Ile Lys Leu Thr Ser Ile Ala Ser Tyr Asn His
Leu Gly Asn Asn 325 330
335Asp Gly Lys Asn Leu Ser Ser Gln Lys Gln Phe Arg Ser Lys Glu Ile
340 345 350Ser Lys Ser Asn Val Val
Asp Asp Met Val Ala Ala Asn Lys Ile Leu 355 360
365Tyr Ala Glu Asp Glu His Pro Asp His Thr Val Val Ile Lys
Tyr Met 370 375 380Pro Ala Val Gly Asp
Asn Lys Arg Ala Leu Asp Glu Tyr Tyr Ala Glu385 390
395 400Ile Phe Met Gly Gly His Gln Thr Ile Ser
Leu Phe Asn Ile Cys Glu 405 410
415Asp Ser Leu Leu Ala Ser Pro Leu Ile Ile Asp Leu Val Leu Ile Ala
420 425 430Glu Met Met Thr Arg
Ile Ser Trp Lys Ser Asp Glu Ala Ala Glu Tyr 435
440 445Lys Gly Phe His Ser Val Leu Ser Val Leu Ser Tyr
Met Leu Lys Ala 450 455 460Pro Leu Thr
Pro Pro Gly Thr Pro Val Val Asn Ser Leu Thr Lys Gln465
470 475 480Arg Ser Ala Leu Thr Asn Ile
Phe Arg Ala Cys Val Gly Leu Gln Pro 485
490 495Glu Ser Glu Met Thr Leu Glu His Lys 500
50515505PRTSesamum indicum 15Phe Lys Val Glu Ser Pro Asn
Val Lys Tyr Thr Glu Gly Glu Ile His1 5 10
15Ser Val Tyr Asn Tyr Glu Thr Thr Glu Leu Val His Glu
Ser Arg Asn 20 25 30Gly Thr
Tyr Gln Trp Ile Val Lys Pro Lys Thr Val Lys Tyr Glu Phe 35
40 45Lys Thr Asp Thr His Val Pro Lys Leu Gly
Val Met Leu Val Gly Trp 50 55 60Gly
Gly Asn Asn Gly Ser Thr Leu Thr Gly Gly Val Ile Ala Asn Arg65
70 75 80Glu Gly Ile Ser Trp Ala
Thr Lys Asp Lys Val Gln Gln Ala Asn Tyr 85
90 95Phe Gly Ser Leu Thr Gln Ala Ser Ser Ile Arg Val
Gly Ser Phe Asn 100 105 110Gly
Glu Glu Ile Tyr Ala Pro Phe Lys Ser Leu Leu Pro Met Val Asn 115
120 125Pro Asp Asp Val Val Phe Gly Gly Trp
Asp Ile Ser Asn Met Asn Leu 130 135
140Ala Asp Ala Met Gly Arg Ala Lys Val Leu Asp Ile Asp Leu Gln Lys145
150 155 160Gln Leu Arg Pro
Tyr Met Glu His Met Val Pro Leu Pro Gly Ile Tyr 165
170 175Asp Pro Asp Phe Ile Ala Ala Asn Gln Gly
Ser Arg Ala Asn Asn Val 180 185
190Ile Lys Gly Thr Lys Lys Glu Gln Val Gln Gln Ile Ile Lys Asp Met
195 200 205Arg Asp Phe Lys Glu Gln Asn
Lys Val Asp Lys Val Val Val Leu Trp 210 215
220Thr Ala Asn Thr Glu Arg Tyr Ser Asn Val Val Val Gly Leu Asn
Asp225 230 235 240Thr Ala
Glu Ser Leu Met Ala Ser Val Glu Arg Asn Glu Ala Glu Ile
245 250 255Ser Pro Ser Thr Leu Tyr Ala
Ile Ala Cys Val Phe Glu Asn Val Pro 260 265
270Phe Ile Asn Gly Ser Pro Gln Asn Thr Phe Val Pro Gly Leu
Ile Asp 275 280 285Leu Ala Ile Gln
Arg Asn Ser Leu Ile Gly Gly Asp Asp Phe Lys Ser 290
295 300Gly Gln Thr Lys Met Lys Ser Val Leu Val Asp Phe
Leu Val Gly Ala305 310 315
320Gly Ile Lys Pro Thr Ser Ile Val Ser Tyr Asn His Leu Gly Asn Asn
325 330 335Asp Gly Met Asn Leu
Ser Ala Pro Gln Thr Phe Arg Ser Lys Glu Ile 340
345 350Ser Lys Ser Asn Val Val Asp Asp Met Val Ala Ser
Asn Gly Ile Leu 355 360 365Tyr Glu
Pro Gly Glu His Pro Asp His Ile Val Val Ile Lys Tyr Val 370
375 380Pro Tyr Val Gly Asp Ser Lys Arg Ala Met Asp
Glu Tyr Thr Ser Glu385 390 395
400Ile Phe Met Gly Gly Lys Ser Thr Ile Val Leu His Asn Thr Cys Glu
405 410 415Asp Ser Leu Leu
Ala Ala Pro Ile Ile Leu Asp Leu Val Leu Leu Ala 420
425 430Glu Leu Ser Thr Arg Ile Gln Leu Lys Ala Glu
Gly Glu Gly Lys Phe 435 440 445His
Ser Phe His Pro Val Ala Thr Ile Leu Ser Tyr Leu Thr Lys Ala 450
455 460Pro Leu Val Pro Pro Gly Thr Pro Val Val
Asn Ala Leu Ser Lys Gln465 470 475
480Arg Ala Met Leu Glu Asn Ile Leu Arg Ala Cys Val Gly Leu Ala
Pro 485 490 495Glu Asn Asn
Met Ile Leu Glu Tyr Lys 500
505162059DNAAspergillus niger 16cttttctctt gtcttttccc tcgttcttct
ctctttcttc tcttctttct ttctctgctt 60cggtccagtc tctcgttctt gtctttactg
accctagtct ttcgtttcgc gtggtctgtc 120gtggtgtcgt atcaaatgat tattattatc
ttctaaccta tccctctgcc tatttgctat 180atatccccaa aactgaccca tacatatcac
atctctccac ctttggttac atatacatac 240attcatacat acatatacac ctctcataca
acaatgaagg ccactagcgc aacggtggct 300ttcctagcca ttgctgctgt ccaagcagcc
aagcacgccc atgaccatgg ccaccaccgt 360agccatcgct cggtggatag ccccgtggtc
aagaagagtt cctcgtgtca gttcccctcc 420ggggctggct tgatccccat cactccccac
gagaccaatg gtggttgggc gatgagccct 480gatcaggagt gcaagcctgg cggatattgt
ccgtatgctt gtccagccgg ccaggtctcc 540atgcagtggg acccggaggc tacttcgtac
acctacccca tgtccatgaa tggtggactg 600tactgcgacg agaatggcga aatacagaag
ccattcccgg accgtcccta ctgcaaggac 660ggcacaggcg tcgtcagcgc gaagaacaag
tgcaaggagc aggtgtcttt ctgccagact 720gttcttccgg gcaatgaagc catgccgatc
cccacgcttg tggaagagcc ggctaccctg 780gctgttccgg atctgagtta ctggtgtgag
actgcggcgc acttctatat caaccctccg 840ggatacaaca ccaagactgc ctgtgtctgg
ggtacctctg agaaccccta cggcaactgg 900tccccgtatg ttgccggtgc caacactgac
ggcgacggca acacctatgt gaagctcaga 960tggaacccga tttacctaga accgactacc
ccgttccgca acgaagtccc tgagttcggt 1020gtcgagatcg agtgtgaggg agacggctgc
aatggactgc catgcaagat cgacccatcc 1080gtgaacggcg tgaacgagat gactggcgac
agctcagtgg gtgcaggcgg tgcatccttc 1140tgtgtggtgg acggtgccca agggcggaaa
ggccaatgtc gttgtcttcg acaagggacg 1200gcggtggact ctaccagcgt gccggtgtcc
agcagcagtg ttagcagcgt cgtcgtgagc 1260agcagcgcca gcaccagcag cacctcgacc
agcagcatcg tccctactac gagcagcacg 1320ccgacgagtg tcagcaccag caccagcacc
agcaccagta ctagcaccag cactagcact 1380agcactagca gcagcacccc ggccccgacg
ccgtccagca ctactactac tatctccagc 1440accactctca gctccagctc caccatcagc
accaccagct ccagcagcat aactccccgg 1500cccaccccca gctggacgcc ctcttccagc
tggaagatca gctcgagcgc agcactgaac 1560tggaccgtgt cggctagcta cacgtacaag
ccacacgtga tggtggagac gggctcctcg 1620cacacgcagc ctgtagctgc tgctgctgtt
gccagtgagg gctcgagcca gacgactgga 1680accgctcaag caacgcagtc agcggttgtg
acggagggag ctgctgtcag cactgccgtg 1740tcgaagctga gtctgattgt ggcagttctt
ggagccattg tcatggtcta gacaatagac 1800catgttgacc atcataccga tcaactgtgt
cggttgcata ctcactcacc atcatcactt 1860ttcttggttc acttctgatg ggcacctttt
tcacacttac acttataccc ttatgattga 1920cgttcttgta tgtgcttgat tgatgagcat
ttatcgataa tgtttactgt taatatagtc 1980attaatttgc ctgtaaattc caagtgccac
ttagcacaga gtagaggtcc aaaaaaaaaa 2040aaaaaaaaaa aaaaaaaaa
205917505PRTAspergillus niger 17Met Lys
Ala Thr Ser Ala Thr Val Ala Phe Leu Ala Ile Ala Ala Val1 5
10 15Gln Ala Ala Lys His Ala His Asp
His Gly His His Arg Ser His Arg 20 25
30Ser Val Asp Ser Pro Val Val Lys Lys Ser Ser Ser Cys Gln Phe
Pro 35 40 45Ser Gly Ala Gly Leu
Ile Pro Ile Thr Pro His Glu Thr Asn Gly Gly 50 55
60Trp Ala Met Ser Pro Asp Gln Glu Cys Lys Pro Gly Gly Tyr
Cys Pro65 70 75 80Tyr
Ala Cys Pro Ala Gly Gln Val Ser Met Gln Trp Asp Pro Glu Ala
85 90 95Thr Ser Tyr Thr Tyr Pro Met
Ser Met Asn Gly Gly Leu Tyr Cys Asp 100 105
110Glu Asn Gly Glu Ile Gln Lys Pro Phe Pro Asp Arg Pro Tyr
Cys Lys 115 120 125Asp Gly Thr Gly
Val Val Ser Ala Lys Asn Lys Cys Lys Glu Gln Val 130
135 140Ser Phe Cys Gln Thr Val Leu Pro Gly Asn Glu Ala
Met Pro Ile Pro145 150 155
160Thr Leu Val Glu Glu Pro Ala Thr Leu Ala Val Pro Asp Leu Ser Tyr
165 170 175Trp Cys Glu Thr Ala
Ala His Phe Tyr Ile Asn Pro Pro Gly Tyr Asn 180
185 190Thr Lys Thr Ala Cys Val Trp Gly Thr Ser Glu Asn
Pro Tyr Gly Asn 195 200 205Trp Ser
Pro Tyr Val Ala Gly Ala Asn Thr Asp Gly Asp Gly Asn Thr 210
215 220Tyr Val Lys Leu Arg Trp Asn Pro Ile Tyr Leu
Glu Pro Thr Thr Pro225 230 235
240Phe Arg Asn Glu Val Pro Glu Phe Gly Val Glu Ile Glu Cys Glu Gly
245 250 255Asp Gly Cys Asn
Gly Leu Pro Cys Lys Ile Asp Pro Ser Val Asn Gly 260
265 270Val Asn Glu Met Thr Gly Asp Ser Ser Val Gly
Ala Gly Gly Ala Ser 275 280 285Phe
Cys Val Val Asp Gly Ala Gln Gly Arg Lys Gly Gln Cys Arg Cys 290
295 300Leu Arg Gln Gly Thr Ala Val Asp Ser Thr
Ser Val Pro Val Ser Ser305 310 315
320Ser Ser Val Ser Ser Val Val Val Ser Ser Ser Ala Ser Thr Ser
Ser 325 330 335Thr Ser Thr
Ser Ser Ile Val Pro Thr Thr Ser Ser Thr Pro Thr Ser 340
345 350Val Ser Thr Ser Thr Ser Thr Ser Thr Ser
Thr Ser Thr Ser Thr Ser 355 360
365Thr Ser Thr Ser Ser Ser Thr Pro Ala Pro Thr Pro Ser Ser Thr Thr 370
375 380Thr Thr Ile Ser Ser Thr Thr Leu
Ser Ser Ser Ser Thr Ile Ser Thr385 390
395 400Thr Ser Ser Ser Ser Ile Thr Pro Arg Pro Thr Pro
Ser Trp Thr Pro 405 410
415Ser Ser Ser Trp Lys Ile Ser Ser Ser Ala Ala Leu Asn Trp Thr Val
420 425 430Ser Ala Ser Tyr Thr Tyr
Lys Pro His Val Met Val Glu Thr Gly Ser 435 440
445Ser His Thr Gln Pro Val Ala Ala Ala Ala Val Ala Ser Glu
Gly Ser 450 455 460Ser Gln Thr Thr Gly
Thr Ala Gln Ala Thr Gln Ser Ala Val Val Thr465 470
475 480Glu Gly Ala Ala Val Ser Thr Ala Val Ser
Lys Leu Ser Leu Ile Val 485 490
495Ala Val Leu Gly Ala Ile Val Met Val 500
505181904DNAAspergillus niger 18aacaaatttt tctcctctta cctttaatca
ttttctttta ttctccttct tcccccccat 60acatcatact ctccgcaata gctctctttc
ttgagtgttt tgtgtcttaa actctactgt 120cccactttcc gcttaatact tacccctcct
ccttttacac attcaccatg gctgcccgtc 180ctcagaacat tggtatcaag gccattgagg
tctacttccc tcgtcagtgt gtcgaccaga 240gcgagcttga gaaattcgat ggcgtgagcg
aaggcaaata caccattggt cttggtcaga 300ccaagatgag cttctgtgat gaccgtgaag
acatctactc cattgctttg accaccttct 360cctcccttct ccgcaagtac aacatcgacc
ccaactccat tggccgcctg gaggtcggta 420ccgagacctt gctggacaag tccaagtccg
tcaagtccgt cctgatgcag cttctggctc 480cccacggaaa caccaacgtt gagggtgttg
acaacgtcaa tgcttgctgc ggtggcacca 540acgctgtttt caacagcatc aactggctcg
agtcctctgc ctgggatggc agagatgccg 600ttgttgtctg cggtgacatt gccctgtacg
ccgagggtgc tgctcgccct accggtggtg 660ctggctgtgt cgccatgctg atcggtcctg
acgcccctat tgtgttcgag cccggtcttc 720gtgcctccta cgtcacccac gcctacgact
tcttcaagcc cgacctgacc agcgagtacc 780ctgtcgtgga tggtcacttc tccctcagat
gctacactga ggctgtcaac gcttgctaca 840aggcctacaa tgctcgtgag aagaccttga
aggagaaggt tcagaacggt accaacggca 900ccgcccagga cgactcccag actgccttgg
accgcttcga atacctctgc taccatgctc 960ctacctgcaa gctggtgcag aaatccttcg
ctcgtatgct gtacaacgac tacctcacaa 1020accccactca ccctgctttc gccgaagtgg
ctcctgagct ccgtgatttg gactacgcca 1080cctctctcac tgacaagaac gtggagaaga
ccttcatggg cctgaccaag aagcgcttcg 1140ctgagcgtgt taagcccgct ctcgaggttg
ccactctttg cggtaacatg tacactgcca 1200ccgtttgggc tggtctggct agcttgatct
ctcacgtccc cttcgatgct agcgagtcca 1260agcgcatcgg tctcttctcc tacggcagtg
gtcttgccag ctccctgctt agcgtaaaga 1320ttgtcggaga cgtgtccaac ctggtggaga
agctcgatct caagaaccgt cttagcaacc 1380gcaacgttct ccctcctcag tcctacgttg
acatgtgtgc cctccgtgag catgctcacc 1440tcaagaagaa cttcaagcct tccggcaaca
ccgagactct ctaccctggt acttactact 1500tgactgaggt ggacgatatg ttccgccgca
agtacgacgt caaggcatga attatgagca 1560tatgatggac ttgctttcga ccttgcttct
ttggacatga ccggttgctt agacggttta 1620actagattcc cttcagcatg cgcattgttt
atttgtggtt cgccttaata gagcttgggg 1680gcagcggaat gctcctacca atttccgggt
ctgcttttct cctttacatt ggttcttaat 1740gtttcatacg ttgttcatgt atcctcctag
ggaggagacc ttctcttgtc cagacaggag 1800ctggaatgca attatataag acgatgacca
ataattccag actcatcaag agtcagaaag 1860aagagtcatg aaaggaaaaa aaaaaaaaaa
aaaaaaaaaa aaaa 190419460PRTAspergillus niger 19Met
Ala Ala Arg Pro Gln Asn Ile Gly Ile Lys Ala Ile Glu Val Tyr1
5 10 15Phe Pro Arg Gln Cys Val Asp
Gln Ser Glu Leu Glu Lys Phe Asp Gly 20 25
30Val Ser Glu Gly Lys Tyr Thr Ile Gly Leu Gly Gln Thr Lys
Met Ser 35 40 45Phe Cys Asp Asp
Arg Glu Asp Ile Tyr Ser Ile Ala Leu Thr Thr Phe 50 55
60Ser Ser Leu Leu Arg Lys Tyr Asn Ile Asp Pro Asn Ser
Ile Gly Arg65 70 75
80Leu Glu Val Gly Thr Glu Thr Leu Leu Asp Lys Ser Lys Ser Val Lys
85 90 95Ser Val Leu Met Gln Leu
Leu Ala Pro His Gly Asn Thr Asn Val Glu 100
105 110Gly Val Asp Asn Val Asn Ala Cys Cys Gly Gly Thr
Asn Ala Val Phe 115 120 125Asn Ser
Ile Asn Trp Leu Glu Ser Ser Ala Trp Asp Gly Arg Asp Ala 130
135 140Val Val Val Cys Gly Asp Ile Ala Leu Tyr Ala
Glu Gly Ala Ala Arg145 150 155
160Pro Thr Gly Gly Ala Gly Cys Val Ala Met Leu Ile Gly Pro Asp Ala
165 170 175Pro Ile Val Phe
Glu Pro Gly Leu Arg Ala Ser Tyr Val Thr His Ala 180
185 190Tyr Asp Phe Phe Lys Pro Asp Leu Thr Ser Glu
Tyr Pro Val Val Asp 195 200 205Gly
His Phe Ser Leu Arg Cys Tyr Thr Glu Ala Val Asn Ala Cys Tyr 210
215 220Lys Ala Tyr Asn Ala Arg Glu Lys Thr Leu
Lys Glu Lys Val Gln Asn225 230 235
240Gly Thr Asn Gly Thr Ala Gln Asp Asp Ser Gln Thr Ala Leu Asp
Arg 245 250 255Phe Glu Tyr
Leu Cys Tyr His Ala Pro Thr Cys Lys Leu Val Gln Lys 260
265 270Ser Phe Ala Arg Met Leu Tyr Asn Asp Tyr
Leu Thr Asn Pro Thr His 275 280
285Pro Ala Phe Ala Glu Val Ala Pro Glu Leu Arg Asp Leu Asp Tyr Ala 290
295 300Thr Ser Leu Thr Asp Lys Asn Val
Glu Lys Thr Phe Met Gly Leu Thr305 310
315 320Lys Lys Arg Phe Ala Glu Arg Val Lys Pro Ala Leu
Glu Val Ala Thr 325 330
335Leu Cys Gly Asn Met Tyr Thr Ala Thr Val Trp Ala Gly Leu Ala Ser
340 345 350Leu Ile Ser His Val Pro
Phe Asp Ala Ser Glu Ser Lys Arg Ile Gly 355 360
365Leu Phe Ser Tyr Gly Ser Gly Leu Ala Ser Ser Leu Leu Ser
Val Lys 370 375 380Ile Val Gly Asp Val
Ser Asn Leu Val Glu Lys Leu Asp Leu Lys Asn385 390
395 400Arg Leu Ser Asn Arg Asn Val Leu Pro Pro
Gln Ser Tyr Val Asp Met 405 410
415Cys Ala Leu Arg Glu His Ala His Leu Lys Lys Asn Phe Lys Pro Ser
420 425 430Gly Asn Thr Glu Thr
Leu Tyr Pro Gly Thr Tyr Tyr Leu Thr Glu Val 435
440 445Asp Asp Met Phe Arg Arg Lys Tyr Asp Val Lys Ala
450 455 46020460PRTAspergillus niger
20Met Ala Ala Arg Pro Gln Asn Ile Gly Ile Lys Ala Ile Glu Val Tyr1
5 10 15Phe Pro Arg Gln Cys Val
Asp Gln Ser Glu Leu Glu Lys Phe Asp Gly 20 25
30Val Ser Glu Gly Lys Tyr Thr Ile Gly Leu Gly Gln Thr
Lys Met Ser 35 40 45Phe Cys Asp
Asp Arg Glu Asp Ile Tyr Ser Ile Ala Leu Thr Thr Phe 50
55 60Ser Ser Leu Leu Arg Lys Tyr Asn Ile Asp Pro Asn
Ser Ile Gly Arg65 70 75
80Leu Glu Val Gly Thr Glu Thr Leu Leu Asp Lys Ser Lys Ser Val Lys
85 90 95Ser Val Leu Met Gln Leu
Leu Ala Pro His Gly Asn Thr Asn Val Glu 100
105 110Gly Val Asp Asn Val Asn Ala Cys Cys Gly Gly Thr
Asn Ala Val Phe 115 120 125Asn Ser
Ile Asn Trp Leu Glu Ser Ser Ala Trp Asp Gly Arg Asp Ala 130
135 140Val Val Val Cys Gly Asp Ile Ala Leu Tyr Ala
Glu Gly Ala Ala Arg145 150 155
160Pro Thr Gly Gly Ala Gly Cys Val Ala Met Leu Ile Gly Pro Asp Ala
165 170 175Pro Ile Val Phe
Glu Pro Gly Leu Arg Ala Ser Tyr Val Thr His Ala 180
185 190Tyr Asp Phe Phe Lys Pro Asp Leu Thr Ser Glu
Tyr Pro Val Val Asp 195 200 205Gly
His Phe Ser Leu Arg Cys Tyr Thr Glu Ala Val Asn Ala Cys Tyr 210
215 220Lys Ala Tyr Asn Ala Arg Glu Lys Thr Leu
Lys Glu Lys Val Gln Asn225 230 235
240Gly Thr Asn Gly Thr Ala Gln Asp Asp Ser Gln Thr Ala Leu Asp
Arg 245 250 255Phe Glu Tyr
Leu Cys Tyr His Ala Pro Thr Cys Lys Leu Val Gln Lys 260
265 270Ser Phe Ala Arg Met Leu Tyr Asn Asp Tyr
Leu Thr Asn Pro Thr His 275 280
285Pro Ala Phe Ala Glu Val Ala Pro Glu Leu Arg Asp Leu Asp Tyr Ala 290
295 300Thr Ser Leu Thr Asp Lys Asn Val
Glu Lys Thr Phe Met Gly Leu Thr305 310
315 320Lys Lys Arg Phe Ala Glu Arg Val Lys Pro Ala Leu
Glu Val Ala Thr 325 330
335Leu Cys Gly Asn Met Tyr Thr Ala Thr Val Trp Ala Gly Leu Ala Ser
340 345 350Leu Ile Ser His Val Pro
Phe Asp Ala Ser Glu Ser Lys Arg Ile Gly 355 360
365Leu Phe Ser Tyr Gly Ser Gly Leu Ala Ser Ser Leu Leu Ser
Val Lys 370 375 380Ile Val Gly Asp Val
Ser Asn Leu Val Glu Lys Leu Asp Leu Lys Asn385 390
395 400Arg Leu Ser Asn Arg Asn Val Leu Pro Pro
Gln Ser Tyr Val Asp Met 405 410
415Cys Ala Leu Arg Glu His Ala His Leu Lys Lys Asn Phe Lys Pro Ser
420 425 430Gly Asn Thr Glu Thr
Leu Tyr Pro Gly Thr Tyr Tyr Leu Thr Glu Val 435
440 445Asp Asp Met Phe Arg Arg Lys Tyr Asp Val Lys Ala
450 455 46021454PRTNeurospora crassa
21Met Ala Thr Arg Pro Gln Asn Ile Gly Ile Lys Ala Ile Glu Ile Tyr1
5 10 15Phe Pro Ser Gln Tyr Val
Glu Gln Ser Glu Leu Glu Lys Phe Asp Gly 20 25
30Val Ser Thr Gly Lys Tyr Thr Ile Gly Leu Gly Gln Thr
Lys Met Ala 35 40 45Phe Cys Asp
Asp Arg Glu Asp Ile Tyr Ser Leu Ala Leu Thr Ala Val 50
55 60Ser Arg Leu Leu Lys Asn Tyr Glu Ile Asp Thr Asn
Thr Ile Gly Arg65 70 75
80Leu Glu Val Gly Thr Glu Thr Leu Leu Asp Lys Ser Lys Ser Val Lys
85 90 95Ser Val Leu Met Gln Leu
Phe Gly Glu Asn Thr Asn Ile Glu Gly Val 100
105 110Asp Thr Ile Asn Ala Cys Tyr Gly Gly Thr Asn Ala
Phe Phe Asn Ser 115 120 125Val Asn
Trp Ile Glu Ser Ser Ala Trp Asp Gly Arg Asp Ala Ile Val 130
135 140Val Ala Gly Asp Ile Ala Leu Tyr Ala Lys Gly
Asn Ala Arg Pro Thr145 150 155
160Gly Gly Ala Gly Cys Val Ala Met Leu Val Gly Pro Asn Ala Pro Ile
165 170 175Ala Val Glu Pro
Gly Leu Arg Gly Ser Tyr Met Ala His Ala Tyr Asp 180
185 190Phe Tyr Lys Pro Asp Leu Thr Ser Glu Tyr Pro
Tyr Val Asp Gly His 195 200 205Tyr
Ser Val Asn Cys Tyr Thr Glu Ala Leu Asp Gly Ala Tyr Arg Ala 210
215 220Tyr Asn Gln Arg Glu Lys Leu Leu Thr Asn
Gly Val Asn Gly His Ser225 230 235
240Glu Asp Ser Thr Lys Thr Pro Leu Asp Arg Phe Asp Tyr Leu Ala
Phe 245 250 255His Ala Pro
Thr Cys Lys Leu Val Gln Lys Ser Tyr Ala Arg Leu Leu 260
265 270Tyr His Asp Tyr Leu Ala Asn Pro Glu Ser
Pro Val Phe Ala Asp Val 275 280
285Pro Pro Glu Val Arg Asp Met Asp Tyr Lys Lys Ser Leu Thr Asp Lys 290
295 300Val Val Glu Lys Thr Phe Met Thr
Leu Thr Lys Lys Arg Phe Gln Glu305 310
315 320Arg Val Asn Pro Ala Ile Gln Val Pro Thr Leu Cys
Gly Asn Met Tyr 325 330
335Cys Gly Ser Val Trp Gly Gly Leu Ala Ser Ile Ile Gly His Val Asp
340 345 350Ser Ala Gln Leu Glu Gly
Lys Arg Ile Gly Leu Phe Ser Tyr Gly Ser 355 360
365Gly Leu Ala Ala Ser Phe Cys Ser Phe Arg Val Thr Gly Ser
Thr Glu 370 375 380Lys Leu Ala Lys Thr
Leu Asn Leu Pro Ala Arg Leu Ala Ala Arg Arg385 390
395 400Ala Val Pro Pro Glu Ser Tyr Asp Ala Met
Cys Asp Leu Arg Lys Gln 405 410
415Ala His Leu Gln Lys Asn Tyr Thr Pro Lys Gly Glu Val Ser Thr Leu
420 425 430Glu Pro Gly Thr Tyr
Tyr Leu Glu Asn Val Asp Asp Met Phe Lys Arg 435
440 445Thr Tyr Ser Ile Lys Ala 450221498DNAAspergillus
niger 22agctcaaatt ctcttccctt tgatctcaac taccattcct taagaagctg tgcttcgtac
60cttcatttcg ccttactttt tttctgctta ctactacaac tccatcactc tctattcttt
120caatatgaag ttcaccggaa tcgctttcgc cggtcttatc ggttacgctg ccgccctgcc
180ggccatgggc gcccagcaag actctgctcc caacggtgtt caggccaccg gagctccctc
240cttccagggt gctgccccct ccggctcccc tcttcccctt ccctcgggtg ctcctcaggg
300ccagggcttc ggaggccagg gcttcggcaa ctctaacggt cagggccagg ctcccaccgt
360gagcttgggc gatgctcctc agcctcctcc cactggctcc gctgcccctg ctccttctgg
420agctcctcgt ggccacaaga ggcgtcagct cgagatcccg gcttccgtct ccaacgtccc
480cgccccgacc ggctccgctg ctgctggagg tgacttcggt ggtgctcctt cgggtcccgc
540tccctctggt gccgctccct ctggcgtcgc tggtggtgac ggcccctctc cttctggttc
600tttcggtggc cagggcggcc agtctggctc tttcggcggc aacggcgccg ctccctctgg
660cattgctggc ggcaatggcc cctctacttc cggctctttc ggtggtgccg ctcctccggt
720gttgctggta gcaatggccc ctctacctcc ggctcttttg gcggccagca gggtcagcag
780ggccagagcg gcttcggcgg ccaggactcc cagtcccagg gccagtccca ggactccaag
840tctcagagct ctaagtccca gaactccagg tctgagggct ctcagtctca ggactcccag
900tcccagggat ctgactctga gggatctcag ggctctttcg agcagggctc ctcctctgag
960cagggctctg gctctagctc tttcggtggt aacggtgctg ctccctccgg tgttgctggt
1020ggcaacggcc cctctccttc cggctctttc ggcggtgctg ctccctccgg tgttgctggc
1080ggcaacggtc cctctccctc tggctccttc ggcggtaacg gcgctgctcc ctctggcgtc
1140gctggtggaa acggcccctc tccttccggc tccttcggtg gtaacggtgc tgctccttct
1200ggtgctgccg gtggtgctcc cgctgcctcg ggcgcccccg ccgccgctcc ctcgggtgct
1260tcttactaag tccatgcgaa agtcttggac ttcgatcgac aataaacacc ttccatatca
1320tctgacgctg atgcaatagt tccaccgagg acatcgacaa tgccattgtg tcggcgtgga
1380ccagaacaac aacaactgga tgaaggtgtg atggattgga cgcttgtgta cattaatcac
1440tcaataacca gtcattcctt tgaccagatc tggtagaaag aaaaaaaaaa aaaaaaaa
149823404PRTAspergillus niger 23Met Lys Phe Thr Gly Ile Ala Phe Ala Gly
Leu Ile Gly Tyr Ala Ala1 5 10
15Ala Leu Pro Ala Met Gly Ala Gln Gln Asp Ser Ala Pro Asn Gly Val
20 25 30Gln Ala Thr Gly Ala Pro
Ser Phe Gln Gly Ala Ala Pro Ser Gly Ser 35 40
45Pro Leu Pro Leu Pro Ser Gly Ala Pro Gln Gly Gln Gly Phe
Gly Gly 50 55 60Gln Gly Phe Gly Asn
Ser Asn Gly Gln Gly Gln Ala Pro Thr Val Ser65 70
75 80Leu Gly Asp Ala Pro Gln Pro Pro Pro Thr
Gly Ser Ala Ala Pro Ala 85 90
95Pro Ser Gly Ala Pro Arg Gly His Lys Arg Arg Gln Leu Glu Ile Pro
100 105 110Ala Ser Val Ser Asn
Val Pro Ala Pro Thr Gly Ser Ala Ala Ala Gly 115
120 125Gly Asp Phe Gly Gly Ala Pro Ser Gly Pro Ala Pro
Ser Gly Ala Ala 130 135 140Pro Ser Gly
Val Ala Gly Gly Asp Gly Pro Ser Pro Ser Gly Ser Phe145
150 155 160Gly Gly Gln Gly Gly Gln Ser
Gly Ser Phe Gly Gly Asn Gly Ala Ala 165
170 175Pro Ser Gly Ile Ala Gly Gly Asn Gly Pro Ser Thr
Ser Gly Ser Phe 180 185 190Gly
Gly Ala Ala Pro Pro Val Leu Leu Val Ala Met Ala Pro Leu Pro 195
200 205Pro Ala Leu Leu Ala Ala Ser Arg Val
Ser Arg Ala Arg Ala Ala Ser 210 215
220Ala Ala Arg Thr Pro Ser Pro Arg Ala Ser Pro Arg Thr Pro Ser Leu225
230 235 240Arg Ala Leu Ser
Pro Arg Thr Pro Gly Leu Arg Ala Leu Ser Leu Arg 245
250 255Thr Pro Ser Pro Arg Asp Leu Thr Leu Arg
Asp Leu Arg Ala Leu Ser 260 265
270Ser Arg Ala Pro Pro Leu Ser Arg Ala Leu Ala Leu Ala Leu Ser Val
275 280 285Val Thr Val Leu Leu Pro Pro
Val Leu Leu Val Ala Thr Ala Pro Leu 290 295
300Leu Pro Ala Leu Ser Ala Val Leu Leu Pro Pro Val Leu Leu Ala
Ala305 310 315 320Thr Val
Pro Leu Pro Leu Ala Pro Ser Ala Val Thr Ala Leu Leu Pro
325 330 335Leu Thr Ser Leu Val Glu Thr
Ala Pro Leu Leu Pro Ala Pro Ser Val 340 345
350Val Thr Val Leu Leu Leu Leu Val Leu Pro Val Val Leu Pro
Leu Pro 355 360 365Arg Ala Pro Pro
Pro Pro Leu Pro Arg Val Leu Leu Thr Lys Ser Met 370
375 380Arg Lys Ser Trp Thr Ser Ile Asp Asn Lys His Leu
Pro Tyr His Leu385 390 395
400Thr Leu Met Gln24763DNAAspergillus niger 24acacaaagca cattccttac
attcacattc gtttcttctt cactccttta cttccctatc 60tttccaatat tcacgatgca
gtggacgaac tttctgtgcc ctctgattgc catgcaggct 120agcctgagtg ctgcctgggg
cacccacgtc aagagaggat cggagaccaa tgccaccctg 180tttgcctatg gacagaactc
ttccgcttac cccattgctt atgggctcag tgacggtctt 240ctctacattg cccaagatcc
ggagaacacc gcggcggacc tgacacccat gtcctgggat 300ctgccctcca tcactgatga
gtgctggatt gtcaacggca cgtttatgaa tggcactcgt 360gcaggatctc tctatatccg
accggatagc aacaactgtc ttggcgtgct gccttttgcc 420caggctaaag gggtgaatgg
cgtggtcacg ggctttggcc tctttgcatc gcagctggtc 480tataacaacg atacccagct
ggaagcacag ttctgggcgt cgaagacaga cactgaagat 540gtctacaaac tggtgtgggt
ggaggactct tcgcaaattg cgagcgaaag ctttcccgtc 600gtggtgaaag cgtccgagga
ctcgacctga aaagaacagc gatccaggga ggtgtgactt 660gggttgttgg ggtggtgctc
cgagcatgat gttgttcatt gccattgcgt ggtaatatat 720ataatagtca ctcgtttata
tttgacaaaa aaaaaaaaaa aaa 76325184PRTAspergillus
niger 25Met Gln Trp Thr Asn Phe Leu Cys Pro Leu Ile Ala Met Gln Ala Ser1
5 10 15Leu Ser Ala Ala
Trp Gly Thr His Val Lys Arg Gly Ser Glu Thr Asn 20
25 30Ala Thr Leu Phe Ala Tyr Gly Gln Asn Ser Ser
Ala Tyr Pro Ile Ala 35 40 45Tyr
Gly Leu Ser Asp Gly Leu Leu Tyr Ile Ala Gln Asp Pro Glu Asn 50
55 60Thr Ala Ala Asp Leu Thr Pro Met Ser Trp
Asp Leu Pro Ser Ile Thr65 70 75
80Asp Glu Cys Trp Ile Val Asn Gly Thr Phe Met Asn Gly Thr Arg
Ala 85 90 95Gly Ser Leu
Tyr Ile Arg Pro Asp Ser Asn Asn Cys Leu Gly Val Leu 100
105 110Pro Phe Ala Gln Ala Lys Gly Val Asn Gly
Val Val Thr Gly Phe Gly 115 120
125Leu Phe Ala Ser Gln Leu Val Tyr Asn Asn Asp Thr Gln Leu Glu Ala 130
135 140Gln Phe Trp Ala Ser Lys Thr Asp
Thr Glu Asp Val Tyr Lys Leu Val145 150
155 160Trp Val Glu Asp Ser Ser Gln Ile Ala Ser Glu Ser
Phe Pro Val Val 165 170
175Val Lys Ala Ser Glu Asp Ser Thr 18026641DNAAspergillus
niger 26actccacctt ttctcatctg tcctctgtac ctagattcct tcttatatct tatccgtggt
60tccttctttt ctggccaaga tcttagccat ctatcaacac gagagaaaac ttattcccat
120cctatcacat cacaatgtct gctgctgctc ctcctgctcc cccggttaac ggtgaccggc
180ccgagacggg tcactcacat ggaaagagtt ccctgtccag caagtcggac ccgaaccagg
240cgttgagagg tgaagaggct gtgtacagcg ttggatcgag tggattctct ctacgctcaa
300tgcagcatcg cgaccgtggg ggcaaaatca tcactgaacc cgacttgtcc aaccctaccc
360gttaccgatt cgagcggccg ctggacacca ttcgatcgtt tgaggcagcc atcgagcgcc
420gtcgtcgtga ggccatgtaa gatgagactt ggcgtgtgaa tatactgcga atgatgttcg
480atttcttgtg attatgtttg ggttcggcgc tggacgacgt atggatatgg acatggacat
540ggatatgagt ttgatttgat tgagcgtgta cattacttca ctgggtatgc ttctggaatg
600ttaccttgtc gatctcttat ttcaaaaaaa aaaaaaaaaa a
64127101PRTAspergillus niger 27Met Ser Ala Ala Ala Pro Pro Ala Pro Pro
Val Asn Gly Asp Arg Pro1 5 10
15Glu Thr Gly His Ser His Gly Lys Ser Ser Leu Ser Ser Lys Ser Asp
20 25 30Pro Asn Gln Ala Leu Arg
Gly Glu Glu Ala Val Tyr Ser Val Gly Ser 35 40
45Ser Gly Phe Ser Leu Arg Ser Met Gln His Arg Asp Arg Gly
Gly Lys 50 55 60Ile Ile Thr Glu Pro
Asp Leu Ser Asn Pro Thr Arg Tyr Arg Phe Glu65 70
75 80Arg Pro Leu Asp Thr Ile Arg Ser Phe Glu
Ala Ala Ile Glu Arg Arg 85 90
95Arg Arg Glu Ala Met 100281343DNAAspergillus niger
28cgactggagc acgagggaca ctgacatgga ctgaaggagt agaaaagatc ttcctctccc
60acctccccag cctttccttc tttgcacctg tgccgtgcac ggtcgagcca ttccttcatt
120ctttgaacat attgcctggc tccgagtagt ctagcatcca ctccttgcaa gagcactttg
180agagaaccgg tcttctcata ctcaaaagtt atacatacac atcacttctc tccgaacaaa
240accgaacaga attcgaagaa cacatacaca atggtctcct tcaagtctct tctgaccgcc
300accaccctgg ccaccgccgt tctggccatc cctcatagtg gccacggcca tggcagccac
360aagcaccgtt ccacccatgt cgcctccaag cggacctctt cctccaagcg tggcgctgcc
420tacaactctg cttccagcgt tcacacgctg acctccggct cctccggcaa cggtaccgtc
480tcctgggcct acgactggaa catgtacgcc gacggcaccc tccccagtaa cgtcgaatac
540gtgcccatgc tgtggggcag caagatgttt ggaggctggt tgaccgccat cgagactgcc
600ctggacagcg gtagcaatta catcatggga ttcaacgagc ctgactcctc ctcccaagcc
660tcgatgactg cttccgaggc cgccagctcc tacaagaatt acatcactcc ttactctggc
720aaggctaagc tcgtcacccc ggccgtgacc agtagcacca cggaaggcga gggtctcagc
780tggatgaagt ccttcctgtc cgaatgcagc gagtgtgaca tgtcggtgct ggcagtccac
840tggtacggca cctcggccga tgagttcaag tccttcgtgc aggaggccat gcaggtggcg
900gacgacaacg gattggacga gacctgggtg acagaattcg ccctcaccag cgacgagtct
960gccggcggcg atgagagttc agcggcggac ttccttgacg aagttttgcc gtggttggac
1020agccagagtg gcgtgggacg gtatgcgtat tacatgtgtg cagatgggta tctgctcagc
1080ggggaggagt tgagctcgag tggaaaggtc tacgttgcat agaacaacca actacctttg
1140gattccatta gatctgcttt accttggact tttatcccgt gatacctttt tgtgctgttt
1200tttattctat tccattctac catccatctc tcacctaaga gggaaagaga gacggacaac
1260cccattttac ccacccactt tactttccaa tatattacaa ttccaatttg aatcaaattc
1320aaatccaaaa aaaaaaaaaa aaa
134329283PRTAspergillus nigerMISC_FEATUREX = gap in homologous sequence
29Met Val Ser Phe Lys Ser Leu Leu Thr Ala Thr Thr Leu Ala Thr Ala1
5 10 15Val Leu Ala Ile Pro His
Ser Gly His Gly His Gly Ser His Lys His 20 25
30Arg Ser Thr His Val Ala Ser Lys Arg Thr Ser Ser Ser
Lys Arg Gly 35 40 45Ala Ala Tyr
Asn Ser Ala Ser Ser Val His Thr Leu Thr Ser Gly Ser 50
55 60Ser Gly Asn Gly Thr Val Ser Trp Ala Tyr Asp Trp
Asn Met Tyr Ala65 70 75
80Asp Gly Thr Leu Pro Ser Asn Val Glu Tyr Val Pro Met Leu Trp Gly
85 90 95Ser Lys Met Phe Gly Gly
Trp Leu Thr Ala Ile Glu Thr Ala Leu Asp 100
105 110Ser Gly Ser Asn Tyr Ile Met Gly Phe Asn Glu Pro
Asp Ser Ser Ser 115 120 125Gln Ala
Ser Met Thr Ala Ser Glu Ala Ala Ser Ser Tyr Lys Asn Tyr 130
135 140Ile Thr Pro Tyr Ser Gly Lys Ala Lys Leu Val
Thr Pro Ala Val Thr145 150 155
160Ser Ser Thr Thr Glu Gly Glu Gly Leu Ser Trp Met Lys Ser Phe Leu
165 170 175Ser Glu Cys Ser
Glu Cys Asp Met Ser Val Leu Ala Val His Trp Tyr 180
185 190Gly Thr Ser Ala Asp Glu Phe Lys Ser Phe Val
Gln Glu Ala Met Gln 195 200 205Val
Ala Asp Asp Asn Gly Leu Asp Glu Thr Trp Val Thr Glu Phe Ala 210
215 220Leu Thr Ser Asp Glu Ser Ala Gly Gly Asp
Glu Ser Ser Ala Ala Asp225 230 235
240Phe Leu Asp Glu Val Leu Pro Trp Leu Asp Ser Gln Ser Gly Val
Gly 245 250 255Arg Tyr Ala
Tyr Tyr Met Cys Ala Asp Gly Tyr Leu Leu Ser Gly Glu 260
265 270Glu Leu Ser Ser Ser Gly Lys Val Tyr Val
Ala 275 2803022PRTAspergillus niger 30Lys Arg Arg
Lys Asp Glu Leu Ala Asp Thr Thr Leu Arg Gln Val Ala1 5
10 15Gln Asn Gln Thr Glu Thr
203116PRTAspergillus niger 31Leu Gly Asp Val Met Ser Ile Ser Ile Asn Pro
Thr Asn Gln Asn Val1 5 10
153229PRTAspergillus niger 32Ser Cys Arg Leu Phe Asp Ile Arg Ala Asp Arg
Glu Leu Asn Thr Tyr1 5 10
15Gln Ser Asp Gln Ile Leu Cys Gly Ile Thr Ser Val Ala 20
25331221DNAAspergillus niger 33taccactcac ctttcgcgca
tcgccatctg cgatcctccc cacaacactc cacctagata 60catacaccat taactgcgct
tctacaacat gcagatcttc gttaagaccc tcaccggcaa 120gacaatcacc ctcgaggtcg
agtccagcga taccatcgac aacgtcaaga ccaagatcca 180ggataaggag ggcatccctc
ccgaccagca gcgtctgatc ttcgccggaa agcagctgga 240ggatggccgc acgcttagtg
actacaacat ccagaaggag tctactctcc atcttgtcct 300ccgcctgcgt ggtggtatgc
agattttcgt caagaccctg accggaaaga ccatcaccct 360tgaggtggag tcttctgaca
ccatcgacaa tgtgaagacc aagattcagg acaaggaggg 420cattcccccg gaccagcagc
gtctgatctt cgctggaaag cagctggagg atggccgtac 480cctgtctgac tacaacattc
aaaaggaatc cacccttcac ctcgtccttc gtctgcgtgg 540tggtatgcag atcttcgtca
agactctcac gggaaagacg atcacattgg aagttgaatc 600ttccgacaca attgataacg
ttaagaccaa gattcaagac aaggaaggca tccccccgga 660ccagcagcgt ttgatcttcg
ctggtaagca gttggaggat ggccgtacct tgtccgacta 720caacatccag aaagaatcca
ctcttcacct tgtccttcgt ctccgtggtg gtatgcagat 780cttcgtgaag actcttaccg
gcaagacgat tacgctggag gtggagagct cggataccat 840tgataacgtc aagactaaga
ttcaagataa ggagggcatt cccccggacc agcagcgtct 900catcttcgct ggtaagcagt
tggaagatgg acgtacgctc tccgattaca acatccagaa 960ggagagcact ctgcacctgg
tgctccgtct ccgtggcgga aactaatgcc ttattttgat 1020ctttcttctt tagcacggct
catctacggt tgagtggcct gcatggcgtt gggacggttg 1080ttttcatcgg tttttatgat
acggataaat tgggcatacc ttagggtcac catcttccat 1140ggtgccttgc gtcattcttt
tacctaggaa tcaattcaat aatcatattc cacctgatat 1200ctaaaaaaaa aaaaaaaacc t
122134236PRTAspergillus niger
34Val Leu Arg Leu Arg Gly Gly Met Gln Ile Phe Val Lys Thr Leu Thr1
5 10 15Gly Lys Thr Ile Thr Leu
Glu Val Glu Ser Ser Asp Thr Ile Asp Asn 20 25
30Val Lys Thr Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro
Asp Gln Gln 35 40 45Arg Leu Ile
Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser 50
55 60Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu
Val Leu Arg Leu65 70 75
80Arg Gly Gly Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile
85 90 95Thr Leu Glu Val Glu Ser
Ser Asp Thr Ile Asp Asn Val Lys Thr Lys 100
105 110Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln
Arg Leu Ile Phe 115 120 125Ala Gly
Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile 130
135 140Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg
Leu Arg Gly Gly Met145 150 155
160Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val
165 170 175Glu Ser Ser Asp
Thr Ile Asp Asn Val Lys Thr Lys Ile Gln Asp Lys 180
185 190Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile
Phe Ala Gly Lys Gln 195 200 205Leu
Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser 210
215 220Thr Leu His Leu Val Leu Arg Leu Arg Gly
Gly Asn225 230 23535235PRTAspergillus
niger 35Val Leu Arg His Ala Asn Asn Leu Ala Val Val Lys Thr Leu Thr Gly1
5 10 15Lys Thr Ile Thr
Leu Glu Val Glu Ser Ser Asp Thr Ile Asp Asn Val 20
25 30Lys Thr Lys Ile Gln Asp Lys Glu Gly Ile Pro
Pro Asp Gln Gln Arg 35 40 45Leu
Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp 50
55 60Tyr Asn Ile Gln Lys Glu Ser Thr Leu His
Leu Val Leu Arg Leu Arg65 70 75
80Gly Gly Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile
Thr 85 90 95Leu Glu Val
Glu Ser Ser Asp Thr Ile Asp Asn Val Lys Ser Lys Ile 100
105 110Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln
Gln Arg Leu Ile Phe Ala 115 120
125Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln 130
135 140Lys Glu Ser Thr Leu His Leu Val
Leu Arg Leu Arg Gly Gly Met Gln145 150
155 160Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr
Leu Glu Val Glu 165 170
175Ser Ser Asp Thr Ile Asp Asn Val Lys Thr Lys Ile Gln Asp Lys Glu
180 185 190Gly Ile Pro Pro Asp Gln
Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu 195 200
205Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu
Ser Thr 210 215 220Leu His Leu Val Leu
Arg Leu Arg Gly Gly Asn225 230
23536404DNAAspergillus niger 36tcgagcggcc gccgggcagg tacctccctt
atgctgctgg atgaagacgt ggttactgcc 60tactatgcgc aagttcccaa ctcggtctac
gtgagcagtg ccggtggtta catctacccc 120tgcaacacca ctcttcccag cttctcgctt
gtcctcggcg agtcgagcct ggccacgatc 180cccggtaacc tgatcaattt ctccaaggtt
ggcaccaaca ccaccaccgg acaggccttg 240tgctttggcg gcattcaatc caacggaaac
acctcgctgc agattctggg cgatattttc 300ctgaaggcct ttttcgttgt cttcgacatg
cgcggcccct cgcttggtgt tgcctctccc 360aagaactagt ttccttttcc tgtacctcgg
ccgcgaccac gcta 40437334DNAAspergillus niger
37acaaagaatt ctccaggact cttgtctgac gtaaaatagg aagaaaagga aaactgaggt
60gatatcgcct gtgtagtgcg gcgattgacg tccttcctcc gcttgcccag cggtggtggg
120tcgagctgag gtgtccgttt atacgtgatg gtagtggtca cgatatggcg cacacaaaag
180gtgtttccat tctcactgac gggtgattcg aagaagcgct gtccccggtc tgatggagta
240aaaggggaac ggagggggtg cgcactccgc ggggacgcag acactggggt aatagaggta
300tggtgcagga aggcgcatgc gctgggcatg aata
334
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