# Patent

## Title
Methods for producing polypeptides in enzyme-deficient mutants of Fusarium venenatum

## Abstract

The present invention relates to methods of producing a polypeptide, comprising: (a) cultivating a mutant of a parent Fusarium venenatum strain in a medium for the production of the polypeptide, wherein the mutant strain comprises a polynucleotide encoding the polypeptide and one or more (several) genes selected from the group consisting of pyrG, amyA, and alpA, wherein the one or more (several) genes are modified rendering the mutant strain deficient in the production of one or more (several) enzymes selected from the group consisting of orotidine-5′-monophosphate decarboxylase, alpha-amylase, and alkaline protease, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions; and (b) recovering the polypeptide from the cultivation medium. The present invention also relates to enzyme-deficient mutants of Fusarium venenatum strains and methods for producing such mutants.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 13/121,254, filed on Jun. 1, 2011, now U.S. Pat. No. 8,647,856, which is a 35 U.S.C. §371 national application of PCT/US2009/059039, filed on Sep. 30, 2009, which claims the benefit of U.S. Provisional Application No. 61/101,250, filed on Sep. 30, 2008, which applications are fully incorporated herein by reference.


REFERENCE TO A SEQUENCE LISTING
This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods of producing polypeptides in enzyme-deficient Fusarium venenatum mutant strains, enzyme-deficient Fusarium venenatum mutant strains, and methods of obtaining the enzyme-deficient Fusarium venenatum mutant strains.
2. Description of the Related Art
Fusarium venenatum has been shown to be useful as a host cell for the recombinant production of polypeptides having biological activity (WO 96/00787, WO 97/26330). Fusarium venenatum hosts with the desirable traits of increased protein expression and secretion may not necessarily have the most desirable characteristics for successful fermentation. The fermentation may not be optimal because of the production of biological substances, e.g., enzymes, detrimental to the production, recovery, or application of a particular polypeptide of interest.
WO 99/60137 discloses trichothecene-deficient mutants of Fusarium venenatum. WO 00/42203 discloses cyclohexadepsipeptide-deficient mutants of Fusarium venenatum. 
The present invention relates to improved Fusarium venenatum hosts that combine the capacity for expression of commercial quantities of a polypeptide of interest while being deficient in the production of enzymes that can complicate recovery and downstream processing of the polypeptide.
SUMMARY OF THE INVENTION
The present invention relates to methods of producing a polypeptide, comprising:
(a) cultivating a mutant of a parent Fusarium venenatum strain in a medium for the production of the polypeptide, wherein the mutant strain comprises a polynucleotide encoding the polypeptide and one or more (several) genes selected from the group consisting of pyrG, amyA, and alpA, wherein the one or more (several) genes are modified rendering the mutant strain deficient in the production of one or more (several) enzymes selected from the group consisting of orotidine-5′-monophosphate decarboxylase, alpha-amylase, and alkaline protease, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions; and
(b) recovering the polypeptide from the cultivation medium.
In one aspect of the methods of producing a polypeptide, the mutant strain further comprises one or both of the genes tri5 and dps1, wherein the one or both genes are modified rendering the mutant strain deficient in the production of one or both enzymes trichodiene synthase and cyclohexadepsipeptide synthetase, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
The present invention also relates to mutants of a parent Fusarium venenatum strain, comprising a polynucleotide encoding a polypeptide and one or more (several) genes selected from the group consisting of pyrG, amyA, and alpA, wherein the one or more (several) genes are modified rendering the mutant strain deficient in the production of one or more (several) enzymes selected from the group consisting of orotidine-5′-monophosphate decarboxylase, alpha-amylase, and alkaline protease, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
In one aspect, the mutants of a parent Fusarium venenatum strain further comprise one or both of the genes tri5 and dps1, wherein the one or both genes are modified rendering the mutant strain deficient in the production of one or both enzymes trichodiene synthase and cyclohexadepsipeptide synthetase, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
The present invention also relates to methods of obtaining mutants of a parent Fusarium venenatum strain, comprising:
(a) modifying one or more (several) genes selected from the group consisting of pyrG, amyA, and alpA; and
(b) identifying a mutant strain from step (a) wherein the one or more (several) genes selected from the group consisting of pyrG, amyA, and alpA are modified rendering the mutant strain deficient in the production of one or more (several) enzymes selected from the group consisting of orotidine-5′-monophosphate decarboxylase, alpha-amylase, and alkaline protease, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
In one aspect, the methods of obtaining mutants of a parent Fusarium venenatum strain further comprise modifying one or both of the genes tri5 and dps1 rendering the mutant strain deficient in the production of one or both enzymes trichodiene synthase and cyclohexadepsipeptide synthetase, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows a restriction map of pDM156.2.
FIG. 2 shows a restriction map of pEmY21.
FIG. 3 shows a restriction map of pEmY23.
FIG. 4 shows a restriction map of pWTY1470-19-07.
FIG. 5 shows a restriction map of pWTY1515-2-01.
FIG. 6 shows a restriction map of pJaL504-[Bam HI].
FIG. 7 shows a restriction map of pJaL504-[Bgl II].
FIG. 8 shows a restriction map of pJaL574.
FIG. 9 shows a restriction map of pWTY1449-02-01.
FIG. 10 shows a restriction map of pJfyS1540-75-5.
FIG. 11 shows a restriction map of pJfyS1579-1-13.
FIG. 12 shows a restriction map of pJfyS1579-8-6.
FIG. 13 shows a restriction map of pJfyS1579-21-16.
FIG. 14 shows a restriction map of pAlLo1492-24.
FIG. 15 shows a restriction map of pJfyS1579-35-2.
FIG. 16 shows a restriction map of pJfyS1579-41-11.
FIG. 17 shows a restriction map of pJfyS1604-55-13.
FIG. 18 shows a restriction map of pJfyS1579-93-1.
FIG. 19 shows a restriction map of pJfyS1604-17-2.
FIG. 20 shows a restriction map of pEJG61.
FIG. 21 shows a restriction map of pEJG69.
FIG. 22 shows a restriction map of pEJG65.
FIG. 23 shows a restriction map of pMStr19.
FIG. 24 shows a restriction map of pEJG49.
FIG. 25 shows a restriction map of pEmY15.
FIG. 26 shows a restriction map of pEmY24.
FIG. 27 shows a restriction map of pDM257.
FIG. 28 shows a restriction map of pDM258.
FIG. 29 shows the relative lactose oxidase yields of transformants of Fusarium venenatum JfyS1643-95-04 (Δtri5 ΔpyrG ΔamyA).
FIG. 30 shows the relative alpha-amylase activity of transformants of transformants of Fusarium venenatum JfyS1643-95-04 (Δtri5 ΔpyrG ΔamyA).
FIG. 31 shows a restriction map of pJfyS1698-65-15.
FIG. 32 shows a restriction map of pJfyS1698-72-10.
FIG. 33 shows the relative alkaline protease activity of transformants of Fusarium venenatum JfyS1763-11-01 (Δtri5 ΔpyrG ΔamyA ΔalpA).
FIG. 34 shows a restriction map of pJfyS1879-32-2.
FIG. 35 shows a restriction map of pJfyS111.



DEFINITIONS
Orotidine-5′-monophosphate decarboxylase: The term “orotidine-5′-monophosphate decarboxylase” is defined herein as a UTP:ammonia ligase (ADP-forming) (EC 6.3.4.2) that catalyzes the conversion of ATP+UTP+NH3 to ADP+phosphate+CTP. For purposes of the present invention, orotidine-5′-monophosphate decarboxylase activity is determined according to the method described by Liberman, 1956, Journal of Biological Chemistry 222: 765-775).
Alpha-amylase: The term “alpha-amylase” is defined herein as an 1,4-α-D-glucan glucanohydrolase (EC 3.2.1.1) that catalyzes the endohydrolysis of 1,4-α-D-glucosidic linkages in polysaccharides containing three or more 1,4-α-linked D-glucose units. For purposes of the present invention, alpha-amylase activity is determined using 4,6-ethylidene (G7)-p-nitrophenyl (G1)-alpha-D-maltoheptaside as substrate and Sigma Chemical Co. Kit 577 (St. Louis, Mo., USA) at pH 7.0.
Alkaline protease: The term “alkaline protease” is defined herein as a serine protease that catalyzes the hydrolysis of peptide bonds in proteins. For purposes of the present invention, alkaline protease activity is determined according to the procedure described in Example 28.
Trichothecenes: The term “trichothecenes” is defined herein as a family of sesquiterpene epoxides produced by a sequence of oxygenations, isomerizations, cyclizations, and esterifications leading from trichodiene to the more complex trichothecenes (Desjardins, Hohn, and McCormick, 1993, Microbiological Reviews 57: 595-604). Trichothecenes include, but are not limited to, 2-hydroxytrichodiene, 12,13-epoxy-9,10-trichoene-2-ol, isotrichodiol, isotrichotriol, trichotriol, isotrichodermol, isotrichodermin, 15-decalonectrin, 3,15-didecalonectrin, deoxynivalenol, 3-acetyldeoxynivalenol, calonectrin, 3,15-d iacetoxyscirpenol, 3,4,15-triacetoxyscirpenol, 4,15-diacetoxyscirpenol, 3-acetylneosolaniol, acetyl T-2 toxin, and T-2 toxin; and derivatives thereof.
Trichodiene synthase: The term “trichodiene synthase” is defined herein as a dextrin 6-alpha-D-glucanohydrolase that catalyses the isomerization-cyclization of farnesylpyrophosphate to form the bicyclic olefin trichodiene. For purposes of the present invention, trichodiene synthase activity is determined according to the procedure described by Hohn and Beremand, 1989, Applied and Environmental Microbiology 55: 1500-1503.
The level of trichothecenes produced by a mutant Fusarium venenatum strain of the present invention may be determined using methods well known in the art (see, for example, Rood et al., 1988, Journal of Agricultural and Food Chemistry 36: 74-79; Romer, 1986, Journal of the Association of Official Analytical Chemists 69: 699-703; McCormick et al., 1990, Applied and Environmental Microbiology 56: 702-706).
Cyclohexadepsipeptides: The term “cyclohexadepsipeptides” is defined herein as a family of peptide-related compounds composed of hydroxy and amino acids linked by amide and ester bonds. The term cyclohexadepsipeptides includes, but is not limited to, enniatins.
Enniatins: The term “enniatins” is defined herein as a family of cyclohexadepsipeptides composed of three D-2-hydroxyisovaleric acid residues joined alternatively to L-amino acids or N-methyl-L-amino acids to produce an 18-membered cyclic structure. Enniatins are cyclohexadepsipeptide phytoxins with ionophoretic properties produced by various species of actinomycetes and filamentous fungi, particularly strains of Fusarium. The enniatins include, but are not limited to, enniatin A, A1, B, B1, B2, B3, B4, C, D, E, and F; and derivatives thereof (Visconte et al., 1992, Journal of Agricultural and Food Chemistry 40: 1076-1082; Tomodo et al., 1992, Journal of Antibiotics 45: 1207-1215), and mixed-type enniatins containing more than one species of amino acid (Zocher et al. 1982, Biochemistry 21: 43-48).
The biosynthesis of enniatins is catalyzed by enniatin synthetase, which is a large multifunctional enzyme that has all the essential functions for assembling enniatins from their primary precursors, i.e., D-2-hydroxyisovaleric acid, a branched chain L-amino acid (e.g., valine, leucine, isoleucine), S-adenosylmethionine, and ATP (Reper et al., 1995, European Journal of Biochemistry 230: 119-126). The precursors (D-2-hydroxyisovaleric acid and branched chain L-amino acid) are activated as thioesters. Covalently bound substrate amino acid residues are methylated under the consumption of S-adenosylmethionine. Then peptide bond formation and cyclization reactions occur.
Cyclohexadepsipeptide synthetase: The term “cyclohexadepsipeptide synthetase” is defined herein as a synthetase that catalyzes the production of a cyclohexadepsipeptide from D-2-hydroxyisovaleric acid, a branched chain L-amino acid (e.g., valine, leucine, isoleucine), S-adenosylmethionine, and ATP. For purposes of the present invention, cyclohexadepsipeptide synthetase activity is determined by measuring the production of a cyclohexadepsipeptide according to the procedure of Zocher et al., 1982, Biochemistry 21: 43-48. Specifically, the cyclohexadepsipeptide synthetase is incubated with 1 mM valine, 0.2 mM S-adenosylmethionine, 0.2 mM D-2-hydroxyisovaleric acid, 4 mM ATP, and 4 mM magnesium acetate in a total volume of 100 μl for 10 minutes at 37° C. in 50 mM MOPS pH 7.0. The amount of cyclohexadepsipeptide is determined as described in WO 2000/92203 based on the method of Visconti et al., 1992, Journal of Agriculture and Food Chemistry 40: 1076-1082. One unit of cyclohexadepsipeptide synthetase activity is defined as 1.0 μmole of cyclohexadepsipeptide produced per minute at 37° C., pH 7.0.
The level of cyclohexadepsipeptides can be determined according to the method of Visconti et al., 1992, Journal of Agriculture and Food Chemistry 40: 1076-1082. Specifically, one ml of Fusarium venenatum cell-free culture broth is extracted twice with 2.0 ml of ethyl acetate. The combined organic extracts are evaporated to dryness under a stream of nitrogen gas and redissolved in 0.5 ml hexane. One microliter samples are analyzed using a Hewlett-Packard 6890 GC/Series MSD system operating in the electron impact (EI) mode. Samples are injected on-column and separated utilizing a DB-5 capillary column (30 m×0.25 mm, 0.25 μm film) employing a temperature program with heating from 120 to 300° C. at a rate of 15° C./minute. For example, enniatins A, A1, B, B1, B2, and B3 are identified by m/z ratios for the (M++H) ion of 682, 668, 640, 654, 626, and 612, respectively.
Deficient: The term “deficient” is defined herein as a Fusarium venenatum mutant strain that produces no detectable activity of one or more (several) enzymes selected from the group consisting of orotidine-5′-monophosphate decarboxylase, alpha-amylase, and alkaline protease and alternatively also one or both of the enzymes trichodiene synthase and cyclohexadepsipeptide synthetase compared to the parent Fusarium venenatum strain when cultivated under identical conditions, or, in the alternative, produces preferably at least 25% less, more preferably at least 50% less, even more preferably at least 75% less, and most preferably at least 95% less of one or more (several) enzymes selected from the group consisting of orotidine-5′-monophosphate decarboxylase, alpha-amylase, and alkaline protease and alternatively also one or both of the enzymes trichodiene synthase and cyclohexadepsipeptide synthetase than the parent Fusarium venenatum strain when cultivated under identical conditions. The level of enzyme produced by a Fusarium venenatum mutant strain of the present invention may be determined using methods described herein or known in the art.
The mutant Fusarium venenatum strain produces preferably at least about 25% less, more preferably at least about 50% less, even more preferably at least about 75% less, most preferably at least about 95% less, and even most preferably no trichothecene than the corresponding parent filamentous fungal cell when cultured under identical conditions. The parent and mutant cells may be compared with regard to production of a trichothecene under conditions conducive for the production of a polypeptide of interest or under conditions conducive for the production of a trichothecene.
The mutant Fusarium venenatum strain produces preferably at least about 25% less, more preferably at least about 50% less, even more preferably at least about 75% less, most preferably at least about 95% less, and even most preferably no cyclohexadepsipeptide than the corresponding parent filamentous fungal cell when cultured under identical conditions. The parent and mutant cells may be compared with regard to production of a cyclohexadepsipeptide under conditions conducive for the production of a polypeptide of interest or under conditions conducive for the production of a cyclohexadepsipeptide.
Isolated polypeptide: The term “isolated polypeptide” as used herein refers to a polypeptide that is isolated from a source. In a preferred aspect, the polypeptide is at least 1% pure, preferably at least 5% pure, more preferably at least 10% pure, more preferably at least 20% pure, more preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, and most preferably at least 90% pure, as determined by SDS-PAGE.
Substantially pure polypeptide: The term “substantially pure polypeptide” denotes herein a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polypeptide material with which it is natively or recombinantly associated. It is, therefore, preferred that the substantially pure polypeptide is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, most preferably at least 99.5% pure, and even most preferably 100% pure by weight of the total polypeptide material present in the preparation. The polypeptides of the present invention are preferably in a substantially pure form, i.e., that the polypeptide preparation is essentially free of other polypeptide material with which it is natively or recombinantly associated. This can be accomplished, for example, by preparing the polypeptide by well-known recombinant methods or by classical purification methods.
Mature polypeptide: The term “mature polypeptide” is defined herein as a polypeptide having enzyme activity that is in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.
Mature polypeptide coding sequence: The term “mature polypeptide coding sequence” is defined herein as a nucleotide sequence that encodes a mature polypeptide having enzyme activity.
Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “identity”.
For purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment)

For purposes of the present invention, the degree of identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the—nobrief option) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment)

Polypeptide fragment: The term “polypeptide fragment” is defined herein as a polypeptide having one or more (several) amino acids deleted from the amino and/or carboxyl terminus of a mature polypeptide or a homologous sequence thereof; wherein the fragment has enzyme activity, e.g., orotidine-5′-monophosphate decarboxylase, alpha-amylase, alkaline protease, trichodiene synthase, or cyclohexadepsipeptide synthetase activity.
Subsequence: The term “subsequence” is defined herein as a nucleotide sequence having one or more (several) nucleotides deleted from the 5′ and/or 3′ end of a mature polypeptide coding sequence or a homologous sequence thereof; wherein the subsequence encodes a polypeptide fragment having enzyme activity, e.g., orotidine-5′-monophosphate decarboxylase, alpha-amylase, alkaline protease, trichodiene synthase, or cyclohexadepsipeptide synthetase activity.
Allelic variant: The term “allelic variant” denotes herein any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
Isolated polynucleotide: The term “isolated polynucleotide” as used herein refers to a polynucleotide that is isolated from a source. In a preferred aspect, the polynucleotide is at least 1% pure, preferably at least 5% pure, more preferably at least 10% pure, more preferably at least 20% pure, more preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, and most preferably at least 90% pure, as determined by agarose electrophoresis.
Substantially pure polynucleotide: The term “substantially pure polynucleotide” as used herein refers to a polynucleotide preparation free of other extraneous or unwanted nucleotides and in a form suitable for use within genetically engineered protein production systems. Thus, a substantially pure polynucleotide contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polynucleotide material with which it is natively or recombinantly associated. A substantially pure polynucleotide may, however, include naturally occurring 5′ and 3′ untranslated regions, such as promoters and terminators. It is preferred that the substantially pure polynucleotide is at least 90% pure, preferably at least 92% pure, more preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, even more preferably at least 98% pure, most preferably at least 99% pure, and even most preferably at least 99.5% pure by weight. The polynucleotides of the present invention are preferably in a substantially pure form, i.e., that the polynucleotide preparation is essentially free of other polynucleotide material with which it is natively or recombinantly associated. The polynucleotides may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.
Coding sequence: When used herein the term “coding sequence” means a nucleotide sequence, which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA, synthetic, or recombinant nucleotide sequence.
cDNA: The term “cDNA” is defined herein as a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic cell. cDNA lacks intron sequences that are usually present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps before appearing as mature spliced mRNA. These steps include the removal of intron sequences by a process called splicing. cDNA derived from mRNA lacks, therefore, any intron sequences.
Nucleic acid construct: The term “nucleic acid construct” as used herein refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic. The term nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention.
Control sequences: The term “control sequences” is defined herein to include all components necessary for expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide.
Operably linked: The term “operably linked” denotes herein a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence such that the control sequence directs expression of the coding sequence of a polypeptide.
Expression: The term “expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
Expression vector: The term “expression vector” is defined herein as a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide of the present invention and is operably linked to additional nucleotides that provide for its expression.
Host cell: The term “host cell”, as used herein, includes any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention.
Modification: The term “modification” is defined herein as an introduction, substitution, or removal of one or more nucleotides in a gene or a control sequence required for the transcription or translation thereof, or gene disruption, gene conversion, gene deletion, or random or specific mutagenesis of amyA, alpA, dps1, pyrG, tri5, or a combination thereof. The deletion of one or more (several) of the amyA, alpA, dps1, pyrG, and tri5 genes may be partial or complete. The modification results in a decrease in or elimination (inactivation) of expression of pyrG, amyA, alpA, tri5, dps1, or a combination thereof. In a preferred aspect, one or more (several) are inactivated.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to methods of producing a polypeptide, comprising: (a) cultivating a mutant of a parent Fusarium venenatum strain in a medium for the production of the polypeptide, wherein the mutant strain comprises a polynucleotide encoding the polypeptide and one or more (several) genes selected from the group consisting of pyrG, amyA, and alpA, wherein the one or more (several) genes are modified rendering the mutant strain deficient in the production of one or more (several) enzymes selected from the group consisting of orotidine-5′-monophosphate decarboxylase, alpha-amylase, and alkaline protease, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions; and (b) recovering the polypeptide from the cultivation medium.
In one aspect, the mutant strain further comprises one or both of the genes tri5 and dps1, wherein the one or both genes are modified rendering the mutant strain deficient in the production of one or both enzymes trichodiene synthase and cyclohexadepsipeptide synthetase, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
An advantage of the present invention is elimination or reduction of one or more (several) enzyme activities, which may be detrimental to the production, downstream processing, e.g., recovery, and/or application of a particular polypeptide of interest
In the methods of the present invention, the parent Fusarium venenatum strain may be a wild-type Fusarium venenatum strain or a mutant thereof. It will be understood that the term “Fusarium venenatum” also includes varieties of Fusarium venenatum (see, for example, Robert A. Samsom and John I. Pitt, editors, Integration of Modern Taxonomic Methods for Penicillium and Aspergillus Classification, Harwood Academic Publishers, The Netherlands). In one aspect, the parent Fusarium venenatum strain is Fusarium venenatum A3/5. In another aspect, the parent Fusarium venenatum strain is Fusarium venenatum NRRL 30747. In another aspect, the parent Fusarium venenatum strain is Fusarium venenatum ATCC 20334. In another aspect, the parent Fusarium venenatum strain is a morphological mutant (WO 97/26330).
The enzyme-deficient Fusarium venenatum mutant strain may be constructed by reducing or eliminating expression of one or more (several) genes selected from the group consisting of pyrG, amyA, and alpA, and alternatively also one or both of the genes tri5 and dps1 using methods well known in the art, such as insertions, disruptions, replacements, or deletions. A portion of the gene can be modified such as the coding region or a control sequence required for expression of the coding region. Such a control sequence of a gene may be a promoter sequence or a functional part thereof, i.e., a part that is sufficient for affecting expression of the gene. For example, a promoter sequence may be inactivated resulting in no expression or a weaker promoter may be substituted for the native promoter sequence to reduce expression of the coding sequence. Other control sequences for possible modification include, but are not limited to, a leader, propeptide sequence, signal sequence, transcription terminator, and transcriptional activator.
The Fusarium venenatum mutant strains may be constructed by gene deletion techniques to eliminate or reduce expression of a gene. Gene deletion techniques enable the partial or complete removal of the gene thereby eliminating their expression. In such methods, deletion of the gene(s) is accomplished by homologous recombination using a plasmid that has been constructed to contiguously contain the 5′ and 3′ regions flanking the gene.
The Fusarium venenatum mutant strains may also be constructed by introducing, substituting, and/or removing one or more (several) nucleotides in the gene or a control sequence thereof required for the transcription or translation thereof. For example, nucleotides may be inserted or removed so as to result in the introduction of a stop codon, the removal of the start codon, or a frame-shift of the open reading frame. Such a modification may be accomplished by site-directed mutagenesis or PCR generated mutagenesis in accordance with methods known in the art. See, for example, Botstein and Shortle, 1985, Science 229: 4719; Lo et al., 1985, Proceedings of the National Academy of Sciences USA 81: 2285; Higuchi et al., 1988, Nucleic Acids Research 16: 7351; Shimada, 1996, Meth. Mol. Biol. 57: 157; Ho et al., 1989, Gene 77: 61; Horton et al., 1989, Gene 77: 61; and Sarkar and Sommer, 1990, BioTechniques 8: 404.
The Fusarium venenatum mutant strains may also be constructed by gene disruption techniques by inserting into a gene a disruptive nucleic acid construct comprising a nucleic acid fragment(s) homologous to the gene that will create a duplication of the region of homology and incorporate construct DNA between the duplicated regions. Such gene disruption can eliminate gene expression if the inserted construct separates the promoter of the gene from the coding region or interrupts the coding sequence such that a non-functional gene product results. A disrupting construct may be simply a selectable marker gene accompanied by 5′ and 3′ regions homologous to the gene. The selectable marker enables identification of transformants containing the disrupted gene.
The Fusarium venenatum mutant strains may also be constructed by the process of gene conversion (see, for example, Iglesias and Trautner, 1983, Molecular General Genetics 189: 73-76). For example, in the gene conversion method, a nucleotide sequence corresponding to the gene(s) is mutagenized in vitro to produce a defective nucleotide sequence, which is then transformed into the parent Fusarium venenatum strain to produce a defective gene. By homologous recombination, the defective nucleotide sequence replaces the endogenous gene. It may be desirable that the defective nucleotide sequence also comprises a marker for selection of transformants containing the defective gene.
The Fusarium venenatum mutant strains may also be constructed by established anti-sense techniques using a nucleotide sequence complementary to the nucleotide sequence of the gene (Parish and Stoker, 1997, FEMS Microbiology Letters 154: 151-157). More specifically, expression of the gene by a Fusarium venenatum strain may be reduced or inactivated by introducing a nucleotide sequence complementary to the nucleotide sequence of the gene, which may be transcribed in the strain and is capable of hybridizing to the mRNA produced in the strain. Under conditions allowing the complementary anti-sense nucleotide sequence to hybridize to the mRNA, the amount of protein translated is thus reduced or eliminated.
The Fusarium venenatum mutant strains may also be constructed by established RNA interference (RNAi) techniques (see, for example, WO 2005/056772).
The Fusarium venenatum mutant strains may be further constructed by random or specific mutagenesis using methods well known in the art, including, but not limited to, chemical mutagenesis (see, for example, Hopwood, The Isolation of Mutants in Methods in Microbiology (J. R. Norris and D. W. Ribbons, eds.) pp 363-433, Academic Press, New York, 1970). Modification of the gene may be performed by subjecting the parent strain to mutagenesis and screening for mutant strains in which expression of the gene has been reduced or inactivated. The mutagenesis, which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, use of a suitable oligonucleotide, or subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing methods.
Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), N-methyl-N′-nitrosogaunidine (NTG) O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues. When such agents are used, the mutagenesis is typically performed by incubating the parent strain to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and selecting for mutants exhibiting reduced or no expression of a gene.
In one aspect, the modification results in the inactivation of one or more (several) genes selected from the group consisting of pyrG, amyA, and alpA, and alternatively also one or both of the genes tri5 and dps1. In another aspect, the modification results in a decrease in expression of one or more (several) genes selected from the group consisting of pyrG, amyA, and alpA, and alternatively also one or both of the genes tri5 and dps1. In another aspect, the modification results in expression of one or more (several) genes selected from the group consisting of pyrG, amyA, and alpA, and alternatively also one or both of the genes tri5 and dps1 being decreased, inactivated, or a combination thereof.
In another aspect, the mutant comprises a modification of pyrG and amyA. In another aspect, the mutant comprises a modification of pyrG and alpA. In another aspect, the mutant comprises a modification of amyA and alpA. In another aspect, the mutant comprises a modification of tri5 and pyrG. In another aspect, the mutant comprises a modification of tri5 and amyA. In another aspect, the mutant comprises a modification of tri5 and alpA. In another aspect, the mutant comprises a modification of tri5 and dps1. In another aspect, the mutant comprises a modification of amyA and dps1. In another aspect, the mutant comprises a modification of alpA and dps1. In another aspect, the mutant comprises a modification of pyrG and dps1.
In another aspect, the mutant comprises a modification of pyrG, amyA, and alpA. In another aspect, the mutant comprises a modification of tri5, pyrG, and amyA. In another aspect, the mutant comprises a modification of tri5, pyrG, and alpA. In another aspect, the mutant comprises a modification of tri5, pyrG, and dps1. In another aspect, the mutant comprises a modification of tri5, amyA, and alpA. In another aspect, the mutant comprises a modification of tri5, amyA, and dps1. In another aspect, the mutant comprises a modification of tri5, alpA, and dps1. In another aspect, the mutant comprises a modification of amyA, alpA, and dps1. In another aspect, the mutant comprises a modification of pyrG, alpA, and dps1. In another aspect, the mutant comprises a modification of pyrG, amyA, and dps1.
In another aspect, the mutant comprises a modification of tri5, pyrG, amyA, and alpA. In another aspect, the mutant comprises a modification of tri5, pyrG, amyA, and dps1. In another aspect, the mutant comprises a modification of tri5, amyA, alpA, and dps1. In another aspect, the mutant comprises a modification of pyrG, amyA, alpA, and dps1. In another aspect, the mutant comprises a modification of tri5, pyrG, alpA, and dps1.
In another aspect, the mutant comprises a modification of pyrG, amyA, alpA, tri5, and dps1.
In one aspect, the pyrG gene comprises a nucleotide sequence encoding a polypeptide having orotidine-5′-monophosphate decarboxylase activity comprising an amino acid sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 44. In another aspect, the pyrG gene comprises a nucleotide sequence encoding a polypeptide having orotidine-5′-monophosphate decarboxylase activity comprising the amino acid sequence of SEQ ID NO: 44. In another aspect, the pyrG gene comprises a nucleotide sequence encoding a polypeptide having orotidine-5′-monophosphate decarboxylase activity consisting of the amino acid sequence of SEQ ID NO: 44.
In another aspect, the pyrG gene comprises a nucleotide sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 43. In another aspect, the pyrG gene comprises the nucleotide sequence of SEQ ID NO: 43. In another aspect, the pyrG gene consists of the nucleotide sequence of SEQ ID NO: 43.
In another aspect, the pyrG gene comprises a nucleotide sequence that hybridizes under preferably very low stringency conditions, more preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with the nucleotide sequence of SEQ ID NO: 43 or its full-length complementary strand.
In another aspect, the orotidine-5′-monophosphate decarboxylase comprises an amino acid sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 44. In another aspect, the orotidine-5′-monophosphate decarboxylase comprises the amino acid sequence of SEQ ID NO: 44. In another aspect, the orotidine-5′-monophosphate decarboxylase consists of the amino acid sequence of SEQ ID NO: 44.
In another aspect, the orotidine-5′-monophosphate decarboxylase is encoded by a polynucleotide comprising a nucleotide sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 43. In another aspect, the orotidine-5′-monophosphate decarboxylase is encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 43. In another aspect, the orotidine-5′-monophosphate decarboxylase is encoded by a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 43.
In another aspect, the orotidine-5′-monophosphate decarboxylase is encoded by a polynucleotide comprising a nucleotide sequence that hybridizes under preferably very low stringency conditions, more preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with the nucleotide sequence of SEQ ID NO: 43 or its full-length complementary strand.
In another aspect, the amyA gene comprises a nucleotide sequence encoding a polypeptide having alpha-amylase activity comprising an amino acid sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 52. In another aspect, the amyA gene comprises a nucleotide sequence encoding a polypeptide having alpha-amylase activity comprising the amino acid sequence of SEQ ID NO: 52. In another aspect, the amyA gene comprises a nucleotide sequence encoding a polypeptide having alpha-amylase activity consisting of the amino acid sequence of SEQ ID NO: 52.
In another aspect, the amyA gene comprises a nucleotide sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 51. In another aspect, the amyA gene comprises the nucleotide sequence of SEQ ID NO: 51. In another aspect, the amyA gene consists of the nucleotide sequence of SEQ ID NO: 51.
In another aspect, the amyA gene comprises a nucleotide sequence that hybridizes under preferably very low stringency conditions, more preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with the nucleotide sequence of SEQ ID NO: 51 or its full-length complementary strand.
In another aspect, the alpha-amylase comprises an amino acid sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 52. In another aspect, the alpha-amylase comprises the amino acid sequence of SEQ ID NO: 52. In another aspect, the alpha-amylase consists of the amino acid sequence of SEQ ID NO: 52.
In another aspect, the alpha-amylase is encoded by a polynucleotide comprising a nucleotide sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 51. In another aspect, the alpha-amylase is encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 51. In another aspect, the alpha-amylase is encoded by a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 51.
In another aspect, the alpha-amylase is encoded by a polynucleotide comprising a nucleotide sequence that hybridizes under preferably very low stringency conditions, more preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with the nucleotide sequence of SEQ ID NO: 51 or its full-length complementary strand.
In another aspect, the alpA gene comprises a nucleotide sequence encoding a polypeptide having alkaline protease activity comprising an amino acid sequence having a preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 84. In another aspect, the alpA gene comprises a nucleotide sequence encoding a polypeptide having alkaline protease activity comprising the amino acid sequence of SEQ ID NO: 84. In another aspect, the alpA gene comprises a nucleotide sequence encoding a polypeptide having alkaline protease activity consisting of the amino acid sequence of SEQ ID NO: 84.
In another aspect, the alpA gene comprises a nucleotide sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 83. In another aspect, the alpA gene comprises the nucleotide sequence of SEQ ID NO: 83. In another aspect, the alpA gene consists of the nucleotide sequence of SEQ ID NO: 83.
In another aspect, the alpA gene comprises a nucleotide sequence that hybridizes under preferably very low stringency conditions, more preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with the nucleotide sequence of SEQ ID NO: 83 or its full-length complementary strand.
In another aspect, the alkaline protease comprises an amino acid sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 84. In another aspect, the alpha-amylase comprises the amino acid sequence of SEQ ID NO: 84. In another aspect, the alpha-amylase consists of the amino acid sequence of SEQ ID NO: 84.
In another aspect, the alkaline protease is encoded by a polynucleotide comprising a nucleotide sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 83. In another aspect, the alkaline protease is encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 83. In another aspect, the alkaline protease is encoded by a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 83.
In another aspect, the alkaline protease is encoded by a polynucleotide comprising a nucleotide sequence that hybridizes under preferably very low stringency conditions, more preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with the nucleotide sequence of SEQ ID NO: 83 or its full-length complementary strand.
In another aspect, the tri5 gene comprises a nucleotide sequence encoding a polypeptide having trichodiene synthase activity comprising an amino acid sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 20. In another aspect, the tri5 gene comprises a nucleotide sequence encoding a polypeptide having trichodiene synthase activity comprising the amino acid sequence of SEQ ID NO: 20. In another aspect, the tri5 gene comprises a nucleotide sequence encoding a polypeptide having trichodiene synthase activity consisting of the amino acid sequence of SEQ ID NO: 20.
In another aspect, the tri5 gene comprises a nucleotide sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 19. In another aspect, the tri5 gene comprises the nucleotide sequence of SEQ ID NO: 19. In another aspect, the tri5 gene consists of the nucleotide sequence of SEQ ID NO: 19.
In another aspect, the tri5 gene comprises a nucleotide sequence that hybridizes under preferably very low stringency conditions, more preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with the nucleotide sequence of SEQ ID NO: 19 or its full-length complementary strand.
In another aspect, the trichodiene synthase comprises an amino acid sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 20. In another aspect, the trichodiene synthase comprises the amino acid sequence of SEQ ID NO: 20. In another aspect, the trichodiene synthase consists of the amino acid sequence of SEQ ID NO: 20.
In another aspect, the trichodiene synthase is encoded by a polynucleotide comprising a nucleotide sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 19. In another aspect, the trichodiene synthase is encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 19. In another aspect, the trichodiene synthase is encoded by a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 19.
In another aspect, the trichodiene synthase is encoded by a polynucleotide comprising a nucleotide sequence that hybridizes under preferably very low stringency conditions, more preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with the nucleotide sequence of SEQ ID NO: 19 or its full-length complementary strand.
In another aspect, the dps1 gene comprises a nucleotide sequence encoding a polypeptide having cyclohexadepsipeptide synthetase activity comprising an amino acid sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 94. In another aspect, the dps1 gene comprises a nucleotide sequence encoding a polypeptide having cyclohexadepsipeptide synthetase activity comprising the amino acid sequence of SEQ ID NO: 94. In another aspect, the dps1 gene comprises a nucleotide sequence encoding a polypeptide having cyclohexadepsipeptide synthetase activity consisting of the amino acid sequence of SEQ ID NO: 94.
In another aspect, the dps1 gene comprises a nucleotide sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 93. In another aspect, the dps1 gene comprises the nucleotide sequence of SEQ ID NO: 93. In another aspect, the dps1 gene consists of the nucleotide sequence of SEQ ID NO: 93.
In another aspect, the dps1 gene comprises a nucleotide sequence that hybridizes under preferably very low stringency conditions, more preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with the nucleotide sequence of SEQ ID NO: 93 or its full-length complementary strand.
In another aspect, the cyclohexadepsipeptide synthetase comprises an amino acid sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 94. In another aspect, the cyclohexadepsipeptide synthetase comprises the amino acid sequence of SEQ ID NO: 94. In another aspect, the cyclohexadepsipeptide synthetase consists of the amino acid sequence of SEQ ID NO: 94.
In another aspect, the cyclohexadepsipeptide synthetase is encoded by a polynucleotide comprising a nucleotide sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 93. In another aspect, the cyclohexadepsipeptide synthetase is encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 93. In another aspect, the cyclohexadepsipeptide synthetase is encoded by a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 93.
In another aspect, the cyclohexadepsipeptide synthetase is encoded by a polynucleotide comprising a nucleotide sequence that hybridizes under preferably very low stringency conditions, more preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with the nucleotide sequence of SEQ ID NO: 93 or its full-length complementary strand.
The nucleotide sequences disclosed herein or subsequences thereof, as well as the amino acid sequences thereof or fragments thereof, may be used to design nucleic acid probes to identify and clone homologous DNA of the genes described above from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, preferably at least 25, and more preferably at least 35 nucleotides in length. Longer probes can also be used. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with 32P, 3H, 35S, biotin, or avidin).
Thus, a genomic DNA or cDNA library prepared from such other organisms may be screened for DNA that hybridizes with the probes described above. Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that is homologous with the nucleotide sequences disclosed herein or subsequences thereof, the carrier material is used in a Southern blot. For purposes of the present invention, hybridization indicates that the nucleic acid sequence hybridizes to a labeled nucleic acid probe corresponding to the nucleotide sequences disclosed herein, its complementary strand, or a subsequence thereof, under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions are detected using X-ray film.
For long probes of at least 100 nucleotides in length, very low to very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures for 12 to 24 hours optimally.
For long probes of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS preferably at 45° C. (very low stringency), more preferably at 50° C. (low stringency), more preferably at 55° C. (medium stringency), more preferably at 60° C. (medium-high stringency), even more preferably at 65° C. (high stringency), and most preferably at 70° C. (very high stringency).
For short probes of about 15 nucleotides to about 70 nucleotides in length, stringency conditions are defined as prehybridization, hybridization, and washing post-hybridization at about 5° C. to about 10° C. below the calculated Tm using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures for 12 to 24 hours optimally.
For short probes of about 15 nucleotides to about 70 nucleotides in length, the carrier material is washed once in 6×SCC plus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C. below the calculated Tm.
A nucleotide sequence homologous or complementary to a gene described herein may be used from other microbial sources to modify the corresponding gene in the Fusarium venenatum strain of choice.
In another aspect, the modification of a gene in the Fusarium venenatum mutant strain is unmarked with a selectable marker.
Removal of the selectable marker gene may be accomplished by culturing the mutants on a counter-selection medium. Where the selectable marker gene contains repeats flanking its 5′ and 3′ ends, the repeats will facilitate the looping out of the selectable marker gene by homologous recombination when the mutant strain is submitted to counter-selection. The selectable marker gene may also be removed by homologous recombination by introducing into the mutant strain a nucleic acid fragment comprising 5′ and 3′ regions of the defective gene, but lacking the selectable marker gene, followed by selecting on the counter-selection medium. By homologous recombination, the defective gene containing the selectable marker gene is replaced with the nucleic acid fragment lacking the selectable marker gene. Other methods known in the art may also be used.
It will be understood that the methods of the present invention are not limited to a particular order for obtaining the Fusarium venenatum mutant strain. The modification of a gene may be introduced into the parent strain at any step in the construction of the strain for the production of a polypeptide of interest. It is preferred that the Fusarium venenatum mutant strain has already been made enzyme-deficient prior to such a construction.
In a further aspect of the present invention, the mutants of Fusarium venenatum strains may contain additional modifications, e.g., deletions or disruptions, of other genes, which may encode substances detrimental to the production, recovery, or application of a polypeptide of interest.
In one aspect, the Fusarium venenatum strain further comprises a modification, e.g., disruption or deletion, of one or more (several) genes encoding a proteolytic activity. In another aspect, the proteolytic activity is selected from the group consisting of an aminopeptidase, dipeptidylaminopeptidase, tripeptidylaminopeptidase, carboxypeptidase, aspergillopepsin, serine protease, metalloprotease, cysteine protease, and vacuolar protease.
In another aspect, the Fusarium venenatum strain further comprises a modification, e.g., disruption or deletion, of one or more (several) additional genes encoding an enzyme selected from the group consisting of a carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, galactosidase, beta-galactosidase, glucose oxidase, glucosidase, haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase, lipase, lyase, mannosidase, oxidase, pectinolytic enzyme, peroxidase, phytase, phenoloxidase, polyphenoloxidase, ribonuclease, transferase, alpha-1,6-transglucosidase, alpha-1,6-transglucosidase, transglutaminase, and xylanase.
In the methods of the present invention, the Fusarium venenatum mutant strain preferably produces at least the same amount of the polypeptide of interest as the corresponding parent Fusarium venenatum strain when cultured under identical production conditions. In another aspect, the mutant strain produces at least 25% more, preferably at least 50% more, more preferably at least 75% more, and most preferably at least 100% more of the polypeptide than the corresponding parent Fusarium venenatum strain when cultured under identical production conditions.
The Fusarium venenatum mutant strains are cultivated in a nutrient medium for production of the polypeptide of interest using methods known in the art. For example, the strain may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). The secreted polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it may be obtained from cell lysates.
The polypeptide of interest may be detected using methods known in the art that are specific for the polypeptide. These detection methods may include use of specific antibodies, high performance liquid chromatography, capillary chromatography, formation of an enzyme product, disappearance of an enzyme substrate, or SDS-PAGE. For example, an enzyme assay may be used to determine the activity of an enzyme. Procedures for determining enzyme activity are known in the art for many enzymes (see, for example, D. Schomburg and M. Salzmann (eds.), Enzyme Handbook, Springer-Verlag, New York, 1990).
The resulting polypeptide may be isolated by methods known in the art. For example, a polypeptide of interest may be isolated from the cultivation medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. The isolated polypeptide may then be further purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
The polypeptide of interest may be any polypeptide native or foreign (heterologous) to the Fusarium venenatum strain. The polypeptide may be encoded by a single gene or two or more genes. The term “polynucleotide encoding the polypeptide” will be understood to encompass one or more (several) genes involved in the production of the polypeptide. The term “heterologous polypeptide” is defined herein as a polypeptide that is not native to the host strain; a native polypeptide in which structural modifications have been made to alter the native polypeptide, e.g., the protein sequence of a native polypeptide; or a native polypeptide whose expression is quantitatively altered as a result of a manipulation of the polynucleotide or host strain by recombinant DNA techniques, e.g., a stronger promoter. Thus, the present invention also encompasses, within the scope of the term “heterologous polypeptides,” such recombinant production of native polypeptides, to the extent that such expression involves the use of genetic elements not native to the Fusarium venenatum strain, or use of native elements that have been manipulated to function in a manner that do not normally occur in the host strain. In one aspect, the polypeptide is a native polypeptide to the Fusarium venenatum strain. In another aspect, the polypeptide is a heterologous polypeptide to the Fusarium venenatum strain.
The polypeptide may be any polypeptide having a biological activity of interest. The term “polypeptide” is not meant herein to refer to a specific length of the encoded product and, therefore, encompasses peptides, oligopeptides, and proteins. The term “polypeptide” also encompasses two or more polypeptides combined to form the encoded product. Polypeptides also include fusion polypeptides, which comprise a combination of partial or complete polypeptide sequences obtained from at least two different polypeptides wherein one or more (several) may be heterologous to the Fusarium venenatum strain. Polypeptides further include naturally occurring allelic and engineered variations of the above-mentioned polypeptides and hybrid polypeptides.
Preferably, the polypeptide is an antibody, antigen, antimicrobial peptide, enzyme, growth factor, hormone, immunodilator, neurotransmitter, receptor, reporter protein, structural protein, or transcription factor.
In one aspect, the polypeptide is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase. In another aspect, the polypeptide is an aminopeptidase, alpha-amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, beta-galactosidase, glucocerebrosidase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, urokinase, or xylanase.
In another aspect, the polypeptide is an albumin, collagen, tropoelastin, elastin, or gelatin.
In the methods of the present invention, the mutant of the Fusarium venenatum strain is a recombinant strain, comprising a polynucleotide encoding a heterologous polypeptide, which is advantageously used in the recombinant production of the polypeptide. The strain is preferably transformed with a vector comprising the polynucleotide encoding the heterologous polypeptide followed by integration of the vector into the chromosome. “Transformation” means introducing a vector comprising the polynucleotide into a host strain so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector. Integration is generally considered to be an advantage as the polynucleotide is more likely to be stably maintained in the strain. Integration of the vector into the chromosome can occur by homologous recombination, non-homologous recombination, or transposition.
The polynucleotide encoding a heterologous polypeptide may be obtained from any prokaryotic, eukaryotic, or other source, e.g., archaeabacteria. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide is produced by the source or by a strain in which a gene from the source has been inserted.
In the methods of the present invention, a mutant Fusarium venenatum strain of the present invention may also be used for the recombinant production of a polypeptide that is native to the Fusarium venenatum strain. The native polypeptide may be produced by recombinant means by, for example, placing a gene encoding the polypeptide under the control of a different promoter to enhance expression of the substance, expediting its export outside the strain by use of, for example, a signal sequence, or increasing the copy number of a gene encoding the polypeptide normally produced by the Fusarium venenatum strain.
The techniques used to isolate or clone a polynucleotide encoding a polypeptide of interest are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of such a polynucleotide from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR). See, for example, Innis et al., 1990, PCR Protocols: A Guide to Methods and Application, Academic Press, New York. The cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the polynucleotide encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a mutant Fusarium venenatum strain of the present invention where multiple copies or clones of the polynucleotide will be replicated. The polynucleotide may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.
In the methods of the present invention, the polypeptide may also be a fused polypeptide or cleavable fusion polypeptide in which a polypeptide is fused at the N-terminus or the C-terminus of another polypeptide or fragment thereof. A fused polypeptide is produced by fusing a nucleotide sequence (or a portion thereof) encoding a polypeptide to another nucleotide sequence (or a portion thereof) encoding another polypeptide. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator.
An isolated polynucleotide encoding a heterologous polypeptide may be manipulated in a variety of ways to provide for expression of the polypeptide in a mutant Fusarium venenatum strain of the present invention. Manipulation of the polynucleotide's sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotide sequences utilizing recombinant DNA methods are well known in the art.
A nucleic acid construct comprising a polynucleotide encoding a polypeptide may be operably linked to one or more (several) control sequences capable of directing expression of the coding sequence in a mutant Fusarium venenatum strain of the present invention under conditions compatible with the control sequences.
The control sequence may be an appropriate promoter sequence, a nucleotide sequence that is recognized by a mutant Fusarium venenatum strain of the present invention for expression of the polynucleotide encoding the polypeptide. The promoter sequence contains transcriptional control sequences that mediate expression of the polypeptide. The promoter may be any nucleotide sequence that shows transcriptional activity in the mutant Fusarium venenatum strain, including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either native or heterologous (foreign) to the mutant Fusarium venenatum strain.
Examples of suitable promoters for directing the transcription of the nucleic acid constructs in the methods of the present invention are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporum trypsin-like protease (WO 96/00787), Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase); and mutant, truncated, and hybrid promoters thereof.
The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a mutant Fusarium venenatum strain of the present invention to terminate transcription. The terminator sequence is operably linked to the 3′ terminus of the nucleotide sequence encoding the heterologous polypeptide. Any terminator that is functional in a Fusarium venenatum strain may be used in the present invention.
Preferred terminators are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.
The control sequence may also be a suitable leader sequence, a nontranslated region of a mRNA that is important for translation by a mutant Fusarium venenatum strain of the present invention. The leader sequence is operably linked to the 5′ terminus of the nucleotide sequence encoding the heterologous polypeptide. Any leader sequence that is functional in the mutant Fusarium venenatum strain may be used in the present invention.
Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3′ terminus of the nucleotide sequence and, when transcribed, is recognized by the mutant Fusarium venenatum strain as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the mutant Fusarium venenatum strain may be used in the present invention.
Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase.
The control sequence may also be a signal peptide coding sequence that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5′ end of the coding sequence of the nucleotide sequence may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the secreted polypeptide. Alternatively, the 5′ end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. The foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, the foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of the mutant Fusarium venenatum strain, i.e., secreted into a culture medium, may be used in the present invention.
Effective signal peptide coding regions for the mutant Fusarium venenatum strains are the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, Humicola insolens endoglucanase V, and Humicola lanuginosa lipase.
The control sequence may also be a propeptide coding region, which codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to a mature, active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from genes for Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila laccase (WO 95/33836).
Where both signal peptide and propeptide sequences are present at the amino terminus of a polypeptide, the propeptide sequence is positioned next to the amino terminus of a polypeptide and the signal peptide sequence is positioned next to the amino terminus of the propeptide sequence.
The nucleic acid constructs may also comprise one or more (several) polynucleotides that encode one or more (several) factors that are advantageous for directing expression of the heterologous polypeptide, e.g., a transcriptional activator (e.g., a trans-acting factor), a chaperone, and a processing protease. Any factor that is functional in the mutant Fusarium venenatum strain may be used in the present invention. The nucleic acids encoding one or more (several) of these factors are not necessarily in tandem with the nucleotide sequence encoding the heterologous polypeptide.
It may also be desirable to add regulatory or control sequences that allow regulation of expression of the polypeptide relative to the growth of the mutant Fusarium venenatum strain. Examples of regulatory systems are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in filamentous fungi such as the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the nucleotide sequence encoding the polypeptide would be operably linked with the regulatory sequence.
In the methods of the present invention, a recombinant expression vector comprising a nucleotide sequence, a promoter, and transcriptional and translational stop signals may be used for the recombinant production of a polypeptide of interest. The various nucleic acids and control sequences described herein may be joined together to produce a recombinant expression vector that may include one or more (several) convenient restriction sites to allow for insertion or substitution of the nucleotide sequence encoding the polypeptide at such sites. Alternatively, the nucleotide sequence may be expressed by inserting the nucleotide sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the nucleotide sequence. The choice of the vector will typically depend on its compatibility with the mutant Fusarium venenatum strain into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.
The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the mutant Fusarium venenatum strain, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the mutant Fusarium venenatum strain, or a transposon, may be used.
The vector preferably contains one or more (several) selectable markers that permit easy selection of transformed mutant Fusarium venenatum strains. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
Examples of selectable markers for use in the mutant Fusarium venenatum strain include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hpt (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in the mutant Fusarium venenatum strain are the amdS gene of Aspergillus nidulans and the bar gene of Streptomyces hygroscopicus. 
The vectors preferably contain an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
For integration into the genome of the mutant Fusarium venenatum strain, the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the mutant Fusarium venenatum strain at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which have a high degree of identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the mutant Fusarium venenatum strain. Furthermore, the integrational elements may be non-encoding or encoding nucleotide sequences. On the other hand, the vector may be integrated into the genome of the mutant Fusarium venenatum strain by non-homologous recombination.
For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the mutant Fusarium venenatum strain. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” is defined herein as a nucleotide sequence that enables a plasmid or vector to replicate in vivo.
Examples of origins of replication useful in the mutant Fusarium venenatum strain are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.
The procedures used to ligate the elements described herein to construct the recombinant expression vectors are well known to one skilled in the art (see, e.g., J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).
A vector comprising the nucleotide sequence can be introduced, e.g., by transformation, into the mutant Fusarium venenatum strain so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector. Integration is generally considered to be an advantage as the nucleotide sequence is more likely to be stably maintained in the strain. Integration of the vector into the chromosome occurs by homologous recombination, non-homologous recombination, or transposition.
The introduction of an expression vector into the mutant Fusarium venenatum strain may involve a process consisting of protoplast formation, transformation of the protoplasts, and regeneration of the strain wall in a manner known per se. Suitable procedures for transformation of Fusarium venenatum strains are described in Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787.
The present invention also relates to methods of obtaining mutants of a parent Fusarium venenatum strain, comprising: (a) modifying one or more (several) genes selected from the group consisting of pyrG, amyA, and alpA; and (b) identifying a mutant strain from step (a) wherein the one or more (several) genes are modified rendering the mutant strain deficient in the production of one or more (several) enzymes selected from the group consisting of orotidine-5′-monophosphate decarboxylase, alpha-amylase, and alkaline protease, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
In one aspect, the methods of obtaining mutants of a parent Fusarium venenatum strain further comprise modifying one or both of the genes tri5 and dps1 rendering the mutant strain deficient in the production of one or both enzymes trichodiene synthase and cyclohexadepsipeptide synthetase, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
The present invention also relates to mutants of a parent Fusarium venenatum strain, comprising a polynucleotide encoding a polypeptide and one or more (several) genes selected from the group consisting of pyrG, amyA, and alpA, wherein the one or more (several) genes are modified rendering the mutant strain deficient in the production of one or more (several) enzymes selected from the group consisting of orotidine-5′-monophosphate decarboxylase, alpha-amylase, and alkaline protease, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
In one aspect, the mutants of a parent Fusarium venenatum strain further comprise one or both of the genes tri5 and dps1, wherein the one or both genes are modified rendering the mutant strain deficient in the production of one or both enzymes trichodiene synthase and cyclohexadepsipeptide synthetase, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.
EXAMPLES
Materials 
Chemicals used as buffers and substrates were commercial products of at least reagent grade. All primers and oligonucleotides were supplied by MWG Biotech, Inc., High Point, N.C., USA.
Fungal Strain
Fusarium strain A3/5, now reclassified as Fusarium venenatum (Yoder and Christianson, 1998, Fungal Genetics and Biology 23: 62-80; O'Donnell et al., 1998, Fungal Genetics and Biology 23: 57-67), was obtained from Dr. Anthony Trinci, University of Manchester, Manchester, England. Deposits of this strain can be obtained from the American Type Culture Collection, Manassas, Va., USA as Fusarium strain ATCC 20334 or the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 University Street, Peoria, Ill., USA as Fusarium strain NRRL 30747.
Media and Solutions
LB plates were composed per liter of 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, and 15 g of Bacto agar.
NZY top agarose was composed per liter of 5 g of NaCl, 5 g of yeast extract, 10 g of NZ amine, 2 g of MgSO4, and 7 g of agarose.
M400 medium was composed per liter of 50 g of maltodextrin, 2 g of MgSO4.7H2O, 2 g of KH2PO4, 4 g of citric acid, 8 g of yeast extract, 2 g of urea, 0.5 g of CaCl2, and 0.5 ml of AMG trace metals solution, pH 6.0.
AMG trace metals solution were composed per liter of 14.3 g of ZnSO4.7H2O, 2.5 g of CuSO4.5H2O, 0.5 g of NiCl2, 13.8 g of FeSO4, 8.5 g of MnSO4, and 3.0 g of citric acid.
2XYT medium was composed per liter of 16 g of tryptone, 10 g of yeast extract, 5 g of NaCl, and 5 g of Bacto agar.
YP medium was composed per liter of 10 g of yeast extract and 20 g of Bacto peptone.
YPG5% medium was composed per liter of 10 g of yeast extract, 20 g of Bacto peptone, and 50 g of glucose.
RA medium was composed per liter of 50 g of succinic acid, 12.1 g of NaNO3, 1 g of glucose, and 20 ml of 50× Vogels salts solution (No C, No NaNO3).
RA+uridine medium was composed per liter of 50 g of succinic acid, 12.1 g of NaNO3, 1 g of glucose, and 20 ml of 50× Vogels salts solution (No C, No NaNO3). After filter sterilization of the RA medium, filter sterilized uridine was added to a final concentration of 10 mM.
RA+BASTATM medium was composed per liter of 50 g of succinic acid, 12.1 g of NaNO3, 1 g of glucose, and 20 ml of 50× Vogels salts solution (No C, No NaNO3). After filter sterilization of the RA medium, filter-sterilized BASTATM (glufosinate, Hoechst Schering AgrEvo, Frankfurt, Germany) was added to a final concentration of 6 mg/ml using a working stock solution of 250 mg/ml.
50× Vogels salts solution (No C, No NaNO3) was composed of per liter of 250 g of KH2PO4, 10 g of MgSO4.7H2O, 5 g of CaCl22H2O, 2.5 ml of biotin solution, and 5 ml of Vogels trace elements solution.
Biotin stock solution was composed of 5 mg of biotin in 100 ml of 50% ethanol.
Vogels trace elements solution was composed per 100 ml of 5 g of citric acid, 5 g of ZnSO4.7H2O, 1 g of Fe(NH4)2(SO4)2.6H2O, 0.25 g of CuSO4.5H2O, 0.05 g of MnSO4.H2O, 0.05 g of H3BO3, and 0.05 g of Na2MoO4.2H2O.
VNO3RLMT plates were composed per liter of 20 ml of 50× Vogels salts solution (25 mM NaNO3), 273.33 g of sucrose, and 15 g of LMT agarose (Sigma, St. Louis, Mo., USA).
50× Vogels salts solution (25 mM NaNO3) was composed per liter of 125 g of sodium citrate, 250 g of KH2PO4, 106.25 g of NaNO3, 10 g of MgSO4.7H2O, 5 g of CaCl22H2O, 2.5 ml of biotin stock solution, and 5 ml of Vogels trace elements solution.
VNO3RLMT-BASTATM plates were composed per liter of 20 ml of 50× Vogels salts solution (25 mM NaNO3), 273.33 g of sucrose, and 15 g of LMT agarose. After autoclaving and cooling BASTATM was added to a final concentration of 6 mg/ml.
STC was composed of 0.8 M sorbitol, 25 or 50 mM Tris pH 8, and 50 mM CaCl2.
SPTC was composed of 40% PEG 4000, 0.8 M sorbitol, 25 or 50 mM Tris pH 8, and 50 mM CaCl2.
SY50 medium (pH 6.0) was composed per liter of 50 g of sucrose, 2.0 g of MgSO4.7H2O, 10 g of KH2PO4, 2.0 g of K2SO4, 2.0 g of citric acid, 10 g of yeast extract, 2.0 g of urea, 0.5 g of CaCl2.2H2O, and 5 ml of 200×AMG trace metals solution (no nickel).
200×AMG trace metals solution (no nickel) was composed per liter of 3.0 g of citric acid, 14.3 g of ZnSO4.7H2O, 2.5 g of CuSO4.5H2O, 13.8 g of FeSO4.7H2O, and 8.5 g of MnSO4.H2O.
20×SSC was composed of 0.3 M sodium citrate pH 7 and 3 M sodium chloride.
DNA Sequencing
DNA sequencing was conducted with an ABI PRIZM® 3700 DNA Analyzer (Applied Biosystems, Inc., Foster City, Calif., USA).
Example 1
Fusarium venenatum Transformation Procedure
One hundred micrograms of each of the deletion cassettes described in the following examples were digested with either Bst Z171/Bam HI (Example 11) or Not I (Examples 14, 16, 28 and 30). Each digestion reaction was purified by 1% agarose gel electrophoresis in TAE buffer and a DNA band was extracted using a QIAQUICK® Gel Extraction Kit. The resulting purified DNA was concentrated in a 1.5 ml microfuge tube by ethanol precipitation with the addition of 10% reaction volume of 3 M sodium acetate pH 5 followed by 2.5 volumes of ice cold ethanol (94%) and incubation on ice for 20 minutes. The tube was then centrifuged at 15,000×g for 10 minutes in an EPPENDORF® 5424 bench-top centrifuge (Eppendorf, Hamburg, Germany). The supernatant was discarded and the pellet washed with 1 ml of ice cold 70% ethanol and centrifuged at 15,000×g for 5 minutes. The supernatant was discarded and the pellet allowed to air dry. The pellet was then resuspended in 70 μl of 10 mM Tris pH 8 buffer. The concentration of the resulting DNA containing solution was determined using a NANODROP® 1000 spectrophotometer (ThermoFischer Scientific, Waltham, Mass., USA).
Protoplasts of the appropriate recipient strain were generated by the following method. Spores were first obtained by inoculating 500 ml of RA medium (Example 11) or RA medium supplemented with 10 mM uridine (Examples 14, 16, 28, and 30) in a 2.8 L Fernbach flask with 15×1 cm2 agar plugs of a 7-day old culture containing VNO3RLMT medium and incubating the flask for 36 hours at 28° C. with shaking at 150 rpm. The spore culture was filtered through sterile MIRACLOTHTM and the spores captured on a MILLIPORE® STERICUP® 0.2 μm filter unit (Millipore, Bellerica, Mass., USA). The spores were washed with 200 ml of sterile glass distilled water and resuspended in 10 ml of sterile glass distilled water.
One ml of the spore solution was used to inoculate 100 ml of YP medium supplemented with 5% glucose (Example 11) or YP medium supplemented with 5% glucose and 10 mM uridine (Examples 14, 16, 28, and 30). The inoculated medium was incubated for 16 hours at 17° C. with shaking at 150 rpm. Cultures were filtered through MIRACLOTHTM to collect mycelia, which were transferred to a 50 ml polypropylene tube using a sterile spatula. The mycelia were resuspended in 20 ml of protoplasting solution, which contained 5 mg of NOVOZYMETM 234 per ml and 5 mg of GLUCANEXTM (both from Novozymes A/S, Bagsvaerd, Denmark) in 1 M MgSO4 per ml and transferred to 50 ml polypropylene tubes. The tubes were incubated at 29.5° C. with shaking at 90 rpm for one hour after which 30 ml of 1 M sorbitol were added. Then the tubes were centrifuged at 800×g for 10 minutes in a Sorvall RT 6000B swinging-bucket centrifuge (ThermoFischer Scientific, Waltham, Mass., USA). The supernatants were discarded and the protoplast pellets were washed twice with 30 ml of 1 M sorbitol. The tubes were centrifuged at 800×g for 5 minutes and the supernatants discarded. The protoplasts were resuspended in a solution of filter-sterilized 9:1:0.1 (v/v) STC:SPTC:DMSO at a concentration of 5×107 per ml and frozen overnight at −80° C. at controlled rate freezing using a NALGENETM Cryo 1° C. Freezing Container (ThermoFischer Scientific, Waltham, Mass., USA).
Transformation was accomplished by thawing the protoplasts on ice and adding 200 μl of the protoplasts to each of four 14 ml tubes. Five μg of DNA (in less than 10 μl) were added to the first three and no DNA was added to the fourth. Then 750 μl of SPTC were added to each tube and the tubes were inverted gently 6 times. The tubes were incubated at room temperature for 30 minutes and 6 ml of STC were added to each tube. Each transformation was divided into three parts and added to 150 mm diameter plates containing VNO3RLMT medium supplemented with 125 μg of hygromycin per ml (Example 11) or VNO3RLMT medium supplemented with 125 μg of hygromycin per ml and 10 mM uridine (Examples 14, 16, 28, and 30) and incubated at room temperature for 7 days.
Example 2
Southern Analyses
Fungal biomass was produced by inoculating 25 ml of M400 medium (Example 11) or M400 medium supplemented with 10 mM uridine (Examples 14, 16, 28 and 30) with four 1 cm agar plugs from 7 day old transformants generated as described in Examples 1 and 11. The cultures were incubated for 3 days at 28° C. with shaking at 150 rpm. Agar plugs were removed and the cultures were filtered through MIRACLOTHTM. Harvested biomass was frozen with liquid nitrogen and the mycelia were ground using a mortar and pestle.
Genomic DNA was isolated using a DNEASY® Plant Maxi Kit (QIAGEN, Valencia, Calif., USA) according to the manufacturer's instructions except the lytic incubation period at 65° C. was extended to 1.5 hours from 10 minutes.
Two μg of genomic DNA were digested with the indicated restriction endonucleases in a 50 μl reaction volume at 37° C. for 22 hours. The digestions were subjected to 1.0% agarose gel electrophoresis in TAE buffer. The DNA was fragmented in the gel by treating with 0.25 M HCl, denatured with 1.5 M NaCl-0.5 M NaOH, neutralized with 1.5 M NaCl-1 M Tris pH 8, and then transferred in 20×SSC to a NYTRAN® Supercharge nylon membrane using a TURBOBLOTTERTM Kit (both from Whatman, Kent, UK). The DNA was UV cross-linked to the membrane using a UV STRATALINKERTM (Stratagene, La Jolla, Calif., USA) and pre-hybridized for 1 hour at 42° C. in 20 ml of DIG Easy Hyb (Roche Diagnostics Corporation, Indianapolis, Ind., USA).
Probes were generated using a PCR Dig Probe Synthesis Kit (Roche Diagnostics Corporation, Indianapolis, Ind., USA) according to the manufacturer's instructions. The probes were purified by 1.2% agarose gel electrophoresis in TAE buffer and the bands corresponding to the probes were excised and agarose-extracted using a MINELUTE® Gel Extraction Kit (QIAGEN Inc., Valencia, Calif., USA). The probes were boiled for 5 minutes and each added to 10 ml of DIG Easy Hyb to produce the hybridization solution. Hybridization was performed at 42° C. for 15-17 hours. The membranes were then washed under high stringency conditions in 2×SSC plus 0.1% SDS for 5 minutes at room temperature followed by two washes in 0.1×SSC plus 0.1% SDS for 15 minutes each at 65° C. The probe-target hybrids were detected by chemiluminescent assay (Roche Diagnostics, Indianapolis, Ind., USA) according to the manufacturer's instructions.
Example 3
Construction of Plasmid pDM156.2 Harboring the Genomic DNA Fragment Incorporating the Fusarium venenatum Orotidine-5′-Monophosphate Decarboxylase (pyrG) Gene
A probe of a Neurospora crassa orotidine-5′-monophosphate decarboxylase (pyr-4) gene (SEQ ID NO: 1 for the DNA sequence and SEQ ID NO: 2 for the deduced amino acid sequence) was prepared by PCR incorporating digoxigenin-labeled deoxyuridine-triphosphate (dUTP) and the primers described below.









Primer (sense):







(SEQ ID NO: 3)









5′-GTCAGGAAACGCAGCCACAC-3′



 



Primer (anti-sense):







(SEQ ID NO: 4)









5′-AGGCAGCCCTTGGACGACAT-3′






Plasmid pFB6 (Buxton et al, 1983, Molecular and General Genetics 190: 403-405) was digested with Hind III and the digestion purified by 1% agarose gel electrophoresis using TAE buffer. A 1.1 kb pyr-4 fragment was excised and agarose-extracted using a QIAQUICK® Gel Extraction Kit according to the manufacturer's suggested protocols.
The amplification reaction (50 μl) was composed of 1× Taq DNA Polymerase Buffer (New England Biolabs Inc., Ipswich, Mass., USA), 5 μl of PCR DIG Labeling Mix (Boehringer Mannheim, Manheim, Germany), 10 ng of the 1.1 kb Hind III pyr-4 fragment, 10 pmol of the sense primer, 10 pmol of the anti-sense primer, and 1 unit of Taq DNA polymerase New England Biolabs Inc., Ipswich, Mass., USA). The reaction was incubated in a ROBOCYCLER® (Stratagene, La Jolla, Calif., USA) programmed for 1 cycle at 95° C. for 3 minutes followed by 35 cycles each at 95° C. for 30 seconds, 55° C. for 1 minute, and 72° C. for 1 minute. A final extension was performed for 5 minutes at 72° C.
The amplification reaction products were purified by 1% agarose gel electrophoresis using TAE buffer. A digoxigenin (DIG) labeled probe of approximately 0.78 kb was excised from the gel and agarose-extracted using a QIAQUICK® Gel Extraction Kit.
A genomic DNA library of Fusarium venenatum strain A3/5 was generated and cloned into lambda vector EMBL4 as described in WO 99/60137.
The DIG-labeled probe was used to screen the genomic library of Fusarium venenatum A3/5 DNA cloned into lambda vector EMBL4. Lambda phages were plated with E. coli K802 cells (New England Biolabs, Ipswich, Mass., USA) onto LB plates with NZY top agarose. Plaque lifts were made to HYBONDTM N nylon membranes (Amersham Biosciences, Buckinghamshire, UK) using the technique of Sambrook et al. (Molecular Cloning, A Laboratory Manual, Second Edition; J. Sambrook, E. F. Fritsch, and T. Maniatis; Cold Spring Harbor Laboratory Press, 1989). DNA was bound to the membranes by UV cross-linking using a UV STRATALINKERTM. Filters were then hybridized with the 0.78 kb DIG-labeled N. crassa pyr-4 probe. Hybridization and detection of pyrG clones were performed according to the GENIUSTM System User's Guide (Boehringer Hammheim, Manheim, Germany) at 42° C. with a hybridization solution composed of 5×SSC, 35% formamide, 0.1% L-lauroylsarcosine, 0.02% SDS, and 1% blocking reagent (Boehringer Hammheim, Manheim, Germany). The concentration of DIG-labeled probe used was 2.5 ng per ml of the hybridization solution. Hybridizing DNA was immuno-detected with an alkaline-phosphatase-conjugated anti-digoxigenin antibody (Boehringer Hammheim, Manheim, Germany) and visualized with Lumiphos 530, a chemiluminescent substrate (Boehringer Hammheim, Manheim, Germany). DNA preparations were made from putative positive lambda clones using a Lambda Midi Kit (QIAGEN Inc., Valencia, Calif., USA).
Lambda DNA from a clone identified above was digested with Eco RI and subjected to 1% agarose gel electrophoresis in TAE buffer. A 3.9 kb fragment was excised and agarose-extracted using a QIAEX Gel Extraction Kit (QIAGEN Inc., Valencia, Calif.). The fragment was then cloned into the Eco RI site of pUC18 (Viera and Messing, 1987, Methods in Enzymology 153: 3-11) and ONE SHOT® TOP10 competent cells (Invitrogen, Carlsbad, Calif., USA) were transformed with 2 μl of the cloning reaction. Plasmid DNA from eight of the resulting transformants was analyzed by DNA sequencing. One clone with the desired sequence was selected and designated pDM156.2 (FIG. 1). The pyrG fragment harbored the entire coding region plus 1.3 kb of the promoter and 1.5 kb of the terminator.
Example 4
Generation of pEmY21
An E. coli hygromycin phosphotransferase (hpt) gene (SEQ ID NO: 5 for the DNA sequence and SEQ ID NO: 6 for the deduced amino acid sequence) was amplified from plasmid pPHTI (Cummings et al., 1999, Current Genetics 36: 371-382) using the following primers:









Forward primer:







(SEQ ID NO: 7)









5′-GGGttcgaaTTCATTTAAACGGCT-3′



 



Reverse primer:







(SEQ ID NO: 8)









5′-GGGagcgctCAATATTCATCTCTC-3′






The restriction enzyme sites Bst BI (forward primer) and Eco 47III (reverse primer) were engineered into the primers, represented by the underlined sequence, for cloning.

The PCR reaction (to amplify the hpt gene) was composed of 1× ThermoPol reaction buffer, 200 μM dNTPs, 50 pmol of the forward and reverse primers, 100 pg of pPHT1, 1 unit of Vent® DNA polymerase (New England Biolabs Inc., Ipswich, Mass. USA), and sterile distilled water in a total volume of 100 μl. The amplification reaction was performed using a ROBOCYCLER® programmed for 1 cycle at 95° C. for 2 minutes; 25 cycles each at 95° C. for 1 minute, 51° C. for 1 minute, and 72° C. for 2 minutes; and 1 cycle at 72° C. for 7 minutes.
PCR products were separated by 1% agarose gel electrophoresis in TAE buffer. A 1.8 kb fragment was excised from the gel and agarose extracted using a QIAQUICK® Gel Extraction Kit. The gel purified fragment was then cloned into pCR®-BluntII-TOPO® (Invitrogen, Carlsbad, Calif., USA) using a TOPO® Blunt Cloning Kit (Invitrogen, Carlsbad, Calif., USA). The resulting plasmid was designated pEmY10.
The Eco RI site was then removed from the coding sequence of the hpt gene in pEmY10 using a QUIKCHANGE® Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif., USA) according to the manufacturer's instructions using the primers shown below, where the lower case letters represent the non-mutated nucleotides of the target Eco RI site and the underlined case letters represent the mutated nucleotides. The resulting plasmid was designated pBK3.









Forward primer:







(SEQ ID NO: 9)









5′-GGGTACCCCAAGGGCgTattcTGCAGATGGG-3′



 



Reverse primer:







(SEQ ID NO: 10)









5′-CCCATCTGCAgaatAcGCCCTTGGGGTACCC-3′






The resulting hpt gene without the Eco RI site was PCR amplified from pBK3 using forward and reverse primers shown below.










Forward primer:







(SEQ ID NO: 11)









5′-GGggtaccTTCATTTAAACGGCTTCAC-3′



 



Reverse primer:







(SEQ ID NO: 12)









5′-GGggtaccCGACCAGCAGACGGCCC-3′






The underlined portions represent introduced Kpn I sites for cloning.

Portions of the Aspergillus oryzae pyrG gene were used to generate direct repeats and were PCR amplified from pSO2 (WO 98/12300) using the following primers:









Repeat 1:



Forward primer:







(SEQ ID NO: 13)









5′-TCCcccgggTCTCTGGTACTCTTCGATC-3′



 



Reverse primer:







(SEQ ID NO: 14)









5′-GGggtaccCGACCAGCAGACGGCCC-3′



 



Repeat 2:



Forward primer:







(SEQ ID NO: 15)









5′-GGggtaccTCTCTGGTACTCTTCGATC-3′



 



Reverse primer:







(SEQ ID NO: 16)









5′-TCCcccgggCGACCAGCAGACGGCCC-3′






The underlined portions represent introduced restriction sites Sma I (cccggg) or Kpn I (ggtacc) for cloning.

The three fragments (hpt, repeat #1 and repeat #2) were amplified in separate reactions (50 μl each) composed of 1× ThermoPol reaction buffer, 200 μM dNTPs, 0.25 μM each primer, 50 ng of template DNA, and 1 unit of Vent® DNA polymerase. The amplification reaction was performed using a ROBOCYCLER® programmed for 1 cycle at 95° C. for 2 minutes; 30 cycles each at 95° C. for 1 minute, 61° C. for 1 minute, and 72° C. for 2 minutes; and 1 cycle at 72° C. for 7 minutes.
The PCR products were separated by 1.5% agarose gel electrophoresis in TAE buffer. The approximately 2 kb amplified hpt fragment and the approximately 0.2 kb repeat fragments were excised from the gels and agarose-extracted using a MINELUTE® Gel Extraction Kit. The two pyrG repeat fragments were digested with Kpn I, dephosphorylated with calf intestine phosphatase (New England Biolabs Inc., Ipswich, Mass., USA), and treated with a MINELUTE® Reaction Cleanup Kit (QIAGEN Inc., Valencia, Calif., USA) according to the manufacturer's instructions. The fragments harboring repeat #1 and hpt were then ligated together using a QUICK LIGATIONTM Kit (New England Biolabs Inc., Ipswich, Mass., USA) according to the manufacturer's instructions, treated with a MINELUTE® Reaction Cleanup Kit and the resulting ligation cloned into pCR®II Blunt using a TOPO® Blunt Cloning Kit. Sequence analysis confirmed one clone in which repeat #1 and the hpt fragment were ligated together in pCR®II Blunt. This plasmid was designated pEmY18.
In order to clone the second repeat into pEmY18, plasmid pEmy18 was digested with Eco RV and the digestion purified by 1% agarose gel electrophoresis in TAE buffer. A 5.6 kb fragment was excised from the gel and agarose-extracted using a QIAQUICK® Gel Extraction Kit. The 0.2 kb Repeat 2 fragment (described above) and digested pEmY18 were ligated together using a QUICK LIGATIONTM Kit. The ligation mixture was used to transform SOLOPACK® Gold Supercompetent Cells (Stratagene, La Jolla, Calif., USA). Sequence analysis identified a plasmid in which the three components (repeat #1, hpt and repeat #2) were in the desired order and orientation and which lacked PCR errors. The resulting plasmid was designated pEmY20.
To insure that subsequent digestion of pEmY20 with Eco RI would liberate a single fragment, an Eco RI site was removed using a QUIKCHANGE® Site-Directed Mutagenesis Kit according to the manufacturer's instructions and forward and reverse primers shown below. The resulting plasmid was designated pEmY21 (FIG. 2) after sequence verification.









Forward primer:







(SEQ ID NO: 17)









5′-GGGTACCCCAAGGGCQTATTCTGCAGATGGG-3′



 



Reverse primer:







(SEQ ID NO: 18)









5′-CCCATCTGCAGAATACGCCCTTGGGGTACCC-3′






Example 5
Creation of the Fusarium venenatum pyrG Deletion Vector pEmY23
The Fusarium venenatum pyrG coding sequence (2,678 bp) was excised from pDM156.2 (Example 3) by digestion with Eco RV and Stu I restriction endonucleases, and the remaining 4,398 bp vector was gel-purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's directions. The Sma I fragment of pEmY21 was isolated and gel-purified using a QIAQUICK® Gel Extraction Kit and the two gel-purified fragments were ligated together. They were screened for insert orientation, sequenced for the absence of errors, and one of the clones with the correct insert sequence was selected and designated pEmY23 (FIG. 3).
Example 6
Construction of Plasmid pWTY1470-19-07
Plasmid pJRoy40 (U.S. Pat. No. 7,332,341), which harbors 5′ and 3′ flanking sequences of a Fusarium venenatum trichodiene synthase (tri5) gene (SEQ ID NO: 19 for the DNA sequence and SEQ ID NO: 20 for the deduced amino acid sequence), was used as template for amplification of a portion of the 5′ tri5 gene flanking sequence. The PCR reaction contained 200 μM dNTPs, 1× Taq DNA polymerase buffer, 125 μg of pJRoy40 DNA, 50 pmol of each primer shown below, and 1 unit of Taq DNA polymerase in a final volume of 50 μl.







Forward primer:







(SEQ ID NO: 21)







5′-GGGAGATCTTCGTTATCTGTGCC-3′


 


Reverse primer:







(SEQ ID NO: 22)







5′-GGGAGATCTTAGTAGTCGGCATTTGAAAC-3′


(Underlined nucleotides indicate introduced Bgl II


sites).






The amplification reaction was incubated in a ROBOCYCLER® programmed for 1 cycle at 95° C. for 3 minutes; 10 cycles each at 95° C. for 30 seconds, 52° C. for 45 seconds, and 7° C. for 2 minutes; 20 cycles each at 95° C. for 30 seconds, 52° C. for 45 seconds, and 72° C. for 5 minutes; and 1 cycle at 72° C. for 7 minutes.
PCR products were separated by 1.5% agarose gel electrophoresis using TBE buffer. A fragment of approximately 600 bp was excised from the gel and agarose-extracted using a MINELUTE® Gel Extraction Kit. The fragment was inserted into pCR®2.1 (Invitrogen, Carlsbad, Calif., USA) using a TOPO® TA Cloning Kit (Invitrogen, Carlsbad, Calif., USA) and ONE SHOT® TOP10 competent cells (Invitrogen, Carlsbad, Calif., USA) were transformed with 2 μl of the TOPO® TA cloning reaction. Plasmid DNA form eight of the resulting transformants was digested with Eco RI and Bgl II in separate reactions and the inserts for three transformants with the correct restriction digestion patterns were confirmed by DNA sequencing. One clone with the desired sequence was selected and designated pWTY1470-09-05.
A 608 bp Bgl II fragment harboring the tri5 gene 5′ repeat was liberated from pWTY1470-09-05 by digestion with Bgl II, purified by 1.0% agarose gel electrophoresis using TBE buffer, excised from the gel, and agarose extracted using a MINELUTE® Gel Extraction Kit.
Plasmid pJRoy40 was linearized by digestion with Bgl II, after which it was dephosphorylated using shrimp alkaline phosphatase (Roche Diagnostics Corporation, Indianapolis, Ind., USA) according to the manufacturer's instructions, and purified using a QIAQUICK® PCR Purification Kit (QIAGEN Inc., Valencia, Calif., USA). Linearized pJRoy40 and the gel-purified Bgl II fragment were ligated together using T4 DNA ligase (New England Biolabs Inc., Ipswich, Mass., USA) according to the manufacturer's instructions. Transformation of E. coli SURE® chemically competent cells (Stratagene, La Jolla, Calif., USA) was performed according to the manufacturer's directions. One transformant was confirmed by DNA sequencing to contain the desired vector, i.e., harboring the tri5 5′ and 3′ flanking sequences and a repeat of a portion of the 5′ flanking sequence. The resulting plasmid was designated pWTY1470-19-07 (FIG. 4).
Example 7
Construction of Plasmid pWTY1515-02-01
Plasmid pWTY1470-19-07 was subjected to in vitro mutagenesis using a QUIKCHANGE® Site-Directed Mutagenesis Kit according to the manufacturer's instructions and forward and reverse primers shown below.







Forward primer:







(SEQ ID NO: 23)







5′-CAAGTAACAGACGCGACAGCTTGCAAAATCTTCGTTATCTGTG-3′


 


Reverse primer:







(SEQ ID NO: 24)







5′-CACAGATAACGAAGATTTTGCAAGCTGTCGCGTCTGTTACTTG-3′






The mutagenesis removed the Bgl II site at 1779 bp and rendered the Bgl II site at 2386 bp unique and usable in subsequent manipulations to insert fragments harboring thymidine kinase (tk) and hygromycin phosphotransferase (hpt) gene cassettes. The mutagenesis reaction was used to transform the kit-supplied E. coli XL10-GOLD® Ultra-competent cells (Stratagene, La Jolla, Calif., USA) according to the manufacturer's suggested protocol.
One transformant harboring the mutations indicated above, as verified by sequence analysis, was designated pWTY1515-02-01 (FIG. 5) and used as the backbone in Example 10.
Example 8
Construction of Plasmid pJaL574
Plasmid pDV8 (U.S. Pat. No. 6,806,062) harbors the Herpes simplex virus type 1 thymidine kinase (HSV1-TK; tk) gene (SEQ ID NO: 29 for the DNA sequence and SEQ ID NO: 30 for the deduced amino acid sequence) as a 1.2 kb Bgl II/Bam HI fragment inserted between a 1.0 kb Xho I/Bgl II fragment of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpdA) promoter and a 1.8 kb Bam HI/Hind III fragment harboring the tri-functional Aspergillus nidulans indoleglycerolphosphate synthase, phosphoribosylanthranilate isomerase, and glutamine amidotransferase (trpC) transcriptional terminator. Plasmid pDV8 was digested with Bam HI, extracted with phenol-chloroform, ethanol precipitated, and then filled in using Klenow polymerase (Stratagene, La Jolla, Calif., USA). The digested plasmid was re-ligated using a QUICK LIGATIONTM Kit following the manufacturer's protocol, treated with a MINELUTE® Gel Extraction Kit, and the resulting ligation products cloned into pCR®4Blunt-TOPO® (Invitrogen, Carlsbad, Calif., USA) using a TOPO® Blunt Cloning Kit according to the manufacturer's instructions. The cloning reaction was transformed into ONE SHOT® chemically competent TOP10 cells (Invitrogen, Carlsbad, Calif., USA) according to the manufacturer's directions. Plasmid DNA was extracted from eight of the resulting transformants using a BIOROBOT® 9600 (QIAGEN Inc, Valencia, Calif., USA) and screened by restriction digestion using XhoI/Bam HI and Xho I/Hind III. DNA sequencing of plasmid DNA from two transformants with the correct restriction digestion pattern confirmed that both harbored the desired sequence. One was named pJaL504-[Bam HI] (FIG. 6).
Plasmid pJaL504-[Bam HI] was digested with Bgl II, extracted with phenol-chloroform, ethanol precipitated, and then filled in using Klenow polymerase. The digested plasmid was re-ligated using a QUICK LIGATIONTM Kit following the manufacturer's protocol, treated with a MINELUTE® Reaction Cleanup Kit, and the resulting ligation cloned into pCR®4Blunt-TOPO® using a TOPO® Blunt Cloning Kit according to the manufacturer's instructions. The cloning reaction was transformed into ONE SHOT® chemically competent E. coli TOP10 cells according to the manufacturer's directions. Plasmid DNA was extracted from eight of the resulting transformants using a BIOROBOT® 9600 and screened by restriction digestion using Xho I/Bgl II and Xho I/Hind III. DNA sequencing of plasmid DNA from two transformants with the correct restriction digestion pattern confirmed that both harbored the desired sequence. One was named pJaL504-[Bgl II] (FIG. 7). Punt et al. (1990, Gene 3: 101-109) have previously shown that 364 bp of the Aspergillus nidulans gpdA promoter could be deleted without affecting the strength of the promoter. Based on these authors' observations, primer #172450 shown below was designed to truncate the Aspergillus nidulans gpdA promoter and reduce the size of the vector.







Primer 172450:







(SEQ ID NO: 25)







5′-GACGAATTCTCTAGAAGATCTCTCGAGGAGCTCAAGCTTCTGTACA


 


GTGACCGGTGACTC-3′






The underlined sequence corresponds to gpdA promoter sequence. The remaining sequence is a handle harboring the following restriction sites: Eco RI, Xba I, Bgl II, Xho I, and Hind III.

For truncating the Aspergillus nidulans trpC terminator (again to reduce vector size), primer #172499, shown below, was designed harboring an Eco RI handle.









Primer 172499:







(SEQ ID NO: 26)









5′-GACGAATTCCGATGAATGTGTGTCCTG-3′






The underlined sequence corresponds to the trpC terminator sequence. Amplification using primers 172499 and 172450 truncates the promoter by 364 bp and the trpC terminator sequence by 239 bp.
PCR was performed with the above two primers using pJaL504-[Bgl II] as template to generate a 2.522 kb fragment composed of a truncated version of the A. nidulans gpdA promoter, the coding sequence of the HSV1-TK gene, and a truncated version of the A. nidulans trpC terminator.
The amplification reaction consisted of 5 μl of 10× Buffer (Promega Corporation, Madison, Wis., USA), 0.4 μl of 25 mM dNTPs, 1.25 μl of primer 172450 (100 ng/μl), 1.25 μl of primer 172499 (100 ng/μl), 0.5 μl of pJaL504-[Bgl II] (100 ng/μl), 2 μl of Pfu DNA polymerase (Promega Corporation, Madison, Wis., USA) (2.5 U/μl), and 39.6 μl of sterile distilled water. The amplification reaction was incubated in a ROBOCYCLER® programmed for 1 cycle at 95° C. for 45 seconds; and 28 cycles each at 95° C. for 45 seconds, 57° C. for 45 seconds, and 72° C. for 5 minutes. A final extension was performed for 10 minutes at 72° C.
The amplification reaction was subjected to 1% agarose gel electrophoresis using low melting temperature agarose gel in 50 mM Tris-50 mM boric acid-1 mM disodium EDTA (TBE) buffer. A 2522 bp fragment was excised from the gel and extracted using a QIAQUICK® Gel Extraction Kit. The gel-purified DNA was then inserted into pCR®4Blunt-TOPO® using a TOPO® Blunt Cloning Kit according to the manufacturer's instructions. The cloning reaction was transformed into ONE SHOT® chemically competent TOP10 cells according to the manufacturer's directions. Plasmid DNA was extracted from eight of the resulting transformants using a BIOROBOT® 9600 and screened by restriction digestion using Eco RI and Bgl II. DNA sequencing of plasmid DNA from two transformants with the correct restriction digestion pattern confirmed that both harbored the desired sequence. One was designated pJaL574 (FIG. 8).
Example 9
Construction of Plasmid pWTY1449-02-01
Plasmid pJaL574 was transformed into competent E. coli SCS110 cells (Stratagene, La Jolla, Calif., USA) following the manufacturer's recommended protocol. Plasmid DNA was extracted from twenty-four of the resulting transformants, using a BIOROBOT® 9600, and then subjected to analytical digestion using Eco RI and Bgl II. Subsequent DNA sequence analysis resulted in the identification of a clone with the correct sequence, which was designated pWTY1449-02-01 (FIG. 9).
Example 10
Generation of the Tris Deletion Vector pJfyS1579-21-16
An E. coli hygromycin phoshotransferase (hpt) gene cassette was PCR amplified from plasmid pEmY23 using an ADVANTAGE® GC Genomic PCR Kit (Clonetech, Palo Alto, Calif., USA) and gene-specific forward and reverse primers shown below. The underlined portion in the reverse primer is a Bgl II site for cloning.







Forward primer:







(SEQ ID NO: 27)







5′-TTGAACTCTCAGATCCCTTCATTTAAACGGCTTCACGGGC-3′


 


Reverse primer:







(SEQ ID NO: 28)







5′-CAGATAACGAAGATCTACGCCCTTGGGGTACCCAATATTC-3′






The PCR reaction contained 362 ng of pEmY23 as DNA template, 200 μm dNTP's, 1.1 mM magnesium acetate, 0.4 μM primers, 1×GC Reaction Buffer (Clonetech, Palo Alto, Calif., USA), 0.5 M GC Melt (Clonetech, Palo Alto, Calif., USA), and 1×GC Genomic Polymerase Mix (Clonetech, Palo Alto, Calif., USA) in a final volume of 50 μl.
The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® (Eppendorf, Munich, Germany) programmed for 1 cycle at 95° C. for 2 minutes; 25 cycles each at 94° C. for 30 seconds and 66° C. for 3 minutes; and 1 cycle at 66° C. for 3 minutes; and hold at 4° C.
PCR products were separated by 1% agarose gel electrophoresis using TAE buffer. A fragment of approximately 1.9 kb was excised from the gel and agarose extracted using a MINIELUTE® Gel Extraction Kit. The fragment was cloned into pCR®2.1 using a TOPO® TA Cloning Kit according to the manufacturer's instructions. ONE SHOT® TOP10 competent cells (Invitrogen, Carlsbad, Calif., USA) were transformed with 2 μl of the TOPO® TA reaction. Sequence analysis of plasmid DNA from 8 transformants confirmed that there were no deviations from the expected sequence and the plasmid was designated pJfyS1540-75-5 (FIG. 10).
The hpt insert was liberated from pJfyS1540-75-05 by digestion with Bam HI and Bgl II and purified by 1% agarose gel electrophoresis in TAE buffer. A fragment of 1.9 kb was excised and agarose-extracted using a MINIELUTE® Gel Extraction Kit. A Rapid DNA Ligation Kit was used to ligate the fragment to Bgl II-linearized empty tri5 deletion vector pWTY1515-02-01 (Example 7) which had been dephosphorylated using calf intestine phosphatase. E. coli SURE® chemically competent cells were transformed with the ligation reaction and plasmid DNA from 24 of the resulting transformants was analyzed by restriction digestion with Eco RI to confirm the orientation of the insert. One of the transformants harboring the insert in the desired orientation was selected and designated pJfyS1579-1-13 (FIG. 11).
A Herpes simplex virus thymidine kinase (tk) gene (SEQ ID NO: 29 for the DNA sequence and SEQ ID NO: 30 for the deduced amino acid sequence) was PCR amplified using pWTY1449-2-1 as template and gene specific forward and reverse primers shown below. The bold sequence represents the introduced Bgl II site.







Forward primer:







(SEQ ID NO: 31)







5′-GCCGACTACTAGATCGACCGGTGACTCTTTCTGGCATGCG-3′


 


Reverse primer:







(SEQ ID NO: 32)







5′-CAGATAACGAAGATCTGAGAGTTCAAGGAAGAAACAGTGC-3′






The PCR reaction contained 1× HERCULASE® reaction buffer (Stratagene, La Jolla, Calif., USA), 200 μM dNTPs, 55 ng of pWTY1449-2-1, 0.2 μM primers, 2% DMSO, and 2.5 units of HERCULASE® DNA polymerase (Stratagene, La Jolla, Calif., USA) in a final volume of 50 μl.
The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 95° C. for 1 minute; 25 cycles each at 94° C. for 30 seconds, 60° C. for 30 seconds, and 68° C. for 2 minutes and 45 seconds; and 1 cycle at 68° C. for 2 minutes and 45 seconds; and a hold at 4° C.
PCR products were separated by 1% agarose gel electrophoresis using TAE buffer. A fragment of approximately 2.8 kb was excised from the gel and purified using a MINIELUTE® Gel Extraction Kit. The fragment was cloned into pCR®2.1 using a TOPO® TA Cloning Kit. ONE SHOT® TOP10 competent cells (Invitrogen, Carlsbad, Calif., USA) were transformed with 2 μl of the TOPO® TA reaction. Sequence analysis of plasmid DNA from one of the transformants identified a mutation in the tk coding sequence (C1621G) resulting in an amino acid change of glycine to alanine. This mutation was corrected using a QUIKCHANGE® II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif., USA) according to the manufacturer's instructions and forward and reverse primers shown below. The lower case letter indicates the desired change. Sequence analysis of 16 clones resulted in the selection of one which was designated pJfyS1579-8-6 (FIG. 12).









Forward primer:







(SEQ ID NO: 33)









5′-CCCTGTTTCGGGgCCCCGAGTTGCTGG-3′



 



Reverse primer:







(SEQ ID NO: 34)









5′-CCAGCAACTCGGGGcCCCGAAACAGGG-3′






Plasmid pJfyS1579-08-06 was digested with Bam HI and Bgl II to liberate the 2.8 kb tk fragment and the fragment was purified as described above. This fragment was ligated to pJfyS1579-1-13, which had been linearized with Bgl II and treated with calf intestine phosphatase, using a QUICK LIGATIONTM Kit and used to transform E. coli SURE® chemically competent cells according to the manufacturer's protocol. The resulting plasmid was designated pJfyS1579-21-16 (FIG. 13) and used as the tri5 deletion cassette.
Example 11
Construction of the Δtri5 Fusarium venenatum Strain JfyS1604-47-02
Fusarium venenatum A3/5 protoplasts were transformed with Bst Z171/Bam HI-linearized pJfyS1579-21-16 using the method described in Example 1. Transformants were selected on VNO3RLMT plates containing 125 μg of hygromycin B (Roche Diagnostics Corporation, Indianapolis, Ind., USA) per ml. After day 7, 48 out of 123 transformants were sub-cultured to a new plate containing the same medium. Eight transformants were then analyzed by Southern analysis as follows. Fungal biomass of these strains was generated by inoculating 25 ml of M400 medium with four 1 cm agar plugs from 7 day old transformants obtained as described above. The cultures were incubated for 3 days at 28° C. with shaking at 150 rpm. Agar plugs were removed and the cultures were filtered through MIRACLOTHTM. Harvested biomass was frozen with liquid nitrogen and the mycelia were ground using a mortar and pestle.
Genomic DNA was isolated using a DNEASY® Plant Maxi Kit according to the manufacturer's instructions, except the lytic incubation period at 65° C. was extended to 1.5 hours from 10 minutes.
Two μg of genomic DNA were digested with 16 units of Sph I and 22 units of Dra I in a 50 μl reaction volume at 37° C. for 22 hours. The digestion was subjected to 1.0% agarose gel electrophoresis in TAE buffer. The DNA was fragmented in the gel by treating with 0.25 M HCl, denatured with 1.5 M NaCl-0.5 M NaOH, neutralized with 1.5 M NaCl-1 M Tris pH 8, and then transferred in 20×SSC to a NYTRAN® Supercharge nylon membrane using a TURBOBLOTTERTM Kit. The DNA was UV cross-linked to the membrane using a UV STRATALINKERTM and pre-hybridized for 1 hour at 42° C. in 20 ml of DIG Easy Hyb.
A PCR probe to the 3′ flanking sequence of the tri5 gene was generated using the following forward and reverse primers.









Forward primer:







(SEQ ID NO: 35)









5′-GTGGGAGGATCTGATGGATCACCATGGGC-3′′



 



Reverse primer:







(SEQ ID NO: 36)









5′-CCGGGTTTCGTTCCGAACGATCTTTACAAGG-3′






The probe was generated using a PCR Dig Probe Synthesis Kit according to the manufacturer's instructions. The probe was purified by 1.2% agarose gel electrophoresis in TAE buffer and the band corresponding to the probe was excised and agarose-extracted using a MINELUTE® Gel Extraction Kit. The probe was boiled for 5 minutes and added to 10 ml of DIG Easy Hyb to produce the hybridization solution. Hybridization was performed at 42° C. for 15-17 hours. The membrane was then washed under high stringency conditions in 2×SSC plus 0.1% SDS for 5 minutes at room temperature followed by two washes in 0.1×SSC plus 0.1% SDS for 15 minutes each at 65° C. The probe-target hybrids were detected by chemiluminescent assay (Roche Diagnostics, Indianapolis, Ind., USA) according to the manufacturer's instructions.
One transformant, Fusarium venenatum JfyS1579-43-23, harboring the deletion cassette in a single copy in the tri5 locus, as determined by Southern analysis, was sporulated by cutting four 1 cm2 plugs using sterile toothpicks from a 7 day-old plate containing VNO3RLMT medium and transferring them to a 125 ml baffled shake flask containing 25 ml of RA medium. The flask was incubated at 28° C. with shaking at 150 rpm for 48 hours. The spore culture was filtered through sterile MIRACLOTHTM and collected in a 50 ml polypropylene tube. The concentration of spores was determined using a hemocytometer and 105 spores (in one ml) were transferred to a 150 mm plate containing VNO3RLMT medium supplemented with 50 μM 5-fluoro-5′-deoxyuridine (FdU) (Sigma Chemical Co., St. Louis, Mo., USA) and incubated for 4 days at 28° C. Spore isolates were picked using sterile toothpicks and transferred to a new plate containing VNO3RLMT medium supplemented with 10 μM FdU and allowed to grow for 7 days at 24-28° C.
Genomic DNA was extracted from 7 spore isolates and Southern analyses performed as described above to insure the cassette's correct excision from the genome. All spore isolates analyzed by Southern blots had excised the cassette leaving behind one repeat as expected. One spore isolate was spore purified once by inducing sporulation in the strain as described in the preceding paragraph, and the spore concentration was determined using a hemocytometer and diluted to 40 spores per ml. One ml of the diluted spore solution was plated to 150 mm plates containing VNO3RLMT medium and the plates were incubated at 28° C. for 4 days. Spore isolates were sub-cultured to new plates containing VNO3RLMT medium and one spore isolate, designated Fusarium venenatum JfyS1604-17-02 (Δtri5), was used as the starting strain for deletion of the pyrG gene.
Example 12
Construction of a Universal Deletion Vector Harboring the Thymidine Kinase (tk) Negative Selection Marker and Hygromycin Phosphotransferase (hpt) Positive Selection Marker
A universal deletion vector harboring both the thymidine kinase (tk) and hygromycin phosphotransferase (hpt) markers was constructed to facilitate assembly of subsequent deletion plasmids. Flanking sequences for 5′ and 3′ regions of the gene targeted for deletion can be easily ligated to the vector following digestion of the latter with Pme I or Asc I (for 5′ flanking sequences) and Sbf I or Swa I (for 3′ flanking sequences).
In order to PCR-amplify the direct repeats derived from the 5′ flanking region of the Fusarium venenatum pyrG gene, 50 picomoles of the primers shown below were used in two PCR reactions containing 50 ng of pDM156.2, 1× Pfx Amplification Buffer (Invitrogen, Carlsbad, Calif., USA), 6 μl of a 10 mM blend of dNTPs, 2.5 units of PLATINUM® Pfx DNA polymerase (Invitrogen, Carlsbad, Calif., USA), and 1 μl of 50 mM MgSO4 in a total volume of 50 μl.







Primers:


Repeat #1


Sense Primer:







(SEQ ID NO: 37)







5′-GTTTAAACGGCGCGCC CGACAAAACAAGGCTACTGCAGGCAGG-3′


 


Antisense Primer:







(SEQ ID NO: 38)







5′-TTGTCGCCCGGG AATACTCCAACTAGGCCTTG-3′


 


Repeat #2


Sense Primer:







(SEQ ID NO: 39)







5′-AGTATTCCCGGG CGACAAAACAAGGCTACTGCA-3′


 


Antisense Primer:







(SEQ ID NO: 40)







5′-ATTTAAATCCTGCAGG AATACTCCAACTAGGCCTTG-3′






The amplification reactions were incubated in an EPPENDORF® MASTERCYCLER® programmed as follows. For repeat #1:1 cycle at 98° C. for 2 minutes; and 5 cycles each at 94° C. for 30 seconds, 55° C. for 30 seconds, and 68° C. for 1 minute. This was followed by 35 cycles each at 94° C. for 30 seconds, 59° C. for 30 seconds, and 68° C. for 1 minute. For repeat #2 the cycling parameters were: 1 cycle at 98° C. for 2 minutes; and 5 cycles each at 94° C. for 30 seconds, 55° C. for 30 seconds, and 68° C. for 1 minute. This was followed by 35 cycles each at 94° C. for 30 seconds, 56° C. for 30 seconds, and 68° C. for 1 minute. After the 35 cycles both reactions (i.e., repeats #1 and #2) were incubated at 68° C. for 10 minutes and then cooled at 10° C. until being further processed.
PCR products from both reactions were separated by 0.8% GTG-agarose (Cambrex Bioproducts, East Rutherford, N.J., USA) gel electrophoresis using TAE buffer. For repeat #1 and repeat #2, fragments of approximately 0.26 kb were excised from the gel and purified using Ultrafree®-DA spin cups (Millipore, Billerica, Mass., USA) according to the manufacturer's instructions. Ten microliters of each purified repeat were then used in a single overlapping PCR reaction containing 1× Pfx Amplification Buffer, 6 μl of a 10 mM blend of dATP, dTTP, dGTP, and dCTP, 2.5 units of PLATINUM® Pfx DNA polymerase, and 1 μl of 50 mM MgSO4 in a total volume of 50 μl.
The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 98° C. for 2 minutes; and 5 cycles each at 94° C. for 30 seconds, 50° C. for 30 seconds, and 68° C. for 1 minute. The reaction was then mixed with a pre-warmed solution containing 50 picomoles of the sense primer for repeat #1 and 50 picomoles of the anti-sense primer for repeat #2, 1× Pfx Amplification Buffer, 6 μl of a 10 mM dNTPs, 2.5 units of PLATINUM® Pfx DNA polymerase, and 1 μl of 50 mM MgSO4 in a final volume of 50 μl.
The new 100 μl amplification reaction was incubated in an EPPENDORF®MASTERCYCLER® programmed for 35 cycles each at 94° C. for 30 seconds, 58° C. for 30 seconds, and 68° C. for 1 minute. After 35 cycles, the reaction was incubated at 68° C. for 10 minutes and then cooled at 10° C. until being further processed. A 0.5 kb PCR product (harboring the repeat assembly) was isolated by 0.8% GTG-agarose gel electrophoresis as described above.
Plasmid pCR4 (Invitrogen, Carlsbad, Calif., USA) was used as the source of the vector backbone for the construction of the universal deletion vector. To remove the non-essential portions of the pCR4 DNA, 2.5 μg of plasmid pTter61C (WO 2005/074647) were digested sequentially with Bsp LU1 II and Bst XI. The digested vector was then treated with Antarctic phosphatase (New England Biolabs Inc., Ipswich, Mass., USA). The 3.1 kb digested backbone was isolated by 0.8% GTG-agarose gel electrophoresis as described above. The purified repeat assembly was then ligated to the purified vector backbone with a Rapid Ligation Kit (Roche Diagnostics Corporation, Indianapolis, Ind., USA). The ligation reaction consisted of: 75 ng of purified vector backbone and 3 μl of the purified repeat assembly. One microliter of this ligation reaction was used to transform chemically competent SOLOPACK® Supercompetent cells (Stratagene, Carlsbad, Calif., USA) using the manufacturer's suggested protocols. Twenty four transformants were analyzed by Nco I/Pme I restriction digestion. Twenty three out of twenty four transformants had the expected restriction digestion pattern. Clone pFvRs #10 was selected at random for sequencing to confirm that there were no PCR-induced errors. Sequencing analysis showed that the repeat assembly in clone pFvRs #10 had the expected sequence, and this was therefore selected as the backbone of the Fusarium venenatum universal vector and designated pAlLo1492-24 (FIG. 14).
The cassette harboring the hygromycin phosphotransferase (hpt) gene was PCR amplified from pEmY23 using the gene-specific forward and reverse primers shown below. The underlined sequence represents a Xma I site and the bold letters represent a Bgl II site. The four “a”s at each 5′ end allow for subsequent digestion of the terminal ends of the PCR product.









Forward primer:







(SEQ ID NO: 41)









5′-aaaacccgggCCTTCATTTAAACGGCTTCACGGGC-3′



 



Reverse primer:







(SEQ ID NO: 42)









5′-aaaacccgggAGATCTACGCCCTTGGGGTACCCAATATTC-3′






The amplification reaction contained 60 ng of pEmY23, 200 μm dNTPs, 1 mM magnesium acetate, 0.4 μM primers, 1× Pfx Amplification Buffer, 0.5 M GC Melt, and 2.5 units of PLATINUM® Pfx polymerase in a final volume of 50 μl. The reaction was incubated in an EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 95° C. for 2 minutes; 10 cycles each at 94° C. for 30 seconds, 60° C. for 30 seconds, and 68° C. for 1 minute 50 seconds; and 1 cycle at 68° C. for 7 minutes followed by holding at 4° C.
PCR products were separated by 1% agarose gel electrophoresis using TAE buffer. A fragment of approximately 1.8 kb was excised from the gel and agarose-extracted using a MINIELUTE® Gel Extraction Kit. The gel-purified PCR product was subsequently digested with Xma I and run on a 1% agarose gel and gel-purified again as above. A QUICK LIGATIONTM Kit was used to ligate the hpt PCR product to Xma I-linearized pAlLo1492-24, which had been treated with calf intestine phosphatase. The resulting plasmid was designated pJfyS1579-35-2 (FIG. 15) and was used as the recipient for the insertion of the thymidine kinase gene.
The source of the Herpes simplex virus tk cassette was plasmid pJfyS1579-8-6 (Example 10), from which the insert was liberated by digestion with Bam HI and Bgl II. The digestion products were separated by 1% agarose gel electrophoresis using TAE buffer, and a fragment corresponding to the 2.8 kb tk gene insert was excised and agarose-extracted using a MINELUTE® Gel Extraction Kit. A QUICK LIGATIONTM Kit was used to ligate the tk gene cassette to Bgl II-linearized pJfyS1579-35-02, which had been treated with calf intestine phosphatase. The resulting plasmid was designated pJfyS1579-41-11 (FIG. 16) and this was used as the starting point for construction of the pyrG, amyA, alpA, and dps1 deletion vectors.
Example 13
Generation of the pyrG Deletion Vector pJfyS1604-55-13
The 3′ flanking sequence of the Fusarium venenatum A3/5 pyrG gene (SEQ ID NO: 43 for the DNA sequence and SEQ ID NO: 44 for the deduced amino acid sequence) was amplified using an EXPAND® High Fidelity PCR System (Roche Diagnostics Corporation, Indianapolis, Ind., USA) and gene-specific forward and reverse primers shown below. The underlined portion is a Sbf I site introduced for cloning and the italicized portion is a Not I site introduced for later digestion to remove the pCR®2.1 portion of the plasmid before transformation.









Forward primer:







(SEQ ID NO: 45)









5′-aaaaaacctgcaggATCCTGCGCGGACTCTTGATTATTT-3′



 



Reverse primer:







(SEQ ID NO: 46)









5′-aaaaaacctgcagggcggccgcAATTCCATTCCTGTAGCTGAGT



ATA-3′






The amplification reaction contained 125 ng of Fusarium venenatum A3/5 genomic DNA, 200 μm dNTP's, 0.4 μM primers, 1× EXPAND® Buffer (Roche Diagnostics Corporation, Indianapolis, Ind., USA) with 5 mM MgCl2, and 2.5 units of EXPAND® DNA polymerase (Roche Diagnostics Corporation, Indianapolis, Ind., USA) in a final volume of 50 μl.
The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 95° C. for 2 minutes; 10 cycles each at 94° C. for 30 seconds, 54° C. for 30 seconds, and 72° C. for 1 minute; and 20 cycles each at 94° C. for 30 seconds, 54° C. for 30 seconds, and 72° C. for 1 minute and 10 seconds.
PCR products were separated by 1% agarose gel electrophoresis using TAE buffer and a 0.7 kb fragment was excised and agarose extracted using a MINELUTE® Gel Extraction Kit.
The 0.7 kb PCR product was digested with Sbf I and purified by 1% agarose gel electrophoresis using TAE buffer. A fragment of approximately 0.7 kb was excised from the gel and further purified using an Ultrafree®-DA spin cup. The 0.7 kb fragment was ligated to pJfyS1579-41-11 (which had been digested with Sbf I and dephosphorylated using calf intestine phosphatase) using a QUICK LIGATIONTM Kit and the ligation mixture used to transform E. coli SURE® chemically competent cells according to the manufacturer's protocol. The resulting plasmid was designated pJfyS1604-35-13.
The 5′ pyrG flanking sequence from pEmY23 (Example 5) was amplified using an EXPAND® High Fidelity PCR System and gene-specific forward and reverse primers shown below. The underlined portion is a Pme I site introduced for cloning and the italicized portion is a Not I site introduced for later digestion to remove the beta-lactamase gene prior to fungal transformation.









Forward primer:







(SEQ ID NO: 47)









5′-aaaaaagtttaaacgcggccgcCTGTTGCCTTTGGGCCAATCAA



TG-3′



 



Reverse primer:







(SEQ ID NO: 48)









5′-aaaaaagtttaaacCTAGTTGGAGTATTGTTTGTTCTT-3′






The amplification reaction contained 20 ng of pEmY23, 200 μm dNTP's, 0.4 μM primers, 1× EXPAND® Buffer with 15 mM MgCl2, and 2.5 units of EXPAND® DNA polymerase.
The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 95° C. for 2 minutes; 10 cycles each at 94° C. for 30 seconds, 53° C. for 30 seconds, and 72° C. for 40 seconds; and 20 cycles each at 94° C. for 30 seconds, 53° C. for 30 seconds, and 72° C. for 40 seconds plus an additional 10 seconds per subsequent cycle.
The PCR product was purified using a MINELUTE® PCR Purification Kit (QIAGEN Inc., Valencia, Calif., USA). The purified PCR products were digested with Pme I and separated by 1% agarose gel electrophoresis using TAE buffer. A fragment of approximately 0.5 kb was excised from the gel and agarose extracted using a MINELUTE® Gel Extraction Kit. The 0.5 kb fragment was ligated to Pme I digested and calf intestine phosphatase treated pJfyS1604-35-13 using a QUICK LIGATIONTM Kit. The ligation reaction contained 50 ng of vector, 20 ng of insert, 1×QUICK LIGATIONTM Reaction Buffer (New England Biolabs Inc., Ipswich, Mass., USA), and 10 units of Quick T4 DNA Ligase (New England Biolabs Inc., Ipswich, Mass., USA) in a 20 μl reaction volume. The reaction was incubated at room temperature for 5 minutes and 2 μl of the ligation were used to transform E. coli SURE® chemically competent cells according to the manufacturer's Instructions. Sequence analysis was used to identify transformants containing the insert in the desired orientation and to confirm the absence of PCR errors. The resulting plasmid was designated pJfyS1604-55-13 (FIG. 17) and was used as the pyrG gene deletion cassette.
Example 14
Generation of ΔTri5 ΔpyrG Fusarium venenatum Strain JfyS1643-18-2
Fifty-one putative transformants of Fusarium venenatum JfyS1604-17-2 (Δtri5), transformed with Not I-digested and gel-purified pJfyS1604-55-13 according to the procedure described in Example 1, were transferred from transformation plates with sterile toothpicks to new plates containing VNO3RLMT medium supplemented with 125 μg of hygromycin B per ml and 10 mM uridine and grown at 24-28° C. for 7 days. Transformants were then analyzed phenotypically by transferring a plug to each of two VNO3RLMT plates, one with and one without uridine (10 mM). Nine transformants displaying no or poor growth on the plates without uridine were then analyzed by Southern analysis. Genomic DNA from each of the 9 transformants was extracted as described in Example 2 and 2 μg of each were digested with 28 units of Mfe I and 14 units of Dra I. A PCR probe to the 3′ flanking sequence of the pyrG gene was generated as described in Example 11, with the following forward and reverse primers:









Forward primer:







(SEQ ID NO: 49)









5′-GGATCATCATGACAGCGTCCGCAAC-3′



 



Reverse primer:







(SEQ ID NO: 50)









5′-GGCATAGAAATCTGCAGCGCTCTCT-3′






Southern analysis indicated that 2 of the 9 uridine auxotrophs harbored the deletion cassette in a single copy while the remainder had sustained ectopic integrations of the cassette. One transformant, Fusarium venenatum JfyS1604-85-5, was sporulated as described in Example 1 in RA medium with 10 mM uridine, and 105 spores were plated to a 150 mm plate containing VNO3RLMT medium supplemented with 50 μM FdU and 0.1 mM uridine. The spore isolates obtained were sub-cultured to a new plate containing VNO3RLMT medium supplemented with 10 μM FdU and 0.1 mM uridine and analyzed subsequently by Southern analysis to insure correct excision from the genome.
The analyzed strains had all excised the cassette correctly and one strain, Fusarium venenatum JfyS1643-10-3, was sporulated as described in the preceding paragraph. The spore concentration was determined using a hemocytometer and the stock solution diluted to a concentration of 40 spores per ml. One ml was plated to 150 mm plates containing VNO3RLMT medium supplemented with 10 mM uridine. Resulting spore colonies were sub-cultured to a new plate containing VNO3RLMT medium supplemented with 10 mM uridine and one spore isolate, Fusarium venenatum JfyS1643-18-2 (Δtri5 ΔpyrG), was used as the strain for deletion of the Fusarium venenatum alpha-amylase A gene (amyA).
Example 15
Generation of the amyA Deletion Vector pJfyS1604-17-2
In order to obtain upstream and downstream flanking sequence information for complete removal of the Fusarium venenatum amyA gene (SEQ ID NO: 51 for the DNA sequence and SEQ ID NO: 52 for the deduced amino acid sequence), a GENOME WALKERTM Universal Kit (Clonetech, Palo Alto, Calif., USA) was used. Each Fusarium venenatum A3/5 genomic DNA library, generated with the kit, was subjected to two rounds of PCR for the 5′ flanking sequence using a 5′ gene-specific primer and a 5′ nested primer shown below. The 3′ flanking sequence was obtained using a 3′ gene-specific primer and a 3′ nested primer shown below.









5′ gene-specific primer:







(SEQ ID NO: 53)









5′-GAGGAATTGGATTTGGATGTGTGTGGAATA-3′



 



5′ nested primer:







(SEQ ID NO: 54)









5′-GGAGTCTTTGTTCCAATGTGCTCGTTGA-3′



 



3′ gene-specific primer:







(SEQ ID NO: 55)









5′-CTACACTAACGGTGAACCCGAGGTTCT-3′



 



3′ nested primer:







(SEQ ID NO: 56)









5′-GCGGCAAACTAATGGGTGGTCGAGTTT-3′






The primary PCR reactions contained 1× HERCULASE® Reaction Buffer, 2 μl of each genomic DNA library (generated as described in the kit), 200 nM kit-supplied AP1 (adaptor primer 1), 200 nM gene specific primer (above), 200 μM dNTPs, and 2.5 units of HERCULASE® DNA polymerase in a 50 μl reaction volume.
The primary amplifications were performed in an EPPENDORF® MASTERCYCLER® programmed for 7 cycles each at 94° C. for 25 seconds and 72° C. for 3 minutes, and 32 cycles each at 94° C. for 25 seconds and 67° C. for 3 minutes, and one cycle at 67° C. for 7 minutes.
The secondary PCR reaction contained 1× HERCULASE® Reaction Buffer, 1 μl of each primary PCR reaction (above), 200 nM kit-supplied AP2 (adaptor primer 2), 200 nM gene specific nested primer (above), 200 μM dNTPs, and 2.5 units of HERCULASE® DNA polymerase in a 50 μl reaction volume.
The secondary amplifications were performed in an EPPENDORF® MASTERCYCLER® programmed for 5 cycles each at 94° C. for 25 seconds, 72° C. for 3 minutes, and 20 cycles each at 94° C. for 25 seconds and 67° C. for 3 minutes, and one cycle at 67° C. for 7 minutes.
PCR products were separated by 1% agarose gel electrophoresis using TAE buffer. A fragment of approximately 0.7 kb was excised from the gel and purified using a MINIELUTE® Gel Extraction Kit according to the manufacturer's instructions. The PCR product was sequenced directly using the corresponding nested primer described above and the kit-supplied primer 2. The obtained sequence was used to design primers to amplify a 1 kb region of the 5′ flanking sequence and a 0.7 kb region of the 3′ flanking sequence of the amyA gene for insertion into the empty deletion vector pJfyS1579-41-11.
The amyA 3′ flanking sequence was PCR amplified from Fusarium venenatum A3/5 genomic DNA using forward and reverse primers shown below.







Forward primer:







(SEQ ID NO: 57)







5′-AAAAAAcctgcaggTAATGGGTGGTCGAGTTTAAAAGTA-3′


 


Reverse primer:







(SEQ ID NO: 58)







5′-AAAAAAcctgcagggcggccgcTTTAAGCATCATTTTTGACTACG


CAC-3′






The underlined letters represent a Not I site for later beta-lactamase removal, and the italicized letters represent a Sbf I site for vector cloning.

The PCR reaction contained 1× HERCULASE® Reaction Buffer, 120 ng of genomic DNA template, 400 nm primers, 200 μM dNTPs, and 2.5 units of HERCULASE® DNA polymerase.
The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 95° C. for 2 minutes; 10 cycles each at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute; and 20 cycles each at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute and 10 seconds.
PCR products were separated by 1% agarose gel electrophoresis using TAE buffer. A fragment of approximately 0.7 kb was excised from the gel and agarose extracted using a MINIELUTE® Gel Extraction Kit. The PCR fragment was digested with Sbf I to produce sticky ends. This fragment was inserted into Sbf I-linearized, calf intestine phosphatase-treated universal deletion vector pJfyS1579-41-11. The ligation reaction contained 80 ng of vector, 80 ng of insert, 1× Quick Ligation Reaction Buffer, and 10 units of Quick T4 DNA Ligase in a 20 μl reaction volume. A 1.5 μl volume of the ligation reaction was used to transform 100 μl of E. coli SURE® chemically competent cells according to the manufacturer's instructions. Clones were screened for insert orientation using restriction analysis with Eco RI and sequence analysis, which identified a clone devoid of PCR errors. This plasmid was designated pJfyS1579-93-1 (FIG. 18) and used as the recipient for insertion of the 5′ amyA flanking sequence.
The 5′ amyA flanking sequence was PCR amplified using forward and reverse primers shown below. The underlined bases represent a Not I site for bla gene removal and the other lower case letters represent a Pme I site to insure the fragment was blunt for cloning into a blunt vector site.







Forward primer:







(SEQ ID NO: 59)







5′-AAAAAAgtttaaacGCGGCCGCTTGATTATGGGATGACCCCAGAC


AAGTGGT-3′


 


Reverse primer:







(SEQ ID NO: 60)







5′-AAAAAAgtttaaacCCGCACGAGCGTGTTTCCTTTTCATCTCG-3′






The PCR amplification was similar to that described above except for different cycling parameters. The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 95° C. for 2 minutes; 10 cycles each at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute 15 seconds; and 20 cycles each at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute 15 seconds with an additional 10 seconds per subsequent cycle.
PCR products were separated by 1% agarose gel electrophoresis using TAE buffer. A fragment of approximately 1 kb was excised from the gel and agarose-extracted using a MINIELUTE® Gel Extraction Kit. The 1 kb fragment was digested with Pme I to create blunt ends and the insert was cloned into Pme I-digested, calf intestine phosphatase-dephosphorylated pJfyS1579-93-1.
The ligation reaction contained 75 ng of vector, 100 ng of insert, 1× Quick Ligation Reaction Buffer, and 10 units of Quick T4 DNA Ligase in a 20 μl reaction volume. After a 5 minute incubation, 2 μl of the ligation reaction were used to transform 100 μl of E. coli SURE® chemically competent cells according to the manufacturer's instruction. Sequence analysis was used to confirm that the insert was in the correct orientation and the absence of PCR errors. The resulting vector identified was designated pJfyS1604-17-2 (FIG. 19).
Example 16
Generation of Δtri5 ΔpyrG ΔamyA Fusarium venenatum Strain JfyS1643-95-4
Five putative transformants of Fusarium venenatum JfyS1643-18-2 (Δtri5 ΔpyrG), transformed with Not I-digested and gel-purified pJfyS1604-17-2 according to the procedure described in Example 1, were transferred from transformation plates with sterile toothpicks to new plates containing VNO3RLMT medium supplemented with 125 μg of hygromycin B per ml and 10 mM uridine and incubated at 24-28° C. for 7 days. For Southern analysis, 2 μg of genomic DNA were digested with 25 units of Ssp I. A DIG probe to the 5′ flanking sequence of the amyA gene was generated as described in Example 11 with forward and reverse primers shown below.









Forward primer:







(SEQ ID NO: 61)









5′-GGATCATCATGACAGCGTCCGCAAC-3′



 



Reverse primer:







(SEQ ID NO: 62)









5′-GGCATAGAAATCTGCAGCGCTCTCT-3′






Southern analysis was performed as described in Example 2 and the results indicated that two of the five transformants had a replaced coding sequence with a single integration of the deletion cassette. A primary transformant designated Fusarium venenatum JfyS1643-73-2 was sporulated as described in Example 1 and 105 spores were plated to a 150 mm diameter plate containing VNO3RLMT medium supplemented with 50 μM FdU and 0.1 mM uridine. Spore isolates obtained were sub-cultured to a new plate containing VNO3RLMT medium supplemented with 10 μM FdU and 0.1 mM uridine.
Two Fusarium venenatum spore isolates (JfyS1643-83-02 and JfyS1643-83-4) were spore purified once resulting in strains JfyS1643-95-1 and JfyS1643-95-2 (from JfyS1643-83-2) and Jfys1643-95-4 (from JfyS1643-83-4). The original spore isolates picked from the FdU plates, as well as their respective one time spore-purified isolates, were analyzed by Southern analysis to insure correct excision from the genome. All analyzed strains had excised the cassette correctly. Fusarium venenatum JfyS1643-95-4 (Δtri5 ΔpyrG ΔamyA) was used as the strain for deletion of the Fusarium venenatum alkaline protease A gene (alpA).
Example 17
Construction of Plasmid pEJG61
Plasmid pEJG61 (FIG. 20) was constructed as described in U.S. Pat. No. 7,368,271, with the exception that the orientation of the bar cassette was reversed (i.e., nucleotides 5901-5210 encode the amdS promoter, nucleotides 5209-4661 encode the bar coding sequence, and nucleotides 4660-4110 encode the Aspergillus niger glucoamylase (AMG) terminator).
Example 18
Construction of Plasmid pEJG69
The Microdochium nivale lactose oxidase (LOx) gene (SEQ ID NO: 63 for the DNA sequence and SEQ ID NO: 64 for the deduced amino acid sequence) was PCR amplified from pEJG33 (Xu et al., 2001, European Journal of Biochemistry 268: 1136-1142) using forward and reverse primers shown below.










Forward Primer:








(SEQ ID NO: 65)









5′-CCCGCATGCGTTCTGCATTTATCTTG-3′



 



Reverse Primer:







(SEQ ID NO: 66)









5′-GGGTTAATTAATTATTTGACAGGGCG-3′






The underlined portions represent introduced Sph I (forward) or Pac I (reverse) sites for cloning.

The PCR contained 200 μM dNTPs, 1 μM each primer, 50 ng of pEJG33, 1× Pwo buffer (Promega, Madison, Wis., USA), and 1 μl of Pwo Hot Start Polymerase (Promega, Madison, Wis., USA) in a final volume of 50 μl.
The amplification reaction was incubated in a ROBOCYCLER® programmed for 1 cycle at 95° C. for 2 minutes; 10 cycles each at 95° C. for 30 seconds, 55° C. for 45 seconds, and 72° C. for 1 minute; 20 cycles each at 95° C. for 30 seconds, 55° C. for 45 seconds, and 72° C. for 1 minutes with an additional 20 second extension for each subsequent cycle; and 1 cycle at 50° C. for 10 minutes.
PCR products were separated by 1% agarose gel electrophoresis using TAE buffer. A fragment of approximately 1.5 kb was excised from the gel and agarose-extracted using a QIAQUICK® Gel Extraction Kit.
The lactose oxidase gene was re-amplified using the same conditions and purified as described above, except that the polymerase and buffer were replaced with Taq DNA polymerase and Taq DNA Polymerase Buffer, respectively, and the gel-purified PCR product above was used as template. The PCR product was cloned into pCR®2.1 using a TOPO® TA Cloning Kit and sequenced to insure the absence of PCR errors. The resulting error-free plasmid was digested with Sph I, treated with T4 DNA polymerase (New England Biolabs Inc., Ipswich, Mass., USA), purified using a QIAQUICK® Nucleotide Removal Kit (QIAGEN Inc., Valencia, Calif., USA), and digested with Pac I. The fragment was purified by 1% agarose gel electrophoresis in TAE buffer, and a fragment of approximately 1.5 kb was excised from the gel and agarose-extracted using a QIAQUICK® Gel Extraction Kit.
Plasmid pEJG61 was digested with Bsp LU11I, treated with Klenow DNA polymerase (New England Biolabs Inc., Ipswich, Mass., USA) according to the manufacturer's directions, and then digested with Pac I. The digested plasmid was purified by 1% agarose gel electrophoresis in TAE buffer and a 8 kb fragment was excised and agarose-extracted using a QIAQUICK® Gel Extraction Kit.
The LOx coding sequence was ligated to the Bsp LU11I- and Pac I-digested pEJG61 using T4 DNA Ligase according to the manufacturer's directions. Plasmids were screened by sequence analysis to insure the absence of PCR errors and a resulting plasmid was identified and designated pEJG69 (FIG. 21).
Example 19
Construction of Plasmid pEJG65
Plasmid pEJG61 (Example 17) was digested with Bsp LU111, treated with Klenow DNA polymerase, and digested with Pac I. The digested plasmid was isolated by 1% agarose gel electrophoresis in TAE buffer and a 8.1 kb fragment was excised and agarose-extracted using a QIAQUICK® Gel Extraction Kit.
The Candida antarctica lipase A coding sequence (SEQ ID NO: 67 for the DNA sequence and SEQ ID NO: 68 for the deduced amino acid sequence) was PCR amplified from pMT1229 (WO 94/01541) using forward and reverse primers shown below.









Forward primer:







(SEQ ID NO: 69)









5′-GCATGCGAGTGTCCTTGCGC-3′



 



Reverse primer:







(SEQ ID NO: 70)









5′-TTAATTAACTAAGGTGGTGTGATG-3′






The PCR reaction contained 200 μM dNTPs, 1 μM each primer, 20 ng of pMT1229, 1× Pwo buffer (Promega, Madison, Wis., USA), and 1 μl of Pwo Hot Start Polymerase.
The amplification reaction was incubated in a ROBOCYCLER® programmed for 1 cycle at 94° C. for 2 minutes; 10 cycles each at 94° C. for 30 seconds, 55° C. for 45 seconds, and 72° C. for 1 minute; 17 cycles each at 94° C. for 30 seconds, 55° C. for 45 seconds, and 72° C. for 1 minutes with an additional 20 second extension for each subsequent cycle; and 1 cycle at 72° C. for 10 minutes.
PCR products were isolated by 1% agarose gel electrophoresis in TAE buffer and a 1.4 kb fragment was excised and agarose extracted using a QIAQUICK® Gel Extraction Kit. The PCR fragment was cloned into pCR®2.1 using a TOPO® TA Cloning Kit and sequenced to verify the absence of PCR errors.
Due to the presence of an internal Sph I site in the coding sequence of the gene, the Candida antarctica lipase A coding sequence was liberated from pCR®2.1 as two separate fragments by separate digestions. To liberate the first fragment (1 kb), the plasmid was digested with Sph I and treated with T4 DNA polymerase. The polymerase was heat-inactivated for 10 minutes at 75° C. and the plasmid was digested with Nhe I. The second fragment (0.4 kb) was liberated from the plasmid with a Nhe I/Pac I digestion. Both digestions were subjected to 1% agarose gel electrophoresis in TAE buffer and a 1 kb fragment from the Sph I/Nhe I digestion and a 0.4 kb fragment from the Nhe I/Pac I digestion were excised and agarose-extracted using a QIAQUICK® Gel Extraction Kit. The two fragments were ligated to digested pEJG61 using T4 DNA ligase. The ligation reaction contained 1× Ligation Buffer (New England Biolabs Inc., Ipswich, Mass., USA), 100 ng of the 1 kb fragment above, 50 ng of the 0.4 kb fragment, 50 ng of digested pEJG61, and 10 units of T4 DNA ligase. The reaction was incubated at room temperature for 16 hours and used to transform E. coli XL10-GOLD® Ultra-competent cells according to manufacturer's instructions. Transformants were screened by sequence analysis and one clone containing a plasmid with the desired error-free coding sequence was identified and designated pEJG65 (FIG. 22).
Example 20
Construction of Plasmid pMStr19
Plasmid pMStr19 was constructed by cloning a Fusarium oxysporum phospholipase gene from pA2Ph10 (WO 1998/26057) into the Fusarium venenatum expression vector pDM181 (WO 2000/56900). PCR amplification was used to isolate the phospholipase gene on a convenient DNA fragment.
The Fusarium oxysporum phospholipase gene was specifically amplified from pA2Ph10 using standard amplification conditions with Pwo DNA polymerase (Roche Molecular Biochemicals, Basel, Switzerland) and an annealing temperature of 45° C. with the primers shown below.









PLMStr10:







(SEQ ID NO: 71)









5′-TCAGATTTAAATATGCTTCTTCTACCACTCC-3′



        SwaI



 



PLMStr11:







(SEQ ID NO: 72)









5′-AGTCTTAATTAAAGCTAGTGAATGAAAT-3′






The resulting DNA fragment was gel-purified and digested with Swa I. Plasmid pDM181 was also digested with Swa I and dephosphorylated. The DNA fragments were then ligated together to produce plasmid pMStr18.
The phospholipase gene in two individual E. coli transformants of pMStr18, #4, and #17 generated using the ligation mixture, were sequenced using standard primer walking methods. Both had acquired single point mutations at different positions in the gene. The mutations were separated by a Nar I site, which cleaves pMStr18 twice. An error-free phospholipase gene was therefore assembled in the Fusarium expression vector pDM181 by digesting both pMStr18#4 and pMStr18#17 with Nar I, isolating the error-free fragments, and ligating them together to produce pMStr19 (FIG. 23). The phospholipase sequence in pMStr19 was confirmed using standard methods.
Example 21
Construction of Plasmid pEJG49
The Fusarium venenatum expression vector pEJG49 was generated by modification of pSheB1 (WO 2000/56900). The modifications included (a) removal of one Bsp LU11I site within the pSheB1 sequence by site-directed mutagenesis; (b) removal of 850 bp of the Fusarium oxysporum trypsin promoter; (c) introduction of a Bsp LU11I site, by ligation of a linker, to aid in the insertion of the 2 kb Fusarium venenatum glucoamylase promoter; and (d) introduction of a Fusarium oxysporum phospholipase gene.
Removal of the Bsp LU11I site within the pSheB1 sequence was accomplished using a QUIKCHANGE® Site-Directed Mutagenesis Kit according to the manufacturer's instructions with the following pairs of mutagenesis primers:








5′-GCAGGAAAGAACAAGTGAGCAAAAGGC-3′
(SEQ ID NO: 73)


 


5′-GCCTTTTGCTCACTTGTTCTTTCCTGC-3′
(SEQ ID NO: 74)






This created pSheB1 intermediate 1.

Removal of 930 bp of the Fusarium oxysporum trypsin promoter was accomplished by digesting pSheB1 intermediate 1 (6,971 bp) with Stu I and Pac I, subjecting the digest to 1% agarose gel electrophoresis using TBE buffer, excising the 6,040 bp vector fragment, and purifying the excised fragment with a QIAQUICK® Gel Extraction Kit. To introduce a new Bsp LU11I site, a linker was created using the following primers:










5′-dCCTACATGTTTAAT-3′
(SEQ ID NO: 75)



      Bsp Lu11I



 



5′-dTAAACATGTAGG-3′
(SEQ ID NO: 76)






Each primer (2 μg each) was heated at 70° C. for 10 minutes and then cooled to room temperature over an hour. This linker was ligated into the Stu I-Pac 1-digested pSheB1 intermediate 1 vector fragment, creating pSheB1 intermediate 2. Vector pSheB1 intermediate 2 was then digested with Bsp Lu11I and Pac I. The digested vector was purified by 1% agarose gel electrophoresis in TBE buffer, excised from the gel, and agarose-extracted using a QIAQUICK® Gel Extraction Kit.
The Fusarium oxysporum phospholipase gene fragment was also generated by PCR using pMSTR19 as template. The following PCR primers were used to introduce a Sph I site at the 5′ end and a Pac I site at the 3′ end of the gene:








5′-GGGGGCATGCTTCTTCTACCACTCC-3′
(SEQ ID NO: 77)


        Sph I


 


5′-GGGGTTAATTAAGAGCGGGCCTGGTTA-3′
(SEQ ID NO: 78)


        Pac I






The conditions for PCR and purification were performed as above. The phospholipase gene fragment was cloned into pCR®-TOPO® according to the manufacturer's instructions. The pCR®-TOPO® phospholipase clone was then digested with Sph I and treated with T4 DNA polymerase to remove the protruding 3′ termini. The fragment was purified using QIAQUICK® Nucleotide Removal Kit and digested with Pac I. The digestion was purified by 1% agarose gel electrophoresis in TBE buffer and a 1 kb band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit.
Plasmid pSheb1 intermediate 2 (above) was digested with Stu I and Bsp Lu11I and purified using a QIAQUICK® Nucleotide Removal Kit. The fragment was then ligated to a 2 kb Stu I-Bsp Lu11I Fusarium venenatum glucoamylase promoter fragment (WO 2000/056900). This vector, known as pSheb1 intermediate 3, was digested with Bsp Lu11I, treated with Klenow fragment to fill in the 5′ overhang, digested with Pac I, and purified using a QIAQUICK® Nucleotide Removal Kit. The fragment was then ligated to the Sph I, blunt-Pac I Fusarium oxysporum phospholipase fragment (described above). The resulting vector, designated pEJG49 (FIG. 24), harbored the phospholipase reporter gene under the transcriptional control of the Fusarium venenatum glucoamylase promoter.
Example 22
Construction of Plasmid pEmY15
Site-directed mutagenesis was used to remove one of each of the Eco RI and Not I restriction sites from expression plasmid pEJG49 and render these restriction sites flanking the bialaphos resistance marker (bar gene) unique. The mutagenesis was completed using forward and reverse primers shown below and a QUIKCHANGE® Site-Directed Mutagenesis Kit.









Forward primer:







(SEQ ID NO: 79)









5′-cctgcatggccgcCgccgcCaattcttacaaaccttcaaca



gtgg-3′



 



Reverse primer:







(SEQ ID NO: 80)









5′-ccactgttgaaggtttgtaagaattGgcggcGgcggccatgc



agg-3′






The uppercase letters indicate the desired changes and the resulting plasmid was designated pEmY15 (FIG. 25).

Example 23
Construction of Plasmid pEmY24
In order to replace the bar gene in expression plasmid pEmY15 with the Fusarium venenatum pyrG gene, the following protocol was performed. Plasmid pEmY15 was digested with Eco RI and Not I and purified by 1% agarose gel electrophoresis in TAE buffer. A 7.1 kb fragment was excised and agarose extracted using a QIAQUICK® Gel Extraction Kit.
A 2.3 kb fragment of the pyrG gene was PCR amplified from pDM156.2 using forward and reverse primers shown below.









Forward primer:







(SEQ ID NO: 81)









5′-ATAAGAATgcggccgcTCCAAGGAATAGAATCACT-3′



 



Reverse primer:







(SEQ ID NO: 82)









5′-CGgaattcTGTCGTCGAATACTAAC-3′






The bold sequence corresponds to an introduced Not I site and Eco RI site for the forward and reverse primers, respectively.

The amplification reaction was composed of 1× ThermoPol Buffer (New England Biolabs, Ipswich, Mass., USA), 200 μM dNTPs, 31 ng of pDM156.2, 1 μM each primer, and 1 unit of VENT® DNA polymerase in a final volume of 50 μl.
The reaction was incubated in an EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 95° C. for 3 minutes; 30 cycles each at 95° C. for 30 seconds, 55° C. for 1 minute; and 72° C. for 3 minutes; and 1 cycle at 72° C. for 7 minutes.
PCR products were isolated by 1% agarose gel electrophoresis in TAE buffer and a 2.3 kb fragment was excised and agarose-extracted using a MINELUTE® Gel Extraction Kit. The fragment was then digested with Eco RI and Not I and the digestion reaction purified using a MINELUTE® Reaction Cleanup Kit. The fragment was ligated to Not I/Eco RI-digested pEmY15 using T4 DNA ligase according to the manufacturer's instructions. The ligation mixture was transformed into E. coli XL1-Blue sub-cloning-grade competent cells (Stratagene, La Jolla, Calif., USA) according to the manufacturer's instructions. Transformants were sequenced to insure the absence of PCR errors and a plasmid was identified containing an error-free pyrG fragment. The resulting plasmid was designated pEmY24 (FIG. 26).
Example 24
Construction of Plasmid pDM257
Plasmid pEmY24 (Example 23) was digested with Afl II and Sna BI. A 6.5 kb fragment was purified by 1% agarose gel electrophoresis in TAE buffer, excised from the gel, and agarose-extracted using a QIAQUICK® Gel Extraction Kit. Plasmid pEJG65 was digested with Afl II and Sna BI. A 3.3 kb fragment was purified by 1% agarose gel electrophoresis in TAE buffer, excised from the gel, and agarose-extracted using a QIAQUICK® Gel Extraction Kit.
The two fragments were ligated together using T4 DNA ligase according to the manufacturer's instructions. The ligation mixture was transformed into E. coli XL1-Blue sub-cloning-grade competent cells according to the manufacturer's instructions. Transformants were screened by sequence analysis and a clone was identified containing a plasmid with the desired fragments. The resulting plasmid was designated pDM257 (FIG. 27).
Example 25
Construction of Plasmid pDM258 
Plasmid pDM257 was digested with Sca I and Afl II and purified by 1% agarose gel electrophoresis in TAE buffer and a 4.1 kb fragment was excised from the gel and agarose-extracted using a QIAQUICK® Gel Extraction Kit. Plasmid pEJG69 was also digested with Sca I and Afl II and purified by 1% agarose gel electrophoresis in TAE buffer and a 5.8 kb fragment was excised from the gel and agarose-extracted as above.
The two fragments were ligated together using T4 DNA ligase according to the manufacturer's instructions. The ligation mixture was transformed into E. coli XL1-Blue sub-cloning-grade competent cells according to the manufacturer's instructions. Transformants were screened by sequence analysis and the desired plasmid was identified and designated pDM258 (FIG. 28).
Example 26
Expression of Lactose Oxidase in Fusarium venenatum Strain JfyS1643-95-4
Protoplasts of Fusarium venenatum JfyS1643-95-04 (Δtri5 ΔpyrG ΔamyA) were generated as described in Example 1. The protoplasts were then transformed according to the procedure described in Example 1 with pDM258, harboring the Microdochium nivale lactose oxidase expression vector, to evaluate the expression potential of the Fusarium venenatum JfyS1643-95-04 strain. Transformants were grown in shake flasks as described in Example 21 except that the flasks were incubated for five days at 28° C. with shaking at 200 rpm.
The shake flask broths were assayed for lactose oxidase activity using an activity assay in conjunction with a BIOMEK® 3000, (Beckman Coulter, Inc, Fullerton, Calif., USA). The lactose oxidase assay was a modified version of the Glucose Oxidase Assay Procedure (K-Glox) (Megazyme, Wicklow, Ireland). Culture supernatants were diluted appropriately in 0.1 M MOPS buffer pH 7.0 (sample buffer) followed by a series dilution from O-fold to 1/3-fold to 1/9-fold of the diluted sample. A lactose oxidase standard (Novozymes A/S, Bagsvaerd, Denmark) was diluted using 2-fold steps starting with a 0.056 mg/ml concentration and ending with a 0.007 mg/ml concentration in the sample buffer. A total of 20 μl of each dilution including standard was transferred to a 96-well flat bottom plate. One hundred microliters of a POD solution (Peroxidase, 4AA, stabilizers in potassium phosphate buffer pH 7 plus p-hydroxybenzoic acid and sodium azide) were added to each well followed by addition of 100 μl of glucose substrate (0.5 M glucose in sample buffer). The rate of reaction was measured at ambient temperature (approximately 26° C.) at 510 nm for a total of 10 minutes. Sample concentrations were determined by extrapolation from a standard curve generated using lactose oxidase as a standard. The highest producing lactose oxidase transformants were selected for growth and analysis in 2 liter fermenters.
The fermentation medium (pH 6) was composed per liter of 20 g of soya flour, 20 g of sucrose, 2.0 g of MgSO4.7H2O, 2.0 g of anhydrous KH2PO4, 2.0 g of K2SO4, 5.0 g of (NH4)2SO4, 1.0 g of citric acid, 0.5 ml of 200×AMG trace metals solution (no nickel), and 0.5 ml of pluronic acid with a 20% maltose feed. The fermentations were run at 29.0+/−1.0° C., 1200 rpm, and 1.0 vvm aeration where % DO was maintained above 30%.
Fermentation broths were assayed for alpha-amylase activity using an Alpha-Amylase Assay Kit (Megazyme International Ireland Ltd., Wicklow, Ireland) in conjunction with a BIOMEK® 3000 and BIOMEK® NX (Beckman Coulter, Inc, Fullerton Calif., USA). Fermentation broths were assayed for lactose oxidase activity as described above.
The resulting top transformant, Fusarium venenatum JfyS1643-95-04, had equivalent lactose oxidase production levels to other Fusarium venenatum transformants without the deletions in 2 liter fermenters (FIG. 29) indicating that deletion of the amyA gene did not have a negative impact on heterologous protein production. The deletion did, however, abolish alpha-amylase activity in the culture broth of this strain and all later strains in this lineage (FIG. 30). Since this transformant had equivalent heterologous protein production capacity to the current production strain, and reduced alpha-amylase levels during fermentation, Fusarium venenatum JfyS1643-95-04 host strain was selected for deletion of an alkaline protease A gene (alpA).
Example 27
Generation of the Fusarium venenatum Alkaline Protease A (alpA) Deletion Vector pJfyS1698-72-10
Upstream flanking sequence for use in the complete removal of the Fusarium venenatum A3/5 alkaline protease A (alpA) gene (SEQ ID NO: 83 for the DNA sequence and SEQ ID NO: 84 for the deduced amino acid sequence) was obtained using a GENOME WALKERTM Universal Kit. Each library generated with the kit was subjected to two rounds of PCR for the 5′ flanking sequence using a 5′ gene-specific primer and a 5′ nested primer shown below.









5′ gene-specific primer:







(SEQ ID NO: 85)









5′-GAGGAATTGGATTTGGATGTGTGTGGAATA-3′



 



5′ nested primer:







(SEQ ID NO: 86)









5′-GGAGTCTTTGTTCCAATGTGCTCGTTGA-3′






Sequence information was obtained from the PCR product using a Nested Adaptor Primer supplied with the GENOME WALKERTM Universal Kit and the 5′ nested primer above. The obtained sequence was used to design primers to amplify a 1 kb region of the 5′ alpA flanking sequence for insertion into the empty deletion vector pJfyS1579-41-11
The alpA 5′ flanking sequence was PCR amplified from Fusarium venenatum A3/5 genomic DNA using region-specific forward and reverse primers shown below. The underlined letters represent a Not I site, for later removal of the pCR®2.1 portion of the vector, and the italicized letters represent an Asc I site for vector cloning.







Forward primer:







(SEQ ID NO: 87)







5′-aaaaaaggcgcgccgcggccgcGTTACGGTGTTCAAGTACATCT


TACA-3′


 


Reverse primer:







(SEQ ID NO: 88)







5′-aaaaaaggcgcgccATTGCTATCATCAACTGCCTTTCTT-3′






The amplification reaction contained 1× HERCULASE® Reaction Buffer, 120 ng of genomic DNA, 400 nm primers, 200 μM dNTPs, and 2.5 units of HERCULASE® DNA polymerase.
The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 95° C. for 2 minutes; 20 cycles each at 94° C. for 30 seconds, 56° C. for 30 seconds, and 72° C. for 1 minute 10 seconds; and 1 cycle at 72° C. for 7 minutes.
A 5 μl portion of the amplified reaction was visualized by 1% agarose gel electrophoresis using TAE buffer to insure the reaction had produced the desired 1 kb band. The insert was then directly cloned into pCR®2.1 from the amplification reaction using a TOPO® TA Cloning Kit according to the manufacturer's instructions. Transformants were screened by restriction analysis with Eco RI to insure the presence of the insert and 5 correct preparations were combined. The insert was liberated from pCR®2.1 by digestion with Asc I and the fragment was purified by agarose gel electrophoresis as described above. The insert was cloned into Asc I-linearized pJfyS1579-41-11 using a QUICK LIGATIONTM Kit and the ligation mixture used to transform E. coli SURE® chemically competent cells according to the manufacturer's protocol. Transformants were screened by sequence analysis to insure the absence of PCR errors. One plasmid containing the flanking sequence without errors was designated pJfyS1698-65-15 (FIG. 31) and used to insert the 3′ flanking sequence.
The 3′ flanking sequence of the alpA gene was amplified from Fusarium venenatum A3/5 genomic DNA using region specific forward and reverse primers shown below. The underlined letters represent a Not I site, for later beta-lactamase removal, and the italicized letters represent a Sbf I site for vector cloning.







Forward primer:







(SEQ ID NO: 89)







5′-aaaaacctgcaggGGATGTGTGTGGAATAGGATATG-3′


 


Reverse primer:







(SEQ ID NO: 90)







5′-aaaaacctgcagggcggccgcCCTCAAGGTGGAGAAATAATCT


GT-3′






The PCR reaction contained 1× HERCULASE® Reaction Buffer, 120 ng of genomic DNA template, 400 nm primers, 200 μM dNTPs, and 2.5 units of HERCULASE® DNA polymerase.
The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 95° C. for 2 minutes; 20 cycles each at 94° C. for 30 seconds, 56° C. for 30 seconds, and 72° C. for 1 minute 10 seconds; and 1 cycle at 72° C. for 7 minutes.
A 5 μl portion of the amplified reaction was visualized on a 1% agarose gel in TAE buffer to insure the reaction had produced the desired 1 kb band. The 1 kb insert, directly from the PCR reaction, was then cloned into pCR®2.1 using a TOPO® TA Cloning Kit. The resulting plasmid was sequenced to identify a colony containing the correct sequence. The fragment was then liberated from this plasmid by Sbf I digestion and purified by 1% agarose gel electrophoresis in TAE buffer. A 1 kb band was excised and agarose-extracted using a MINELUTE® Gel Extraction Kit.
This fragment was then ligated to Sbf I linearized pJfyS1698-65-15 (treated with calf intestine phosphatase) using a QUICK LIGATIONTM Kit and the ligation mixture was used to transform E. coli SURE® chemically competent cells according to the manufacturer's instructions. Transformants were screened by restriction analysis with Not I to insure the fragment had been inserted in the correct orientation and sequenced to insure no deviations from the expected sequence. The resulting plasmid pJfyS1698-72-10 (FIG. 32) was used for deletion of the alpA gene.
Example 28
Generation of Δtri5 ΔpyrG ΔamyA ΔalpA Fusarium venenatum Strain JfyS1763-11-1
Three transformants of Fusarium venenatum JfyS1643-95-4 (Δtri5 ΔpyrG ΔamyA) (Example 16) transformed with Not I-digested and gel-purified pJfyS1698-72-10 according to the procedure described in Example 1 were transferred from transformation plates with sterile toothpicks to new plates containing VNO3RLMT medium supplemented with 125 μg of hygromycin B per ml and 10 mM uridine and incubated at room temperature for 7 days. For Southern analysis, 2 μg of Fusarium venenatum genomic DNA from each of the 3 transformants were digested with 34 units of Sph I. A DIG probe to the 5′ flanking sequence of the alpA gene was generated according to the method described in Example 11 using the forward and reverse primers shown below.









Forward primer:







(SEQ ID NO: 91)









5′-GCACGTTAGGCTCAAGCCAGCAAGG-3′



 



Reverse primer:







(SEQ ID NO: 92)









5′-GAGGCTCATGGATGTGGCGTTAATG-3′






Southern analysis performed as described in Example 11 indicated that one of the three transformants contained a single copy of the deletion cassette at the alpA gene locus and this transformant was designated Fusarium venenatum JfyS1698-83-2.
Fusarium venenatum JfyS1698-83-2 was sporulated as described in Example 1 and 105 spores were plated onto a 150 mm diameter plate containing VNO3RLMT medium supplemented with 50 μM FdU and 0.1 mM uridine. Spore isolates obtained were sub-cultured to a new plate containing VNO3RLMT medium supplemented with 10 μM FdU and 0.1 mM uridine. The resulting spore isolates were analyzed by Southern analysis as described in Example 2 and one spore isolate was identified that had correctly excised the cassette. The isolate was designated Fusarium venenatum JfyS1698-94-04. Fusarium venenatum JfyS1698-94-04 was spore-purified once as described in Example 11 and one spore isolate was picked and designated Fusarium venenatum JfyS1763-11-01 (Δtri5 ΔpyrG ΔamyA ΔalpA).
Protoplasts of Fusarium venenatum JfyS1763-11-01 were generated and transformed as described in Example 1 with pDM258. Transformants were analyzed as described in Example 26 and shake flask broths were assayed for alkaline protease activity. A PROTAZYME® AK tablet (Megazyme, Wicklow, Ireland) was suspended in 2.0 ml of 0.01% TRITON® X-100 by gentle stirring. Five hundred microliters of this suspension and 500 μl of assay buffer supplied with the PROTAZYME® AK tablet were mixed in an EPPENDORF® tube and placed on ice. Twenty microliters of protease sample (diluted in 0.01% TRITON® X-100) were added. The assay was initiated by transferring the EPPENDORF® tube to an EPPENDORF® thermomixer, which was set to the assay temperature. The tube was incubated for 15 minutes on the EPPENDORF® thermomixer at 1300 rpm. The incubation was stopped by transferring the tube back to an ice bath. Then the tube was centrifuged at 16,000×g in an ice cold centrifuge for a few minutes and 200 μl of supernatant was transferred to a microtiter plate. The absorbance at 650 nm was read as a measure of protease activity.
As with the amyA deletion, deletion of the alpA gene did not have a positive impact on lactose oxidase expression. However, the alkaline protease side activity in the fermentation supernatants was reduced 10-fold (FIG. 33).
Example 29
Generation of the Dps1 Deletion Vector pJfyS111
The 3′ flanking sequence for the Fusarium venenatum depsipeptide synthase (dps1) gene (SEQ ID NO: 93 for the DNA sequence and SEQ ID NO 94 for the deduced amino acid sequence) was PCR amplified from Fusarium venenatum JfyS1763-11-01 genomic DNA using the forward and reverse primers shown below. The underlined portion in the primer represents the introduced Sbf I site for cloning and the italicized portion corresponds to an introduced Not I site for later beta-lactamase removal.







Forward primer:







(SEQ ID NO: 95)







5′-GACTAAGCCCTGCAGGTTGGTCTCAATCGTCGCGACAG-3′


 


Reverse primer:







(SEQ ID NO: 96)







5′-AGTCTACCCCTGCAGGCGGCCGCTGGCATCGGTGGACGTAACA


CGC-3′






The amplification reaction contained 1× HERCULASE® Reaction Buffer, 400 nM each primer, 200 μM dNTPs, 100 ng of genomic DNA, and 1.5 units of HERCULASE® DNA polymerase in a final volume of 50 μl. The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 95° C. for 2 minutes; 25 cycles each at 95° C. for 30 seconds, 57° C. for 30 seconds, and 72° C. for 1 minute and 20 seconds; and 1 cycle at 72° C. for 7 minutes.
The amplification reaction was purified using a MINELUTE® PCR Purification Kit. The purified reaction was then digested with Sbf I and submitted to 1% agarose gel electrophoresis using TAE buffer. A 1 kb band was excised from the gel and agarose-extracted using a MINELUTE® Gel Extraction Kit. The digested vector was then ligated to Sbf I-digested pJfyS1579-41-11 (Example 12) (which had been dephosphorylated with calf intestine phosphatase) using a QUICK LIGATIONTM Kit according to the manufacturer's suggested protocols. Resulting clones were analyzed by restriction analysis with Eco RI (to check for insert presence and orientation) and sequence analysis (to insure the absence of PCR errors), and the resulting plasmid was designated pJfyS1879-32-2 (FIG. 34).
In order to obtain flanking sequence on the 5′ end of the dps1 gene, a GENOME WALKERTM Universal Kit was used as described in Example 15 with gene-specific and gene-specific nested primers shown below.









Gene-Specific primer:







(SEQ ID NO: 97)









5′-GCTATTGAGGGGACTATCTCCATGACTACA-3′



 



Gene-Specific nested primer:







(SEQ ID NO: 98)









5′-GCCTACCATCGACAGCAGTAAGATATTCC-3′






The 5′ dps1 flanking sequence was amplified from Fusarium venenatum JfyS1763-11-1 genomic DNA using forward and reverse primers indicated below. The underlined portion in the forward primer represents an introduced Asc I site for cloning and the italicized portion corresponds to an introduced Not I site for later beta-lactamase removal. The amplification reaction and cycling parameters were identical to those described above except the primers used were those below, the annealing temperature used was 53° C., and the extension time was 1 minute and 15 seconds.







Forward primer:







(SEQ ID NO: 99)







5′-ATGTGCTACAGGCGCGCCGCGGCCGCGAGTTCCAACATGTC


TTATTATCC-3′


 


Reverse primer:







(SEQ ID NO: 100)







5′-TACTGTACCGGCGCGCCATCTGAGCCAAGAGACTCATTCAT-3′






The PCR reaction was purified using a MINELUTE® PCR Purification Kit. The purified reaction was digested with Asc I, and subjected to 1% agarose gel electrophoresis using TAE buffer. A 0.7 kb band was excised from the gel and agarose-extracted as described above. The 0.7 kb band was ligated to pJfyS1879-32-2 (digested with Asc I and dephosphorylated with calf intestine phosphatase) using a QUICK LIGATIONTM Kit. Resulting clones were analyzed by sequence analysis to insure the absence of PCR errors, and the resulting plasmid was designated pJfyS111 (FIG. 35) and used to delete the Fusarium venenatum dps1 gene.
Example 30
Generation of Δtri5 ΔpyrG ΔamyA ΔalpA Δdps1 Fusarium venenatum Strain JfyS1879-57-01
When Fusarium venenatum JfyS1763-11-01 protoplasts were transformed with Not I-digested and gel-purified pJfyS111 (according to the procedure described in Example 1), 77 transformants were obtained. Of those 48 were transferred from transformation plates with sterile toothpicks to new plates containing VNO3RLMT medium supplemented with 125 μg of hygromycin B per ml and 10 mM uridine and incubated at room temperature for 7 days.
Fungal biomass was produced by inoculating 25 ml of M400 medium supplemented with 10 mM uridine with four 1 cm agar plugs from 7 day old transformants generated as described in Example 1. The cultures were incubated for 3 days at 28° C. with shaking at 150 rpm. Agar plugs were removed and the cultures were filtered through MIRACLOTHTM. Harvested biomass was frozen with liquid nitrogen and the mycelia were ground using a mortar and pestle.
Genomic DNA was isolated using a DNEASY® Plant Maxi Kit according to the manufacturer's instructions, except the lytic incubation period at 65° C. was extended to 1.5 hours from 10 minutes.
Two μg of genomic DNA were digested with 28 units each of Nco I and Spe I in a 50 μl reaction volume at 37° C. for 22 hours. The digestion was subjected to 1.0% agarose gel electrophoresis in TAE buffer. The DNA was fragmented in the gel by treating with 0.25 M HCl, denatured with 1.5 M NaCl-0.5 M NaOH, neutralized with 1.5 M NaCl-1 M Tris pH 8, and then transferred in 20×SSC to a NYTRAN® Supercharge nylon membrane using a TURBOBLOTTERTM Kit. The DNA was UV cross-linked to the membrane using a UV STRATALINKERTM and pre-hybridized for 1 hour at 42° C. in 20 ml of DIG Easy Hyb.
A DIG probe to the 3′ flanking sequence of the dps1 gene was generated according to the method described in Example 11 using the forward and reverse primers shown below.









Forward primer:







(SEQ ID NO: 101)









5′-CTTGACTATTATCTCACGTTGTCAG-3′



 







Reverse primer:









(SEQ ID NO: 102)



5′-TCAAGTGTTGTGTAATGTTGGAACA-3′






Southern analysis performed as described in Example 11 indicated that three of the 8 transformants contained the deletion fragment in a single copy at the dps1 locus. One was named Fusarium venenatum JfyS1879-43-05.
Southern analysis indicated that three of the 8 transformants contained the deletion fragment in a single copy at the dps1 locus. One of these was named Fusarium venenatum JfyS1879-43-5.
Fusarium venenatum JfyS1879-43-5 was sporulated as described in Example 1 and 105 spores were plated onto a 150 mm diameter plate containing VNO3RLMT medium supplemented with 50 μM FdU and 0.1 mM uridine. Spore isolates obtained were sub-cultured to new plates containing VNO3RLMT medium supplemented with 50 μM FdU and 0.1 mM uridine. The resulting spore isolates were analyzed by Southern analysis as described in Example 2 and one spore isolate was identified that had correctly excised the cassette. The isolate was designated Fusarium venenatum JfyS1879-52-3. Fusarium venenatum JfyS1879-52-3 was spore purified once as described in Example 11 and one spore isolate was picked and designated Fusarium venenatum JfyS1879-57-1 (Δtri5 ΔpyrG ΔamyA ΔalpA Δdps1).
The present invention is further described by the following numbered paragraphs:
[1] A method of producing a polypeptide, comprising:
(a) cultivating a mutant of a parent Fusarium venenatum strain in a medium for the production of the polypeptide, wherein the mutant strain comprises a polynucleotide encoding the polypeptide and one or more (several) genes selected from the group consisting of pyrG, amyA, and alpA, wherein the one or more (several) genes are modified rendering the mutant strain deficient in the production of one or more (several) enzymes selected from the group consisting of orotidine-5′-monophosphate decarboxylase, alpha-amylase, and alkaline protease, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions; and
(b) recovering the polypeptide from the cultivation medium.
[2] The method of paragraph 1, wherein the mutant strain comprises a modification of a pyrG gene.
[3] The method of paragraph 1, wherein the mutant strain comprises a modification of an amyA gene.
[4] The method of paragraph 1, wherein the mutant strain comprises a modification of an alpA gene.
[5] The method of paragraph 1, wherein the mutant strain comprises a modification of a pyrG gene and an amyA gene.
[6] The method of paragraph 1, wherein the mutant strain comprises a modification of a pyrG gene and an alpA gene.
[7] The method of paragraph 1, wherein the mutant strain comprises a modification of an amyA gene and an alpA gene.
[8] The method of paragraph 1, wherein the mutant strain comprises a modification of a pyrG gene, an amyA gene, and an alpA gene.
[9] The method of any of paragraphs 1-8, wherein the mutant strain further comprises one or both of the genes tri5 and dps1, wherein the one or both of the genes are modified rendering the mutant strain deficient in the production of one or both enzymes trichodiene synthase and cyclohexadepsipeptide synthetase, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
[10] The method of paragraph 9, wherein the mutant strain comprises a modification of a tri5 gene.
[11] The method of paragraph 9, wherein the mutant strain comprises a modification of a dps1 gene.
[12] The method of paragraph 9, wherein the mutant strain comprises a modification of a tri5 gene and a dps1 gene.
[13] The method of any of paragraph 9-12, wherein the mutant strain produces at least 25% less of the one or both enzymes of trichodiene synthase and cyclohexadepsipeptide synthetase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
[14] The method of any of paragraph 9-12, wherein the mutant strain is completely deficient in the one or both enzymes trichodiene synthase and cyclohexadepsipeptide synthetase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
[15] The method of any of paragraphs 1-14, wherein the polypeptide is native or foreign to the Fusarium venenatum strain.
[16] The method of paragraph 15, wherein the polypeptide is selected from the group consisting of an antigen, enzyme, growth factor, hormone, immunodilator, neurotransmitter, receptor, reporter protein, structural protein, and transcription factor.
[17] The method of paragraph 16, wherein the enzyme is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase.
[18] The method of any of paragraphs 1-17, wherein the mutant strain comprises at least two copies of the polynucleotide encoding the polypeptide.
[19] The method of any of paragraphs 1-18, wherein the mutant strain produces at least 25% less of the one or more (several) enzymes selected from the group consisting of orotidine-5′-monophosphate decarboxylase, alpha-amylase, and alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
[20] The method of any of paragraphs 1-18, wherein the mutant strain is completely deficient in the one or more (several) enzymes selected from the group consisting of an orotidine-5′-monophosphate decarboxylase, alpha-amylase, and alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
[21] The method of any of paragraphs 1-20, wherein the pyrG gene encoding a polypeptide having orotidine-5′-monophosphate decarboxylase activity is selected from the group consisting of:
(a) a gene encoding a polypeptide having orotidine-5′-monophosphate decarboxylase activity comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 44;
(b) a gene that hybridizes under at least low stringency conditions with (i) SEQ ID NO: 43 or its full-length complementary strand; and
(c) a gene comprising a nucleotide sequence having at least 60% sequence identity to SEQ ID NO: 43.
[22] The method of paragraph 21, wherein the pyrG gene encodes a polypeptide having orotidine-5′-monophosphate decarboxylase activity comprising or consisting of SEQ ID NO: 44 or a fragment thereof having orotidine-5′-monophosphate decarboxylase activity.
[23] The method of any of paragraphs 1-20, wherein the amyA gene encoding a polypeptide having alpha-amylase activity is selected from the group consisting of:
(a) a gene encoding a polypeptide having alpha-amylase activity comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 52;
(b) a gene that hybridizes under at least low stringency conditions with (i) SEQ ID NO: 51 or its full-length complementary strand; and
(c) a gene comprising a nucleotide sequence having at least 60% sequence identity to SEQ ID NO: 51.
[24] The method of paragraph 23, wherein the amyA gene encodes a polypeptide having alpha-amylase activity comprising or consisting of SEQ ID NO: 52 or a fragment thereof having alpha-amylase activity.
[25] The method of any of paragraphs 1-20, wherein the alpA gene encoding a polypeptide having alkaline protease activity is selected from the group consisting of:
(a) a gene encoding a polypeptide having alkaline protease activity comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 84;
(b) a gene that hybridizes under at least low stringency conditions with (i) SEQ ID NO: 83 or its full-length complementary strand; and
(c) a gene comprising a nucleotide sequence having at least 60% sequence identity to SEQ ID NO: 83.
[26] The method of paragraph 25, wherein the alpA gene encodes a polypeptide having alkaline protease activity comprising or consisting of SEQ ID NO: 84 or a fragment thereof having alkaline protease activity.
[27] The method of any of paragraphs 1-20, wherein the tri5 gene encoding a polypeptide having trichodiene synthase activity is selected from the group consisting of:
(a) a gene encoding a polypeptide having trichodiene synthase activity comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 20;
(b) a gene that hybridizes under at least low stringency conditions with (i) SEQ ID NO: 19 or its full-length complementary strand; and
(c) a gene comprising a nucleotide sequence having at least 60% sequence identity to SEQ ID NO: 19.
[28] The method of paragraph 27, wherein the tri5 gene encodes a polypeptide having trichodiene synthase activity comprising or consisting of SEQ ID NO: 20 or a fragment thereof having trichodiene synthase activity.
[29] The method of any of paragraphs 1-20, wherein the dps1 gene encoding a polypeptide having cyclohexadepsipeptide synthetase activity is selected from the group consisting of:
(a) a gene encoding a polypeptide having cyclohexadepsipeptide synthetase activity comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 94;
(b) a gene that hybridizes under at least low stringency conditions with (i) SEQ ID NO: 93 or its full-length complementary strand; and
(c) a gene comprising a nucleotide sequence having at least 60% sequence identity to SEQ ID NO: 93.
[30] The method of paragraph 29, wherein the dps1 gene encodes a polypeptide having cyclohexadepsipeptide synthetase activity comprising or consisting of SEQ ID NO: 94 or a fragment thereof having cyclohexadepsipeptide synthetase activity.
[31] A mutant of a parent Fusarium venenatum strain, comprising a polynucleotide encoding a polypeptide and one or more (several) genes selected from the group consisting of pyrG, amyA, and alpA, wherein the one or more (several) genes are modified rendering the mutant strain deficient in the production of one or more (several) enzymes selected from the group consisting of orotidine-5′-monophosphate decarboxylase, alpha-amylase, and alkaline protease, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
[32] The mutant strain of paragraph 31, which comprises a modification of a pyrG gene.
[33] The mutant strain of paragraph 31, which comprises a modification of an amyA gene.
[34] The mutant strain of paragraph 31, which comprises a modification of an alpA gene.
[35] The mutant strain of paragraph 31, which comprises a modification of a pyrG gene and an amyA gene.
[36] The mutant strain of paragraph 31, which comprises a modification of a pyrG gene and an alpA gene.
[37] The mutant strain of paragraph 31, which comprises a modification of an amyA gene and an alpA gene.
[38] The mutant strain of paragraph 31, which comprises a modification of a pyrG gene, an amyA gene, and an alpA gene.
[39] The mutant strain of any of paragraphs 31-38, which further comprises one or both of the genes tri5 and dps1, wherein the one or both of the genes are modified rendering the mutant strain deficient in the production of one or both enzymes trichodiene synthase and cyclohexadepsipeptide synthetase, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
[40] The mutant strain of paragraph 39, which comprises a modification of a tri5 gene.
[41] The mutant strain of paragraph 39, which comprises a modification of a dps1 gene.
[42] The mutant strain of paragraph 39, which comprises a modification of a tri5 gene and a dps1 gene.
[43] The mutant strain of any of paragraphs 39-42, which produces at least 25% less of the one or both enzymes of trichodiene synthase and cyclohexadepsipeptide synthetase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
[44] The mutant strain of any of paragraphs 39-42, which is completely deficient in the one or both enzymes trichodiene synthase and cyclohexadepsipeptide synthetase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
[45] The mutant strain of any of paragraphs 31-44, wherein the polypeptide is native or foreign to the Fusarium venenatum strain.
[46] The mutant strain of any of paragraphs 31-45, wherein the polypeptide is selected from the group consisting of an antigen, enzyme, growth factor, hormone, immunodilator, neurotransmitter, receptor, reporter protein, structural protein, and transcription factor.
[47] The mutant strain of paragraph 46, wherein the enzyme is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase.
[48] The mutant strain of any of paragraphs 31-47, which produces at least 25% less of the one or more (several) enzymes selected from the group consisting of orotidine-5′-monophosphate decarboxylase, alpha-amylase, and alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
[49] The mutant strain of any of paragraphs 31-47, which is completely deficient in the one or more (several) enzymes selected from the group consisting of an orotidine-5′-monophosphate decarboxylase, alpha-amylase, and alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
[50] The mutant strain of any of paragraphs 31-49, wherein the pyrG gene encoding a polypeptide having orotidine-5′-monophosphate decarboxylase activity is selected from the group consisting of:
(a) a gene encoding a polypeptide having orotidine-5′-monophosphate decarboxylase activity comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 44;
(b) a gene that hybridizes under at least low stringency conditions with (i) SEQ ID NO: 43 or its full-length complementary strand; and
(c) a gene comprising a nucleotide sequence having at least 60% sequence identity to SEQ ID NO: 43.
[51] The mutant strain of paragraph 50, wherein the pyrG gene encodes a polypeptide having orotidine-5′-monophosphate decarboxylase activity comprising or consisting of SEQ ID NO: 44 or a fragment thereof having orotidine-5′-monophosphate decarboxylase activity.
[52] The mutant strain of any of paragraphs 31-49, wherein the amyA gene encoding a polypeptide having alpha-amylase activity is selected from the group consisting of:
(a) a gene encoding a polypeptide having alpha-amylase activity comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 52;
(b) a gene that hybridizes under at least low stringency conditions with (i) SEQ ID NO: 51 or its full-length complementary strand; and
(c) a gene comprising a nucleotide sequence having at least 60% sequence identity to SEQ ID NO: 51.
[53] The mutant strain of paragraph 52, wherein the amyA gene encodes a polypeptide having alpha-amylase activity comprising or consisting of SEQ ID NO: 52 or a fragment thereof having alpha-amylase activity.
[54] The mutant strain of any of paragraphs 31-49, wherein the alpA gene encoding a polypeptide having alkaline protease activity is selected from the group consisting of:
(a) a gene encoding a polypeptide having alkaline protease activity comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 84;
(b) a gene that hybridizes under at least low stringency conditions with (i) SEQ ID NO: 83 or its full-length complementary strand; and
(c) a gene comprising a nucleotide sequence having at least 60% sequence identity to SEQ ID NO: 83.
[55] The mutant strain of paragraph 54, wherein the alpA gene encodes a polypeptide having alkaline protease activity comprising or consisting of SEQ ID NO: 84 or a fragment thereof having alkaline protease activity.
[56] The mutant strain of any of paragraphs 31-49, wherein the tri5 gene encoding a polypeptide having trichodiene synthase activity is selected from the group consisting of:
(a) a gene encoding a polypeptide having trichodiene synthase activity comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 20;
(b) a gene that hybridizes under at least low stringency conditions with (i) SEQ ID NO: 19 or its full-length complementary strand; and
(c) a gene comprising a nucleotide sequence having at least 60% sequence identity to SEQ ID NO: 19.
[57] The mutant strain of paragraph 56, wherein the tri5 gene encodes a polypeptide having trichodiene synthase activity comprising or consisting of SEQ ID NO: 20 or a fragment thereof having trichodiene synthase activity.
[58] The mutant strain of any of paragraphs 31-49, wherein the dps1 gene encoding a polypeptide having cyclohexadepsipeptide synthetase activity is selected from the group consisting of:
(a) a gene encoding a polypeptide having cyclohexadepsipeptide synthetase activity comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 94;
(b) a gene that hybridizes under at least low stringency conditions with (i) SEQ ID NO: 93 or its full-length complementary strand; and
(c) a gene comprising a nucleotide sequence having at least 60% sequence identity to SEQ ID NO: 93.
[59] The mutant strain of paragraph 58, wherein the dps1 gene encodes a polypeptide having cyclohexadepsipeptide synthetase activity comprising or consisting of SEQ ID NO: 94 or a fragment thereof having cyclohexadepsipeptide synthetase activity.
[60] The mutant strain of any of paragraphs 31-59, which comprises a polynucleotide encoding a polypeptide foreign to the mutant strain.
[61] A method for obtaining a mutant of a parent Fusarium venenatum strain, comprising:
(a) modifying one or more (several) genes selected from the group consisting of pyrG, amyA, and alpA; and
(b) identifying a mutant strain from step (a) wherein the one or more (several) genes selected from the group consisting of pyrG, amyA, and alpA are modified rendering the mutant strain deficient in the production of one or more (several) enzymes selected from the group consisting of orotidine-5′-monophosphate decarboxylase, alpha-amylase, and alkaline protease, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
[62] The method of paragraph 61, wherein the mutant strain comprises a modification of a pyrG gene.
[63] The method of paragraph 61, wherein the mutant strain comprises a modification of an amyA gene.
[64] The method of paragraph 61, wherein the mutant strain comprises a modification of an alpA gene.
[65] The method of paragraph 61, wherein the mutant strain comprises a modification of a pyrG gene and an amyA gene.
[66] The method of paragraph 61, wherein the mutant strain comprises a modification of a pyrG gene and an alpA gene.
[67] The method of paragraph 61, wherein the mutant strain comprises a modification of an amyA gene and an alpA gene.
[68] The method of paragraph 61, wherein the mutant strain comprises a modification of a pyrG gene, an amyA gene, and an alpA gene.
[69] The method of any of paragraphs 61-68, further comprising modifying one or both of the genes tri5 and dps1, rendering the mutant strain deficient in the production of one or both enzymes trichodiene synthase and cyclohexadepsipeptide synthetase, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
[70] The method of paragraph 69, wherein the mutant strain comprises a modification of a tri5 gene.
[71] The method of paragraph 69, wherein the mutant strain comprises a modification of a dps1 gene.
[72] The method of paragraph 69, wherein the mutant strain comprises a modification of a tri5 gene and a dps1 gene.
[73] The method of any of paragraphs 69-72, wherein the mutant strain produces at least 25% less of the one or both enzymes of trichodiene synthase and cyclohexadepsipeptide synthetase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
[74] The method of any of paragraphs 69-72, wherein the mutant strain is completely deficient in the one or both enzymes trichodiene synthase and cyclohexadepsipeptide synthetase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
[75] The method of any of paragraphs 61-74, wherein the mutant strain produces at least 25% less of the one or more (several) enzymes selected from the group consisting of orotidine-5′-monophosphate decarboxylase, alpha-amylase, and alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
[76] The method of any of paragraphs paragraph 61-74, wherein the mutant strain is completely deficient in the one or more (several) enzymes selected from the group consisting of an orotidine-5′-monophosphate decarboxylase, alpha-amylase, and alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.
[77] The method of any of paragraphs 61-76, wherein the pyrG gene encoding a polypeptide having orotidine-5′-monophosphate decarboxylase activity is selected from the group consisting of:
(a) a gene encoding a polypeptide having orotidine-5′-monophosphate decarboxylase activity comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 44;
(b) a gene that hybridizes under at least low stringency conditions with (i) SEQ ID NO: 43 or its full-length complementary strand; and
(c) a gene comprising a nucleotide sequence having at least 60% sequence identity to SEQ ID NO: 43.
[78] The method of paragraph 77, wherein the pyrG gene encodes a polypeptide having orotidine-5′-monophosphate decarboxylase activity comprising or consisting of SEQ ID NO: 44 or a fragment thereof having orotidine-5′-monophosphate decarboxylase activity.
[79] The method of any of paragraphs 61-76, wherein the amyA gene encoding a polypeptide having alpha-amylase activity is selected from the group consisting of:
(a) a gene encoding a polypeptide having alpha-amylase activity comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 52;
(b) a gene that hybridizes under at least low stringency conditions with (i) SEQ ID NO: 51 or its full-length complementary strand; and
(c) a gene comprising a nucleotide sequence having at least 60% sequence identity to SEQ ID NO: 51.
[80] The method of paragraph 79, wherein the amyA gene encodes a polypeptide having alpha-amylase activity comprising or consisting of SEQ ID NO: 52 or a fragment thereof having alpha-amylase activity.
[81] The method of any of paragraphs 61-76, wherein the alpA gene encoding a polypeptide having alkaline protease activity is selected from the group consisting of:
(a) a gene encoding a polypeptide having alkaline protease activity comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 84;
(b) a gene that hybridizes under at least low stringency conditions with (i) SEQ ID NO: 83 or its full-length complementary strand; and
(c) a gene comprising a nucleotide sequence having at least 60% sequence identity to SEQ ID NO: 83.
[82] The method of paragraph 81, wherein the alpA gene encodes a polypeptide having alkaline protease activity comprising or consisting of SEQ ID NO: 84 or a fragment thereof having alkaline protease activity.
[83] The method of any of paragraphs 61-76, wherein the tri5 gene encoding a polypeptide having trichodiene synthase activity is selected from the group consisting of:
(a) a gene encoding a polypeptide having trichodiene synthase activity comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 20;
(b) a gene that hybridizes under at least low stringency conditions with (i) SEQ ID NO: 19 or its full-length complementary strand; and
(c) a gene comprising a nucleotide sequence having at least 60% sequence identity to SEQ ID NO: 19.
[84] The method of paragraph 83, wherein the tri5 gene encodes a polypeptide having trichodiene synthase activity comprising or consisting of SEQ ID NO: 20 or a fragment thereof having trichodiene synthase activity.
[85] The method of any of paragraphs 61-76, wherein the dps1 gene encoding a polypeptide having cyclohexadepsipeptide synthetase activity is selected from the group consisting of:
(a) a gene encoding a polypeptide having cyclohexadepsipeptide synthetase activity comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 94;
(b) a gene that hybridizes under at least low stringency conditions with (i) SEQ ID NO: 93 or its full-length complementary strand; and
(c) a gene comprising a nucleotide sequence having at least 60% sequence identity to SEQ ID NO: 93.
[86] The method of paragraph 85, wherein the dps1 gene encodes a polypeptide having cyclohexadepsipeptide synthetase activity comprising or consisting of SEQ ID NO: 94 or a fragment thereof having cyclohexadepsipeptide synthetase activity.
The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control. Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

## Claims

1. A mutant of a parent Fusarium venenatum strain, comprising a polynucleotide encoding a polypeptide and amyA and alpA genes, wherein the amyA and alpA genes are modified rendering the mutant strain deficient in the production of alpha-amylase and alkaline protease, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

2. The mutant strain of claim 1, which further comprises one or both of the genes tri5 and dps1, wherein the one or both of the genes are modified rendering the mutant strain deficient in the production of one or both enzymes trichodiene synthase and cyclohexadepsipeptide synthetase, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

3. The mutant strain of claim 2, which produces at least 25% less of the one or both enzymes of trichodiene synthase and cyclohexadepsipeptide synthetase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

4. The mutant strain of claim 2, which is completely deficient in the one or both enzymes trichodiene synthase and cyclohexadepsipeptide synthetase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

5. The mutant strain of claim 1, wherein the polypeptide is native or foreign to the Fusarium venenatum strain.

6. The mutant strain of claim 1, which produces at least 25% less of the enzymes alpha-amylase and alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

7. The mutant strain of claim 1, which is completely deficient in the enzymes alpha-amylase and alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

8. A method for obtaining the mutant of a parent Fusarium venenatum strain of claim 1, comprising:
(a) modifying amyA and alpA genes in the parent Fusarium venenatum strain; and
(b) identifying a mutant strain from step (a) wherein the amyA and alpA genes are modified rendering the mutant strain deficient in the production of alpha-amylase and alkaline protease, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

9. The method of claim 8, further comprising modifying one or both of the genes tri5 and dps1, rendering the mutant strain deficient in the production of one or both enzymes trichodiene synthase and cyclohexadepsipeptide synthetase, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

10. The method of claim 9, wherein the mutant strain produces at least 25% less of the one or both enzymes of trichodiene synthase and cyclohexadepsipeptide synthetase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

11. The method of claim 9, wherein the mutant strain is completely deficient in the one or both enzymes trichodiene synthase and cyclohexadepsipeptide synthetase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

12. The method of claim 8, wherein the mutant strain produces at least 25% less of the enzymes alpha-amylase and alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

13. The method of claim 8, wherein the mutant strain is completely deficient in the enzymes alpha-amylase and alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

14. A method of producing a polypeptide, comprising:
(a) cultivating the mutant of a parent Fusarium venenatum strain of claim 1 in a medium for the production of the polypeptide, wherein the mutant strain comprises a polynucleotide encoding the polypeptide and amyA and alpA genes, wherein the amyA and alpA genes are modified rendering the mutant strain deficient in the production of alpha-amylase and alkaline protease, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions; and
(b) recovering the polypeptide from the cultivation medium.

15. The method of claim 14, wherein the mutant strain further comprises one or both of the genes tri5 and dps1, wherein the one or both of the genes are modified rendering the mutant strain deficient in the production of one or both enzymes trichodiene synthase and cyclohexadepsipeptide synthetase, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

16. The method of claim 15, wherein the mutant strain produces at least 25% less of the one or both enzymes of trichodiene synthase and cyclohexadepsipeptide synthetase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

17. The method of claim 15, wherein the mutant strain is completely deficient in the one or both enzymes trichodiene synthase and cyclohexadepsipeptide synthetase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

18. The method of claim 14, wherein the polypeptide is native or foreign to the Fusarium venenatum strain.

19. The method of claim 14, wherein the mutant strain produces at least 25% less of the enzymes alpha-amylase and alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

20. The method of claim 14, wherein the mutant strain is completely deficient in the enzymes alpha-amylase and alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.