BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention relates to secondary metabolite production by fungi. More particularly, the invention relates to modulation of secondary metabolite production by fungi through genetic manipulation of such fungi.

[0003] 1. Description of the Related Art

[0004] Secondary metabolite production by various fungi has been an extremely important source of a variety of therapeutically significant pharmaceuticals. β-lactam antibacterials such as penicillin and cephalosporin are produced by Penicillium chrysogenum and Acremonium cirysogenum, respectively, and these compounds are by far the most frequently used antibacterials (reviewed in Luengo and Penalva (1994), Prog. Ind. Microbiol. 29: 603-38; Jensen and Demain (1995), Biotechnology 28: 239-68; Brakhage (1998), Microbiol. Mol. Biol. Rev. 62: 547-85). Cyclosporin A, a member of a class of cyclic undecapeptides, is produced by Tolypocladium inflatum. Cyclosporin A dramatically reduces morbidity and increases survival rates in transplant patients (Borel (1986), Prog. Allergy 38: 9-18). In addition, several fungal secondary metabolites are cholesterol lowering drugs, including lovastatin, which is made by Aspergillus terreus and several other fungi (Alberts et al. (1980), Proc. Natl. Acad. Sci. USA 77: 3957-3961). These and many other fungal secondary metabolites have contributed greatly to health care throughout the world (see Demain (1992), Ciba Found. Symp. 171: 3-16; Bentley (1991), Crit. Rev. Biotechnol. 19: 1-40).

[0005] Unfortunately, many challenges are encountered between the detection of a secondary metabolite activity to production of significant quantities of pure drug. Thus, efforts have been made to improve the production of secondary metabolites by fungi. Some of these efforts have attempted to improve production by modification of the growth medium or the bioreactor used to carry out the fermentation. Buckland et al. (1989), in Topics in Industrial Microbiology: Novel Microbial Products for Medicine and Agriculture, Elsevier, Amsterdam, pp. 161-169, discloses improved lovastatin production by modification of carbon source and also teaches the superiority of a hydrofoil axial-flow impeller in the bioreactor. Other efforts have involved strain improvements, either through re-isolation or random mutagenesis. Agathos et al. (1986), J. Ind. Microbiol. 1: 39-48, teaches that strain improvement and process development together resulted in a ten-fold increase in cyclosporin A production. While important, studies of these types have still left much room for improvement in the production of secondary metabolites.

[0006] More recently, strains have been improved by manipulation of the genes encoding the biosynthetic enzymes that catalyze the reactions required for production of secondary metabolites. Penalva et al. (1998), Trends Biotechnol. 16: 483-489 discloses that production strains of P. chrysogenum have increased copy number of the penicillin synthesis structural genes. Other studies have modulated expression of other biosynthetic enzyme-encoding genes, thereby affecting overall metabolism in the fungus. Mingo et al. (1999), J. Biol. Chem. 21: 14545-14550, demonstrate that disruption of phacA, an enzyme in A. nidulans that catalyzes phenylacetate 2-hydroxylation, leads to increased penicillin production, probably by elimination of competition for the substrate phenylacetate. Similarly, disruption of the gene encoding aminoadipate reductase in P. chrysogenum increased penicillin production, presumably by eliminating competition for the substrate alpha-aminoadipate (Casquiero et al. (1999), J. Bacteriol. 181: 1181-1188).

[0007] Thus, genetic manipulation holds promise for improving production of secondary metabolites. Genetic manipulation to increase the activity of biosynthetic enzymes for secondary metabolite production or to decrease the activity of competing biosynthetic pathways has proven effective for improving production. Maximum benefit can be achieved by combining several strategies of manipulation. For example, modulating the expression of genes that regulate the biosynthetic enzyme-encoding genes can improve production. In addition, genetic manipulation can be used to impact upon the challenges that are encountered in the fermentor run or downstream processing (e.g., energy cost, specific production of desired metabolite, maximal recovery of metabolite, cost of processing waste from fermentations). There is, therefore, a need for methods for improving secondary metabolite production in a fungus, comprising modulating the expression of a gene involved in regulation of secondary metabolite production in fungi.

[0008] One challenge is to identify the types of genes that would be useful for such modulation. Todd and Andrianopoulos (1997), Fungal Genetics and Biology 21: 388-405, teaches that Zn(II)2Cys6 proteins (zinc binuclear cluster proteins, or “ZBC proteins”) are involved in a wide range of processes, including primary and secondary metabolism. This reference teaches that such proteins are primarily, though not exclusively, transcriptional activators. Chang et al. (1995), Applied Environ. Microbiol. 61: 2372-2377, teaches that increased expression of aflR, a ZBC protein, relieves nitrate inhibition of aflatoxin biosynthesis in Aspergillus parasiticus. PCT Publication WO 00/37629, teaches that over-expressing lovE, another ZBC protein, increases lovastatin production in Aspergillus terreus. Noel et al (1998), Mol. Microbiol. 27: 131-142, teaches that xinR, a ZBC protein, induces expression of xylanolytic extracellular enzymes in Aspergillus niger. Hasper et al. (2000), Mol. Microbiol. 36: 193-200, teaches that xlnR also regulates D-xylose reductase gene expression in Aspergillus niger. D'Alessio and Brandriss (2000), J. Bacteriology 182: 3748-3753, discloses that Gal4p, a ZBC protein, can activate the PUT (proline utilization) genes in a Saccharomyces cerevisiae strain lacking the normal gene for regulation of this pathway, PUT3. PCT Publication WO 00/20596 discloses that prtT, a ZBC protein, activates extracellular proteases in Aspergillus niger.

[0009] Numerous studies have examined the effects of mutations in genes that encode ZBC proteins. Crowley et al (1998), J. Bacteriol. 180: 4177-4183, discloses that a single missense mutation in UPC2, a ZBC gene, results in pleiotropic effects in Saccharomyces cerevisiae. Friden et al. (1989), Mol. Cell. Biol. 9: 4056-4060, teaches that a large internal deletion in Leu3p, a ZBC protein, in Sacclzaromyces cerevisiae causes the protein to be a constitutive transcriptional activator. Oestreicher and Scazzocchio (1995), J. MoL Biol. 249: 693-699, discloses that a single amino acid change in Yc462, a ZBC protein, leads to constitutive, hyperinducible and derepressed expression of at least three genes in Aspergillus nidulans. Wang et al. (1999), J. Biol. Chem. 274: 19017-19024, discloses that nine distinct missense mutations in LEU3 affect the masking of the activation domain of that ZBC protein. Dickson et al. (1990), Nucleic Acids Res. 18: 5213-5217, discloses that single amino acid changes in the C terminal region of Gal4p and Lac9p, two ZBC proteins, lead to constitutive expression of target genes. Marczak and Brandriss (1991), MoL CelL Biol. 11: 2609-2619, teaches that single point mutations in PUT3, a ZBC gene from Saccharomyces cerevisiae, lead to either constitutive or uninducible expression of proline utilization genes. Carvajal et al. (1997), Mol. Gen. Genet. 256: 406-415, teaches that single amino acid substitutions in Pdr1p, a ZBC protein from Saccharomyces cerevisiae, are responsible for over-expression of three transporter genes associated with multiple drug resistance. Nourani et al. (1997), Mol. Gen. Genet. 256: 397-405, teaches that substitutions in a conserved region of Pdr3p, a ZBC protein from Saccharomyces cerevisiae, leads to gain of function mutations. Zhou et al. (1990), Nucleic Acids Res. 18: 291-298, discloses that deletion of all or part of the linker region of Leu3p results in unmodulated activation of Leu3p target genes. Herlich et al. (1998), Fungal Genetics Biol. 23: 1807-1845, teaches that deletion of three amino acids in the C terminus of Af1R results in increased expression of the aflatoxin pathway.

[0010] These studies demonstrate that ZBC genes can be manipulated in beneficial ways and may have promise as regulators of secondary metabolism. Unfortunately, no one has been able to create a commercial process in which production of a useful secondary metabolite has been significantly increased through the action of a ZBC protein. There is, therefore, a need for new commercial processes using ZBC proteins, or variants thereof, to significantly increase useful secondary metabolite production.

SUMMARY OF THE INVENTION

[0011] The invention relates to secondary metabolite production by fungi. More particularly, the invention relates to modulation of secondary metabolite production by fungi through genetic manipulation of such fungi. The invention provides new commercial processes using ZBC proteins, or variants thereof, to significantly improve the production of useful secondary metabolites. Generally, the methods according to the invention comprise expressing in a fungus a ZBC protein or a variant thereof.

[0012] In a first aspect, the invention provides methods for improving production of a secondary metabolite by a fungus by increasing the yield of the secondary metabolite produced by the fungus. The methods according to this aspect of the invention comprise modulating the expression of a ZBC gene or gene variant in a manner that improves the yield of the secondary metabolite.

[0013] In a second aspect, the invention provides methods for improving production of a secondary metabolite by a fungus by increasing productivity of the secondary metabolite in the fungus, the methods comprising modulating the expression of a ZBC gene or gene variant in a manner that improves the productivity of the secondary metabolite.

[0014] In a third aspect, the invention provides methods for improving production of a secondary metabolite in a fungus by increasing efflux or excretion of the secondary metabolite, the method comprising modulating the expression of a ZBC gene or gene variant in a manner that increases efflux or excretion of the secondary metabolite.

[0015] In a fourth aspect, the invention provides methods for improving production of a secondary metabolite in a fungus by decreasing production of side products or non-desired secondary metabolites, the method comprising modulating the expression of a ZBC gene or gene variant in a manner that decreases production of side products or non-desired secondary metabolites.

[0016] In a fifth aspect, the invention provides methods for improving production of a secondary metabolite in a fungus by altering the characteristics of the fungus in a manner that is beneficial to the production of the secondary metabolite, the method comprising modulating the expression of a ZBC gene or gene variant in a manner that alters the characteristics of the fungus.

[0017] In a sixth aspect, the invention provides methods for improving production of a secondary metabolite in a fungus by causing conditional lysis of the fungus, the method comprising modulating the expression of a ZBC gene or gene variant in a manner that causes conditional lysis.

[0018] In a seventh aspect, the invention provides methods for improving production of a secondary metabolite in a fungus by increasing the resistance of the fungus to the deleterious effects of exposure to a secondary metabolite made by the same organism, the method comprising modulating the expression of a ZBC gene or gene variant in a manner that increases resistance to the deleterious effects of exposure to a secondary metabolite.

[0019] In an eighth aspect, the invention provides methods for improving production of a secondary metabolite in a fungus by modulating the expression of one or more genes, the method comprising modulating the expression of a ZBC gene or gene variant that does not normally modulate the expression of such gene or genes.

[0020] In a ninth aspect, the invention provides genetically modified fungi, wherein the genetically modified fungi have an ability to produce secondary metabolites and the ability of the genetically modified fungus to produce secondary metabolites has been improved by any of the methods according to the invention.

[0021] In a tenth aspect, the invention provides a method for making a secondary metabolite, the method comprising culturing a genetically modified fungus according to the invention under conditions suitable for the production of secondary metabolites.

DETAILED DESCRIPTION

[0022] The invention relates to secondary metabolite production by fungi. More particularly, the invention relates to modulation of secondary metabolite production by fungi through genetic manipulation of such fungi. All issued patents, published and pending patent applications, and other references cited herein reflect the level of knowledge in this field and are hereby incorporated by reference in their entirety. In case of any conflict between the teachings of a cited reference and this specification, the latter shall prevail.

[0023] The invention provides new commercial processes using ZBC proteins, or variants thereof, to significantly increase useful secondary metabolite production. Generally, the methods according to the invention comprise expressing in a fungus a ZBC protein or a variant thereof. All aspects of the invention contemplate the modulation of one or more ZBC genes in a fungal cell of interest.

[0024] In a first aspect, the invention provides methods for improving production of a secondary metabolite by a fungus by increasing the yield of the secondary metabolite produced by the fungus. The methods according to this aspect of the invention comprise modulating the expression of a ZBC gene or gene variant in a manner that improves the yield of the secondary metabolite.

[0025] As used for all aspects of the invention, the term “improving production of a secondary metabolite” means positively impacting one or more of the variables that affect the process of production of secondary metabolites in a fungal fermentation. These variables include, without limitation, the amount of secondary metabolite being produced, the volume required for production of sufficient quantities, the cost of raw materials and energy, the time of fermentor run, the amount of waste that must be processed after a fermentor run, the specific production of the desired metabolite, the percent of produced secondary metabolite that can be recovered from the fermentation broth, and the resistance of an organism producing a secondary metabolite to possible deleterious effects of contact with the secondary metabolite. Also for all aspects, the term “secondary metabolite” means a compound, derived from primary metabolites, that is produced by an organism, is not a primary metabolite, is not ethanol or a fusel alcohol, and is not required for growth under standard conditions. Secondary metabolites are derived from intermediates of many pathways of primary metabolism. These pathways include, without limitation, pathways for biosynthesis of amino acids, the shikimic acid pathway for biosynthesis of aromatic amino acids, the polyketide biosynthetic pathway from acetyl coenzyme A (CoA), the mevalonic acid pathway from acetyl CoA, and pathways for biosynthesis of polysaccharides and peptidopolysaccharides. Secondary metabolism involves all primary pathways of carbon metabolism (Fungal Physiology, Chapter 9, pp 246-274, Griffm (ed.), John Wiley & Sons, Inc., New York, (1994)). “Secondary metabolites” also include intermediate compounds in the biosynthetic pathway for a secondary metabolite that are dedicated to the pathway for synthesis of the secondary metabolite. “Dedicated to the pathway for synthesis of the secondary metabolite” means that once the intermediate is synthesized by the cell, the cell will not convert the intermediate to a primary metabolite. “Intermediate compounds” also include secondary metabolite intermediate compounds which can be converted to useful compounds by subsequent chemical conversion or subsequent biotransformation. Nevertheless, providing improved availability of such intermediate compounds still leads to improved production of the ultimate useful compound, which itself may be referred to herein as a secondary metabolite. The yeast Saccharomyces cerevisiae is not known to produce secondary metabolites. The term “primary metabolite” means a natural product that has an obvious role in the functioning of the relevant organism. Primary metabolites include, without limitation, compounds involved in the biosynthesis of lipids, carbohydrates, proteins, and nucleic acids. The term “increasing the yield of the secondary metabolite” means increasing the quantity of the secondary metabolite present in the fermentation broth per unit volume of fermentation broth.

[0026] The term “ZBC gene” means any gene encoding a protein having as part of its structure Cys-(Xaa)₂-Cys-(Xaa)₆-Cys-(Xaa)₅-₁₆-Cys-(Xaa )₂-Cys-(Xaa)6-₈-Cy s (see e.g., Todd and Andrianopoulos (1997), Fungal Genetics and Biology 21: 388-405), wherein Xaa is any amino acid, each of which can be the same or different. Preferred ZBC genes according to this aspect of the invention include, without limitation, those genes identified in Table 1, below, and any fungal homologs thereof. 1

[0027] A “fungal homolog” of a reference gene is a fungal gene encoding a product that is capable of performing at least a portion of the function of the product encoded by the reference gene, which is substantially identical to the reference gene, and/or which encodes a product which is substantially identical to the product encoded by the reference gene. “Substantially identical” means a polypeptide or nucleic acid exhibiting at least 25%, preferably 50%, more preferably 80%, and most preferably 90%, or even 95% identity to a reference amino acid sequence or nucleic acid sequence. For polypeptides, the length of comparison sequences is generally at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids or greater. For nucleic acids, the length of comparison sequences is generally at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides or greater. Sequence identity is typically measured using sequence analysis software (for example, the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison Wis. 53705; or the BLAST, BEAUTY, or PILEUP/PRETTYBOX programs). For determining percentages of identity, a gap existence penalty of 11 and a gap extension penalty of 1 may be employed in such programs. For determining sequence similarity, such software assigns degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0028] For this aspect of the invention, when the secondary metabolite is aflatoxin or sterigmatocystin, the ZBC gene is not the aflR gene from Aspergillus spp.; and when the secondary metabolite is lovastatin, the ZBC gene is not the Aspergillus terreus lovE gene.

[0029] The term “ZBC gene or gene variant” means any ZBC gene, or any useful mutant form of such gene. Many useful mutations of ZBC genes and/or proteins are contemplated by the present invention, including various dominant mutations. A “dominant mutation” is an allele of a gene that encodes a protein capable of changing the phenotype of an organism more than a non-mutated form of the gene. Preferred dominant mutations include dominant negative mutations, dominant positive mutations, and dominant neomorphic mutations. A “dominant negative mutation” is a dominant mutation that achieves its phenotypic effect by interfering with some function of the gene or gene product from which it was derived, or from a homolog thereof. A “dominant positive mutation” is a dominant mutation that achieves its phenotypic effect by activating some function of the gene or gene product from which it was derived, or from a homolog thereof. A “dominant neomorphic mutation” is a dominant mutation that achieves the phenotypic effect of providing a novel function to the gene or gene product from which it was derived, or from a homolog thereof. Preferred dominant mutations according to this aspect of the invention include:

[0030] (1) Mutations that result in increased or decreased stability of the transcript of a gene.

[0031] (2) Mutations that result in increased or decreased stability of the product of translation: For example, specific sequences near the amino terminus of a protein have been shown to cause increased or decreased protein stability. Similarly, sequences elsewhere in the protein, such as those required for ubiquitin-dependent degradation, can be mutated to increase the stability of a protein.

[0032] (3) Amino acid substitutions that mimic post-translational modifications: For example, phosphorylation has been demonstrated to positively or negatively regulate the activity of a variety of proteins, including transcription factors and kinases. Phosphorylation most commonly occurs on serine, threonine, and tyrosine residues; in some instances residues such as aspartate and histidine can be phosphorylated. Mutations that mimic constitutive dephosphorylation can be produced by mutating the coding sequence of the phosphorylated residue to the coding sequence of an amino acid that cannot be phosphorylated and does not have a negatively charged side chain (e.g., alanine). Alternatively, substitutions that result in changing serine, threonine, or tyrosine residues to charged amino acids such as glutamate or aspartate can result in an allele that mimics constitutive phosphorylation.

[0033] Proteolytic cleavage is another post-translational mechanism for regulating the activity of a protein. Mutations that result in truncation of a protein can mimic activation by proteolysis. Mutations that change amino acids required for proteolysis can activate proteins that are negatively regulated by proteolysis.

[0034] (4) Amino acid substitutions that promote or inhibit the binding of small molecules such as ATP, cAMP, GTP or GDP: For example, Nucleotides are co-factors for many enzymes, and the nucleotide-binding domains of such proteins are highly conserved. Lysine to arginine substitutions in the nucleotide binding domain frequently result in the inhibition of enzymatic activity. Enzymatically inactive proteins can be dominant inactive molecules, acting by sequestering substrates from functional enzymes.

[0035] (5) Mutations in portions of genes that encode regulatory domains of proteins: For example, many proteins, including kinases, contain regulatory domains that function as mechanisms to control the timing of activation.

[0036] Mutations in these domains might result in constitutive activation. Regulatory domains include linker regions and C terminal regions in the case of some ZBC proteins.

[0037] (6) Mutations that create a new protein function: For example, a mutation in a ZBC protein could result in altered DNA recognition specificity, such that the mutated ZBC protein can modulate the activity of pathways that it does not usually regulate.

[0038] (7) Fusion of the ZBC protein or variants thereof to a transcriptional activation domain: Transcriptional activation domains (TADS) are defined as discrete regions of proteins that promote gene expression by a variety of mechanisms that ultimately result in the activation of RNA polymerase. A TAD generally is defined as the minimal motif that activates transcription when fused to a DNA-binding domain (Webster et al. (1988), Cell 52: 169-178; Fischer et al. (1988), Nature 332: 853-856; Hope et al. (1988), Nature 333: 635-640).

[0039] As used for all aspects of the invention, the term “modulating the expression of a gene” means affecting the function of a gene's product, preferably by increasing or decreasing protein activity or creating a new protein activity through mutation; increasing or decreasing transcription; increasing or decreasing translation; increasing, decreasing or changing post-translational modification; altering intracellular localization; increasing or decreasing translocation; increasing or decreasing protein activity by fusion or by interaction of the protein with another molecule; and/or creating a new protein activity by interaction of the protein with another molecule. In some cases, such modulation is achieved by allowing or causing the expression of an exogenously supplied nucleic acid or gene, e.g., by transformation. In some cases, other exogenously supplied molecules can mediate the modulation. The modulation is not achieved, however, by simply randomly mutagenizing the fungus, either spontaneously or by chemical means. In certain embodiments, the ZBC gene is from an organism in which it is not present within a biosynthetic cluster, or the ZBC gene is not present in the biosynthetic cluster of the desired secondary metabolite to be regulated. In certain embodiments, the ZBC gene is from an organism other than the production fungus, preferably from a different species or genus. In certain embodiments, the ZBC gene in its native locus regulates a different secondary metabolite than the desired secondary metabolite produced by the production fungus. In certain embodiments, the ZBC gene in its native locus does not regulate secondary metabolism. “Native locus” means the chromosomal locus in the original organism from which the gene was cloned.

[0040] As used for all aspects of the invention, “mutation” means an alteration in DNA sequence, either by site-directed or random mutagenesis. Mutation encompasses point mutations as well as insertions, deletions, or rearrangements.

[0041] As used for all aspects of the invention, “mutant” means an organism containing one or more mutations.

[0042] In certain embodiments of the methods according to this aspect of the invention, the modulation is over-expression of the gene. “Over-expression of the gene” means transcription and/or translation and/or gene product maturation at a rate that exceeds by at least two-fold, preferably at least five-fold, and more preferably at least ten-fold, the level of such expression that would be present under similar growth conditions in the absence of the modulation of expression of the gene. “Similar growth conditions” means similar sources of nutrients such as carbon, nitrogen, and phosphate, as well as similar pH, partial oxygen pressure, temperature, concentration of drugs or other small molecules, and a similar substrate for growth, whether solid, semi-solid, or liquid.

[0043] In certain embodiments of the methods according to this aspect of the invention, the modulation is expression of a dominant mutation of the gene. The term “dominant mutation” is as used before. Preferred dominant mutations according to this aspect of the invention are as used before.

[0044] In certain embodiments of the methods according to this aspect of the invention, the modulation is conditional expression of the gene. “Conditional expression” of a gene means expression under certain growth conditions, but not under others. Such growth conditions that may be varied include, without limitation, carbon source, nitrogen source, phosphate source, pH, temperature, partial oxygen pressure, the presence or absence of small molecules such as drugs, and the presence or absence of a solid substrate.

[0045] In certain embodiments of the methods according to this aspect of the invention, the secondary metabolite is an anti-bacterial. An “anti-bacterial” is a molecule that has cytocidal or cytostatic activity against some or all bacteria. Preferred anti-bacterials include, without limitation, β-lactams. Preferred β-lactams include, without limitation, penicillins and cephalosporins. Preferred penicillins and biosynthetic intermediates include, without limitation, isopenicillin N, 6-aminopenicillanic acid (6-APA), penicillin G, penicillin N, and penicillin V. Preferred cephalosporins and biosynthetic intermediates include, without limitation, deacetoxycephalosporin V (DAOC V), deacetoxycephalosporin C (DAOC), deacetylcephalosporin C (DAC), 7-aminodeacetoxy-cephalosporanic acid (7-ADCA), cephalosporin C, 7-β-(5-carboxy-5-oxopentanamido)-cephalosporanic acid (keto-AD-7ACA), 7-β-(4-carboxybutanamido)-cephalosporanic acid (GL-7ACA), and 7-aminocephalosporanic acid (7ACA).

[0046] In certain embodiments of the methods according to this aspect of the invention, the secondary metabolite is an anti-hypercholesterolemic. An “anti-hypercholesterolemic” is a drug administered to a patient diagnosed with elevated cholesterol levels, for the purpose of lowering the cholesterol levels. Preferred anti-hypercholesterolemics include, without limitation, lovastatin, mevastatin, simvastatin, and pravastatin.

[0047] In certain embodiments of the methods according to this aspect of the invention, the secondary metabolite is an immunosuppressant. An “immunosuppressant” is a molecule that reduces or eliminates an immune response in a host when the host is challenged with an immunogenic molecule, including immunogenic molecules present on transplanted organs, tissues or cells. Preferred immunosuppressants include, without limitation, members of the cyclosporin family and beauverolide L. Preferred cyclosporins include, without limitation, cyclosporin A and cyclosporin C.

[0048] In certain embodiments of the methods according to this aspect of the invention, the secondary metabolite is an ergot alkaloid. An “ergot alkaloid” is a member of a large family of alkaloid compounds that are most often produced in the sclerotia of fungi of the genus Claviceps. An “alkaloid” is a small molecule that contains nitrogen and has basic pH characteristics. The classes of ergot alkaloids include clavine alkaloids, lysergic acids, lysergic acid amides, and ergot peptide alkaloids. Preferred ergot alkaloids include, without limitation, ergotamine, ergosine, ergocristine, ergocryptine, ergocornine, ergotaminine, ergosinine, ergocristinine, ergocryptinine, ergocorninine, ergonovine, ergometrinine, and ergoclavine.

[0049] In certain embodiments of the methods according to this aspect of the invention, the secondary metabolite is an inhibitor of angiogenesis. An “angiogenesis inhibitor” is a molecule that decreases or prevents the formation of new blood vessels. Angiogenesis inhibitors have proven effective in the treatment of several human diseases including, without limitation, cancer, rheumatoid arthritis, and diabetic retinopathy. Preferred inhibitors of angiogenesis include, without limitation, fumagillin and ovalicin.

[0050] In certain embodiments of the methods according to this aspect of the invention, the secondary metabolite is a glucan synthase inhibitor. A “glucan synthase inhibitor” is a molecule that decreases or inhibits the production of 1,3-β-D-glucan, a structural polymer of fungal cell walls. Glucan synthase inhibitors are a class of antifungal agents. Preferred glucan synthase inhibitors include, without limitation, echinocandin B, pneumocandin B, aculeacin A, and papulacandin.

[0051] In certain embodiments of the methods according to this aspect of the invention, the secondary metabolite is a member of the gliotoxin family of compounds. The “gliotoxin family of compounds” are related molecules of the epipolythiodioxopiperazine class. Gliotoxins display diverse biological activities, including, without limitation, antimicrobial, antifungal, antiviral, and immunomodulating activities. Preferred members of the “gliotoxin family of compounds” include, without limitation, gliotoxin and aspirochlorine.

[0052] In certain embodiments of the methods according to this aspect of the invention, the secondary metabolite is a fungal toxin. A “fungal toxin” is a compound that causes a pathological condition in a host, either plant or animal. Fungal toxins could be mycotoxins present in food products, toxins produced by phytopathogens, toxins from poisonous mushrooms, or toxins produced by zoopathogens. Preferred fungal toxins include, without limitation, aflatoxins, patulin, zearalenone, cytochalasin, griseofulvin, ergochrome, cercosporin, marticin, xanthocillin, coumarins, tricothecenes, fusidanes, sesterpenes, amatoxins, malformin A, phallotoxins, pentoxin, HC toxin, psilocybin, bufotenine, lysergic acid, sporodesmin, pulcheriminic acid, sordarins, fumonisins, ochratoxin A, and fusaric acid.

[0053] In certain embodiments of the methods according to this aspect of the invention, the secondary metabolite is a modulator of cell surface receptor signaling. As used herein, the term “cell surface receptor” means a molecule that resides at or in the plasma membrane, binds an extracellular signaling molecule, and transduces this signal to propagate a cellular response. Modulators of cell surface receptor signaling might function by one of several mechanisms including, without limitation, acting as agonists or antagonists; sequestering a molecule that interacts with a receptor, such as a ligand; or stabilizing the interaction of a receptor and a molecule with which it interacts. Preferred modulators of cell surface signaling include, without limitation, the insulin receptor agonist L-783,281 and the cholecystokinin receptor antagonist asperlicin.

[0054] In certain embodiments of the methods according to this aspect of the invention, the secondary metabolite is a plant growth regulator. A “plant growth regulator” is a molecule that controls growth and development of a plant by affecting processes that include, without limitation, division, elongation, and differentiation of cells. Preferred plant growth regulators include, without limitation, cytokinin, auxin, gibberellin, abscisic acid, and ethylene.

[0055] In certain embodiments of the methods according to this aspect of the invention, the secondary metabolite is a pigment. A “pigment” is a substance that imparts a characteristic color. Preferred pigments include, without limitation, melanins and carotenoids.

[0056] In certain embodiments of the methods according to this aspect of the invention, the secondary metabolite is an insecticide. An “insecticide” is a molecule that is toxic to at least some insects. A preferred insecticide, without limitation, is nodulisporic acid.

[0057] In certain embodiments of the methods according to this aspect of the invention, the secondary metabolite is an anti-neoplastic compound. An “anti-neoplastic” compound is a molecule that prevents or reduces tumor formation. Preferred anti-neoplastic compounds include, without limitation, taxol (paclitaxel) and related taxoids.

[0058] In certain embodiments of the methods according to this aspect of the invention, the methods further comprise purifying the secondary metabolite from a culture of the fungus. “Purifying” means obtaining the secondary metabolite in substantially pure form. “Substantially pure” means comprising at least 90%, more preferably at least 95%, and most preferably at least 99%, of the purified composition on a dry-weight basis.

[0059] In a second aspect, the invention provides methods for improving production of a secondary metabolite by a fungus by increasing productivity of the secondary metabolite in the fungus, the methods comprising modulating the expression of a ZBC gene or gene variant in a manner that improves the productivity of the secondary metabolite.

[0060] “Improves the productivity” means to increase the quotient of the concentration of the secondary metabolite divided by the product of the fermentor run-time multiplied by the fermentation volume multiplied by the grams of the dry cell weight of biomass (Productivity=concentration metabolite/(time×volume ×gDCW)).

[0061] Significant advantages that might result from increasing productivity include, without limitation, a decrease in fermentor run-time, a decrease in the size of the fermentor required for production of equivalent amounts of secondary metabolite, or a decrease in the biomass required for production, which translates into decreased waste that must be handled in downstream processing. Preferably, such increased productivity is by at least ten percent, more preferably at least 50 percent, and most preferably at least two-fold.

[0062] “Modulating the expression of a ZBC gene” is as used before. In certain embodiments of the methods according to this aspect of the invention, the modulation is over-expression of the ZBC gene. “Over-expression of the gene” is as used before. In certain embodiments of the methods according to this aspect of the invention, the modulation is expression of a dominant mutation of the gene. The term “dominant mutation” is as used before. Preferred dominant mutations according to this aspect of the invention are as used before. In certain embodiments of the methods according to this aspect of the invention, the modulation is conditional expression of the gene. The term “conditional expression” of a gene is as used before.

[0063] In the methods according to this aspect of the invention, the term “secondary metabolite” is as used previously and preferred secondary metabolites include, without limitation, those discussed previously. In certain embodiments of the methods according to this aspect of the invention, the methods further comprise purifying the secondary metabolite from a culture of the fungus. The term “purifying” is as used before.

[0064] In a third aspect, the invention provides methods for improving production of a secondary metabolite in a fungus by increasing efflux or excretion of the secondary metabolite, the method comprising modulating the expression of a ZBC gene or gene variant in a manner that increases efflux or excretion of the secondary metabolite. “Increasing efflux or excretion of the secondary metabolite” means that, without lysing a fungal cell, a greater quantity of the secondary metabolite passes from the inside of the fungal cell to the outside of the fungal cell per unit time. “Outside of the fungal cell” is defined as being no longer contained wholly within the lipid bilayer of the cell and/or extractable from the cell with methods which do not release a majority of intracellular contents. “Modulating the expression of a ZBC gene” is as used before, except that the ZBC gene can be the Aspergillus spp. aflR gene when the secondary metabolite is aflatoxin or sterigmatocystin, and the ZBC gene can be lovE when the secondary metabolite is lovastatin. In certain embodiments of the methods according to this aspect of the invention, the modulation is over-expression of the gene. “Over-expression of the gene” is as used before. In certain embodiments of the methods according to this aspect of the invention, the modulation is expression of a dominant mutation of the gene. The term “dominant mutation” is as used before. Preferred dominant mutations according to this aspect of the invention are as used before. In certain-embodiments of the methods according to this aspect of the invention, the modulation is conditional expression of the gene. The term “conditional expression” of a gene is as used before.

[0065] In the methods according to this aspect of the invention, the term “secondary metabolite” is as used previously and preferred secondary metabolites include, without limitation, those discussed previously. In certain embodiments of the methods according to this aspect of the invention, the methods further comprise purifying the secondary metabolite from a culture of the fungus. The term “purifying” is as used before.

[0066] In a fourth aspect, the invention provides methods for improving production of a secondary metabolite in a fungus by decreasing production of side products or non-desired secondary metabolites, the method comprising modulating the expression of a ZBC gene or gene variant in a manner that decreases production of side products or non-desired secondary metabolites. “Decreasing production of side products or non-desired secondary metabolites” means reducing the amount of such side products or non-desired secondary metabolites that are synthesized or which are retained within the cells or the media surrounding the cells. Preferably, such reduction is at least by 25%, more preferably by at least 50%, even more preferably by at least 2-fold, and most preferably by at least 5-fold. “Modulating the expression of a ZBC gene” is as used for the third aspect of the invention. In certain embodiments of the methods according to this aspect of the invention, the modulation is over-expression of the gene. “Over-expression of the gene” is as used before. In certain embodiments of the methods according to this aspect of the invention, the modulation is expression of a dominant mutation of the gene. The term “dominant mutation” is as used before. Preferred dominant mutations according to this aspect of the invention are as used before. In certain embodiments of the methods according to this aspect of the invention, the modulation is conditional expression of the gene. The term “conditional expression” of a gene is as used before.

[0067] In the methods according to this aspect of the invention, the term “secondary metabolite” is as used previously and preferred secondary metabolites include, without limitation, those discussed previously. In certain embodiments of the methods according to this aspect of the invention, the methods further comprise purifying the secondary metabolite from a culture of the fungus. The term “purifying” is as used before.

[0068] In a fifth aspect, the invention provides methods for improving production of a secondary metabolite in a fungus by altering the characteristics of the fungus in a manner that is beneficial to the production of the secondary metabolite, the method comprising modulating the expression of a ZBC gene or gene variant in a manner that alters the characteristics of the fungus.

[0069] “Altering the characteristics” means changing the morphology or growth traits of the fungus. Preferred alterations include, without limitation, alterations that result in transition of the fungus from the hyphal to the yeast form; alterations that result in transition of the fungus from the yeast to the hyphal form; alterations that lead to more or less hyphal branching; alterations that increase or decrease flocculence, adherence, cell buoyancy, surface area of the fungus, cell wall integrity and/or stability, pellet size, vacuole formation, and/or ability to grow at higher or lower temperatures; and alterations that increase the saturating growth density of a culture or rate of pellet formation. “Modulating the expression of a ZBC gene” is as used for the third aspect of the invention. In certain embodiments of the methods according to this aspect of the invention, the modulation is over-expression of the gene. “Over-expression of the gene” is as used before. In certain embodiments of the methods according to this aspect of the invention, the modulation is expression of a dominant mutation of the gene. The term “dominant mutation” is as used before. Preferred dominant mutations according to this aspect of the invention are as used before. In certain embodiments of the methods according to this aspect of the invention, the modulation is conditional expression of the gene. The term “conditional expression” of a gene is as used before.

[0070] In the methods according to this aspect of the invention, the term “secondary metabolite” is as used previously and preferred secondary metabolites include, without limitation, those discussed previously. In certain embodiments of the methods according to this aspect of the invention, the methods further comprise purifying the secondary metabolite from a culture of the fungus. The term “purifying” is as used before.

[0071] In a sixth aspect, the invention provides methods for improving production of a secondary metabolite in a fungus by causing conditional lysis of the fungus, the method comprising modulating the expression of a ZBC gene or gene variant in a manner that causes conditional lysis. “Causing conditional lysis” means causing the fungus to grow without lysis under a first set of growth conditions and to lyse under a second and different set of conditions, which are not lytic to the unmodified fungus. In preferred embodiments, the conditions that can be altered between the first and second growth conditions include, without limitation, the source or amount of nutrients such as carbon, nitrogen, and phosphate; the source or amount of specific enzymes; the source or amount of specific components found in cell walls; the amount of salts or osmolytes; the pH of the medium; the partial oxygen pressure; temperature; and the amount of specific small molecules.

[0072] “Modulating the expression of a ZBC gene” is as used for the third aspect of the invention. In certain embodiments of the methods according to this aspect of the invention, the modulation is over-expression of the gene. “Over-expression of the gene” is as used before. In certain embodiments of the methods according to this aspect of the invention, the modulation is expression of a dominant mutation of the gene. The term “dominant mutation” is as used before. Preferred dominant mutations according to this aspect of the invention are as used before. In certain embodiments of the methods according to this aspect of the invention, the modulation is conditional expression of the gene. The term “conditional expression” of a gene is as used before.

[0073] In the methods according to this aspect of the invention, the term “secondary metabolite” is as used previously and preferred secondary metabolites include, without limitation, those discussed previously. In certain embodiments of the methods according to this aspect of the invention, the methods further comprise purifying the secondary metabolite from a culture of the fungus. The term “purifying” is as used before.

[0074] In a seventh aspect, the invention provides methods for improving production of a secondary metabolite in a fungus by increasing the resistance of the fungus to the deleterious effects of-exposure to a secondary metabolite made by-the same organism, the method comprising modulating the expression of a ZBC gene or gene variant in a manner that increases resistance to the deleterious effects of exposure to a secondary metabolite. As used herein, the phrase “increasing the resistance of the fungus to the deleterious effects of exposure to a secondary metabolite” means to allow the fungus to survive, grow, or produce the secondary metabolite in conditions that otherwise would be toxic to the fungus or prevent the production of the secondary metabolite. In particular, the growth of a fungus that produces a secondary metabolite can be limited, in part, by the toxic effects of the secondary metabolite itself. In the absence of resistance mechanisms to protect the fungi from the toxic effects of these metabolites, decreased yields of the metabolite can be observed. For example, Alexander et al. (1999), Mol. Gen. Genet. 261: 977-84, have shown that the trichothecene efflux pump of Fusarium sporotrichiodes, encoded by the gene TR112, is required both for high level production of, and resistance to the toxic effects of, trichothecenes produced by this fungus. Thus, modifications that increase the resistance of a fungus to a toxic secondary metabolite that it produces can increase the saturation density and extend the metabolically active lifetime of the producing fungus. In a bioreactor, such attributes will have the beneficial effect of increasing the yield and productivity of a metabolite. Regulators of secondary metabolite production whose expression can be modulated to increase resistance of a fungus to toxic metabolites can include, without limitation, transporters that promote efflux of the metabolite from cells, enzymes that alter the chemical structure of the metabolite within cells to render it non-toxic, target(s) of the metabolite that mediate its toxicity, and gene products that alter cellular processes to counteract the toxic effects of a metabolite. Additional benefits of increasing efflux of secondary metabolites include increasing the amount of metabolite available for purification from the fermentation broth and mitigation of feedback inhibition of secondary metabolism that may be mediated by the metabolite itself. Indeed, feedback inhibition of a biosynthetic pathway by a product of that pathway is well documented in many microorganisms, and this inhibition can act at the transcriptional, translational, and post-translational levels. Several well-documented examples in yeast include the transcriptional repression of lysine biosynthetic genes by lysine (Feller et al. (1999), Eur. J. Biochen. 261: 163-70), the decreased stability of both the mRNA encoding the uracil permease Fur4p and the permease itself in the presence of uracil (Seron et al. (1999), J. Bacteriol. 181: 1793-800), and the inhibition of alpha-isopropyl malate synthase, a key step in leucine biosynthesis, by the presence of leucine (Beltzer et aL (1988), J. Biol. Chem. 263:368-74).

[0075] Transcription factors that regulate the expression of efflux pumps could also be used to increase efflux of a drug from a fungal cell to increase the yields of a metabolite and decrease the toxicity of the secondary metabolite in a fermentation. Such transcription factors include, but are not limited to, ZBC genes such as PDR1, and PDR3 from S. cerevisiae and their homologs. Over-expression of each of these genes has been shown to up-regulate expression of transporters and cause increased resistance of S. cerevisiae to toxic compounds (for examples, see Reid et al. (1997), J. Biol. Chem. 272: 12091-9; Katzmann et al. (1994), Mol. Cell. Biol. 14: 4653-61; Wendler et al. (1997), J. Biol. Chem. 272: 27091-8).

[0076] Increases in resistance to the toxic effects of secondary metabolites will vary with the metabolite. For example, amatoxins kill cells by inhibiting the function of the major cellular RNA polymerase, RNA polymerase II, in eucaryotic cells. Mutant forms of RNA polymerase II resistant to the effects of alpha-amanitin have been described (Bartolomei et al. (1988), Mol. Cell. Biol. 8: 330-9; Chen et al. (1993), Mol. Cell. Biol. 13: 4214-22). Similarly, mutations affecting HMG CoA reductase, the target enzyme for the secondary metabolite lovastatin, have been identified. Increased levels of HMG CoA Reductase can also cause resistance to lovastatin (Ravid et al. (1999), J. Biol. Chem. 274: 29341-51; Lum et al. (1996), Yeast 12: 1107-24). Taxol (paclitaxel), causes lethality by increasing microtubule stability, thus preventing exit from mitosis. Dominant mutations affecting β-tubulin that confer resistance to taxol have been characterized (for example, see Gonzalez et aL (1999), J. Biol. Chem. 274:23875-82) and could prove to be useful to confer resistance to this toxic metabolite in production strains. The pneumocandin and echinocandin families of metabolites are fungal secondary metabolites that inhibit the enzyme 1,3-β-D-glucan synthase. Dominant mutations in the C. albicans glucan synthase gene, FKSI, have been shown to confer resistance to candins (Douglas et al. (1997), Antinzicrob. Agents Chemother. 41: 2471-9). Glucan synthase mutations such as these could be used to generate fungal production strains with increased resistance to the candin class of antifungals. S. cerevisiae mutants resistant to the growth-inhibitory effects of the fungal secondary metabolite cyclosporin A have also been described (Cardenas et al. (1995), EMBO J. 14: 2772-83). These mutants were shown to harbor mutations in CNA1, the gene encoding the catalytic subunit of the heterodimeric calcium-calmodulin dependent phosphatase, calcineurin A. Fumagillin, an antiangiogenic agent, binds to and inhibits the N-terminal aminopeptidases in a wide variety of both procaryotes and eucaryotes (Sin et al. (1997), Proc. Natl. Acad. Sci. USA 94: 6099-103, Lowther et al. (1998), Proc. Natl. Acad Sci. USA 95: 12153-7). Mutations in this enzyme that block fumagillin binding and/or inhibitory activity could well prove useful in enhancing the resistance of fungal production strains to the growth inhibitory effects of this secondary metabolite. “Modulating the expression of a ZBC gene” is as used for the third aspect of the invention. In certain embodiments of the methods according to this aspect of the invention, the modulation is over-expression of the gene. “Over-expression of the gene” is as used before. In certain embodiments of the methods according to this aspect of the invention, the modulation is expression of a dominant mutation of the gene. The term “dominant mutation” is as used before. Preferred dominant mutations according to this aspect of the invention are as used before. In certain embodiments of the methods according to this aspect of the invention, the modulation is conditional expression of the gene. The term “conditional expression” of a gene is as used before.

[0077] In the methods according to this aspect of the invention, the term “secondary metabolite” is as used previously and preferred secondary metabolites include, without limitation, those discussed previously. In certain embodiments of the methods according to this aspect of the invention, the methods further comprise purifying the secondary metabolite from a culture of the fungus. The term “purifying” is as used before.

[0078] In an eighth aspect, the invention provides methods for improving production of a secondary metabolite in a fungus by modulating the expression of one or more genes, the method comprising modulating the expression of a ZBC gene or gene variant that does not normally modulate the expression of such gene or genes.

[0079] In a ninth aspect, the invention provides genetically modified fungi, wherein the genetically modified fungi have an ability to produce secondary metabolites and the ability of the genetically modified fungus to produce secondary metabolites has been improved by any of the methods according to the invention.

[0080] In a tenth aspect, the invention provides a method for making a secondary metabolite, the method comprising culturing a genetically modified fungus according to the invention under conditions suitable for the production of secondary metabolites.

[0081] Nine ZBC genes (and derivatives thereof) have been tested to date for effects on either lovastatin yield in A. terreus or penicillin production in P. chrysogenum, and in some cases both. Of these genes (lovE, lovU, An 13, At 18, CAT8, SIP4, LYS14, tamA, and YAF1) three of the nine (lovU, At18 and LYS14), or 33%, have demonstrable positive effects on metabolite production. Details of the lovU, At18 and LYS14 results are described in the examples presented below.

[0082] The following examples illustrate some preferred modes of practicing the present invention, but are not intended to limit the scope of the claimed invention. Alternative materials and methods may be utilized to obtain similar results.