Method of producing antihypercholesterolemic agents

Abstract

A method of increasing the production of lovastatin or monacolin J in a iovastatin-producing or non-lovastatin-producing organism is disclosed. In one embodiment, the method comprises the steps of transforming an organism with the A. terreus D4B segment, wherein the segment is translated and where an increase in lovastatin production occurs.

Description

CROSS-REFERENCES TO RELATED APPLICATION

[0002] Not applicable.

BACKGROUND OF THE INVENTION

[0003] Cholesterol and other lipids are transported in body fluids by low-density lipoproteins (LDL) and high-density lipoproteins (HDL). Substances that effectuate mechanisms for lowering LDL-cholesterol may serve as effective antihypercholesterolemic agents because LDL levels are positively correlated with the risk of coronary artery disease.

[0004] MEVACOR (lovastatin; mevinolin) and ZOCOR (simvastatin) are members of a group of active antihypercholesterolemic agents that function by inhibiting the rate-limiting step in cellular cholesterol biosynthesis, namely the conversion of hydroxymethylglutarylcoenzyme A (HMG-CoA) into mevalonate by HMG-CoA reductase.

[0005] The general biosynthetic pathway of a naturally occurring HMG-CoA reductase inhibitor has been outlined by Moore, et al., who showed that the biosynthesis of mevinolin (lovastatin) by Aspergillus terreus ATCC 20542 begins with acetate and proceeds via a polyketide pathway (R. N. Moore, et al., J. Amer. Chem. Soc. 107:3694-3701, 1985). Endo, et al. described similar biosynthetic pathways in Pencillium citrinum NRRL 8082 and Monascus ruber M-4681 (A. Y. Endo, et al., J. Antibiot. 38:444-448, 1985).

[0006] The recent commercial introduction of microbial HMG-CoA reductase inhibitors has fostered a need for high yielding production processes. Methods of improving process yield have included scaling up the process, improving the culture medium and simplifying the isolation.

[0007] Previous attempts to increase the biosynthesis of HMG-CoA reductase inhibitors at the level of gene expression have focused on increasing the concentration triol polyketide synthase (TPKS), a multifunctional protein with at least six activities as evidenced by the product of the enzymatic activity (Moore, supra, 1985). TPKS is believed to be the rate-limiting enzymatic activity(ies) in the biosynthesis of the HMG-CoA reductase inhibitor compounds.

[0008] U.S. Pat. No. 5,744,350 identifies a DNA encoding triol polyketide synthase (TPKS) from Aspergillus terreus. “NPKS” is now preferred to TPKS as the acronym for “nonaketide polyketide synthase.”

SUMMARY OF THE INVENTION

[0009] In one embodiment, the present invention is a method of increasing the production of lovastatin in a lovastatin-producing organism. The method comprises the steps of transforming the organism with a nucleic acid sequence comprising the D4B segment, preferably comprising nucleotides 579-33,000 of SEQ ID NO: 18 and 1-5,349 of SEQ ID NO: 19. The nucleic acid sequence is transcribed and translated and an increase in lovastatin production occurs. Preferably, this increase is at least 2-fold.

[0010] In a preferred form of the present invention, the lovastatin-producing organism is selected from the group consisting A. terreus ATCC 20542 and ATCC 20541.

[0011] In another embodiment, the method comprises the step of transforming the organism with the corresponding D4B segment isolated from a non-A. terreus lovastatin-producing organism.

[0012] In another embodiment, the present invention is a method of increasing the production of lovastatin in a lovastatin-producing organism, comprising the step of transforming the organism with the LovE gene, wherein the nucleic acid sequence is transcribed and translated and wherein an increase in lovastatin production occurs.

[0013] In another embodiment of the present invention, one may increase the production of monacolin J in a non-lovastatin-producing organism comprising the steps of transforming the organism with a nucleic acid sequence comprising the D4B segment. As a further step, one may additionally transform the organism with an entire LovF gene. If the entire LovF gene is added to the D4B segment, the organism will produce lovastatin.

[0014] In another embodiment, the present invention is the lovastatin production gene cluster, preferably SEQ ID NOs: 18 and 19, and the individual genes comprising that cluster.

[0015] It is an object of the present invention to provide a method for increasing lovastatin and monacolin J production in both lovastatin-producing and non-lovastatin producing organisms.

[0016] Other objects, features and advantages of the present invention will become apparent after review of the specification, claims and drawings.

DESCRIPTION OF DRAWINGS

[0017] FIG. 1 is a diagram of lovastatin production genes.

[0018] FIG. 2 is a schematic diagram of a hypothetical mevinolin/lovastatin biosynthesis pathway.

[0019] FIG. 3 is a comparative diagram of statins.

[0020] FIG. 4 is a schematic drawing of plasmid pWHM1264/CB24A.

[0021] FIG. 5 is a schematic drawing of plasmid pWHM1424.

[0022] FIG. 6 is a schematic drawing of plasmid CD4B/pWHM1263.

DESCRIPTION OF THE INVENTION

[0023] In General

[0024] The Examples below disclose the cloning and sequencing of a cluster of 17 genes from A. terreus ATCC 20542, a strain that natively produces lovastatin (See FIG. 1). These genes flank the NPKS gene, which is known to be required for lovastatin production (see, for example, U.S. Pat. No. 5,744,350).

[0025] The DNA sequence of the cluster has been determined and is disclosed below at SEQ ID NOs: 18 and 19. Mutations in four of the genes (P450I/LovA, SEQ ID NO: 22; dehydrogenase/LovC, SEQ ID NO: 24; esterase/LovD, SEQ ID NO: 25; and ScPKS/LovF, SEQ ID NO: 29) have been isolated and demonstrate that each of these four individual genes is required for lovastatin production. These genes are indicated with an X symbol in FIG. 1 and referred to herein as the “A. terreus lovastatin gene cluster.”

[0026] Another of the genes (Zn Finger I/LovE, SEQ ID NO: 27) is thought to regulate the transcription of the other genes and causes a notable increase in lovastatin production when reintroduced into A. terreus ATCC 20542.

[0027] Applicants have used the following convention in naming the genes and proteins of the present invention. The genes and proteins are first named with either an “ORF” or “Lov” prefix and then named either numerically or alphabetically. “Lov” signifies genes shown to be essential for lovastatin production. Applicants have also included a descriptor name that describes a probable function of the protein. For example, SEQ ID NO: 1 is described as the “ORF1/esterase-like protein” because Applicants have compared the amino acid sequence to known esterases.

[0028] The portion of the gene cluster between ORF1/esterase-like protein and the mid-region of LovF/SCPKS is referred to as the “D4B segment”. The A. terreus D4B segment is contained within a plasmid clone deposited as ATCC 98876. As described below, other lovastatin-producing organisms contain an analogous D4B segment comprising analogous genes. The present invention comprises a “D4B segment” isolated from other lovastatin-producing organisms. The arrangment of the genes within the D4B segment may be different in other organisms. We predict that the genes within these other segments will have at least 80% homology, at the nucleic acid level, with the genes disclosed herein. We envision that each of these lovastatin-producing organisms will comprise within their genomes a LovA, LovB, LovC, LovD, LovE and LovF gene.

[0029] We have determined that the D4B segment will confer production of monocolin J if the genes are all expressed, as we show below in an example using A. nidulans. We envision that adding the LovF gene to the D4B segment genes will result in the production of lovastatin in a non-lovastatin-producing organism.

[0030] Table 1, below, summarizes information regarding the different protein and nucleic acid sequences of the present invention. SEQ ID NOs: 1-17 are predicted translation products of various members of the gene cluster. SEQ ID NOs: 18 and 19 are the entire DNA sequence of the gene cluster. SEQ ID NOs: 21-36 are the genomic DNA sequences of the various members of the gene cluster and include the introns. These DNA sequences are reported in the Sequence Listing in the 5′- 3′ orientation, although, as FIG. 1 indicates, some of these DNA sequences are in the inverted orientation in the actual cluster.

TABLE 1
SEQ ID NO.	DESCRIPTION	COMMENTS
SEQ ID NO: 1	Predicted amino acid sequence	Translation of 6 EXONS 6865-6568,
	of ORF1/Esterase-like protein	6462-5584, 5520-4822, 4774-3511,
		3332-2372, 2301-1813
		(reverse complement)
		FROM SEQ ID NO: 18
SEQ ID NO: 2	Predicted amino acid sequence	Translation of 1 EXON 7616-8602
	of ORF2	FROM SEQ ID NO: 18
SEQ ID NO: 3	Predicted amino acid sequence	Translation of 1 EXON 10951-9980
	of LovA/P4501 protein	(reverse complement)
		FROM SEQ ID NO: 18
SEQ ID NO: 4	Predicted amino acid sequence	Translation of 1 EXON 22760-21990
	of ORF5	(reverse complement)
		FROM SEQ ID NO: 18
SEQ ID NO: 5	Predicted amino acid sequence	Translation of 3 EXONS 23158-23717,
	of LovC/Dehydrogenase	23801-23912, 23991-24410
		FROM SEQ ID NO: 18
SEQ ID NO: 6	Predicted amino acid sequence	Translation of 3 EXONS 26203-26080,
	of LovD/Esterase	26005-25017, 24938-24810
		(reverse complement)
		FROM SEQ ID NO: 18
SEQ ID NO: 7	Predicted amino acid sequence	Translation of 5 EXONS 30062-29882,
	of ORF8/HMG CoA Reductase	29803-29745, 29664-27119,
		27035-26779, 26722-26559 (reverse
		complement)
		FROM SEQ ID NO: 18
SEQ ID NO: 8	Predicted amino acid sequence	Translation of 1 EXON 31360-32871
	of LovE/Zn Finger I	FROM SEQ ID NO: 18
SEQ ID NO: 9	Predicted amino acid sequence	Translation of 8 EXONS 1400-1452,
	of ORF10/Metabolite transport	1619-1695, 1770-1996, 2065-2088,
		2154-2225, 2332-2865, 2939-3099,
		3180-3560
		FROM SEQ ID NO: 19
SEQ ID NO: 10	Predicted amino acid sequence	Translation of 7 EXONS 4430-4627,
	of LovF/ScPKS	4709-4795, 4870-4927, 4985-5318,
		5405-5912, 5986-6565, 6631-12464
		FROM SEQ ID NO: 19
SEQ ID NO: 11	Predicted amino acid sequence	Translation of 3 EXONS 13596-13496,
	of ORF12	13451-13063, 12968-12709
		(reverse complement)
		FROM SEQ ID NO: 19
SEQ ID NO: 12	Predicted amino acid sequence	Translation of 5 EXONS 16608-16463
	of ORF13/Zn Finger II	16376-15572, 15519-15346,
		15291-14825, 14767-14131 (reverse
		complement)
		FROM SEQ ID NO: 19
SEQ ID NO: 13	Predicted amino acid sequence	Translation of 7 EXONS 19642-19571,
	of ORF14/Acetyl CoA	19502-19427, 19352-19227,
	transport protein	19158-19011, 18956-18663, 18587-18438,
		18380-18341 (reverse
		complement)
		FROM SEQ ID NO: 19
SEQ ID NO: 14	Predicted amino acid sequence	Translation of 2 EXONS 20332-20574,
	of ORF15	20631-21860
		FROM SEQ ID NO: 19
SEQ ID NO: 15	Predicted amino acid sequence	Translation of 5 EXONS 24521-24054,
	of ORF16/Membrane transport	23996-23936, 23876-23184,
	protein	23111-22977, 22924-22818 (reverse
		complement)
		FROM SEQ ID NO: 19
SEQ ID NO: 16	Predicted amino acid sequence	Translation of 3 EXONS 28525-27673,
	of ORF17/P450II protein	27606-27284, 27211-26837
		(reverse complement)
		FROM SEQ ID NO: 19
SEQ ID NO: 17	Predicted amino acid sequence	Translation of 2 EXONS 29826-30995,
	of ORF18 (incomplete)	31054-31328 (incomplete)
		FROM SEQ ID NO: 19
SEQ ID NO: 18	DNA sequence of gene cluster-
	first 33,000 nucleotides
SEQ ID NO: 19	DNA sequence of cluster-
	nucleotides 33,001-64,328
	(renumbered 1-31,328)
SEQ ID NO: 20	DNA sequence of	Start = 6865
	ORF1/Esterase-like gene	Stop = 1813
		SEQ ID NO: 18
SEQ ID NO: 21	DNA sequence of ORF2	Start = 7616
		Stop = 8602
		SEQ ID NO: 18
SEQ ID NO: 22	DNA sequence of LovA/P450I	Start = 10951
	gene	Stop = 9980
		SEQ ID NO: 18
SEQ ID NO: 23	DNA sequence of ORF5	Start = 22760
		Stop = 21990
		SEQ ID NO: 18
SEQ ID NO: 24	DNA sequence of	Start = 23158
	LovC/Dehydrogenese	Stop = 24410
		SEQ ID NO: 18
SEQ ID NO: 25	DNA sequence of	Start = 24810
	LovD/Esterase	Stop = 26203
		SEQ ID NO: 18
SEQ ID NO: 26	DNA sequence of ORF8/HMG	Start = 30062
	CoA Reductase	Stop = 26559
		SEQ ID NO: 18
SEQ ID NO: 27	DNA sequence of LovE/Zn	Start = 31360
	Finger I	Stop = 32871
		SEQ ID NO: 18
SEQ ID NO: 28	DNA sequence of	Start = 1400
	ORF10/Metabolite transport	Stop = 3560
		SEQ ID NO: 19
SEQ ID NO: 29	DNA sequence of LovF/ScPKS	Start = 4430
		Stop = 12464
		SEQ ID NO: 19
SEQ ID NO: 30	DNA sequence of ORF12	Start = 13596
		Stop = 12709
		SEQ ID NO: 19
SEQ ID NO: 31	DNA sequence of ORF13/Zn	Start = 16608
	Finger II	Stop = 14131
		SEQ ID NO: 19
SEQ ID NO: 32	DNA sequence of	Start = 19642
	ORF14/Acetyl CoA transport	Stop = 18341
	gene	SEQ ID NO: 19
SEQ ID NO: 33	DNA sequence of ORF15	Start = 20332
		Stop = 21860
		SEQ ID NO: 19
SEQ ID NO: 34	DNA sequence of	Start = 24521
	ORF16/Membrane transport	Stop = 22818
	protein	SEQ ID NO: 19
SEQ ID NO: 35	DNA sequence of	Start = 28525
	ORF17/P450II gene	Stop = 26837
		SEQ ID NO: 19
SEQ ID NO: 36	DNA sequence of ORF18	Start = 29826
	(incomplete)	to 31328 (incomplete)
		SEQ ID NO: 19

[0031] Table 1 also notes the translation start and stop points in the various gene sequences.

[0032] The sequence of the NPKS gene is not listed in SEQ ID NOs: 21-36. This gene is characterized in U.S. Pat. No. 5,744,350. However, SEQ ID NOs: 18 and 19 do contain the sequence of the NPKS gene within the context of the entire gene cluster.

[0033] To perform many embodiments of the present invention, one will need to recreate various genes or a portion of the gene cluster described herein. Applicants have provided sequence data in the Sequence Listing sufficient to allow one of skill in the art to construct numerous probes suitable to recreate the genes from an A. terreus genomic library. Applicants have also described below various methods for isolating A. terreus DNA.

[0034] Additionally, Applicants have deposited ATCC Accession No. ATCC 98876, which contains clone pWHM1263 (cD4B) and ATCC Accession No. ATCC 98877 which contains clone pWHM1265 (CB2A4). Both plasmids are described in more detail below. FIG. 4 describes clone CB2A4/pWHM1265, and FIG. 6 describes clone CB4B/pWHM1263. FIG. 1 also indicates the boundaries of the D4B and B2A4 clones.

[0035] The clones and their inserts may be prepared from the ATCC deposits by methods known to those of skill in the art. The DNA from the clones may be isolated and any gene within the gene cluster may be isolated and utilized.

[0036] Increasing the Production of Lovastatin by Lovastatin-Producing Fungi and Yeast

[0037] In one embodiment, the present invention is a method of increasing the production of lovastatin in a lovastatin-producing fungi and yeast, preferably A. terreus ATCC20542 and ATCC20541. Other examples of suitable lovastatin-producing fungi and yeast are listed in Table 2, below.

TABLE 2
Microorganisms other than A. terreus reported
to produce lovastatin (mevinolin)
Monascus (17 of 124 strains screened) species1
M. ruber
M. purpureus
M. pilosus
M. vitreus
M. pubigerus
Penicillium sp.1
Hypomyces sp.
Doratomyces sp.
Phoma sp.
Eupenicillium sp.
Gymnoascus sp.
Trichoderma sp.
Pichia labacensis2
Candida cariosilognicola
Aspergilus oryzea3
Doratomyces stemonitis
Paecilomyces virioti
Penicillium citrinum
Penicillin chrysogenum
Scopulariopsis brevicaulis
Trichoderma viride
1P. Juzlova, L. Martinkova, V. Kren. Secondary Metabolites of the fungus Monascus: a J. Ind. Microbiol. 16: 163-170 and references cited therein (1996).
2N. Gunde-Cimerman, A. Plemenitas and A. Cimerman. A hydroxymethylglutaryl-CoA inhibitor synthesized by yeasts. FEMS Microbiol. Lett. 132: 39-43 (1995).
3A. A. Shindia. Mevinolin production by some fungi. Folio Microbiol. 42: 477-480 (1997

[0038] By “increasing the production” we mean that the amount of lovastatin produced is increased by at least 2-fold, preferably by at least 5-fold. The examples below demonstrate two preferred methods for analyzing strains for lovastatin production. In method A, the spore suspension is inoculated into a flask of SEED medium and grown. The resulting seed culture is used to inoculate FM media and grown for six days. In fermentation method B, one inoculates 50 ml of RPM media and grows this larger culture for 7 days.

[0039] Both cultures are extracted, pH adjusted, mixed with ethyl acetate and shaken for two hours. For analysis, 1 ml of the ethyl acetate layer is dried under a nitrogen stream and resuspended in methanol. For TLC analysis, a small amount of the extract is run on C18 reverse phase TLC plates in a solvent system of methanol; 0.1% phosphoric acid. The TLC plates are developed by spraying with phosphomolybdic acid in methanol and heating with a heat gun. The extracts are compared with authentic lovastatin, monacolin J, monacolin L and dihydromonocolon L.

[0040] If one wishes HPLC analysis, the examples below describe the use of a Waters Nova-Pak C18 column used with a solvent system of acetonitrile and phosphoric acid. A Waters 996 Photodiode Array Detector will detect the metabolites. Lovastatin was detected at 238 nm.

[0041] In one embodiment, one would transform a lovastatin-producing fungi or yeast with the lovE/zinc finger I gene, preferably comprising the nucleotides of SEQ ID NO: 27. The examples below predict that this will result in an increase of at least 5-7 fold. Preferably, the increase will be at least 2.0-fold.

[0042] One may also transform a lovastatin-producing fungi or yeast with the LovE gene isolated from other lovastatin-producing fungi or yeast. One may obtain this gene by use of a probe derived from SEQ ID NO: 27 by methods known to those of skill in the art.

[0043] One may also transform lovastatin-producing fungi and yeast with the D4B segment of the lovastatin production gene cluster (see FIG. 1), preferably as found in ATCC accession number 98876. Alternatively, one may transform lovastatin-producing fungi or yeast with the entire gene cluster, as diagramed in FIG. 1.

[0044] We envision that to successfully increase lovastatin production, one may also wish to transform less than the entire gene cluster. Preferably, one may determine what the smallest possible segment is by deleting various portions of the gene cluster and determining whether lovastatin production is continually increased. Similarly, if one begins with the D4B segment, one may delete various portions for the segment and determine whether lovastatin production is continually increased by at least 2-fold.

[0045] Modification of the LovB/NPKS gene would produce other HMG CoA inhibitors. For example, FIG. 3 diagrams the relationship between mevastatin, lovastatin, simvastatin and pravastatin. In one example, the methyl transferase domain of the NPKS gene may be replaced with an inactive form to make pravastatin. The HMG-CoA reductase inhibitors within this invention include, but are not limited to, compactin (ML-236B), lovastatin, simvastatin, pravastatin and mevastatin.

[0046] In another embodiment of the present invention, one may transform a lovastatin-producing organism with the genes described above and obtain the production of an HMG CoA reductase inhibitor with a structure different from monacolin J. monacolin L or lovastatin. Alterations in the side chain attached to C8 are the most likely possibility but other alterations may occur. These alterations would happen through the native biochemistry of the organism.

[0047] If one wishes to express the A. terreus genes in yeast, one may wish to consult examples in which others have engineered fungal secondary metabolism genes for expression in yeast. (See for example, J. T. Kealey, et al., Proc. Natl. Acad. Sci. USA 95:505-509 (1998)). The exact approach could be used with the NPKS (LovB) and ScpKS (LovF) genes, and a somewhat simpler approach with the other lovastatin genes in their cDNA forms.

[0048] Production of HMG-CoA Reductase Inhibitors by Fungi and Yeast that do not Natively Produce Inhibitors.

[0049] In another embodiment, the present invention is the production of HMG-CoA reductase inhibitors, such as lovastatin, by fungi and yeast that do not natively produce lovastatin. An example of a suitable fungi or yeast is A. nidulans and S. cerevisiae, respectively.

[0050] For this embodiment one preferably transforms the genes within the D4B segment into the non-inhibitor-producing strain. By this method, one would produce monacolin J (See FIG. 2) which could be chemically converted to lovastatin by one of skill in the art.

[0051] Monacolin J, in its lactone form obtained by treatment with anhydrous acid under dehydrative conditions, is preferably treated with a derivative of (2S)-2-methybutyric acid, in which the carboxyl group has been suitable activated for undergoing esterification, and the resulting lovastatin is isolated by conventional methods. For example, see WO 33538, U.S. Pat. No. 4,444,784 and J. Med. Chem. 29:849 (1986). These are citations for synthesis of simvastatin from monacolin J. One would use the same method, but use the (2S)-2-methylbutyrate derivative to make lovastatin.

[0052] In another embodiment of the present invention, one would transform the genes within the D4B segment, including an entire LovF/SCPKS gene, into the non-inhibitor-producing organism. By this method, one would produce lovastatin in a non-lovastatin-producing organism.

[0053] In another embodiment of the present invention, one may transform a non-lovastatin-producing organism with the genes described above and obtain the production of an HMG CoA reductase inhibitor with a structure different from monacolin J, monacolin L or lovastatin, as described above.

[0054] Modification of the LovB/NPKS gene would produce other inhibitors. For example, FIG. 3 diagrams the relationship between mevastatin, lovastatin, simvastatin and pravastatin. In one example, the methyl transferase domain of the NPKS gene may be replaced with an inactive form to make pravastatin. The HMG-CoA reductase inhibitors within this invention include, but are not limited to, compactin (ML-236B), lovastatin, simvastatin, pravastatin and mevastatin.

[0055] Production of Intermediate Materials

[0056] In another embodiment, the present invention is a method of isolating intermediate materials in the production of lovastatin and analogs such as mevastatin and simvastatin. For example, the Examples below demonstrate the disruption of the lovastatin projection gene cluster with mutagenized LovC, LovD, LovF, LovA or LovB genes. Disruption of many of these genetic elements of the lovastatin production gene cluster will result in accumulation of intermediate materials. Therefore, to practice this embodiment of the present invention, one would transform a suitable lovastatin-producing host with a mutagenized gene within the D4B segment, as described below.

[0057] Many other mutations would be suitable to destroy the function of LovC, LovD, LovF, LovA or LovB. All that is necessary is these genes be disrupted to the extent that they are non-functional.

[0058] Production of Lovastatin Analogs

[0059] In another embodiment, the present invention provides a method for engineering the production of lovastatin analogs in such organisms as fungi or yeast, using monacolin J as the starting point.

[0060] Isolated DNA Segments

[0061] In another embodiment, the present invention is a DNA segment capable of conferring lovastatin or monacolin J production or increase in lovastatin or monacolin J production in yeast or fungi. In a preferred example, this segment is the “D4B segment” that is deposited at ATCC 98876. The nucleotide sequence of this segment is found in residues 579-33,000 of SEQ ID NO: 18 and residues 1-5,349 of SEQ ID NO: 19.

[0062] In another embodiment, the present invention is the entire A. terreus lovastatin gene cluster, as exemplified by SEQ ID NOs: 18 and 19 and ATCC deposits 98876 and 98877.

[0063] The present invention is also the individual genes that make up the A. terreus lovastatin gene cluster. Therefore, the present invention is a nucleic acid segment selected from the group of consisting of SEQ ID NOs: 20-36. Preferably, the present invention is the coding region found within SEQ ID NOs: 20-36 and described in Table 1. The present invention is also a mutagenized version of SEQ ID NOs: 22, 24, 25 and 29, wherein the gene is mutagenized to be non-functional in terms of lovastatin or monacolin J production.

[0064] Organisms with Increased Lovastatin or Monacolin J Production

[0065] In another embodiment, the present invention are the organisms described above. These organisms include lovastatin-producing organisms, preferably yeast and fungi, that have been engineered to display at least a 2-fold increase in lovastatin or monacolin J production. The organisms also include non-lovastatin-producing organisms, preferably yeast or fungi, that have been engineered to produce monacolin J or lovastatin.

[0066] Antifungal Compounds

[0067] Applicants note that lovastatin, monocolin J, monocolin L and dihydromonocolin L all have varying degrees of antifungal activity. Applicants envision that the present invention is also useful for providing antifungal compounds and organisms engineered to express antifungal compounds. Preferably, one would measure the antifungal properties of a compound in the manner of N. Lomovskaya, et al., Microbiology 143:875-883, 1997. Measurement of inhibition of yeast growth can be found in R. Ikeura, et al., J. Antibiotics 41:1148, 1988. The same general methods could be used for all fungi. Both of these references are hereby incorporated by reference.

EXAMPLES

[0068] 1. General Methods and Procedures

[0069] Construction of an A. terreus ATCC20542 Genomic Library.

[0070] A. terreus ATCC20542 genomic DNA was partially digested with Sau3AI so as to produce an average fragment size of 40-50 kb. The partially digested genomic DNA was then separated on a sucrose gradient and the 40-50 kb fraction was collected. Cosmid AN26 (Taylor and Borgmann, Fungal Genet. Newsletter 43, 1996) was prepared by digestion with ClaI, dephosphorylated with CIP, then digested with BamHI to create the two cosmid arms. Ligation reactions with genomic DNA fragments and cosmid arms were optimized and packaged using Gigapack III XL packaging extract (Stratagene). The packaged cosmid library was infected into E. coli JM109 and plated out onto LB agar (Sambrook, et al., Molecular Cloning. A Laboratory Manual. 2nd ed. Cold Spring Harbour Laboratory Press, 1989; other standard methods used can be found here also) with ampicillin (50 μg/ml) plates. After checking for the presence of insert DNA in a selection of clones, 5000 colonies were picked into LB plus 50 μg/ml ampicillin filled microtitre plates and grown overnight at 37° C. The colonies were replica plated onto nylon membranes (Amersham Hybond-N). Glycerol was added at a final concentration of 15% (v/v) to the microtitre plates and these were stored at −70° C.

[0071] Isolation of Genomic Clones Containing the Lovastatin Biosynthesis Cluster.

[0072] A 2.8 kb EcoRI fragment from pTPKS100 containing part of the NPKS gene (Vinci, et al., U.S. Pat. No. 5,744,350) was gel-isolated and labelled with digoxigenin using the Genius Kit II (Boehringer Mannheim). This labelled fragment was hybridized (65° C., 5×SSC) with the nylon membranes containing the A. terreus genomic library, then washed (65° C, 0.1×SSC). Two positive clones were identified, pWHM1263 (cD4B) and pWHM1264 (cJ3A). Two of these clones, pWHM1263 (cD4B) and pWHM1265 (cB2A4), have been deposited in the ATCC (American Type Culture Collection, 10801 University Boulevard, Menassas, Va. 20110) at accession number ATCC 98876 and 98877, respectively, under the terms and conditions of the Budapest Treaty. The presence of the NPKS gene was confirmed initially by restriction digestion and later by DNA sequencing overlapping clones were found by repeating the hybridization process using labelled fragments from both ends of the insert in pWHM1263. This resulted in the isolation of pWHM1265-1270 (cB2A4, cL3E2, cJ3B5, cO2B5, cR3B2, cW3B1) from downstream of the NPKS gene and pWHM1271 (cQ1F1) from upstream of NPKS. All these clones were transformed into E. coli strain STBL2 (Stratagene) to help prevent rearrangements.

[0073] FIG. 4 is a diagram of the cB2A4/pWHM1265 clone. This clone contains an insert of approximately 43 kb in AN26 and includes the nucleotide sequence from at least nucleotides 4988 of SEQ ID NO: 19 to nucleotide 31,328 of SEQ ID NO: 19 and 10-14 kb of uncharacterized DNA. FIG. 6 is a schematic diagram of cD4B/pWHM1263. This clone contains a 37,770 bp insert in AN26 and contains nucleotides 579-33,000 of SEQ ID NO: 18 and nucleotides 1-5,349 of SEQ ID NO: 19.

[0074] Sequencing Strategy and Analysis.

[0075] A series of overlapping subclones (pWHM1272-pWHM1415) were constructed in pSPORT1 (Gibco-BRL) and pGEM3 (Promega). Plasmid DNAs for sequencing were prepared using the QiaPrep spin miniprep kit (Qiagen). Cycle sequencing was carried out using the AmpliTaq FS or BigDye reagents (ABI) and were analyzed using a ABI model 373 or 377 DNA Sequencer. Primer walking was performed by synthesis of 18-22 bp oligonucleotide primers based on the sequenced DNA strand, with the help of the Oligo 4.05 program (National Biosciences, Inc.). Every region of DNA was sequenced at least once on both strands. Direct sequencing of cosmids and PCR products was used to confirm adjoining regions where no overlapping clones existed. DNA sequence analysis and manipulations were performed using SeqMan (DNASTAR) and SeqEd (ABI) software. Assignments of putative ORFS, including putative introns, were performed with the aid of BLAST 2.0 searches (Atschul, et al., Nucl. Acids Res. 25:3389-3402, 1997), and the Genetics Computer Group (GCG) programs (Program Manual for the Wisconsin Package, Version 8, September 1994, Genetics Computer Group, Madison, Wis.), version 8.1.

[0076] Isolation and Characterization of lovF (ScPKS, ORF11), lovD (EST1, ORF7), lovC (DH, ORF6), and lovA (P450I, ORF3) Mutants.

[0077] lovF

[0078] To disrupt the polyketide synthase gene, lovF, a 1.7 kb EcoRI fragment internal to the lovF gene was subcloned from pWHM1265 into pSPORT1 to give pWHM1291. The ScPKS fragment was then subcloned from this vector, as an Acc65I—HindIII fragment, into pPLOA (Vinci, et al., U.S. Pat. No. 5,744,350) to give pWHM1416. This vector contains the phleomycin (Zeocin, obtained from InVitrogen) resistance gene for selection in A. terreus. A. terreus ATCC20542 was then transformed to Zeocin resistance with this plasmid as described below. Transformants were screened for lovastatin production as described below (Method A). In one of the transformants, WMH1731, lovastatin production was abolished and a new compound accumulated. This new compound comigrated with monacolin J on TLC and HPLC according to the methods described below. Semi-preparative HPLC was used to partially purify the major product which was then analyzed by HPLC—MS. The same mass and fragmentation pattern as authentic monacolin J was observed. To confirm the disruption of the lovF gene, total genomic DNA was prepared from wild-type A. terreus ATCC20542 and the WMH1731 mutant strain. The genomic DNA was digested with BamHI and HindIII, electrophoresed on an agarose gel and capillary blotted onto a nylon membrane. The membrane was hybridized with the 1.7 kb EcoRI fragment from pWHM1416 labelled using the Genius II kit (Boehringer Mannheim) using the conditions described previously. The wild-type strain had hybridizing bands at 4.2 kb for BamHI and 11.5 kb for HindIII. As predicted, the WMH1731 mutant strain had hybridizing bands at 6.5 kb and 2.2 kb for DamHI and 11 kb and 7.8 kb for HindIII confirming the homologous integration of a single copy of pWHM1416 at the lovF locus.

[0079] lovD

[0080] To disrupt the putative esterase/carboxypeptidase-like gene, lovD, a 4.8 kb NotI—EcoRI fragment from pWHM1263 was subcloned into pSPORTI to give pWHM1274. This plasmid was digested with HindIII and BsiWI and a 1.8 kb fragment was isolated. The plasmid was also digested with HindIII and BamHI and the 6.6 kb fragment was isolated. pPLOA was digested with BamHI and Acc65I and the 2.1 kb fragment containing the phleomycin resistance marker was purified. These three fragments were ligated together and used to transform competent E. coli cells. The expected plasmid, pWHM1417, containing the phleomycin resistance gene flanked by the beginning and the end of the lovD gene was isolated. This plasmid was linearized by digestion with XbaI or RsrII before being used to transform A. terreus ATCC20542 to Zeocin resistance. Transformants were screened for lovastatin production as described below (Method A). In one of the transformants, WMH1732, lovastatin production was abolished and a new compound accumulated. This new compound comigrated with monacolin J on TLC and HPLC according to the methods described below. Semi-preparative HPLC was used to partially purify the major product which was then analyzed by HPLC—MS. The same mass and fragmentation pattern as authentic monacolin J was observed. To confirm the disruption of the lovD gene, total genomic DNA was prepared from wild type A. terreus ATCC20542 and the WMH1732 mutant strain. The genomic DNA was digested with ApaI, run out on an agarose gel and capillary blotted onto a nylon membrane. The membrane was hybridized with the 4.8 kb NotI—EcoRI fragment from pWHM1274 labelled using the Genius II kit using the conditions described previously. The wild-type strain had hybridizing bands at 9 kb, 8.4 kb and 1.5 kb. As predicted the mutant strain had hybridizing bands at 9 kb, 8 kb, 3 kb and 1.5 kb confirming the homologous integration of a single copy of pWHM1417 at the lovD locus.

[0081] lovA

[0082] To disrupt the cytochrome P450 I gene, lovA, an 11 kb Acc65I—EcoRI fragment from pWHM1263 was subcloned into pGEM3 to give pWHM1272. From this plasmid a 2.1 kb ApaI—SnaBI fragment was purified and ligated to ApaI—EcoRV digested pPLOA to give p450Phleo (pWHM1418). From this plasmid a 4.2 kb ApaI—NotI fragment was purified and ligated with a 1.8 kb EagI—KpnI fragment from pWHM1272 and ApaI—KpnI digested pGEM7 to give p45ODphleo (pWHM1419) which contains the lovA gene disrupted by the phleomycin resistance gene. This plasmid was then digested with KpnI and ApaI and the resulting fragment was used to transform A. terreus ATCC20542 to Zeocin resistance. Transformants were screened for lovastatin production as described below (Method A). In one of the transformants, WMH1733, lovastatin production was abolished and two new compounds accumulated. Genomic DNA was prepared from this strain and from A. terreus ATCC20542, digested with EagI, run out on an agarose gel, and capillary blotted onto a nylon membrane. The membrane was hybridized with the 6 kb ApaI—KpnI fragment from pWHM1419 labelled using the Genius II kit using the conditions described previously. The wild-type strain had hybridizing bands at 2.0 kb, 1.9 kb and 1.1 kb. Mutant strain WMH1733 had hybridizing bands at 2.5 kb, 2.0 kb, 1.1 kb and 0.7 kb confirming the homologous integration of a single copy of the fragment from pWHM1419 at the lovA locus.

[0083] lovC

[0084] To disrupt the dehydrogenase-like gene, lovC, a 2 kb EcoRI—BglII fragment from pTPKS100 was ligated with a 1.7 kb EcoRI—Sacd fragment from pWHM1274 and BglII—SacI digested litmus 28 (New England Biolabs) to produce pDH1 (pWHM1420). Another plasmid pDH2 (pWHM1421) was constructed from a 2.2 kb Acc65I—SadI fragment from pWHM1274, a 2.1 kb HindIII—SadI fragment from pPLOA containing the phleomycin resistance gene and HindIII—Acc65I digested litmus 28. The disruption vector pDH-dis (pWHM1422) was constructed by ligating together a 2.5 kb BglII—HpaI fragment from pWHM1420, a 4.3 kb EcoRV—KpnI fragment from pWHM1421 and BglII—KpnI digested litmus 28. This plasmid was digested with BglII and KpnI and the resulting 6.8 kb fragment was used to transform A. terreus ATCC20542 to Zeocin resistance. Transformants were screened for lovastatin production as described below (Method A). In two of the transformants, WMH1734 and WMH1735, lovastatin production was abolished. Genomic DNA was prepared from these strains and from A. terreus ATCC20542, digested with EagI, run out on an agarose gel, and capillary blotted onto a nylon membrane. The membrane was hybridized with the 6.8 kb BglII—KpnI fragment from pWHM1422 labelled using the Genius II kit using the conditions described previously. The wild type strain had hybridizing bands at 5 kb, 1.5 kb and 1.3 kb. Mutant strain WMH1734 had hybridizing bands at 4.9 kb, 1.3 kb, 1.0 kb and 0.7 kb confirming the homologous integration of a single copy of the fragment from pWHM1422 at the lovC locus. The other mutant strain, WMH1735, had a similar banding pattern but with additional hybridizing bands indicating that multiple integration events had occurred, one of which was at the lovC locus.

[0085] Construction and Characterization of the A. terreus Strain with Extra Copies of lovE.

[0086] A 10.4 kb NotI—EcoRI fragment containing the putative regulatory gene, lovE was subcloned from pWHM1263 to pSPORT1 to give pWHM1276. From this plasmid a 3.9 kb HindIII—BamHI fragment was subcloned into pGEM7 to give pWHM1423. The regulatory gene was subcloned from this vector into pPLOA as an SstI—XbaRI fragment to give pWHM1424 (FIG. 5). pWHM1424 contains nucleotides 30,055-33,000 from SEQ ID NO: 18 and nucleotides 1-1,026 from SEQ ID NO: 19.

[0087] Extra copies of the regulatory gene were introduced into A. terreus ATCC20542 by transformation to Zeocin resistance with pWHM1424. Transformants were fermented (method A) and screened for lovastatin production initially by TLC analysis. Most of the transformants appeared to be producing significantly more lovastatin than the wild-type strain. The yields of lovastatin from the two transformant strains, WMH1736 and WMH1737, which had the most elevated levels compared to the wild-type was quantified by HPLC as described below. These were found to produce 7-fold and 5-fold more lovastatin than the A. terreus ATCC20542 strain.

[0088] Because of the way that the DNA integrates (ectopically), each transformant is or can be unique, genotypically and phenotypically. However, some will be overproducers; others may exhibit no difference, for unknown reasons.

[0089] Heterologous Expression of the Lovastatin Biosynthesis Genes.

[0090] To place the NPKS gene (lovB) under the control of the inducible alcA promoter, the 11.5 kb KpnI—AvrII fragment from pTPKS100 containing the NPKS open reading frame was ligated into pAL3 (Waring, et al., Gene 79:119, 1989) previously digested with KpnI and XbaI. The resulting plasmid was designated pAL3TPKS (WHM1425). The polymerase chain reaction was used to amplify the NPKS gene sequence between the NPKS promoter region just upstream of the translational start codon and a AgeI site internal to NPKS. The design of the forward primer introduced a KpnI site 31 bases from the translational start codon allowing the NPKS to be placed against the alcA promoter but also incorporating upstream elements from the A. terreus system. Amplification was performed using Vent DNA polymerase with pTPKS100 as template and 1 μmol of each primer in a final volume of 100 μl using the manufacturer's buffer recommendations. After an initial denaturation cycle of 10 minutes at 95° C. amplification was achieved with 30 cycles of 95° C. for 1 minute; 55° C. for 1 minute and 72° C. for 1.5 minutes. The final cycle was followed by 10 minutes at 72° C. to ensure complete polymerization. The amplified product (1.7 kb) was digested with KpnI and AgeI and ligated into pWHM1425 that had been digested with the same enzymes and gel isolated. The resulting plasmid was designated pAL3TPKSNT (pWHM1426). The region introduced by PCR was sequenced on a ABI automated DNA sequencer to ensure sequence fidelity. This plasmid was then used to transform A. nidulans strain A722 (Fungal Genetics Stock Centre) to uridine prototrophy.

[0091] Transformants were grown by inoculating 0.5 ml of spore suspension (108 c.f.u./ml) in 50 ml YEPD in a 250 ml unbaffled flask. This was then grown for 20 hours at 250 rpm and 37° C. (New Brunswick Scientific Series 25 Incubator Shaker). The mycelia were then harvested by filtration through Miracloth (Calbiochem), rinsed with sterile, distilled water, and inoculated into fresh 250 ml unbaffled flasks containing 50 ml AMM+lactose+10 mM cyclopentanone and grown for a further 20 hours under the same conditions. The mycelia were harvested by filtration using Miracloth (Calbiochem), squeezed as dry as possible and frozen in liquid nitrogen. Protein extracts for SDS-PAGE and western analysis were prepared as described in Kennedy and Turner, Molec. Gen. Genet. (1996), 253:189-197, 1996.

[0092] One transformant, WMH1738, was shown to have a large protein (>200 kDa) visible on a SDS-PAGE gel that cross reacted with the affinity purified NPKS antibodies (Panlabs). This strain WMH1738 was transformed to hygromycin B resistance with pWHM1263. Transformant colonies were screened for lovastatin resistance and for the production of new metabolites as described below and two strains WMH1739 and WMH1740 were chosen for further analysis. Both of these strains were found to be significantly resistant (up to 100 μg/ml on solid media) to lovastatin compared with the host strain. This was analyzed by streaking 10 μl of a spore suspension on solid AMM plates containing lovastatin at 0, 0.1, 0.5, 1, 5, 10, 50 and 100 μg/ml and incubating at 37° C. Strains WMH1739 and WMH1740 were compared to strains WMH1741 and WMH1742 which were derivatives of WMH1738 transformed to hygromycin resistance with AN26. Strains WMH1739 and −1740 exhibited no inhibition of growth at any of these lovastatin concentrations whereas strains WMH1741 and −1742 showed slight inhibition of grown at 5 μg/ml and almost complete growth inhibition at 50 μg/ml. The two lovastatin resistant strains were fermented in lovastatin-producing conditions using fermentation method B and extracts were analyzed for lovastatin related metabolites as described below. Both strains were found to produce new metabolites. One compound that was common to both comigrated with monacolin J on TLC and HPLC analysis by the methods described below. Semi-preparative HPLC was used to partially purify some of this compound, which was then analyzed by HPLC—MS. It had the same mass and fragmentation pattern as authentic monacolin J. The other compound, found in only one of the strains, comigrated with monacolin L on TLC and HPLC.

[0093] Methods

[0094] Solid Medium for Growth of A. terreus

[0095] For the generation of spore suspensions A. terreus strains were grown on CM agar at 30° C. for 4 to 5 days.

[0096] CM Agar (for CM liquid medium the agar was omitted):

[0097] 50 ml Clutterbuck's salts (Vinci, et al., U.S. Pat. No. 5,744,350)

[0098] 2 ml Vogel's trace elements (Vinci, et al., U.S. Pat. No. 5,744,350)

[0099] 0.5% Difco Bacto tryptone

[0100] 0.5% Difco Bacto yeast extract

[0101] 1% glucose

[0102] 2% Difco Bacto agar

[0103] in 1 liter of distilled water

[0104] Clutterbuck's salts:

[0105] 12% NaNO3

[0106] 1.02% KCl

[0107] 1.04% MgSO4.7H2O

[0108] 3.04% KH2PO4

[0109] Vogel's trace elements:

[0110] 0.004% ZnCl2

[0111] 0.02% FeCl3

[0112] 0.001% CuCl2

[0113] 0.001% MnCl2.4H2O

[0114] 0.001% Na2B4O7.1OH2O

[0115] 0.001% (NH4)6Mo7O24.7H2O

[0116] For long term storage A. terreus spores were suspended in SSS (10% glycerol, 5% lactose) and stored at −70° C.

[0117] For the generation of spore stocks A. nidulans was grown on the following solid growth medium (ACM) for 3 to 4 days at 37° C.

[0118] ACM:

[0119] 2% Difco Bacto malt extract

[0120] 0.1% Difco Bacto peptone

[0121] 2% glucose

[0122] 2% agar (Difco, Detroit, Mich.)

[0123] For strains which required para-aminobenzoic acid (PABA) for growth, PABA was added to a final concentration of 1 μg/ml. For strains which required uracil and uridine these were added at 20 mM and 10 mM, respectively. Spores were suspended in Tween 80—saline solution (0.025% Tween 80, 0.8% NaCl) and stored at 4° C.

[0124] AMM:

[0125] 0.6% (w/v) NaNO3

[0126] 0.052% (w/v) KCl

[0127] 0.152% (w/v) KH2PO4

[0128] 0.052% (w/v) MgSO4.7H2O

[0129] 1% (w/v) glucose

[0130] 0.1% (v/v) AMM trace elements solution pH to 6.5 and make up to 1 liter with distilled water.

[0131] For preparation of plates 2% (w/v) Difco Bacto agar was added. If required the glucose can be omitted and an alternative carbon source (e.g., lactose added at the same concentration). For the preparation of transformation plates KCl was added at 4.47% (w/v) (0.6 M).

[0132] AMM trace elements solution:

[0133] 0.1% (w/v) FeSO4.7H2O

[0134] 0.88% (w/v) ZnSO4.7H2O

[0135] 0.04% (w/v) CuSO4.5H2O

[0136] 0.015% (w/v) MnSO4.4H2O

[0137] 0.01% (w/v) Na2B4O7.10H2O

[0138] 0.005% (NH4)6Mo7O24.7H2O distilled water to 1 liter

[0139] Large Scale Genomic DNA Preparation from A. terreus for Genomic Library Construction.

[0140] A 2.5 ml aliquot of spore suspension (108 c.f.u./ml) was used to inoculate 500 ml of liquid CM medium and grown for 20 hours at 30° C. and 200 rpm. The mycelium was harvested by filtration through Miracloth (Calbiochem) and rinsed extensively with water then TSE [150 mM NaCl, 100 mM Na2EDTA, 50 mM Tris-HCl pH 8.0]. The mycelium was squeezed dry, broken into small pellets and frozen in liquid nitrogen then ground to a fine powder in a pre-chilled pestle and mortar followed by transferral to a 500 ml flask. Fifty ml of extraction buffer [150 mM NaCl, 100 mM Na2EDTA, 50 mM Tris-HCl pH 8.0, 2% (w/v) SDS] and 10 ml of toluene was added to the flask which was shaken at 60 rpm for 72 hours. This mixture was centrifuged at 1000×g for 15 minutes and the supernatant was removed and extracted with an equal volume of chloroform:isoamyl alcohol (24:1 vol/vol). This mixture was centrifuged at 10,000×g for 30 minutes at 15° C. The aqueous layer was carefully removed and 1.1 volumes of ethanol was layered on top. The DNA was spooled out from the resulting suspension and resuspended in 5 ml TE [10 mM Tris-HCl pH 8.0, 1 mM EDTA]+50 μg/ml RNase and 100 μg/ml proteinase K then incubated at 37° C. for 2 hours. The mixture was extracted again with chloroform:isoamyl alcohol (24:1) and the DNA was spooled out as before. Following resuspension in 1 ml of TE the DNA was extracted once with phenol:chloroform:isoamyl alcohol (25:24:1, vol/vol), once with chloroform:isoamyl alcohol (24:1) and precipitated with 0.6 volumes isopropanol. The DNA clot was removed, dried briefly and resuspended in 0.5 ml TE.

[0141] Small Scale Genomic DNA Preparation from A. terreus for Southern Blot.

[0142] A 0.5 ml aliquot of spore suspension (108 c.f.u./ml) was used to inoculate 100 ml of liquid CM and grown for 20 hours at 30° C. and 200 rpm. The mycelium was harvested by filtration through Miracloth (Calbiochem) and rinsed extensively with water then TSE [150 mM NaCl, 100 mM Na2EDTA, 50 mM Tris-HCl pH 8.0]. The mycelium was squeezed dry, broken into small pellets and frozen in liquid nitrogen. The mycelium was ground to a fine powder in a pre-chilled pestle and mortar and transferred to a mortar pre-heated to 65° C. Three ml of lysis buffer [0.5 M NaCl, 10 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% (w/v) SDS] at 65° C. was added and 0.3 ml of 10% (w/v) cetyltrimethylammonium bromide in 0.7 M NaCl. After thorough mixing to form a slurry, 3 ml of phenol:chloroform:isoamyl alcohol (25:24:1) was added. This mixture was transferred to a Corex tube and incubated at 65° C. for 15 minutes. Following centrifugation at 12,000×g for 15 minutes at 4° C. the aqueous phase was carefully removed and re-extracted once with phenol, once with phenol:chloroform:isoamyl alcohol (25:24:1) and once with chloroform:isoamyl alcohol (24:1). The DNA was precipitated from the extract by addition of 0.1 volume of 3 M sodium acetate pH 5 and 0.6 volumes isopropanol then collected by centrifugation (10,000×g, 10 minutes, 4° C.). After washing with 70% ethanol the pellet was briefly dried and resuspended in TE+RNase (50 μg/ml).

[0143] Transformation of A. terreus.

[0144] A 0.5 ml aliquot of spore suspension (108 c.f.u./ml) was used to inoculate 100 ml of liquid CM and grown for 20 hours at 30° C. and 200 rpm. The mycelium was harvested by centrifugation at 2000×g for 15 minutes at 4° C. and washed twice with an aqueous solution containing 0.27 M CaCl2 and 0.6 M NaCl. To produce protoplasts the washed mycelia was resuspended in 20 ml of the same solution containing 5 mg/ml Novozym 234 (NovoNordisk) and incubated at 30° C. for 1-3 hours with gentle agitation. Protoplasts were separated from undigested mycelia by filtration through Miracloth (Calbiochem). The protoplast suspension was diluted with an equal volume of STC1700 [1.2 M sorbitol, 10 mM Tris-HCl pH 7.5, 35 mM NaCl] and incubated on ice for 10 minutes. The protoplasts were collected by centrifugation (2000×g, 10 minutes, 4° C.), washed with STC1700 and resuspended in 1 ml STC1700. Plasmid DNA, purified using Qiagen columns, (2-5 μg in 10 μl) was added to 150 μl of protoplast suspension and incubated at room temperature for 25 minutes. PEG solution [60% (w/v) polyethylene glycol 4000, 50 mM CaCl2, 10 mM Tris-HCl pH 7.5] was added to the DNA/protoplasts mixture in three steps: 250 μl, 250 μl, and 850 μl with mixing after each addition. The suspension was incubated at room temperature for 25 minutes then diluted to 10 ml with STC1700. Protoplasts were collected by centrifugation as above and diluted with 500 μl STC1700. 100 μl aliquots of this mixture were plated onto osmotically stabilized plates [CM medium containing 3% (w/v) Difco Bacto agar and 23.4% (w/v) mannitol, 15 ml of agar per plate]. After 4 hours growth at 30° C., 25 ml of OL agar [1% (w/v) Difco Bacto peptone, 1% (w/v) Difco Bacto agar, 200 μg/ml Zeocin] was overlayered onto each dish. The plates were incubated for 3-4 days at 30° C. before transformant colonies were picked. These were streaked to single colonies twice on selective media (CM+100 μg/ml Zeocin) before spore suspensions were prepared.

[0145] Transformation of A. nidulans.

[0146] A 0.5 ml aliquot of spore suspension (108 c.f.u./ml) was used to inoculate 100 ml of YEPD [2% (w/v) Difco Bacto yeast extract, 2% (w/v) glucose, 0.1% Difco Bacto peptone] liquid medium including necessary supplements and grown for 20 hours at 37° C. and 200 rpm. The mycelia was harvested by centrifugation (2000×g, 10 minutes, 4° C.) and washed twice with 0.6 M KCl. To generate protoplasts the mycelia was resuspended in 20 ml of 0.6 M KCl containing 5 mg/ml Novozym 234 and incubated at 30° C. for 1-2 hours with gentle shaking. Protoplasts were separated from undigested mycelia by filtration through Miracloth (Calbiochem). The protoplasts were harvested by centrifugation as described above and washed twice with 0.6 M KCl, then resuspended in 10 ml 0.6 M KCl+50 mM CaCl2. After counting in a haemocytometer the protoplasts were harvested by centrifugation as before and resuspended to a final concentration of 5×108 protoplasts/ml. To 50 μl of protoplast suspension, 5 μl of DNA (2-5 μg, purified using Qiagen columns) was added, then 12.5 μl of PEG solution [25% (w/v) PEG 6000, 50 mM CaCl2, 10 mM Tris—HCl pH 7.5] and the mixture was incubated on ice for 20 minutes. A further 0.5 ml of PEG solution was added and the mixture was incubated on ice for a further 5 minutes. A 1 ml aliquot of 0.6 M KCl+50 mM CaCl2 was added and the protoplasts were plated out in 50 μl, 200 μl, and 400 μl aliquots. For transformation to uridine prototrophy, protoplasts were plated out onto AMM+0.6 M KCl plates without adding uridine or uracil supplements. Plates were incubated at 37° C. for 3-4 days when transformants were picked. For transformation to hygromycin B resistance protoplasts were plated out onto AMM +0.6 M KCl plates (15 ml) and incubated for 4 hours at 30° C. 30 ml of 1% peptone, 1% agar, 1 mg/ml hygromycin B was then used to overlay the plates, which were incubated for 3-4 days when transformants were picked. Transformants from both methods were streaked out to single colonies on selective media (i.e., lacking uridine/uracil supplements or containing 1 μg/ml hygromycin B) twice before spore suspensions were made.

[0147] Analysis of Strains for Lovastatin Production.

[0148] Two fermentation methods were used for the analysis of lovastatin production. In Method A, 0.5 ml of spore suspension (108 c.f.u./ml) was inoculated into 25 ml of SEED medium in 250 ml unbaffled flasks and grown for 18 hours at 250 rpm and 30° C. (New Brunswick Scientific Model 25 incubator/shaker). A 1 ml portion of the resulting seed culture was used to inoculate 25 ml of FM in a 250 ml unbaffled flask and grown for 6 days in the conditions described above. Fermentation Method B involved inoculating 50 ml of RPM in a 250 ml unbaffled flask with 0.5 ml of spore suspension (108 c.f.u./ml) and growing at 30° C. and 250 rpm for 7 days in a New Brunswick Scientific Series 25 Incubator Shaker.

[0149] SEED medium:

[0150] 0.5% (w/v) Sigma corn steep liquor

[0151] 4% (w/v) tomato paste

[0152] 1% (w/v) oat flour

[0153] 1% (w/v) glucose

[0154] 1% (v/v) Vogel's trace elements

[0155] distilled water to 1 l

[0156] FM:

[0157] 4.5% (w/v) glucose

[0158] 2.4% (w/v) Sigma peptonized milk

[0159] 0.25% (w/v) Difco Bacto yeast extract

[0160] 0.25% (w/v) polyethylene glycol 2000

[0161] distilled water up to 1 l

[0162] RPM:

[0163] 4% (w/v) lactose

[0164] 0.3% (w/v) rapeseed meal

[0165] 0.2% (w/v) KNO3

[0166] 0.3% (w/v) KH2PO4

[0167] 0.05% (w/v) MgSO4.7H2O

[0168] 0.05% (w/v) NaCl

[0169] 0.05% (v/v) Sigma antifoam B

[0170] 0.05% (v/v)trace elements solution

[0171] pH to 6.5 and made up to 1 l with distilled water.

[0172] Trace elements solution is:

[0173] 0.16% (w/v) MnSO4

[0174] 0.34% (w/v) ZnSO4.7H2O

[0175] 0.2% (w/v) CoCl2.6H2O

[0176] 0.5% (w/v) FeSO4.7H2O

[0177] made up to 1 liter with distilled water.

[0178] The cultures were extracted by adjusting the pH of the media to 3 with HCl, adding an equal volume of ethyl acetate, and shaking the mixture on a New Brunswick Scientific Series 25 incubator/shaker at 250 rpm for 2 hours. For analysis, 1 ml of the ethyl acetate layer was dried under a nitrogen stream and resuspended in 0.1 ml of methanol. For TLC analysis 10 μl of this extract was run on C-18 reverse phase TLC plates (RP-18 F254—Merck) in a solvent system of methanol:0.1% phosphoric acid (9:1). TLC plates were developed by spraying with 10% phosphomolybdic acid in methanol and heating with a heat gun. Extracts were compared with authentic lovastatin, monacolin J. monacolin L, and dihydromonacolin L (acid and lactone forms). For HPLC analysis a Waters Nova-Pak C18 (3.9×150 mm) column was used with a solvent system of acetonitrile (B) and 0.1% phosphoric acid (A). The column was eluted with a preprogrammed gradient of 0 to 100% B into A over 25 minutes using gradient 7 (Waters Millenium Software) with a flow rate of 1.5 ml/min and metabolites were detected with a Waters 996 Photodiode Array Detector; lovastatin was detected at 238 nm. For purification of metabolites a Waters Prep Nova-Pak HR C18 (7.8×300 mm) column was used. The same solvent system as above was used with gradient of 0 to 100% B in A over 75 minutes at a flow rate of 4.5 ml/min. Fractions were collected manually, back extracted with ethyl acetate and dried. For HPLC-MS an Aquapore OD-300 7 micron (1.0×100 mm) column was used with a gradient of 0 to 100% acetonitrile into A (0.05% TFA) over 30 minutes at a flow rate of 0.02 ml/min.

Claims

1. A method of increasing the production of lovastatin in a lovastatin-producing organism, comprising the steps of transforming the organism with the D4B segment, wherein the segment is transcribed and translated, and wherein an increase in lovastatin production occurs.

2. The method of claim 1 wherein the D4B segment is the A. terreus D4B segment.

3. The method of claim 1, wherein the D4B segment is identical to nucleotides 579-33,000 of SEQ ID NO: 18 and 1-5,349 of SEQ ID NO: 19.

4. The method of claim 1, wherein the lovastatin-producing organism is selected from the group consisting of A. terreus ATCC 20542 and ATCC 20541.

5. The method of claim 1, wherein the organism is selected from the group consisting of fungi and yeast.

6. The method of claim 1 wherein the increase is at least 2-fold.

7. The method of claim 1 wherein the nucleic acid sequence is identical to a sequence isolated from ATCC 98876.

8. The method of claim 1 additionally comprising transforming the organism with the entire A. terreus lovastatin gene cluster.

9. The method of claim 8 wherein the gene cluster comprises SEQ ID NOs: 18 and 19.

10. The method of claim 8 wherein the nucleic acid sequence of the gene cluster is identical to sequences isolated from ATCC 98876 and 98877.

11. A method of increasing the production of monacolin J in a lovastatin-producing organism, comprising the steps of transforming the organism with the D4B segment, wherein the segment is translated, and wherein an increase monacolin J production occurs.

12. A method of increasing the production of lovastatin in a lovastatin-producing organism, comprising the step of transforming the organism with the LovE gene, wherein the nucleic acid sequence is translated, and wherein an increase in lovastatin production occurs.

13. The method of claim 12 wherein the increase is at least 2.0-fold.

14. The method of claim 13 wherein the increase is at least 5-fold.

15. The method of claim 12 wherein the nucleotide sequence of the LovE gene comprises SEQ ID NO: 27.

16. A method of increasing the production of lovastatin in a lovastatin-producing organism comprising the steps of transforming the organism with a nucleic acid sequence comprising a truncated version of the A. terreus D4B segment, wherein the nucleic acid sequence is transcribed and translated and wherein an increase in lovastatin production occurs.

17. A method of increasing the production of lovastatin in a lovastatin-producing organism comprising the steps of transforming the organism with a nucleic acid sequence comprising a truncated version of the A. terreus lovastatin-producing gene cluster, wherein the nucleic acid sequence is transcribed and translated and wherein an increase in lovastatin production occurs.

18. A method of increasing or conferring the production of monacolin J in a non-lovastatin-producing organism comprising the steps of transforming the organism with a nucleic acid sequence comprising the D4B segment, wherein the nucleic acid sequence is transcribed and translated and wherein an increase in monacolin J production occurs.

19. The method of claim 18 wherein the D4B segment is the A. terreus D4B segment.

20. The method of claim 18 wherein the D4B segment comprises nucleotides 579-33,000 of SEQ ID NO: 18 and 1-5,349 of SEQ ID NO: 19.

21. The method of claim 18 additionally comprising the step of converting the monacolin J into lovastatin.

22. The method of claim 18 additionally comprising the step of transforming the organism with a nucleic acid sequence comprising the LovF gene, wherein the nucleic acid sequence is transcribed and translated and wherein an increase in lovastatin production occurs.

23. An isolated nucleic acid sequence selected from the group consisting of SEQ ID NOs: 20-36.

24. A lovastatin-producing organism, wherein the organism has been genetically modified to have increased lovastatin production, wherein the increase is at least 2-fold.

25. The organism of claim 24, wherein the organism is a yeast or a fungi.

26. A non-lovastatin producing organism, wherein the organism has been genetically modified to produce monacolin J.

27. The organism of claim 26, wherein the organism is a yeast or a fungi.

28. A non-lovastatin producing organism, wherein the organism has been genetically modified to produce lovastatin.

29. The organism of claim 28 wherein the organism is a yeast or a fungi.