IMPROVED STATIN PRODUCTION

Field of the invention

The present invention relates to a method for fermentation of statins.

Background of the invention

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. Cholesterol lowering agents of the statin class are medically very important drugs as they lower the cholesterol concentration in the blood by inhibiting HMG-CoA reductase. The latter enzyme catalyses the rate limiting step in cholesterol biosynthesis, i.e. the conversion of (3S)-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) to mevalonate. As can be seen from the scheme below, there are several types of statins on the market, amongst which atorvastatin, compactin (V), lovastatin (3), simvastatin (4) and pravastatin (6). Whilst atorvastatin is made via chemical synthesis, the other statins mentioned above are produced either via direct fermentation or via precursor fermentation. These (precursor) fermentations are carried out by fungi of the genera Penicillium, Aspergillus and Monascus. There is a common problem while fermenting these compounds as part of the carbon supplied in the fermentation ends up in the form of useless side-chain less molecules (such as compounds 2 in case of compactin production, 5 in case of lovastatin and simvastatin production and 7 in case of pravastatin production). Depending on the exact strain and fermentation conditions this can be quite a significant portion of the total statins produced, resulting in a loss of product. This phenomenon has been described by various publications, for both 2-methylbutyric ester and 2,2- dimethylbutyric ester statins (see for example Serizawa, N., Nakagawa, K., Tsujita, Y., Terahara, A., Kuwano, H. and Tanaka, M. (1983) J. Antibiot. 7:918-920; Endo, A., Kuroda, M. and Tsujita, Y. (1976) J. Antibiot. 12: 1346-1348; Schimmel, T.G., Borneman,

W.S. and Condor, MJ. (1997) Appl. Environm. Microbiol. 63:1307-131 1 ; Xie X, Watanabe K, Wojcicki WA, Wang CC, Tang Y., Chem Biol. 2006, 13(1 1 ):1 161-1169). Moreover, this problem was shown to be very relevant while producing compactin in a heterologous host (WO07122249). So far, solutions to this problem have not been disclosed and there is thus room for improved statin production.

1 (compactin) H

H,

H,

5 (monacolin J) H CH,

6 (pravastatin) OH

H OH

Description of the invention

The object of the present invention is to provide a method to solve some of the problems encountered in prior art processes. Thus, provided is a method in which the degradation of statins, like hydrolysis of compactin, pravastatin, lovastatin and/or simvastatin is reduced by inactivating a selected set of fungal enzymes. More specifically a process for reducing the degradation of compactin, pravastatin, lovastatin

and/or simvastatin is provided, characterized in that the fermentation process is carried out in media not inducing the enzymes that catalyze the hydrolysis of compactin, pravastatin, lovastatin and/or simvastatin. Preferably, a process is provided which makes use of microorganisms in which the genes encoding the hydrolyzing enzymes are inactivated or deleted. In the context of this invention the terms 'inactivated' and 'inactivation' are used to describe the various methods by which a gene can be modified in order to produce less active enzyme. This includes: inactivation by base pair mutation resulting in a(n early) stop or frame shift; mutation of critical amino acids (such as the catalytic triad for hydrolases); mutations causing a decreased half-life of the enzyme; modifying the mRNA molecule in such away that the mRNA half-life is decreased; insertion of a second sequence (Ae. a selection marker gene) disturbing the open reading frame; a partial or complete removal of the gene; removal/mutation of the promoter of the gene; using anti-sense DNA or comparable RNA inhibition methods to lower the effective amount of mRNA in the cell. Preferred methods are gene deletion and prevention of transcription as described in Example 9 of the present invention. The net result of these variant methods is that the overall esterase activity is reduced. Throughout the present invention hydrolyzing enzymes are also referred to as esterases. However, the scope of the invention is not limited to esterases but is also meant to include other classes capable of hydrolyzing esters, such as hydrolases, lipases, proteases and amidases (Ae. hydrolases, EC class 3.x.x.x; more preferably enzymes capable of cleaving ester bonds, EC class 3.1.x.x; enzymes capable of cleaving peptide bonds (peptidases), EC class 3.4.x.x; enzymes acting on carbon-nitrogen bonds other than peptide bonds, EC class 3.5.x.x; or even more preferably Carboxylic Ester Hydrolases, EC class 3.1.1.x; Thioester Hydrolases, EC class 3.1.2.x; Enzymes acting on carbon-nitrogen bonds, other than peptide bonds in Linear Amides, EC class 3.5.1.x).

In the first aspect the present invention provides a method of preventing the hydrolysis of statin molecules, like compactin, pravastatin, lovastatin and/or simvastatin into their side-chain-less variants, 2 (ML-236A), 7, and 5 (monacolin J). The structures V- 7 given in the scheme above show the various molecules in the so-called closed form, Ae. bearing a lactone ring. This ring can however also exist in the so-called open form, Ae. bearing a carboxylic acid function and a hydroxyl group and as a matter of fact under many circumstances there is equilibrium between the two forms. In the context of the present invention, both open and closed forms are meant to be included. Thus, the

present invention provides a method for the fermentative production of a compound of interest chosen from the list consisting of compactin, lovastatin, pravastatin and simvastatin comprising culturing a mutant host capable of producing said compound of interest, characterized in that esterase activity in said mutant host is more than 25% below the activity of said esterase in the parent host. Preferably said esterase activity in said mutant host is more than 50% below the activity of said esterase in the parent host, more preferably said esterase activity in said mutant host is more than 75% below the activity of said esterase in the parent host. In the context of this invention the phrase 'parent host' is used to describe any organism capable of producing compactin, lovastatin, pravastatin and/or simvastatin, either via direct fermentation or by bioconversion (Ae. feeding compactin in order to hydroxylate towards pravastatin, or feeding monacolin J or lovastatin with and without dimethylbutyric esters in order to produce simvastatin). In the context of this invention the phrase 'mutant host' is used to describe any organism derived from the parent host by genetic engineering or classical mutagenesis.

In one embodiment esterase activity in said parent host is lowered by means of suppressing activity of at least one esterase, for instance by means of classical mutagenesis and/or inactivation of said esterase. Advantageously, the esterase of which the activity is lowered preferably is a 2-methylbutyric esterase and/or a 2,2-dimethylbutyric esterase. Lowering the activity of 2-methylbutyric esterase is particularly advantageous for hosts producing compactin and/or lovastatin and/or pravastatin. Lowering the activity of 2,2-dimethylbutyric esterase is particularly advantageous for hosts producing simvastatin. In the context of the present invention the term 2-methylbutyric esterase refers to an enzyme capable of hydrolyzing an ester of 2-methylbutyric acid; the term 2,2-dimethylbutyric esterase refers to an enzyme capable of hydrolyzing an ester of 2,2-dimethylbutyric acid.

By feeding compactin, pravastatin, lovastatin and/or simvastatin to active cells it was shown that an active enzyme or enzymes hydrolyze these molecules, producing the side-chain less variants. Preferably, the active cell producing statins is a microorganism, more preferably a yeast, a fungus or a bacterium, such as Streptomyces carbophilus, Escherichia coli expressing LovD or a homologue thereof, Saccharaothrix, Actinomadura, Amycolatopsis orientalis, Streptomyces flavidovirens, Streptomyces sp., Pseudonocardia sp., Micromonospora, Streptomyces exfoliatus, most preferably a filamentous fungus. Suitable examples are Penicillium chrysogenum, Penicillium

citrinum, Penicillium brevicompactum, Aspergillus terreus, Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Monascus paxii, Monascus pilosus, Monascus purpureus and Monascus ruber.

In another embodiment of the invention production organisms that have reduced capabilities to hydrolyze compactin, pravastatin, lovastatin and/or simvastatin in their side-chain-less variants, 2 (ML-236A), 7 and 5 (monacolin J) are isolated as specific mutants. Typically, Organisms incapable of hydrolyzing compactin, pravastatin, lovastatin and/or simvastatin' refers to organisms wherein the production ratio between compactin, lovastatin, pravastatin and/or simvastatin on the one hand and their hydrolyzed congeners 2 (ML-236A), 5 (monacolin J), 7 and 5 (monacolin J) respectively on the other hand, is from 0.75-50 (w/w), preferably from 1-200 (w/w), more preferably from 1.5-500 (w/w), most preferably from 2-1000 (w/w). Specifically, the present invention discloses mutant hosts producing compactin in 80-99.9% of total statins.

In still another embodiment transcriptomics (with DNA MicroArrays), proteomics (with 2D gel electrophoresis) or comparable methods are used to identify genes and/or enzymes responsible for the hydrolysis of compactin, pravastatin, lovastatin and simvastatin. This can be done by preparing samples from fermentations with cells grown under non-inducing conditions and inducing conditions (Ae. without and with urea), but also under statin producing and non-producing conditions (Ae. with and without compactin gene cluster). After analysis and comparison of the results target genes can be identified, which can be inactivated to destroy any hydrolysis activity in future strains.

In yet another embodiment lipases and/or esterases from different sources

(commercial samples, deduced from fungal genome sequences, derived from protein databases) are tested in vitro for the hydrolysis of statins, like compactin into ML-236A. Gene sequences encoding the enzymes that do hydrolyze statins can be used as probes to identify the homologous genes in the statin producing species, which can be inactivated to destroy any hydrolysis activity in future strains. Alternatively, the genome of the statin producing organism can be determined and after sequence analysis all selected genes encoding putative hydrolyzing enzymes can be inactivated and the mutants can be tested for their ability to hydrolyze statins. As an alternative approach the promoter activity of such genes can be modified to decrease the amount of mRNA transcribed and thus protein obtained, thereby reducing the overall hydrolyzing activity. Another approach is to identify transcriptional regulators that control the transcription of

genes encoding the hydrolyzing enzymes and modify the transcription level of these transcription regulators in such a way that the hydrolyzing activity is decreased.

To inactivate a gene encoding such hydrolysis activity in statin producing strains one can apply several methods. One approach is a temporary one using an anti-sense molecule or RNAi molecule (Kamath et al. 2003. Nature 421 :231-237). Another is using a regulatable promoter system, which can be switched off using external triggers like tetracycline (see Park and Morschhauser, 2005, Eukaryot. Cell. 4:1328-1342). Yet another one is to apply a chemical inhibitor or a protein inhibitor or a physical inhibitor (see Tour et al. 2003. Nat Biotech 21 :1505-1508). The most preferred situation is to remove part of or the complete gene(s) encoding the hydrolysis activity. To obtain such a mutant one can apply state of the art methods like Single Cross-Over Recombination or Double Homologous Recombination. For this one needs to construct an integrative cloning vector that may integrate at the predetermined target locus in the chromosome of the host cell. In a preferred embodiment of the invention, the integrative cloning vector comprises a DNA fragment, which is homologous to a DNA sequence in a predetermined target locus in the genome of host cell for targeting the integration of the cloning vector to this predetermined locus. In order to promote targeted integration, the cloning vector is preferably linearized prior to transformation of the host cell. Linearization is preferably performed such that at least one but preferably either end of the cloning vector is flanked by sequences homologous to the target locus. The length of the homologous sequences flanking the target locus is preferably at least 0.1 kb, even preferably at least 0.2 kb, more preferably at least 0.5 kb, even more preferably at least 1 kb, most preferably at least 2 kb. The length that finally is best suitable in an experiment depends on the organism, the sequence and length of the target DNA. The efficiency of targeted integration of a nucleic acid construct into the genome of the host cell by homologous recombination, i.e. integration in a predetermined target locus, is preferably increased by augmented homologous recombination abilities of the host cell. Such phenotype of the cell preferably involves a deficient hdfA or hdfB gene as described in WO 05/95624. WO 05/95624 discloses a preferred method to obtain a filamentous fungal cell comprising increased efficiency of targeted integration by preventing non-homologous random integration of DNA fragments into the genome. The vector system may be a single vector or plasmid or two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell.

Fungal cells may be transformed by protoplast formation, protoplast transformation, and regeneration of the cell wall. Suitable procedures for transformation of fungal host cells are described in EP 238023 and Yelton et al. (1984. Proc. Nat. Acad. Sci. USA 81 :1470-1474). Suitable procedures for transformation of filamentous fungal host cells using Agrobacterium tumefaciens are described by de Groot MJ. et al. (1998. Nat. Biotechnol. 16:839-842. Erratum in: Nat. Biotechnol. 1998. 16:1074). Other methods like electroporation, described for Neurospora crassa, may also be applied.

Fungal cells are transfected using co-transformation, i.e. along with gene(s) of interest also a selectable marker gene is transformed. This can be either physically linked to the gene of interest (Ae. on a plasmid) or on a separate fragment. Following transfection transformants are screened for the presence of this selection marker gene and subsequently analyzed for the integration at the preferred predetermined genomic locus. A selectable marker is a product, which provides resistance against a biocide or virus, resistance to heavy metals, prototrophy to auxotrophs and the like. Useful selectable markers include, but are not limited to, amdS (acetamidase), argB (ornithine- carbamoyltransferase), bar (phosphinothricinacetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC or sutB (sulfate adenyltransferase), trpC (anthranilate synthase), ble (phleomycin resistance protein), as well as equivalents thereof. The most preferred situation is providing a DNA molecule comprising a first DNA fragment comprising a desired replacement sequence (i.e. the selection marker gene) flanked at its 5' and 3' sides by DNA sequences substantially homologous to sequences of the chromosomal DNA flanking the target sequence. Cells wherein the target sequence in the chromosomal DNA sequence is replaced by the desired replacement sequence can be selected by the presence of the selectable marker of the first DNA fragment. To increase the relative frequency of selecting the correct mutant microbial strain, a second DNA fragment comprising an expression cassette comprising a gene encoding a selection marker and regulatory sequences functional in the eukaryotic cell can be operably linked to the above described fragment (i.e. 5'-flank of target locus + selection marker gene + 3'-flank of target locus) and cells wherein the target sequence in the chromosomal DNA sequence is replaced by the desired replacement sequence can be selected by the presence of the selectable marker of the first DNA fragment and the absence of the second selection marker gene.

In another embodiment, mutants can be selected after classical mutagenesis and subsequent screening. The screening can be at random, by growing the randomly isolated mutants in liquid media and incubate them with compactin. The mutants that do not (or at least to a lesser extend than the parent strain) hydrolyze compactin into ML-236A are the preferred mutants. As their might be substrate specificity amongst the different esterases (i.e. simvastatin is a dimethylbutyric ester and pravastatin has a hydroxyl group on the statin core) one or more different esterases may be targeted for inactivation depending on the product of interest. To this end this screen may also be done with pravastatin, lovastatin and/or simvastatin. Also, use of selective compounds can target the screening and enrich the obtained mutant population for the right mutants. One example of such a compound is tributyrin. Upon hydrolysis of this compound by lipase activity a so-called halo is formed around the colony on an agar-plate. After mutagenesis and screening, colonies with a smaller or no halo at all can be isolated as preferred mutants, which possibly have a reduced activity on statins too. This approach will result in statin producing strains that have reduced levels of the hydrolysis enzymes; hydrolysis enzymes with deteriorated enzyme kinetics and/or hydrolysis enzymes that are targeted to wrong compartments in the cell. Such a classical approach can be performed in a strain containing one or more of the above targeted mutations, but also in a strain without such a targeted mutation. Moreover, both the molecular and/or the classical approach can be used in iterative rounds to reduce the rate of hydrolysis step- by-step until mutants are obtained with a very low level of or no hydrolysis of statins at all.

In a second aspect, the present invention provides a mutant host having 2-methylbutyric esterase or 2,2-dimethylbutyric esterase activity which is more than 25% below the activity of said esterase in the parent host. Said mutant host can be obtained according to the method of the first aspect of the invention.

In one embodiment, the present invention provides a polypeptide having an amino acid sequence that is substantially homologous to the sequence of SEQ ID NO 24 and which displays 2-methylbutyric esterase and/or 2,2-dimethylbutyric esterase activity. The invention further provides for polypeptides of SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22 or polypeptides that are substantially homologous to these sequences. Examples of this latter category are SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 32, SEQ ID NO 34,

SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40 SEQ ID NO 42, SEQ ID NO 44 or polypeptides that are substantially homologous to these sequences. The present invention further provides for polynucleotides encoding the provided polypeptides. In particular, a specific DNA sequence is provided encoding the polypeptide of SEQ ID NO 12, i.e. SEQ ID NO 11 , or encoding the polypeptide of SEQ ID NO 14, i.e. SEQ ID NO 13, or encoding the polypeptide of SEQ ID NO 16, i.e. SEQ ID NO 15, or encoding the polypeptide of SEQ ID NO 18, i.e. SEQ ID NO 17, or encoding the polypeptide of SEQ ID NO 20, i.e. SEQ ID NO 19, or encoding the polypeptide of SEQ ID NO 22, i.e. SEQ ID NO 21 , or encoding the polypeptide of SEQ ID NO 24, i.e. SEQ ID NO 23, encoding the polypeptide of SEQ ID NO 26, i.e. SEQ ID NO 25, or encoding the polypeptide of SEQ ID NO 28, i.e. SEQ ID NO 27, or encoding the polypeptide of SEQ ID NO 30, i.e. SEQ ID NO 29, or encoding the polypeptide of SEQ ID NO 32, i.e. SEQ ID NO 31 , or encoding the polypeptide of SEQ ID NO 34, i.e. SEQ ID NO 33, or encoding the polypeptide of SEQ ID NO 36, i.e. SEQ ID NO 35, or encoding the polypeptide of SEQ ID NO 38, i.e. SEQ ID NO 37, or encoding the polypeptide of SEQ ID NO 40, i.e. SEQ ID NO 39, or encoding the polypeptide of SEQ ID NO 42, i.e. SEQ ID NO 41 , or encoding the polypeptide of SEQ ID NO 44, i.e. SEQ ID NO 43.

To provide for a mutant host having 2-methylbutyric esterase and/or 2,2-dimethylbutyric esterase activity which is more than 25% below the activity of said esterase in the parent host, said polypeptide and/or polynucleotide sequences should be inactivated according to the method of the first aspect of the invention. In a preferred embodiment the genes or homologous genes described by the nucleotide sequences of SEQ ID NO 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37 or 39 are inactivated (i.e. modified, disrupted or deleted). In the most preferred embodiment the gene or homologous genes described by the nucleotide sequences of SEQ ID NO 23 is inactivated (Ae. modified, disrupted or deleted).

A polypeptide having an amino acid sequence that is "substantially homologous" to the sequence of SEQ ID NO 12 is defined as a polypeptide having an amino acid sequence possessing a degree of identity to the specified amino acid sequence of at least 75%, preferably at least 80%, more preferably at least 85%, still more preferably at least 90%, still preferably at least 95%, still more preferably at least 96%, still more preferably at least 97%, still more preferably at least 98% and most preferably at least 99%, the substantially homologous peptide displaying 2-methylbutyric esterase and/or

2,2-dimethylbutyιϊc esterase activity. A polypeptide having an amino acid sequence that is "substantially homologous" to the sequence of SEQ ID NO 14 is defined as a polypeptide having an amino acid sequence possessing a degree of identity to the specified amino acid sequence of at least 90%, preferably at least 92%, more preferably at least 95%, still more preferably at least 96%, still more preferably at least 97%, still more preferably at least 98% and most preferably at least 99%, the substantially homologous peptide displaying 2-methylbutyric esterase and/or 2,2-dimethylbutyric esterase activity. A polypeptide having an amino acid sequence that is "substantially homologous" to the sequence of SEQ ID NO 18 is defined as a polypeptide having an amino acid sequence possessing a degree of identity to the specified amino acid sequence of at least 65%, preferably at least 70%, more preferably at least 75%, still more preferably at least 80%, still preferably at least 85%, still more preferably at least 90%, still more preferably at least 95%, still more preferably at least 98% and most preferably at least 99%, the substantially homologous peptide displaying 2-methylbutyric esterase and/or 2,2-dimethylbutyric esterase activity. A polypeptide having an amino acid sequence that is "substantially homologous" to the sequence of SEQ ID NO 22 is defined as a polypeptide having an amino acid sequence possessing a degree of identity to the specified amino acid sequence of at least 50%, preferably at least 60%, more preferably at least 70%, still more preferably at least 75%, still preferably at least 80%, still more preferably at least 85%, still more preferably at least 90%, still more preferably at least 95% and most preferably at least 99%, the substantially homologous peptide displaying 2-methylbutyric esterase and/or 2,2-dimethylbutyric esterase activity. A polypeptide having an amino acid sequence that is "substantially homologous" to the sequence of SEQ ID NO 24 is defined as a polypeptide having an amino acid sequence possessing a degree of identity to the specified amino acid sequence of at least 75%, preferably at least 80%, more preferably at least 85%, still more preferably at least 90%, still preferably at least 95%, still more preferably at least 96%, still more preferably at least 97%, still more preferably at least 98% and most preferably at least 99%, the substantially homologous peptide displaying 2-methylbutyric esterase and/or 2,2-dimethylbutyric esterase activity. A polypeptide having an amino acid sequence that is "substantially homologous" to the sequence of SEQ ID NO 42 is defined as a polypeptide having an amino acid sequence possessing a degree of identity to the specified amino acid sequence of at least 40%, preferably at least 50%, more preferably at least 60%, still more preferably at least 70%, still preferably at least 80%, still more

preferably at least 90%, still more preferably at least 95%, still more preferably at least 98% and most preferably at least 99%, the substantially homologous peptide displaying 2-methylbutyric esterase and/or 2,2-dimethylbutyric esterase activity. A polypeptide having an amino acid sequence that is "substantially homologous" to the sequence of SEQ ID NO 44 is defined as a polypeptide having an amino acid sequence possessing a degree of identity to the specified amino acid sequence of at least 50%, preferably at least 60%, more preferably at least 70%, still more preferably at least 75%, still preferably at least 80%, still more preferably at least 85%, still more preferably at least 90%, still more preferably at least 95% and most preferably at least 99%, the substantially homologous peptide displaying 2-methylbutyric esterase and/or 2,2-dimethylbutyric esterase activity. A substantially homologous polypeptide may encompass polymorphisms that may exist in cells from different populations or within a population due to natural allelic or intra-strain variation. A substantially homologous polypeptide may further be derived from a fungus other than the fungus where the specified amino acid and/or DNA sequence originates from, or may be encoded by an artificially designed and synthesized DNA sequence. DNA sequences related to the specified DNA sequences and obtained by degeneration of the genetic code are also part of the invention. Homologues may also encompass biologically active fragments of the full-length sequence. Functional equivalents maybe identified as exemplified in example 7 and the definition of "substantially homologous" sequences as described above can be applied to the sequences identified in this way.

In the present invention, the degree of identity between two amino acid sequences refers to the percentage of amino acids that are identical between the two sequences. The degree of identity is determined using the BLAST algorithm, which is described in Altschul, et al., J. MoI. Biol. 215: 403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11 , the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

Substantially homologous polypeptides may contain only conservative substitutions of one or more amino acids of the specified amino acid sequences or substitutions, insertions or deletions of non-essential amino acids. Accordingly, a non-

essential amino acid is a residue that can be altered in one of these sequences without substantially altering the biological function. For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al. (Science 247:1306-1310 (1990)) wherein the authors indicate that there are two main approaches for studying the tolerance of an amino acid sequence to change. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selects or screens to identify sequences that maintain functionality. As the authors state, these studies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which changes are likely to be permissive at a certain position of the protein. For example, most buried amino acid residues require non-polar side chains, whereas few features of surface side chains are generally conserved. Other such phenotypically silent substitutions are described in Bowie et al, and the references cited therein. The term "conservative substitution" is intended to mean that a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain. These families are known in the art and include amino acids with basic side chains (e.g. lysine, arginine and histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagines, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine).

In one embodiment of the invention, a modified polypeptide is provided. In the context of the present invention, the term "modified polypeptide" refers to a 2-methylbutyric esterase or 2,2-dimethylbutyric esterase protein wherein at least the 2-methylbutyric esterase or 2,2-dimethylbutyric esterase activity is decreased. The 2-methylbutyric esterase or 2,2-dimethylbutyric esterase selection as exemplified in examples 1-3 provides for an assay to measure their activity in vivo and thus modified proteins may easily be selected. Modified polypeptides of the invention thus are characterized by the fact that they show less activity in cleaving off the 2-methylbutyric or 2,2-dimethylbutyric side chains of statins.

Particularly useful modified polypeptides are modified MIcH (SEQ ID NO 49), MkF (SEQ ID NO 51 ) and LovD (SEQ ID NO 53) proteins of the compactin and

lovastatin biosynthetic pathways. MIcH, MkF and LovD are proteins involved in the attachment of the 2-methylbutyric and/or 2,2-dimethylbutyric side chain on compactin, pravastatin, lovastatin and/or simvastatin. The genes encoding these enzymes are named mlcH (SEQ ID NO 48) mkF (SEQ ID NO 50) and lovD (SEQ ID NO 52), respectively (Abe Y, Suzuki T, Ono C, Iwamoto K, Hosobuchi M, Yoshikawa H., MoI Genet Genomics. 2002, 267(5):636-646; Genbank Accession NO DQ176595; Kennedy J, Auclair K, Kendrew SG, Park C, Vederas JC, Hutchinson CR., Science. 1999, 284(5418):1368-1372). Although, enzymes like MIcH, MkF and LovD are described to catalyze the forward reaction they may function under specific conditions also in the opposite direction and thereby hydrolyze compactin, pravastatin, lovastatin and/or simvastatin (Xie X, Watanabe K, Wojcicki WA, Wang CC, Tang Y., Chem Biol. 2006, 13(1 1 ):1 161-1 169). This should be prevented either by modification of the cultivation conditions (see below) or by improving the synthesis:hydrolysis ratio (S/H ratio) of the enzyme via methods like classical mutagenesis, directed evolution or protein engineering. Preferred methods for directed evolution are described in WO03010183 and WO03010311. This is not limited to the currently known enzymes MIcH, MkF and LovD, but also their homologues in various parent and mutant hosts.

The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The specific sequences disclosed herein can be readily used to isolate the complete gene from filamentous fungi, in particular Penicillium chrysogenum, which in turn can easily be subjected to further sequence analyses thereby identifying sequencing errors.

Unless otherwise indicated, all sequences determined by sequencing a DNA molecule were determined using an automated DNA sequencer and all amino acid sequences of polypeptides encoded by DNA molecules were predicted by translation of a DNA sequence determined as above. Therefore, as is known for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence causes a frame shift in translation of the nucleotide sequence such that the predicted amino acid

sequence encoded by a determined nucleotide sequence will differ completely from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion. The skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors.

In a third aspect, the present invention provides for methods to decrease 2-methylbutyric esterase and/or 2,2-dimethylbutyric esterase activity with more than 25% by the use of specific media components.

In one embodiment selected nitrogen sources are used to obtain this result. Although many nitrogen sources are known to the person skilled in the art, urea is a well-known example of a nitrogen source used in fermentations. Performing fermentations in the absence of specific nitrogen sources, like for example urea, led to a marked reduction in formation of side-chain-less molecules, indicating a reduced activity of 2-methylbutyric esterase and/or 2,2-dimethylbutyric esterase. In general any media component inducing the enzyme(s) catalyzing the hydrolysis of compactin, pravastatin, lovastatin and/or simvastatin should be omitted from the fermentation media. Preferred media are defined and mineral media, as described in US20020039758. Most preferably components like urea are omitted from the media.

In a second embodiment media are used that lack components that repress the biosynthesis of compactin, pravastatin, lovastatin and/or simvastatin. Otherwise these components should be kept at a concentration below the value that triggers repression of the biosynthetic pathways. Examples of such repressing agents are carbon source, pH, sulfur source, phosphorous source, dissolved oxygen concentration, carbon dioxide levels, nitrogen levels, temperature and the like. In a third embodiment media are used that comprise components that stimulate the biosynthesis of compactin, pravastatin, lovastatin and/or simvastatin. These components typically increase the availability of building blocks derived from primary metabolism. Examples of these are, but not limited to, carboxylic acids or salts thereof (including thioesters) such as for instance methyl butyrate, dimethyl butyrate, malonate, methylmalonate, propionate, acetate, oleate, and the like.

The classical mutagenesis and screening approach as described to isolate mutants as described in the first aspect could also be applied to screen for general mutants, in which the nitrogen-dependent regulation of the hydrolyzing enzyme(s) and/or statin biosynthesis pathways are deregulated. This is not limited to nitrogen, but also

mutants not sensitive to or even induced by specific media components can be isolated in such a way (i.e. glucose derepression mutants).

Legends to the figures

Figure 1 shows the stability of compactin, pravastatin and lovastatin in the presence Penicillium chrysogenum. Legend. Y-axis = statin concentration in g/L; X-axis

= time in days after addition of statins; ♦ = compactin; ▲ = pravastatin; D = lovastatin. Figure 2 shows the conversion of compactin into ML-236A in the presence

Penicillium chrysogenum. Legend. Y-axis = relative concentration in peak area; X-axis = time in days after addition of compactin; ♦ = compactin; O = ML-236A.

Figure 3 shows the conversion of lovastatin into monacolin J in the presence of growing Penicillium chrysogenum. Legend. Y-axis = relative concentration in peak area; X-axis = time in days after addition of lovastatin; D = lovastatin; • = monacolin J.

Figure 4 shows the elution pattern in the Akta Purifier with 1 ml Butyl FF HiTrap HIC column. Legend: Y-axis = saturated (NhU) ₂ SO ₄ (%); x-axis = elution volume (ml).

Figure 5 shows the SDS-PAGE gel of selected fractions as shown in table 5. The 2 marked areas in lane 9 were cut from gel and identified with LC-MS. The arrow indicates the 1 ,4-butanediol diacrylate esterase (Pc15g00720) at 4OkDa identified by LC- MS. Legend, lane 1 = MW marker; Lane 2 = Fraction 14; Lane 3 = Fraction 16; Lane 4 = Fraction 18; Lane 5 = Fraction 20; Lane 6 = Fraction 22; Lane 7 = Fraction 23; Lane 8 = Fraction 24; Lane 9 = Fraction 28; Lane 10 = 40% saturated sample.

Figure 6 shows the steps involved in deleting the Penicillium chrysogenum gene Pc15g00720. Legend: solid arrow, Pc15g00720 promoter; open box with Pc15g00720, Pc15g00720 gene; open arrow, Pc15g00720 terminator; hatched box, trpC terminator; dashed box, ccdA gene; solid box, lox site; crosses, recombination event; downwards arrows, subsequent steps in the procedure; REKR and KRAM, overlapping nonfunctional amdS selection marker fragments; REKRAM, functional amdS selection marker gene.. Numbers indicate the SEQ ID NO's of the oligonucleotides.

EXAMPLES

General materials and methods

Standard DNA procedures were carried out as described elsewhere (Sambrook, J. et al., 1989, Molecular cloning: a laboratory manual, 2 ^nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) unless otherwise stated. DNA was amplified using the proofreading enzyme Herculase polymerase (Stratagene). Restriction enzymes were from Invitrogen or New England Biolabs. Fungal growth was performed in a mineral medium, containing (g/L): glucose (5); lactose (80); urea (4.5); (NH ₄ ) ₂ SO ₄ (1.1 ); Na ₂ SO ₄ (2.9); KH ₂ PO ₄ (5.2); K ₂ HPO ₄ (4.8) and 10 ml_/L of a trace element solution A containing citric acid (150); FeSO ₄ -7H ₂ O (15); MgSO ₄ -7H ₂ O (150); H ₃ BO ₃ (0.0075); CuSO ₄ -5H ₂ O (0.24); CoSO ₄ -7H ₂ O (0.375); ZnSO ₄ -7H ₂ O (5); MnSO ₄ -H ₂ O (2.28); CaCI ₂ -2H ₂ O (0.99); pH before sterilization 6.5. Also, a version without urea was applied, having the same constituents and pH as above however without urea and with 2.5 g/L rather than 1.1 g/L (NH ₄ ) ₂ SO ₄ . As rich medium YEPD was used, containing (g/L): yeast extract (10); peptone (10); glucose (20). The cultures are incubated at 25°C in an orbital shaker at 280 rpm for 72-168 h. Although the Examples given below are illustrative for Penicillium chrysogenum, they are not meant to exclude other organism. In particular the skilled person will be able to repeat the invention for microorganisms such as Aspergillus terreus, Penicillium citrinum and Streptomyces carbophilus.

Example 1 Hydrolysis of statins by fungi

Penicillium chrysogenum was cultivated in mineral medium (25 mL) for 96 h at 25°C. Next, different statins were added in concentrations ranging from 0.10-0.15 g/L. After 24 and 48 h broth samples (0.25 mL) were mixed with an equal volume of CH ₃ OH, followed by vigorous vortexing (at least 1 min per sample) and then analyzed for compactin, pravastatin and lovastatin. Care was taken that all material in the tube was brought into suspension, and no clumps remained during extraction. The tubes were centrifuged at 13.000 rpm for 5 min, and the clear supernatant was transferred to HPLC sample tubes.

The HPLC analysis conditions were as follows:

Eluens: A: 33% CH ₃ CN, 0.025% CF ₃ CO ₂ H in milliQ water

B: 80% CH ₃ CN in milliQ water Gradient: Time (min) A B

0-6 100% 0

6.1-10 0 100%

10.1-12 100% 0

Column: Waters XTerra RP 18

Column Temperature: room temperature

Flow: 1 mL/min

Injection volume: 10 μl

Tray Temperature: room temperature

Instrument: Waters Alliance 2695

Detector: Waters 996 Photo Diode Array

Wavelength: 238 nm

Retention times (min): Pravastatin (3.2), compactin (8.6), lovastatin (8.8), simvastatin (9.0)

Figure 1 shows that compactin , pravastatin and lovastatin are degraded by Penicillium chrysogenum.

Example 2

Removal of the 2-methylbutyric moiety of compactin by fungal hydrolase activity

Penicillium chrysogenum was cultivated in mineral medium (US 2002/0039758) for 96 h at 25°C. After that, compactin was added to the broth in a final concentration of 0.10 g/L. After 24 and 48 hours samples were taken for analysis of compactin and ML-236A. Broth samples (0.25 ml.) were mixed with an equal volume of methanol, followed by vigorous vortexing, at least 1 minute per sample. Care was taken that all material in the Eppendorf tube was brought into suspension, and no clumps remained during extraction. The tubes were centrifuged at 13000 rpm for 5 minutes, and the clear supernatant was transferred to HPLC sample tubes. The HPLC analysis conditions were the same as in Example 1 ; Retention times (min): ML-236A (2.6), compactin (8.6). Figure 2 shows that the methyl butyrate side chain is readily hydrolyzed from compactin by Penicillium chrysogenum and the side-chain-less ML-236A accumulates.

Example 3 Removal of the 2-methylbutyric moiety of lovastatin by fungal hydrolase activity

Penicillium chrysogenum was cultivated in mineral medium (as described in US 2002/0039758) for 96 hours 25°C in 25 ml_. From that time point lovastatin was added to the total broth in at a final concentration of 0.10 g/L. After 24 and 48 hours following the addition samples were taken for analysis of lovastatin and monacolin J. Broth samples (0.25 ml.) were mixed with an equal volume of methanol, followed by vigorous vortexing, at least 1 minute per sample. Care was taken that all material in the Eppendorf tube was brought into suspension, and no clumps remained during extraction. The tubes were centrifuged at 13000 rpm for 5 minutes, and the clear supernatant was transferred to HPLC sample tubes. The HPLC analysis conditions were the same as in Example 1 ; Retention times (min): Monacolin J (6.5), lovastatin (8.8). Figure 3 shows that the methyl butyrate side chain is readily removed from lovastatin by Penicillium chrysogenum and the side-chain-less monacolin J accumulates.

Example 4 Use of media devoid of urea to inactivate statin hydrolysis during fermentation

Penicillium chrysogenum devoid of the β-lactam biosynthesis pathway and equipped with the compactin biosynthesis pathway (WO07122249) was cultivated in standard mineral medium (as described in US 2002/0039758), urea-less mineral medium and YEPD. After 72 hours of cultivation samples from the YEPD cultures were taken and after 12O h of cultivation samples were taken from the mineral media. As a control the natural compactin producer Penicillium citrinum NRRL 8082 was used. A sample of 1 mL was mixed with 1 mL methanol and vortexed vigorously for 1 minute. Following this extraction procedure the tubes were centrifuged at 13000 rpm for 5 minutes to spin down the cell debris and the clear supernatant was transferred to HPLC sample tubes. HPLC was performed using the following equipment and conditions:

Eluens: A: 33% CH ₃ CN , 0.025% CF ₃ CO ₂ H in milliQ water

B: 80% CH ₃ CN in milliQ water

C: milliQ water

Gradient: Time (min) A B C

0.0-8.0 100% 0% 0%

8.0-8.1 100→0% 0→100% 0%

8.1-12 0% 100% 0%

12.0-13.0 0→100% 100→0% 0%

13.0-14.0 100% 0% 0% Column, column temperature, flow rate, injection volume, tray temperature, instrument, detector and wavelength as in Example 1. Retention times (min): ML-236A (2.6), hydrolyzed compactin (10.4) The wild type Penicillium citrinum strains barely produce any statins, while the Penicillium chrysogenum compactin transformants produce significant amounts (see Table 1 ). Moreover, the ratio between compactin and ML-236A is shifted towards compactin when urea is left out of the medium. In conclusion, omitting urea from fermentation media increases the ratio between compactin and ML-236A.

Table 1 Statin production in shake flasks (in mg/L)

Table 2 Statin production in shake flasks with Penicillium chrysogenum compactin producers using different nitrogen sources (duplicate experiments).

Example 5

Use of specific nitrogen sources to inactivate statin hydrolysis during fermentation

Penicillium chrysogenum devoid of the β-lactam biosynthesis pathway and equipped with the compactin biosynthesis pathway (WO07122249) was cultivated in duplicate in standard mineral medium (as described in US 2002/0039758) wherein the nitrogen source was varied. After 96 hours of cultivation samples from the cultures were taken. A sample of 1 ml. was mixed with 1 ml. methanol and vortexed vigorously for 1 minute.

Following this extraction procedure the tubes were centrifuged at 13000 rpm for 5 minutes to spin down the cell debris and the clear supernatant was transferred to HPLC sample tubes. HPLC was performed using equipment, conditions and retention times as mentioned in Example 4. In the 'standard' medium, so with urea, the Penicillium chrysogenum compactin strains produce significant amounts of ML-236A (see Table 2).

But, the ratio between compactin and ML-236A is shifted towards compactin when urea is left out and other nitrogen sources are used. Most preferred nitrogen sources are:

(NH ₄ ) ₂ SO ₄ , lysine and/or glutamate. In conclusion, use of specific nitrogen sources in fermentation media increases the ratio between compactin and ML-236A.

Example 6 Use of specific carbon sources to inactivate statin hydrolysis during fermentation

Penicillium chrysogenum devoid of the β-lactam biosynthesis pathway and equipped with the compactin biosynthesis pathway (WO07122249) was cultivated in duplicate in standard mineral medium (as described in US 2002/0039758) wherein the carbon source was varied. After 144 hours of cultivation samples from the cultures were taken. A sample of 1 mL was mixed with 1 mL methanol and vortexed vigorously for 1 minute. Following this extraction procedure the tubes were centrifuged at 13000 rpm for 5 minutes to spin down the cell debris and the clear supernatant was transferred to HPLC sample tubes. HPLC was performed using equipment, conditions and retention times as mentioned in Example 4. In 'standard' fermentation media glucose is most often used as carbon source. From Table 3 it is clear that this (in batch cultivation) is not the best choice: (a) the total level of statins is not high and (b) the ratio between ML-236B and ML-236A is very low. These date suggest (partial) glucose repression of the compactin pathway and (partial) induction of the hydrolyzing enzyme(s). So, when changing the glucose for lactose, the Penicillium chrysogenum compactin strains produce significant

amounts of statins, but also significant amounts of ML-236A are obtained (see Table 3). But, the ratio between compactin and ML-236A is shifted towards compactin when for example ethanol and/or acetate are used as carbon sources. In conclusion, use of specific carbon sources in fermentation media increases the ratio between compactin and ML-236A.

Table 3 Statin production in shake flasks with Penicillium chrysogenum compactin producers using different carbon sources (duplicate experiments).

Example 7

Identification of commercial enzymes that hydrolyze statins.

40 mg of freeze-dried commercial enzyme was suspended in 475 μl of 50 mM potassium phosphate buffer at pH 7.5. Screening was set up using 1 mg/mL compactin and samples were incubated for 24 h in microtiterplates at room temperature, while shaking. After incubation, the microtiterplates were centrifuged and samples were diluted with 500 μl buffer. After dilution, the microtiterplates were centrifuged again, in order to perform HPLC analysis (protocol: Example 6). Table 4 shows the hydrolysis in several

samples. A Bacillus subtilis enzyme sample from Novozymes (Subtilisin A) shows the highest hydrolysis rate: 39.7% in 24 h. Using blastP and the known protein sequence of this enzyme (SEQ ID NO 45) several genes encoding homologous enzymes in various fungi, including Penicillium chrysogenum, or bacteria, including Streptomyces carbophilus and Amycolatopsis orientalis, can be identified; see SEQ ID NO 15, 19, 41 and 43 for the DNA sequences of such genes and SEQ ID NO 16, 20, 42 and 44 for the putative protein sequences encoded by such genes. Using other positive examples as listed in Table 4 in a blastP analysis (Lypozym Im45 and liquid CaIB Novo525, respectively SEQ ID NO 46 and 47) several other genes encoding homologous enzymes in Penicillium chrysogenum, can be identified; see SEQ ID NO 1 1 , 13, 17 and 21 for the DNA sequences of such genes and SEQ ID NO 12, 14, 18 and 22 for the putative protein sequences encoded by such genes. The above sequences can be used to identify relevant homologous sequences in other species like Aspergillus niger (SEQ ID NO 25-30), Aspergillus terreus (SEQ ID NO 31-34) and Penicillium citrinum (SEQ ID NO 35-40).

Table 4 Compactin hydrolysis using commercially available esterases/lipases.

Hydrolysis

Origin Supplier Description

(%)

Candida antarctica Novozymes liquid CaIB Novo525 5.6 Aspergillus niger Amano Protease A-2 5.7 Bacillus subtilus Novozymes Subtilisin A 39.7 Bacillus licheniformis DSM Devolase/Pescalase (protease) 8.5 Bacillus subtilus Novozymes Alcalase 5.0 Bacillus subtilus Novozymes Alcalase 5.8 Bacillus subtilus Novozymes Lipase SP 539 11.3 Rhizomucor miehei Novozymes Lypozym Im45 (L-214) 5.3 Bacillus sp. Amano Proleather (protease) 5.3 Bacillus sp. Amano Proleather 5.6 Aspergillus Amano Prozyme 6 1 1.3 hog liver Sigma Fluka Esterase porcine liver 7.0 Bacillus sp. Nagase Protease C L- 15 12.6

Example 8 Isolation and identification of P. chrysogenum enzymes that hydrolyze statins

Fractionation of lipase/esterase:

Penicillium chrysogenum was cultivated in standard media containing urea as nitrogen source. After 96 hours the cells were filtrated and rapidly washed with icecald physiological salt (0.9% NaCI). The pellet was quickly frozen in liquid nitrogen and

subsequentely freeze-dried. Cell Free Extract (CFE) of Penicillium chrysogenum was prepared by mortaring 240 mg freeze dried sample on ice and dissolving in 15 ml cold 50 mM Tris-HCI buffer pH 8. After dissolving, the sample was centrifuged for 10 min at 14.000 rpm and 4°C. The supernatant (CFE) was stored at -20 ⁰ C in small aliquots. Before use, a sample (one eppendorf cup) was thawed on ice (samples are always kept on ice unless stated otherwise). The sample was precipitated by slowly adding (NhU) ₂ SO ₄ (226 mg/ml) up to 40% saturation at 0 ⁰ C (precipitation with 40% saturated (NhU) ₂ SO ₄ is sufficient to precipitate other proteins while the active protein is still soluble). After 30 min precipitation, the sample was centrifuged for 10 min at 14.000 rpm and 4°C. The HIC column (1 ml Butyl FF HiTrap) was equilibrated with 40% saturated (NhU) ₂ SO ₄ on the Akta purifier. After applying -1.4 ml of the precipitated sample on the column, a gradient of 40% saturated to 0% saturated (NhU) ₂ SO ₄ was initiated with a flow of 1 ml/min as shown in Figure 4. Fractions of 1 ml were collected. 100 μl was used for the compactin assay. The other part was (partially) used for TCA precipitation and SDS- PAGE.

Compactin assay:

10μl (10 x diluted in ethanol; 2 mg/ml) compactin was added to 100 μl sample and incubated at 37°C for 2 h. 50μl of sample was added to 50 μl methanol and centrifuged for 5 min at 14.000 rpm. 50 μl of the supernatant was used for the HPLC compactin assay. As shown in Table 5, the main activity is found in cell free extract and in fraction 28. However, the protein concentration of every sample differs and the results have not been corrected for the amount of protein, explaining the high activity of CFE compared to fraction 28. The samples were precipitated by adding 20% TCA to a final concentration of 10%, kept on ice for 1 h and were centrifuged for 10 min at 14.000 rpm and 4°C. The pellet was washed with 80% ethanol and again centrifuged for 10 min at 14.000 rpm and 4°C. Supernatant was discarded and the pellet was dried to air for 5 min. 5 μl sample buffer (Invitrogen NuPAGE ^® LDS Sample Buffer, pH 8.4) was added to the pellet and heated for 10 min at 70 ⁰ C. 2 μl reducing agent (Invitrogen NuPAGE ^® Sample Reducing Agent) and 13 μl milliQ was added to the sample to a total volume of 20 μl and again heated for 5 min at 70 ⁰ C. Electrophoresis was performed using MOPS buffer and an 4- 12% Bis-Tris PAGE gel (Figure 6).

Table 5 Compactin hydrolysis activity of various HIC fractions

ML-236A Compactin

Sample Percentage ML-236A Peak area Peak Area

Fraction 13 141566 2664742 5.3 Fraction 14 152021 2864236 5.3 Fraction 15 162888 3077368 5.3 Fraction 16 157140 2957027 5.3 Fraction 17 157225 2977385 5.3 Fraction 18 150828 2832126 5.3 Fraction 19 155835 2952001 5.3 Fraction 20 155395 2943975 5.3 Fraction 21 157219 2998133 5.2 Fraction 22 157035 2945314 5.3 Fraction 23 165608 3005715 5.5 Fraction 24 181393 2851906 6.4 Fraction 28 268145 2742386 9.8 CFE 442983 2810811 15.8

40% precipitant 185563 3032108 6.1 40% saturated sample 147608 3017276 4.9

In-gel and in-vitro digestion and protein identification by LC-MS/MS: The active fraction (lane 9) has slightly different protein bands on -7OkDa and -4OkDa compared to the other lanes. The 2 spots were cut from gel (Figure 5), digested, and identified with LC-MS. For analyzing peptide mixtures, a survey scan was used. This method, in which each scan consists of two segments, was defined as follows:

1. full MS scan analysis, selecting precursor ions for MS/MS based on defined criteria

2. MS/MS of the selected ions to obtain amino acid sequence information With this procedure one precursor ion at a time was selected for MS/MS within certain thresholds, which results in MS/MS datasets of peptides, all presenting the amino acid sequence of a part of a protein. All MS/MS spectra were used for protein identification by database searching using the MASCOT search engine in the MSDB database. MS identification: Both 7OkDa and 4OkDa samples were identified with LC-MS. The 7OkDa band could not be identified with LC-MS. Reanalysis with MALDI gave a hit. The results of MS identification:

• -4OkDa band: Pc15g00720 ss 1 ,4-butanediol diacrylate esterase (48% sequence coverage) BDA1 from B. linens (theoretical molecular weight = 43kDa).

• -7OkDa band: Pc22g01380 ss beta-1 ,3-exoglucanase exgi c from C. carbonum (theoretical molecular weight = 86kDa).

The esterase identified on the 4OkDa spot is definitely a good candidate for degrading ML-236B (see SEQ ID NO 23 for the DNA sequence of this gene and SEQ ID NO 24 for

the protein sequence encoded by this gene). The exoglucanase on the 7OkDa spot is unlikely to be involved in the degradation of ML-236B.

Example 9 Deletion of P. chrysogenum genes encoding enzymes that hydrolyze statins.

The gene Pc15g00720, encoding an esterase, was identified as the putative 2-methylbutyric esterase. In order to prevent transcription of this gene a selection marker gene was inserted between the promoter and the terminator. To this end the promoter and the terminator were PCR amplified using the oligonucleotides SEQ ID NO 1 plus 2 and SEQ ID NO 3 plus 4, respectively. Phusion Hot-Start Polymerase (Finnzymes) was used to PCR amplify the fragments from Penicillium chrysogenum genomic DNA (strain Wisconsin54-1255). Both fragments are approximately 1800 bp long (SEQ ID NO 5 and 6) and contain a 14 bp tail suitable for the STABY cloning method (Eurogentec). From the standard STABY vector, pSTC1.3, two derivatives were obtained. One, pSTamdSL, was used for cloning the PCR amplified Pc15g00720 promoter. The other, pSTamdSR, was used for cloning the PCR amplified Pc15g00720 terminator. pSTamdSL was constructed by insertion of an inactive part of the amdS selectionmarker gene (cf. the PgpdA-amdS cassette of pHELY-A1 in WO 04106347) by PCR amplification of the last 2/3 of the gene {amdS) and cloning it in the Hind\\\-BamH\ sites of pSTC1.3. pSTamdSR was constructed by insertion of another inactive part of the amdS selectionmarker gene (cf. the PgpdA-amdS cassette of pHELY-A1 in WO 04106347) by PCR amplification of the PgpdA promoter and the first 2/3 of the gene wherein the EcoRV sites where removed and cloning it in the Hind\\\-Pme\ sites of pSTC1.3. Also, a strong terminator was inserted in front of the PgpdA-amdS; the trpC terminator was PCR amplified and inintroduced via the Sbf\-Not\ sites of the PgpdA-amdS fragment. Both vectors do contain an overlapping but non-functional fragment of the fungal selectionmarker gene amdS, encoding acetamidase and allowing recipient cells that recombine the two fragments into a functional selectionmarker to grow on agar media with acetamide as the sole nitrogen source (EP 635,574; WO 9706261 ; Tilburn et al., 1983, Gene 26: 205- 221 ). The PCR fragments were ligated into the vectors overnight using T4 ligase (Invitrogen) at 16 ⁰ C, according to the STABY-protocol (Eurogentec) and transformed to chemically competent CYS21 cells (Eurogentec). Ampicillin resistant clones were isolated and used to PCR amplify the cloned fragments fused to the two non-functional amdS fragments (Figure 7). This was done using the oligonucleotides SEQ ID NO 7 and

8. The thus obtained PCR fragments (SEQ ID NO 9 and 10) were combined and used to transform a Penicillium chrysogenum strain with the hdfA gene deleted (WO05095624). In this strain the non-homologous end-joining pathway is disturbed and therefore the random integration of DNA is drastically reduced. As the combined PCR fragments themselves should recombine also to form a functional amdS selection marker gene (Ae. the so-called bipartite or split-marker method), correct targeted integrants should undergo a triple homologous recombination event (Figure 6).

Table 6 Compactin hydrolysis activity of various Penicillium chrysogenum strains (i.e. Pd 5g00720 deletions) after 24 hours of incubation

Strain ML-236A Compactin Compactin

(peak area) (peak area) (% of total)

K01 163,754 14,029,890 98.8

KO2 4,489,876 8,627,420 65.8

KO3 153,886 13,282,392 98.9

KO4 143,323 12,655,952 98.9

KO5 207,446 12,863,580 98.4

KO6 223,121 13,431 ,032 98.4

KO7 163,672 13,471 ,123 98.8

KO8 213,895 12,840,465 98.4

KO9 181 ,969 12,895,948 98.6

KO10 246,818 12,631 ,598 98.1

KO1 1 2,589,830 10,872,225 80.8

Penicillium chrysogenum 12,861 ,096 250,288 1.9

Eleven transformants were obtained on acetamide containing agar (EP 635,574; WO 9706261 ). These were transferred to a second acetamide selectionplate to induce sporulation. Spores from these plates were used to inoculate synthetic media with urea as the nitrogen source (Examples 1 and 2) to investigate if the urea-inducible statin hydrolysis activity was decreased or absent. After three days of cultivation 0.1 g/l compactin was added to the cultures and samples were taken after 4 and 24 hours. Analysis of the samples was perfomed as described in Examples 1 and 2. While the remaining compactin in the parent strain was almost zero (1.9%, see Table 6), this remaining compactin was much higher in the mutant strains: 65.8-98.9%. This data confirms that the gene Pc15g00720 encodes a 2-methylbutyric esterase and that by deleting this gene the specific esterase activity was decreased with more than 25%.