WO2009056539A1

WO2009056539A1 - Fermentative production of simvastatin

Info

Publication number: WO2009056539A1
Application number: PCT/EP2008/064595
Authority: WO
Inventors: Van Den Marco Alexander Berg; Marcus Hans; Axel Christoph Trefzer
Original assignee: Dsm Ip Assets B.V.
Priority date: 2007-10-30
Filing date: 2008-10-28
Publication date: 2009-05-07

Abstract

The present invention relates to the biotechnological production of the HMG-CoA reductase inhibitor simvastatin by us ing a microorganism capable of producing monacolin J, a transesterase and the addition of 2,2-dimethylbutyric acid or a salt or an ester thereof.

Description

FERMENTATIVE PRODUCTION OF SIMVASTATIN

Field of the invention

The present invention relates to the biotechnological production of the HMG-CoA reductase inhibitor simvastatin.

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. Today, simvastatin is amongst the most prescribed drugs in cholesterol lowering applications. The synthesis of simvastatin is not straightforward as it involves a semi-synthetic approach starting from the natural product lovastatin (synthesized by Aspergillus terreus).

Name R

Monacolin J H

Simvastatin

The molecular difference between lovastatin and simvastatin resides in the side chain on the C-8 position. Here, lovastatin carries a 2-methylbutyrate moiety, while simvastatin has a 2,2-dimethylbutyrate moiety on this position. Numerous chemical syntheses of simvastatin have been reported worldwide since its discovery in 1984. One route, as described in US 4,444,784, involves de-esterification of the 2-methylbutyrate side chain of lovastatin, followed by several distinct chemical steps that involve lactonization, hydroxy group protection/deprotection and re-esterification with the appropriate 2,2-dimethylbutyrate side chain. This process results in a low overall yield. Another method, described in US 4,582,915, involves direct methylation of the lovastatin side chain using a metal alkyl amide and methyl halide. This method suffers from problems concerning the purity of the final product as it leads to several by-products that have to be separated from the target compound. Furthermore, the chemicals used are toxic and/or carcinogenic making implementation on an industrial scale difficult and hazardous. Likewise, yet another method described in US 4,820,850, while addressing the problems of low overall yield and purity, involves as many as six chemical steps that also utilize reagents that are unsafe to handle on an industrial scale. The more recent method described in US 5,763,646 involves conversion of lovastatin to simvastatin using fewer chemical steps. However, this method employs expensive chemical reagents and also results in low overall yield. As an alternative for the existing chemical routes WO 03/010324 describes the metabolic engineering of one of both polyketide synthases (LovF) involved in the biosynthesis of lovastatin. However, the problem underlying the direct fermentative production of simvastatin in a host is the incorporation of the unnatural side chain, 2,2-dimethylbutyrate. By exchanging modules of the LovF polyketide synthase for modules from other sources this problem is not solved because, in order to synthesize the 2,2-dimethylbutyrate side chain, the modified LovF enzyme requires a substrate (methylmalonyl-CoA) that is not present in lovastatin producing organisms such as Aspergillus terreus. In WO 00/037629 it is suggested to modify the lovB gene for the production of other HMG-CoA reductase inhibitors. As the lovB gene is involved in the biosynthesis of the monacolin J core indeed other HMG-CoA reductase inhibitors are accessible through modification of this gene, however not simvastatin as the formation of the side chain at position C-8 does not reside in the lovB gene.

More recently other biotechnological methods for production of simvastatin have been explored. The use of esterases for deacetylation of lovastatin and removal of protection groups has been described in WO 05/040107. The esterase is first used to remove the side-chain of lovastatin. Following chemical acylation of protected monacolin J the same or another enzyme is used to remove protecting groups yielding simvastatin. Another approach utilizing an enzyme for attachment of the di-methyl butyrate by a whole cell biocatalyst expressing the transesterase LovD was described. This method requires addition of the immediate precursor monacolin J and the dimethylbutyrate side chain in an activated thioester form (Xie et al. Chem. Biol. 13, 1161 -1 169 (2006); Appl. Environ. Microbiol. 73, 2054-2060 (2007); Metabolic Engineering 9, 379-386 (2007)). All these methods suffer from the need of producing monacolin J from lovastatin by chemical or enzymatic hydrolysis. This is then followed by a separate conversion step of monacolin J to simvastatin. The use of the LovD enzyme for this final transformation is hampered by the fact that its activity is limited by substrate inhibition of the enzyme by monacolin J, This only allows use of low concentrations of substrate limiting the amount of product that can be produced by the enzyme. The use of this biocatalyst is further hampered by its ability to also catalyze the reverse reaction resulting in hydrolysis of simvastatin to monacolin J and dimethylbutyrate.

Detailed description of the invention

The term statin biosynthetic gene encompasses any of the wild type genes from, but not limited to, Aspergillus terreus, Penicillium citήnum, Monascus ruber, or Monascus pupurea including also modified, inactive and truncated variants plus homologous enzymes and non-homologous enzymes with the same function or similar functions involved in biosynthesis of statins or similar compounds (i.e. the lovastatin and compactin enzymes systems). The term 2,2-dimethylbutyrate encompasses all bio-available molecules containing the CH₃CH₂C(CH₃)₂C(O)-R moiety, in which R can be OH, O X⁺ wherein X⁺ represents a cation such as a metal cation, ammonia or other nitrogen derived cations. Particularly suitable compounds are those wherein R represents an activated group. Any activated group known to the skilled person is suitable. Particularly suitable activated groups are for instance S-coenzyme A (SCoA) or derivatives thereof, S-Λ/-acetylcysteamine (SNAC) or derivatives thereof, S-methylthioglycolate (SMTG), mercaptopropionate and mercaptobutyrate and other thioalkyl groups and the like.

A polypeptide having an amino acid sequence that is "substantially homologous" to the lovastatin biosynthetic genes is defined as a polypeptide having an amino acid sequence possessing a degree of identity to the specified amino acid sequence of at least 30%, preferably at least 40%, more preferably at least 50%, still more preferably at least 60%, still preferably at least 70%, still more preferably at least 80%, still more preferably at least 90%, still more preferably at least 98% and most preferably at least 99%, the substantially homologous peptide displaying activity towards the synthesis of lovastatin and/or simvastatin. 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 species 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. For the purpose of 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, J. U. 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).

The first aspect of this invention is to provide a host with a supply of monacolin J and the capability for direct conversion of such an intermediate to simvastatin. Using LC-

MS analysis it was demonstrated that simvastatin is not synthesized or present in natural lovastatin producers (e.g. Aspergillus terreus). Secondly, in wild type Aspergillus terreus, grown under lovastatin producing conditions, neither 2,2-dimethylbutyrate nor simvastatin can be detected, intra-or extracellularly. Taken this together, it is shown that Aspergillus terreus lacks methylmalonyl-CoA to synthesize 2,2-dimethylbutyrate.

One embodiment of the invention describes engineering of a microorganism for production of monacolin J harboring a complete or part of a statin biosynthetic gene cluster. Suitable organisms are prokaryotes such as those from Bacillus, Escherichia, Myxococcus, Pseudomonas, Saccharopolyspora, Sorangium, Stigmatella, Amycolatopsis, Actinomyces and Streptomyces and particularly those chosen from the group consisting of Bacillus amolyquefaciens, Bacillus subtilis, Streptomyces coelicolor, Streptomyces albus, Streptomyces carbophilus, Saccharopolyspora erythraea, Amycolatopsis orientalis, Actinomyces spec, Nocardia autotrophica, Myxococcus xanthus, Stigmatella aurantiaca, Sorangium cellulosum, Pseudomonas putida, Pseudomonas fluoresceins and Escherichia coli or eukaryotes such as Aspergillus, Monascus, Kluyveromyces, Penicillium and Saccharomyces and particularly those chosen from the group consisting of Aspergillus nidulans, Aspergillus terreus, Aspergillus niger, Penicillium citrinum, Penicillium brevicompactum, Penicillium chrysogenum, Penicillium patulum, Penicillium aurantiogriseum, Monascus ruber, Monascus purpurea, Eupenicillium javanicum, Paecilomyces viridis, Penicillium decumbens, Saccharomyces cerevisiae and Kluyveromyces lactis. As described by Abe et al. (Molecular Genetics and Genomics 267, 636-646 (2002)) this can be achieved by heterologous expression of part of the lovastatin biosynthetic gene cluster. An alternative approach is to delete or inactivate the methyl butyrate biosynthetic enzyme (e.g. LovF or homologue) in a statin producing organism (e.g. Aspergillus terreus, Penicillium citrinum) or to engineer another statin biosynthetic gene cluster (e.g. compactin gene cluster from Penicillium citrinum) towards the production of monacolin J in a heterologous microorganism. In one specific embodiment the expression level and pattern of the complete or part of various statin biosynthetic gene cluster as described can be optimized. One option is to modify at the level of transcription. This can be done in various ways: 1) Improve the effectiveness of the specific transcriptional regulator (i.e. lovE), as described in US 2004/191877; 2) Improve the codon usage of the specific transcriptional regulator (i.e. lovE), as described in EP 1841861 ; 3) Remove the original transcriptional activator and replace all promoters of the individual genes by promoters suitable for the specific purpose (i.e. high transcription level via strong promoters, or specific induction via specific inductors, like the tetracycline system)

Another option is to modify the pattern of transcription. This will allow for an optimal process design, for example: (1 ) separating growth and production phases by the use of specific promoters; (2) limit the number of gene copies to introduce by using specific strong (and preferably inducible) promoters; (3) growth phase specific promoters which are switched on during fermentation when production is wanted.

Yet another alternative is to utilize a microorganism capable or engineered for the specific enzymatic conversion of a statin molecule to monacolin J. This can be achieved by expressing a suitable enzyme or by activating expression of such an enzyme by specific inducing growth conditions of an organism harboring a gene encoding such an enzyme. Suitable enzymes for such a conversion are hydrolases including esterases, amidases, or proteases. One such enzyme was discovered in Penicillium chrysogenum and its polypeptide sequence is given in SEQIDNO. 3. This polypeptide is encoded by the DNA given in SEQIDNO. 2. The production of this polypeptide can be controlled by certain culture conditions including but not limited to the nitrogen source. Alternative ways to control the production of this polypeptide is the use of regulable (inducible or repressible) promoter sequences. This transcriptional regulation allows for a delicate control of the fermentation process and the products made. One can, however not limited to, switch from direct synthesis of monacolin J towards a biphasic production wherein the organism first produces lovastatin and upon induction of the suitable enzyme system converts it into monacolin J. Penicillium chrysogenum produces another polypeptide which could be used for the same application: polypeptide sequence SEQIDNO. 6. This polypeptide is encoded by the DNA sequences given in SEQIDNO. 5. Another such enzyme has been described in WO 05/040107. The scope of this invention is not limited to these two polypeptides, but covers all substantially homologous polypeptides catalyzing similar conversions. Optimization of expression cassettes (i.e. codon optimization and optimal Kozak sequences) are described in PCT/EP2007/05594 and EP 1841861 . In one specific embodiment this enzyme is included in and thus expressed by the host. In another embodiment this enzyme can be added in several forms (see also below) to lovastatin fermentation broths to convert the lovastatin formed to monacolin J.

This engineered organism is further equipped with an enzymatic system suitable for attachment of the dimethylbutyrate side chain present in simvastatin. One such system is provided by the enzyme LovD capable of transferring the dimethylbutyrate from an extracellularly added suitable thioester such as but not limited to Coenzyme A thioesters, Λ/-acetylcysteamine thioesters, methyl-thioglycolate thioesters, mercapto- propionate thioesters and mercapto-butyrate thioesters. Similar enzymes can be found in other statin biosynthetic pathways as exemplified by MIcH which is encoded in the compactin biosynthetic gene cluster present in Penicillium cithnum (Abe et al., Molecular Genetics and Genomics 267, 636-646 (2002)). It is also conceivable to provide an activated thioester intracellular^ by expressing a modified polyketide synthase engineered for the production of dimethylbutyrate. Alternatively the microorganism is further engineered with an enzyme capable of converting dimethylbutyrate to a suitable thioester. One such enzyme is a Coenzyme A ligase that converts dimethylbutyrate to dimethylbutyrate-CoA. It has been demonstrated (Reger et al., Biochemistry 46, 6536- 6546 (2007)) that the substrate specificity of CoA-ligases can be altered by protein engineering. The organism is grown under conditions suitable for production of monacolin J (e.g. similar to lovastatin conditions as described in WO 98/37179) and dimethylbutyrate or a suitable dimethylbutyrate precursor is added to the culture resulting in the one step production of simvastatin. Alternative enzyme systems to be used are the enzymes named above for the conversion of lovastatin into monacolin J to be applied for the reverse reaction and specified for example in SEQIDNO. 3 and SEQIDNO. 6. Another embodiment of the invention describes the fermentative production of monacolin J and the in-situ conversion of monacolin J to simvastatin by an exogenously added enzyme system for attachment of the dimethylbutyrate side chain and addition of a dimethylbutyrate side chain precursor and required co-substrates for its activation (if necessary). This enzyme system minimally consists of a trans-esterase capable of attaching the dimethylbutyrate side chain to monacolin J. The enzyme system may also contain a dimethylbutyrate activating enzyme such as a CoA-ligase for in-situ generation of the activated dimethylbutyrate precursor. Such an enzyme system may be provided in the form of pure enzymes, crude lysates or whole cell biocatalyst(s) consisting of one or more organisms. One transesterase described for the conversion of monacolin J to simvastatin is the transesterase LovD (see above). To facilitate complete conversion of monacolin J to simvastatin substrate inhibition of the transesterase has to be overcome and catalysis of the reverse reaction (hydrolysis of simvastatin to monacolin J by LovD) has to be limited. For the LovD enzyme it has been suggested that using a whole cell system alleviates these problems. Another method to control substrate inhibition is the use of an immobilized enzyme thus facilitating efficient separation of enzyme, substrate and product. This will allow limiting both the local substrate and product concentrations in contact with the enzyme to optimal concentrations maximizing turnover and yield. Addition of a suitable agent(s) capable of specifically binding monacolin J and/ or simvastatin to the reaction mix is another way of efficiently separating enzyme and product resulting in suitable local substrate and product concentrations in contact with the enzyme. Protein engineering of LovD by rational or random methods provides another solution to create variants of LovD not suffering from product inhibition and/ or being unable to catalyze the hydrolysis of simvastatin. Additionally, biosynthetic gene clusters that are not homologous, but follow the same biosynthetic building principle for statin synthesis can be used.

When 2,2-dimethylbutyrate was fed to an organism with one or more lovastatin biosynthetic genes, for example an intact nonaketide synthase gene (such as the lovB gene), a mixture of lovastatin and simvastatin can be obtained, So, surprisingly, the lovastatin enzyme system exhibits, besides the natural preference for 2-methylbutyrate, a relatively high substrate tolerance towards 2,2-dimethylbutyrate. A further improvement can be achieved by addition of a lovB gene optimized by methods known in the art (e.g. directed evolution, gene shuffling or site directed mutagenesis) with a preference for attaching 2,2-dimethylbutyrate to the monacolin J core yielding simvastatin. Preferably, this is combined with inactivating the wild type lovF gene of Aspergillus terreus. Feeding of 2,2-dimethylbutyrate to such an optimized microorganism resulted in improved simvastatin production.

Claims

1. A method for the fermentative production of simvastatin comprising the combined use of a microorganism capable of producing monacolin J, a transesterase and the addition of 2,2-dimethylbutyric acid or a salt or an ester thereof.

2. Method according to claim 1 wherein 2,2-dimethylbutyric acid ester is produced in situ by a 2,2-dimethylbutyric acid activating enzyme.

3. Method according to anyone of claims 1 to 2 wherein said 2,2-dimethylbutyric acid ester is a thioester.

4. Method according to anyone of claims 1 to 3 wherein said transesterase is expressed in said microorganism capable of producing monacolin J.

5. Method according to anyone of claims 1 to 3 wherein said transesterase is added to a mixture comprising said microorganism capable of producing monacolin J.

6. Method according to anyone of claims 1 to 5 wherein product inhibition is reduced.

7. Method according to anyone of claims 1 to 6 wherein said microorganism capable of producing monacolin J is capable of enzymatic conversion of lovastatin into monacolin J.

8. Method according to anyone of claims 1 to 6 wherein said microorganism capable of producing monacolin J is capable of producing monacolin J de novo.

9. A microorganism capable of producing monacolin J and harboring an enzyme system for conversion of monacolin J into simvastatin.