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WO2003010324A2 - Methode de preparation de simvastatine - Google Patents

Methode de preparation de simvastatine Download PDF

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Publication number
WO2003010324A2
WO2003010324A2 PCT/IN2002/000157 IN0200157W WO03010324A2 WO 2003010324 A2 WO2003010324 A2 WO 2003010324A2 IN 0200157 W IN0200157 W IN 0200157W WO 03010324 A2 WO03010324 A2 WO 03010324A2
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Prior art keywords
pks
host
simvastatin
dna
plasmid
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PCT/IN2002/000157
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English (en)
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WO2003010324A3 (fr
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Anand Ranganathan
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International Centre For Genetic Engineering And Biotechnology
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Priority to AU2002337601A priority Critical patent/AU2002337601A1/en
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Publication of WO2003010324A3 publication Critical patent/WO2003010324A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein

Definitions

  • the field of the invention relates to a process for the preparation of simvastatin and analogues thereof through expression of customised hybrid modular polyketide synthases in a host, preferably one containing the lovastatin-producing polyketide synthase gene cluster.
  • the compounds lovastatin, pravastatin, mevastatin and simvastatin are highly potent anticholesterolaemic agents that act as inhibitors of HMG-CoA reductase, an enzyme involved in the rate-limiting step in cholesterol biosynthesis in humans.
  • simvastatin that is a synthetically made drug
  • the above-mentioned compounds are naturally occurring and are obtained through fermentation.
  • Lovastatin a product of Aspergillus terreus, possesses a 2-methylbutyrate side chain linked at C-8 position of the hexahydronaphthalene ring system.
  • Simvastatin on the other hand possesses a 2,2-dimethylbutyrate side chain at the C-8 position. This subtle sutructural difference has been shown to render simvastatin much more potent than lovastatin.
  • simvastatin The increased potency of simvastatin over its analogues has necessitated a need for a chemical synthesis that is efficient, eco-friendly, economically viable and high yielding. Numerous chemical syntheses of simvastatin have been reported worldwide since its discovery in 1984.
  • Another method involves direct methylation of the lovastatin side chain using a metal alkylamide and methylhalide. 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.
  • Another method (Verhoeven et al, 1989), while addressing the problems of low overall yield and purity, involves as many as six chemical steps that utilize reagents that are unsafe to handle on industrial scale.
  • the recent method of Kumar et al (1998) involves conversion of lovastatin to simvastatin using fewer chemical steps. However, this method employs expensive chemical reagents and also results in low overall yield.
  • the object of the present invention to provide a process for the preparation of simvastatin or analogues or derivatives thereof which avoids the drawbacks of prior art methods.
  • Another aspect of the invention relates to a polyketide synthase gene that is customised to obtain the 2,2-dimethylbutyrate side chain of simvastatin.
  • Another aspect of the invention relates to the host being a eukaryote or a prokaryote. Another aspect of the invention relates to the host being a lovastain-producer.
  • Another aspect of the invention relates to the host being selected from Aspergillus spp..
  • Another aspect of the invention relates to the Aspergillus spp. being the lovastain- producing Aspergillus terreus. Another aspect of the invention relates to the host being the lovastain-producing
  • Aspergillus terreus mutant that is unable to produce the 2-methylbutyrate side chain.
  • Another aspect of the invention relates to the host being Aspergillus terreus/ARl.
  • Another aspect of the invention relates to a method wherein said domains for the customised polyketide synthzase are obtained from fatty acid synthase.
  • Another aspect of the invention relates to a method wherein the selected domain nucleic acids are selected from the group comprising type I modular polyketide synthase, type II PKS and type I iterative PKS.
  • Another aspect of the invention relates to a method wherein the selected domain nucleic acids are type I modular PKS.
  • Another aspect of the invention relates to a method wherein the polyketide synthase gene is one selected from the group provided in Figures 6.
  • Another aspect of the invention relates to a method wherein the polyketide synthase gene is one selected from the group provided in Figures 8.
  • the invention also provides for simvastatin or analogs or derivatives thereof whenever prepared by the novel process.
  • the invention also provides for a host as modified according to the objectives of the invention.
  • the invention also provides for a construct encoding for a polyketide synthase according to the objectives of the invention. Description of the drawings
  • Figure 1 The anticholesterolaemic compounds that act as HMG-CoA reductase inhibitors.
  • Figure 2 The primary organization of genes and their corresponding proteins of the erythromycin-producing type I modular polyketide synthase.
  • Figure 3 The primary organization of genes and their corresponding proteins of the lovastatin-producing polyketide synthase. The hypothetical iterative functioning of the
  • LNKS is illustrated by analogy to the ery PKS.
  • the diketide is synthesised by LDKS.
  • FIG. 5 The composition of hybrid modular polyketide synthase A template (HMPKSA) that is customised to synthesise the 2,2-dimethylbutyrate side chain (22DMB).
  • Figure 6 The hybrid modular polyketide synthase Al-4 (HMPKSA1-4) constructed to synthesise 22DMB; the engineered sequence that is at the junctions of the assembled fragments is shown in capitals.
  • FIG 7 The composition of the hybrid modular polyketide synthase B template (HMPKSB) that is customised to synthesise analogues of 22DMB.
  • Figure 8 The hybrid modular polyketide synthase Bl-2 (HMPKSB1-2) constructed to synthesise analogue of 22DMB.
  • Figure 9 Integration of plasmid pSIM10H4S through homologous double recombination into the wild-type A. terreus strain resulting in the construction of a lovF mutant.
  • the invention describes a process for the synthesis of simvastatin.
  • the process comprises of the following steps: i). construction of a customised gene coding for a polyketide synthase (PKS) that is geared to synthesise the 2,2-dimethylbutyrate side chain of simvastatin. ii). transforming a host, preferably one containing the lovastatin-producing PKS gene cluster with a plasmid bearing the above said PKS gene and fermentation thereof to produce simvastatin.
  • PKS polyketide synthase
  • the host selected may be any prokaryote or a eukaryote that expresses the lovastatin PKS. Therefore, any host in addition to those know to express lovastatin PKS may also be employed for the transformation thereof with a plasmid bearing the 2,2- dimethylbutyrate synthesizing PKS gene.
  • the process described herein dispenses with the number of steps normally required for the synthesis of simvastatin as compared with the chemical syntheses of the target that have been reported so far in the scientific literature.
  • the primary factor responsible for this is an aspect of the invention that involves the synthesis of the 2,2-dimethylbutyrate side chain of simvastatin through the use of customised PKS enzymes in a host, especially a lovastatin-producing one.
  • the use of such enzymes for synthesis of the side chain followed by its in vivo priming of the hexahydronaphthalene ring of simvastatin circumvents the production thereof by the lengthy chemical synthesis route.
  • the cost of synthesising the drug is drastically reduced when compared with chemical synthesis.
  • the tight stereocontrol that is normally the feature of a PKS-catalysed reaction provides an advantage over chemical synthesis.
  • PKSs Polyketide synthases
  • type I PKS are giant multienzymes (with a molecular weight ranging from 100- lOOOKDa) that synthesise medicinally valuable drugs like the antibacterial erythromycin, immunosuppressant rapamycin and the anticancer epothilone B.
  • Type I PKSs are composed of enzymes like the ketoacyl synthase (KS), acyl carrier protein (ACP), acyl transferase (AT), dehydratase (DH), enoyl reductase (ER), keto reductase (KR) and thioesterase (TE; Donadio et al, 1991; Schwecke et al, 1995).
  • KS ketoacyl synthase
  • ACP acyl carrier protein
  • AT acyl transferase
  • DH dehydratase
  • ER enoyl reductase
  • KR keto reductase
  • TE thioesterase
  • the multienzymes through composite domains, catalyse the condensation and subsequent processing of small carbon units, ultimately giving rise to complex polyketides like erythromycin and rapamycin.
  • processing it is meant that chemical groups belonging to the carbon units are altered or removed.
  • the third class of PKSs are the type I iterative. These are systems where the polyketide part of the molecule is synthesised by just one module (that has covalently linked domains) that functions iteratively. Lovastatin and methylsalicylic acid are synthesised by this class of polyketide synthases.
  • the type I iterative module shows significant homology to animal fatty acid synthases, and like the latter, it possesses all the reduction-domains (DH, ER and KR).
  • lovastatin biosynthetic gene cluster has recently been isolated from A. terreus and sequenced (Figure 3; Kennedy et al., 1999).
  • Two large open reading frames lovB and lovF have been identified as those coding for the nonaketide-producing 'lovastatin nonaketide synthase' (LNKS) and 'lovastatin diketide synthase' (LDKS) respectively.
  • LNKS has been shown to be a type I iterative module while LDKS is predicted to be non- iterative, much like the type I DEBS modules that synthesise erythromycin ( Figure 2).
  • lovD has been proposed to code for a transesterase that could help in bringing the two polyketide chains together.
  • An A. terreus mutant with the inactivated LDKS gene (lovF) was shown to accumulate monacolin J, the immediate precursor to lovastatin that is devoid of the side chain, thus directly implicating LDKS in 2-methylbutyrate synthesis (Kenedy et al, 1999).
  • the LDKS-catalysed polyketide biosynthesis starts through the uptake of a small carbon unit called starter acid that primes the KS (defined as KS in LDKS, Figure 4) of the PKS and is one of the units involved in the first condensation reaction between the said starter acid (in this case acetyl-CoA) and extension acid (malonyl-CoA) that is selected by the AT domain immediately downstream of the KS.
  • starter acid in this case acetyl-CoA
  • extension acid malonyl-CoA
  • the sequential order of domains present in LDKS is as shown in Figure 4.
  • the presence of a methyltransferase domain (MeT) in LDKS argues well for the addition of a methyl group at the C-2 position being catalysed by this enzyme.
  • PKS modules containing such "embedded" MeT domains have been reported for the yerseniabactin and epothilone PKSs (Suo et al, 2001; Mol
  • polyketide biosynthesis starts through the uptake of the starter acid that primes the first AT (defined as ATO in HMPKSB, Figure 7) of the PKS and, like in LDKS, it is one of the units involved in the first condensation reaction between the said starter acid and extension acid that is selected by the AT domain immediately downstream of the first AT.
  • the choice of the starter acid is dependent upon the degree of stringency that the first AT domain employs in the uptake.
  • the first AT domain of the erythromycin-producing PKS specifically selects propionyl coenzyme A (propionylCoA) as the starter acid whereas the first AT domain of the avermectin-producing PKS has been shown to accept a wide variety of starter acids (Dutton et al, 1991).
  • the above said avermectin PKS domain is thus most suited for generating polyketide molecules that require to be synthesised from starter acids that are not used naturally by any of the PKS enzymes known in the art. Therefore, preferred first AT domains in some of the customised type I PKSs envisaged by the invention is from the avermectin-producing PKS.
  • the 2,2-dimethylbutyrate side chain of simvastatin is synthesised through the use of customised PKSs.
  • the customisation is based on identification of the stereochemical features of the said side chain.
  • customisation it is meant that the domain selection is carried out in view of the identified stereochemical features.
  • the DNA fragments that correspond to the desired domains are Ugated with each other to give a gene that is expressed in a suitable host in order to obtain the above said side chain.
  • the blueprint for the customised PKS therefore is the customised gene that is incorporated in a host.
  • the desired PKS DNA fragments are assembled by any one of the methods known in the art. Particularly preferred is the method taught in Ranganathan (2000) as it is a time and cost-effective alternative to the conventional methods, especially suited for multi- component DNA assembly.
  • the incorporation of the customised PKS genes into host organisms is accomplished through the use of molecular biology techniques that are known to a person of skill (see Methods).
  • Hosts that are envisaged for the synthesis of the simvastatin side chain are of the prokaryotic and eukaryotic variety, such as actinomycetes, Aspregillus spp., s/9 line of insect cells, P. pastoris, and especially Aspregillus spp.
  • the invention discloses the transformation of a mutant strain of the lovastatin-producing Aspregillus terreus that is unable to synthesize the LDKS protein with the plasmid bearing the customised PKS gene.
  • This procedure results in the replacement of the 2-methylbutyrate side chain with the 2,2-dimethylbutyrate side chain in the above said lovastatin-producing strain, ultimately resulting in the production of only simvastatin from the same strain.
  • simvastatin was carried out by keeping to the following sequence: i). Synthesis of the 2,2-dimethylbutyrate side chain (22DMB) of simvastatin. ii). Transformation of a mutant of A. terreus with a plasmid bearing the 22DMB- producing PKS gene. Example 1.1 Synthesis of 22DMB
  • the HMPKS A template ( Figure 5) comprises of the following enzymatic domains in the given sequence: keto synthase - KS1, acyltransf erase - ATI, dehydratase - DH1, methyltransferase - MeTl, enoylreductase - ER1, ketoreductase - KR1 and acyl carrier protein - ACPI.
  • the domains are covalently linked with each other, to give a multi- enzyme HMPKS A template.
  • HMPKSAl hybrid modular polyketide synthase Al
  • pARHMPKSAl requires a multi-component DNA assembly
  • the method of Ranganthan (2000) was employed for the purpose.
  • the DNA sequences corresponding to the constituent domains of HMPKSAl were amplified by PCR to incorporate the two recognition sequences for Xbal (5'TCTAGA3' and 5'TCTAGATC3') at the 5' and 3' ends of the DNA fragment respectively.
  • the DNA sequence 5OATC3' is recognised by the Dam methylase gene of E. coli (Geier and Modrich, 1979). All PCR products were phosphorylated and ligated to Smal-cut, dephosphorylated pUC18 vector and then used to transform E. coli DH10B electrocompetent cells.
  • the desired plasmids containing the amplified DNA fragments were isolated and sequenced using standard pUC forward and reverse primers. No mistakes in the amplified products were detected. All four plasmids were then used to transform the E.coli ET12567 dam " strain (MacNeil et al, 1992). Isolated DNA was then digested with Xbal and the desired fragments isolated and purified. Fragment 4 was then ligated to Xbal-cut pCJR24HS vector that is derived from the S. erythraea expression vector pCJR24 (Rowe et al, 1998).
  • the pCJR24HS vector has a unique Xbal site and contains hygromycin as well as thiostrepton-resistance gene as markers for identifying successful integrands. Construction of pCJR24HS is described in the Methods section. The ligation products were used to transform E. coli DH10B electrocompetent cells and the desired plasmid was isolated using ampicillin as a resistance marker. This plasmid (pSIMAl) can only be singly cleaved with Xbal despite possessing two Xbal recognition sequences, as one of the sites (situated at the 3 ' end of 4) has been methylated by the E. coli Dam methylase.
  • Plasmid pSIMAl was digested with Xbal, ligated to fragment 3, and the ligation products treated as mentioned above to yield pSIMA2. DNA fragment 2 was then added to finally yield plasmid pSIMA3. This plasmid was then digested with Ndel and Xbal and ligated with the KS1 fragment (1) previously digested with the same two enzymes. The ligated products were used to transform E. coli DH10B electrocompetent cells and the final expression plasmid pARHMPKSAl, containing the customised HMPKSAl gene, was isolated. The junctions where the domains were joined are shown in Figure 6. iv). Expression of plasmid pARHMPKSAl in a host
  • Plasmid pARHMPKSAl was used to transform the mutant strain A. terreus/ARl (for the construction of this strain see Methods 1.3). Particularly preferred was the transformation procedure of Punt et al. (1992). Thiostrepton-resistant colonies were selected upon integration of the vector into the A. terreus chromosome. Single transformants were picked and grown on plates supplemented with hygromycin and thiostrepton. v). Growth of host The above said single transformants were grown in one litre of liquid media for seven days. Media composition was based on the findings of Szakacs (1998). Cells were removed by centrifugation and the supernatant collected. vi).
  • HMPKSA2 hybrid modular polyketide synthase A2
  • HMPKSA2 namely fragments 1, 3 and 4 had been prepared earlier, as described in Example 1.1.2.
  • the nucleotide positions in the template are shown according to the same disclosed in prior art (Cole et al., 1998).
  • the source of DNA, nucleotide positions of the template and the oligonucleotide primers (shown as 5' to 3') used for each PCR were as follows:
  • Isolated DNA was digested with Xbal and the desired fragments isolated and purified. Fragment 4 was then cloned in Xbal-c t pCJR24HS vector using the procedure described in Example 1.1.2.
  • the resulting plasmid pSIMAl was digested with Xbal and the other DNA fragments, namely, 3 and 5 were sequentially added to yield plasmid pSIMA23.
  • This plasmid was then digested with Ndel and Xbal and ligated with fragment 1 previously digested with the same two enzymes. The ligated products were used to transform E.
  • Plasmid pARHMPKSA2 was used to transform the mutant strain A. terreus/ARl. Particularly preferred was the transformation procedure of Punt et al. (1992). Thiostrepton-resistant colonies were selected upon integration of the vector into the A. terreus chromosome. Single transformants were picked and grown on plates supplemented with hygromycin and thiostrepton. v). Growth of host
  • HMPKSA3 hybrid modular polyketide synthase A3
  • fragment 6 contained DNA sequences corresponding to two domains ( Figure 6).
  • the source of DNA, nucleotide positions of the template and the oligonucleotide primers (shown as 5' to 3') used for each PCR were as follows:
  • pARHMPKSA3 For the construction of pARHMPKSA3, the method of Ranganthan (2000) was employed, as described earlier in Example 1.1.2.
  • the PCR products were phosphorylated and ligated to Smal-c t, dephosphorylated pUC18 vector and then used to transform E. coli DH10B electrocompetent cells.
  • the desired plasmids containing the amplified DNA fragments were isolated and sequenced using standard pUC forward and reverse primers. No mistakes in the amplified products were detected.
  • the plasmids were then used to transform the E.coli ET12567 dam " strain. Isolated DNA was digested with Xbal and the desired fragments isolated and purified.
  • Fragment 4 was then cloned in Xbal-cut pCJR24HS vector using the procedure described in Example 1.1.2.
  • the resulting plasmid pSIMAl was digested with Xbal and the other DNA fragments, namely 6 and 2 were sequentially added to finally yield plasmid pSEVIA33.
  • This plasmid was then digested with Ndel and Xbal and ligated with fragment 1 previously digested with the same two enzymes.
  • the ligated products were used to transform E. coli DH10B electrocompetent cells and the final expression plasmid pARHMPKSA3, containing the customised HMPKS A3 gene, was isolated.
  • the junctions where the domains were joined are shown in Figure 6. iv). Expression of plasmid pARHMPKSA3 in a host
  • Plasmid pARHMPKSA3 was used to transform the mutant strain terreus/ARl. Particularly preferred was the transformation procedure of Punt et al. (1992). Thiostrepton-resistant colonies were selected upon integration of the vector into the A. terreus chromosome. Single transformants were picked and grown on plates supplemented with hygromycin and thiostrepton. v). Growth of host The above said single transformants were grown in one litre of liquid media for seven days. Media composition was based on the findings of Szakacs (1998). Cells were removed by centrifugation and the supernatant collected. vi). Isolation of simvastatin
  • HMPKSA4 hybrid modular polyketide synthase A4
  • pARHMPKSA4 For the construction of pARHMPKSA4, the method of Ranganthan (2000) was employed, as described earlier in Example 1.1.2.
  • the PCR products were phosphorylated and ligated to Sw ⁇ l-cut, dephosphorylated pUC18 vector and then used to transform E. coli DH10B electrocompetent cells.
  • the desired plasmids containing the amplified DNA fragments were isolated and sequenced using standard pUC forward and reverse primers.
  • the plasmids were then used to transform the E.coli ET12567 dam " strain. Isolated DNA was digested with Xbal and the desired fragments isolated and purified. Fragment 4 was then cloned in Xbal-cvA. pCJR24HS vector using the procedure described in Example 1.1.2. The resulting plasmid pSIMAl was digested with Xbal and the other DNA fragments, namely, 8 and 7 were sequentially added to finally yield plasmid pSIMA43. This plasmid was then digested with Ndel and Xbal and ligated with fragment 1 previously digested with the same two enzymes. The ligated products were used to transform E. coli DH10B electrocompetent cells and the final expression plasmid pARHMPKSA4, containing the customised HMPKS A4 gene, was isolated. The junctions where the domains were joined are shown in
  • Plasmid pARHMPKSA4 was used to transform the mutant strain terreus/ARl.
  • HMPKSB hybrid modular polyketide synthase B
  • the HMPKSB template ( Figure 7) comprises of the following enzymatic domains in the given sequence: acyltransferase - ATO, acyl carrier protein - ACP0, ketosynthase -
  • KS2 acyltransferase - AT2, dehydratase - DH2, methyltransferase - MeT2, enoylreductase - ER2, ketoreductase - KR2 and acyl carrier protein - ACP2.
  • the domains are covalently linked with each other, to give a multi-enzyme HMPKSB template.
  • HMPKSB For the construction of HMPKSB 1, the selection of domains from publicly available DNA sequences was as follows:
  • pARHMPKSBl For the construction of pARHMPKSBl, the method of Ranganthan (2000) was employed, as described earlier in Example 1.1.2.
  • the PCR products were phosphorylated and ligated to Sm ⁇ l-cut, dephosphorylated pUC18 vector and then used to transform E. coli DH10B electrocompetent cells.
  • the desired plasmids containing the amplified DNA fragments were isolated and sequenced using standard pUC forward and reverse primers. No mistakes in the amplified products were detected.
  • the plasmids were then used to transform the E.coli ET12567 dam " strain. Isolated DNA was then digested with Xbal and the desired fragments isolated and purified.
  • Fragment 4 was then cloned in Xbal-c t pCJR24HS vector using the procedure described in Example 1.1.2.
  • the resulting plasmid pSIMAl was digested with Xbal and the other DNA fragments, namely, 6, 2, and 9 were sequentially added to finally yield plasmid pSIMB14.
  • This plasmid was then digested with Ndel and Xbal and ligated with fragment 10 previously digested with the same two enzymes.
  • the ligated products were used to transform E. coli DH10B electrocompetent cells and the final expression plasmid pARHMPKSBl, containing the customised HMPKSB 1 gene, was isolated.
  • the junctions where the domains were joined are shown in Figure 8. iv). Expression of plasmid pARHMPKSBl in a host
  • Plasmid pARHMPKSBl was used to transform the mutant strain A. terreus/ARl. Particularly preferred was the transformation procedure of Punt et al. (1992). Thiostrepton-resistant colonies were selected upon integration of the vector into the A. terreus chromosome. Single transformants were picked and grown on plates supplemented with hygromycin and thiostrepton. v). Growth of host
  • HMPKSB2 hybrid modular polyketide synthase B2
  • the above said PKS domain DNA sequences were amplified from genomic DNA using PCR as described in Example 1.1.2.
  • the total number of fragments that were amplified were five (11, 12, 13, 14 and 15; Figure 8).
  • DNA fragment 7, required for the construction of HMPKSB2 had been prepared earlier as described in Example 1.1.5.
  • the nucleotide positions in the template are shown according to the same disclosed in prior art (Donadio et al., 1991).
  • the source of DNA, nucleotide positions of the template and the oligonucleotide primers (shown as 5' to 3') used for each PCR were as follows:
  • pARHMPKSB2 For the construction of pARHMPKSB2, the method of Ranganthan (2000) was employed, as described earlier in Example 1.1.2.
  • the PCR products were phosphorylated and ligated to Smal-cut, dephosphorylated pUC18 vector and then used to transform E. coli DH10B electrocompetent cells.
  • the desired plasmids containing the amplified DNA fragments were isolated and sequenced using standard pUC forward and reverse primers. No mistakes in the amplified products were detected.
  • the plasmids were then used to transform the E.coli ET12567 dam " strain. Isolated DNA was digested with Xbal and the desired fragments isolated and purified.
  • Fragment 15 was then cloned in .X7j> ⁇ l-cut pCJR24HS vector using the procedure described in Example 1.1.2.
  • the resulting plasmid pSIMB21 was digested with Xbal and the other DNA fragments, namely, 14, 13, 7 and 12 were sequentially added to finally yield plasmid pSIMB25.
  • This plasmid was then digested with Ndel and Xbal and ligated with fragment 11 previously digested with the same two enzymes.
  • the ligated products were used to transform E. coli DH10B electrocompetent cells and the final expression plasmid pARHMPKSB2, containing the customised HMPKSB2 gene, was isolated.
  • the junctions where the domains were joined are shown in Figure 8. iv). Expression of plasmid pARHMPKSB2 in a host
  • Plasmid pARHMPKSB2 was used to transform the mutant strain A. terreus/ AR1. Particularly preferred was the transformation procedure of Punt et al. (1992). Thiostrepton-resistant colonies were selected upon integration of the vector into the A. terreus chromosome. Single transformants were picked and grown on plates supplemented with hygromycin and thiostrepton. v). Growth of host The above said single transformants were grown in one litre of liquid media for seven days. Media composition was based on the findings of Szakacs (1998). Cells were removed by centrifugation and the supernatant collected. vi).
  • GC-MS Gas chromatography mass spectometry
  • HPLC-MS Analytical reverse phase high performance liquid chromatography mass spectrometry
  • A. terreus/ARl LovF mutant gene over monacolin J expression producer
  • the strain A. terreus/ARl was constructed by integrating vector pSEVI10H4S into the A. terreus wild-type chromosome.
  • the desired DNA sequences were amplified from plasmid DNA, using PCR as described in Example 1.1.2.
  • the nucleotide positions in the template are shown according to the same disclosed in prior art (Quandt and Hynes, 1993; Mahenthiralingam et al, 1998).
  • the source of DNA, nucleotide positions of the template and the oligonucleotide primers (shown as 5' to 3') used for each PCR were as follows:
  • pSIM10H4S For the construction of pSIM10H4S, the method of Ranganthan (2000) was employed, as described earlier in Example 1.1.2.
  • the PCR products were phosphorylated and ligated to Smal-c ⁇ , dephosphorylated pUC18 vector and then used to transform E. coli DH10B electrocompetent cells.
  • the desired plasmids containing the amplified DNA fragments were isolated and sequenced using standard pUC forward and reverse primers. No mistakes in the amplified products were detected.
  • the plasmids were then used to transform the E.coli ET12567 dam " strain. Isolated DNA was then digested with Xbal and the desired fragments isolated and purified.
  • DNA fragment S was then cloned in .ATj l-cut pUC18 vector using the procedure described in Example 1.1.2.
  • the resulting plasmid pSEVISl was digested with Xbal and the other DNA fragments, namely, 4, H and O10 were inserted as Xbal fragments to finally yield plasmid pSIM10H4S.
  • the integrity of the plasmid was confirmed by restriction enzyme mapping and selection on hygromycin.
  • Plasmid pSIM10H4S was used to transform the wild-type lovastatin-producing strain A terreus ATCC20542. Particularly preferred was the transformation procedure of Punt et al. (1992). Hygromycin-resistant colonies were selected upon integration of the vector into the A. terreus chromosome. Single transformants were picked and grown on plates supplemented with hygromycin, following which the colonies were selected on plates containing 10% sucrose. v). Growth of host The above said single transformants were grown in one litre of liquid media for seven days. Media composition was based on the findings of Szakacs (1998). Cells were removed by centrifugation and the supernatant collected. vi).
  • pCJR24HS For the construction of pCJR24HS, the method of Ranganthan (2000) was employed, as described earlier in Example 1.1.2.
  • DNA fragment S (Method 1.3.1) was cloned in Xbal-cut pCJR24 vector using the procedure described in Example 1.1.2.
  • the resulting plasmid pCJR24S was digested with Xbal and the other DNA fragment, namely the hygromycin-resistance gene H (Method 1.3.1) was inserted as an Xbal fragment to finally yield plasmid pCJR24HS.
  • the integrity of the plasmid was confirmed by restriction enzyme mapping and selection on hygromycin. References Ballance, D.J, Turner, G. Gene. 1985, 36, 321.
  • MacNeil D.I, Gewain, KM, Ruby, C.L, Dezeny, G, Gibbons P.H, MacNeil, T. Gene. 1992, 111, 61.

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  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Cette invention concerne une méthode de préparation de simvastatine, ou des analogues ou dérivés de simvastatine, par expression biologique. La méthode consiste à: a) mettre en oeuvre un hôte présentant un gène personnalisé codant pour une polycétide synthase; b) fermenter ledit hôte afin d'obtenir simvastatine ou des analogues ou dérivés de simvastatine.
PCT/IN2002/000157 2001-07-25 2002-07-25 Methode de preparation de simvastatine WO2003010324A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002337601A AU2002337601A1 (en) 2001-07-25 2002-07-25 Process for the preparation of simvastatin

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN796/DEL/01 2001-07-25
IN796DE2001 2001-07-25

Publications (2)

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WO2003010324A2 true WO2003010324A2 (fr) 2003-02-06
WO2003010324A3 WO2003010324A3 (fr) 2003-08-21

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AU (1) AU2002337601A1 (fr)
WO (1) WO2003010324A2 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007147801A1 (fr) * 2006-06-20 2007-12-27 Dsm Ip Assets B.V. Procédé de production de la simvastatine
WO2009056539A1 (fr) * 2007-10-30 2009-05-07 Dsm Ip Assets B.V. Production de la simvastatine par fermentation
EP2032715A4 (fr) * 2006-05-24 2010-12-15 Univ California Méthodes et matières pour l'élaboration de simvastatine et de ses composés
CN102703539A (zh) * 2003-10-21 2012-10-03 维莱尼姆公司 制备新伐他汀及中间体的方法
US8981056B2 (en) 2009-10-08 2015-03-17 The Regents Of The University Of California Variant LovD polypeptide
US9499803B2 (en) 2009-10-08 2016-11-22 The Regents Of The University Of California Variant LovD polypeptide

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6391583B1 (en) * 1998-12-18 2002-05-21 Wisconsin Alumni Research Foundation Method of producing antihypercholesterolemic agents

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102703539A (zh) * 2003-10-21 2012-10-03 维莱尼姆公司 制备新伐他汀及中间体的方法
EP2032715A4 (fr) * 2006-05-24 2010-12-15 Univ California Méthodes et matières pour l'élaboration de simvastatine et de ses composés
US8211664B2 (en) 2006-05-24 2012-07-03 The Regents Of The University Of California Methods and materials for making simvastatin and related compounds
US8951754B2 (en) 2006-05-24 2015-02-10 The Regents Of The University Of California Methods and materials for making simvastatin and related compounds
US9970037B2 (en) 2006-05-24 2018-05-15 The Regents Of The University Of California Methods and materials for making simvastatin and related compounds
US10793884B2 (en) 2006-05-24 2020-10-06 The Regents Of The University Of California Methods and materials for making simvastatin and related compounds
WO2007147801A1 (fr) * 2006-06-20 2007-12-27 Dsm Ip Assets B.V. Procédé de production de la simvastatine
WO2009056539A1 (fr) * 2007-10-30 2009-05-07 Dsm Ip Assets B.V. Production de la simvastatine par fermentation
US8981056B2 (en) 2009-10-08 2015-03-17 The Regents Of The University Of California Variant LovD polypeptide
US9499803B2 (en) 2009-10-08 2016-11-22 The Regents Of The University Of California Variant LovD polypeptide
US10246689B2 (en) 2009-10-08 2019-04-02 The Regents Of The University Of California Variant LovD polypeptide
US10689628B2 (en) 2009-10-08 2020-06-23 The Regents Of The University Of California Method of making variant LovD polypeptides

Also Published As

Publication number Publication date
AU2002337601A1 (en) 2003-02-17
WO2003010324A3 (fr) 2003-08-21

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