Genes from a gene cluster

Abstract

ML-236B is an inhibitor of HMG-CoA reductase and useful in preparing another such inhibitor, pravastatin. The preparation of ML-236B using an ML-236B-producing microorganism is enhanced using a polynucleotide encoding a gene related to the polyketide synthase cluster occurring in such a microorganism.

Description

CROSS-REFERENCE TO RELATED APPLICATION [0001] This application is a Divisional application of application Ser. No. 09 / 836,705 filed Apr. 17, 2001.FIELD OF THE INVENTION [0002] The present invention relates to a gene cluster, and more particularly to genes from a gene cluster. [0003] More particularly the invention relates to polynucleotides, such as DNA, which accelerate the biosynthesis of a HMG-CoA reductase inhibitor, ML-236B, in an ML-236B producing micro-organism when introduced in the ML-236B producing micro-organism. The invention further relates to vector into which said polynucleotides are incorporated, host cells transformed by said vectors, proteins expressed by said vectors, a method for producing ML-236B using said polynucleotides and / or proteins when the method comprises recovering ML-236B from the culture of said host cell, and the invention further relates to other associated aspects. BACKGROUND OF THE INVENTION [0004] Pravastatin is an HMGCoA reductase inhibitor. Pr...

AI Technical Summary

Benefits of technology

[0015] We have discovered that the incorporation of mlcE or cDNA corresponding to mlcE can accelerate the biosynthesis of ML-236B, and the incorporation of mlcR or cDNA corresponding to mlcR can accelerate the biosynthesis of ML-236B. Furthermore, mlcR stimulates transcriptional expression of mlcA to D, mlcA, B, C and D are involved in the production of ML-236B, independently or in combination, as shown by gene disruption studies.

[0058] Culturing of a ML-236B producing micro-organism can be performed under conditions suitable for the particular ML-236B producing micro-organisms. For example, culturing of Penicillium citrinum, a preferred ML-236B producing micro-organism, can be performed by inoculating the cells in MBG3-8 medium [composition: 7 % (w / v) glycerin, 3 % (w / v) glucose, 1% (w / %) soybean powder, 1% (w / v) peptone (manufactured by Kyokuto Seiyaku Kogyo corporation), 1% (w / v) Corn steep liqueur (manufactured by Honen corporation), 0.5% (w / v) sodium nitrate, 0.1% (w / v) magnesium sulfate heptahydrate (pH 6.5)], and incubating at 22 to 28° C. with shaking for 3 to 7 days. A slant for storage of the bacterium can be prepared by pouring melted PGA agar medium [composition: 200 g / L potato extract, 15% (w / v) glycerin, 2% (w / v) agar] into a test tube, and allowing the agar to solidify at an angle. Penicillium citrinum may then be inoculating into the slant using a platinum needle, followed by incubation at 22 to 28° C. for 7 to 15 days. Micro-organisms or bacteria grown in this way can be continuously maintained on the slant by reserving the slant at 0 to 4° C.

[0071] The primer for PCR are suitably designed to comprise nucleotide sequences which encode amino acid sequences that are highly conserved within PKS genes. Methods to identify nucleotide sequences corresponding to a given amino acid sequence include deduction on the basis of the codon usage of the host cell, and methods of making mixed oligonucleotide sequences using multiple codons (hereinafter referred to as a ‘degenerate oligonucleotides’). In the latter case, the multiplicity of oligonucleotides can be reduced by introducing hypoxanthine to their nucleotide sequences.

[0133] Confirmation of functional expression of the ML-236B biosynthesis accelerating cDNAs obtained using the above methods in an ML-236B producing micro-organism can be obtained by cloning the cDNA into a DNA vector suitable for functional expression in an ML-236B producing micro-organism. Suitable cells are then transformed with the recombinant DNA vector, and the ML-236B biosynthesis ability of the transformed cells and non transformed host cells compared. If ML-236B biosynthesis accelerating cDNA is functionally expressed in the transformed cell, then the ML-236B biosynthesis ability of the transformed cell is improved compared with that of a host cell.

[0147] As described above, ML-236B can be efficiently produced by inoculating a Penicillium citrinum transformant obtained from a slant as above, and having a regenerated cell wall, into MBG 3-8 medium, followed by incubation at 22 to 28° C. for 7 to 12 days with shaking. Penicillium citrinuim as a host can be cultured in liquid medium as well to produce ML-236B.

Problems solved by technology

However, to date, there has been insufficient molecular biological analysis into the biosynthesis of ML-236B, and factors regulating it.

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