title: Construction of a novel expression vector in Pseudonocardia autotrophica and its application to efficient biotransformation of compactin to pravastatin, a specific HMG-CoA reductase inhibitor.
abstract: The novel plasmid vector (pTAOR4-Rev) suitable for gene expression in actinomycete strains of Pseudonocardia autotrophica was constructed from 2 P. autotrophica genetic elements, the novel replication origin and the acetone-inducible promoter. The replication origin was isolated from the endogenous plasmid of strain DSM 43082 and the acetone-inducible promoter was determined by analysis of the upstream region of an acetaldehyde dehydrogenase gene homologue in strain NBRC 12743. P. autotrophica strains transformed with pTAOR4-P450, carrying a gene for cytochrome P450 monooxygenase, expressed P450 from the acetone-inducible promoter, as verified by SDS-PAGE and spectral analysis. The biotransformation test of acetone-induced resting cells prepared from a strain of P. autotrophica carrying pTAOR4 that harbors a compactin (CP)-hydroxylating P450 gene revealed 3.3-fold increased production of pravastatin (PV), a drug for hypercholesterolemia. Biotransformation of CP by the same strain in batch culture yielded PV accumulation of 14.3 g/l after 100 h. The expression vector pTAOR4-Rev and its function-enhancing derivatives provide a versatile approach to industrial biotransformation by Pseudonocardia strains, which can be good hosts for P450 monooxygenase expression.
authors.affiliation: Bioresource Laboratories, Mercian Corporation, 1808 Nakaizumi, Iwata, Shizuoka 438-0078, Japan.
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Fujii
authors.forename: Koji
authors.initials: K
authors.lastname: Norihisa
authors.forename: Tadashi
authors.initials: T
authors.lastname: Fujii
authors.forename: Yasuhide
authors.initials: Y
authors.lastname: Aritoku
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Kagawa
authors.forename: Khalid Ibrahim
authors.initials: KI
authors.lastname: Sallam
authors.forename: Osamu
authors.initials: O
authors.lastname: Johdo
authors.forename: Akira
authors.initials: A
authors.lastname: Arisawa
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Tamura
mesh_terms: D000096:Acetone / Q000494:pharmacology; D001483:Base Sequence; D001711:Biotransformation; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics*; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000378:metabolism*; D007218:Industrial Microbiology / Q000379:methods; D008148:Lovastatin / Q000031:analogs & derivatives* / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017035:Pravastatin / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D018741:Replication Origin; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*
chemical_list: D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D000096:Acetone; C012258:mevastatin; D003577:Cytochrome P-450 Enzyme System; D008148:Lovastatin; D017035:Pravastatin