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

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作者：

Y Fujii，K Norihisa，T Fujii，Y Aritoku，Y Kagawa，KI Sallam，O Johdo，A Arisawa，T Tamura

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摘要：

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 14.3g/l after 100h. 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.

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关键词：

HMG-CoA3-hydroxy-3-methylglutaryl-Coenzyme A 3-hydroxy-3-methylglutaryl-Coenzyme A Vdhvitamin D3 hydroxylase vitamin D3 hydroxylase Fdxferredoxin ferredoxin Fdrferredoxin-NADP+ reductase ferredoxin-NADP+ reductase SDSsodium dodecyl sulfate sodium dodecyl sulfate