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 Yoshikazu Fujii a,d , Koji Norihisa b , Tadashi Fujii a , Yasuhide Aritoku a , Yusuke Kagawa d , Khalid Ibrahim Sallam c,1 , Osamu Johdo a , Akira Arisawa a,⇑ , Tomohiro Tamura c,d,⇑ a Bioresource Laboratories, Mercian Corporation, 1808 Nakaizumi, Iwata, Shizuoka 438-0078, Japan b Biotechnical Development Center, Mercian Corporation, 1808 Nakaizumi, Iwata, Shizuoka 438-0078, Japan c Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan d Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan article info Article history: Received 25 November 2010 Available online 6 December 2010 Keywords: Pseudonocardia autotrophica Pravastatin Rolling-circle type replication Acetone-inducible promoter Biotransformation abstract The novel plasmid vector (pTAOR4-Rev) suitable for gene expression in actinomycete strains of Pseudono- cardia 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 biotransforma- tion 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. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction Host-vector systems are indispensable for the expression of re- combinant proteins for research or industrial purposes. In the industrial application of heterologous gene expression, bacterial strains are used preferentially as hosts because they are easy to handle and their bioprocesses are easily manipulated. In addition to the general Ecsherichia coli system, streptomycete host-vector systems are noteworthy because the actinomycete strains have the potential to produce industrially valuable bioactive materials such as antibiotics, antitumor agents, and statins via fermentation or biotransformation [1]. The streptomycete vectors are generally incompatible and of limited utility in non-streptomycete hosts such as Pseudonocardia autotrophica, which is utilized for biotrans- formation of vitamin D 3 (VD 3 ) to calcitriol (CT) [2]. P. autotrophica catalyze hydroxylation of VD 3 at C-25, and then C-1a to CT. In our previous study of efficient biocatalytic processes of VD 3 hydroxyl- ation, we identified cytochrome P450 monooxygenase (P450) as the hydroxylase responsible for VD 3 transformation. This P450, designated as Vdh, catalyzed the 25- and 1a-hydroxylations. The Vdh gene (vdh) was cloned and expressed in E. coli, in which the enzymatic properties of the recombinant protein were determined. E. coli expression and biotransformation systems were established and enabled to increase the hydroxylation activity of Vdh by direc- ted evolution [3]. Improved biotransformation was obtained with P. autotrophica than with Vdh-expressing E. coli (data not shown). A Pseudonocardia-specific expression system was required for expressing improved vdh genes to enhance VD 3 hydroxylation activity. 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.12.013 Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl-Coenzyme A; Vdh, vita- min D 3 hydroxylase; Fdx, ferredoxin; Fdr, ferredoxin-NADP + reductase; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; HPLC, high-performance liquid chromatography. ⇑ Corresponding authors. Address: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsuki- samu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan (T. Tamura). Fax: +81 538 21 1135 (A. Arisawa), fax: +81 11 857 8980 (T. Tamura). E-mail addresses: arisawa-a@mercian.co.jp (A. Arisawa), t-tamura@aist.go.jp (T. Tamura). 1 Present address: Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Mansoura University, Mansoura, Egypt. Biochemical and Biophysical Research Communications 404 (2011) 511–516 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc