Characterization of polyketide ketoreductase gene (MPKR) from midecamycin-producing strain (Streptomyces mycarofaciens 1748)

Abstract

This paper presents the results about the expression of the polyketide ketoreductase gene from midecamycin-producing strain (S. mycarofaciens 1748), gene localization and nucleotide sequences analysis. A BamHI-BamHI 4.0 kg fragment isolated from a genomic library of midecamycin-producing strains containing the actIII homologous DNA was inserted into E. coli-Streptomyces shuttle vector pWHM3. A recombinant plasmid pCB4 was obtained and introduced into a 2-hydroxyaklavinone producer S. galilaeus ATCC 31671 that was a polyketide ketoreductase gene deficient mutant. The transformant produced aklavinone according to TLC and HPLC analyses. The BamHI-BamHI 4.0-kb fragment was inserted into pWHM3 in the reverted orientation with pCB4 and a recombinant plasmid pCBR4 was obtained. Introduction pCBR4 into S. galilaeus ATCC 31671 also resulted in the production of aklavinone. Thus we demonstrated that this gene encoded a polyketide ketoreductase which results in deoxygenation of 2-hydroxyaklavinone in C-2 position and that this gene has its own promoter. Restriction map analysis of pCB4 indicated that there were no sites for EcoRI and HindIII, but there were sites for BssHII, SalI, SphI and XhoI on the cloned gene fragment. The polyketide ketoreductase gene was located on a BssHII-BamHI 1.3-kb fragment from Southern hybridization result, using actIII gene as a probe, and that was confirmed by gene expression in S. galilaeus ATCC 31671. The nucleotide sequence analysis showed that the BssHII-BamHI 1.3-kb fragment contained an open reading frame (ORF). The protein coding region was assumed to start with an ATG start codon, end at an TAG stop codon. There was a 5nt (GGAGG) SD sequence at the upstream of start codon. A presumptive promoter consisted of 7 nt AACCGGA at the -10 region and 5 nt TTCGA at the -35 region. The deduced amino acid sequences of MPKR gene consisted of 261 aa residues. Its amino acid were compared with actIII gene coding protein sequences. The similarity was 77.4% and the identity was 66.7%.