麦迪霉素产生菌酮基还原酶基因的研究

将麦迪霉素产生菌基因文库中与放线紫红索酮基还原酶基因actⅢ有同源性的4·0kb DNA片段克隆到质粒载体pWHM3中，构成重组质粒pCB4。将质粒pCB4转入酮基还原酶基因缺陷菌株——加利利链霉菌ATCC3167l中，得到转化子。转化子发酵产物经TLC和HPLC分析证明是阿克拉菌酮，与加利利链霉菌原株ATCC31133的产物相同，说明麦迪霉素产生菌酮基还原酶基因互补了加利利链霉菌ATCC31671中缺陷的酮基还原酶基因，使其恢复了产生阿克拉菌酮的能力。4．Okb DNA片段插入方向相反的重组质粒pCBR4在加利利链霉菌ATCC31671中发酵产物经TLC分析证明也是阿克拉菌酮，这说明4．0kbDNA片段中麦迪霉素产生菌酮基还原酶基因具有自身的启动子。对4．0kb DNA片段进行了限制酶酶切分析，建立了其酶切图谱。以actⅢ基因为探针，经分子杂交以及亚克隆和DNA转化实验，将麦迪霉索产生菌酮基还原酶基因定位于BssH Ⅱ—BamH Ⅰ 1．3kb DNA片段上。对1．3kb DNA片段核苷酸序列分析结果表明：此1．3kbDNA片段中含有一个独立的ORF，起始密码ATG，终止密码TAG，含783bp；在起始密码上游有GGAGG5个核苷酸SD序列；此ORF编码260个氨基酸，与actⅢ基因编码的261个氨基酸相似性为77．4%，相同性为66．7％，对麦迪霉素产生苗酮基还原酶基因的可能作用进行了讨论。

Characterization of Polyketide Ketoreductase Gene from Midecamycin Producing Strain

This paper presents the results about the expression of the polyketide ke-toreductase gene (PKG) from midecamycin producing strain (S. mycarofaciens 1748), gene localization and nucleotide sequences analysis of the PKG. A BamH I -BamHI 4.0kb fragment isolated from genomic library of midecamycin producing strain containing the act I -homologous DNA was inserted into E. coli-Strep-tomyces shuttle vector pWHM3. A recombinant plasmid pCBR4 was obtained and introduced into a 2-hydroxyaklavinone producer 5. galilaeus ATCC31671 that was a polyketide reductase 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 5. galilaeus ATCC31671 also resulted in the production of aklavinone. Thus we demonstrated that this gene encode a polyketide ketoreductase which results in deoxygenation of 2-hydroxyaklavinoe in C-2 position and this gene has own promoter itself. Restriction analysis of pCB4 indicated that there was no sites for EcoRI, Hind III , but there were sites for Sail, SphI, Xho I , BssH II on the cloned gene fragment. The polyketide ketoreductase gene was located on a BssH I -BamHI 1. 3kb fragment from Southern hybrization result, using act I gene as a probe, and that was confirmed by gene expression experiment in S. galilaeus ATCC31671. The nucleotide sequences analysis showed that the BssH I -BamHI 1. 3kb 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 5 nt (GGAGG) SD sequence at the upstream of proposed 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 the polyketide ketoreductase gene from midecamycin producer consisted of 260 aa residues. Its amion acid sequences were compared with act IB gene coding protein sequences. The percentage of similarity was 77.4 and percentage identity was 66. 7.