The Function Of Polyketide Ketoreductase Gene From Midecamycin Producing Strain (S.mycarofaciens 1748)

This paper presents the results about the polyketide ketoreductase (MKR) gene from midecamycin producing strain (S.mycarofaciens 1748), including studies on the gene expression, gene localization, nucleotide sequences analysis and function.A BamHI-BamHI 4.0kb fragment isolated from genomic library of midecamycin producing strain containing the actIII -homologous DNA was inserted into E.coli-Streptomyces shuttle vector pWHM3 and a recombinant plasmid pCB4 was obtained. The actlll homologous region was located on a BssHII-BamHI 1.3kb fragment from Southern hybrization result, using actlll gene as a probe. The 1.3kb fragment was ligated into vector pIJ702 and a recombinant plasmid pWX2 was obtained. The recombinant plasmid pWX2 was introduced by transformation into actlll deficient mutant ofStr.coelicolor TK18. The transformant restored blue pigment (actinorhodin) formation on agar plates. A 2-hydroxyaklavinone producer S.galilaeus ATCC31671 that was a polyketide ketoreductase gene deficient mutant was transformed with the recombinant plasmid pWX2. The transformant produced aklavinone according to TLC analyses. Actinorhodin and aklavinone were synthesized from polyketide chains of different lengths. Actinorhodin is a octaketide and aklavinone is a decaketide. The ketoreductase from Str. mycarofaciens 1748 is able to function with the PKS enzymes in the Str.coelicolor TK18 actinorhodin pathway and the S.galilaeus ATCC31671 aklavinone pathway. This suggested that the MKR can recognize and reduce polyketide chains of different length.We have found that the 3.0kb DNA fragment upstream of the MKR gene could enhance the production of antibiotics. When plasmid pCB4 and pWX1 were transfer into Str. galilaeus ATCC31671 respectively, the aklavinone production of the transformant containing pCB4 is 2-3 fold more than the transformant containing pWX1. The 3.0kb DNA fragment was ligated into vector pWHM3 and a recombinant plasmid pWX3 was obtained. The 2-hydroxyaklavinone production of the transformant containing pWX3 is 1 fold more than the transformant containing...