Effect Of BkdF And NsdA Gene Disruption On The Secondary Metabolism Of Streptomyces Avermitilis

bkdF and nsdA gene found in Streptomyces avermitilis genome are both closely related to secondary metabolism and morphological differentiation. bkdF gene encoding E1a subunit of branched-chain a keto acid dehydrogenase(BCDH) is involved in the biosynthesis of avermectin starter unit. Deletion of bkdF resulted in a S.avermitilis bkd mutant which lacked the ability to produce natural avermectins. nsdA gene is a globally negative regulator gene extensively existent in streptomyces genome. S.coelicolor nsdA null mutants produce more actinorhodin, CD A, methyleonomycin and spores than wild type strain. The goal of the study is to disrupt bkdF and nsdA gene by using gene replacement method, and investigate their effect on secondary metabolism of 5. avermitilis.In this study, a novel PCR-mediated gene replacement protocol was used to construct bkdF gene disruption strain. Firstly, a pair of long (58,59nt) primers were prepared, which have at 5'end 39nt matching the flanking sequence of bkdF, and the 3'sequence matching the right or left end of the apramycin resistant cassettes ( aac(3)IV+oriT ) .The linear DNA PCR-amplified by these primers was electro-transformed to the strain BW25113/ pIJ790/ pXW1224 which expresses λRed enzyme and contains target plasmid, then a positive recombined plasmid (pXW1230) was obtained. The 4kb fragment from pXW1230 restricted by Bgl II was inserted into pHZ1351, and introduced into S. avermitilis BIB9903 by conjugal transfer. A ApraRThios isolate BIB0423 was obtained and bkdF disruption was confirmed by PCR. HPLC and MS analysis of fermentation culture indicated that the avermectins production was blocked and the only product of BIB9903 was oligomycin A.Using as gene replacement vector, pHL214 was introduced into S. avermitilis BIB9903 by conjugal transfer. NsdA null mutant BIB0515 was obtained by screening and nsdA disruption was confirmed by PCR analysis. The phenotype of the mutant changed greatly. After cultivated on YMS plate, it produced less spores and more melanin pigment than wild strain. HPLC analysis of fermentation culture indicated that the avermectins production of nsdA disruption strain decreased sharply. The result doesn't accord with the anticipation, and the reason remains to elucidate.