Effect Of NsdA And BldA Gene Disruption On The Morphological Differentiation And Secondary Metabolism Of Streptomyces Bingchenggensis

Recently, Streptomyces bingchenggensis was isolated from a soil sample collected in Harbin, China in our lab, which produced at least two kinds of antibiotics, the polyether nanchangrnycin and macrolide compound milbemycin A3 and A4. With outstanding activity against various kinds of fungi, insects and acarids, non-toxic to mammals, plants easily degraded in nature, these antibiotics possess potent anthelmintic and nematocidal activities.nsdA was first found in Streptomyces coelicolor A3 (2), negatively regulating sporulation and antibiotic production. Subsequent studies showed that nsdA homologous gene was wildly existent and conserved in Streptomyces. bldA gene encodes the only tRNA for UUA, a rare leucine codon. Controlling the genes containing TTA codon, bldA might have some general effects on antibiotic production. Obviously, the regulation of bldA was very complex at a variety of different levels. Accounting for the complexity of regulation in Stremtomysecs and the novelty of S.bingchenggensis, functions of regulatory genes nsdA and bldA in the morphological differentiation and antibiotic production of S. bingchenggensis were studied.Primers were designed according to the data obtained from the S. coelicolor and S. avermitilis genome sequences. nsdA and bldA genes from S. bingchenggensis₂26541 were cloned, sequenced and submitted to GenBank (EU779992; FJ380950). Sequence analysis showed that the nsdA gene has a 75 % identity in the nucleotide sequence compared with that of other Streptomyces, while bldA gene shares a 86% identity in the nucleotide sequence compared with that of other Streptomyces.In this study,λRed-mediated PCR-targeting gene replacement technique was applied in the the process of constructing nsdA and bldA gene disruption vectors, which were introduced into S. bingchenggensis₂26541 by conjugal transfer. The double cross-over recombination mutants were obtained and gene disruptions were confirmed by PCR. Both pigment amounts and eventual sporulation levels in the nsdA disruption mutant BC29 appeared to be greater than those of the ancestral strain. HPLC assays on BC29 revealed that disruption of nsdA remarkably increased yields of milbemycin and bingchengmycin by 250% and 67%, respectively. Changes of phenotype and antibiotic production were reversed to the ancestral level by the introduction of nsdA back into BC29, excluding potential polar effects on adjacent genes as an explanation. These results suggested that nsdA played a negative role in morphological differentiation and antibiotic production in S. bingchenggensis₂26541. Compared with the ancestral strain, bldA disruption mutant LC29 appeared to be"bald", had no milbemycin and bingchengmycin in the fermentation broth by HPLC analysis. Changes of phenotype and antibiotic production were reversed to the ancestral level by the introduction of bldA back into LC29. There might be some genes containing TTA codon, involved in the biosynthesis of antibiotic.These results were consistent with presumption. Further functional studies may be helpful to elucidate the exact roles of nsdA and bldA genes in secondary metabolism and morphological differentiation, then adapt them to increase antibiotic yield.