Construction Of The Conjugal Transfer System Of Streptomyces Cinnamonensis And Effect Of PCR-mediated NsdA Gene Disruption On Yield Of Monensin

Conjugal transfer, a powerful trans-gene tool, has been successfully applied in variousStreptomyces stains for genetic manipulation. This method protects foreign DNA fromnuclease-induced degradation thereby avoiding restriction of host cells and largelyenhancing the gene transfer efficiency. At present, the genetic manipulation ofStreptomyces cinnamonensis was mainly accomplished by protoplast transformation, andapplication of conjugation methodology has not been reported. In this study, weinvestigated the conjugation between E. coli and Streptomyces cinnamonensis for the firsttime. Using E. coli-Streptomyces shuttle plasmid to transform E. coli strain ET12567(pUZ8002) followed by conjugal transfer into Streptomyces cinnamonensis, the optimizedcondition was obtained.nsdA gene (negative regulator of streptomyces differentiation) is a global negativeregulator for antibiotic production, presenting conserved sequence in the streptomycesgenome. However, the application of nsdA in streptomyces industrial strain has not beenreported till now. In present work, the disruption of nsdA in production strain wasaccomplished at first time. The process is as following:Analyzing the bio-information of nsdA encoding gene and flanking sequence, universalprimer P1, P2 was designed to successfully amplified and verified 4.8kb lengthcorresponding fragments from Streptomyces cinnamonensis BIB2005. Firstly, a pair oflong (58,59nt) primers were prepared, which have at 5'end 39nt matching the flankingsequence of ndsA, and the 3'sequence matching the right or left end of the apramycinresistant cassettes (aac(3)Ⅳ+oriT). The linear DNA PCR-amplified by these primers waselectro-transformed to the strain BW25113/pIJ790/ pBIB118 which expressesλRedenzyme and contains target plasmid, then a positive recombined plasmid (pBIB929) wasobtained. The 4kb fragment from pXW1230 restricted by BglⅡwas inserted intopHZ1351, and introduced into S. cinnamonensis BIB2005 by conjugal transfer. A Apra~RKan~S isolate BIB309 was obtained and nsdA disruption was confirmed by PCR.Compared with the start strain, the yield of monensin of the nsdA mutant BIB309increased 270 percent in the level of flask.