Effect Of NsdA Gene Disruption And Integrative Expression Of OrfX Gene On The Secondary Metabolism Of Streptomyces

nsdA (negative regulator of Streptomyces differentiation) gene is a globally negativeregulator gene, which widely exists in Streptomyces. S. coelicolor nsdA null mutantsproduced more actinorhodin, CDA, methylenomycin and spores than wild type strain.Compared with the start strain of Streptomyces avermitilis NRRL8165, the level ofavermectin produced by S. avermitilis nsdA disruption strain increased 3-5 folds, orfXgene is a regulator gene discovered in the genome of S. avermitilis in 2003. The level ofactinorhodin and methylenomycin produced by S. lividans TK21 increased dramaticallywhen the DNA fragment containing orfX gene was transformed into it. When therecombinant plasmid containing the orfX gene, which was derived from multicopy vector,was transformed into S. avermitilis, the level of avermectin was elevated prominently. Inthe past, the study on the nsdA and orfX gene started with the wild type strains. Thefunction of the two genes was only investigated in theory and the possible industrialapplication was not carried out. The goal of this study is to test the effect of nsdA genedisruption and integrative expression of orfX gene on the secondary metabolism of S.avermitilis, and investigate the application of the two genes in the industrial breeding ofStreptomyces.In this study, PCR-mediated gene replacement technique was applied in theconstruction nsdA gene disruption strain. Firstly, a pair of long (58,59nt) primers wereprepared, which have at 5'end 39nt matching the flanking sequence of nsdA, and the3'sequence matching the right or left end of the apramycin resistant cassettes(aac(3)Ⅳ+oriT). The linear DNA PCR-amplified by these primers waselectro-transformed to the strain BW25113/pIJ790/pBIB0501 which expressesλ-Redenzyme and contains target plasmid, then a positive recombined plasmid (pBIB0502) wasobtained. The 4.5kb fragment from pBIB0502 digested by BglⅡwas inserted intopHL212, and then introduced into S. avermitilis BIB9903 by conjugal transfer. AnApra~RThio~S isolate BIB3009 was obtained and nsdA disruption was confirmed by PCR.Compared with the start strain, BIB3009 produced fewer spores and less pigment onYMS plate. HPLC assays of the fermentation broth of BIB3009 indicated that the level ofavermectin decreased dramatically, and only 296μg/ml avermectin was produced, whichreached 1/4-1/5 of the yield of the start strain.In this study, the PCR primers P1 and P2 were designed according to the predictedgene sequences of orfX gene in the S. avermitilis genome published in the GeneBank. orfX structural gene was amplified with genomic DNA of BIB9903 as template and P1and P2 as primers. Recombinant plasmid pBIB827, which contained the structural gene oforfX and ermEp~*, was derived from pIJ8600. The recombinant strain BIB0827 wasobtained by introducing pBIB827 into the industrial strain BIB9903 through intergenericgene transfer. Compared with the start strain, BIB0827 produced more spores and lesspigment on YMS plate. Fermentation assays indicated the biomass of the fermentationbroth of BIB0827 increased slightly when compared with the start strain. HPLC assaysshowed the level of avermectin produced by BIB0827 reached 3357μg/ml, 1.5-foldincreasing when compared with the start strain. The characteristic of high-level producingstrain BIB0827 was stable after ten rounds of non-selective growth, which could beapplied in the industrial production of avermectin.