头霉素C基因簇大片段敲除促进克拉维酸合成

Large fragment knockout of cephamycin C gene cluster promoting the synthesis of clavulanic acid

目的本研究以棒状链霉菌(Streptomyces clavuligerus)F613-1为出发菌株,构建头霉素C基因簇中orf10、blp、lat、pcbAB和pcbC 5个完整基因片段(cep)的基因缺失突变菌株,探究头霉素C和克拉维酸(clavulanic acid,CA)生物合成的关系。方法通过生物活性测定法检测出发菌株F613-1和两株cep片段基因缺失突变菌株(△cep::apra-2和△cep::apra-4)中的头霉素C产量,同时通过HPLC检测上述菌株的生物量以及CA产量,分析头霉素C和CA生物合成之间的关系。结果与出发菌株F613-1相比,△cep::apra-2和△cep::apra-4菌株的表型没有明显变化;固体发酵168h,F613-1菌株中头霉素C产量为0.8875g/L,△cep::apra-2和△cep::apra-4菌株中的头霉素C产量均为0;液体发酵144h,突变菌株△cep::apra-2和△cep::apra-4中CA产量的分别为4.28和4.26g/L,较F613-1(3.16g/L)分别提高了35%和34%。结论在头霉素C生物合成能力缺失的情况下,CA产量明显提高,说明头霉素C和CA的生物合成存在明显的竞争作用,这对于探究CA与头霉素C生物合成之间的关系提供有力的证据,并且为提高CA产量提供新思路,对CA的工业生产有一定的指导意义。

Objective In this study,Streptomyces clavuligerus F613-1 was used as the parental strain to construct gene deletion mutations of five complete gene fragments(cep)including orf10,blp,lat,pcbAB,and pcbC in the cephamycin C gene cluster.This study aims to explore the relationship between cephamycin C and clavulanic acid(CA)biosynthesis.Methods The production of cephamycin C in the parental strain F613-1 and two cep fragment gene deletion mutant strains(△cep::apra-2 and△cep::apra-4)was detected by the biological activity assay,and the biomass and the CA yields of the above strains were detected by HPLC.The relationship between cephamycin C and CA biosynthesis was also analyzed.Results Compared with the parental strain F613-1,the phenotypes of△cep::apra-2 and△cep::apra-4 strains did not change significantly.When cultured for 168h under solid fermentation,the yield of cephamycin C in F613-1 strain was 0.8875g/L,and the production of cephamycin C in the both cef deletion mutant strains△cep::apra-2 and△cep::apra-4 was 0;while cultured for 144h in liquid fermentation,the CA yields in the mutant strains△cep::apra-2 and△cep::apra-4 were respectively 4.28 and 4.26g/L,which were 35%and 34%higher than F613-1(3.16g/L),respectively.Conclusion In the absence of cephamycin C biosynthesis ability,CA yield is significantly increased,indicating that there is a clear competitive effect of cephamycin C and CA biosynthesis.This study provides strong evidence to explore the relationship between CA and cephamycin C biosynthesis.In addition,this study provides new ideas for increasing CA yield,and provides certain guiding significance for CA industrial production.