核糖体工程技术选育卡伍尔链霉菌高产菌株

Screening of High Yield Antibiotic Producing Strptomyces cavourensis Strain by Ribosome Engineering Technology

通过赋予前期分离获得的一株卡伍尔链霉菌菌株TJ430利福平抗性,达到迅速提高菌株产抗生素能力的目的;采用核糖体工程技术使菌株TJ430的ropB基因发生突变从而获得利福平抗性,通过构建好的高通量筛选模型结合高效液相色谱技术,筛选正向和负向突变菌株,利用液体发酵复筛进一步确认初筛结果,通过对突变株进行基因测序寻找突变株ropB基因的突变位点;构建的高通量筛选模型能准确、快速筛选出突变菌株,分离到5株正向突变株和2株负向突变株。在负向突变株ropB基因的第280 bp处发现一个点突变,但是在正向突变株测序片段中未发现突变位点。采用的引物序列只能对ropB基因的部分序列进行测序,从正向突变菌株抗生素产量较原始菌株提高2.5倍以上,且菌丝形态发生显著变化来推测,正向突变菌株的突变位点应位于ropB基因中未测序片段。在完成原始菌株全基因组测序基础上,可重新设计引物对该菌株的全ropB基因进行测序,寻找突变位点。

To increase antibiotic production, the rifampicin resistance was conferred on a Strptomyces cavourensis strain which was screened in the early stage. Ribosome engineering technology was used to enablethe strain to get rifampicin resistance by ropB gene mutation of strain TJ430. Positive and negative mutantstrains were screened by high throughput screening model combined with HPLC. Liquid fermentation was usedto further confirm the preliminary screening results. The mutation site of ropB gene was confirmed by genesequencing. A high throughput screening model was constructed to screen the mutant strains accurately andrapidly. Five positive and two negative mutant strains were isolated. A mutation site was found in the 280 bp ofthe negative mutant ropB gene, but no mutation site was found in the sequenced fragment of the positivemutant. Partial sequence of ropB gene could be sequenced by the primer sequences. Compared with theoriginal strain, the antibiotic yield of positive strain increased more than two and a half times, and themorphological changes of the hyphae were greatly changed, thus, the mutation site of the positive mutationstrain should be located in the other fragment of ropB gene which was not sequenced. Based on the wholegenome sequencing of the original strain, the whole ropB gene of the strain could be sequenced by redesigningprimers to search for the mutation site.