反相高效液相谱型分析检测吸水链霉菌17997变株发酵新产物

以吸水链霉菌17997为出发菌株。分别阻断格尔德霉索（geldanamycin，GA）生物合成酶基因簇中的Ⅰ型聚酮合酶（type Ⅰ polyketide synthase，pks）基因的第6模块，单加氧酶（monooxygenase，gdmM）基因和氨甲酰基转移酶（carbamoyltransferase，et）基因得到3种变株（pks^-）、（gdmM^-）和（ct^-）。比较变株与原株发酵产物的HPLC谱型，结合紫外吸收图谱分析。检测结果表明各变株的GA生物合成均被阻断。并产生3个不同于原始菌株的新物质。这证明基因操作所涉及的pks，gdmM和ct基因是GA生物合成的必需基因；这些基因的阻断可产生不同于原株的新物质。HPLC谱型比较可以在多样化代谢产物的产生菌中快速、准确地发现基因工程变株发酵产物的变化。指导新化合物的分离鉴定，样品用量甚微。是化学早期鉴别的有力工具。

Application of RP-HPLC pattern analysis to detect new products of Streptomyces hygroscopicus 17997 mutants

In order to detect the new fermentation products of several Streptomyces hygroscopicus 17997 mutants, the RP-HPLC pattern analysis in correlation with UV absorption determination of the fermentation crude extract was applied. Three genes pks model 6, gdmM and ct likely involved in GA biosynthesis were targeted to obtain the gene disruption mutants by conjugation. Their fermentation ethylacetate crude extract was analyzed by TLC and RP-HPLC and the HPLC pattern of the mutants was compared with the parent strain. By this method, three new compounds with different appearance in retention time were detected in these mutants, and the biosynthesis of GA in these mutants was disrupted in the meanwhile. Thus we concluded that the genes of pks model 6 gdmM and ct were necessary for the GA biosynthesis. The fermentation product of the ct gene disrupted mutant was very likely to be a derivative of GA and showed very high antibacterial activity. HPLC pattern analysis was proved to be a rapid indication for detecting the new fermentation products in gene engineered mutants. It was a powerful means for early directing to chemical isolation and purification in the multiple secondary metabolite producer.