糖多孢红霉菌λC3-SRR突变体的构建及其产物鉴定

Construction of Saccharopolyspora erythraea λC3-SRR mutant and identification of its product

目的 :构建糖多孢红霉菌KR6酶域基因失活突变体 ,探讨SRR氨基酸相应 9核苷酸去除突变体与合成酮内酯类化合物 3 脱氧 3 羰基 红霉内酯B的关系。方法 :以糖多孢红霉菌基因组DNA为模板 ,用重叠PCR技术扩增出缺少SRR氨基酸相应 9核苷酸的KR6酶域DNA片段 ,并克隆到载体pWHM3上 ,构建了同源重组质粒pWHM3 SRR。将pWHM3 SRR质粒转化糖多孢红霉菌λC3菌株 ,筛选出质粒整合到染色体上红霉素合成基因位点的整合体λC3 A、B和C。λC3 A在无硫链丝菌肽 (Thio)的R3M斜面上生长两代后 ,制备的原生质体涂R3M平皿 ,挑选出不能在含Thio的R3M斜面上生长、发酵液也无抑制枯草芽孢杆菌活性的突变菌株λC3 SRR。结果 :部分基因组DNA序列分析表明 ,糖多孢红霉菌λC3 SRR染色体上SRR氨基酸相应 9核苷酸已经敲除 ,ZabspecFab质谱分析证实λC3 SRR菌株合成了 3 脱氧 3 羰基 红霉内酯B。结论 :糖多孢红霉菌KR6酶域SRR氨基酸相应 9核苷酸敲除的λC3 SRR突变菌株可以合成 3 脱氧 3 羰基 红霉内酯B ,SRR为KR6酶域上NADPH 2′ 磷酸结合位点。

Objective: To construct Saccharopolyspora erythraea mutant in which KR6 domain of polyketide synthase lost its function and to probe into the relationship between SRR amino acid codes deletion mutation in the chromosome of S.erythraea and biosynthesis of 3-deoxy-3-oxo-erythronolide B. Methods: Taking genomic DNA of S.erythraea as a template, KR6 domain DNA fragment without the nine nucleotides corresponding to SRR amino acids was amplified by overlapping PCR techniques and cloned into homologous recombinant vector pWHM3 to build plasmid pWHM3-SRR. Plasmid pWHM3-SRR was introduced into protoplasts of S.erythraea λC3 and integrated into the gene locus for erythromycin biosynthesis in the chromosome, and three integrants, λC3-A, B and C, were selected out. After the integrant λC3-A grew for at least two generations on R3M media without thiostrepton, the protoplasts were prepared and made to single colonies on R3M plates. One mutant, named as S.erythraea λC3-SRR, which couldn′t grow on the R3M media with thiostrepton and couldn′t restrain Bacillus subtilis PUB110′s growth, was picked out. Results: DNA sequencing proved that the nine nucleotides corresponding to SRR amino acids in the chromosome of S.erythraea λ C3-SRR had been knocked out, and mass spectrometry showed that λC3-SRR could synthesize 3-deoxy-3-oxo-erythronolide B. Conclusion: S.erythraea λC3-SRR with SRR codes knocked out in KR6 domain could synthesize 3-deoxy-3-oxo-erythronolide B, and SRR of KR6 is the NADPH′s 2′-phosphate-binding site.