过表达根瘤血红蛋白基因对活跃链霉菌那西肽产量的影响

摘要

[目的]采用过表达根瘤血红蛋白基因改善活跃链霉菌的溶氧表达，以提高那西肽的产量。[方法]通过全基因组合成的方法得到根瘤菌血红蛋白基因（sm），连接到整合型质粒pIB139上，构建表达载体pIB139－sm。通过大肠杆菌－链霉菌结合转移的方法将sm基因整合到活跃链霉菌基因组上，通过PCR验证各转化子目的片段已整合到活跃链霉菌基因组上，并进行大量筛选得到重组菌株。[结果]经SDS－PAGE和CO结合差示光谱分析，结果显示，目的蛋白均已成功表达且具有相应的蛋白活性。摇瓶培养结果显示，SmfHb表达对菌体生长和那西肽产量有明显的促进作用，过表达SmfHb的重组菌株那西肽产量达1245．7μg／mL，比原始菌株提高39．6％。[结论]过表达sm基因提高那西肽产量的作用优于vhb。%Abs rt act [ Objective] To improve the dissolved oxygen expression of Streptomyces actuosus by overexpressing hemoprotein Gene from Sinorhi-zobium meliloti, and to enhance the yield of nosiheptide.[ Method] Sinorhizobium meliloti hemoglobin gene ( sm) was obtained through the whole gene synthesis method, which were cloned into the plasmid pIB139 to construct expression vectors pIB139-sm.The plasmid containing sm was introduced into Streptomyces actuosus through conjugal transfer between E.coli and Streptomces.The transformants were verified by PCR, and then were purified by several cycles of single-colony screen.[ Result] The expression and activity of SmfHb was confirmed by SDS-PAGE and CO-difference spectrum analysis, respectively.Flask culture experiment results showed that the nosiheptide yield in SmfHb -overex-pressing strain (1 245.7 μg/mL) was increased by 39.6% ( P<0.05) compared with the control strain (892.5 μg/mL).[Conclusion] Overexpressing sm gene is superior to vhb in enhancing the yield of nosiheptide yield.