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

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p IB139上,构建表达载体p IB139-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,Smf Hb表达对菌体生长和那西肽产量有明显的促进作用,过表达Smf Hb的重组菌株那西肽产量达1 245.7μg/m L,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。

Effects of Overexpressing Hemoprotein Gene from Sinor hizob ium me liloti on Nosih ept id e Yile d of Streptomyces actuosus

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.