透明颤菌血红蛋白基因在龟裂链霉菌中的表达及其对土霉素合成的影响

目的 考察透明颤菌血红蛋白基因(vgb)在大肠埃希菌(E. coli)和龟裂链霉菌(S. rimosus)中的表达，研究在不同溶氧条件下表达的透明颤菌血红蛋白(VHb)对龟裂链霉菌生长和土霉素合成的影响。方法 将vgb插入表达型质粒pET-28a中，导入E. coli BL21(DE3)用以表达VHb并测定其活性。以整合型质粒pSET152为载体，利用ermE*启动子组成型表达vgb基因，将构建的质粒通过接合转移整合到S. rimosus M4018基因组上，利用CO差示光谱法和SDS-PAGE鉴定两个重组菌中的血红蛋白。在5L罐上考察不同溶氧条件下VHb的表达对重组龟裂链霉菌生长和土霉素合成的影响。结果 重组大肠埃希菌和重组龟裂链霉菌都能够表达具有生物活性的VHb，发酵结果表明，在117h时与对照菌相比，重组龟裂链霉菌在高氧和低氧条件下的干重分别提高了6%和32%，同时单位干重土霉素的产量分别提高了41%和93%。结论 VHb在重组龟裂链霉菌中成功表达，改善了菌体的生长和土霉素的合成，为解决工业发酵过程中氧限制问题提供了策略。

Effects of Vitreoscilla hemoglobin gene expression on the growth and oxytetracycline biosynthesis by Streptomyces rimosus

Objective To investigate the expression of Vitreoscilla hemoglobin gene (vgb) in E. coli and Streptomyces and study the effects of expressed Vitreoscilla hemoglobin (VHb) on the growth and oxytetracycline (OTC) production in S. rimosus grown under different dissolved oxygen concentrations. Methods The vgb gene was inserted into the expression plasmid pET-28a, and the generated plasmid was introduced into E. coli BL21 (DE3) to express VHb. In addition, a pSET152-derived integrative plasmid was constructed with vgb gene expression under the control of a constitutive promoter PermE*, and it was introduced into S. rimosus M4018 chromosome through conjugation. The VHb was identified with CO difference spectrum analysis and SDS-PAGE. The effects of VHb expression on the growth and OTC biosynthesis of recombinant S. rimosus grown under conditions with different dissolved oxygen were studied in a 5L fermentor. Results Both recombinant E. coli and S. rimosus were able to express biologically active VHb. Compared with the control strain M4018, the dry weights of recombinant S. rimosus cultivated with high or low dissolved oxygen concentrations were increased by 6% and 32% at 117h, respectively. Meanwhile, the OTC productions per dry cell weight were increased by 41% and 93% at 117h, respectively. Conclusion The successful expression of VHb in the recombinant S. rimosus could improve its growth and OTC yield, providing an effective strategy to solve the oxygen limitation problem during the fermentation