聚磷酸激酶基因的阻断对刺糖多孢菌生长及多杀菌素合成的影响

摘要

聚磷酸激酶基因（Polyphosphate kinase gene，ppk）是放线霉菌中的一种影响抗生素合成的全局性负调控因子，阻断该基因能显著提高其次级代谢产物产量。本文利用 PCR扩增了刺糖多孢菌中的ppk基因中间片段，经酶切连接技术将其克隆到大肠杆菌-链霉菌穿梭载体pOJ260上，构建阻断型载体pOJ260-ppk；通过接合转移将该功能质粒导入刺糖多孢菌中，获得了遗传性能稳定的重组菌株 S. sp-△ppk。对工程菌株的PCR检测结果显示，ppk基因片段已整合到刺糖多孢菌染色体上并成功阻断了该基因的表达。摇瓶发酵结果显示，工程菌株多杀菌素产量较原始菌株提高了122%。阻断聚磷酸激酶基因的表达对刺糖多孢菌的菌丝形态及生长发育产生了影响，并有效地促进该菌多杀菌素的生物合成。%Previous report revealed that polyphosphate kinase of Streptomyces played a negative role in antibiotic production. Here, we investigated whether polyphosphate kinase of Saccharopolyspora spinosa also negatively regulates the production of spinosad. Polyphosphate kinase encoding gene (ppk) was disrupted by homologous recombination. Primer pair used for amplification of ppk gene from Saccharopolyspora spinosa was designed according to the homologous ppk gene of S. spinosa. PCR product of partial sequence of ppk gene in S. spinosa was cloned into Escherichia coli-Streptomyces shuttle vector pOJ260 to generate pOJ260-ppk, which was transformed into S. spinosa by conjugation. Apramycin resistant colonies were picked up and identified by PCR. Positive transconjugants of which ppk gene was successfully disrupted were chosen for further study. Mycelia in S. sp-Δppk displayed a much higher degree of fragment and fewer branches when compared with parental strain. The growth rate of S. sp-Δppk was delayed and its biomass was reduced. Shake flask fermentation demonstrated that spinosad yield increased by 122% in S. sp-Δppk strain compared to that of parental strain. S. sp-Δppk exhibited highly genetic stability. Thus, we concluded that the product of ppk gene could negatively regulate the biosynthesis of spinosad and disruption of ppk gene could affect the mycelial morphology and growth of S. spinosa.