亮氨酰氨肽酶基因的阻断对刺糖多孢菌生长及次级代谢产物合成的影响

Disruption of leucyl aminopeptidase gene affects phenotypes and second metabolite production of Saccharopolyspora spinosa

摘要:

[目的] 构建亮氨酰氨肽酶基因(pepA)被阻断的刺糖多孢菌工程菌株,并鉴定该基因对刺糖多孢菌菌丝形态、生物量、菌体全蛋白表达水平及产多杀菌素能力的影响,探究该基因调控多杀菌素合成的可能机制。[方法] 利用PCR扩增刺糖多孢菌中的pepA基因同源片段,经酶切连接技术构建敲除载体pOJ260-pepA;通过接合转移和单交换同源重组将该载体整合至刺糖多孢菌染色体中,获得工程菌株S. sp-△pepA;利用培养特征、形态学、高效液相色谱、SDS-PAGE等方法对菌株进行研究分析。[结果] 工程菌株S. sp-△pepA菌丝片段化程度加剧,生长态势被延缓且生物量降低,但有效促进了多杀菌素的生物合成。阻断亮氨酰胺肽酶基因的表达使刺糖多孢菌菌体全蛋白表达情况发生明显改变,找到表达水平显著上调的差异蛋白核糖体蛋白亚基和醛基脱氢酶,核糖体蛋白亚基通过影响蛋白质代谢对菌体生长产生影响;醛基脱氢酶则可与乙醇脱氢酶、乙酰辅酶A的合成酶相互作用影响辅酶A合成,而辅酶A是合成多杀菌素的重要底物。[结论] 在刺糖多孢菌合成多杀菌素的次级代谢过程中,pepA基因作为负调控因子发挥作用。

关键词:刺糖多孢菌;亮氨酰氨肽酶基因;阻断;多杀菌素;接合转移

Abstract:

[Objective] In order to investigate effects of leucyl aminopeptidase on mycelia morphology, growth rate, spinosad yield and protein expression in Saccharopolyspora spinosa by disrupting its encoding gene pepA and analyzing the characteristics of engineered S. spinosa. [Methods] The pepA gene of S. spinosa was amplified based on the conserved sequence and cloned into Escherichia coli-Streptomyces shuttle vector pOJ260 to generate pOJ260-pepA, which was transformed into S. spinosa by conjugation. Mycelium observation, SDS-PAGE and HPLC were used to analyze the engineered strain. [Results] Mycelia in S. sp-ΔpepA displayed a much higher degree of fragmentation and fewer branches compared to that of parental strain. Meanwhile, the growth rate of S. sp-ΔpepA was retarded and its biomass was reduced. Shake-flask fermentation demonstrated that spinosad yield increased by 122% in S. sp-ΔpepA strain compared to that of parental strain. SDS-PAGE analysis showed that protein expression profile of the engineered strain significantly changed. [Conclusion] The pepA gene negatively regulates the biosynthesis of spinosad and disruption of pepA gene could affect the mycelial morphology and growth of S. spinosa.

Key words:Saccharopolyspora spinosa;pepA gene;disruption;spinosad;conjugational transfer