利用代谢工程技术强化肉桂地链霉菌莫能菌素的生物合成

Metabolic engineering of Streptomyces cinnamonensis enhances biosynthesis of monensin

摘要:

【目的】莫能菌素(monensin)是由肉桂地链霉菌(Streptomyces cinnamonensis)产生的聚醚类抗生素。本研究以肉桂地链霉菌2110为研究材料，利用代谢工程技术开展其重要前体甲基丙二酰CoA合成途径强化的研究，以此提高莫能菌素产量。【方法】首先过表达巴豆酰CoA还原酶(crotonyl-CoA reductase, CCR)强化乙酰乙酰CoA途径，接着过表达甲基丙二酰CoA变位酶(methylmalonyl-CoA mutase, MCM)强化琥珀酰CoA途径，最后串联过表达CCR和MCM并评估工程菌株的发酵性能。【结果】过表达CCR能促进菌体生长，同时提高莫能菌素产量，摇瓶发酵10 d后过表达菌株的生物量和发酵效价分别提高10.4%和19.0%；过表达MCM未能促进菌体生长，但提高了莫能菌素产量，过表达菌株摇瓶发酵10 d时效价提升9.9%；串联过表达CCR和MCM也使菌株的生物量和发酵效价提高，工程菌株2110-CCR-MCM在摇瓶发酵10 d时生物量和发酵效价分别提升9.4%和26.8%，在5 L罐上发酵6 d时达到最高的生物量和发酵效价分别为54.6 g/L和11.3 kU/mL，比原始菌株分别增加12.7%和36.2%。【结论】CCR和MCM的确介导了莫能菌素生物合成的关键代谢途径，串联过表达CCR和MCM更有效地促进了莫能菌素的合成。本研究为其他聚酮类化合物高产工程菌株的构建提供技术借鉴。

Abstract:

[Objective] Monensin is a polyether antibiotic produced by Streptomyces cinnamonensis. To enhance the production of monensin by microbial fermentation, we employed metabolic engineering to strengthen the synthesis pathway of the key precursor methylmalonyl-CoA in S. cinnamonensis 2110. [Methods] Firstly, crotonyl-CoA reductase (CCR) was overexpressed to strengthen the acetoacetyl-CoA pathway. Subsequently, methylmalonyl-CoA mutase (MCM) was overexpressed to improve the succinyl-CoA pathway. Finally, an engineered strain with tandem overexpression of CCR and MCM was constructed and evaluated for the fermentation performance. [Results] The overexpression of CCR increased the strain biomass and monensin titer by 10.4% and 19.0%, respectively, after 10 days of shake-flask fermentation. The overexpression of MCM increased the monensin titer by 9.9%, whereas it did not increase the strain biomass after 10 days of shake-flask fermentation. The tandem overexpression of CCR and MCM increased the biomass and monensin titer by 9.4% and 26.8%, respectively, after 10 days of shake-flask fermentation. In a 5 L bioreactor, the engineered strain 2110-CCR-MCM reached the highest biomass of 54.6 g/L and monensin titer of 11.3 kU/mL, which increased by 12.7% and 36.2%, respectively, compared with those of the starting strain 2110. [Conclusion] CCR and MCM mediated the key metabolic pathway of monensin biosynthesis in S. cinnamonensis, and the overexpression of CCR and MCM was highly favorable for monensin synthesis. This study provides technical reference for the engineering of strains with high yields of other polyketides.