乙酸合成途径阻断及NADH氧化酶表达对于谷氨酸棒杆菌生产乙偶姻的影响

摘要

[目的]研究乙酸合成途径阻断及NADH氧化酶表达对于谷氨酸棒杆菌生产乙偶姻的影响.[方法]在谷氨酸棒杆菌CGF2中异源表达alsSD操纵子构建乙偶姻生产菌株CGT1,考察敲除乙酸生成途径cat和pqo对乙偶姻的影响.然后引入短乳杆菌的NADH氧化酶,在优化的溶氧条件下研究其对乙偶姻产量的影响.[结果]CGT1在摇瓶发酵中可积累6.27 g/L乙偶姻,敲除cat使乙偶姻产量显著提高30.94％,达到8.21 g/L;双敲除cat和pqo没有进一步提高产量.通过优化发酵的溶氧水平,乙偶姻产量达到10.06 g/L.在高溶氧水平下引入NADH氧化酶导致菌株的生长和糖代谢速率提高,但乙偶姻产量略有降低.在分批补料发酵中,重组菌株乙偶姻产量达到40.51 g/L,产率为0.51 g/(L·h).[结论]在谷氨酸棒杆菌中阻断乙酸合成途径cat能够有效提高乙偶姻产量,NADH氧化酶在高溶氧水平下表达不利于乙偶姻的合成,需要进一步调节表达水平以确定其效果.%[Objective] We researched the effects of inactivation of acetate synthetic pathway and expression of NADH oxidase on acetoin production in Corynebacterium glutamicum.[Methods]Heterologous expression of alsSD operon in C.glutamicum CGF2 resulted in acetoin production strain CGT1,and disruption of acetate synthetic pathway gene cat and pqo was investigated.Subsequently,NADH oxidase from Lactobacillus brevis was developed for acetoin production under optimal oxygen condition.[Results] CGT1 accumulated 6.27 g/L acetoin in flask cultivation.Deletion of cat increased acetion production by 30.94％ compared with CGT1,which was 8.21 g/L.Deletion of cat and pqo had no improvement on production.Moreover,the production of acetoin enhanced 10.06 g/L by optimizing the concentration of dissolved oxygen.Under high dissolved oxygen,expression of NADH oxidase contributed to an increase in biomass and glucose consumption rate,but the titer of acetoin decreased slightly.In intermittent fed-batch fermentation,recombination strain produced 40.51 g/L acetoin with a productivity of 0.51 g/(L·h).[Conclusion] The inactivation of acetate synthetic pathway gene cat could efficiently increase the production of acetoin in C.glutamicum.However,expression of NADH oxidase had disadvantage of acetion,and its expression level required more regulation to confirm the effect.