产丁二酸谷氨酸棒状杆菌基因缺失代谢工程菌株的构建

Construction a metabolic engineering strain to produce succinic acid from Corynebacterium glutamicum by gene deletion mutation

摘要:

【目的】提高谷氨酸棒状杆菌(Corynebacterium glutamicum) ATCC13032厌氧条件下的丁二酸产量, 并降低发酵产物中副产物的含量。【方法】以谷氨酸棒状杆菌(Corynebacterium glutamicum) ATCC13032 为出发菌, 首先敲除乳酸形成的关键酶乳酸脱氢酶基因(ldh), 构建ldh缺失株谷氨酸棒状杆菌 ATCC13032Δldh; 然后以缺失株谷氨酸棒状杆菌ATCC13032Δldh 为出发菌, 敲除该菌的丙酮酸脱氢酶系的E1p酶基因(aceE), 构建一株双缺失突变菌株谷氨酸棒状杆菌ATCC13032ΔldhΔaceE。【结果】与供试菌比较, 谷氨酸棒状杆菌ATCC13032Δldh的丁二酸产量和转化率分别提高了94.9%和32%, 并且主要的副产物乳酸产量由出发菌产量的63.5 g/L降低到很微量的程度。丙酮酸脱氢酶的失活并不能完全消除副产物乙酸的形成, 但乙酸的产量较ATCC13032Δldh降低了37.9%, 丁二酸的产量略有提高。【结论】该重组菌具有较强的丁二酸生产工业化潜力, 并且该研究方法为微生物代谢育种提供参考。

Abstract:

[Objective] In order to improve the production of succinic acid and decrease the accumulation of by-products under the oxygen deprivation by Corynebacterium glutamicum 13032. [Methods] From the C. glutamicum ATCC13032, firstly, the gene of lactic dehydrogenase (ldh) was knocked-out and the single deletion mutation strain C. glutamicum ATCC13032Δldh was constructed. Secondly, we interrupted the pathway of pyruvic acid flux to acetic acid by marker-free deletion the E1p gene (aceE) which encode the essential part of the pyruvate dehydrogenase complex (PDHC). [Results] The production and yield of succinic acid of recombination strain improved 94.9% and 32%, respectively, compared to the parent strain C. glutamicum ATCC13032 and what’s more, the main by-product lactic acid was eliminated to none from 63.5 g/L. The inactive of the pyruvate dehydrogenase complex could not prevent the formation of acetic acid absolutely, but the production of acetic acid decreased 37.9%, compared to C. glutamicum ATCC13032Δldh and the production of succinic acid improved a little. [Conclusion] This recombination bacteria has a potential in industrial applications, and this study methods could provide reference for microbial metabolism breeding.