不同辅因子NADPH水平对谷氨酸棒杆菌生长及产物合成的影响

Different NADPH Levels Affected Growth and Metabolites Accumulation in Corynebacterium glutamicum

由谷氨酸棒杆菌(Corynebacterium glutamicum)分泌的初级代谢产物赖氨酸,其合成与胞内NADPH水平密切相关,但目前对谷氨酸棒杆菌中赖氨酸的生物合成与辅因子NADPH之间调节机制的理解仍然有限。作者以赖氨酸高产菌C.glutamicum XQ-5为出发菌株,通过阻断磷酸戊糖途径构建了低NADPH水平的重组菌C.glutamicum XQ-5Δzwf::pgi,使胞内每1万个细胞的NADPH量从3.57×10^(-8)μmol降低至1.12×10^(-8)μmol。接着通过强化磷酸戊糖途径构建了高NADPH水平的工程菌C.glutamicum XQ-5Δpgi::(zwf-gnd),使胞内每1万个细胞的NADPH量从3.57×10^(-8)μmol提高至1.80×10^(-7)μmol。通过摇瓶发酵,不同NADPH水平下菌株的菌体生长情况、菌体量和胞内副产物积累明显不同。利用转录组学和其他实验,发现重组菌C.glutamicum XQ-5Δpgi::(zwf-gnd)中的panD基因表达水平明显提高。当panD基因被敲除后,重组菌C.glutamicum XQ-5ΔpanDΔpgi::(zwf-gnd)的赖氨酸质量浓度从41 g/L提高至55 g/L,比出发菌C.glutamicum XQ-5提高了14.3%。数据表明,通过过表达磷酸戊糖途径来提高NADPH水平导致赖氨酸水平下降的原因之一,是panD基因表达水平的提高。这为进一步探究辅因子NADPH对赖氨酸生物合成的调控机制提供了坚实基础。

Lysine,as a primary metabolite,is normally produced by Corynebacterium glutamicum,and its synthesis is closely related to intracellular NADPH levels.However,the regulatory mechanisms between L-lysine biosynthesis and the cofactor NADPH in C.glutamicum are still poorly understood.In this paper,the lysine-producing strain C.glutamicum XQ-5 was used as the starting strain,and the recombinant strain C.glutamicum XQ-5Δzwf::pgi with low NADPH levels was constructed by blocking the pentose phosphate pathway,reducing the intracellular NADPH level in each 104 cells from 3.57×10^(-8)μmol to 1.12×10^(-8)μmol.In addition,the recombinant strain C.glutamicum XQ-5Δpgi::(zwf-gnd)with high NADPH level was constructed by enhancing the pentose phosphate pathway.It increased the intracellular NADPH level in each 104 cells from 3.57×10^(-8)μmol to 1.8×10^(-7)μmol.Cell growth,bacterial mass and intracellular by-product accumulation of strains under different NADPH levels were significantly different through shake-flask fermentation.Based on the results of transcriptomics and other experiments,the expression level of the panD gene in the recombinant strain C.glutamicum XQ-5Δpgi::(zwf-gnd)was significantly increased.The lysine production of recombinant strain C.glutamicum XQ-5ΔpanDΔpgi::(zwf-gnd)with inactivated panD gene increased to 55 g/L,an increase of 14.3%to the original strain C.glutamicum XQ-5,which suggested that the increased expression level of panD gene was one of the reasons for the decline in lysine levels based on the over-expression of pentose phosphate pathway.This study provides a solid theoretical basis for further studies on the regulation mechanism of cofactor NADPH on L-lysine biosynthesis.