该文通过敲除谷氨酸棒杆菌(C. glutamicum)中参与NADPH合成的葡萄糖-6-磷酸脱氢酶基因(zwf)、苹果酸酶基因(malE),并将自身NADP+-依赖型异柠檬酸脱氢酶基因(icdCg)替换为变形链球菌(Streptococcus mutans)NAD+-依赖型异柠檬酸脱氢酶基因(icdSm),构建重组菌株C. glutamicum LYS Δzwf ΔmalE ΔicdCg::icdSm,并研究阻断NADPH合成途径对胞内腺嘌呤核苷酸、吡啶核苷酸、细胞生长、葡萄糖消耗速率、产物合成的影响。与出发菌C. glutamicum LYS相比,重组菌胞内NADH提高38.58%,NADH/NAD+提高53.84%。而NADPH降低89.51%,明显低于出发菌,NADPH/NADP+降低93.13%。摇瓶结果表明,重组菌葡萄糖代谢能力明显减弱,生长也相对缓慢,菌体量降低4.36%。同时L-赖氨酸产量降低87.69%,而在胞内积累了大量副产物,如丙酮酸、乳酸等。为进一步研究NADPH调控L-赖氨酸产生菌胞内微环境的生理机制提供了研究基础。
NADPH plays important roles in regulating cell growth and L-lysine synthesis in Corynebacterium glutamicum. A recombinant strain with blocked NADPH biosynthesis was constructed by deleting the genes of glucose-6-phosphate dehydrogenase (zwf) and malic enzyme (malE) and by replacing NADP+-dependent isocitrate dehydrogenase gene (icdCg) with Streptococcus mutans NAD+-dependent isocitrate dehydrogenase gene (icdSm). Compared with C. glutamicum LYS, the levels of intracellular NADH and NADH/NAD+ in the recombinant strain increased by 38.58% and 53.84%, respectively, while the intracellular NADPH significantly decreased by 89.51%, and intracellular NADPH/NADP+ decreased by 93.13%. Meanwhile, the recombinant strain showed slower cell growth and its glucose metabolism significantly lowered during shake-flask fermentation. As a result, the biomass reduced 4.36% and the yield of L-lysine decreased by 87.69%, together with more by-products (pyruvate and lactate etc.) accumulated in cells. This study provides a basis for further studies on physiological mechanisms of NADPH that regulates intracellular microenvironment of L-lysine producing strains.
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