Research Institute of Innovative Technology for the Earth, Kizugawadai, Kizugawa, Kyoto, Japan.
Appl Environ Microbiol. 2012 Feb;78(3):865-75. doi: 10.1128/AEM.07056-11. Epub 2011 Dec 2.
Production of L-valine under oxygen deprivation conditions by Corynebacterium glutamicum lacking the lactate dehydrogenase gene ldhA and overexpressing the L-valine biosynthesis genes ilvBNCDE was repressed. This was attributed to imbalanced cofactor production and consumption in the overall L-valine synthesis pathway: two moles of NADH was generated and two moles of NADPH was consumed per mole of L-valine produced from one mole of glucose. In order to solve this cofactor imbalance, the coenzyme requirement for L-valine synthesis was converted from NADPH to NADH via modification of acetohydroxy acid isomeroreductase encoded by ilvC and introduction of Lysinibacillus sphaericus leucine dehydrogenase in place of endogenous transaminase B, encoded by ilvE. The intracellular NADH/NAD(+) ratio significantly decreased, and glucose consumption and L-valine production drastically improved. Moreover, L-valine yield increased and succinate formation decreased concomitantly with the decreased intracellular redox state. These observations suggest that the intracellular NADH/NAD(+) ratio, i.e., reoxidation of NADH, is the primary rate-limiting factor for L-valine production under oxygen deprivation conditions. The L-valine productivity and yield were even better and by-products derived from pyruvate further decreased as a result of a feedback resistance-inducing mutation in the acetohydroxy acid synthase encoded by ilvBN. The resultant strain produced 1,470 mM L-valine after 24 h with a yield of 0.63 mol mol of glucose(-1), and the L-valine productivity reached 1,940 mM after 48 h.
在缺乏乳酸脱氢酶基因 ldhA 并过表达 L-缬氨酸生物合成基因 ilvBNCDE 的谷氨酸棒杆菌中，在缺氧条件下生产 L-缬氨酸受到抑制。这归因于整个 L-缬氨酸合成途径中辅酶的产生和消耗不平衡：从 1 摩尔葡萄糖生产 1 摩尔 L-缬氨酸时，会产生 2 摩尔 NADH 和 2 摩尔 NADPH。为了解决这种辅酶失衡问题，通过修饰编码 ilvC 的乙酰羟酸异构还原酶，将 L-缬氨酸合成的辅酶要求从 NADPH 转换为 NADH，并引入来自解淀粉芽孢杆菌的亮氨酸脱氢酶替代内源性转氨酶 B（由 ilvE 编码）。细胞内 NADH/NAD(+) 比值显著降低，葡萄糖消耗和 L-缬氨酸产量大幅提高。此外，随着细胞内氧化还原状态的降低，L-缬氨酸产量增加，琥珀酸形成减少。这些观察结果表明，细胞内 NADH/NAD(+) 比值，即 NADH 的再氧化，是缺氧条件下 L-缬氨酸生产的主要限速因素。由于乙酰羟酸合酶编码基因 ilvBN 中的反馈抗性诱导突变，L-缬氨酸的生产力和产率甚至更好，并且来自丙酮酸的副产物进一步减少。该菌株在 24 小时后生产 1,470 mM L-缬氨酸，产率为 0.63 mol/mol 葡萄糖（-1），48 小时后 L-缬氨酸生产力达到 1,940 mM。
