Regulation of the NADH pool and NADH/NADPH ratio redistributes acetoin and 2,3-butanediol proportion in Bacillus subtilis
Regulation of the NADH pool and NADH/NADPH ratio redistributes acetoin and 2,3-butanediol proportion in Bacillus subtilis
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NADH 库和 NADH/NADPH 比例的调节可重新分配枯草芽孢杆菌中的乙偶姻和 2,3-丁二醇比例
DOI：
10.1002/biot.201400577
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发表时间：
2015-08-01
影响因子：
4.7
通讯作者：
Yang, Shangtian
中科院分区：
文献类型：
--
作者：
Bao, Teng;Zhang, Xian;Yang, Shangtian
枯草芽孢杆菌的主要产物是乙酰托因以及一些nadh依赖的副产物，特别是2,3-丁二醇。在本研究中，NADH库的下调和NADH/NADPH的重新分配是通过外部和遗传过程来定位的，作为一种重新分配代谢通量的手段，有利于乙酰蛋白的合成。首先，发现使用不同氧化态的碳源会导致细胞内NADH/NAD（+）比率的差异，这决定了乙酰素的总工艺产率。葡萄糖和葡萄糖酸盐的混合物作为底物产生相对较低的NADH/NAD+比率，并导致乙酰蛋白的产量增加，而副产物显著减少。使用葡萄糖作为底物的代谢工程方法可以产生类似的效果。过氧化磷酸戊糖途径的过度表达显著增强了乙酰胆碱的产生：葡萄糖-6-磷酸脱氢酶的表达增加导致细胞内NADH/NADPH比值（1.9倍）和NADH/NAD（+）比值（1.7倍）降低。在补料间歇培养中，工程菌株的乙酰素浓度为43.3 g L-1，而2,3-丁二醇的产量仅为1.7 g L-1。本文探讨了通过外部和遗传手段操纵辅因子水平来重新分配碳通量的概念，为改善工业乙酰素发酵提供了一种新的策略。
Bacillus subtilis produces acetoin as a major product along with several NADH-dependent byproducts, especially 2,3-butanediol. In this study, the down-regulation of the NADH pool and the redistribution of NADH/NADPH were targeted using external and genetic processes, as a means by which to redistribute the metabolic flux in favor of acetoin synthesis. First, it was found that the use of carbon sources of different oxidation states resulted in very different intracellular NADH/NAD(+) ratios that dictated the total process yield of acetoin. A mixture of glucose and gluconate as substrate produced a relatively low NADH/NAD+ ratio, and resulted in an increase in acetoin production while byproducts significantly decreased. Metabolic engineering methods using glucose as a substrate could yield a similar effect. Acetoin production was significantly enhanced by overexpression of the oxidative pentose phosphate pathway: increased expression of glucose-6-phosphate dehydrogenase resulted in a decrease in the intracellular NADH/NADPH ratio (1.9-fold) and NADH/NAD(+) ratio (1.7-fold). In fed-batch culture the engineered strain yielded an acetoin concentration of 43.3 g L-1, while the production of 2,3-butanediol was only 1.7 g L-1. The concept of the manipulation of cofactor levels to redistribute carbon flux by external and genetic means as explored in this paper provides a novel strategy for improving industrial acetoin fermentation.