Metabolic engineering of Bacillus subtilis for redistributing the carbon flux to 2,3-butanediol by manipulating NADH levels.
Metabolic engineering of Bacillus subtilis for redistributing the carbon flux to 2,3-butanediol by manipulating NADH levels.
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枯草芽孢杆菌的代谢工程通过操纵 NADH 水平将碳通量重新分配给 2,3-丁二醇
DOI：
10.1186/s13068-015-0320-1
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发表时间：
2015
影响因子：
6.3
通讯作者：
Yang ST
中科院分区：
文献类型：
--
作者：
Yang T;Rao Z;Hu G;Zhang X;Liu M;Dai Y;Xu M;Xu Z;Yang ST
背景：乙偶姻还原酶（Acr）催化乙偶姻转化为2，3-丁二醇（2，3-BD），同时将NADH氧化为NAD（+）。因此，细胞内2，3-BD的产生可能受限速因子Acr和/或NADH的量控制。以前，我们表明，高水平的Acr是有益的2，3-BD accumulation.Results：代谢工程策略提出了重新分配碳通量2，3-BD通过操纵NADH水平。通过在枯草芽孢杆菌中插入甲酸脱氢酶基因来破坏NADH氧化酶（YodC，由yodC编码）比通过插入Cat表达盒来破坏YodC更有效地提高2，3-BD生产和减少乙偶姻形成。这是因为前者导致重组菌株AFY中引入了额外的NADH再生系统，同时破坏了NADH氧化酶。在菌株AFY的发酵中，相对于亲本菌株，2，3-BD的最高浓度增加了19.9%，而乙偶姻滴度降低了71.9%。然而，主要副产物乳酸的浓度增加了47.2%。为了进一步提高2，3-BD的碳通量和NADH，使用插入突变技术破坏乳酸脱氢酶基因ldhA来阻断乳酸途径。得到的工程菌株B.结论：通过提高NADH的利用率和降低副产物的浓度，为利用整合型重组宿主生产2，3-BD提供了一种重要的代谢工程策略。
Background:Acetoin reductase (Acr) catalyzes the conversion of acetoin to 2,3-butanediol (2,3-BD) with concomitant oxidation of NADH to NAD(+). Therefore, intracellular 2,3-BD production is likely governed by the quantities of rate-limiting factor(s) Acr and/or NADH. Previously, we showed that a high level of Acr was beneficial for 2,3-BD accumulation.Results:Metabolic engineering strategies were proposed to redistribute carbon flux to 2,3-BD by manipulating NADH levels. The disruption of NADH oxidase (YodC, encoded by yodC) by insertion of a formate dehydrogenase gene in Bacillus subtilis was more efficient for enhancing 2,3-BD production and decreasing acetoin formation than the disruption of YodC by the insertion of a Cat expression cassette. This was because the former resulted in the recombinant strain AFY in which an extra NADH regeneration system was introduced and NADH oxidase was disrupted simultaneously. On fermentation by strain AFY, the highest 2,3-BD concentration increased by 19.9 % while the acetoin titer decreased by 71.9 %, relative to the parental strain. However, the concentration of lactate, the main byproduct, increased by 47.2 %. To further improve carbon flux and NADH to 2,3-BD, the pathway to lactate was blocked using the insertional mutation technique to disrupt the lactate dehydrogenase gene ldhA. The resultant engineered strain B. subtilis AFYL could efficiently convert glucose into 2,3-BD with little acetoin and lactate accumulation.Conclusions:Through increasing the availability of NADH and decreasing the concentration of unwanted byproducts, this work demonstrates an important strategy in the metabolic engineering of 2,3-BD production by integrative recombinant hosts.