Evolutionary engineering of Escherichia coli for improved anaerobic growth in minimal medium accelerated lactate production
Evolutionary engineering of Escherichia coli for improved anaerobic growth in minimal medium accelerated lactate production
复制标题
大肠杆菌的进化工程可改善基本培养基中的厌氧生长，加速乳酸的产生
DOI：
10.1007/s00253-018-09588-9
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发表时间：
2019
影响因子：
5
通讯作者：
Zhao Xueming
中科院分区：
文献类型：
--
作者：
Wang Baowei;Zhang Xiaoxia;Yu Xinlei;Cui Zhenzhen;Wang Zhiwen;Chen Tao;Zhao Xueming
厌氧发酵是微生物生产乙醇、有机酸等大宗化学品的有利工艺。厌氧发酵生产能力低是制约厌氧发酵工艺发展的瓶颈，严重影响了生产菌的技术竞争力。提高生产菌株的生长速率可以加快总的生产周期，并可能最终提高厌氧过程的生产力。本研究采用进化工程的方法对野生型大肠杆菌W3110进行改造，以提高其在无机盐培养基中的厌氧生长。在进化菌株WE 269中实现了指数生长速率和静止细胞密度的显著增加，并且在使用M9基本培养基的该菌株的分批发酵中也观察到乳酸生产率增加了96.5%。以WE 269为平台，构建了乳酸工程菌BW 100，在5L的生物反应器中发酵48 h，乳酸产量达到98.3g/L（D-乳酸光学纯度大于95%），生产能力为2.05g/（L.h）。初步研究表明，sucD（sucD M245 I）（编码琥珀酰辅酶A合成酶）、ilvG（ilvG Δ1bp）（编码乙酰乳酸合成酶2催化亚基）和rpoB（rpoB T1037 P）（编码RNA聚合酶β亚基）的突变显著提高了E.杆菌ilvG和sucD双基因突变恢复了进化菌株WE 269的大部分生长潜力。这项工作表明，改善生产宿主的厌氧生长可以提高乳酸等有机酸的生产率，并且特定突变使能的改善生长也可以应用于代谢工程以生产其他大宗化学品。
Anaerobic fermentation is a favorable process for microbial production of bulk chemicals like ethanol and organic acids. Low productivity is the bottleneck of several anaerobic processes which has significant impact on the technique competitiveness of production strain. Improving growth rate of production strain can speed up the total production cycle and may finally increase productivity of anaerobic processes. In this work, evolutionary engineering of wild-type strain Escherichia coli W3110 was adopted to improve anaerobic growth in mineral medium. Significant increases in exponential growth rate and stationary cell density were achieved in evolved strain WE269, and a 96.5% increase in lactate productivity has also been observed in batch fermentation of this strain with M9 minimal medium. Then, an engineered strain for lactate production (BW100) was constructed by using WE269 as a platform and 98.3 g/L lactate (with an optical purity of D-lactate above 95%) was produced in a 5-L bioreactor after 48 h with a productivity of 2.05 g/(L·h). Finally, preliminary investigation demonstrated that mutation in sucD (sucD M245I) (encoding succinyl-CoA synthetase); ilvG (ilvG Δ1bp) (encoding acetolactate synthase 2 catalytic subunit), and rpoB (rpoB T1037P) (encoding RNA polymerase β subunit) significantly improved anaerobic growth of E. coli. Double-gene mutation in ilvG and sucD resumed most of the growth potential of evolved strain WE269. This work suggested that improving anaerobic growth of production host can increase productivity of organic acids like lactate, and specific mutation-enabled improved growth may also be applied to metabolic engineering for production of other bulk chemicals.