Spatial modulation and cofactor engineering of key pathway enzymes for fumarate production in Candida glabrata
Spatial modulation and cofactor engineering of key pathway enzymes for fumarate production in Candida glabrata
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光滑念珠菌富马酸生产关键途径酶的空间调控和辅因子工程
DOI：
10.1002/bit.26906
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发表时间：
2019-03-01
影响因子：
3.8
通讯作者：
Liu, Liming
中科院分区：
文献类型：
--
作者：
Chen, Xiulai;Li, Yang;Liu, Liming
富马酸盐是一种天然存在的有机酸，是三羧酸（TCA）循环的中间体，在食品、制药和化学工业中有着广泛的应用。然而，微生物从可再生原料中生产富马酸盐受到代谢物运输和辅因子失衡所导致的合成途径固有的低效率的限制。本研究通过对TCA还原途径中关键途径酶的空间调控和辅因子工程，培养了一株能够高效生产富马酸盐的光假丝酵母菌株。具体而言，首先构建并优化了DNA引导支架系统，以调节丙酮酸羧化酶，苹果酸脱氢酶和富马酸酶，将富马酸滴度从0.18提高到11.3 g/L。然后，通过控制二磷酸腺苷依赖性磷酸烯醇丙酮酸羧激酶和NAD+依赖性甲酸脱氢酶的表达强度，组合调节辅因子平衡，导致富马酸产量大幅增加，最高可达18.5 g/L。最后，工程菌株T.G‐4G‐S（1:1:2）‐P(M)‐F(H)能够在5‐L间歇式生物反应器中产生21.6 g/L富马酸盐。这里描述的这种策略为开发高效的细胞工厂以生产其他工业上有用的化学品铺平了道路。
Fumarate is a naturally occurring organic acid that is an intermediate of the tricarboxylic acid (TCA) cycle and has numerous applications in food, pharmaceutical, and chemical industries. However, microbial fumarate production from renewable feedstock is limited by the intrinsic inefficiency of its synthetic pathway caused by week metabolites transportation and cofactor imbalance. In this study, spatial modulation and cofactor engineering of key pathway enzymes in the reductive TCA pathway were performed for the development of a Candida glabrata strain capable of efficiently producing fumarate. Specifically, DNA‐guided scaffold system was first constructed and optimized to modulate pyruvate carboxylase, malate dehydrogenase, and fumarase, increasing the fumarate titer from 0.18 to 11.3 g/L. Then, combinatorially tuning cofactor balance by controlling the expression strengths of adenosine diphosphate‐dependent phosphoenolpyruvate carboxykinase and NAD+‐dependent formate dehydrogenase led to a large increase in fumarate production up to 18.5 g/L. Finally, the engineered strain T.G‐4G‐S(1:1:2)‐P(M)‐F(H) was able to produce 21.6 g/L fumarate in a 5‐L batch bioreactor. This strategy described here, paves the way to develop efficient cell factories for the production of the other industrially useful chemicals.