Pathway engineering ofEscherichia colifor α-ketoglutaric acid production
Pathway engineering ofEscherichia colifor α-ketoglutaric acid production
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DOI：
10.1002/bit.27456
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发表时间：
2020-06-27
影响因子：
3.8
通讯作者：
Liu, Liming
中科院分区：
文献类型：
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作者：
Chen, Xiulai;Dong, Xiaoxiang;Liu, Liming
α-酮戊二酸(α-KG)是三羧酸(TCA)循环中的一种多功能二元酸，但由于其生物合成途径的内在低效，微生物工程生产α-酮戊二酸的经济效益不高。本研究采用途径工程的方法来提高α-KG在大肠杆菌中的生产途径效率。首先，从丙酮酸开始，重新布线TCA循环，以生产α-KG，并改造Straine。ColiW3110 Delta 4-PCA1产量15.66g/Lα-Kg。然后，通过设计不同强度的丙酮酸羧化酶和异柠檬酸脱氢酶表达框来优化重新连接的TCA循环，使α-KG的产量大幅提高(24.66g/L)。此外，通过过量表达乙酰辅酶A合成酶，提高了乙酰辅酶A(乙酰辅酶A)的利用率，使α-KG产量达到28.54g/L。ColiW3110 Delta 4-P((H))CaI((H))A在5-L补料分批生物反应器中能够生产32.20g/Lα-KG。这一策略为开发微生物生产α-KG的有效途径铺平了道路。
alpha-Ketoglutaric acid (alpha-KG) is a multifunctional dicarboxylic acid in the tricarboxylic acid (TCA) cycle, but microbial engineering for alpha-KG production is not economically efficient, due to the intrinsic inefficiency of its biosynthetic pathway. In this study, pathway engineering was used to improve pathway efficiency for alpha-KG production inEscherichia coli. First, the TCA cycle was rewired for alpha-KG production starting from pyruvate, and the engineered strainE. coliW3110 Delta 4-PCAI produced 15.66 g/L alpha-KG. Then, the rewired TCA cycle was optimized by designing various strengths of pyruvate carboxylase and isocitrate dehydrogenase expression cassettes, resulting in a large increase in alpha-KG production (24.66 g/L). Furthermore, acetyl coenzyme A (acetyl-CoA) availability was improved by overexpressing acetyl-CoA synthetase, leading to alpha-KG production up to 28.54 g/L. Finally, the engineered strainE. coliW3110 Delta 4-P((H))CAI((H))A was able to produce 32.20 g/L alpha-KG in a 5-L fed-batch bioreactor. This strategy described here paves the way to the development of an efficient pathway for microbial production of alpha-KG.