Shen Yu-Ping, Liao Yu-Ling, Lu Qian, He Xin, Yan Zhi-Bo, Liu Jian-Zhong
Institute of Synthetic Biology, Biomedical Center, Guangdong Province Key Laboratory of Improved Variety Reproduction in Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, People's Republic of China.
College of Chemistry and Bioengineering, Hunan University of Science and Engineering, Yongzhou, 425199, China.
Biotechnol Biofuels. 2021 Apr 20;14(1):100. doi: 10.1186/s13068-021-01954-6.
4-Hydroxyphenylacetic acid (4HPAA) is an important raw material for the synthesis of drugs, pesticides and biochemicals. Microbial biotechnology would be an attractive approach for 4HPAA production, and cofactors play an important role in biosynthesis.
We developed a novel strategy called cofactor engineering based on clustered regularly interspaced short palindromic repeat interference (CRISPRi) screening (CECRiS) for improving NADPH and/or ATP availability, enhancing the production of 4HPAA. All NADPH-consuming and ATP-consuming enzyme-encoding genes of E. coli were repressed through CRISPRi. After CRISPRi screening, 6 NADPH-consuming and 19 ATP-consuming enzyme-encoding genes were identified. The deletion of the NADPH-consuming enzyme-encoding gene yahK and the ATP-consuming enzyme-encoding gene fecE increased the production of 4HPAA from 6.32 to 7.76 g/L. Automatically downregulating the expression of the pabA gene using the Esa-P quorum-sensing-repressing system further improved the production of 4HPAA. The final strain E. coli 4HPAA-∆yfp produced 28.57 g/L of 4HPAA with a yield of 27.64% (mol/mol) in 2-L bioreactor fed-batch fermentations. The titer and yield are the highest values to date.
This CECRiS strategy will be useful in engineering microorganisms for the high-level production of bioproducts.
4-羟基苯乙酸（4HPAA）是合成药物、农药和生物化学品的重要原料。微生物生物技术是生产4HPAA的一种有吸引力的方法，辅因子在生物合成中起重要作用。
我们开发了一种基于成簇规律间隔短回文重复序列干扰（CRISPRi）筛选（CECRiS）的辅因子工程新策略，用于提高NADPH和/或ATP的可用性，增强4HPAA的产量。通过CRISPRi抑制大肠杆菌所有消耗NADPH和消耗ATP的酶编码基因。经过CRISPRi筛选，鉴定出6个消耗NADPH的酶编码基因和19个消耗ATP的酶编码基因。缺失消耗NADPH的酶编码基因yahK和消耗ATP的酶编码基因fecE，使4HPAA的产量从6.32 g/L提高到7.76 g/L。使用Esa-P群体感应抑制系统自动下调pabA基因的表达，进一步提高了4HPAA的产量。最终菌株大肠杆菌4HPAA-∆yfp在2-L生物反应器分批补料发酵中产生了28.57 g/L的4HPAA，产率为27.64%（mol/mol）。滴度和产率是迄今为止的最高值。
这种CECRiS策略将有助于工程改造微生物以实现生物产品的高水平生产。
