ATP and NADPH engineering of Escherichia coli to improve the production of 4-hydroxyphenylacetic acid using CRISPRi

Biotechnology for Biofuels

Jian-Zhong Liu¹  Zhi-Bo Yan¹  Xin He¹  Qian Lu¹  Yu-Ling Liao¹  Yu-Ping Shen² 

[1] 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, 510275, Guangzhou, People’s Republic of China;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, 510275, Guangzhou, People’s Republic of China;College of Chemistry and Bioengineering, Hunan University of Science and Engineering, 425199, Yongzhou, China;

关键词: 4-Hydroxyphenylacetic acid;    ATP engineering;    NADPH engineering;    CRISPRi;    Escherichia coli;   

DOI  :  10.1186/s13068-021-01954-6

来源: Springer

【 摘 要 】

Background4-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.ResultsWe 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-PesaS 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.ConclusionThis CECRiS strategy will be useful in engineering microorganisms for the high-level production of bioproducts.

【 授权许可】

CC BY