ATP and NADPH Engineering of Escherichia Coli to Improve the Production of 4-hydroxyphenylacetic Acid Using CRISPRi

Overview

Journal Biotechnol Biofuels

Publisher Biomed Central

Specialty Biotechnology

Date 2021 Apr 21

PMID 33879249

Citations 6

Authors

Yu-Ping Shen

Yu-Ling Liao

Qian Lu

Xin He

Zhi-Bo Yan

Jian-Zhong Liu

Affiliations

Soon will be listed here.

Abstract

Background: 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.

Results: 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.

Conclusion: This CECRiS strategy will be useful in engineering microorganisms for the high-level production of bioproducts.

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