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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 esaS  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.    Supplementary Information  The online version contains supplementary material available at 10.1186/s13068-021-01954-6.

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