Abstract
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.
Highlights
All NADPH-consuming and ATP-consuming enzymeencoding genes of E. coli were repressed through clustered regularly interspaced short palindromic repeat interference (CRISPRi), and the target genes that should be modified were identified to enhance the production of 4-Hydroxyphenylacetic acid (4HPAA)
To avoid the auxotrophic phenotype caused by the knockout of the pabA gene, the Esa-PesaS QS-repressing system was used to automatically downregulate the expression of the pabA gene based on cell density in this study
The deletion of the NADPHconsuming enzyme-encoding gene yahK and the ATP-consuming enzyme-encoding gene fecE increased the production of 4HPAA by 22.8%
Summary
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, enhanc‐ ing 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. Downregulating the expression of the pabA gene using the Esa-PesaS quorum-sensing-repressing system further improved the produc‐ tion 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
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