Issue 18, 2022

Improving isoprenol production via systematic CRISPRi screening in engineered Escherichia coli

Abstract

Engineering synthetic pathways in microbial hosts could afford bioproduction of industrially important fuels, chemicals or materials from renewable carbon sources. However, the sophisticated biochemical network and metabolic burden within microbial hosts always compromise the pathway performance and product yields of desired chemicals, inevitably limiting their translation into industrial bioprocesses. In this work, using the mevalonate-based isoprenol pathway as a proof-of-principle demonstration, we applied the CRISPR/dCas9 interference (CRISPRi) screening to identify endogenous gene targets intervening or promoting isoprenol biosynthesis in Escherichia coli. via CRISPRi screening in an engineered mevalonate-overproducing E. coli strain, we identified genes involved in the phosphate intermediate hydrolysis (aphA, yieH and yggV), carbon flux redirection (accA), and isoprenol tolerance (astE) pathways, whose over-expression or repression enabled 68.2%, 28.8%, and 62.1% increase of isoprenol titers, respectively. The combinatorial repression of yggV and accA permitted the highest isoprenol titer of 3.63 g L−1 in shake flask based batch cultivation, with the yield reaching 57% of the theoretical maximal pathway yield. Further shake flask based fed-batch cultivation increased the isoprenol titer to 8.51 g L−1, during which the isoprenol yield within the stationary growth phase could reach 85% of the theoretical maximal pathway yield. This work demonstrated the application of CRISPRi screening in the systematic optimization of the synthetic pathway performance in microbial hosts and enabled high-yield isoprenol production in metabolically engineered E. coli.

Graphical abstract: Improving isoprenol production via systematic CRISPRi screening in engineered Escherichia coli

Supplementary files

Article information

Article type
Paper
Submitted
14 Jun 2022
Accepted
08 Aug 2022
First published
22 Aug 2022

Green Chem., 2022,24, 6955-6964

Improving isoprenol production via systematic CRISPRi screening in engineered Escherichia coli

J. Wang, T. Jiang, S. Milligan, J. Zhang, C. Li and Y. Yan, Green Chem., 2022, 24, 6955 DOI: 10.1039/D2GC02255A

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