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- Title
Engineering global regulators for enhanced tolerance to multiple inhibitors by CRISPR‐enabled trackable genome engineering.
- Authors
Song, Xin; Zheng, Yangyang; Li, Shuting; Choudhury, Alaksh; Liu, Xunli; Chen, Tao; Gill, Ryan T.; Wang, Zhiwen
- Abstract
Strain engineering for industrial production requires the improvement of tolerance to multiple unfavorable conditions. Here, we report global regulator libraries based on the CRISPR‐enabled trackable genome engineering to engineer tolerance against multiple inhibitors in Escherichia coli. Deep mutagenesis libraries were rationally designed, and constructed to target 34,340 mutations across 23 global regulators. Sixty‐nine specific mutations, respectively, conferred tolerance to acetate, NaCl, furfural, and high temperature were isolated, confirmed, and evaluated. Among them, 31 novel reconstructed mutations exhibited better tolerance to the corresponding inhibitors. The most dramatic mutation CRP‐E182D conferred high cross‐tolerance to acetate, NaCl, and isobutanol. According to the reconstruction and transcriptome analysis, the tolerance of mutant strains to acetate is a complex whole‐cell process. In particular, it was confirmed for the first time that upregulation of ilvA and nadA‐pnuC, deletion of potF or the small RNA sgrS, and adding exogenous valine or isoleucine significantly increased acetate tolerance.
- Subjects
GENOME editing; FURFURAL; ENGINEERING tolerances; NON-coding RNA; ACETATES; INDUSTRIAL engineering; ESCHERICHIA coli
- Publication
AIChE Journal, 2023, Vol 69, Issue 4, p1
- ISSN
0001-1541
- Publication type
Article
- DOI
10.1002/aic.18031