Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering

Metabolic Engineering
Rongming LiuRyan T Gill

Abstract

Strain engineering for industrial production requires a targeted improvement of multiple complex traits, which range from pathway flux to tolerance to mixed sugar utilization. Here, we report the use of an iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method to engineer rapid glucose and xylose co-consumption and tolerance to hydrolysate inhibitors in E. coli. Deep mutagenesis libraries were rationally designed, constructed, and screened to target ~40,000 mutations across 30 genes. These libraries included global and high-level regulators that regulate global gene expression, transcription factors that play important roles in genome-level transcription, enzymes that function in the sugar transport system, NAD(P)H metabolism, and the aldehyde reduction system. Specific mutants that conferred increased growth in mixed sugars and hydrolysate tolerance conditions were isolated, confirmed, and evaluated for changes in genome-wide expression levels. We tested the strain with positive combinatorial mutations for 3-hydroxypropionic acid (3HP) production under high furfural and high acetate hydrolysate fermentation, which demonstrated a 7- and 8-fold increase in 3HP productivity relative to the parent strain, respectiv...Continue Reading

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Citations

Oct 11, 2019·Nucleic Acids Research·Anna J SimonIlya J Finkelstein
Jul 29, 2020·Frontiers in Bioengineering and Biotechnology·Liya LiangCarrie A Eckert
Jan 21, 2019·Applied Microbiology and Biotechnology·Kun ZhangHailei Wang
Jul 23, 2020·Frontiers in Bioengineering and Biotechnology·Wentao DingShuobo Shi
Mar 7, 2020·Frontiers in Bioengineering and Biotechnology·Jia-Wei LiYun-Peng Bai
Nov 6, 2020·Current Opinion in Biotechnology·Jacob A Fenster, Carrie A Eckert
Apr 17, 2020·ACS Synthetic Biology·Alaksh ChoudhuryRyan T Gill
Jul 30, 2020·Current Opinion in Biotechnology·Mingfeng CaoHuimin Zhao
Jun 16, 2021·ACS Synthetic Biology·Yangyang ZhengZhiwen Wang
Aug 4, 2021·Nature Reviews. Microbiology·José Montaño LópezJosé L Avalos

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