초록
<P><B>Abstract</B></P> <P>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 <I>E. coli</I>. 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, respectively.</P> <P><B>Highlights</B></P> <P> <UL> <LI> iCREATE strategy was used for complex phenotype engineering. </LI> <LI> Designer libraries targeting ~40,000 mutations, across 30 genes were constructed. </LI> <LI> 3HP fermentation was tested in the best producing quadruple mutant strain BGHPht. </LI> <LI> BGHPht had a 6.3-fold increase of productivity with high furfural hydrolysate. </LI> <LI> BGHPht had a 7-fold increase of productivity with high acetate hydrolysate. </LI> </UL> </P>