Multiomic fermentation with lignocellulosic hydrolysate and synthetic hydrolysate to study the effect of lignocellulose-derived inhibitors on an engineered Escherichia coli ethanologen for its conversion of lignocellulosic biomass to ethanol

Lignocellulose-derived inhibitors (Lignotoxins, or LTs) generate major barriers to efficient conversion of sugars to biofuels. The complex chemical composition of biomass hydrolysates, however, makes it difficult to identify cellular targets of these inhibitors and the mechanism of their effects on microbial sugar conversion. We have developed an approach to study these questions in hydrolysate prepared from AFEX-pretreated corn stover (ACSH), by devising a chemically defined synthetic hydrolysate (SynHv3) that largely replicates the properties of ACSH including a cocktail of LTs identified in ACSH. We used a multiomic dissection of the effects of SynH and LTs on an engineered Escherichia coli ethanologen to identify key stress responses in ACSH caused by LTs. We found that the SynHv3 allowed us to test the effects of LTs on glucose and xylose conversion, independent of other stress-inducing components. Specifically, we found that two most abundant phenolic amides in ACSH were the principal inhibitors of cell growth and xylose utilization in E. coli. Multiomic analysis indicated that SynHv3 + LTs largely recapitulates the growth and physiology of E. coli grown in ACSH. Using it, we identified 4 major stress regulons of high relevance to ACSH and LT-inhibition of xylose conversion, the AaeR, MarR/MarA, YqhC, and FrmR regulons. These stress responses were associated with a depletion of NADPH/NADH, inefficient ethanol synthesis, and pyruvate pooling.  We also used modified SynHv3 to study the inhibitory mechanism of LT on xylose utilization by comparative fermentation with SynHv3 containing different C5 and C6 sugars, such as arabinose and mannose.