Inhibitor tolerance and bioethanol fermentability of levoglucosan-utilizing Escherichia coli were enhanced by overexpression of stress-responsive gene ycfR: The proteomics-guided metabolic engineering

Pretreatment of lignocellulosic biomass is crucial for the release of biofermentable sugars for biofuels production, which could greatly alleviate the burgeoning environment and energy crisis caused by the massive usage of traditional fossil fuels. Pyrolysis is a cost-saving pretreatment process that can readily decompose biomass into levoglucosan, a promising anhydrosugar; however, many undesired toxic compounds inhibitory to downstream microbial fermentation are also generated during the pyrolysis, immensely impeding the bioconversion of levoglucosan-containing pyrolysate. Here, we took the first insight into the proteomic responses of a levoglucosan-utilizing and ethanol-producing Escherichia coli to three representative biomass-derived inhibitors, identifying large amounts of differentially expressed proteins (DEPs) that could guide the downstream metabolic engineering for the development of inhibitor-resistant strains. Fifteen up- and eight down-regulated DEPs were further identified as the biomarker stress-responsive proteins candidate for cellular tolerance to multiple inhibitors. Among these biomarker proteins, YcfR exhibiting the highest expression fold-change level was chosen as the target of overexpression to validate proteomics results and develop robust strains with enhanced inhibitor tolerance and fermentation performance. Finally, based on four plasmid-borne genes encoding the levoglucosan kinase, pyruvate decarboxylase, alcohol dehydrogenase, and protein YcfR, a new recombinant strain E. coli LGE-ycfR was successfully created, showing much higher acetic acid-, furfural-, and phenol-tolerance levels compared to the control without overexpression of ycfR. The specific growth rate, final cell density, ethanol concentration, ethanol productivity, and levoglucosan consumption rate of the recombinant were also remarkably improved. From the proteomics-guided metabolic engineering and phenotypic observations, we for the first time corroborated that YcfR is a stress-induced protein responsive to multiple biomass-derived inhibitors, and also developed an inhibitors-resistant strain that could produce bioethanol from levoglucosan in the presence of inhibitors of relatively high concentration. The newly developed E. coli LGE-ycfR strain that could eliminate the commonly-used costly detoxicification processes, is of great potential for the in situ cost-effective bioethanol production from the biomass-derived pyrolytic substrates.