Mechanism of tolerance to the lignin-derived inhibitor p-benzoquinone and metabolic modification of biorefinery fermentation strains

Abstract

p-Benzoquinone (BQ) is a lignin-derived inhibitor of biorefinery fermentation strains produced during pretreatment of lignocellulose. Unlike the wellstudied inhibitors furan aldehydes, weak acids, and phenolics, the inhibitory properties of BQ, the microbial tolerance mechanism, and the detoxification strategy for this inhibitor have not been clearly elucidated. Here, BQ was identified as a byproduct generated during acid pretreatment of various lignocellulose feedstocks, including corn stover, wheat straw, rice straw, tobacco stem, sunflower stem, and corncob residue. BQ at 20 to 200 mg/liter severely inhibited the cell growth and fermentability of various bacteria and yeast strains used in biorefinery fermentations. The BQ tolerance of the strains was found to be closely related to their capacity to convert BQ to nontoxic hydroquinone (HQ). To identify the key genes responsible for BQ tolerance, transcription levels of 20 genes potentially involved in the degradation of BQ in Zymomonas mobilis were investigated using real-time quantitative PCR in BQ-treated cells. One oxidoreductase gene, one hydroxylase gene, three reductase genes, and three dehydrogenase genes were found to be responsible for the conversion of BQ to HQ. Overexpression of the five key genes in Z. mobilis (ZMO1696, ZMO1949, ZMO1576, ZMO1984, and ZMO1399) accelerated its cell growth and cellulosic ethanol production in BQ-containing medium and lignocellulose hydrolysates. IMPORTANCE This study advances our understanding of BQ inhibition behavior and the mechanism of microbial tolerance to this inhibitor and identifies the key genes responsible for BQ detoxification. The insights here into BQ toxicity and tolerance provide the basis for future synthetic biology to engineer industrial fermentation strains with enhanced BQ tolerance.