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 well-studied 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 by-product 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.

Stress modulation as a means to improve yeasts for lignocellulose bioconversion

“…As such, phenolic compounds have been associated with (i) increased membrane fluidity, (ii) increased cell leakage, (iii) disturbed ion and sugar transport, (iv) DNA damage, and (v) disrupting biological membrane integrity. More recently, quinone derivatives such as p-benzoquinone, hydroquinone, and methoxyhydroquinone have also been implicated as microbial inhibitors present in pretreated lignocellulose biomass (Cavka et al 2015;Jönsson and Martín 2016;Yan et al 2019). These compounds result from the oxidation of lignin-derived phenolic compounds and strongly inhibit the fermentation ability of S. cerevisiae.…”

“…These compounds result from the oxidation of lignin-derived phenolic compounds and strongly inhibit the fermentation ability of S. cerevisiae. In particular, p-benzoquinone has been documented to completely inhibit various ethanologens including S. cerevisiae at concentrations between 20 and 200 mg/L (Larsson et al 2000;Cavka et al 2015;Yan et al 2019). In yeast, p-benzoquinone can induce (i) ROS formation and accumulation and (ii) DNA damage (Yan et al 2019).…”

Engineering of Saccharomyces cerevisiae as a consolidated bioprocessing host to produce cellulosic ethanol: Recent advancements and current challenges

“…However, the extent of inhibition depends upon the type of acids (formic acid > levulinic acid > acetic acid) present in the lignocellulosic hydrolysates. Finally, phenolic compounds consisting of several complex functional groups like aldehyde and ketone strongly affect the electro-chemical gradient of the cellular membranes causing impaired membrane functioning (Fig 2C) (Gu et al, 2019;Yan et al, 2019). S. cerevisiae possess a natural ability to metabolize some of these compounds in low concentration, for example, HMF and furfural can be reduced in a NAD(P)H dependent manner to their less toxic form (alcohols) under aerobic and anaerobic conditions; similarly, some of the phenolic compounds can be assimilated by native PAD1 gene catalysing the decarboxylation of phenyl acrylic acids such as ferulic acid, cinnamic acid etc.…”

Origin, Impact and Control of Lignocellulosic Inhibitors in Bioethanol Production—A Review

“…Some examples are benzyl alcohol, cinnamaldehyde and benzoic acid [20,23]. The last subgroup is benzoquinones, which contains for example inhibitors such as p-Benzoquinone and 2,6-Dimethoxybenzoquinone [25].…”

“…These are lignin-derived inhibitors, which can disable growth and metabolism of microorganisms. In the study by Yan et al [25], a degradation ability of Zymomonas mobilis was discovered, which enabled a reduction of p-benzoquinone to hydroquinone. Additionally, five key genes were detected and overexpressed, which subsequently increased Z. mobilis tolerance against p-benzoquinone [25].…”