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Directed evolution of Zymomonas mobilis sugar facilitator Glf to overcome glucose inhibition.

ABSTRACT: Cellular import of D-xylose, the second most abundant sugar in typical lignocellulosic biomass, has been evidenced to be an energy-depriving process in bacterial biocatalysts. The sugar facilitator of Zymomonas mobilis, Glf, is capable of importing xylose at high rates without extra energy input, but is inhibited by D-glucose (the primary biomass sugar), potentially limiting the utility of this transporter for fermentation of sugar mixtures derived from lignocellulose. In this work we developed an Escherichia coli platform strain deficient in glucose and xylose transport to facilitate directed evolution of Glf to overcome glucose inhibition. Using this platform, we isolated nine Glf variants created by both random and site-saturation mutagenesis with increased xylose utilization rates ranging from 4.8-fold to 13-fold relative to wild-type Glf when fermenting 100 g l-1 glucose-xylose mixtures. Diverse point mutations such as A165M and L445I were discovered leading to released glucose inhibition. Most of these mutations likely alter sugar coordinating pocket for the 6-hydroxymethyl group of D-glucose. These discovered glucose-resistant Glf variants can be potentially used as energy-conservative alternatives to the native sugar transport systems of bacterial biocatalysts for fermentation of lignocellulose-derived sugars.

SUBMITTER: Kurgan G

PROVIDER: S-EPMC9118996 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Directed evolution of Zymomonas mobilis sugar facilitator Glf to overcome glucose inhibition.

Kurgan Gavin G Onyeabor Moses M Holland Steven C SC Taylor Eric E Schneider Aidan A Kurgan Logan L Billings Tommy T Wang Xuan X

Journal of industrial microbiology & biotechnology 20220401 2

Cellular import of D-xylose, the second most abundant sugar in typical lignocellulosic biomass, has been evidenced to be an energy-depriving process in bacterial biocatalysts. The sugar facilitator of Zymomonas mobilis, Glf, is capable of importing xylose at high rates without extra energy input, but is inhibited by D-glucose (the primary biomass sugar), potentially limiting the utility of this transporter for fermentation of sugar mixtures derived from lignocellulose. In this work we developed ...[more]

PMID: 34529081

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Project description:The excessive consumption of sugars can cause health issues. Different strategies have been developed to reduce sugars in the diets. However, sugars in fruits and commercial products may be difficult to reduce, limiting their usage among certain populations of people. Zymomonas mobilis is a generally recognized as safe (GRAS) probiotic bacterium with the capability to produce levan-type prebiotics, and thrives in high-sugar environments with unique characteristics to be developed for lignocellulosic biofuel and biochemical production. In this study, the sugar reduction capabilities of Z. mobilis ZM4 were examined using two fruits of pear and persimmon and three high-sugar-content commercial products of two pear pastes (PPs) and one Chinese traditional wine (CTW). Our results demonstrated that Z. mobilis ZM4 can utilize sugars in fruits with about 20 g/L ethanol and less than 5 g/L sorbitol produced within 22 h using pears, and about 45 g/L ethanol and 30 g/L sorbitol produced within 34 h using persimmons. When PPs made from pears were used, Z. mobilis can utilize nearly all glucose (ca. 60 g/L) and most fructose (110 g/L) within 100 h with 40 ~ 60 g/L ethanol and more than 20 g/L sorbitol produced resulting in a final sorbitol concentration above 80 g/L. In the high-sugar-content alcoholic Chinese traditional wine, which contains mostly glucose and ethanol, Z. mobilis can reduce nearly all sugars with about 30 g/L ethanol produced, resulting in a final ethanol above 90 g/L. The ethanol yield and percentage yield of Z. mobilis in 50 ~ 60% CTW were 0.44 ~ 0.50 g/g and 86 ~ 97%, respectively, which are close to its theoretical yields-especially in 60% CTW. Although the ethanol yield and percentage yield in PPs were lower than those in CTW, they were similar to those in fruits of pears and persimmons with an ethanol yield around 0.30 ~ 0.37 g/g and ethanol percentage yield around 60 ~ 72%, which could be due to the formation of sorbitol and/or levan in the presence of both glucose and fructose. Our study also compared the fermentation performance of the classical ethanologenic yeast Saccharomyces cerevisiae BY4743 to Z. mobilis, with results suggesting that Z. mobilis ZM4 had better performance than that of yeast S. cerevisiae BY4743 given a higher sugar conversion rate and ethanol yield for sugar reduction. This work thus laid a foundation for utilizing the advantages of Z. mobilis in the food industry to reduce sugar concentrations or potentially produce alcoholic prebiotic beverages.Supplementary informationThe online version contains supplementary material available at 10.1186/s40643-021-00467-2.

Project description:BackgroundDuring the pretreatment of biomass feedstocks and subsequent conditioning prior to saccharification, many toxic compounds are produced or introduced which inhibit microbial growth and in many cases, production of ethanol. An understanding of the toxic effects of compounds found in hydrolysate is critical to improving sugar utilization and ethanol yields in the fermentation process. In this study, we established a useful tool for surveying hydrolysate toxicity by measuring growth rates in the presence of toxic compounds, and examined the effects of selected model inhibitors of aldehydes, organic and inorganic acids (along with various cations), and alcohols on growth of Zymomonas mobilis 8b (a ZM4 derivative) using glucose or xylose as the carbon source.ResultsToxicity strongly correlated to hydrophobicity in Z. mobilis, which has been observed in Escherichia coli and Saccharomyces cerevisiae for aldehydes and with some exceptions, organic acids. We observed Z. mobilis 8b to be more tolerant to organic acids than previously reported, although the carbon source and growth conditions play a role in tolerance. Growth in xylose was profoundly inhibited by monocarboxylic organic acids compared to growth in glucose, whereas dicarboxylic acids demonstrated little or no effects on growth rate in either substrate. Furthermore, cations can be ranked in order of their toxicity, Ca++ > > Na+ > NH4+ > K+. HMF (5-hydroxymethylfurfural), furfural and acetate, which were observed to contribute to inhibition of Z. mobilis growth in dilute acid pretreated corn stover hydrolysate, do not interact in a synergistic manner in combination. We provide further evidence that Z. mobilis 8b is capable of converting the aldehydes furfural, vanillin, 4-hydroxybenzaldehyde and to some extent syringaldehyde to their alcohol forms (furfuryl, vanillyl, 4-hydroxybenzyl and syringyl alcohol) during fermentation.ConclusionsSeveral key findings in this report provide a mechanism for predicting toxic contributions of inhibitory components of hydrolysate and provide guidance for potential process development, along with potential future strain improvement and tolerance strategies.

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Directed evolution of Zymomonas mobilis sugar facilitator Glf to overcome glucose inhibition.

Publications

Directed evolution of Zymomonas mobilis sugar facilitator Glf to overcome glucose inhibition.

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