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

Gavin Kurgan, Moses Onyeabor, Steven C. Holland, Eric Taylor, Aidan Schneider, Logan Kurgan, Tommy Billings, Xuan Wang

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Article numberkuab066
JournalJournal of Industrial Microbiology and Biotechnology
Volume49
Issue number2
DOIs
StatePublished - Mar 1 2022
Externally publishedYes

Keywords

  • Directed evolution
  • Lignocellulose
  • Sugar facilitator Glf

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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