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 E. 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.

Construction and characterization of a promoter library with varying strengths to enhance acetoin production from xylose in Serratia marcescens

“…The Glf sugar transporter protein from Z. mobilis efficiently imports xylose without requiring extra energy. This mutant (L445I) effectively increases the rate of xylose transport 23 . Gene synthesis was used to obtain the glf L445I target gene.…”

“…mutant (L445I) effectively increases the rate of xylose transport. 23 Gene synthesis was used to obtain the glf L445I target gene. The plasmid pBBR1MCS-P6-mKate2 served as a template to prepare a plasmid backbone with a strong endogenous promoter using PCR amplification with primers A-X-F and P6-R2.…”

“…This study aimed to identify a robust endogenous constitutive promoter from S. marcescens HBQA‐7 through transcriptome analysis and reporter gene assay. The identified promoter was then utilized for high‐level expression of the sugar transporter protein Glf L445I from Zymomonas mobilis , 23 along with α‐acetyl lactate synthase (AlsS) and α‐acetyl lactate decarboxylase (AlsD). This approach was adopted to enhance xylose utilization rate and improve acetoin production in the strain.…”

Engineering the Metabolic Landscape of Microorganisms for Lignocellulosic Conversion

“…To increase the economic feasibility of plant biomass conversion, all molecular fractions of hydrolysates should be suitable as substrates. Therefore, Kurgan et al [ 117 ] tested the capacity of major facilitator proteins (MFS) to be adapted through laboratory evolution to increase the diffusion of xylose into the cells. Direct evolution was used to increase xylose transport by Glf, a transmembrane sugar uniporter.…”

Characterization and Application of the Sugar Transporter Zmo0293 from Zymomonas mobilis

“…Furthermore, metabolic engineering approaches have enabled xylose and arabinose metabolism in Z. mobilis [ 24 , 25 ]. Recently, transporter engineering strategies have played an increasingly important role in enhancing carbohydrate uptake, eliminating CCR, and improving hydrolysate inhibitor tolerance [ 26 ]. Although the main sugar transporter Glf has been identified in Z. mobilis , more sugar transporters still need to be characterized, and it is advantageous to understand their regulatory mechanism and develop efficient transporter engineering.…”