Directed evolution of xylose specific transporters to facilitate glucose-xylose co-utilization.
A highly active xylose specific transporter without glucose inhibition is highly desirable in cost-effective production of biofuels from lignocellulosic biomass. However, currently available xylose specific transporters suffer from low overall activity and most are inhibited by glucose. In this study, we applied a directed evolution strategy to engineer the xylose specific transporter AN25 from Neurospora crassa with improved xylose transportation capacity. After four rounds of directed evolution using two different strategies, we obtained an AN25 mutant AN25-R4.18 with 43-fold improvement in terms of xylose transportation capacity while maintaining its high xylose specificity. In addition, glucose inhibition was almost completely eliminated in the final evolved mutant. We demonstrated that improved xylose transportation of AN25 mutants in the exponential growth phase led to significant improvement of xylose consumption in high cell-density fermentation. Finally, we showed that AN25 mutant AN25-R4.18 can enable relatively efficient glucose-xylose co-utilization in high concentrations of mixed sugars.
Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801.
Meng
M
Wang
School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801.
Chenzhao
C
Yu
Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801. zhao5@illinois.edu.|Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801. zhao5@illinois.edu.|Departments of Chemistry, Bioengineering, and Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801. zhao5@illinois.edu.
Huimin
H
Zhao
D019020:Directed Molecular Evolution; D005947:Glucose / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009492:Neurospora crassa / Q000235:genetics / Q000378:metabolism*; D014994:Xylose / Q000378:metabolism*
D026901:Membrane Transport Proteins; D050505:Mutant Proteins; D014994:Xylose; D005947:Glucose
directed evolution; glucose-xylose co-utilization; xylose specific transporter