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Open Access Highly Accessed Research

Directed evolution of a cellobiose utilization pathway in Saccharomyces cerevisiae by simultaneously engineering multiple proteins

Dawn T Eriksen12, Pei Chiun Helen Hsieh1, Patrick Lynn3 and Huimin Zhao1234*

Author Affiliations

1 Department of Chemical and Biomolecular Engineering, Institute for Genomic Biology, University of Illinois-Urbana Champaign, Urbana, IL 61801, USA

2 Energy Biosciences Institute, Urbana, IL 61801, USA

3 Department of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA

4 Departments of Chemistry, Biochemistry, and Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA

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Microbial Cell Factories 2013, 12:61  doi:10.1186/1475-2859-12-61

Published: 26 June 2013

Abstract

Background

The optimization of metabolic pathways is critical for efficient and economical production of biofuels and specialty chemicals. One such significant pathway is the cellobiose utilization pathway, identified as a promising route in biomass utilization. Here we describe the optimization of cellobiose consumption and ethanol productivity by simultaneously engineering both proteins of the pathway, the β-glucosidase (gh1-1) and the cellodextrin transporter (cdt-1), in an example of pathway engineering through directed evolution.

Results

The improved pathway was assessed based on the strain specific growth rate on cellobiose, with the final mutant exhibiting a 47% increase over the wild-type pathway. Metabolite analysis of the engineered pathway identified a 49% increase in cellobiose consumption (1.78 to 2.65 g cellobiose/(L · h)) and a 64% increase in ethanol productivity (0.611 to 1.00 g ethanol/(L · h)).

Conclusions

By simultaneously engineering multiple proteins in the pathway, cellobiose utilization in S. cerevisiae was improved. This optimization can be generally applied to other metabolic pathways, provided a selection/screening method is available for the desired phenotype. The improved in vivo cellobiose utilization demonstrated here could help to decrease the in vitro enzyme load in biomass pretreatment, ultimately contributing to a reduction in the high cost of biofuel production.

Keywords:
Cellobiose utilization; β-glucosidase; Cellodextrin transporter; Directed evolution; Protein engineering; Pathway engineering; Pathway optimization; Pathway libraries