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

Dawn T Eriksen,Pei Chiun Helen Hsieh,Patrick Lynn,Huimin Zhao

doi:10.1186/1475-2859-12-61

Abstract

BackgroundThe 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.ResultsThe 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)).ConclusionsBy 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.

Highlights

The optimization of metabolic pathways is critical for efficient and economical production of biofuels and specialty chemicals
Β-glucosidases hydrolyze cellobiose into two glucose monomers, which are subsequently fermented by the cell
The cellobiose is transported into the cell via the cellodextrin transporter and the intracellular phosphorylase cleaves the disaccharide with an inorganic phosphate, producing a glucose molecule and an α-glucose-1-phosphate, which is quickly metabolized

Summary

Introduction

The optimization of metabolic pathways is critical for efficient and economical production of biofuels and specialty chemicals. The first strategy involves cell-surface display of extracellular β-glucosidases [4,5,6,7,8] In this strategy, the cellobiose is hydrolyzed extracellularly and the glucose is transported into the cell and metabolized. The hydrolytic pathway is a third strategy, which involves heterologous expression of a cellodextrin transporter and an intracellular β-glucosidase [12,13,14]. The hydrolytic cellobiose utilization pathway has recently been investigated by our laboratory for optimization through combinatorial transcriptional engineering [15] Though this method and other transcriptional engineering techniques have been successful in optimizing metabolic pathways [15,16,17,18], optimal gene expression cannot overcome the inherent inefficiencies of the proteins within the pathway [19]. We sought to apply protein engineering strategies to improve the pathway performance

Methods

Results

Discussion

Conclusion