Identification of Important Amino Acids in Gal2p for Improving the L-arabinose Transport and Metabolism in Saccharomyces cerevisiae.

Chengqiang Wang,Yu Shen,Jinjin Ma,Yanqin Ding,Binghai Du,Chenxi Qiu,Yanwei Li,Qingzhu Zhang,Shihao Wang,Xiaoming Bao

doi:10.3389/fmicb.2017.01391

Chengqiang Wang, Yu Shen + Show 8 more

Open Access

https://doi.org/10.3389/fmicb.2017.01391

Copy DOI

Abstract

Efficient and cost-effective bioethanol production from lignocellulosic materials requires co-fermentation of the main hydrolyzed sugars, including glucose, xylose, and L-arabinose. Saccharomyces cerevisiae is a glucose-fermenting yeast that is traditionally used for ethanol production. Fermentation of L-arabinose is also possible after metabolic engineering. Transport into the cell is the first and rate-limiting step for L-arabinose metabolism. The galactose permease, Gal2p, is a non-specific, endogenous monosaccharide transporter that has been shown to transport L-arabinose. However, Gal2p-mediated transport of L-arabinose occurs at a low efficiency. In this study, homologous modeling and L-arabinose docking were used to predict amino acids in Gal2p that are crucial for L-arabinose transport. Nine amino acid residues in Gal2p were identified and were the focus for site-directed mutagenesis. In the Gal2p transport-deficient chassis cells, the capacity for L-arabinose transport of the different Gal2p mutants was compared by testing growth rates using L-arabinose as the sole carbon source. Almost all the tested mutations affected L-arabinose transport capacity. Among them, F85 is a unique site. The F85S, F85G, F85C, and F85T point mutations significantly increased L-arabinose transport activities, while, the F85E and F85R mutations decreased L-arabinose transport activities compared to the Gal2p-expressing wild-type strain. These results verified F85 as a key residue in L-arabinose transport. The F85S mutation, having the most significant effect, elevated the exponential growth rate by 40%. The F85S mutation also improved xylose transport efficiency and weakened the glucose transport preference. Overall, enhancing the L-arabinose transport capacity further improved the L-arabinose metabolism of engineered S. cerevisiae.

Highlights

Fuel ethanol is an important renewable energy source, and there is a growing demand for the production of this fuel (Farrell et al, 2006; Mabee, 2007)
To construct L-arabinose transport-deficient chassis and study the L-arabinose transport mechanism, the endogenous and highly efficient L-arabinose transporter Gal2 of BSW4AP was knocked out using the KanMX4 marker to generate the strain BSW5AP
It was clear that BSW5AP-A lost the ability to grow on L-arabinose

Summary

Introduction

Fuel ethanol is an important renewable energy source, and there is a growing demand for the production of this fuel (Farrell et al, 2006; Mabee, 2007). Future large-scale production of fuel ethanol will need lignocellulosic materials, which are renewable and abundant, to replace sugar and grain (Hahn-Hägerdal et al, 2006). Key Residues for Gal Transport requires co-fermentation of all the main hydrolyzed sugars from lignocellulose, including glucose, xylose, and L-arabinose (Wisselink et al, 2007; Seiboth and Metz, 2011; Wang et al, 2017). Saccharomyces cerevisiae is a traditional ethanol production strain that ferments glucose and could ferment xylose and L-arabinose by introducing the initial metabolic pathways (Kuyper et al, 2004; Hahn-Hägerdal et al, 2007; Wisselink et al, 2009; Peng et al, 2012; Kim et al, 2013; Wang et al, 2013). Transport into the cell is the first step and one of the ratelimiting steps for L-arabinose utilization, and the transport efficiency needs to be increased (Subtil and Boles, 2012; Shin et al, 2015)

Methods

Results

Discussion

Conclusion