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

摘要

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 .