Dataset Information

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

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.

SUBMITTER: Wang C

PROVIDER: S-EPMC5519586 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Publications

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

Wang Chengqiang C Li Yanwei Y Qiu Chenxi C Wang Shihao S Ma Jinjin J Shen Yu Y Zhang Qingzhu Q Du Binghai B Ding Yanqin Y Bao Xiaoming X

Frontiers in microbiology 20170721

Efficient and cost-effective bioethanol production from lignocellulosic materials requires co-fermentation of the main hydrolyzed sugars, including glucose, xylose, and L-arabinose. <i>Saccharomyces cerevisiae</i> 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-spe ...[more]

PMID: 28785254

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Project description:Shiga toxins (Stx1 and Stx2) are produced by E. coli O157:H7, which is a leading cause of foodborne illness. The A subunits of Stx1 (Stx1A) and Stx2 (Stx2A) are ribosome inactivating proteins (RIPs) that inhibit translation by removing an adenine from the highly conserved α-sarcin ricin loop (SRL) of the large rRNA. Here, we used mutagenesis in Saccharomyces cerevisiae to identify residues critical for cytotoxicity of Stx1A and Stx2A. The A subunits depurinated the SRL, inhibited translation and caused apoptotic-like cell death in yeast. Single mutations in Asn75, Tyr77, Glu167 and Arg176 reduced the cytotoxicity of both toxins around 10-fold. However, Asn75 and Tyr77 were more critical for the depurination activity of Stx2A, while Arg176 was more critical for the depurination activity of Stx1A. The crystal structures of the two proteins lack electron density for some surface loops, including one which is adjacent to the active site in both molecules. Modeling these loops changed neither the secondary nor the tertiary structures of the rest of the protein. Analysis of solvent accessible surface areas indicated that Asn75 and Tyr77 are more exposed in Stx2A, while Arg176 is more exposed in Stx1A, indicating that residues with higher surface exposure were more critical for enzymatic activity. Double mutations at Glu167 and Arg176 eliminated the depurination activity and cytotoxicity of both toxins. C-terminal deletions of A chains eliminated cytotoxicity of both toxins, but showed functional differences. Unlike Stx1A, cytotoxicity of Stx2A was lost before its ability to depurinate ribosomes. These results identify residues that affect enzymatic activity and cytotoxicity of Stx1A and Stx2A differently and demonstrate that the function of these residues can be differentiated in yeast. The extent of ribosome depurination and translation inhibition did not correlate with the extent of cell death, indicating that depurination of the SRL and inhibition of translation are not entirely responsible for cell death.

Dataset Information

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

Publications

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

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