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

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...

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Main Authors: Chengqiang Wang, Yanwei Li, Chenxi Qiu, Shihao Wang, Jinjin Ma, Yu Shen, Qingzhu Zhang, Binghai Du, Yanqin Ding, Xiaoming Bao
Format: Article
Language:English
Published: Frontiers Media S.A. 2017-07-01
Series:Frontiers in Microbiology
Subjects:
Online Access:http://journal.frontiersin.org/article/10.3389/fmicb.2017.01391/full
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spelling doaj-3ea7a04aac844e6cb773e4e08043ee972020-11-24T22:37:44ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2017-07-01810.3389/fmicb.2017.01391279957Identification of Important Amino Acids in Gal2p for Improving the L-arabinose Transport and Metabolism in Saccharomyces cerevisiaeChengqiang Wang0Chengqiang Wang1Yanwei Li2Chenxi Qiu3Chenxi Qiu4Shihao Wang5Shihao Wang6Jinjin Ma7Yu Shen8Qingzhu Zhang9Binghai Du10Yanqin Ding11Xiaoming Bao12Xiaoming Bao13College of Life Sciences/Shandong Key Laboratory of Agricultural Microbiology, Shandong Agricultural UniversityTai’an, ChinaThe State Key Laboratory of Microbial Technology/Environment Research Institute, Shandong UniversityJinan, ChinaThe State Key Laboratory of Microbial Technology/Environment Research Institute, Shandong UniversityJinan, ChinaCollege of Life Sciences/Shandong Key Laboratory of Agricultural Microbiology, Shandong Agricultural UniversityTai’an, ChinaThe State Key Laboratory of Microbial Technology/Environment Research Institute, Shandong UniversityJinan, ChinaCollege of Life Sciences/Shandong Key Laboratory of Agricultural Microbiology, Shandong Agricultural UniversityTai’an, ChinaThe State Key Laboratory of Microbial Technology/Environment Research Institute, Shandong UniversityJinan, ChinaCollege of Life Sciences/Shandong Key Laboratory of Agricultural Microbiology, Shandong Agricultural UniversityTai’an, ChinaThe State Key Laboratory of Microbial Technology/Environment Research Institute, Shandong UniversityJinan, ChinaThe State Key Laboratory of Microbial Technology/Environment Research Institute, Shandong UniversityJinan, ChinaCollege of Life Sciences/Shandong Key Laboratory of Agricultural Microbiology, Shandong Agricultural UniversityTai’an, ChinaCollege of Life Sciences/Shandong Key Laboratory of Agricultural Microbiology, Shandong Agricultural UniversityTai’an, ChinaThe State Key Laboratory of Microbial Technology/Environment Research Institute, Shandong UniversityJinan, ChinaCollege of Bioengineering, Qilu University of TechnologyJinan, ChinaEfficient 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.http://journal.frontiersin.org/article/10.3389/fmicb.2017.01391/fullL-arabinose transportGal2psite-directed mutagenesiskey residuemetabolismbudding yeast
collection DOAJ
language English
format Article
sources DOAJ
author Chengqiang Wang
Chengqiang Wang
Yanwei Li
Chenxi Qiu
Chenxi Qiu
Shihao Wang
Shihao Wang
Jinjin Ma
Yu Shen
Qingzhu Zhang
Binghai Du
Yanqin Ding
Xiaoming Bao
Xiaoming Bao
spellingShingle Chengqiang Wang
Chengqiang Wang
Yanwei Li
Chenxi Qiu
Chenxi Qiu
Shihao Wang
Shihao Wang
Jinjin Ma
Yu Shen
Qingzhu Zhang
Binghai Du
Yanqin Ding
Xiaoming Bao
Xiaoming Bao
Identification of Important Amino Acids in Gal2p for Improving the L-arabinose Transport and Metabolism in Saccharomyces cerevisiae
Frontiers in Microbiology
L-arabinose transport
Gal2p
site-directed mutagenesis
key residue
metabolism
budding yeast
author_facet Chengqiang Wang
Chengqiang Wang
Yanwei Li
Chenxi Qiu
Chenxi Qiu
Shihao Wang
Shihao Wang
Jinjin Ma
Yu Shen
Qingzhu Zhang
Binghai Du
Yanqin Ding
Xiaoming Bao
Xiaoming Bao
author_sort Chengqiang Wang
title Identification of Important Amino Acids in Gal2p for Improving the L-arabinose Transport and Metabolism in Saccharomyces cerevisiae
title_short Identification of Important Amino Acids in Gal2p for Improving the L-arabinose Transport and Metabolism in Saccharomyces cerevisiae
title_full Identification of Important Amino Acids in Gal2p for Improving the L-arabinose Transport and Metabolism in Saccharomyces cerevisiae
title_fullStr Identification of Important Amino Acids in Gal2p for Improving the L-arabinose Transport and Metabolism in Saccharomyces cerevisiae
title_full_unstemmed Identification of Important Amino Acids in Gal2p for Improving the L-arabinose Transport and Metabolism in Saccharomyces cerevisiae
title_sort identification of important amino acids in gal2p for improving the l-arabinose transport and metabolism in saccharomyces cerevisiae
publisher Frontiers Media S.A.
series Frontiers in Microbiology
issn 1664-302X
publishDate 2017-07-01
description 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.
topic L-arabinose transport
Gal2p
site-directed mutagenesis
key residue
metabolism
budding yeast
url http://journal.frontiersin.org/article/10.3389/fmicb.2017.01391/full
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