Development of a GIN11/FRT-based multiple-gene integration technique affording inhibitor-tolerant, hemicellulolytic, xylose-utilizing abilities to industrial Saccharomyces cerevisiaestrains for ethanol production from undetoxified lignocellulosic hemicelluloses

Microbial Cell Factories

Research

Yoshimi Hori¹  Takatoshi Sakamoto¹  Akihiko Kondo²  Tomohisa Hasunuma³  Haruyo Hatanaka⁴  Misa Ochiai⁴ 

[1] Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, 657-8501, Nada, Kobe, Japan;Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, 657-8501, Nada, Kobe, Japan;Biomass Engineering Program, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Kanagawa, Japan;Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, 657-8501, Nada, Kobe, Japan;Suntory Research Center, 1-1-1 Wakayamadai, Shimamoto-cho, Osaka, 618-8503, Mishima-gun, Japan;

关键词: Bioethanol;    Cell surface display;    Inhibitor tolerance;    Lignocellulose;    Maker recycling;    Saccharomyces cerevisiae;   

DOI  :  10.1186/s12934-014-0145-9

 received in 2014-07-29, accepted in 2014-10-03,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundBioethanol produced by the yeast Saccharomyces cerevisiae is currently one of the most promising alternatives to conventional transport fuels. Lignocellulosic hemicelluloses obtained after hydrothermal pretreatment are important feedstock for bioethanol production. However, hemicellulosic materials cannot be directly fermented by yeast: xylan backbone of hemicelluloses must first be hydrolyzed by heterologous hemicellulases to release xylose, and the yeast must then ferment xylose in the presence of fermentation inhibitors generated during the pretreatment.ResultsA GIN11/FRT-based multiple-gene integration system was developed for introducing multiple functions into the recombinant S. cerevisiae strains engineered with the xylose metabolic pathway. Antibiotic markers were efficiently recycled by a novel counter selection strategy using galactose-induced expression of both FLP recombinase gene and GIN11 flanked by FLP recombinase recognition target (FRT) sequences. Nine genes were functionally expressed in an industrial diploid strain of S. cerevisiae: endoxylanase gene from Trichoderma reesei, xylosidase gene from Aspergillus oryzae, β-glucosidase gene from Aspergillus aculeatus, xylose reductase and xylitol dehydrogenase genes from Scheffersomyces stipitis, and XKS1, TAL1, FDH1 and ADH1 variant from S. cerevisiae. The genes were introduced using the homozygous integration system and afforded hemicellulolytic, xylose-assimilating and inhibitor-tolerant abilities to the strain. The engineered yeast strain demonstrated 2.7-fold higher ethanol titer from hemicellulosic material than a xylose-assimilating yeast strain. Furthermore, hemicellulolytic enzymes displayed on the yeast cell surface hydrolyzed hemicelluloses that were not hydrolyzed by a commercial enzyme, leading to increased sugar utilization for improved ethanol production.ConclusionsThe multifunctional yeast strain, developed using a GIN11/FRT-based marker recycling system, achieved direct conversion of hemicellulosic biomass to ethanol without the addition of exogenous hemicellulolytic enzymes. No detoxification processes were required. The multiple-gene integration technique is a powerful approach for introducing and improving the biomass fermentation ability of industrial diploid S. cerevisiae strains.

【 授权许可】

Unknown   
© Hasunuma et al.; licensee BioMed Central Ltd. 2014. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.