Abstract
Bioethanol 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. A 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. The 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.
Highlights
Bioethanol produced by the yeast Saccharomyces cerevisiae is currently one of the most promising alternatives to conventional transport fuels
Construction of recombinant yeast strains An industrial diploid strain, sun049, was previously selected as one of superior strains for high-temperature xylose fermentation after the implementation of xylose assimilation pathway consisting of S. stipitis xylose reductase (XR) and xylitol dehydrogenase (XDH) and overexpression of endogenous XK through genetic engineering [16], which is used for the parent strain for the multiple-gene integration in this study
Expression units for genes of interest were integrated by homologous recombination into inter-ORF regions in S. cerevisiae genomic DNA with GIN11 controlled by the GAL1 promoter and a marker gene such as G418r and natMX, which are located between FLP recombinase recognition target (FRT) sequences
Summary
Bioethanol 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. The utilization of lignocellulosic biomass for the production of fuels has received particular attention in recent years. Bioethanol is produced by the fermentation of biomass by the yeast Saccharomyces cerevisiae and is currently one of the most promising alternatives to conventional transport fuels. S. cerevisiae is a superior ethanol producer with demonstrated fast sugar consumption, high ethanol yield from glucose, and high resistance to ethanol. Lignocellulosic biomass such as corn stover, rice and wheat straw, sugarcane bagasse, wood chips and other agricultural residues comprise mainly cellulose, hemicelluloses and lignin. If the economic success of lignocellulosic ethanol is to be realized, both cellulose and hemicelluloses must be utilized for ethanol production
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