Development of a GIN11/FRT-based Multiple-gene Integration Technique Affording Inhibitor-tolerant, Hemicellulolytic, Xylose-utilizing Abilities to Industrial Saccharomyces Cerevisiae Strains for Ethanol Production from Undetoxified Lignocellulosic...

Background: 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.

Results: 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.

Conclusions: 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.