New genotypes of industrial yeast Saccharomyces cerevisiae engineered with YXI and heterologous xylose transporters improve xylose utilization and ethanol production

Genetically engineered Saccharomyces cerevisiae strains for renewable biofuels conversion often show limited xylose utilization and no satisfactory strain is currently available for sustainable cellulosic ethanol production. Using a genetically engineered industrial yeast strain NRRL Y-50049-YXI with a chromosomally integrated unique codon-optimized xylose isomerase as a host, we developed six new genotypes using heterologous xylose transporter genes from Scheffersomyces stipitis namely Y-50049-YXI-XUT4, -XUT5, -XUT6, -XUT7, -RGT2, and -SUT4, respectively. While a functional YXI is necessary to enable xylose utilization for Y-50049, introduction of the heterologous xylose transporter genes further improved cell growth and fermentation for all newly developed genotypes. Under aerobic conditions, all new genotypes displayed significant improvement of cell growth and xylose consumption. Among which, Genotypes Y-50049-YXI-RGT2, -XUT7, and -XUT6 showed a volumetric consumption rate ranging from 0.399 to 0.535 g/L/h that is comparable with the native xylose utilizing yeast Scheffersomyces stipitis. Under oxygen-limited fermentation conditions, genotypes Y-50049-YXI-XUT7, -RGT2, and -SUT4 displayed approximately 50% increase of ethanol productivity and ethanol yield compared with their parental strain. We found RGT2 is closely related to GXS1, a glucose-xylose symporter from Candida intermedia, and functioned well as a xylose transporter gene. No obvious inhibition of xylose utilization by glucose was observed in the mixed sugars of glucose and xylose for all genotypes examined. Enhanced expression of YXI by most characterized xylose transporters suggested Y-50049-YXI as a promising host to improve xylose utilization for the next generation biocatalyst development.