Yeast Scheffersomyces Stipitis Research Articles

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.

Immobilization of Scheffersomyces stipitis cells with calcium alginate beads: A sustainable method for hemicellulosic ethanol production from sugarcane bagasse hydrolysate

Lignocellulosic ethanol has shown promising alternative to gasoline however expensive and cumbersome bioprocessing limits the commercialization of biofuels. The major impediment toward the economic ethanol production is the bioconversion of sugars into ethanol via microbial fermentation. Application of immobilized cells in fermentation of hemicellulosic hydrolysate could minimize the ethanol production cost. This study evaluated the conditions for cell immobilization for the yeast Scheffersomyces stipitis NRRL Y-7124 by the method of entrapment in calcium alginate gel. A full factorial design (23) was designed to investigate the effect of three process variables i.e. sodium alginate concentration (1.0, 1.5 and 2.0%), calcium chloride concentration (0.1, 0.15 and 0.2 M) and conditioning time (12, 18 and 24 h). Twelve numbers of experiments were performed with central points in quadruplicates. The range of ethanol production in all experiments was observed from 4.88 g/L (Yp/s, 0.16 g/g) to 9.9 g/L ethanol (Yp/s, 0.29 g/g). Statistical analysis revealed that immobilization conditions (2.0% sodium alginate concentration, 0.1M calcium chloride concentration and 12 h conditioning time) were responsible for high stability of immobilized cells which in-turn enabled the maximum ethanol production (7.2 g/L, Yp/s, 0.26 g/g) from hemicellulosic hydrolysate of sugarcane bagasse

Enhanced ethanol production from wheat straw by integrated storage and pre-treatment (ISP)

Integrated storage and pre-treatment (ISP) combines biopreservation of moist material under airtight conditions and pre-treatment. Moist wheat straw was inoculated with the biocontrol yeast Wickerhamomyces anomalus, the xylan degrading yeast Scheffersomyces stipitis or a co-culture of both. The samples and non-inoculated controls were stored at 4 or 15°C. The non-inoculated controls were heavily contaminated with moulds, in contrast to the samples inoculated with W. anomalus or S. stipitis. These two yeasts were able to grow on wheat straw as sole source of nutrients. When ethanol was produced from moist wheat straw stored for four weeks at 4°C with S. stipitis, an up to 40% enhanced yield (final yield 0.15g ethanol per g straw dry weight) was obtained compared to a dry sample (0.107g/g). In all other moist samples, stored for four weeks at 4°C or 15°C, 6–35% higher yields were obtained. Thus, energy efficient bio-preservation can improve the pre-treatment efficiency for lignocellulose biomass, which is a critical bottleneck in its conversion to biofuels.

Reconstruction and analysis of a genome-scale metabolic model for Scheffersomyces stipitis

BackgroundFermentation of xylose, the major component in hemicellulose, is essential for economic conversion of lignocellulosic biomass to fuels and chemicals. The yeast Scheffersomyces stipitis (formerly known as Pichia stipitis) has the highest known native capacity for xylose fermentation and possesses several genes for lignocellulose bioconversion in its genome. Understanding the metabolism of this yeast at a global scale, by reconstructing the genome scale metabolic model, is essential for manipulating its metabolic capabilities and for successful transfer of its capabilities to other industrial microbes.ResultsWe present a genome-scale metabolic model for Scheffersomyces stipitis, a native xylose utilizing yeast. The model was reconstructed based on genome sequence annotation, detailed experimental investigation and known yeast physiology. Macromolecular composition of Scheffersomyces stipitis biomass was estimated experimentally and its ability to grow on different carbon, nitrogen, sulphur and phosphorus sources was determined by phenotype microarrays. The compartmentalized model, developed based on an iterative procedure, accounted for 814 genes, 1371 reactions, and 971 metabolites. In silico computed growth rates were compared with high-throughput phenotyping data and the model could predict the qualitative outcomes in 74% of substrates investigated. Model simulations were used to identify the biosynthetic requirements for anaerobic growth of Scheffersomyces stipitis on glucose and the results were validated with published literature. The bottlenecks in Scheffersomyces stipitis metabolic network for xylose uptake and nucleotide cofactor recycling were identified by in silico flux variability analysis. The scope of the model in enhancing the mechanistic understanding of microbial metabolism is demonstrated by identifying a mechanism for mitochondrial respiration and oxidative phosphorylation.ConclusionThe genome-scale metabolic model developed for Scheffersomyces stipitis successfully predicted substrate utilization and anaerobic growth requirements. Useful insights were drawn on xylose metabolism, cofactor recycling and mechanism of mitochondrial respiration from model simulations. These insights can be applied for efficient xylose utilization and cofactor recycling in other industrial microorganisms. The developed model forms a basis for rational analysis and design of Scheffersomyces stipitis metabolic network for the production of fuels and chemicals from lignocellulosic biomass.

RNA-Seq of the xylose-fermenting yeast Scheffersomyces stipitis cultivated in glucose or xylose

Xylose is the second most abundant lignocellulosic component besides glucose, but it cannot be fermented by the widely used ethanol-producing yeast Saccharomyces cerevisiae. The yeast Scheffersomyces stipitis, however, is well known for its high native capacity to ferment xylose. Here, we applied next-generation sequencing technology for RNA (RNA-Seq) to generate two high-resolution transcriptional maps of the S. stipitis genome when this yeast was grown using glucose or xylose as the sole carbon source. RNA-Seq revealed that 5,176 of 5,816 annotated open reading frames had a uniform transcription and that 214 open reading frames were differentially transcribed. Differential expression analysis showed that, compared with other biological processes, carbohydrate metabolism and oxidation-reduction reactions were highly enhanced in yeast grown on xylose. Measurement of metabolic indicators of fermentation showed that, in yeast grown on xylose, the concentrations of cysteine and ornithine were twofold higher and the concentrations of unsaturated fatty acids were also increased. Analysis of metabolic profiles coincided with analysis of certain differentially expressed genes involved in metabolisms of amino acid and fatty acid. In addition, we predicted protein-protein interactions of S. stipitis through integration of gene orthology and gene expression. Further analysis of metabolic and protein-protein interactions networks through integration of transcriptional and metabolic profiles predicted correlations of genes involved in glycolysis, the tricarboxylic acid cycle, gluconeogenesis, sugar uptake, amino acid metabolism, and fatty acid β-oxidation. Our study reveals potential target genes for xylose fermentation improvement and provides insights into the mechanisms underlying xylose fermentation in S. stipitis.

A mutation in the COX5 gene of the yeast Scheffersomyces stipitis alters utilization of amino acids as carbon source, ethanol formation and activity of cyanide insensitive respiration

Scheffersomyces stipitis PJH was mutagenized by random integrative mutagenesis and the integrants were screened for lacking the ability to grow with glutamate as sole carbon source. One of the two isolated mutants was damaged in the COX5 gene, which encodes a subunit of the cytochrome c oxidase. BLAST searches in the genome of Sc. stipitis revealed that only one singular COX5 gene exists in Sc. stipitis, in contrast to Saccharomyces cerevisiae, where two homologous genes are present. Mutant cells had lost the ability to grow with the amino acids glutamate, proline or aspartate and other non-fermentable carbon sources, such as acetic acid and ethanol, as sole carbon sources. Biomass formation of the mutant cells in medium containing glucose or xylose as carbon source was lower compared with the wild-type cells. However, yields and specific ethanol formation of the mutant were much higher, especially under conditions of higher aeration. The mutant cells lacked both cytochrome c oxidase activity and cyanide-sensitive respiration, whereas ADH and PDC activities were distinctly enhanced. SHAM-sensitive respiration was obviously essential for the fermentative metabolism, because SHAM completely abolished growth of the mutant cells with both glucose or xylose as carbon source.

Improving L-arabinose utilization of pentose fermenting Saccharomyces cerevisiae cells by heterologous expression of L-arabinose transporting sugar transporters

BackgroundHydrolysates of plant biomass used for the production of lignocellulosic biofuels typically contain sugar mixtures consisting mainly of D-glucose and D-xylose, and minor amounts of L-arabinose. The yeast Saccharomyces cerevisiae is the preferred microorganism for the fermentative production of ethanol but is not able to ferment pentose sugars. Although D-xylose and L-arabinose fermenting S. cerevisiae strains have been constructed recently, pentose uptake is still a limiting step in mixed sugar fermentations.ResultsHere we described the cloning and characterization of two sugar transporters, AraT from the yeast Scheffersomyces stipitis and Stp2 from the plant Arabidopsis thaliana, which mediate the uptake of L-arabinose but not of D-glucose into S. cerevisiae cells. A yeast strain lacking all of its endogenous hexose transporter genes and expressing a bacterial L-arabinose utilization pathway could no longer take up and grow with L-arabinose as the only carbon source. Expression of the heterologous transporters supported uptake and utilization of L-arabinose especially at low L-arabinose concentrations but did not, or only very weakly, support D-glucose uptake and utilization. In contrast, the S. cerevisiae D-galactose transporter, Gal2, mediated uptake of both L-arabinose and D-glucose, especially at high concentrations.ConclusionsUsing a newly developed screening system we have identified two heterologous sugar transporters from a yeast and a plant which can support uptake and utilization of L-arabinose in L-arabinose fermenting S. cerevisiae cells, especially at low L-arabinose concentrations.

A convenient method for the dry preservation of living fungal or bacterial cultures

Integrated storage and pre-treatment (ISP) combines biopreservation of moist material under airtight conditions and pre-treatment. Moist wheat straw was inoculated with the biocontrol yeast Wickerhamomyces anomalus, the xylan degrading yeast Scheffersomyces stipitis or a co-culture of both. The samples and non-inoculated controls were stored at 4 or 15 °C. The non-inoculated controls were heavily contaminated with moulds, in contrast to the samples inoculated with W. anomalus or S. stipitis. These two yeasts were able to grow on wheat straw as sole source of nutrients. When ethanol was produced from moist wheat straw stored for four weeks at 4 °C with S. stipitis, an up to 40% enhanced yield (final yield 0.15 g ethanol per g straw dry weight) was obtained compared to a dry sample (0.107 g/g). In all other moist samples, stored for four weeks at 4 °C or 15 °C, 6–35% higher yields were obtained. Thus, energy efficient bio-preservation can improve the pre-treatment efficiency for lignocellulose biomass, which is a critical bottleneck in its conversion to biofuels.