The STF2p Hydrophilin from Saccharomyces cerevisiae Is Required for Dehydration Stress Tolerance

The yeast Saccharomyces cerevisiae is able to overcome cell dehydration; cell metabolic activity is arrested during this period but restarts after rehydration. The yeast genes encoding hydrophilin proteins were characterised to determine their roles in the dehydration-resistant phenotype, and STF2p was found to be a hydrophilin that is essential for survival after the desiccation-rehydration process. Deletion of STF2 promotes the production of reactive oxygen species and apoptotic cell death during stress conditions, whereas the overexpression of STF2, whose gene product localises to the cytoplasm, results in a reduction in ROS production upon oxidative stress as the result of the antioxidant capacity of the STF2p protein

Amylolytic enzymes from the yeast Lipomyces kononenkoae

The Lipomyces kononenkoae ?-amylases LKA1 and LKA2 belong to the glycoside hydrolase family 13 and exhibit specificity towards ∝-1,4 and ∝-1,6 linkages in starch and related substrates. LKA1 exhibits specificity towards ∝-1,4 and ∝-1,6 linkages and large amounts of reducing sugars are liberated from highly branched amylopectin and glycogen and linear amylose. LKA2, on the other hand, shows high reactivity towards lintner starch, dextrin and amylase, although only small amounts of reducing sugars are liberated from branched substrates, such as amylopectin and glycogen. These enzymes share the four conserved segments of the catalytic domain found in other members of the family, but have some major variant amino acids within these segments. In addition, LKA1 consists of an N-terminal starch-binding domain (SBD). This is the only ∝-amylase known to possess this N-terminal domain and it exhibits homology to the N-terminal SBD of Rhizopus oryzae glucoamylase. It shares no homology with the C-terminal starch-binding domains present in the cyclodextrin glucanotransferases, glucoamylases or ∝-amylases. The evolutionary tree based on the sequence alignment of SBDs reveals that the N-terminal SBDs are separated from the C-terminal SBDs.8 page(s

Molecular cloning and functional expression of a novel Neurospora crassa xylose reductase in Saccharomyces cerevisiae in the development of a xylose fermenting strain

The development of a xylose-fermenting Saccharomyces cerevisiae yeast would be of great benefit to the bioethanol industry. The conversion of xylose to ethanol involves a cascade of enzymatic reactions and processes. Xylose (aldose) reductases catalyse the conversion of xylose to xylitol. The aim of this study was to clone, characterise and express a cDNA copy of a novel aldose reductase {NCAR-X) from the filamentous fungus Neurospora crassa in S. cerevisiae. NCAR-X harbours an open reading frame (ORF) of 900 nucleotides. This ORF encodes a protein (NCAR-X, assigned NCBI protein accession ID: XP_956921) consisting of 300 amino acids, with a predicted molecular weight of 34 kDa. The NVAR-X-encoded aldose reductase showed significant homology to the xylose reductases of Candida tenuis and Pichia stipitis. When NCAR-X was expressed under the control of phosphoglycerate kinase I gene (PGK1) regulatory sequences in S. cerevisiae, its expression resulted In the production of biologically active xylose reductase. Small-scale oxygen-limited xylose fermentation with the NCAR-X containing S. cerevisiae strains resulted In the production of less xylitol and at least 15% more ethanol than the strains transformed with the P. stipitis xylose reductase gene (PsXYL1). The NCAR-X-encoded enzyme produced by S. cerevisiae was NADPH-dependent and no activity was observed in the presence of NADH. The co-expression of the NCAR-X and PsXYL1 gene constructs in S. cerevisiae constituted an important part of an extensive research program aimed at the development of xylolytic yeast strains capable of producing ethanol from plant biomass.9 page(s

Enhancing volatile phenol concentrations in wine by expressing various phenolic acid decarboxylase genes in Saccharomyces cerevisiae

Phenolic acids, which are generally esterified with tartaric acid, are natural constituents of grape must and wine and can be released as free acids (principally p-coumaric, caffeic, and ferulic acids) by certain cinnamoyl esterase activities during the wine-making process. Some of the microorganisms present in grape can metabolize the free phenolic acids into 4-vinyl and 4-ethyl derivatives. These volatile phenols contribute to the aroma of wine. The Saccharomyces cerevisiae phenyl acrylic acid decarboxylase gene (PAD1) is steadily transcribed, but its encoded product, Pad1p, shows low activity. In contrast, the phenolic acid decarboxylase (PADC) from Bacillus subtilis and the p-coumaric acid decarboxylase (PDC) from Lactobacillus plantarum display substrate-inducible decarboxylating activity in the presence of phenolic acids. In an attempt to develop wine yeasts with optimized decarboxylation activity on phenolic acids, the padc, pdc, and PAD1 genes were cloned under the control of S. cerevisiae's constitutive phosphoglyceratekinase I gene promoter (PGK1P) and terminator (PGK1T) sequences. These gene constructs were integrated into the URA3 locus of a laboratory strain of S. cerevisiae, Σ1278b. The overexpression of the two bacterial genes, padc and pdc, in S. cerevisiae showed high enzyme activity. However, this was not the case for PAD1. The padc and pdc genes were also integrated into an industrial wine yeast strain, S. cerevisiae VIN13. As an additional control, both alleles of PAD1 were disrupted in the VIN13 strain. In microvinification trials, all of the laboratory and industrial yeast transformants carrying the padc and pdc gene constructs showed an increase in volatile phenol formation as compared to the untransformed host strains (Σ1278b and VIN13). This study offers prospects for the development of wine yeast starter strains with optimized decarboxylation activity on phenolic acids and the improvement of wine aroma in the future.7 page(s

Development and characterisation of a recombinant Saccharomyces cerevisiae mutant strain with enhanced xylose fermentation properties

The purpose of this study was to help lay the foundation for further development of xylose-fermenting Saccharomyces cerevisiae yeast strains through an approach that combined metabolic engineering and random mutagenesis in a recombinant hap-loid strain that overexpressed only two genes of the xylose pathway. Previously, S. cerevisiae strains, overexpressing heterologous genes encoding xylose reductase, xylitol dehydrogenase and the endogenous XKS1 xylulokinase gene, were randomly mutagenised to develop improved xylose-fermenting strains. In this study, two gene cassettes (ADHI p-PsXYL1-ADH1T- and PGKlP-PsXYL2-PGK1T) containing the xylose reductase (PsXYL1) and xylitol dehydrogenase (PsXYL2) genes from the xylose-fermenting yeast, Pichia stipitis, were integrated into the genome of a haploid S. cerevisiae strain (CEN.PK 2-1D). The resulting recombinant strain (YUSM 1001) over-expressing the P. stipitis XYL1 and XYL2 genes (but not the endogenous XKS1 gene) was subjected to ethyl methane sulfonate (EMS) mutagenesis. The resulting mutants were screened for faster growth rates on an agar medium containing xylose as the sole carbon source. A mutant strain (designated Y-X) that showed 20-fold faster growth in xylose medium in shake-flask cultures was isolated and characterised. In anaerobic batch fermentation, the Y-X mutant strain consumed 2.5-times more xylose than the YUSM 1001 parental strain and also produced more ethanol and glycerol. The xylitol yield from the mutant strain was lower than that from the parental strain, which did not produce glycerol and ethanol from xylose. The mutant also showed a 50% reduction in glucose consumption rate. Transcript levels of XYL1, XYL2 and XKS1 and the GPD2 glycerol 3-phosphate dehydrogenase gene from the two strains were compared with real-time reverse transcription polymerase chain reaction (RT-PCR) analysis. The mutant showed 10-40 times higher relative expression of these four genes, which corresponded with either the higher activities of their encoded enzymes or by-product formation during fermentation. Furthermore, no mutations were observed in the mutant's promoter sequences or the open reading frames of some of its key genes involved in carbon catabolite repression, glycerol production and redox balancing. The data suggest that the enhancement of the xylose fermentation properties of the Y-X mutant was made possible by increased expression of the xylose pathway genes, especially the XKS1 xylulokinase gene.9 page(s