Pierre van Rensburg

Engineering yeast for efficient cellulose degradation

Saccharomyces cerevisiae produces several β‐1,3‐glucanases, but lacks the multicomponent cellulase complexes that hydrolyse the β‐1,4‐linked glucose polymers present in cellulose‐rich biomass as well as in haze‐forming glucans in certain wines and beers. We have introduced into S. cerevisiae a functional cellulase complex for efficient cellulose degradation by cloning the Endomyces fibuliger cellobiase (BGL1) gene and co‐expressing it with the Butyrivibrio fibrisolvens endo‐β‐1,4‐glucanase (END1), the Phanerochaete chrysosporium cellobiohydrolase (CBH1) and the Ruminococcus flavefaciens cellodextrinase (CEL1) gene constructs in this yeast. The END1, CBH1 and CEL1 genes were inserted into yeast expression/secretion cassettes. Expression of END1, CBH1 and CEL1 was directed by the promoter sequences derived from the alcohol dehydrogenase II (ADH2), the phosphoglycerate kinase I (PKG1) and the alcohol dehydrogenase I (ADH1) genes, respectively. In contrast, BGL1 was expressed under the control of its native promoter. Secretion of End1p and Cel1p was directed by the signal sequence of the yeast mating pheromone α‐factor (MFα1), whereas Cbh1p and Bgl1p were secreted using their authentic leader peptides. The construction of a fur1 ura3 S. cerevisiae strain allowed for the autoselection of this multicopy URA3‐based plasmid in rich medium. S. cerevisiae transformants secreting biologically active endo‐β‐1,4‐glucanase, cellobiohydrolase, cellodextrinase and cellobiase were able to degrade various substrates including carboxymethylcellulose, hydroxyethylcellulose, laminarin, barley glucan, cellobiose, polypectate, birchwood xylan and methyl‐β‐d‐glucopyranoside. This study could lead to the development of industrial strains of S. cerevisiae capable of converting cellulose in a one‐step process into commercially important commodities.

Characterization of selected South African young cultivar wines using FTMIR Spectroscopy, Gas chromatography, and multivariate data analysis

The powerful combination of analytical chemistry and chemometrics and its application to wine analysis provide a way to gain knowledge and insight into the inherent chemical composition of wine and to objectively distinguish between wines. Extensive research programs are focused on the chemical characterization of wine to establish industry benchmarks and authentication systems. The aim of this study was to investigate the volatile composition and mid-infrared spectroscopic profiles of South African young cultivar wines with chemometrics to identify compositional trends and to distinguish between the different cultivars. Data were generated by gas chromatography and FTMIR spectroscopy and investigated by using analysis of variance (ANOVA), principal component analysis (PCA), and linear discriminant analysis (LDA). Significant differences were found in the volatile composition of the cultivar wines, with marked similarities in the composition of Pinotage wines and white wines, specifically for 2-phenylethanol, butyric acid, ethyl acetate, isoamyl acetate, isoamyl alcohol, and isobutyric acid. Of the 26 compounds that were analyzed, 14 had odor activity values of >1. The volatile composition and FTMIR spectra both contributed to the differentiation between the cultivar wines. The best discrimination model between the white wines was based on FTMIR spectra (98.3% correct classification), whereas a combination of spectra and volatile compounds (86.8% correct classification) was best to discriminate between the red wine cultivars.

Over-expression of the Saccharomyces cerevisiae exo-β-1,3-glucanase gene together with the Bacillus subtilis endo-β-1,3-1,4-glucanase gene and the Butyrivibrio fibrisolvens endo-β-1,4-glucanase gene in yeast

The EXG1 gene encoding the main Saccharomyces cerevisiae exo-beta-1,3-glucanase was cloned and over-expressed in yeast. The Bacillus subtilis endo-1,3-1,4-beta-glucanase gene (beg1) and the Butyrivibrio fibrisolvens endo-beta-1,4-glucanase gene (end1) were fused to the secretion signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1S) and inserted between the yeast alcohol dehydrogenase II gene promoter (ADH2P) and terminator (ADH2T). Constructs ADH2P-MF alpha 1S-beg1-ADH2T and ADH2P-MF alpha 1S-end 1-ADH2T designated BEG1 and END1, respectively, were expressed separately and jointly with EXG1 in S. cerevisiae. The construction of fur 1 ura3 S. cerevisiae strains allowed for the autoselection of these multicopy URA3-based plasmids in rich medium. Enzyme assays confirmed that co-expression of EXG1, BEG1 and END1 enhanced glucan degradation by S. cerevisiae.

Co-expression of a Phanerochaete chrysosporium cellobiohydrolase gene and a Butyrivibrio fibrisolvens endo-β-1,4-glucanase gene in Saccharomyces cerevisiae

Abstract A cDNA fragment encoding the Phanerochaete chrysosporium cellobiohydrolase (cbh1-4) was amplified and cloned with the aid of the polymerase chain reaction (PCR) technique. The cbh1-4 gene and the Butyrivibrio fibrisolvens endo-β-1,4-glucanase (end1) gene were successfully expressed in Saccharomyces cerevisiae under the control of the phosphoglycerate kinase-I (PGK1) and alcohol dehydrogenase-II (ADH2) gene promoters and terminators, respectively. The native P. chrysosporium signal sequence mediated secretion of cellobiohydrolase in S. cerevisiae, whereas secretion of the endo-β-1,4-glucanase was directed by the signal sequence of the yeast mating pheromone α-factor (MFα1S). These constructs, designated CBH1 and END1, respectively, were expressed separately and jointly in S. cerevisiae. The construction of fur1 ura3 S. cerevisiae strains allowed for the autoselection of these multicopy URA3-based plasmids in rich medium. Enzyme assays confirmed that co-expression of CBH1 and END1 synergistically enhanced cellulose degradation by S. cerevisiae.

Cloning and characterization of a second α‐amylase gene (LKA2) from Lipomyces kononenkoae IGC4052B and its expression in Saccharomyces cerevisiae

Lipomyces kononenkoae secretes a battery of highly effective amylases (i.e. α‐amylase, glucoamylase, isoamylase and cyclomaltodextrin glucanotransferase activities) and is therefore considered as one of the most efficient raw starch‐degrading yeasts known. Previously, we have cloned and characterized genomic and cDNA copies of the LKA1 α‐amylase gene from L. kononenkoae IGC4052B (CBS5608T) and expressed them in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Here we report on the cloning and characterization of the genomic and cDNA copies of a second α‐amylase gene (LKA2) from the same strain of L. kononenkoae. LKA2 was cloned initially as a 1663 bp cDNA harbouring an open reading frame (ORF) of 1496 nucleotides. Sequence analysis of LKA2 revealed that this ORF encodes a protein (Lka2p) of 499 amino acids, with a predicted molecular weight of 55 307 Da. The LKA2‐encoded α‐amylase showed significant homology to several bacterial cyclomaltodextrin glucanotransferases and also to the α‐amylases of Aspergillus nidulans, Debaryomyces occidentalis, Saccharomycopsis fibuligera and Sz. pombe. When LKA2 was expressed under the control of the phosphoglycerate kinase gene promoter (PGK1p) in S. cerevisiae, it was found that the genomic copy contained a 55 bp intron that impaired the production of biologically active Lka2p in the heterologous host. In contrast to the genomic copy, the expression of the cDNA construct of PGK1p–LKA2 in S. cerevisiae resulted in the production of biologically active α‐amylase. The LKA2‐encoded α‐amylase produced by S. cerevisiae exhibited a high specificity towards substrates containing α‐1,4 glucosidic linkages. The optimum pH of Lka2p was found to be 3.5 and the optimum temperature was 60 °C. Besides LKA1, LKA2 is only the second L. kononenkoae gene ever cloned and expressed in S. cerevisiae. The cloning, characterization and co‐expression of these two genes encoding these highly efficient α‐amylases form an important part of an extensive research programme aimed at the development of amylolytic strains of S. cerevisiae for the efficient bioconversion of starch into commercially important commodities. The nucleotide sequence of the LKA2 gene has been assigned GenBank Accession No. AF443872. Copyright

Regulation of endo‐polygalacturonase activity in Saccharomyces cerevisiae

Pectolytic activity in Saccharomyces cerevisiae is due to the secretion of an endo-polygalacturonase encoded by the PGU1 gene. The ability to degrade polygalacturonic acid has been shown to vary between different strains. In this study, we attempted to elucidate how pectolytic activity is regulated in S. cerevisiae and to determine whether the means of regulation differ between strains. Saccharomyces cerevisiae strains from different genetic backgrounds, with varying ability to degrade pectin, were compared. Activity was found not to be regulated by sequence differences in the PGU1 gene, but by the transcription level of the gene. Expression of PGU1 was found to be determined by the transcription level of its two transcription factors TEC1 and STE12. The activation of PGU1 transcription by galactose was found to be strain specific, independent of the strain being an industrial or a domesticated one. The EUROSCARF yeast deletion library was screened for genes encoding inhibitors and activators of polygalacturonase activity. Fourteen strains were identified, in which deletion of a specific gene resulted in a recovery of polygalacturonase activity; these genes were identified as encoding inhibitors of polygalacturonase activity, and two activators were identified.

Expression of the Ruminococcus flavefaciens cellodextrinase gene in Saccharomyces cerevisiae

SummaryA recombinant strain of Saccharomyces cerevisiae secreting bacterial cellodextrinase was constructed. The Ruminococcus flavefaciens cellodextrinase gene (celA) was inserted between a yeast expression-secretion cassette and yeast gene terminator, and cloned into a yeast-centromeric shuttle vector. Enzyme assays revealed growth-associated production of biologically active cellodextrinase by S. cerevisiae transformants.

Epigenetic regulation of PGU1 transcription in Saccharomyces cerevisiae

The PGU1 gene of Saccharomyces cerevisiae has been shown to encode a polygalacturonase. The polygalacturonase activity in S. cerevisiae is strain specific. There are no significant differences in the PGU1 promoter regions of strains with and without polygalacturonase activity. The PGU1 gene is subtelomeric because it is located within 25 kb of the right telomere of chromosome X. Expressions of genes located in subtelomeric regions in the yeast S. cerevisiae are inhibited compared with the rest of the genome. In this study, we showed that the deletion of genes involved in telomere silencing enhances polygalacturonase activity. PGU1 transcription and polygalacturonase activity are increased when PGU1 is shifted to a different location in the genome, away from the telomere located close to this gene, and the depletion of the histone H4 leads to an increase in PGU1 transcription. We concluded that PGU1 is silenced in strains without polygalacturonase activity due to an epigenetic effect. The results of this study suggest that PGU1 is silenced by being folded into a heterochromatin-like structure at its subtelomeric position on chromosome X. Formation of this silent structure is dependent on the Isw2p chromatin remodeling complex, its histone fold motif containing subunit Dls1p and the N-terminal tail of the H4 histone.

Coexpression of α‐l‐arabinofuranosidase and β‐glucosidase in Saccharomyces cerevisiae

Monoterpenes are important aroma compounds in grape varieties such as Muscat, Gewürztraminer and Riesling, and are present as either odourless, glycosidically bound complexes or free aromatic monoterpenes. Commercial enzymes can be used to release the monoterpenes, but they commonly consist of crude extracts that often have unwanted and unpredictable side-effects on wine aroma. This project aims to address these problems by the expression and secretion of the Aspergillus awamoriα-l-arabinofuranosidase in combination with either the β-glucosidases from Saccharomycopsis fibuligera or from Aspergillus kawachii in the industrial yeast Saccharomyces cerevisiae VIN13. The concentration of five monoterpenes was monitored throughout alcoholic fermentation of Gewürztraminer grapes. The recombinant yeast strains that caused an early boost in the geraniol concentration led to a reduction in the final geraniol levels due to the downregulation of the sterol biosynthetic pathway. Monoterpene concentrations were also analysed 9 and 38 days after racking and the performance of the VB2 and VAB2 recombinant strains was similar, and in many cases, better than that of a commercial enzyme used in the same experiment. The results were backed by sensorial analysis, with the panel preferring the aroma of the wines produced by the VAB2 strain.

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-fermentingSaccharomyces cerevisiae yeast strains through an approach that combined metabolic engineering and random mutagenesis in a recombinant haploid strain that overexpressed only two genes of the xylose pathway. Previously,S. cerevisiae strains, overexpressing heterologous genes encoding xylose reductase, xylitol dehydrogenase and the endogenousXKS1 xylulokinase gene, were randomly mutagenised to develop improved xylose-fermenting strains. In this study, two gene cassettes (ADH1p-PsXYL1-ADH1T andPGK1p-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 haploidS. cerevisiae strain (CEN.PK 2-1D). The resulting recombinant strain (YUSM 1001) over-expressing theP. stipitis XYL1 andXYL2 genes (but not the endogenousXKS1 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 ofXYL1, XYL2 andXKS1 and theGPD2 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 theXKS1 xylulokinase gene.