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.Articl

The effect of flocculation on the efficiency of raw-starch fermentation by Saccharomyces cerevisiae producing the Lipomyces kononenkoae LKA1-encoded α-amylase

A major limitation of most industrially important Saccharomyces yeast strains are their inability to efficiently convert starch-rich substrates into commercially important commodities, such as bioethanol, low carbohydrate beer and grain whiskey. In an attempt to overcome this impediment, we have previously expressed in Saccharomyces cerevisiae the LKA1 α-amylase-encoding gene from an efficient raw-starch degrading yeast, Lipomyces kononenkoae. Although the engineered S. cerevisiae strain was capable of utilising starch, the growth rate was much slower than in glucose-containing media and the ethanol yield in batch fermentations was nowhere near the levels required for an economically viable bioconversion process. The purpose of the present study was to further improve the fermentation performance of the engineered yeast by expressing the LKA1 gene in a flocculent and non-flocculent genetic background. Despite producing similar levels of α-amylase activities in the extracellular culture media, the flocculent S. cerevisiae transformants degraded starch at an earlier hydrolytic window than the non-flocculent transformants. In small-scale batch fermentations, the non-flocculent strain consumed 76% of the starch supplied in the culture medium and produced 4.61 g l-1 of ethanol after 90 h, while the flocculent strain utilised 82% of the starch and produced 5.1 g l-1 of ethanol after 90 h. Flow-cell system and atomic force microscopy revealed that the 'tighter' interaction between the flocculent cells and the starch granules might contribute to the better performance of the flocculent transformant.Articl

Optimization of a rapid method for studying the cellular location of β-glucosidase activity in wine yeasts

Aims: To improve a method for determining β-glucosidase activity and to apply it in yeasts isolated from wine ecosystems from 'La Mancha' region and to know its cellular location. Methods and Results: A total of 82 wine yeasts were identified (PCR/RFLP) and evaluated for their β-glucosidase activity. First, they were qualitatively evaluated by growth on YNB cellobiose, the activity was quantified using different culture media, under aerobic and anaerobic conditions and cells after 24-72 h of growth. To study the location activity, five fractions were obtained (supernatant, whole cell, cell wall, cytosol and cell membrane). The enzymatic assays were optimized, being: growth in YP cellobiose for 72 h in aeration conditions and, after cell removing, enzyme analysis with 128 g 1-1 of cellobiose as substrate, for 30 min at 30°C. The genus that displayed the greatest activity were Pichia, Hanseniaspora and Rhodotorula, and the activity was intracellular. Conclusions: The study showed that β-glucosidase activity was induced by the carbon source and was aerobic dependent. The non-Saccharomyces species displayed the greatest activity, which was intracellular and strain-dependent. Significance and Impact of the Study: This study developed a reliable method for screening β-glucosidase activity in yeasts isolated from wine ecosystems. This activity is very important in the release of monoterpenols from glycoside precursors for the enhancement of wine aromas. © 2005 The Society for Applied Microbiology.Articl

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.Articl

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 © 2002 John Wiley & Sons, Ltd.Revie

Molecular analysis of a Saccharomyces cerevisiae mutant with improved ability to utilize xylose shows enhanced expression of proteins involved in transport, initial xylose metabolism, and the pentose phosphate pathway

Differences between the recombinant xylose-utilizing Saccharomyces cerevisiae strain TMB 3399 and the mutant strain TMB 3400, derived from TMB 3399 and displaying improved ability to utilize xylose, were investigated by using genome-wide expression analysis, physiological characterization, and biochemical assays. Samples for analysis were withdrawn from chemostat cultures. The characteristics of S. cerevisiae TMB 3399 and TMB 3400 grown on glucose and on a mixture of glucose and xylose, as well as of S. cerevisiae TMB 3400 grown on only xylose, were investigated. The strains were cultivated under chemostat conditions at a dilution rate of 0.1 h-1, with feeds consisting of a defined mineral medium supplemented with 10 g of glucose liter-1, 10 g of glucose plus 10 g of xylose liter-1 or, for S. cerevisiae TMB 3400, 20 g of xylose liter-1. S. cerevisiae TMB 3400 consumed 31% more xylose of a feed containing both glucose and xylose than S. cerevisiae TMB 3399. The biomass yields for S. cerevisiae TMB 3400 were 0.46 g of biomass g of consumed carbohydrate-1 on glucose and 0.43 g of biomass g of consumed carbohydrate-1 on xylose. A Ks value of 33 mM for xylose was obtained for S. cerevisiae TMB 3400. In general, the percentage error was <20% between duplicate microarray experiments originating from independent fermentation experiments. Microarray analysis showed higher expression in S. cerevisiae TMB 3400 than in S. cerevisiae TMB 3399 for (i) HXT5, encoding a hexose transporter; (ii) XKS1, encoding xylulokinase, an enzyme involved in one of the initial steps of xylose utilization; and (iii) SOL3, GND1, TAL1, and TKL1, encoding enzymes in the pentose phosphate pathway. In addition, the transcriptional regulators encoded by YCR020C, YBR083W, and YPR199C were expressed differently in the two strains. Xylose utilization was, however, not affected in strains in which YCR020C was overexpressed or deleted. The higher expression of XKS1 in S. cerevisiae TMB 3400 than in TMB 3399 correlated with higher specific xylulokinase activity in the cell extracts. The specific activity of xylose reductase and xylitol dehydrogenase was also higher for S. cerevisiae TMB 3400 than for TMB 3399, both on glucose and on the mixture of glucose and xylose.Articl

Engineering of an oenological Saccharomyces cerevisiae strain with pectinolytic activity and its effect on wine

A pectinolytic industrial yeast strain of Saccharomyces cerevisiae was generated containing the S. cerevisiae endopolygalacturonase gene (PGU1) constitutively expressed under the control of the 3-phosphoglycerate kinase gene (PGK1) promoter. The new strain contains DNA derived exclusively from yeast and expresses a high polygalacturonic acid hydrolyzing activity. Yeast transformation was carried out by an integrative process targeting a dispensable upstream region of the acetolactate synthase locus (ILV2), which determines sulfometuron methyl resistance. Microvinification assays were performed on white and red musts with the transformed UCLMS-1M strain and with the same strain untransformed. It was found that the changes in the pectic polysaccharide contents did not directly affect the taste or flavor of the wine. From the data reported, it is deduced that the chief advantage of using the modified strain is that it improves the yield of must/wine extraction, while it also positively affects some variables relating to appearance. © 2005 Elsevier B.V. All rights reserved.Articl

Expression of the Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae and its potential applications in wine production

There is a growing consumer demand for wines containing lower levels of alcohol and chemical preservatives. The objectives of this study were to express the Aspergillus niger gene encoding a glucose oxidase (GOX; β-D-glucose:oxygen oxidoreductase, EC 1.1.3.4) in Saccharomyces cerevisiae and to evaluate the transformants for lower alcohol production and inhibition of wine spoilage organisms, such as acetic acid bacteria and lactic acid bacteria, during fermentation. The A. niger structural glucose oxidase (gox) gene was cloned into an integration vector (YIp5) containing the yeast mating pheromone α-factor secretion signal (MFα1S) and the phosphoglycerate-kinase-1 gene promoter (PGK1P) and terminator (PGK1T). The PGK1P-MFα1S-gox-PGK1T cassette (designated GOX1) was introduced into a laboratory strain (∑1278) of S. cerevisiae. Yeast transformants were analysed for the production of biologically active glucose oxidase on selective agar plates and in liquid assays. The results indicated that the recombinant glucose oxidase was active and was produced beginning early in the exponential growth phase, leading to a stable level in the stationary phase. The yeast transformants also displayed antimicrobial activity in a plate assay against lactic acid bacteria and acetic acid bacteria. This might be explained by the fact that a final product of the GOX enzymatic reaction is hydrogen peroxide, a known antimicrobial agent. Microvinification with the laboratory yeast transformants resulted in wines containing 1.8-2.0% less alcohol. This was probably due to the production of D-glucono-δ-lactone and gluconic acid from glucose by GOX. These results pave the way for the development of wine yeast starter culture strains for the production of wine with reduced levels of chemical preservatives and alcohol.Articl

Generation of the improved recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3400 by random mutagenesis and physiological comparison with Pichia stipitis CBS 6054

The recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3399 was constructed by chromosomal integration of the genes encoding D-xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK). S. cerevisiae TMB 3399 was subjected to chemical mutagenesis with ethyl methanesulfonate and, after enrichment, 33 mutants were selected for improved growth on D-xylose and carbon dioxide formation in Durham tubes. The best-performing mutant was called S. cerevisiae TMB 3400. The novel, recombinant S. cerevisiae strains were compared with Pichia stipitis CBS 6054 through cultivation under aerobic, oxygen-limited, and anaerobic conditions in a defined mineral medium using only D-xylose as carbon and energy source. The mutation led to a more than five-fold increase in maximum specific growth rate, from 0.0255 h-1 for S. cerevisiae TMB 3399 to 0.14 h-1 for S. cerevisiae TMB 3400, whereas P. stipitis grew at a maximum specific growth rate of 0.44 h-1. All yeast strains formed ethanol only under oxygen-limited and anaerobic conditions. The ethanol yields and maximum specific ethanol productivities during oxygen limitation were 0.21, 0.25, and 0.30 g ethanol g xylose-1 and 0.001, 0.10, and 0.16 g ethanol g biomass-1 h-1 for S. cerevisiae TMB 3399, TMB 3400, and P. stipitis CBS 6054, respectively. The xylitol yield under oxygen-limited and anaerobic conditions was two-fold higher for S. cerevisiae TMB 3399 than for TMB 3400, but the glycerol yield was higher for TMB 3400. The specific activity, in U mg protein-1, was higher for XDH than for XR in both S. cerevisiae TMB 3399 and TMB 3400, while P. stipitis CBS 6054 showed the opposite relation. S. cerevisiae TMB 3400 displayed higher specific XR, XDH and XK activities than TMB 3399. Hence, we have demonstrated that a combination of metabolic engineering and random mutagenesis was successful to generate a superior, xylose-utilizing S. cerevisiae, and uncovered distinctive physiological properties of the mutant. © 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.Articl

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.Articl