The effect of polysaccharide-degrading wine yeast transformants on the efficiency of wine processing and wine flavour

Commercial polysaccharase preparations are applied to winemaking to improve wine processing and quality. Expression of polysaccharase-encoding genes in Saccharomyces cerevisiae allows for the recombinant strains to degrade polysaccharides that traditional commercial yeast strains cannot. In this study, we constructed recombinant wine yeast strains that were able to degrade the problem-causing grape polysaccharides, glucan and xylan, by separately integrating the Trichoderma reesei XYN2 xylanase gene construct and the Butyrivibrio fibrisolvens END1 glucanase gene cassette into the genome of the commercial wine yeast strain S. cerevisiae VIN13. These genes were also combined in S. cerevisiae VIN13 under the control of different promoters. The strains that were constructed were compared under winemaking conditions with each other and with a recombinant wine yeast strain expressing the endo-beta-1,4-glucanase gene cassette (END1) from B. fibrisolvens and the endo-beta-1,4-xylanase gene cassette (XYN4) from Aspergillus niger, a recombinant strain expressing the pectate lyase gene cassette (PEL5) from Erwinia chrysanthemi and the polygalacturonase-encoding gene cassette (PEH1) from Erwinia carotovora. Wine was made with the recombinant strains using different grape cultivars. Fermentations with the recombinant VIN13 strains resulted in significant increases in free-flow wine when Ruby Cabernet must was fermented. After 6 months of bottle ageing significant differences in colour intensity and colour stability could be detected in Pinot Noir and Ruby Cabernet wines fermented with different recombinant strains. After this period the volatile composition of Muscat d'Alexandria, Ruby Cabernet and Pinot Noir wines fermented with different recombinant strains also showed significant differences. The Pinot Noir wines were also sensorial evaluated and the tasting panel preferred the wines fermented with the recombinant strains.     

Local comparison of the genomes of Mycobacterium tuberculosis and Mycobacterium leprae using the polymerase chain reaction

Abstract A gene fusion between the Saccharomyces cerevisiae actin gene promoter and the cDNA of the Fusarium solani f. sp. pisi pelA gene has been constructed. This expression cassette has been introduced into the industrial wine yeast strain T₇₃. The resulting recombinant strain is able to secrete active PELA enzyme into the culture medium. In preliminary microvinification experiments the wine produced by this pectinolytic strain is indistinguishable from wine produced using the non-transformed strain on the basis of the chemical analyses. Large scale fermentations need to be carried out in order to assess the effects on filtrability.     

Construction of Aspergillus nidulans strains producing enzymes of potential use in enology

Two different Aspergillus nidulans recombinant strains producing either the Aspergillus nidulans a-L-arabinofuranosidase A or a Candida molischiana b-glucosidase have been constructed. Depending on the growing conditions, the modified strains produce up to 4 or 18 times more b-glucosidase or a-L-arabinofuranosidase activity levels, respectively, than the wild type strain.     

Construction of self-cloning, indigenous wine strains of Saccharomyces cerevisiae with enhanced glycerol and glutathione production

To improve wine taste and flavor stability, a novel indigenous strain of Saccharomyces cerevisiae with enhanced glycerol and glutathione (GSH) production for winemaking was constructed. ALD6 encoding an aldehyde dehydrogenases of the indigenous yeast was replaced by a GPD1 and CUP1 gene cassette, which are responsible for NAD-dependent glycerol-3-phosphatase dehydrogenase and copper resistance, respectively. Furthermore, the α-acetohydroxyacid synthase gene ILV2 of the indigenous yeast was disrupted by integration of the GSH1 gene which encodes γ-glutamylcysteine synthetase and the CUP1 gene cassette. The fermentation capacity of the recombinant was similar to that of the wild-type strain, with an increase of 21 and 19?% in glycerol and GSH production. No heterologous DNA was harbored in the recombinant in this study.     

Enhanced Production of β-Carotene by Recombinant Industrial Wine Yeast Using Grape Juice as Substrate

In this study, both recombinant Saccharomyces cerevisiae T73-63 and FY-09 derived from the industrial wine yeast T73-4 and laboratory yeast FY1679-01B, respectively, were constructed and compared for their β-carotene production in real grape juice. The results showed that highest β-carotene content (5.89 mg/g) was found in strain T73-63, which was 2.1 fold higher than that of strain FY-09. Although the cell growth was inhibited by the metabolic burden induced by the production of heterogeneous β-carotene, the pigment yield in T73-63 was still 1.7 fold higher than that of FY-09. Furthermore, high contents of ergosterol and fatty acid were also observed in T73-63. These results suggest that industrial wine yeast has highly active metabolic flux in mevalonate pathway, which leads to more carbon flux into carotenoid branch compared to that of laboratory yeast. The results of this study collectively suggest that in the application of recombinant strains to produce carotenoid using agro-industrial by-products as substrate, the suitable host strains should have active mevalonate pathway. For this purpose, the industrial wine yeast is a suitable candidate.     

一种两步基因同源重组敲除酵母靶标蛋白基因的方法

目的:建立一种在代谢工程改造的毕赤酵母菌中高效敲除靶标蛋白基因的方法。方法:构建敲除载体,以粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因为靶基因,以尿嘧啶合成关键酶URA3基因为营养缺陷型筛选标记,第一步重组利用ura3正筛选获得敲除载体在酵母染色体中的定点整合,第二步通过5-氟乳清酸(5-FOA)筛选到表型为ura3-的克隆,ura3基因被剔除的同时,目的基因GM-CSF也随之丢失,实现第二次重组;利用基因组PCR和蛋白电泳进行鉴定。结果:通过两步基因同源重组,敲除了毕赤酵母GJK01的报告蛋白GM-CSF的编码基因388 bp,PCR结果显示该基因已完全丢失,SDS-PAGE分析无GM-CSF表达。结论:仅通过2周时间的筛选、鉴定,在毕赤酵母GJK01中敲除了报告蛋白GM-CSF的编码基因,突变菌株的阳性率达到50%,最终建立了以URA3为筛选标记的两步基因同源重组敲除目的基因的方法。     

Genetically engineered wine yeast produces a high concentration of L-lactic acid of extremely high optical purity

For mass production of lactic acid, we newly constructed a transgenic wine yeast strain that included six copies of the bovine L-lactate dehydrogenase gene on the genome. On fermentation in inexpensive cane juice-based medium, L-lactate production of this recombinant reached 122 g/liter and the optical purity was 99.9% or higher.     

The heterologous expression of polysaccharidase-encoding genes with oenological relevance in Saccharomyces cerevisiae

AIMS: The main objective of this study was to develop polysaccharide-degrading wine strains of Saccharomyces cerevisiae, which are able to improve aspects of wine processing and clarification, as well as colour extraction and stabilization during winemaking. METHODS AND RESULTS: Two yeast expression/secretion gene cassettes were constructed, namely (i) a pectinase gene cassette (pPPK) consisting of the endo-polygalacturonase gene (pelE) from Erwinia chrysanthemi and the pectate lyase gene (peh1) from Erwinia carotovora and (ii) a glucanase/xylanase gene cassette (pEXS) containing the endo-beta-1,4-glucanase gene (end1) from Butyrivibrio fibrisolvens and the endo-beta-1,4-xylanase gene (xynC) from Aspergillus niger. The commercial wine yeast strain, VIN13, was transformed separately with these two gene cassettes and checked for the production of pectinase, glucanase and xylanase activities. Pinot Noir, Cinsaut and Muscat d'Alexandria grape juices were fermented using the VIN13[pPPK] pectinase- and the VIN13[pEXS] glucanase/xylanase-producing transformants. Chemical analyses of the resultant wines indicated that (i) the pectinase-producing strain caused a decrease in the concentration of phenolic compounds in Pinot Noir whereas the glucanase/xylanase-producing strain caused an increase in phenolic compounds presumably because of the degradation of the grape skins; (ii) the glucanase/xylanase-producing strain caused a decrease in wine turbidity, especially in Pinot Noir wine, as well as a clear increase in colour intensity and (iii) in the Muscat d'Alexandria and Cinsaut wines, the differences between the control wines (fermented with the untransformed VIN3 strain) and the wines produced by the two transformed strains were less prominent showing that the effect of these polysaccharide-degrading enzymes is cultivar-dependent. CONCLUSIONS: The recombinant wine yeasts producing pectinase, glucanase and xylanase activities during the fermentation of Pinot Noir, Cinsaut and Muscat d'Alexandria grape juice altered the chemical composition of the resultant wines in a way that such yeasts could potentially be used to improve the clarity, colour intensity and stability and aroma of wine. SIGNIFICANCE AND IMPACT OF THE STUDY: Aspects of commercial-scale wine processing and clarification, colour extraction and stabilization, and aroma enhancement could potentially be improved by the use of polysaccharide-degrading wine yeasts without the addition of expensive commercial enzyme preparations. This offers the potential to further improve the price:quality ratio of wine according to consumer expectations.     

Construction of a recombinant wine yeast strain expressing beta-(1,4)-endoglucanase and its use in microvinification processes.

A genetic transformation system for an industrial wine yeast strain is presented here. The system is based on the acquisition of cycloheximide resistance and is a direct adaptation of a previously published procedure for brewing yeasts (L. Del Pozo, D. Abarca, M. G. Claros, and A. Jiménez, Curr. Genet. 19:353-358, 1991). Transformants arose at an optimal frequency of 0.5 transformant per microgram of DNA, are stable in the absence of selective pressure, and produce wine in the same way as the untransformed industrial strain. By using this transformation protocol, a filamentous fungal beta-(1,4)-endoglucanase gene has been expressed in an industrial wine yeast under the control of the yeast actin gene promoter. Endoglucanolytic wine yeast secretes the fungal enzyme to the must, producing a wine with an increased fruity aroma.     

Engineering the Saccharomyces cerevisiae isoprenoid pathway for de novo production of aromatic monoterpenes in wine

Grape musts contain a variety of terpenols that significantly affect wine aroma. The amounts of these metabolites depend on the grape variety, and many cultivars are non-aromatic. Yeasts like Saccharomyces cerevisiae cannot produce and excrete monoterpenes efficiently, mainly due to their lack of monoterpene synthases. By metabolic engineering we have modified the isoprenoid biosynthesis pathway in a wine yeast strain of S. cerevisiae expressing the Clarkia breweri S-linalool synthase gene. Under microvinification conditions, without compromising other desirable and useful fermentative traits, the recombinant yeast efficiently excreted linalool to levels exceeding the threshold of human perception. Bearing in mind the possibility of (co-)expressing other genes that encode enzymes leading to the production of various aroma compounds and the feasibility of controlling the levels of their expression, the potential of this achievement for future genetic manipulation of wine varietal aroma or for use in other alcoholic drinks seems very promising.     

代谢工程改善野生酵母利用木糖产乙醇的性能

从256个自然样品中筛选得到1株可高效转化D-木糖的酵母。通过生理生化和分子生物学方法鉴定, 证实该菌株是属于Candida tropicalis。以该酵母为研究对象, 增加木糖醇脱氢酶表达量, 通过改变代谢流以达到提高酒精产率的目的。以pXY212-XYL2质粒为基础载体, 构建了含有潮霉素抗性的pYX212-XYL2-Hygro, 电击转化进入野生型C. tropicalis, 潮霉素抗性筛选, 得到含高拷贝木糖醇脱氢酶基因的重组菌株C. tropicalis XYL2-7。重组菌的比酶活达到0.5 u/mg protein, 比原始菌株提高了3倍。实验表明, 重组菌木糖醇得率比原始菌株降低了3倍, 酒精得率提高了5倍。首次通过实验验证了热带假丝酵母利用木糖产乙醇的可行性, 这对研究酵母利用秸秆、麦糠、谷壳等纤维质农业废弃物生产燃料乙醇具有重要启示。     

代谢工程改善野生酵母利用木糖产乙醇的性能

从256个自然样品中筛选得到1株可高效转化D-木糖的酵母。通过生理生化和分子生物学方法鉴定, 证实该菌株是属于Candida tropicalis。以该酵母为研究对象, 增加木糖醇脱氢酶表达量, 通过改变代谢流以达到提高酒精产率的目的。以pXY212-XYL2质粒为基础载体, 构建了含有潮霉素抗性的pYX212-XYL2-Hygro, 电击转化进入野生型C. tropicalis, 潮霉素抗性筛选, 得到含高拷贝木糖醇脱氢酶基因的重组菌株C. tropicalis XYL2-7。重组菌的比酶活达到0.5 u/mg protein, 比原始菌株提高了3倍。实验表明, 重组菌木糖醇得率比原始菌株降低了3倍, 酒精得率提高了5倍。首次通过实验验证了热带假丝酵母利用木糖产乙醇的可行性, 这对研究酵母利用秸秆、麦糠、谷壳等纤维质农业废弃物生产燃料乙醇具有重要启示。     

Fermentative capacity of dry active wine yeast requires a specific oxidative stress response during industrial biomass growth

Induction of the oxidative stress response has been described under many physiological conditions in Saccharomyces cerevisiae, including industrial fermentation for wine yeast biomass production where cells are grown through several batch and fed-batch cultures on molasses. Here, we investigate the influence of aeration on the expression changes of different gene markers for oxidative stress and compare the induction profiles to the accumulation of several intracellular metabolites in order to correlate the molecular response to physiological and metabolic changes. We also demonstrate that this specific oxidative response is relevant for wine yeast performance by construction of a genetically engineered wine yeast strain overexpressing the TRX2 gene that codifies a thioredoxin, one of the most important cellular defenses against oxidative damage. This modified strain displays an improved fermentative capacity and lower levels of oxidative cellular damages than its parental strain after dry biomass production.     

Adaptive evolution of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> to generate strains with enhanced glycerol production

The development of new wine yeast strains with improved characteristics is critical in the highly competitive wine market, which faces the demand of ever-changing consumer preferences. Although new strains can be constructed using recombinant DNA technologies, consumer concerns about genetically modified (GM) organisms strongly limit their use in food and beverage production. We have applied a non-GM approach, adaptive evolution with sulfite at alkaline pH as a selective agent, to create a stable yeast strain with enhanced glycerol production; a desirable characteristic for wine palate. A mutant isolated using this approach produced 41% more glycerol than the parental strain it was derived from, and had enhanced sulfite tolerance. Backcrossing to produce heterozygous diploids revealed that the high-glycerol phenotype is recessive, while tolerance to sulfite was partially dominant, and these traits, at least in part, segregated from each other. This work demonstrates the potential of adaptive evolution for development of novel non-GM yeast strains, and highlights the complexity of adaptive responses to sulfite selection.     

Transcriptional profiling of wine yeast in fermenting grape juice: regulatory effect of diammonium phosphate

The nitrogen composition of grape musts affects fermentation kinetics and production of aroma and spoilage compounds in wine. It is common practice in wineries to supplement grape musts with diammonium phosphate (DAP) to prevent nitrogen-related fermentation problems. Laboratory strains of Saccharomyces cerevisiae preferentially use rich nitrogen sources, such as ammonia, over poor nitrogen sources. We used global gene expression analysis to monitor the effect of DAP addition on gene expression patterns in wine yeast in fermenting Riesling grape must. The expression of 350 genes in the commercial wine yeast strain VIN13 was affected; 185 genes were down-regulated and 165 genes were up-regulated in response to DAP. Genes that were down-regulated encode small molecule transporters and nitrogen catabolic enzymes, including those linked to the production of urea, a precursor of ethyl carbamate in wine. Genes involved in amino acid metabolism, assimilation of sulfate, de novo purine biosynthesis, tetrahydrofolate one-carbon metabolism, and protein synthesis were up-regulated. The expression level of 86 orphan genes was also affected by DAP.     

Interaction of PRK1 Receptor-like Kinase with a Putative elF2B β-Subunit in Tobacco

PRK1, a receptor-like kinase that is expressed in pollen, pollen tubes, and ovaries, has been shown to play important roles in pollen development and embryo sac development in Petunia inflata. We have used the kinase domain of PRK1 as a bait in the yeast two-hybrid system to identify PRK1-interacting proteins. The screening resulted in isolation of a cDNA encoding a protein highly homologous to the human and yeast beta-subunit of translation initiation factor 2B (eIF2B-beta), which was designated NeIF2Bbeta. eIF2B is a guanine nucleotide exchange protein that functions in the regulation of translation in eukaryotic cells. Deletion mutants of NeIF2Bbeta were analyzed for their interaction with PRK1, and the results suggested that the N-terminal half of NeIF2Bbeta, especially the region between residue 103 and 235, is important for the interaction. This protein association was confirmed by in vitro binding assay of the recombinant NeIF2Bbeta and PRK1 proteins. Despite high sequence homology between NeIF2Bbeta and its yeast counterpart, the NeIF2Bbeta cDNA could not rescue the phenotype of the yeast mutant strain lacking the GCD7 gene encoding eIF2B-beta, when transferred into the mutant strain.