Rapid identification of Saccharomyces cerevisiae, Zygosaccharomyces bailii and Zygosaccharomyces rouxii.

Most strains of Saccharomyces cerevisiae, Zygosaccharomyces bailii and Zygosaccharomyces rouxii have been found to contain plasmid DNA. The sequences of the plasmids from these three yeasts are known to be different. We have used two primers within the plasmid from each yeast species in a multiplex polymerase chain reaction (PCR) to discriminate between these three yeasts. The primers were designed to give easily distinguishable fragment sizes when run on a simple agarose gel. Due to the sensitivity of PCR, crude cells can be used with no need to isolate DNA. The method is rapid when compared with current methods.     

生物吸附剂耐盐性鲁氏酵母培养基的优化

耐盐性鲁氏酵母可以用作吸附水中重金属的生物吸附剂。为了获得大量的耐盐性鲁氏酵母,采用响应面分析法对鲁氏酵母CICC1379培养基进行了优化。单因素试验结果表明,最佳碳源为葡萄糖和麦芽糖、最佳氮源为酵母膏。部分因子试验结果表明,葡萄糖、麦芽糖和酵母膏对生物量影响显著,硫酸镁、氯化钠、磷酸二氢钾和氯化钙对生物量影响不显著。由中心组合试验及响应面分析优化确定培养基组成:葡萄糖5.0%,麦芽糖3.3%,酵母膏2.8%,硫酸镁0.3%和氯化钠0.6%,得到二次拟合回归模型,并预测最大生物量为10.13 mg/mL。验证试验表明了该模型的正确性。利用该优化培养基培养得到的生物量比麦芽汁、YEPD、YM培养基分别增加了44.0%、82.8%和102.6%,提升了鲁氏酵母CICC1379作为生物吸附剂的应用潜力。     

甘油与鲁氏酵母共固定在高盐稀态酱油酿造中的应用

本文旨在研究相容性溶质之一的甘油对高渗透压环境下鲁氏结合酵母的保护作用,以及将甘油与鲁氏结合酵母共固定对高盐稀态酱油发酵的影响。添加不同浓度甘油至含3 mol/L氯化钠的YPD培养基中,观察酵母菌生长情况。同时,共固定甘油与鲁氏结合酵母,应用于高盐稀态酱醪发酵。结果显示,1.37 mmol/L甘油即可有效缓解高盐环境对鲁氏结合酵母的生长抑制作用,生长延滞期缩短达4~6 h。高盐稀态酱油发酵中试结果显示,与空白对照组相比,试验组蛋白质转化率提高幅度达2.23~5.64%,头油中各理化指标平均提前15 d达到空白组相同水平。综上所述,甘油作为一种相容性溶质可有效保护高盐环境下鲁氏结合酵母的生长繁殖,及其在高盐稀态酱醪发酵中的代谢性能,甘油与鲁氏接合酵母共固定技术能够提高酱油发酵的原料利用率、缩短发酵周期,在酱油酿造领域具有一定的实际应用价值。     

Molecular cloning and sequence analysis of Zygosaccharomyces rouxii ADE2 gene encoding a phosphoribosyl-aminoimidazole carboxylase.

The nucleotide sequence of a 2.8 kb fragment containing the ADE2 gene of the osmotolerant yeast Zygosaccharomyces rouxii has been determined. The gene was cloned from a Z. rouxii genomic DNA library by complementation of the Saccharomyces cerevisae ade2 mutant strain. The sequenced DNA fragment contains a 1710 bp open reading frame predicting a protein of 570 amino acids. The deduced amino acid sequence shares a high degree of homology with Ade2p homologues in five other yeast species.     

鲁氏酵母脱除水溶液中Cd~(2+)的研究

研究了鲁氏酵母(Zygosaccharomyces rouxii)CICC1379作为吸附剂对水溶液中镉的脱除作用。结果表明:温度和振荡频率对Cd2+脱除率影响不大,初始pH和菌体浓度则影响很大。初始pH3-5,脱除率均随pH增大而增大。pH5-7是鲁氏酵母对低、高2组溶液的脱除Cd2+的最适pH。Cd2+脱除率随时间变化的曲线表明,酵母在10.22mg/L和53.39mg/LCd2+溶液中,在5min内完成大部分吸附并在30min内达到吸附平衡。     

Cloning,sequencing and characterization of a gene encoding dihydroxyacetone kinase from Zygosaccharomyces rouxii NRRL2547

The dihydroxyacetone pathway, an alternative pathway for the dissimilation of glycerol via reduction by glycerol dehydrogenase and subsequent phosphorylation by dihydroxyacetone (DHA) kinase, is activated in the yeasts Saccharomyces cerevisiae and Zygosaccharomyces rouxii during osmotic stress. In experiments aimed at investigating the physiological function of the DHA pathway in Z. rouxii, a typical osmotolerant yeast, we cloned and characterized a DAK gene encoding dihydroxyacetone kinase from Z. rouxii NRRL 2547. Sequence analysis revealed a 1761 bp open reading frame, encoding a peptide composed of 587 deduced amino acids with the predicted molecular weight of 61 664 Da. As the amino acid sequence was most closely homologous (68% identity) to the S. cerevisiae Dak1p, we named the gene and protein ZrDAK1 and ZrDak1p, respectively. A putative ATP binding site was also found but no consensus element associated with osmoregulation was found in the upstream region of the ZrDAK1 gene. The ZrDAK1 gene complemented a S. cerevisiae W303-1A dak1delta dak2 delta strain by improving the growth of the mutant on 50 mmol/l dihydroxyacetone and by increasing the tolerance to dihydroxyacetone in a medium containing 5% sodium chloride, suggesting that it is a functional homologue of the S. cerevisiae DAK1. However, expression of the ZrDAK1 gene in the S. cerevisiae dak1delta dak2 delta strain had no significant effect on glycerol levels during osmotic stress. The ZrDAK1 sequence has been deposited in the public data bases under Accession No. AJ294719; regions upstream and downstream of ZrDAK1are deposited as Accession Nos AJ294739 and AJ294720, respectively.     

基于响应面法的鲁氏酵母发酵培养基优化

鲁氏酵母（Zygosaccharomyces rouxii ）是酱油及酱类生产中风味形成的重要微生物之一。为了获得大量菌体,采用响应面法（RSM）对鲁氏酵母CCTCC M 2013310培养基进行了优化,Plackett-Burman（PB）设计对培养基中相关影响因素的效应进行评价,结果表明,葡萄糖、玉米浆和磷酸二氢钾对生物量影响显著。由中心组合及响应面分析优化确定优化培养基为葡萄糖12.03%,玉米浆2.24%,酵母粉1%,磷酸二氢钾0.26%,甘油3%,VB1 0.001%。优化培养基的生物量为28.28 g/L,比未优化前提高了4.5倍。     

Characterization of the Na+-ATPase gene (ZENA1) from the salt-tolerant yeast Zygosaccharomyces rouxii

In order to clarify the relationship between the salt tolerance of Zygosaccharomyces rouxii and the function of Na+-ATPase, a gene which exhibited homology to the Na+-ATPase gene (ZENA1) of Saccharomyces cerevisiae was isolated from Z. rouxii. This newly isolated gene (ZENA1) encoded a product of 1048 amino acids. The predicted amino-acid sequence of Zena1p was highly homologous to that of S. cerevisiae Ena1p and Ena2p, and Schwanniomyces occidentalis Ena1p and Ena2p, but showed low homology to that of Zpma1p, which is the product of the Z. rouxii plasma membrane H+.ATPase gene (ZENA1). Zena1p shares the peptide motifs which have been suggested to participate in the function of ATPase. Expression of ZENA1 was observed, but was independent of NaCl shock. When ZENA1 was expressed in salt-sensitive S. cerevisiae under the regulation of a GAL1 promoter by using the expression vector pYES2, salt tolerance of the transformants was observed. The growth characteristics of Zena1Delta-disruptants of Z. rouxii and the pH profiles of their plasma membrane ATPase activity were almost the same as those of the wild-type strain, indicating that the function of Zena1p is of little relevance to the salt tolerance property of Z. rouxii. By considering the close relationship between the salt tolerance of Z. rouxii and the function of its Na+/H+-antiporter, we concluded that the extrusion of Na+ across the plasma membrane in Z. rouxii cells might be carried out mainly via the function of the Na+/H+-antiporter in a high salinity environment.     

Characterization of Na+/H+-antiporter gene closely related to the salt-tolerance of yeast Zygosaccharomyces rouxii

In order to clarify the relationship between salt-tolerance of Zygosaccharomyces rouxii and the function of Na⁺/H⁺-antiporter, a gene was isolated from Z. rouxii which exhibited homology to the Na⁺/H⁺-antiporter gene (sod2) from Schizosaccharomyces pombe. This newly isolated gene (Z-SOD2) encoded a product of 791 amino acids, which was larger than the product encoded by its Sz. pombe homologue. The predicted amino-acid sequence of Z-Sod2p was highly homologous to that of the Sz. pombe protein, but included an extra-hydrophilic stretch in the C-terminal region. The expression of Z-SOD2 was constitutive and independent of NaCl-shock. Z-SOD2-disruptants of Z. rouxii did not grow in media supplemented with 3 M -NaCl, but grew well in the presence of 50% sorbitol, indicating that the function of Z-SOD2 was closely related to the salt-tolerance of Z. rouxii. Several genes are also compared and discussed in relation to the salt-tolerance of Z. rouxii. The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the following accession number: D43629.     

鲁氏酵母发酵剂喷雾干燥参数优化及动力学研究

为制备鲁氏酵母发酵剂,研究喷雾干燥工艺对鲁氏酵母发酵剂活菌存活率的影响。通过单因素及正交试验,确定最佳喷雾干燥工艺参数;以最重要因素进口温度为基础,研究喷雾干燥过程的动力学特性,应用Sigma Plot软件构建理论与经验结合的喷雾干燥动力学模型。结果显示,鲁氏酵母喷雾干燥工艺最优参数为进口温度120℃、进料流量17%(蠕动泵转速占比)、保护剂与菌泥比例1∶1(g/L)。在喷雾干燥过程中,活菌存活率符合Polynomial模型函数,在温度范围内,存活率变化符合二次线性函数模型。     

Effect of Tetragenococcus halophilus,Zygosaccharomyces rouxii,and Torulopsis versatilis addition sequence on soy sauce fermentation

The aim of this study was to investigate the effect that the sequences of ingredient addition had on soy sauce fermentation. Five strategies were assessed for the inoculation of Tetragenococcus halophilus, Zygosaccharomyces rouxii, and Torulopsis versatilis. The results of physicochemical analysis and levels of flavor compounds demonstrated that the best strategy for soy sauce fermentation was to inoculate T. halophilus (2 × 10⁵ CFU/mL) on the 15th day, Z. rouxii (1.0 × 10⁶ CFU/mL) on the 30th day, and T. versatilis (1.0 × 10⁶ CFU/mL) on the 45th day; this enhanced its fruity, sauce, alcoholic, and caramel-like flavors by 64.3%, 22.7%, 43.1%, and 36.2%, respectively, and saline taste by 64.3%. Spearman correlation analysis revealed the content of ethanol positively correlated with acetic acid (r = 0.82) and succinic acid (r = 0.80), while the level of formic acid negatively correlated with ethyl phenylacetate (r = −0.88). In summary, these results provided fundamental evidence for strain addition sequence for producing flavor-enhanced soy sauce with potential application in the future.     

Genome shuffling of Zygosaccharomyces rouxii to accelerate and enhance the flavour formation of soy sauce

BACKGROUND: The purpose of this study was to achieve rapid improvement of the flavour of soy sauce by increasing the salt stress resistance of Zygosaccharomyces rouxii. Here, we describe genome shuffling to improve the salt tolerance of Z. rouxii while simultaneously enhancing and accelerating flavour formation of soy sauce. RESULTS: A mutant, S3‐2, with a stronger resistance to salt, was selected after three rounds of genome shuffling. S3‐2 not only grew well in peptone/yeast extract/dextrose medium containing a high salt content with wide range of pH, but also exhibited stronger stress resistance to potassium chloride and lithium chloride. In high‐salt liquid fermentation, S3‐2 obviously accelerated flavour formation of soy sauce, thus decreasing the total time required for development of the aroma. In addition, S3‐2 gave high amino acid nitrogen and good flavour. In particular, the ethyl acetate content was 2.38 times that in the control. S3‐2 distinctly improved the formation of 4‐hydroxy‐2 (or 5) ‐ethyl‐5 (or 2) ‐methyl‐3 (2H) ‐furanone by up to 75%. Another important flavour component, 4‐ethylguaiacol, was also detected. CONCLUSIONS: Genome shuffling was successfully used to achieve significant improvements in flavour formation. The selected strain improved the main flavour components and amino acid nitrogen, thereby enhancing the quality of soy sauce. Copyright © 2009 Society of Chemical Industry     

A set of plasmids carrying antibiotic resistance markers and Cre recombinase for genetic engineering of nonconventional yeast Zygosaccharomyces rouxii

The so-called nonconventional yeasts are becoming increasingly attractive in food and industrial biotechnology. Among them, Zygosaccharomyces rouxii is known to be halotolerant, osmotolerant, petite negative, and poorly Crabtree positive. These traits and the high fermentative vigour make this species very appealing for industrial and food applications. Nevertheless, the biotechnological exploitation of Z. rouxii has been biased by the low availability of genetic engineering tools and the recalcitrance of this yeast towards the most conventional transformation procedures. Centromeric and episomal Z. rouxii plasmids have been successfully constructed with prototrophic markers, which limited their usage to auxotrophic strains, mainly derived from the Z. rouxii haploid type strain Centraalbureau voor Schimmelcultures (CBS) 732^T. By contrast, the majority of industrially promising Z. rouxii yeasts are prototrophic and allodiploid/aneuploid strains. In order to expand the genetic tools for manipulating these strains, we developed two centromeric and two episomal vectors harbouring KanMX^R and ClonNAT^R as dominant drug resistance markers, respectively. We also constructed the plasmid pGRCRE that allows the Cre recombinase-mediated marker recycling during multiple gene deletions. As proof of concept, pGRCRE was successfully used to rescue the kanMX–loxP module in Z. rouxii ATCC 42981 G418-resistant mutants previously constructed by replacing the MATα^P expression locus with the loxP–kanMX–loxP cassette.     

外源相容性溶质对S酵母耐盐性及酱油风味形成的研究

研究外源相容性溶质对S酵母(Zygosaccharomyces rouxii)耐盐性的影响,并探讨在高盐稀态酿造工艺中添加海藻糖对酱油风味的影响.结果表明,在酵母培养基中添加0.005mol/L海藻糖、甜菜碱和山梨醇,均可以显著改善S酵母在高盐培养基中的生长情况,缩短渗透胁迫条件下S酵母的延滞期,增加生物量,其中海藻糖的渗透保护效果最佳;添加海藻糖可使酱油的主体香味物质HEMF(4-羟基-2-乙基-5-甲基-3-呋喃酮)和乙酸乙酯的含量显著增加,同时促进S酵母合成酱油中典型风味物质4-EG (4-乙基愈创木酚).在酱油高盐稀态酿造中添加相容性溶质,如海藻糖,可有效改善酱油的风味和品质.