Expression of the AZR1 gene (ORF YGR224w), encoding a plasma membrane transporter of the major facilitator superfamily, is required for adaptation to acetic acid and resistance to azoles in Saccharomyces cerevisiae

In this work, we report results on the functional analysis of Saccharomyces cerevisiae ORF YGR224w, predicted to code for an integral membrane protein, with 14 potential transmembrane segments, belonging to the major facilitator superfamily (MFS) of transporters which are required for multiple-drug resistance (MDR). This MFS-MDR homologue is required for yeast adaptation to high stress imposed by low-chain organic acids, in particular by acetic acid, and for resistance to azoles, especially to ketoconazole and fluconazole; the encoding gene was thus named the AZR1 gene. These conclusions were based on the higher susceptibility to these compounds of an azr1Delta deletion mutant strain compared with the wild-type and on the increased resistance of both azr1Delta and wild-type strains upon increased expression of the AZR1 gene from a centromeric plasmid clone. AZR1 gene expression reduces the duration of acetic acid-induced latency, although the growth kinetics of adapted cells under acetic acid stress is apparently independent of AZR1 expression level. Fluorescence microscopy observation of the distribution of the Azr1-GFP fusion protein in yeast living cells indicated that Azr1 is a plasma membrane protein. Studies carried out to gain some understanding of how this plasma membrane putative transporter facilitates yeast adaptation to acetic acid did not implicate Azr1p in the alteration of acetic acid accumulation into the cell through the active efflux of acetate.     

提高乙酸耐受性酿酒酵母JJJ1突变株构建及转录组学分析

目的 构建JJJ1突变株以提高酿酒酵母在发酵过程中的乙酸耐受性,提高发酵效率。方法 本研究采用CRISPR-Cas9基因编辑技术构建酿酒酵母JJJ1Δ突变株,检测突变对酿酒酵母菌株的乙酸耐受性影响,基于转录组学分析突变株耐受乙酸的分子机制。结果 在含有5 g/L乙酸的液体培养基中,酿酒酵母JJJ1Δ存活率是野生型菌株的4.44倍,发酵72 h后酿酒酵母突变株JJJ1Δ的细胞生物量是野生型菌株的1.15倍,酿酒酵母JJJ1Δ的生长延滞期比野生型菌株缩短了30 h;转录组学研究表明,敲除JJJ1基因增强酿酒酵母的代谢、生物调控、膜流动性以及转运活性和电子转移活性,减少酿酒酵母细胞生理过程、细胞连接和拟核功能以及细胞结合功能。结论 敲除JJJ1基因的酿酒酵母突变株通过提高能量代谢和氨基酸合成,降低核糖体生物发生减少细胞生理活动,增强酿酒酵母菌株乙酸耐受性。     

己酸菌对高产酯酿酒酵母酒精发酵及酯醇代谢的影响

将己酸菌与高产酯酿酒酵母在高粱汁培养基中进行混合发酵,研究己酸菌对高产酯酿酒酵母酒精发酵及酯醇代谢的影响。结果表明,己酸菌菌悬液对高产酯酿酒酵母的生长及代谢没有直接影响;己酸菌的代谢产物对高产酯酿酒酵母的发酵速度及酒精产量有所促进,在培养基己酸浓度为220mg/L时,酒精产量提高了6.63%;在己酸浓度为45~220mg/L时,高产酯酿酒酵母的乙酸酯类和主要高级醇受到的抑制作用逐渐增强,乙酸乙酯最多降低65.32%,乙酸异丁酯最多降低91.32%,乙酸异戊酯最多降低82.14%,高级醇最多降低71.14%;在己酸浓度为220mg/L时,与己酸浓度为0mg/L时对比,高产酯酿酒酵母共有1032个基因转录水平上调,有1037个基因转录水平下调,这些转录水平变化的基因主要涉及到高产酯酿酒酵母的氮代谢、糖酵解、苯丙氨酸代谢、碳代谢等多条代谢途径。     

Isolation and characterization of the ATF2 gene encoding alcohol acetyltransferase II in the bottom fermenting yeast Saccharomyces pastorianus

The ATF2 gene encodes alcohol acetyltransferase II, which catalyses the synthesis of isoamyl acetate from acetyl coenzyme A and isoamyl alcohol. To characterize the ATF2 gene from the bottom fermenting yeast Saccharomyces pastorianus, the S. pastorianus ATF2 gene was cloned by colony hybridization using the S. cerevisiae ATF2 gene as a probe. When an atf1 null mutant strain was transformed with a multi-copy plasmid carrying the S. pastorianus ATF2 gene, the AATase activity of this strain was increased by 2.5-fold compared to the control. The S. pastorianus ATF2 gene has 99% nucleic acid homology in the coding region and 100% amino acid homology with the S. cerevisiae ATF2 gene. Southern blot analysis of chromosomes separated by pulse-field gel electrophoresis indicated that the ATF2 gene probe hybridized to chromosome VII in S. cerevisiae and to the 1100 kb chromosome in S. pastorianus. As S. pastorianus is thought to be a hybrid of S. cerevisiae and S. bayanus, the S. bayanus-type gene, which has a relatively low level of homology with the S. cerevisiae-type gene, is also usually detected. Interestingly, an S. bayanus-type ATF2 gene could not be detected. These results suggested that the cloned ATF2 gene was derived from S. cerevisiae. Analysis using an ATF2-lacZ fusion gene in S. pastorianus showed that expression of the ATF2 gene was relatively lower than that of the ATF1 gene and that it is repressed by aeration but activated by the addition of unsaturated fatty acids. The S. pastorianus ATF1, Lg-ATF1 and ATF2 Accession Numbers in the DDBJ Nucleotide Sequence Database are D63449, D63450 and D86480, respectively.     

Decreasing acetic acid accumulation by a glycerol overproducing strain of Saccharomyces cerevisiae by deleting the ALD6 aldehyde dehydrogenase gene

Glycerol is a major fermentation product of Saccharomyces cerevisiae that contributes to the sensory character of wine. Diverting sugar to glycerol overproduction and away from ethanol production by overexpressing the glycerol 3-phosphate dehydrogenase gene,GPD2, caused S. cerevisiae to produce more than twice as much acetic acid as the wild-type strain (S288C background) in anaerobic cell culture. Deletion of the aldehyde dehydrogenase gene, ALD6, in wild-type and GPD2 overexpressing strains (GPD2-OP) decreased acetic acid production by three- and four-fold, respectively. In conjunction with reduced acetic acid production, the GPD2-OP ald6Delta strain produced more glycerol and less ethanol than the wild-type. The growth rate and fermentation rate were similar for the modified and wild-type strains, although the fermentation rate for the GPD2 ald6Delta strain was slightly less than that of the other strains from 24h onwards. Analysis of the metabolome of the mutants revealed that genetic modification affected the production of some secondary metabolites of fermentation, including acids, esters, aldehydes and higher alcohols, many of which are flavour-active in wine. Modification of GPD2 and ALD6 expression represents an effective strategy to increase the glycerol and decrease the ethanol concentration during fermentation, and alters the chemical composition of the medium such that, potentially, novel flavour diversity is possible. The implications for the use of these modifications in commercial wine production require further investigation in wine yeast strains.     

Polymorphism of the MPR1 gene required for toxic proline analogue resistance in the Saccharomyces cerevisiae complex species

We recently discovered, on the chromosome of Saccharomyces cerevisiae sigma 1278b, novel MPR1 and MPR2 genes required for resistance to a toxic analogue of L-proline, L-azetidine-2-carboxylic acid. The MPR genes, which were absent in the S. cerevisiae genome project strain S288C, encoded a novel acetyltransferase of 229 amino acids that detoxifies the analogue by acetylating it. The MPR1 gene homologue found in Schizosaccharomyces pombe was also shown to encode a similar acetyltransferase. To further analyse the origin and the physiological role of the yeast novel gene, we report here the comparative analysis of the MPR1 gene in the S. cerevisiae complex spp. which belong to the Saccharomyces sensu stricto group. Only the type strain of S. paradoxus exhibited resistance and acetyltransferase activity to L-azetidine-2-carboxylic acid. PCR was then used to isolate the new MPR1 homologue (Spa MPR1) from S. paradoxus with the primers based on the sequence of the MPR1 gene. Gene expression and enzymatic analysis showed that the cloned Spa MPR1 gene encodes an L-azetidine-2-carboxylic acid acetyltransferase of 231 amino acids, which has 87% identity to the MPR1 protein. We also found in the protein databases that S. bayanus contains a DNA fragment that is partly homologous to the MPR1 gene. However, the gene product was considered to lose the enzymatic activity, possibly due to the gene truncation or the base substitution(s) at the important region for catalysis. Further, genomic PCR analysis showed that most of the S. cerevisiae complex spp. have the sequence highly homologous to the MPR1 gene.     

Acetate ester formation in wine by mixed cultures in laboratory fermentations

Two non-Saccharomyces wine yeast strains, Hanseniaspora guilliermondii 11104 and Pichia anomala 10590, selected as good producers of acetate esters when grown on synthetic microbiological medium, have been tested in wine fermentations as mixed cultures together with Saccharomyces cerevisiae. Wines produced using mixed cultures showed levels of acetaldehyde, acetic acid, glycerol and total higher alcohols within the ranges described for wine, whereas an increase in acetate ester concentrations was found. Ethyl acetate was the main ester produced, and isoamyl acetate and 2-phenylethyl acetate made up the next largest group of ester compounds in the wines analysed. H. guilliermondii 11104 was found to be a strong producer of 2-phenylethyl acetate in both pure and mixed cultures whereas S. cerevisiae was the best producer of ethyl esters. Mixed cultures did not influence ethyl ester levels at all.     

Effects of Saccharomyces cerevisiae boulardii (CNCM I-1079) on feed intake,blood parameters,and production during early lactation

The periparturient period is a metabolically demanding time for dairy animals because of increased nutrient requirements for milk yield. The objective of this study was to investigate the effect of feeding Saccharomyces cerevisiae boulardii (CNCM I-1079), a commercial active dry yeast (ADY), in dairy cows on productive and metabolic measures during the periparturient period. Primiparous (n = 33) and multiparous (n = 35) cows were fed a close-up total mixed ration (TMR) before calving and a lactation TMR postpartum. Three weeks before expected calving time, animals were blocked by parity and body weight and then randomly assigned to either control group (control; n = 34) or treatment (ADY; n = 34). All animals were housed in a tie-stall barn with individual feed bunks; the ADY animals received supplementary Saccharomyces cerevisiae boulardii (CNCM I-1079), top dressed daily at a predicted dosage of 1.0 × 10¹⁰ cfu (12.5 g) per head. Blood samples were collected weekly along with milk yield and milk composition data; feed intake data were collected daily. Serum samples were analyzed for glucose, nonesterified fatty acid, β-hydroxybutyrate, haptoglobin (Hp), and the cytokines tumor necrosis factor-α, IL-6, and IL-18. Colostrum samples collected within the first 6 to 10 h were analyzed for somatic cell score and IgG, IgA, and IgM concentrations. Data were analyzed using PROC GLIMMIX in SAS with time as a repeated measure; model included time, parity, treatment, and their interactions. The ADY groups had greater milk yield (39.0 ± 2.4 vs. 36.7 ± 2.3 kg/d) and tended to produce more energy-corrected milk with better feed efficiency. There was no difference in plasma glucose, serum nonesterified fatty acid, serum β-hydroxybutyrate, Hp, IL-6, or IL-18 due to ADY treatment. The tumor necrosis factor-α increased in ADY-supplemented animals (1.17 ± 0.69 vs. 4.96 ± 7.7 ng/mL), though week, parity, and their interactions had no effect. Serum amyloid A tended to increase in ADY-supplemented animals when compared to control animals and was additionally affected by week and parity; there were no significant interactions. No difference in colostrum IgG, IgA, and IgM was observed between treatments. Supplementing transition cow TMR with ADY (CNCM I-1079) improved milk production and tended to improve efficiency in early lactation; markers of inflammation were also influenced by ADY treatment, though the immunological effect was inconsistent.     

Isolation of an acetyl-CoA synthetase gene (ZbACS2) from Zygosaccharomyces bailii

A gene homologous to Saccharomyces cerevisiae ACS genes, coding for acetyl-CoA synthetase, has been cloned from the yeast Zygosaccharomyces bailii ISA 1307, by using reverse genetic approaches. A probe obtained by PCR amplification from Z. bailii DNA, using primers derived from two conserved regions of yeast ACS proteins, RIGAIHSVVF (ScAcs1p; 210-219) and RVDDVVNVSG (ScAcs1p; 574-583), was used for screening a Z. bailii genomic library. Nine clones with partially overlapping inserts were isolated. The sequenced DNA fragment contains a complete ORF of 2027 bp (ZbACS2) and the deduced polypeptide shares significant homologies with the products of ACS2 genes from S. cerevisiae and Kluyveromyces lactis (81% and 82% identity and 84% and 89% similarity, respectively). Phylogenetic analysis shows that the sequence of Zbacs2 is more closely related to the sequences from Acs2 than to those from Acs1 proteins. Moreover, this analysis revealed that the gene duplication producing Acs1 and Acs2 proteins has occurred in the common ancestor of S. cerevisiae, K. lactis, Candida albicans, C. glabrata and Debaryomyces hansenii lineages. Additionally, the cloned gene allowed growth of S. cerevisiae Scacs2 null mutant, in medium containing glucose as the only carbon and energy source, indicating that it encodes a functional acetyl-CoA synthetase. Also, S. cerevisiae cells expressing ZbACS2 have a shorter lag time, in medium containing glucose (2%, w/v) plus acetic acid (0.1-0.35%, v/v). No differences in cell response to acetic acid stress were detected both by specific growth and death rates. The mode of regulation of ZbACS2 appears to be different from ScACS2 and KlACS2, being subject to repression by a glucose pulse in acetic acid-grown cells.     

能源甜菜发酵生产酒精的工艺研究

利用ADY(Saccharomyces cerevisiae)对能源甜菜NY0503进行酒精发酵.应用Plackett-Burman设计法从底物浓度、料液比、加菌量、营养盐、加磷量、pH、转速、发酵温度和发酵时间9个因素中筛选出加磷量、发酵温度和底物浓度为主要影响因素.应用响应面分析法求得回归方程,得出最佳工艺为加磷量1.88%、发酵温度30.66℃、底物浓度11.53%,此工艺条件下酒精的最高理论转化率为96.54%.优化后按下列条件:底物浓度12%、料液比1∶1、加菌量15%、营养盐0.5、加磷量1.9%、pH5.0、转速130 r/min、发酵温度31℃和发酵时间44 h进行5批次验证实验,酒精的平均转化率为95.48%,与模型的理论值96.54%的差值仅占理论值的1.09%,进一步说明所建立的模型是切实可行的.     

高糖胁迫下槲皮素对酿酒酵母胞内损伤的保护作用与机制

以酿酒酵母S288c为模型,分析高糖胁迫下槲皮素对其胞内损伤的保护作用及机制。结果表明:与对照相比,高糖胁迫不影响酵母胞内活性氧(reactive oxygen species,ROS)水平,但显著降低了胞内酶比活力(P0.05);槲皮素处理后,与对照组和高糖组相比,酿酒酵母胞内ROS水平、超氧化物歧化酶和过氧化氢酶活力均显著下降(P0.05),而过氧化物酶(peroxidase,POD)比活力极显著升高(P0.01),说明POD比活力对高糖耐受性反应更为灵敏,可作为衡量高糖胁迫应激机制的重要生理指标,槲皮素可通过调节胞内POD比活力来提高机体的抗氧化能力。另外,实时荧光定量聚合酶链式反应结果表明高质量浓度葡萄糖显著抑制了酵母中GPD2和SUC2的表达水平(P0.05),并极显著提高了HXT1的表达水平(P0.01),而对GUT1的表达影响不显著;槲皮素处理后,高糖胁迫下酵母中GPD2、SUC2和HXT1的表达水平显著提高(P0.05),而GUT1无显著变化,说明槲皮素可能通过高渗透甘油途径、菊糖水解途径和己糖转运途径等来促进葡萄糖的分解代谢,从而达到保护机体细胞免受伤害的作用。结果表明槲皮素对高糖诱导的酿酒酵母胞内损伤具有保护作用,其作用机制可能与自身的抗氧化作用以及利用调节机体内高渗透甘油途径与糖的分解和转运途径存在一定的关联性。     

Restarting incomplete fermentations: the effect of high concentrations of acetic acid

A high concentration of acetic acid has been suggested to be a cause of incomplete fermentation. Acetic acid can reduce the growth and fermentation activity of Saccharomyces cerevisiae , and incomplete ferments can contain a high concentration of acetic acid, although the origin is often not clear. Here we examined the concentration of acetic acid which could hinder or prevent the restarting of an incomplete Cabernet Sauvignon fermentation by three acclimatized wine yeast cultures. By taking advantage of new technology which combines reverse osmosis and ion exchange, the concentration of acetic acid in an incomplete commercial ferment was adjusted to between 0.5 and 4.0 g/L. At a concentration up to 4 g/L, acetic acid did not prevent the restarting of the incomplete ferment. No lag period was observed in any of the fermentations. The amount of sugar fermented after 20–21 days was dependent on the strain of Sacch. cerevisiae and was correlated with the initial concentration of acetic acid at the time of restarting, between 0.5 and 2.0 g/L, with the concentration of residual sugar increasing with the concentration of acetic acid. In ferments which contained 4 g acetic acid/L, however, the rate of sugar consumption was sufficiently low after 20–21 days to suggest that complete fermentation would not occur, even with prolonged incubation. Effective treatment of the incomplete fermentation was therefore possible in the presence of up to 2 g acetic acid/L. Cell viability and fermentation kinetics were also strongly dependent on the strain of yeast.     

Application of molecular methods for analysing the distribution and diversity of acetic acid bacteria in Chilean vineyards

The presence of acetic acid bacteria populations on grape surfaces from several Chilean valleys is reported. The bacteria were analysed at both the species and the strain level by molecular methods such as RFLP-PCR 16S rRNA gene, RFLP-PCR ITS 16S-23S rRNA gene regions and Arbitrary Primed (AP) PCR. Our results show that there are limited numbers of species of acetic acid bacteria in the grapes and that there is a need for an enrichment medium before plating to recover the individual colonies. In the Northernmost region analysed, the major species recovered was a non-acetic acid bacteria, Stenotrophomonas maltophila. Following the North-South axis of Chilean valleys, the observed distribution of acetic acid bacteria was zonified: Acetobacter cerevisiae was only present in the North and Gluconobacter oxydans in the South. Both species were recovered together in only one location. The influence of the grape cultivar was negligible. Variability in strains was found to be high (more than 40%) for both Acetobacteraceae species.     

固体超强酸S2O82-/Fe2O3-ZnO催化合成乙酸苄酯

以乙酸和苄醇为原料,固体超强酸S2O82-/Fe2O3-ZnO为催化剂,催化合成乙酸苄酯.实验确定的最佳工艺条件为:n(苄醇):n(乙酸)=1.7,催化剂用量为0.8 g(以0.2 mol乙酸为准),带水剂环己烷用量为12 mL,反应时间为2.5 h.在此条件下,乙酸苄酯收率为85.2%.     

一次敲除BAT2同时过表达Lg-ATF1对啤酒酵母产醇酯的影响

乙酸乙酯和高级醇是啤酒中的重要风味物质,为了探究BAT2基因和Lg-ATF1基因对啤酒酵母产醇酯能力的影响,进而解决啤酒中存在的醇高酯低的问题。本研究通过酶切连接法构建重组质粒p UC-PLABBK,采用醋酸锂转化法和胞内同源重组技术,以多倍体啤酒酵母菌株S5为出发菌株,Kan MX基因作为筛选标记,最终获得过量表达Lg-ATF1基因同时敲除BAT2基因的重组菌株S5-Lg。通过啤酒发酵实验和数字PCR探究重组酵母菌株S5-Lg与出发菌株S5醇酯含量与相关基因表达量的变化。结果显示,与出发菌株相比,S5-Lg的总高级醇生成量降低9.12%,其中异丁醇和异戊醇的生成量分别降低了10.63%和9.55%,乙酸乙酯生成量提升了26.81%,BAT2基因表达量降低45.72%,Lg-ATF1基因表达量大幅提升。BAT2基因和Lg-ATF1基因可以影响啤酒酵母产生高级醇和乙酸乙酯的含量,对改善啤酒风味有重要参考意义。     

4 株本土非酿酒酵母的发酵特性

对分离于我国3 个葡萄酒产区的葡萄汁有孢汉逊酵母（Hanseniaspora uvarum）CVE-HU36、葡萄园有孢汉逊酵母（H. vineae）CVE-HV6、美极梅奇酵母（Metschnikowia pulcherrima）CVE-MP20和陆生伊萨酵母（Issatchenkia terricola）CVE-IT8共4 株非酿酒酵母菌株进行纯种发酵实验,以酿酒酵母BDX为对照,比较4 株非酿酒酵母的发酵和产香特点。结果表明,4 株非酿酒酵母菌株中CVE-HV6菌株生长能力最强、具有最大的发酵速率,并高产甘油和乙醇;CVE-HU36菌株合成乙酸能力最强;CVE-MP20菌株具有最低的乙醇产率;CVE-IT8菌株苹果酸产量较高。不同非酿酒酵母菌株的产香能力存在明显差异：CVE-HV6菌株产乙酸苯乙酯、苯乙醇和癸酸能力显著高于酿酒酵母和其他3 株非酿酒酵母,其中乙酸苯乙酯含量为酿酒酵母的3.04 倍;CVE-HU36菌株的β-香茅醇含量高于酿酒酵母（2.05 倍）和其他3 株非酿酒酵母;CVE-MP20菌株在5 株酵母菌中产乙酸乙酯和α-萜品醇能力最高,且高产异丁醇;CVE-IT8菌株高产辛酸和4-乙基愈创木酚。4 株非酿酒酵母具有不同的发酵性能和产香特性,综合来看,CVE-HV6菌株的酿造特性要优于其他3 株菌,在提升葡萄酒香气和改善品质方面具有较好的应用潜力。