Double mutation of the <Emphasis Type="Italic">PDC1</Emphasis> and <Emphasis Type="Italic">ADH1</Emphasis> genes improves lactate production in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing the bovine lactate dehydrogenase gene

Expression of a heterologous l-lactate dehydrogenase (l-ldh) gene enables production of optically pure l-lactate by yeast Saccharomyces cerevisiae. However, the lactate yields with engineered yeasts are lower than those in the case of lactic acid bacteria because there is a strong tendency for ethanol to be competitively produced from pyruvate. To decrease the ethanol production and increase the lactate yield, inactivation of the genes that are involved in ethanol production from pyruvate is necessary. We conducted double disruption of the pyruvate decarboxylase 1 (PDC1) and alcohol dehydrogenase 1 (ADH1) genes in a S. cerevisiae strain by replacing them with the bovine l-ldh gene. The lactate yield was increased in the pdc1/adh1 double mutant compared with that in the single pdc1 mutant. The specific growth rate of the double mutant was decreased on glucose but not affected on ethanol or acetate compared with in the control strain. The aeration rate had a strong influence on the production rate and yield of lactate in this strain. The highest lactate yield of 0.75 g lactate produced per gram of glucose consumed was achieved at a lower aeration rate.     

Biological acidification during grape must fermentation using mixed cultures of <Emphasis Type="Italic">Kluyveromyces thermotolerans</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Four mixed culture fermentations of grape must were carried out with Kluyveromyces thermotolerans strain TH941 and Saccharomyces cerevisiae strain SCM952. In the first culture, both yeasts were added together, whereas in the remaining three cultures S. cerevisiae was added 1, 2, and 3 days after the inoculation of K. thermotolerans. The growth and survival of the K. thermotolerans strain and the amount of the produced l-lactic acid depend on the time of inoculation of the S. cerevisiae strain and provided an effective acidification during alcoholic fermentation. The four cultures contained, respectively, at the end of fermentation 0.18, 1.80, 4.28, and 5.13 g l-lactic acid l⁻¹. The grape must with an initial pH of 3.50 was effectively acidified (70% increase in titratable acidity, 0.30 pH unit decrease) by the production of 5.13 g l-lactic acid l⁻¹.     

松萝内生酿酒酵母菌株耐酸生理特性与分子机制探究

【目的】对我国西藏地区来源的不同酵母菌株进行有机酸发酵性能测试,此外,对具有良好产酸性能的分离自松萝内部的酿酒酵母菌株Saccharomyces cerevisiae 2-2进行耐酸性能分析,并探究其耐酸较强的分子机制。【方法】比较不同糖浓度培养基液体发酵培养过程中pH的变化,并比较低pH胁迫条件下菌株的生长,检测酿酒酵母菌株的产酸潜力和耐酸特性;对菌株2-2和模式酵母菌株S288C进行比较基因组分析,并利用实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction,RT-qPCR)分析关键基因的转录,探究菌株2-2耐酸分子机制。【结果】松萝内生酿酒酵母2-2在所有检测的菌株中产酸潜力较大,耐酸性能较好。在菌株2-2中与胁迫耐受性相关的基因PDR15、PDR12和SUR1在低pH胁迫条件下存在显著的上调或下调,但这些基因转录变化趋势与菌株S288C相反。【结论】松萝内生酿酒酵母2-2是一株产酸耐酸性能较好的菌株,对其独特的调节机制进行深入分析,有希望选育性能更好的产酸酵母菌株。     

URA3基因影响青蒿酸酿酒酵母工程菌中试发酵产量

CRISPR/Cas9基因编辑技术已经被广泛应用于工程酿酒酵母的基因插入、基因替换和基因敲除,通过使用选择标记进行基因编辑具有简单高效的特点。前期利用CRISPR/Cas9系统敲除青蒿酸生产菌株酿酒酵母(Saccharomyces cerevisiae) 1211半乳糖代谢负调控基因GAL80,获得菌株S. cerevisiae 1211-2,在不添加半乳糖诱导的情况下,青蒿酸摇瓶发酵产量达到了740 mg/L。但在50 L中试发酵实验中,S. cerevisiae 1211-2很难利用对青蒿酸积累起到决定性作用的碳源-乙醇,青蒿酸的产量仅为亲本菌株S.cerevisiae 1211的20%–25%。我们推测因遗传操作所需的筛选标记URA3突变,影响了其生长及青蒿酸产量。随后我们使用重组质粒pML104-KanMx4-u连同90 bp供体DNA成功恢复了URA3基因,获得了工程菌株S. cerevisiae 1211-3。S. cerevisiae 1211-3能够在葡萄糖和乙醇分批补料的发酵罐中正常生长,其青蒿酸产量超过20g/L,与亲本菌株产量相当。研究不但获得了不加半乳糖诱导的青...     

Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

Isolation and identification of killer yeasts from Agave sap (aguamiel) and pulque

Wild killer yeasts have been identified as inhibitory to strains used as starters in the production of alcoholic beverages such as beer and wine; therefore, killer or killer-resistant strains have been sought for use in alcoholic fermentations. In the current paper a total of 16 strains belonging to six species were isolated. From two samples of Agave sap (aguamiel) the following yeast strains were isolated: Candida lusitaneae (1), Kluyveromyces marxianus var. bulgaricus (2), and Saccharomyces cerevisiae (capensis) (1). Additionally, in seven samples of pulque (the fermented product), the species C. valida (six strains), S. cerevisiae (chevalieri) (4), S. cerevisiae (capensis) (1), and K. marxianus var. lactis (1) were found. The killer strains were C. valida and K. marxianus var. lactis from pulque and K. marxianus var. bulgaricus from aguamiel. One strain of S. cerevisiae (chevalieri) isolated from pulque which did not show killer activity was, on the other hand, resistant to other killer strains and it had a remarkable ethanol tolerance, suggesting that this strain could be used for alcohol production.     

Influence of wine-related physicochemical factors on the growth and metabolism of non-<Emphasis Type="Italic">Saccharomyces</Emphasis> and <Emphasis Type="Italic">Saccharomyces</Emphasis> yeasts in mixed culture

The influence of two physicochemical factors involved in winemaking, temperature and SO₂, on the kinetics and metabolic behavior of Kloeckera apiculata and Saccharomyces cerevisiae was examined. Highest biomass was reached at 15 and 25°C for K. apiculata and S. cerevisiae, respectively. Pure cultures of K. apiculata died off early with increasing temperature, but in co-culture with S. cerevisiae it showed higher viability and a change in the death curve from exponential to linear. Statistical analysis revealed that metabolite production was significantly different for the three cultures and also at the different fermentation temperatures. Besides, the interaction between culture type and temperature was significant. At temperatures from 15 to 30°C the mixed culture showed similar ethanol and lower acetic acid production compared with a pure culture of K. apiculata. SO₂ addition slightly increased survival of the non-Saccharomyces species in pure and mixed cultures. Statistical evaluation indicated that culture type and SO₂ addition significantly affected metabolite production, but the interaction between culture and SO₂ was not significant. These results contribute to current knowledge of enological factors and their effect on prevalence and fermentative activities of the composite yeast flora and the statistical significance emphasizes the importance of the combined influence of the culture type and physicochemical factors on the production of fermentation metabolites.     

Screening of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> wine strains for the production of acetic acid

In this study 80 wine strains of Saccharomyces cerevisiae were characterized for the production of acetic acid. A significant variability in the production levels was determined among the strains, which produced from a few mg/l to more than 1 g/l. Fifteen strains, differing in acetic acid production, were tested in fermentation of grape musts of different varieties (Aglianico Basilicata, Aglianico Apulia, Cannonau, Bombino nero, Nero d'Avola, Vermentino, Fiano). The results emphasized a great strain variability, but the best strain behaviour was strictly related to the must composition, which is a determinant factor on the expression of the best strain potentiality. Therefore, this study, confirming the high/low production of acetic acid as a strain characteristic, demonstrated also that the inoculated fermentation becomes more advantageous when the starter culture is chosen in relation to the interaction of yeast strain/vine variety.     

Pseudohyphal growth is induced in Saccharomyces cerevisiae by a combination of stress and cAMP signalling

In Saccharomyces cerevisiae pseudohyphae formation may be triggered by nitrogen deprivation and is stimulated by cAMP. It was observed that even in a medium with an adequate nitrogen supply, cAMP can induce pseudohyphal growth when S. cerevisiae uses ethanol as carbon source. This led us to investigate the effects of the carbon source and of a variety of stresses on yeast morphology. Pseudohyphae formation and invasive growth were observed in a rich medium (YP) with poor carbon sources such as lactate or ethanol. External cAMP was required for the morphogenetic transition in one genetic background, but was dispensable in strain 1278b which has been shown to have an overactive Ras2/cAMP pathway. Pseudohyphal growth and invasiveness also took place in YPD plates when the yeast was subjected to different stresses: a mild heat-stress (37 °C), an osmotic stress (1 m NACl), or addition of compounds which affect the lipid bilayer organization of the cell membrane (aliphatic alcohols at 2%) or alter the glucan structure of the cell wall (Congo red). We conclude that pseudohyphal growth is a physiological response not only to starvation but also to a stressful environment; it appears to require the coordinate action of a MAP kinase cascade and a cAMP-dependent pathway.     

Effect of <Emphasis Type="Italic">Agave tequilana</Emphasis> age,cultivation field location and yeast strain on tequila fermentation process

The effect of yeast strain, the agave age and the cultivation field location of agave were evaluated using kinetic parameters and volatile compound production in the tequila fermentation process. Fermentations were carried out with Agave juice obtained from two cultivation fields (CF1 and CF2), as well as two ages (4 and 8 years) and two Saccharomyces cerevisiae yeast strains (GU3 and AR5) isolated from tequila fermentation must. Sugar consumption and ethanol production varied as a function of cultivation field and agave age. The production of ethyl acetate, 1-propanol, isobutanol and amyl alcohols were influenced in varying degrees by yeast strain, agave age and cultivation field. Methanol production was only affected by the agave age and 2-phenylethanol was influenced only by yeast strain. This work showed that the use of younger Agave tequilana for tequila fermentation resulted in differences in sugar consumption, ethanol and volatile compounds production at the end of fermentation, which could affect the sensory quality of the final product.     

ATP limitation in a pyruvate formate lyase mutant of <Emphasis Type="Italic">Escherichia coli</Emphasis> MG1655 increases glycolytic flux to <Emphasis Type="SmallCaps">d</Emphasis>-lactate

A derivative strain of Escherichia coli MG1655 for d-lactate production was constructed by deleting the pflB, adhE and frdA genes; this strain was designated “CL3.” Results show that the CL3 strain grew 44% slower than its parental strain under nonaerated (fermentative) conditions due to the inactivation of the main acetyl-CoA production pathway. In contrast to E. coli B and W3110 pflB derivatives, we found that the MG1655 pflB derivative is able to grow in mineral media with glucose as the sole carbon source under fermentative conditions. The glycolytic flux was 2.8-fold higher in CL3 when compared to the wild-type strain, and lactate yield on glucose was 95%. Although a low cell mass formed under fermentative conditions with this strain (1.2 g/L), the volumetric productivity of CL3 was 1.31 g/L h. In comparison with the parental strain, CL3 has a 22% lower ATP/ADP ratio. In contrast to wild-type E. coli, the ATP yield from glucose to lactate is 2 ATP/glucose, so CL3 has to improve its glycolytic flux in order to fulfill its ATP needs in order to grow. The aceF deletion in strains MG1655 and CL3 indicates that the pyruvate dehydrogenase (PDH) complex is functional under glucose-fermentative conditions. These results suggest that the pyruvate to acetyl-CoA flux in CL3 is dependent on PDH activity and that the decrease in the ATP/ADP ratio causes an increase in the flux of glucose to lactate.     

Efficient, galactose-free production of Candida antarctica lipase B by GAL10 promoter in Δgal80 mutant of Saccharomyces cerevisiae

An efficient yeast gene expression system with GAL10 promoter that does not require galactose as an inducer was developed using Δgal80 mutant strain of Saccharomyces cerevisiae. We constructed several combinations of gal mutations (Δgal1, Δgal80, Δmig1, Δmig2, and Δgal6) of S. cerevisiae and tested for their effect on efficiency of recombinant protein production by GAL10 promoter using a lipase, Candida antarctica lipase B (CalB), as a reporter. While the use of Δgal1 mutant strain required the addition of a certain amount of galactose to the medium, Δgal80 mutant strain did not require galactose. Furthermore, it was found that the recombinant CalB could be produced more efficiently (1.6-fold at 5 L-scale fermentation) in Δgal80 mutant strain than in the Δgal1 mutant. The Δgal80 mutant strain showed glucose repressible mode of expression of GAL10 promoter. Using Δgal80 mutant strain of S. cerevisiae, CalB was efficiently produced in a glucose-only fermentation at volumes up to 500 L.     

Physiological role of d-amino acid-N-acetyltransferase of Saccharomyces cerevisiae: detoxification of d-amino acids

Saccharomyces cerevisiae is sensitive to d-amino acids: those corresponding to almost all proteinous l-amino acids inhibit the growth of yeast even at low concentrations (e.g. 0.1 mM). We have determined that d-amino acid-N-acetyltransferase (DNT) of the yeast is involved in the detoxification of d-amino acids on the basis of the following findings. When the DNT gene was disrupted, the resulting mutant was far less tolerant to d-amino acids than the wild type. However, when the gene was overexpressed with a vector plasmid p426Gal1 in the wild type or the mutant S. cerevisiae as a host, the recombinant yeast, which was found to show more than 100 times higher DNT activity than the wild type, was much more tolerant to d-amino acids than the wild type. We further confirmed that, upon cultivation with d-phenylalanine, N-acetyl-d-phenylalanine was accumulated in the culture but not in the wild type and hpa3Δ cells overproducing DNT cells. Thus, d-amino acids are toxic to S. cerevisiae but are detoxified with DNT by N-acetylation preceding removal from yeast cells.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid by <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> under oxygen deprivation

In mineral salts medium under oxygen deprivation, Corynebacterium glutamicum exhibits high productivity of l-lactic acid accompanied with succinic and acetic acids. In taking advantage of this elevated productivity, C. glutamicum was genetically modified to produce d-lactic acid. The modification involved expression of fermentative d-lactate dehydrogenase (d-LDH)-encoding genes from Escherichia coli and Lactobacillus delbrueckii in l-lactate dehydrogenase (l-LDH)-encoding ldhA-null C. glutamicum mutants to yield strains C. glutamicum ΔldhA/pCRB201 and C. glutamicum ΔldhA/pCRB204, respectively. The productivity of C. glutamicum ΔldhA/pCRB204 was fivefold higher than that of C. glutamicum ΔldhA/pCRB201. By using C. glutamicum ΔldhA/pCRB204 cells packed to a high density in mineral salts medium, up to 1,336 mM (120 g l⁻¹) of d-lactic acid of greater than 99.9% optical purity was produced within 30 h.     

<Emphasis Type="Italic">MPK1</Emphasis> gene is required for filamentous growth induced by isoamyl alcohol in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains from the alcoholic fermentation

The aim of this study was to evaluate the MPK1 (SLT2) gene deletion upon filamentous growth induced by isoamyl alcohol (IAA) in two haploid industrial strains of Saccharomyces cerevisiae using oligonucleotides especially designed for a laboratory S. cerevisiae strain. The gene deletion was performed by replacing part of the open reading frames from the target gene with the KanMX gene. The recombinant strains were selected by their resistance to G418, and after deletion confirmation by polymerase chain reaction, they were cultivated in a yeast extract peptone dextrose medium + 0.5% IAA to evaluate the filamentous growth in comparison to wild strains. Mpk1 derivatives were obtained for both industrial yeasts showing the feasibility of the oligonucleotides especially designed for a laboratory strain (Σ1278b) by Martinez-Anaya et al. (In yeast, the pseudohyphal phenotype induced by isoamyl alcohol results from the operation of the morphogenesis checkpoint. J Cell Sci 116:3423–3431, 2003). The filamentation rate in these derivatives was significantly lower for both strains, as induced by IAA. This drastic reduction in the filamentation ability in the deleted strains suggests that the gene MPK1 is required for IAA-induced filamentation response. The growth curves of wild and derivative strains did not differ substantially. It is not known yet whether the switch to filamentous growth affects the fermentative characteristics of the yeast or other physiological traits. A genetically modified strain for nonfilamentous growth would be useful for these studies, and the gene MPK1 could be a target gene. The feasibility of designed oligonucleotides for this deletion in industrial yeast strains is shown.     

Killer-toxin-resistantkre12 mutants ofSaccharomyces cerevisiae: genetic and biochemical evidence for a secondary K1 membrane receptor

TheSaccharomyces cerevisiae killer toxin K1 is a secreted α/β-heterodimeric protein toxin that kills sensitive yeast cells in a receptor-mediated two-stage process. The first step involves toxin binding to β-1,6-d-glucan-components of the outer yeast cell surface; this step is blocked in yeast mutants bearing nuclear mutations in any of theKRE genes whose products are involved in synthesis and/or assembly of cell wall β-d-glucans. After binding to the yeast cell wall, the killer toxin is transferred to the cytoplasmic membrane, subsequently leading to cell death by forming lethal ion channels. In an attempt to identify a secondary K1 toxin receptor at the plasma membrane level, we mutagenized sensitive yeast strains and isolated killer-resistant (kre) mutants that were resistant as spheroplasts. Classical yeast genetics and successive back-crossings to sensitive wild-type strain indicated that this toxin resistance is due to mutation(s) in a single chromosomal yeast gene (KRE12), renderingkrel2 mutants incapable of binding significant amounts of toxin to the membrane. Sincekrel2 mutants showed normal toxin binding to the cell wall, but markedly reduced membrane binding, we isolated and purified cytoplasmic membranes from akrel2 mutant and from an isogenicKre12+ strain and analyzed the membrane protein patterns by 2D-electrophoresis using a combination of isoelectric focusing and SDS-PAGE. Using this technique, three different proteins (or subunits of a single multimeric protein) were identified that were present in much lower amounts in thekre12 mutant. A model for K1 killer toxin action is presented in which the gene product ofKRE12 functions in vivo as a K1 docking protein, facilitating toxin binding to the membrane and subsequent ion channel formation.     

Biodiversity of wild strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> as tool to complement and optimize wine quality

The principal agent in winemaking is the yeast Saccharomyces cerevisiae, which is characterized by a significant strain biodiversity. Here we report the characterization of 80 wild S. cerevisiae strains, isolated from grapes of different varieties in southern Italy, for genetic and technological variability. By PCR amplification with M13 primer a significant polymorphism was recorded and 12 different biotypes were identified among the strains. The specific strain-pattern could be used to follow the dynamics of different biotypes during the fermentation process. The analysis of experimental wines obtained by inoculated fermentations with the 80 strains showed significant differences among the wines. The level of each compound was a function of the strain performing the fermentative process. The main variables for the strain differentiation were the production of acetaldehyde and acetic acid, which ranged from 53 to 282 mg/l and from 0.20 to 1.88 g/l, respectively. Selected strains were tested in fermentation with two different grape musts, yielding experimental wines differing in the levels of secondary compounds and polyphenol content, in function of the interaction “grape must composition/yeast strain”. This finding has an applicative value for the potentiality of utilizing the resource of strain variability as a tool to individuate suitable starter cultures, which are able to complement and optimize grape quality.     

A comparison of clinical and food Saccharomyces cerevisiae isolates on the basis of potential virulence factors

Saccharomyces cerevisiae is the most widely used yeast in industrial/commercial food and beverage production and is even consumed as a nutritional supplement. Various cases of fungemia caused by this yeast species in severely debilitated traumatized or immune-deficient patients have been reported in recent years, suggesting that this species could be an opportunistic pathogen in such patients. To determine whether the industrial S. cerevisiae strains can be included in this virulent group of strains, we carried out a comparative study between clinical and industrial yeasts based on the various phenotypic traits associated with pathogenicity in two other yeast species (Candida albicans and Cryptococcus neoformans). The majority of the clinical isolates were found to secrete higher levels of protease and phospholipase, grow better at 42°C and show strong pseudohyphal growth relative to industrial yeasts. However three industrial yeast strains, one commercial wine strain, baker’s yeast and one commercial strain of S. cerevisiae (var. boulardii), were exceptions and based on their physiological traits these yeasts would appear to be related to clinical strains.     

贺兰山东麓优良本土酿酒酵母筛选及发酵特性分析

【背景】商业酵母的使用造成葡萄酒同质化问题严重,发掘优良本土酿酒酵母具有十分重要的意义。【目的】从168株宁夏本土酿酒酵母菌株中筛选出性能优良、具有出色葡萄酒发酵能力的菌株。【方法】基于杜氏管发酵试验和乙醇、高糖等耐受性试验分析产H2S能力及生长曲线测定的方法,筛选出发酵力好、耐受性强、低产H2S的本土酿酒酵母进行赤霞珠葡萄酒发酵试验,测定葡萄酒样基础理化指标、酚类物质和挥发性成分,探究筛选出的酿酒酵母发酵特性。【结果】初步筛选出发酵快速,能适应13%乙醇、350 g/L葡萄糖、250 mg/L SO2、pH 1.0的生存环境且低产H2S的4株本土酿酒酵母YC-E8、QTX-D17、QTX-D7、YQY-E18。菌株YC-E8产甘油能力强,所发酵酒样香气与商业酵母XR、F33最为接近,适用于赤霞珠葡萄酒的发酵。菌株QTX-D17发酵酒样中酒精、单宁、总酚和花色苷含量最高,表现出本土酿酒酵母优良的发酵特性。菌株QTX-D7所发酵酒样香气中乙酸乙酯、辛酸乙酯、1-壬醇等物质含量较高,赋予了葡萄酒香蕉味、苹果味、菠萝味、椰子味等愉悦花果香。【结论】最终筛选出3株优良本土酿酒酵母QTX-D17...