Lactose/whey utilization and ethanol production by transformed Saccharomyces cerevisiae cells

Strains of Saccharomyces cerevisiae transformed with a multicopy expression vector bearing both the Escherichia coli beta-galactosidase gene under the control of the upstream activating sequence of the GAL1-10 genes and the GAL4 activator gene release part of beta-galactosidase in the growth medium. This release is due to cell lysis of the older mother cells; the enzyme maintains its activity in buffered growth media. Fermentation studies with transformed yeast strains showed that the release of beta-galactosidase allowed an efficient growth on buffered media containing lactose as carbon source as well as on whey-based media. The transformed strains utilized up to 95% of the lactose and a high growth yield was obtained in rich media. High productions of ethanol were also observed in stationary phase after growth in lactose minimal media.     

Kinetics of lactose fermentation using a recombinant Saccharomyces cerevisiae strain

This work presents a multi-route, non-structural kinetic model for interpretation of ethanol fermentation of lactose using a recombinant flocculent Saccharomyces cerevisiae strain expressing both the LAC4 (coding for beta-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces lactis. In this model, the values of different metabolic pathways are calculated applying a modified Monod equation rate in which the growth rate is proportional to the concentration of a key enzyme controlling the single metabolic pathway. In this study, three main metabolic routes for S. cerevisiae are considered: oxidation of lactose, reduction of lactose (producing ethanol), and oxidation of ethanol. The main bioprocess variables determined experimentally were lactose, ethanol, biomass, and dissolved oxygen concentrations. Parameters of the proposed kinetic model were established by fitting the experimental data obtained in a small lab-scale fermentor with the initial lactose concentrations ranging from 5 g/dm3 to 50 g/dm3. A very good agreement between experimental data and simulated profiles of the main variables (lactose, ethanol, biomass, and dissolved oxygen concentrations) was achieved.     

Intergeneric yeast fusants with efficient ethanol production from cheese whey powder solution: Construction of a Kluyveromyces marxianus and Saccharomyces cerevisiae AY‐5 hybrid

Due to its high content of lactose and abundant availability, cheese whey powder (CWP) has received much attention for ethanol production in fermentation processes. However, lactose‐fermenting yeast strains including Kluyveromyces marxianus can only produce alcohol at a relatively low level, while the most commonly used distiller yeast strain Saccharomyces cerevisiae cannot ferment lactose since it lacks both β‐galactosidase and the lactose permease system. To combine the unique aspects of these two yeast strains, hybrids of K. marxianus TY‐22 and S. cerevisiae AY‐5 were constructed by protoplast fusion. The fusants were screened and characterized by DNA content, β‐galactosidase activity, ethanol tolerance, and ethanol productivity. Among the genetically stable fusants, the DNA content of strain R‐1 was 6.94%, close to the sum of the DNA contents of TY‐22 (3.99%) and AY‐5 (3.51%). The results obtained by random‐amplified polymorphic DNA analysis suggested that R‐1 was a fusant between AY‐5 and TY‐22. During the fermentation process with CWP, the hybrid strain R‐1 produced 3.8% v/v ethanol in 72 h, while the parental strain TY‐22 only produced 3.1% v/v ethanol in 84 h under the same conditions.     

Transport of fructose by a proton symport in a brewing yeast

Abstract Saccharomyces cerevisiae IGC4261, a brewing strain, transported fructose and sorbose but not glucose by a high-affinity, low-capacity proton symport. The symport was not subject to glucose repression and coexisted with the facilitated diffusion system for glucose, fructose, sorbose and other sugars. Transport by the symport was accumulative. The stoichiometry was one proton per molecule of fructose. Maltose acted as a non-competitive inhibitor.     

Heterologous expression in Saccharomyces cerevisiae of an Arabidopsis thaliana cDNA encoding mevalonate diphosphate decarboxylase

Sequence comparison with the mevalonate diphosphate decarboxylase (MVD) amino acid sequence of Saccharomyces cerevisiae identified an EST clone corresponding to a cDNA that may encode Arabidopsis thaliana MVD (AtMVD1). This enzyme catalyses the synthesis of isopentenyl diphosphate, the building block of sterol and isoprenoid biosynthesis, and uses mevalonate diphosphate as a substrate. Sequencing of the full-length cDNA was performed. The predicted amino acid sequence presents about 55% identity with the yeast, human and rat MVDs. The sequence of the genomic region of A. thaliana MVD was also obtained and Southern blot analysis on genomic DNA showed that A. thaliana could have at least one homologous MVD gene. In order to allow heterologous expression in S. cerevisiae, the MVD open reading frame (ORF) was then cloned under the control of the yeast PMA1 strong promoter. When expressed in yeast, the A. thaliana cDNA complemented both the thermosensitive MN19-34 strain deficient in MVD, and the lethal phenotype of an ERG19 deleted strain. However, the wild-type sterol content was not fully restored suggesting that the A. thaliana MVD activity may not be optimal in yeast. A two-hybrid assay was also performed to evaluate homodimer formation of the A. thaliana MVD and heterodimer formation between the plant and yeast heterologous enzymes.     

琥珀酸对酵母细胞转录组的影响

寻找抗衰老活性小分子并研究其作用机制是衰老药物学研究的重点和热点。本文报道了一种新的抗衰老活性小分子琥珀酸,发现琥珀酸可以显著延缓芽殖酵母细胞的衰老并增强细胞的压力抗性。随后,利用DNA Microarray技术及生物信息学手段较系统分析了琥珀酸处理对基因表达谱、基因本体聚类及相关信号通路的影响。结果显示,琥珀酸处理对细胞转录组产生了显著影响,共导致3 485个基因的差异表达(P 0.05),其中1 335个基因显著上调,2 150个基因显著下调。进一步对基因本体聚类及信号通路分析显示,线粒体及核糖体生物合成相关的分子功能、细胞组分、生物学过程和信号通路可能是琥珀酸作用的主要靶点,其他可能的作用靶点还包括蛋白酶体、细胞内吞、过氧化物酶体代谢及细胞自噬等。本研究为进一步阐明琥珀酸介导的寿命及压力调控机制提供了理论参考和研究线索。     

啤酒酵母菌对无机硒的有机转化

利用啤酒酵母菌对无机硒(亚硒酸钠)进行有机转化。通过在培养基中加入不同浓度的无机硒溶液和不同时间加入无机硒溶液,于28℃、220 r/min摇床条件下培养5 d,离心得菌细胞,测定前样品预处理:破碎菌细胞,显微镜下计数,计算破碎率,破碎后的菌体装入透析袋于蒸馏水中透析除去无机硒。准确测定无机硒,用浓硫酸-高氯酸的消化体系消化样品后,紫外分光光度法于335 nm处测量吸光度,在标准曲线上查出硒含量,计算无机硒的转化率。啤酒酵母菌的最佳加硒时间为24 h,亚硒酸钠浓度大于12μg/mL对啤酒酵母菌转化无机硒有明显抑制作用,啤酒酵母菌对无机硒的摄入率约为62%,转化率约为53%;超生波细胞粉碎仪破碎细胞的破碎率为55%左右。结果表明,啤酒酵母菌可以转化无机硒。     

A comparative study of the wine fermentation performance of Saccharomyces paradoxus under different nitrogen concentrations and glucose/fructose ratios

Aims:  The main goal of the present study is to determine the effects of different nitrogen concentrations and glucose/fructose ratios on the fermentation performance of Saccharomyces paradoxus , a nonconventional species used for winemaking.
Methods and Results:  Ethanol yield, residual sugar concentration, as well as glycerol and acetic acid production were determined for diverse wine fermentations conducted by S. paradoxus . Experiments were also carried out with a commercial Saccharomyces cerevisiae wine strain used as control. The values obtained were compared to test significant differences by means of a factorial anova and the Scheffé test. Our results show that S. paradoxus strain was able to complete the fermentation even in the nonoptimal conditions of low nitrogen content and high fructose concentration. In addition, the S. paradoxus strain showed significant higher glycerol synthesis and lower acetic acid production than S. cerevisiae in media enriched with nitrogen, as well as a lower, but not significant, ethanol yield.
Conclusions:  The response of S. paradoxus was different with respect to the commercial S. cerevisiae strain, especially to glycerol and acetic acid synthesis.
Significance and Impact of the Study:  The present study has an important implication for the implementation of S. paradoxus strains as new wine yeast starters exhibiting interesting enological properties.     

Isopentenyl diphosphate and dimethylallyl diphosphate/isopentenyl diphosphate ratio measured with recombinant isopentenyl diphosphate isomerase and isoprene synthase

Isopentenyl diphosphate (IDP) and its isomer dimethylallyl diphosphate (DMADP) are building units for all isoprenoids; thus, intracellular pool sizes of IDP and DMADP play important roles in living organisms. Several methods have been used to quantify the amount of DMADP or the combined amount of IDP plus DMADP, but measuring the DMADP/IDP ratio has been difficult. In this study, a method was developed to measure the ratio of DMADP/IDP. Catalyzed by a recombinant IDP isomerase (IDI) together with a recombinant isoprene synthase (IspS), IDP was converted to isoprene, which was then detected by chemiluminescence. With this method, the in vitro equilibrium ratio of DMADP/IDP was found to be 2.11:1. IDP and DMADP pools were significantly increased in Escherichia coli transformed with methylerythritol 4-phosphate pathway genes; the ratio of DMADP/IDP was 3.85. An E. coli strain transformed with IspS but no additional IDI had a lower DMADP level and a DMADP/IDP ratio of 1.05. Approximately 90% of the IDP and DMADP pools in light-adapted kudzu leaves were light dependent and so presumably were located in the chloroplasts; the DMADP/IDP ratios in chloroplasts and cytosol were the same as the in vitro ratio (2.04 in the light and 2.32 in the dark).     

Sizing and counting of saccharomyces cerevisiae floc populations by image analysis, using an automatically calculated threshold

A standardized image analysis method has been developed permitting determination of the number of yeast flocs and their size distribution. The method includes image grabbing, image enhancement, automatic determination of the appropriate threshold, curve fitting of the areahistogram, determination of the mean single floc area and its standard deviation, and floc counting. The extension of the method to other applications is immediate and straightforward. Two Saccharomyces cerevisiae floc Populations (with ages of 48 and 72 h) were analyzed. The results showed a variation around the mean of 9%-12% for the single floc mean area, 6%-7% for the number of single flocs, and 5%-6% for the total number of flocs. Aggregates of two flocs (doublets) and three flocs (triplets) were enumerated. The correctness of the method was checked by analyzing the parameters of interest as a function of the threshold. The constant correlation between the parameters and the threshold showed the validity and consistency of the method. (c) 1996 John Wiley & Sons, Inc.     

Flux analysis of recombinant Saccharomyces cerevisiae YPB-G utilizing starch for optimal ethanol production

Genetically modified Saccharomyces cerevisiae strain (YPB-G) which secretes a bifunctional fusion protein that contains both Bacillus subtilis -amylase and Aspergillus awamori glucoamylase activities was used for the direct conversion of starch into ethanol. Starch was either supplied initially to different nutrient media or added instantaneously to the reactor at various discrete time instants (pulse feeding). Stoichiometric modeling was used to investigate the effects of initial substrate concentration and growth rate of the recombinant yeast culture on ethanol production. Reaction stoichiometries describing both the anabolism and catabolism of the microorganism were used as an input to flux balance analysis (FBA), the preferred metabolic modeling approach since the constructed stoichiometric network was underdetermined. Experiments for batch and fed-batch systems at different substrate concentrations were analyzed theoretically in terms of flux distributions using ethanol production rate as the maximization criteria. Calculated ethanol rates were in agreement with experimental measurements, suggesting that this recombinant microorganism is sufficiently evolved to optimize its ethanol production. The function of the main pathways of yeast metabolism (PPP, EMP, TCA) are discussed together with the node analyses of glucose-6-P and pyruvate branch points. Theoretical node analysis revealed that if the split ratio in G6P branch point is changed by genetic manipulations, the ethanol yield would be affected considerably.     

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.     

啤酒酵母代谢工程研究进展

啤酒工业上应用的啤酒酵母菌株在生产中都会存在着某些方面的缺陷。通过分析啤酒酵母某些代谢产物的代谢途径,寻找改变其代谢流量的方法,然后用分子生物学手段对其代谢流量加以改变,来调节啤酒酵母某些产物的代谢水平已经成为啤酒酵母育种的新方式。对酵母的底物利用、可操作性、控制有害副产物的产量及改善啤酒风味等方面的研究成果进行了综述。     

Effects of xylulokinase activity on ethanol production from D-xylulose by recombinant Saccharomyces cerevisiae

AIMS: Recombinant Saccharomyces cerevisiae strains harbouring different levels of xylulokinase (XK) activity and effects of XK activity on utilization of xylulose were studied in batch and fed-batch cultures. METHODS AND RESULTS: The cloned xylulokinase gene (XKS1) from S. cerevisiae was expressed under the control of the glyceraldehyde 3-phosphate dehydrogenase promoter and terminator. Specific xylulose consumption rate was enhanced by the increased specific XK activity, resulting from the introduction of the XKS1 into S. cerevisiae. In batch and fed-batch cultivations, the recombinant strains resulted in twofold higher ethanol concentration and 5.3- to six-fold improvement in the ethanol production rate compared with the host strain S. cerevisiae. CONCLUSIONS: An effective conversion of xylulose to xylulose 5-phosphate catalysed by XK in S. cerevisiae was considered to be essential for the development of an efficient and accelerated ethanol fermentation process from xylulose. SIGNIFICANCE AND IMPACT OF THE STUDY: Overexpression of the XKS1 gene made xylulose fermentation process accelerated to produce ethanol through the pentose phosphate pathway.     

A C6/C5 co‐fermenting Saccharomyces cerevisiae strain with the alleviation of antagonism between xylose utilization and robustness

During second‐generation bioethanol production from lignocellulosic biomass, the desired traits for fermenting microorganisms, such as Saccharomyces cerevisiae, are high xylose utilization and high robustness to inhibitors in lignocellulosic hydrolysates. However, as observed previously, these two traits easily showed the antagonism, one rising and the other falling, in the C6/C5 co‐fermenting S. cerevisiae strain. In this study, LF1 obtained in our previous study is an engineered budding yeast strain with a superior co‐fermentation capacity of glucose and xylose, and was then mutated by atmospheric and room temperature plasma (ARTP) mutagenesis to improve its robustness. The ARTP‐treated cells were grown in 50% (v/v) leachate from lignocellulose pretreatment with high inhibitors content for adaptive evolution. After 30 days, the generated mutant LF1‐6 showed significantly enhanced tolerance, with a six‐fold increase in cell density in the above leachate. Unfortunately, its xylose utilization dropped markedly, indicating the recurrence of the negative correlation between xylose utilization and robustness. To alleviate this antagonism, LF1‐6 cells were iteratively mutated with ARTP mutagenesis and then anaerobically grown using xylose as the sole carbon source, and xylose utilization was restored in the resulting strain 6M‐15. 6M‐15 also exhibited increased co‐fermentation performance of xylose and glucose with the highest ethanol productivity reported to date (0.525 g g^?1 h^?1) in high‐level mixed sugars (80 g L^?1 glucose and 40 g L^?1 xylose) with no inhibitors. Meanwhile, its fermentation time was shortened by 8 h compared to that of LF1. During the fermentation of non‐detoxified lignocellulosic hydrolysate with high inhibitor concentrations at pH ~3.5, 6M‐15 can efficiently convert glucose and xylose with an ethanol yield of 0.43 g g^?1. 6M‐15 is also regarded as a potential chassis cell for further design of a customized strain suitable for production of second‐generation bioethanol or other high value‐added products from lignocellulosic biomass.     

Optimization of pro-urokinase secretion from recombinant Saccharomyces cerevisiae

Secretion of a nonglycosylated form of human pro-urokinase, also known as single-chain urinary plasminogen activator (scu-PA), from Saccharomyces cerevisiae is described. A "supersecreting" yeast strain harboring multiple copies of integrated plasmids was grown batchwise and at constant respiratory quotient (RQ) in 20-L fermenters. Because the promoters used to drive expression of the pro-urokinase genes are not tightly regulated, secretion into the culture supernatant was growth associated. Although the final cell density achieved in the perturbed-batch fermentation (45 g dry wt/L) was less than that observed in the RQ-controlled culture (77 g dry wt/L), the scu-PA titer in the perturbed-batch fermentation (1863 IU/mL) was nearly twice that attained at constant RQ (1108 IU/mL). The effects on cell growth and scu-PA titer of other process variables (pH, temperature, phosphate concentration, and medium composition) are also discussed.     

Reductive biotransformations of organic compounds by saccharomyces cerevisiae: study of oxidoreductases involved using electrophoretic zymograms

The role of oxidoreductases in reduction of carbonyl compounds was investigated by application of zymogram techniques. Eight bands were observed using ethanol with nicotinamide adenine dinucleotide (NAD) as coenzyme. Bands observed with lactic acid and (R)-(-)-phenyl-1,2-ethanediol with nicotinamide adenine dinucleotide phosphate (NADP) had similar R(m) values. 2-Hydroxyvalerate and malate manifested bands having similar R(m) values and were active with both NAD and NADP. Based on their structural similarity and identical R(m) values, oxidation of 1,4-cyclooctanediol (band #2) and cis-1,5-cyclooctanediol may be due to a common enzyme. The PAGE-zymogram technique may be used on a preparative scale to facilitate purification and full characterization on the observed stained bands.     

两株高糖分利用能力的酿酒酵母呼吸突变体选育

以酿酒酵母乙醇发酵高产工业菌株MF1002为始发菌株,对其营养细胞进紫外线诱变,筛选得到两株性状稳定的呼吸缺陷型突变体MF15c和MF11a。菌体细胞对2,3,5-氯化三苯四氮唑(TTC)显色测定呼吸强度的结果表明,两突变菌株的相对呼吸强度分别只有始发菌株的57.77%和47.25%。与现有报道的呼吸缺陷突变体不同,两株突变体的细胞生长速率只在培养初期略低于始发菌株,总体生长速率与始发菌株几乎没有差异,在YPD平板上培养也不形成小菌落。比较蔗糖发酵试验表明,两株突变体的乙醇产量较始发菌株只分别略提高6.48%-6.59%(MF15c)和1.66%-1.97%(MF11a),但发酵终止的残总糖含量却显著低于始发菌株,分别减少34.85%和19.70%,发酵效率较始发菌株分别显著提高6.69%和4.71%,表明这两株突变体为新型的呼吸缺陷型突变。鉴于提高乙醇发酵的发酵效率可显著降低生产成本,认为这两株突变菌株具有较高的潜在工业利用价值。