Production and Characterization Te-Peptide by Induced Autolysis of Saccharomyces Cerevisiae

Recently, the interest in mimicking functions of chalcogen-based catalytic antioxidants like selenoenzymes, has been increased. Various attempts had been done with selenium, but very few attempts were carried out with tellurium. Bio-complex formation and characterization of tellurium was not tried earlier by using any organism. The present study was focused on tellurium peptide production, characterization, and bioactivity assessment especially Mimetic to glutathione peroxidase (GPx). The production was achieved by the autolysis of total proteins obtained from Saccharomyces cerevisiae ATCC 7752 grown with inorganic tellurium. The GPx-like activity of the hydrolyzed tellurium peptide was increased when prepared by autolysis, but decreased when prepared by acid hydrolysis. Tellurium peptide produced by autolysis of the yeast cell showed increased GPx-like activity as well as tellurium content. Tellurium peptide showed little toxicity, compared to highly toxic inorganic tellurium. The results showed the potential of tellurium peptide as an antioxidant that can be produced by simple autolysis of yeast cells.     

Metabolic Engineering of Escherichia coli Cells for Ethanol Production under Aerobic Conditions

The ethanol production by recombinant Escherichia coli introducing of pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) from Zymomonas mobilis was investigated under aerobic conditions. In aerobic culture (K_La = 1.5 min^-1), the cells expressing pdc and adhB produced 0.4 g l^-1 ethanol when cultured for 18 h. This value was improved in BW25113Δpta/pHfdh/pTadhB-pdc following 4 g l^-1 formate feeding at 0.8 g l^-1 ethanol. In higher oxygenation level (K_La = 6.1 min^-1), the production of ethanol was further enhanced at 1.79 g l^-1 ± 0.37 g l^-1 after 24 h cultivation. Formate was found not detectable at the end of culture, indicating complete degradation this organic acid to regenerate NADH from NAD⁺. The culture strategy was effective to inactivate lactate dehydrogenase, which is major competitor for ethanol production in utilizing NADH.     

Ethanol Production by Fermentation Using Immobilized Cells of Saccharomyces cerevisiae in Cashew Apple Bagasse

In this work, cashew apple bagasse (CAB) was used for Saccharomyces cerevisiae immobilization. The support was prepared through a treatment with a solution of 3% HCl, and delignification with 2% NaOH was also conducted. Optical micrographs showed that high populations of yeast cells adhered to pre-treated CAB surface. Ten consecutive fermentations of cashew apple juice for ethanol production were carried out using immobilized yeasts. High ethanol productivity was observed from the third fermentation assay until the tenth fermentation. Ethanol concentrations (about 19.82–37.83 g L^?1 in average value) and ethanol productivities (about 3.30–6.31 g L^?1 h^?1) were high and stable, and residual sugar concentrations were low in almost all fermentations (around 3.00 g L^?1) with conversions ranging from 44.80% to 96.50%, showing efficiency (85.30–98.52%) and operational stability of the biocatalyst for ethanol fermentation. Results showed that cashew apple bagasse is an efficient support for cell immobilization aiming at ethanol production.     

Kinetics and Thermodynamics of Ethanol Production by Saccharomyces cerevisiae MLD10 Using Molasses

In this study, we have used ultraviolet (UV) and γ-ray induction to get a catabolite repression resistant and thermotolerant mutant with enhanced ethanol production along with optimization of sugar concentration and temperature of fermentation. Classical mutagenesis in two consecutive cycles of UV- and γ-ray-induced mutations evolved one best catabolite-resistant and thermotolerant mutant Saccharomyces cerevisiae MLD10 which showed improved ethanol yield (0.48?±?0.02 g g^?1), theoretical yield (93?±?3 %), and extracellular invertase productivity (1,430?±?50 IU l^?1 h^?1), respectively, when fermenting 180 g sugars l^?1 in molasses medium at 43 °C in 300 m³ working volume fermenter. Ethanol production was highly dependent on invertase production. Enthalpy (ΔH*) (32.27 kJ M^?1) and entropy (ΔS*) (?202.88 J M^?1 K^?1) values at 43 °C by the mutant MLD10 were significantly lower than those of β-glucosidase production by a thermophilic mutant derivative of Thermomyces lanuginosus. These results confirmed the enhanced production of ethanol and invertase by this mutant derivative. These studies proved that mutant was significantly improved for ethanol production and was thermostable in nature. Lower fermentation time for ethanol production and maintenance of ethanol production rates (3.1 g l^?1 h^?1) at higher temperature (43 °C) by this mutant could decrease the overall cost of fermentation process and increase the quality of ethanol production.     

Effect of Initial Cell Concentration on Ethanol Production by Flocculent Saccharomyces cerevisiae with Xylose-Fermenting Ability

Different initial cell concentrations of a recombinant flocculent Saccharomyces cerevisiae MA-R4 were evaluated for their effects on xylose fermentation and glucose–xylose cofermentation. A high initial cell concentration greatly increased both the substrate utilization and ethanol production rates. During xylose fermentation, the highest rates of xylose consumption (2.58 g/L h) and ethanol production (0.83 g/L h) were obtained at an initial cell concentration of 13.1 g/L. During cofermentation, the highest rates of glucose consumption (14.4 g/L h), xylose consumption (2.79 g/L h), and ethanol production (6.68 g/L h) were obtained at an initial cell concentration of 12.7 g/L. However, a high initial cell density had no positive effect on the maximum ethanol concentration and ethanol yield mainly due to the increased amount of by-products including xylitol. The ethanol yield remained almost constant (0.34 g/g) throughout xylose fermentation (initial cell concentration range, 1.81–13.1 g/L), while it was slightly lower at high initial cell concentrations (9.87 and 12.7 g/L) during cofermentation. The determination of the appropriate initial cell concentration is necessary for the improvement of substrate utilization and ethanol yield.     

Adaptive Evolution of Saccharomyces cerevisiae with Enhanced Ethanol Tolerance for Chinese Rice Wine Fermentation

High tolerance towards ethanol is a desirable property for the Saccharomyces cerevisiae strains used in the alcoholic beverage industry. To improve the ethanol tolerance of an industrial Chinese rice wine yeast, a sequential batch fermentation strategy was used to adaptively evolve a chemically mutagenized Chinese rice wine G85 strain. The high level of ethanol produced under Chinese rice wine-like fermentation conditions was used as the selective pressure. After adaptive evolution of approximately 200 generations, mutant G85X-8 was isolated and shown to have markedly increased ethanol tolerance. The evolved strain also showed higher osmotic and temperature tolerances than the parental strain. Laboratory Chinese rice wine fermentation showed that the evolved G85X-8 strain was able to catabolize sugars more completely than the parental G85 strain. A higher level of yeast cell activity was found in the fermentation mash produced by the evolved strain, but the aroma profiles were similar between the evolved and parental strains. The improved ethanol tolerance in the evolved strain might be ascribed to the altered fatty acids composition of the cell membrane and higher intracellular trehalose concentrations. These results suggest that adaptive evolution is an efficient approach for the non-recombinant modification of industrial yeast strains.     

啤酒酵母废菌体吸附Pd2+的物理化学特性

以啤酒酿造厂的啤酒酵母废菌体为生物吸附剂,研究死的啤酒酵母菌体从PdCl₂溶液中吸附Pd²⁺的物理化学特性.结果表明,该菌体吸附Pd²⁺受吸附时间、溶液pH值、菌体浓度和Pd²⁺起始浓度等因素的影响.菌体吸附Pd²⁺是个快速的过程,吸附45min时吸附量达最大,但在最初的3min内,吸附量可达到最大吸附量的92%.在5～60℃范围内,吸附作用不受温度影响.吸附作用的最适pH值为3.5.在Pd²⁺起始质量浓度为30～300mg/L范围内和菌体质量浓度为2g/L的条件下,菌体对Pd²⁺的吸附作用符合Langmuir和Freundlich等温吸附模型.在pH=3.5,Pd²⁺与菌体质量比为0.2和30℃条件下吸附60min,吸附量达94.5mg/g.从废钯催化剂处理液回收钯,吸附量为32.2mg/g.XPS分析表明,该菌体能吸附水溶液中的Pd²⁺.TEM结果表明,在无外加电子供体时,死的啤酒酵母废菌体能够吸附和还原溶液中的Pd²⁺成Pd⁰微粒,Pd⁰微粒可进一步形成有一定形状的钯晶粒;该菌体还能使吸附在γ-Al₂O₃上的Pd²⁺还原成Pd⁰.     

Effect of Acetic Acid on Saccharomyces Carlsbergensis ATCC 6269 Batch Ethanol Production Monitored by Flow Cytometry

Bioethanol produced from lignocellulosic materials has been considered a sustainable alternative fuel. Such type of raw materials have a huge potential, but their hydrolysis into mono-sugars releases toxic compounds such as weak acids, which affect the microorganisms' physiology, inhibiting the growth and ethanol production. Acetic acid (HAc) is the most abundant weak acid in the lignocellulosic materials hydrolysates. In order to understand the physiological changes of Saccharomyces carlsbergensis when fermenting in the presence of different acetic acid (HAc) concentrations, the yeast growth was monitored by multi-parameter flow cytometry at same time that the ethanol production was assessed. The membrane potential stain DiOC₆(3) fluorescence intensity decreased as the HAc concentration increased, which was attributed to the plasmic membrane potential reduction as a result of the toxic effect of the HAc undissociated form. Nevertheless, the proportion of cells with permeabilized membrane did not increase with the HAc concentration increase. Fermentations ending at lower external pH and higher ethanol concentrations depicted the highest proportions of permeabilized cells and cells with increased reactive oxygen species levels. Flow cytometry allowed monitoring, near real time (at-line), the physiological states of the yeast during the fermentations. The information obtained can be used to optimize culture conditions to improve bioethanol production.     

微藻高油脂化基因工程研究策略

石化能源危机与全球气候变化已成为人类的两大重要难题。生物柴油作为可替代普通柴油的环境友好且可再生的能源受到普遍关注。相比植物油和动物脂肪生产,藻类油脂产率高且容易培养,被认为是未来生物柴油发展的重要原料之一。通过转基因技术强化油脂代谢途径,提高富油微藻含油量,已经成为新的研究热点。已有植物研究表明,增强甘油酯酰基转移酶表达,可以提高Kennedy途径代谢中间体通量,从而增加甘油三酯(TAG)的积累。本文综述了微藻油脂代谢途径的国内外研究现状和提高油脂积累的代谢调控策略;详细阐述了基于植物油脂合成强化的成功经验,通过增强微藻Kennedy途径对提高TAG生物合成的重要作用;讨论了当前转基因微藻的遗传转化方法及其需要解决的关键性科学技术问题;分析了基因工程技术调控微藻脂类代谢途径生产高油脂的可能性,并对该研究的发展进行了展望。     

Yeasts as Producers of Polyhydroxyalkanoates: Genetic Engineering of Saccharomyces cerevisiae

The genes of the poly(β-hydroxybutyrate) (PHB) synthesis pathway in Ralstonia eutropha and Methylobacterium extorquens were successfully established in the yeast Saccharomyces cerevisiae. Expression of just the polyhydroxyalkanoate (PHA) synthase gene in some experiments, and all three PHB genes (i.e., the genes encoding β-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase) in others, were detected in S. cerevisiae. Thus, it can be used as a “cell factory” for the production of PHB. The maximum amount of polyester accumulated was 6.7% (wt./wt.) when all three genes were expressed. The amount of polymer accumulated in the transgenic yeast harboring just the PHA synthase gene was similar (5.2%), but slightly lower, indicating the necessity of expressing all three genes for high PHB contents in the cells. For viable production of the polymer in yeasts, more needs to be learned about the metabolism of the yeast, especially about the pathways and intermediates competing with formation of the biopolymer. Another host probably needs to be chosen.

Bacteria (on the top) with PHB inclusions and yeasts with storage compounds (on the bottom).     

Efficient Production of Ethanol from Empty Palm Fruit Bunch Fibers by Fed-Batch Simultaneous Saccharification and Fermentation Using Saccharomyces cerevisiae

The concentration of ethanol produced from lignocellulosic biomass should be at least 40 g l^?1 [about 5 % (v/v)] to minimize the cost of distillation process. In this study, the conditions for the simultaneous saccharification and fermentation (SSF) at fed-batch mode for the production of ethanol from alkali-pretreated empty palm fruit bunch fibers (EFB) were investigated. Optimal conditions for the production of ethanol were identified as temperature, 30 °C; enzyme loading, 15 filter paper unit g^?1 biomass; and yeast (Saccharomyces cerevisiae) loading, 5 g l^?1 of dry cell weight. Under these conditions, an economical ethanol concentration was achieved within 17 h, which further increased up to 62.5 g l^?1 after 95 h with 70.6 % of the theoretical yield. To our knowledge, this is the first report to evaluate the economic ethanol production from alkali-pretreated EFB in fed-batch SSF using S. cerevisiae.     

Improved Production of Ethanol by Novel Genome Shuffling in <Emphasis Type=Italic>Saccharomyces cerevisiae</Emphasis>

Fermentation properties under the control of multiple genes of industrial Saccharomyces cerevisiae strain are difficult to alter with traditional methods. Here, we describe efficient and reliable genome shuffling to increase ethanol production through the rapid improvement of stress resistance. The strategy is carried out using yeast sexual and asexual reproduction by itself instead of polyethylene glycol-mediated protoplast fusion. After three rounds of genome shuffling, the best performing strain S3-10 was obtained on the special plate containing a high ethanol concentration. It exhibits substantial improvement in multiple stress tolerance to ethanol, glucose, and heat. The cycle of fermentation of S3-10 was not only shortened, but also, ethanol yield was increased by up to 10.96% compared with the control in very-high-gravity (VHG) fermentations. In total, S3-10 possesses optimized fermentation characteristics, which will be propitious to the development of bioethanol fermentation industry.     

Isolation of a Dosage Dependent Lethal Mutation in Ubiquitin Gene of Saccharomyces Cerevisiae

Summary: Ubiquitin is a small protein with a highly conserved sequence, playing a pivotal role in ubiquitin proteasome system (UPS). Considering the central role UPS has in cellular homeostasis, several drugs have been developed to target UPS to remove cells responsible for cancer and other neurodegenerative diseases. As an alternative to the above approach, in the present study we have isolated dose dependent lethal form of ubiquitin gene by in vitro evolution. In vitro evolution is a powerful tool for developing proteins with novel and desirable properties. The ubiquitin gene of Saccharomyces cerevisiae was subjected to in vitro evolution and lethal mutations were selected. The ubiquitin of S. cerevisiae differs only by three residues from human ubiquitin. The mutants were selected by expressing the protein in temperature sensitive ubi4 deletion mutants of ubiquitin. Most of the mutations in ubiquitin gene failed to complement UBI4 phenotype under heat shock. Only one of the mutants caused cell lysis, even at permissive temperature. Interestingly, expression of the same protein in wild type S. cerevisiae cells left them unaffected, establishing the mutant protein as a competitive inhibitor for UPS. Sequencing of the mutant gene showed four completely novel amino acid substitutions. They are namely, Ser20 to Phe, Ala46 to Ser, Leu50 to Pro and Ile61 to Thr. Construction of the mutant ubiquitin gene and characterization of the mutant phenotype along with the nature and location of the mutations are presented.     

Further Improvements in Isomerization of Olefins in Solvent-Free Conditions

Isomerization of safrole and ethyleugenol are performed quasi-quantitatively within 5 minutes at 80°C using KOH or KOtBu and catalytic amount of transfer agent in the absence of solvent. Transfer agent can be avoided in the case of KOtBu with eventually increase in reaction time. Isolated yields of 99% are obtained from 10 g of materials.     

酿酒酵母凋亡细胞的毛细管电泳行为研究

用醋酸诱导酿酒酵母(saccharomyces cerevisiae)作为细胞凋亡过程,用扫描电镜和流式细胞术两种检测技术确证其细胞凋亡特征：用800mmol／L乙酸诱导21h后的细胞在电镜图上出现明显表面皱缩、细胞壁内陷;流式细胞图上出现典型凋亡峰——亚二倍体峰。详细探讨其高效毛细管电泳的行为,发现凋亡细胞在300nm处的强吸收峰消失;凋亡细胞核在220nm处,12～18min之间出现了一个新的分离峰。研究表明,毛细管电泳非常适于对细胞凋亡过程进行动态特征监测。     

Kinetic Modeling of Ethanol Production by Scheffersomyces stipitis from Xylose

This work focuses on the kinetics of ethanol production by Scheffersomyces stipitis on xylose with the development of a mathematical model considering the effect of substrate and product concentrations on growth rate. Experiments were carried out in batch and continuous modes, with substrate concentration varying from 7.2 to 145 g L^?1. Inhibitory effects on cell growth, substrate uptake, and ethanol production rates were found to be considerable. Kinetic parameters were obtained through linear and non-linear regression methods. Experiments in continuous mode were performed at different dilution rates to evaluate the inhibitory effect of ethanol. A mixed mathematical model which combined Andrews and Levenspiel's models, combining substrate and product inhibition, was used. A quasi-Newton routine was applied to obtain a more accurate fitting of kinetic parameters. The parameters such as cell to product factor (Y _P/X) and limiting cell yield (Y _X) were shown to be dependent on substrate concentration. The kinetic model fitted satisfactorily the experimental data.     

Ethanol production by Saccharomyces cerevisiae grown in sugarcane blackstrap molasses through a fed-batch process

We studied the effect of reactor filling time (T) (3-5 h), initial mass of inoculum (M) (1000-2100 g), and exponential time decay constant for the substrate feed rate (K) (0.6-1.6 h-1) on ethanol production by Saccharomyces cerevisiae grown in sugarcane blackstrap molasses through a fed-batch culture. The highest ethanol productivity (16.9 g/[L x h]) occurred at T = 3 h, K = 1.6 h-1, and M = 1300 g. In addition, productivity was affected by both M (for T = 3 and 4 h) and K (for T = 3 h) and varied inversely with T under any value fixed for M and K. By the quadratic regression multivariable analysis method, equations were determined to estimate ethanol yield and productivity as function of the variables studied (T, K, and M).     

Effects of Propionic Acid and pH on Ethanol Fermentation by Saccharomyces cerevisiae in Cassava Mash

The effects of propionic acid on ethanol and glycerol production by Saccharomyces cerevisiae in cassava mash were examined along with the influence of pH (4.0, 5.0, and 6.0) and of dissolved solids content (22%, 25%, and 27%). Inhibition by propionic acid increased as solids content increased and medium pH declined. Complete inhibition of ethanol fermentation was observed in mashes at pH 4.0 (60 mM propionic acid for 22% solids and 45 mM for 25% and 27%). Glycerol production linearly decreased with increased undissociated propionic acid concentration in all mashes at all pH levels, which partly contributed to increased final ethanol production when propionic acid concentration in mashes was low (≤ 30 mM).