Saccharomyces cerevisiae strains with different degrees of ethanol tolerance exhibit different adaptive responses to produced ethanol

Two Saccharomyces cerevisiae strains with different degrees of ethanol tolerance adapted differently to produced ethanol. Adaptation in the less ethanol-tolerant strain was high and resulted in a reduced formation of ethanol-induced respiratory deficient mutants and an increased ergosterol content of the cells. Adaptation in the more ethanol-tolerant strain was less pronounced. Journal of Industrial Microbiology & Biotechnology (2000) 24, 75–78. Received 22 June 1999/ Accepted in revised form 06 October 1999     

Improved ethanol tolerance and production in strains of Clostridium thermocellum

Two Clostridium thermocellum strains were improved for ethanol tolerance, to 5% (v/v), by gradual adaptation and mutation. The best mutant gave an ethanol yield of 0.37 g/g substrate, with a growth yield 1.5 times more than its parent. Accumulation of acids and reducing sugars by the mutant strain with 5% (v/v) ethanol was lower than that of the parent strain with 1.5% (v/v) ethanol.     

Stress effect of ethanol on fermentation kinetics by stationary-phase cells of Saccharomyces cerevisiae

When 4% (v/v) ethanol was added progressively to two strains exhibiting different fermentative abilities, K1 (a commercial wine strain) and V5 (a strain derived of a wine yeast), the fermentation rate correlated directly to the ethanol concentration for both strains. In contrast, the effect of sudden addition of 2%, 4% or 6% (v/v) ethanol was different depending on the strain. While the same effect was observed for K1 whatever the way of ethanol addition, V5 required an adaptation period after the shock addition of ethanol.     

Engineering membrane asymmetry to increase medium-chain fatty acid tolerance in Saccharomyces cerevisiae

Saccharomyces cerevisiae is an attractive chassis for the production of medium-chain fatty acids, but the toxic effect of these compounds often prevents further improvements in titer, yield, and productivity. To address this issue, Lem3 and Sfk1 were identified from adaptive laboratory evolution mutant strains as membrane asymmetry regulators. Co-overexpression of Lem3 and Sfk1 [Lem3(M)-Sfk1(H) strain] through promoter engineering remodeled the membrane phospholipid distribution, leading to an increased accumulation of phosphatidylethanolamine in the inner leaflet of the plasma membrane. As a result, membrane potential and integrity were increased by 131.5% and 29.2%, respectively; meanwhile, the final OD₆₀₀ in the presence of hexanoic acid, octanoic acid, and decanoic acid was improved by 79.6%, 73.4%, and 57.7%, respectively. In summary, this study shows that membrane asymmetry engineering offers an efficient strategy to enhance medium-chain fatty acids tolerance in S. cerevisiae, thus generating a robust industrial strain for producing high-value biofuels.     

乙醇耐受性酿酒酵母菌株选育的研究进展

酿酒酵母是工业发酵生产乙醇的重要菌种,但是其发酵产物乙醇对酿酒酵母有明显的抑制作用.选育乙醇耐受性酿酒酵母是克服高浓度乙醇的抑制作用,提高乙醇产量的一条重要途径.本文对近年来国内外选育乙醇耐受性酵母的研究作一综述,旨在为乙醇耐受性酵母的选育提供参考.     

Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid–derived hydrocarbons

Fatty acid–derived hydrocarbons attract increasing attention as biofuels due to their immiscibility with water, high‐energy content, low freezing point, and high compatibility with existing refineries and end‐user infrastructures. Yeast Saccharomyces cerevisiae has advantages for production of fatty acid–derived hydrocarbons as its native routes toward fatty acid synthesis involve only a few reactions that allow more efficient conversion of carbon substrates. Here we describe major biosynthetic pathways of fatty acid–derived hydrocarbons in yeast, and summarize key metabolic engineering strategies, including enhancing precursor supply, eliminating competing pathways, and expressing heterologous pathways. With recent advances in yeast production of fatty acid–derived hydrocarbons, our review identifies key research challenges and opportunities for future optimization, and concludes with perspectives and outlooks for further research directions.     

酿酒酵母乙酸耐性分子机制的功能基因组进展

提高工业酿酒酵母对高浓度代谢产物及原料中的毒性底物等环境胁迫因素的耐受性,对提高工业生产效率具有重要的意义。乙酸是纤维素原料水解产生的主要毒性副产物之一,其对酵母细胞的生长和代谢都具有较强的抑制作用,因此,对酿酒酵母乙酸耐性分子机制的研究可为选育优良菌种提供理论依据。近年来,通过细胞全局基因表达分析和代谢组分析,以及对单基因敲除的所有突变体的表型组研究,对酿酒酵母乙酸耐性的分子机制有了更多新的认识,揭示了很多新的与乙酸毒性适应性反应和乙酸耐性提高相关的基因。综述了近年来酿酒酵母乙酸耐性的基因组规模的研究进展,以及在此基础上构建乙酸耐性提高的工业酵母菌的代谢工程操作。结合本课题组的研究,对金属离子锌在酿酒酵母乙酸耐性中的作用进行了深入分析。未来对酿酒酵母乙酸耐性分子机理的认识及改造将深入到翻译后修饰和合成生物学等新的水平,所获得的认知,将为选育可高效进行纤维素原料生物转化、高效生产生物燃料和生物基化学品的工业酿酒酵母的菌株奠定理论基础。     

酿酒酵母 L-阿拉伯糖转化乙醇代谢工程的研究进展简

L-阿拉伯糖是木质纤维素原料中一种重要的五碳糖组分,但传统的乙醇生产菌株酿酒酵母（ Saccharomyces cerevisiae）不能利用L 阿拉伯糖。通过代谢途径工程手段,在酿酒酵母中引入L 阿拉伯糖初始代谢途径可以获得能利用L 阿拉伯糖乙醇发酵的重组菌株。并且,通过代谢途径的疏通以及吸收系统的优化可以强化重组菌株代谢L 阿拉伯糖的能力。笔者从以上角度综述了近年来酿酒酵母转化L 阿拉伯糖生产乙醇的研究进展。     

利用SPT3的定向进化提高工业酿酒酵母乙醇耐受性

利用对转录因子的定向进化可对多基因控制的性状进行有效的代谢工程改造。本研究对酿酒酵母负责胁迫相关基因转录的SAGA复合体成分SPT3编码基因进行易错PCR随机突变,并研究了SPT3的定向进化对酿酒酵母乙醇耐性的影响。将SPT3的易错PCR产物连接改造的pYES2.0表达载体并转化酿酒酵母Saccharomyces cerevisiae4126,构建了突变体文库。通过筛选在高浓度乙醇中耐受性提高的突变株,获得了一株在10%(V/V)乙醇中生长较好的突变株M25。该突变株利用125g/L的葡萄糖进行乙醇发酵时,终点乙醇产量比对照菌株提高了11.7%。由此表明,SPT3是对酿酒酵母乙醇耐性进行代谢工程改造的一个重要的转录因子。     

三种选育高乙醇耐受性工业酿酒酵母方法的比较

由于乙醇耐性受多基因控制,因此需要从全基因组水平进行改造以期得到高乙醇耐受的突变体。文中分别使用紫外诱变、等离子体诱变及人工转录因子3种方法对工业酿酒酵母Sc4126进行改造,获得了乙醇耐性提高的突变体,并比较了3种方法的正突变率。人工转录因子文库转化的方法获得了最多数量的乙醇耐性突变体,高出紫外诱变和等离子体诱变方法1~2个数量级,且遗传稳定。研究结果表明,人工转录因子技术可以用于对工业酿酒酵母快速进行基因组工程改造。     

Enhancing ethanol tolerance of a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae by Mg2+ via reduction in plasma membrane permeability

Mg²⁺ at 3.5 mM increased the tolerance of a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae to ethanol. After 9 h of exposure to 20% (v/v) ethanol at 30 °C, all cells died whereas over 50% remained viable for the cells grown with Mg²⁺. The effect of Mg²⁺ is closely related to its ability to decrease plasma membrane permeability of cells subjected to ethanol stress.     

Stress tolerance of pressure-shocked Saccharomyces cerevisiae

Pressure shock treatment induced synthesis of heat shock protein (hsp104) and tolerance against various stresses such as high temperature, high pressure and high concentration of ethanol in Saccharomyces cerevisiae. The optimum pressures that induced maximal tolerance against these stresses were in the range of 50–75 MPa and depended on the type of stress. However, pressure shock did not stimulate trehalose production in the cells. © Rapid Science Ltd. 1998     

Comparisons of five Saccharomyces cerevisiae strains for ethanol production from SPORL‐pretreated lodgepole pine

The performances of five yeast strains under three levels of toxicity were evaluated using hydrolysates from lodgepole pine pretreated by Sulfite Pretreatment to Overcome the Recalcitrance of Lignocelluloses (SPORL). The highest level of toxicity was represented by the whole pretreated biomass slurry, while intermediate toxicity was represented by the hydrolysate with partial loading of pretreatment spent liquor. The zero toxicity was represented using the enzymatic hydrolysate produced from thoroughly washed SPORL lodgepole pine solids. The results indicate that strains D5A and YRH400 can tolerate the whole pretreated biomass slurry to produce 90.1 and 73.5% theoretical ethanol yield. Strains Y1528, YRH403, and FPL450 did not grow in whole hydrolysate cultures and were observed to have lower ethanol productivities than D5A and YRH400 on the hydrolysate with intermediate toxicity. Both YRH400 and YRH403 were genetically engineered for xylose fermentation but were not able to consume xylose efficiently in hydrolysate. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1076–1083, 2014     

Accelerated ethanol fermentation by Saccharomyces cerevisiae with addition of activated carbon

Fermentation with the addition of activated carbon at 100 g l^–1 promoted the glucose consumption and ethanol production rates of Saccharomyces cerevisiae by 1.3 and 1.1 times, respectively. With fermentation using spent medium, the consumption rate was maintained at 90% of that in the fresh medium with the addition of activated carbon, while the rate without any addition decreased to about 70%.     

Improved ethanol tolerance of Saccharomyces cerevisiae in mixed cultures with Kluyveromyces lactis on high-sugar fermentation

The influence of non-Saccharomyces yeast, Kluyveromyces lactis, on metabolite formation and the ethanol tolerance of Saccharomyces cerevisiae in mixed cultures was examined on synthetic minimal medium containing 20% glucose. In the late stage of fermentation after the complete death of K. lactis, S. cerevisiae in mixed cultures was more ethanol-tolerant than that in pure culture. The chronological life span of S. cerevisiae was shorter in pure culture than mixed cultures. The yeast cells of the late stationary phase both in pure and mixed cultures had a low buoyant density with no significant difference in the non-quiescence state between both cultures. In mixed cultures, the glycerol contents increased and the alanine contents decreased when compared with the pure culture of S. cerevisiae. The distinctive intracellular amino acid pool concerning its amino acid concentrations and its amino acid composition was observed in yeast cells with different ethanol tolerance in the death phase. Co-cultivation of K. lactis seems to prompt S. cerevisiae to be ethanol tolerant by forming opportune metabolites such as glycerol and alanine and/or changing the intracellular amino acid pool.     

转醛醇酶基因差异表达影响酵母发酵木糖及耐受乙酸性能

【目的】木糖发酵是纤维素燃料乙醇生产的一个关键瓶颈,同时木质纤维素水解液中的乙酸严重抑制酿酒酵母的木糖发酵过程,因此通过基因工程手段提高菌株对木糖的利用以及对乙酸的耐受性具有重要意义。本研究以非氧化磷酸戊糖途径(PPP途径)中关键基因转醛醇酶基因(TAL1)为研究对象,探讨了3种不同启动子PTDH3、PAHP1和PUBI4,控制其表达对菌株利用木糖和耐受乙酸的影响。【方法】通过同源重组用3种启动子替换酿酒酵母基因工程菌NAPX37的TAL1基因的启动子PTAL1,再通过孢子分离和单倍体交配构建了纯合子,利用批次发酵比较了在以木糖为唯一碳源和混合糖(葡萄糖和木糖)为碳源条件下,3种启动子控制TAL1基因表达导致的发酵和乙酸耐受能力的差异。【结果】启动子PTDH3、PAHP1和PUBI4在不同程度上提高了TAL1基因的转录水平,提高了菌株对木糖的利用速率及乙酸耐受能力,提高了菌株在60 mmol/L乙酸条件下的葡萄糖利用速率。在以木糖为唯一碳源且无乙酸存在、以及混合糖为碳源的条件下,PAHP1启动子控制TAL1表达菌株的发酵结果优于PTDH3和PUBI4启动子的菌株,PAHP1启动子控制的TAL1基因的转录水平比较合适。在木糖为唯一碳源且乙酸为30 mmol/L时,PUBI4启动子控制TAL1基因表达的菌株发酵结果则优于PAHP1和PTDH3启动子菌株,此时PUBI4启动子控制的TAL1的转录水平比较合适。【结论】启动子PTDH3、PAHP1和PUBI4不同程度地提高TAL1基因的表达,在不同程度上改善了酵母菌株的木糖发酵速率和耐受乙酸性能,改善程度受发酵条件的影响。