絮凝酵母海藻糖合成酶基因TPS1启动子区的克隆和乙醇胁迫下启动子活性的变化

提高生物能源生产菌株对各种胁迫因素的耐受性对于提高生产过程的经济性和高效生产生物能源具有重要的意义。对酿酒酵母乙醇耐性的分子机制的研究,可揭示影响其耐受性的关键基因,并通过代谢工程操作定向提高酵母菌的乙醇耐受性,从而提高燃料乙醇的生产效率。海藻糖对酵母菌在多种环境胁迫下的细胞活性具有保护作用,但其对乙醇耐性分子机制的研究还不够深入。克隆了自絮凝酵母Saccharomyces cerevisiae flo的海藻糖-6-磷酸合成酶基因TPS1的启动子区域,利用pYES2.0载体骨架,构建了PTPS1启动绿色荧光蛋白EGFP标记基因的报告载体,并转化酿酒酵母ATCC4126。对酵母转化子在含有7%和10%乙醇的生长培养基中的EGFP的表达情况进行相对荧光定量分析,发现PTPS1活性在7%乙醇存在下受到强烈诱导。EGFP表达量对高温和高糖胁迫无明显差别,显示了TPS1启动子对乙醇浓度的特异响应。研究结果表明,絮凝酵母海藻糖的合成是对乙醇胁迫的保护性反应。     

适于二代燃料乙醇生产酿酒酵母的精准构制

以木质纤维素为原料的二代燃料乙醇工业生产对发酵微生物的基本要求,一是可对木质纤维素组分中的全糖发酵,二是对预处理过程产生的毒性物质具有高耐受性。酿酒酵母(Saccharomyces cerevisiae)是具有优良生产性能的传统乙醇发酵生产菌株,是适合包括二代燃料乙醇等生物基化合物转化的理想底盘细胞。近30年来,利用理性代谢工程改造、非理性适应性进化以及新兴起的合成生物学等策略,对酿酒酵母进行精准构制,极大地提高了其二代燃料乙醇生产的产业化性能。综述了适于二代燃料乙醇生产酿酒酵母精准构制过程中的己糖和戊糖代谢途径工程、辅酶工程、糖转运蛋白、抗性元件发掘以及产业化推进等方面的研究进展。     

酿酒酵母耐热元器件的筛选

以提高酿酒酵母耐热性、降低乙醇发酵过程控温能耗成本为目的,通过分析嗜热栖热菌(Thermus thermophiles)HB8热激蛋白基因,设计并构建了5个热激蛋白元器件,并导入酿酒酵母。通过梯度升温培养筛选出性能较好的耐热元器件FBA1p-groes-SLM5t,并利用恒定高温培养进一步验证了含有该元器件的酿酒酵母工程菌S.c-Gro ES具有良好的耐热性,研究表明在42℃培养48h的存活率是对照的3倍。此外,FBA1p-groes-SLM5t还能提高酵母的抗氧化性,42℃下菌株S.c-Gro ES的ROS水平比对照低37.6%,H2O2处理1 h后存活率是对照的1.62倍,说明耐热元器件在缓解热胁迫的同时对细胞的抗氧化性也有帮助。耐热工程酿酒酵母S.c-Gro ES,其40℃发酵乙醇产量相对于30℃对照和40℃对照分别提高了25%和13.8%。嗜热菌热激蛋白的引入可以明显提高酿酒酵母的耐热性及其乙醇合成效率。     

酿酒酵母乙醇耐性的分子机制及基因工程改造

提高工业微生物对毒性代谢产物及高温等环境胁迫因素的耐受性对工业生产具有重要的意义。发酵过程中产生的乙醇对酵母细胞的生长和代谢都具有较强的抑制作用,是酿酒酵母的重要环境胁迫因素之一。对酿酒酵母乙醇耐性的分子机制的研究可为选育具有较强乙醇耐受性的酵母菌种提供理论基础。近年来,通过细胞全局基因转录分析和基因功能分析,对酿酒酵母乙醇耐性的分子机制有了更多新的认识,揭示了很多新的与乙醇耐性相关的基因,并在此基础上,通过对相关基因进行过量表达或敲除,成功提高了酵母菌的乙醇耐性。以下综述了近年来酵母菌乙醇耐性的生物化学与分子生物学机制的研究进展,以及构建具有较高乙醇耐性的酵母菌的基因工程操作。这些研究不仅加深了对酿酒酵母乙醇耐性的机理认识,也可为高效进行生物转化生产生物质能源奠定理论基础。     

定向进化Rho1提高酿酒酵母的乙醇耐受性和超高浓度乙醇发酵性能

燃料乙醇发酵过程中酿酒酵母细胞活性被高浓度乙醇严重抑制而导致发酵提前终止，生产强度严重降低，因此构建同时具有高耐受性、高发酵性能的菌株一直是发酵工业追求的目标。选取酿酒酵母细胞形态调节关键基因小GTP酶家族成员Rho1,构建易错PCR产物文库，以酿酒酵母S288c为出发菌株采取“富集-自然生长-复筛”的筛选策略，成功筛选得到两株乙醇胁迫耐受性与发酵性能均提高的突变株M2和M5。测序发现突变株过表达的Rho1序列出现了3～5个氨基酸的突变和大片段的缺失突变。以300 g/L起始葡萄糖进行乙醇发酵，72 h时，M2和M5的乙醇滴度比对照菌株分别提高了19.4%和22.3%,超高浓度乙醇发酵能力显著提高。本研究为利用蛋白定向进化方法改良酵母菌复杂表型提供了新的作用靶点。     

酿酒酵母耐受单萜类化合物的机理研究进展

增强酿酒酵母对单萜的耐受性对于利用其生产单萜和利用含有单萜的生物质均具有重要意义.深入了解酿酒酵母应对单萜胁迫机理有助于构建一株较高单萜耐受性的酵母菌株,该菌株将有助于更高效率的单萜生产效率.研究表明,单萜会破坏酿酒酵母体内的氧化还原平衡,造成活性氧积累并进而导致菌体死亡.为了应对单萜诱发氧胁迫造成的损伤,酿酒酵母需要系统提升其抗氧化能力.本文归纳了酿酒酵母耐受多种典型单萜化合物胁迫机制的研究进展,并从酿酒酵母自身抗氧化机制方面,介绍了酿酒酵母应对氧胁迫的策略,并提出了进一步研究的方向.     

酿酒酵母在纤维素乙醇生产中对毒性化合物的耐受机理研究进展

利用木质纤维素生产燃料乙醇的过程中,前期预处理所产生的抑制剂会影响酵母的正常生长和后续的发酵过程。为减小抑制剂的影响所采取的一些脱毒策略往往造成糖的损失和生产成本的增加,这在实际生产与经济上是不可行的。因此,具有强的抑制剂耐受性的酿酒酵母菌株对于提高纤维素乙醇产率是十分重要的。近十年来,对于酿酒酵母胁迫耐受机制的研究取得了一些重要的进展,着重介绍目前酿酒酵母对抑制剂耐受机制的研究现状,包括一些关键性基因的表达及代谢通路过程分析等。同时也介绍一些应对抑制剂提高酵母发酵能力的措施。     

酿酒酵母对酚类抑制物耐受性研究

通过短期驯化策略提高酿酒酵母对木质纤维素预处理产生的酚类物质的耐受性。考察酚酸对酵母的抑制作用,比较驯化菌株在酚酸中生理指标,通过流式细胞仪分析酵母细胞膜完整性。单一酚酸低浓度对酿酒酵母生长和发酵没有明显抑制作用,而高浓度抑制强烈。混合酚酸具有更强的抑制作用,特别对乙醇发酵影响显著。相比原始菌株,短期驯化菌株在混合酚酸胁迫下的生长发酵动力学参数明显提高,细胞膜保持良好的完整性。酚酸对酿酒酵母生长有直接抑制作用,短期驯化能提高酵母酚酸耐受性,这与细胞膜应激反应保持其完整性有关。     

过表达长链鞘氨醇激酶基因LCB4提高酿酒酵母抑制物耐受性

木质纤维素预处理过程中产生的有毒副产物严重影响了纤维素乙醇发酵,提高酿酒酵母抑制物耐受性是提高纤维素乙醇发酵效率的有效方法。文中通过过表达LCB4基因,研究了重组菌株S288C-LCB4在乙酸、糠醛和香草醛胁迫下的细胞生长和乙醇发酵性能。结果表明,LCB4过表达菌株在分别含有10 g/L乙酸、1.5 g/L糠醛和1 g/L香草醛的平板中生长均优于对照菌株;在分别含有10 g/L乙酸、3 g/L糠醛和2 g/L香草醛的液体乙醇发酵过程中,重组菌株S288C-LCB4乙醇发酵产率分别为0.85 g/(L·h)、0.76 g/(L·h)和1.12 g/(L·h),比对照菌株提高了34.9%、85.4%和330.8%;且糠醛和香草醛胁迫下发酵时间分别缩短了30 h和44 h。根据发酵终点发酵液代谢物分析发现重组菌株比对照菌株产生了更多甘油、海藻糖和琥珀酸,这些物质有利于增强菌株的抑制物耐受性。综上所述,LCB4基因过表达可显著提高酿酒酵母S288C在乙酸、糠醛和香草醛胁迫下的乙醇发酵性能。     

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.     

Stress tolerance in a yeast sterol auxotroph: role of ergosterol, heat shock proteins and trehalose

The role of ergosterol in yeast stress tolerance, together with heat shock proteins (hsps) and trehalose, was examined in a sterol auxotrophic mutant of Saccharomyces cerevisiae. Ergosterol levels paralleled viability data, with cells containing higher levels of the sterol exhibiting greater tolerances to heat and ethanol. Although the mutant synthesised hsps and accumulated trehalose upon heat shock to the same levels as the wild-type cells, these parameters did not relate to stress tolerance. These results indicate that the role of ergosterol in stress tolerance is independent of hsps or trehalose.     

Trehalose promotes the survival of Saccharomyces cerevisiae during lethal ethanol stress, but does not influence growth under sublethal ethanol stress

Trehalose is known to protect cells from various environmental assaults; however, its role in the ethanol tolerance of Saccharomyces cerevisiae remains controversial. Many previous studies report correlations between trehalose levels and ethanol tolerance across a variety of strains, yet variations in genetic background make it difficult to separate the impact of trehalose from other stress response factors. In the current study, investigations were conducted on the ethanol tolerance of S. cerevisiae BY4742 and BY4742 deletion strains, tsl1 Δ and nth1 Δ, across a range of ethanol concentrations. It was found that trehalose does play a role in ethanol tolerance at lethal ethanol concentrations, but not at sublethal ethanol concentrations; differences of 20–40% in the intracellular trehalose concentration did not provide any growth advantage for cells incubated in the presence of sublethal ethanol concentrations. It was speculated that the ethanol concentration-dependent nature of the trehalose effect supports a mechanism for trehalose in protecting cellular proteins from the damaging effects of ethanol.     

酿酒酵母乙醇耐受性机理研究进展

酿酒酵母(Sacchromyces cerevisiae)一直是主要的生物乙醇和酿酒业发酵菌株, 具有发酵速度快、乙醇产量高特性。然而, 产物乙醇积累造成的毒性效应是限制乙醇产量的主要因素之一, 研究酿酒酵母乙醇耐受性为解决这一工业难题奠定了理论基础。本文从乙醇对酵母细胞生理、细胞结构和组分的影响, 以及酿酒酵母乙醇耐受性的遗传基础方面综述了酿酒酵母乙醇耐受性机理的研究进展。     

Changes of Saccharomyces cerevisiae cell membrane components and promotion to ethanol tolerance during the bioethanol fermentation

During bioethanol fermentation process, Saccharomyces cerevisiae cell membrane might provide main protection to tolerate accumulated ethanol, and S. cerevisiae cells might also remodel their membrane compositions or structure to try to adapt to or tolerate the ethanol stress. However, the exact changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation still remains poorly understood. This study was performed to clarify changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation. Both cell diameter and membrane integrity decreased as fermentation time lasting. Moreover, compared with cells at lag phase, cells at exponential and stationary phases had higher contents of ergosterol and oleic acid (C_18:1) but lower levels of hexadecanoic (C_16:0) and palmitelaidic (C_16:1) acids. Contents of most detected phospholipids presented an increase tendency during fermentation process. Increased contents of oleic acid and phospholipids containing unsaturated fatty acids might indicate enhanced cell membrane fluidity. Compared with cells at lag phase, cells at exponential and stationary phases had higher expressions of ACC1 and HFA1. However, OLE1 expression underwent an evident increase at exponential phase but a decrease at following stationary phase. These results indicated that during bioethanol fermentation process, yeast cells remodeled membrane and more changeable cell membrane contributed to acquiring higher ethanol tolerance of S. cerevisiae cells. These results highlighted our knowledge about relationship between the variation of cell membrane structure and compositions and ethanol tolerance, and would contribute to a better understanding of bioethanol fermentation process and construction of industrial ethanologenic strains with higher ethanol tolerance.     

Accumulation of trehalose by overexpression of tps1, coding for trehalose-6-phosphate synthase, causes increased resistance to multiple stresses in the fission yeast schizosaccharomyces pombe

Recent studies have shown that heat shock proteins and trehalose synthesis are important factors in the thermotolerance of the fission yeast Schizosaccharomyces pombe. We examined the effects of trehalose-6-phosphate (trehalose-6P) synthase overexpression on resistance to several stresses in cells of S. pombe transformed with a plasmid bearing the tps1 gene, which codes for trehalose-6P synthase, under the control of the strong thiamine-repressible promoter. Upon induction of trehalose-6P synthase, the elevated levels of intracellular trehalose correlated not only with increased tolerance to heat shock but also with resistance to freezing and thawing, dehydration, osmostress, and toxic levels of ethanol, indicating that trehalose may be the stress metabolite underlying the overlap in induced tolerance to these stresses. Among the isogenic strains transformed with this construct, one in which the gene coding for the trehalose-hydrolyzing enzyme, neutral trehalase, was disrupted accumulated trehalose to a greater extent and was more resistant to the above stresses. Increased trehalose concentration is thus a major determinant of the general stress protection response in S. pombe.     

Induction of acetic acid tolerance and trehalose accumulation by added and produced ethanol in Saccharomyces cerevisiae

Both added (49.6 g/l) and produced ethanol (46.2 g/l) caused an increase in the acetic acid tolerance of Saccharomyces cerevisiaegrown in an anaerobic chemostat; added ethanol, however, to a less extent than produced ethanol. The ethanol induced acetic acid tolerance of the cells was linked with an accumulation of trehalose within the cells. These results indicate that trehalose plays a role in the ethanol induced acetic acid tolerance of S. cerevisiae.     

Accumulation of Trehalose by Overexpression of tps1, Coding for Trehalose-6-Phosphate Synthase, Causes Increased Resistance to Multiple Stresses in the Fission Yeast Schizosaccharomyces pombe

Recent studies have shown that heat shock proteins and trehalose synthesis are important factors in the thermotolerance of the fission yeast Schizosaccharomyces pombe. We examined the effects of trehalose-6-phosphate (trehalose-6P) synthase overexpression on resistance to several stresses in cells of S. pombe transformed with a plasmid bearing the tps1 gene, which codes for trehalose-6P synthase, under the control of the strong thiamine-repressible promoter. Upon induction of trehalose-6P synthase, the elevated levels of intracellular trehalose correlated not only with increased tolerance to heat shock but also with resistance to freezing and thawing, dehydration, osmostress, and toxic levels of ethanol, indicating that trehalose may be the stress metabolite underlying the overlap in induced tolerance to these stresses. Among the isogenic strains transformed with this construct, one in which the gene coding for the trehalose-hydrolyzing enzyme, neutral trehalase, was disrupted accumulated trehalose to a greater extent and was more resistant to the above stresses. Increased trehalose concentration is thus a major determinant of the general stress protection response in S. pombe.     

代谢工程与全基因组重组构建酿酒酵母抗逆高产乙醇菌株

将酿酒酵母海藻糖代谢工程与全基因组重组技术相结合,改良工业酿酒酵母菌株的抗逆性和乙醇发酵性能。对来源于二倍体出发菌株Zd4的两株优良单倍体Z1和Z2菌株进行杂交获得基因组重组菌株Z12,并对Z1和Z2先进行(1)过表达海藻糖-6-磷酸合成酶基因 (TPS1) ,(2)敲除海藻糖水解酶基因 (ATH1), (3)同时过表达 TPS1和敲除ATH1, 经此三种基因工程操作后再进行杂交获得代谢工程菌株的全基因组重组菌株Z12ptps1、Z12 Δath1和Z12pTΔA。与亲株Zd4相比,Z12及结合代谢工程获得的菌株在高糖、高乙醇浓度与高温条件下生长与乙醇发酵性能都有不同程度的改进。对比研究结果表明:在高糖发酵条件下,同时过表达 TPS1和敲除ATH1 的双基因操作工程菌株胞内海藻糖积累、乙醇主发酵速率和乙醇产量相对于亲株的提高幅度要大于只过表达 TPS1,或敲除ATH1 的工程菌。结合了全基因组重组后获得的二倍体工程菌株Z12pTΔA,与原始出发菌株Zd4及重组子Z12相比,主发酵速率分别提高11.4%和6.3%,乙醇产量提高7.0%和4.1%,与其胞内海藻糖含量高于其它菌株、在胁迫条件下具有更强耐逆境能力相一致。结果证明,海藻糖代谢工程与杂交介导的全基因组重组相结合,是提高酿酒酵母抗逆生长与乙醇发酵性能的有效策略与技术途径。     

A novel strategy to construct yeast Saccharomyces cerevisiae strains for very high gravity fermentation

Very high gravity (VHG) fermentation is aimed to considerably increase both the fermentation rate and the ethanol concentration, thereby reducing capital costs and the risk of bacterial contamination. This process results in critical issues, such as adverse stress factors (ie., osmotic pressure and ethanol inhibition) and high concentrations of metabolic byproducts which are difficult to overcome by a single breeding method. In the present paper, a novel strategy that combines metabolic engineering and genome shuffling to circumvent these limitations and improve the bioethanol production performance of Saccharomyces cerevisiae strains under VHG conditions was developed. First, in strain Z5, which performed better than other widely used industrial strains, the gene GPD2 encoding glycerol 3-phosphate dehydrogenase was deleted, resulting in a mutant (Z5ΔGPD2) with a lower glycerol yield and poor ethanol productivity. Second, strain Z5ΔGPD2 was subjected to three rounds of genome shuffling to improve its VHG fermentation performance, and the best performing strain SZ3-1 was obtained. Results showed that strain SZ3-1 not only produced less glycerol, but also increased the ethanol yield by up to 8% compared with the parent strain Z5. Further analysis suggested that the improved ethanol yield in strain SZ3-1 was mainly contributed by the enhanced ethanol tolerance of the strain. The differences in ethanol tolerance between strains Z5 and SZ3-1 were closely associated with the cell membrane fatty acid compositions and intracellular trehalose concentrations. Finally, genome rearrangements in the optimized strain were confirmed by karyotype analysis. Hence, a combination of genome shuffling and metabolic engineering is an efficient approach for the rapid improvement of yeast strains for desirable industrial phenotypes.     

Minimization of glycerol synthesis in industrial ethanol yeast without influencing its fermentation performance

To synthesize glycerol, a major by-product during anaerobic production of ethanol, the yeast Saccharomyces cerevisiae would consume up to 4% of the sugar feedstock in typical industrial ethanol processes. The present study was dedicated to decreasing the glycerol production mostly in industrial ethanol producing yeast without affecting its desirable fermentation properties including high osmotic and ethanol tolerance, natural robustness in industrial processes. In the present study, the GPD1 gene, encoding NAD+-dependent glycerol-3-phosphate dehydrogenase in an industrial ethanol producing strain of S. cerevisiae, was deleted. Simultaneously, a non-phosphorylating NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Bacillus cereus was expressed in the mutant deletion of GPD1. Although the resultant strain AG1A (gpd1△ P(PGK)-gapN) exhibited a 48.7±0.3% (relative to the amount of substrate consumed) lower glycerol yield and a 7.6±0.1% (relative to the amount of substrate consumed) higher ethanol yield compared to the wild-type strain, it was sensitive to osmotic stress and failed to ferment on 25% glucose. However, when trehalose synthesis genes TPS1 and TPS2 were over-expressed in the above recombinant strain AG1A, its high osmotic stress tolerance was not only restored but also improved. In addition, this new recombinant yeast strain displayed further reduced glycerol yield, indistinguishable maximum specific growth rate (μ(max)) and fermentation ability compared to the wild type in anaerobic batch fermentations. This study provides a promising strategy to improve ethanol yields by minimization of glycerol production.