呼吸缺陷型耐糖耐高温酒精酵母菌种的筛选

采用紫外诱变和2,3,5-氯化三苯基四氮唑(TTC)鉴别性培养基,从市售酒精活性酵母粉中分离出13株呼吸缺陷型突变株.通过进一步筛选,最终筛选出一株呼吸缺陷型耐糖、耐高温的高效酒精酵母菌株,编号为T11.该菌株菌落表面湿润,乳白色,小且圆;细胞椭圆形(2.5～5.0μ×3.75～6.25μ),出芽繁殖,在25%(W/V)葡萄糖发酵液,起始pH 5.5,40℃条件下发酵,酒精发酵产酒精率和糖利用率分别可达45.54%和98.01%.     

酵母菌小菌落突变株酒精发酵的研究——Ⅰ.菌种的选育及其生理特性

酵母菌小菌落呼吸缺陷型突变株(Petite mutant)的研究国外已有许多报道。Bacila等证明ρ~-突变株在5L发酵罐中酒精发酵产量比野生型ρ~+约提高10%左右。我们用诱变剂对酒精工业上使用的酵母菌进行处理,得到呼吸缺陷型突变株,这些菌与氯     

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。     

降低光滑球拟酵母电子传递链活性加速丙酮酸合成

光滑球拟酵母CCTCCM2 0 2 0 19经溴化乙锭诱变 ,挑选假阳性呼吸缺陷型菌株共 4 0株。对其中 7株丙酮酸产量提高的突变株进行发酵性底物 (葡萄糖 )和非发酵性底物 (甘油、乙酸 )的利用能力测试 ,鉴定得到 3株呼吸缺陷型突变株RD 16、RD 17和RD 18。相对于出发菌株 ,呼吸缺陷型突变株生长速率下降 ,最终菌体浓度降低 2 1%～2 9% ,胞内ATP含量下降 15 %～ 2 1% ,但单位细胞耗葡萄糖能力和单位细胞产丙酮酸能力分别提高了 2 0 7%～30 7%和 30 7%～ 5 5 5 %。进一步研究发现 ,呼吸缺陷型突变株线粒体复合体Ⅰ、Ⅰ Ⅲ、Ⅱ Ⅲ和Ⅳ的活性分别下降了 34%～ 4 1%、38 6 %～ 5 2 6 %、2 1%～ 2 5 %、15 0 %～ 6 30 % ,表明线粒体电子传递链氧化NADH的功能受到抑制。为使酵解产生的NADH正常氧化 ,在RD 18菌株的对数生长期流加 2 1mmol L外源电子受体乙醛。发现细胞合成丙酮酸能力提高 2 1 6 % ,且葡萄糖消耗速度明显加快 ,发酵周期缩短 14h。结果表明适当削弱能量代谢能够提高真核微生物中心代谢途径的速度     

适合甘蔗汁发酵高产酒精酵母的选育

目的:选育出适合发酵甘蔗汁生产燃料乙醇的高产酿酒酵母的菌株.方法:以酵母菌株 YS,作为出发菌株,将酶解破壁后获得的原生质体进行紫外诱变,通过初筛和复筛进行选育.结果:获得一株高产酒精的酿酒酵母突变株 YSs-1,该突变株发酵甘蔗汁的乙醇含量可达12.6%(V/V),较出发菌株的 11.6%(V/V)提高了 8.6%,其糖的转化率高达 94.5%,高于出发菌株的 87.0%.结论:通过 5 次连续传代培养后其突变株的乙醇产量保持稳定,表明该突变株完全可以用于发酵甘蔗汁生产燃料乙醇.     

清酒酵母与酿酒醇母原生质体融合的研究

清酒酵母（ＳａｃｃｈａｒｏｍｙｃｅｓｓａｋｅＹａｂｅ）是日本清酒的生产菌株．耐酒精能力强;Ｋ氏酿酒酵母（ＳａｃｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅＫ）是酒精生产的常用菌株,发酵力强。本文应用原生质体融合技术进行了二菌株原生质体融合的研究。通过硫酸二乙酯（ＤＥＳ）诱变得到营养缺隐型菌株Ｑ（ａｒｇ－）和Ｋ（ｌｙｓ－,ρ－）,其融合率为１．２５×１０－５。检出的融合子其酒精发酵特性、细胞形态、体积大小都不同于双亲菌株。比较了在２８℃培养条件下,出发余株清酒酵母,Ｋ氏酿酒酵母和融合子Ｆ１、Ｆ２的发酵速度曲线、乙醇产量和酒精耐量等,得到一株在２８℃培养条件下,乙醇产量为７．４％（Ｖ／Ｖ）,酒精耐量为１５％的融合株Ｆ１。     

酿酒酵母α-磷酸甘油脱氢酶变异株的鉴定及其高渗敏感性状的遗传分析

本室检测到一株酿酒酵母α-磷酸甘油脱氢酶(α-glycerophosphate dehydrogenase,简称α-GPD)变异株,在所试条件下,变异株的α-GPD酶活比野生型菌株低5倍多。遗传分析表明,该变异株的α-GPD变异是由单一结构基因发生突变,使其所编码的α-GPD分子的构象发生变化而导致酶活显著下降,这也就使它在高渗条件下不能正常生长而表现为高渗敏感菌株。但在一般培养条件下,变异株的呼吸能力、生长量和发酵力与野生型菌株均无明显差异。此外,本文还对酿酒酵母的α-GPD活性与高渗敏感性状的关系进行了讨论。     

长期水淹对‘中山杉118’幼苗呼吸代谢的影响

中山杉(Taxodium‘Zhongshansha’)具有极强的耐淹性,但其耐淹机理仍没有明确。该研究以‘中山杉118’(Taxodium‘Zhongshansha 118’)幼苗为对象,在经过93天不同水淹处理(对照、水浸、浅淹、深淹)后测定中山杉叶片和根系的无氧呼吸酶活性、淀粉及可溶性糖含量、生物量以及根系活力,从能量消耗的角度初步探索了中山杉的耐淹性。结果表明:长期水淹使中山杉叶片与根系中3种无氧呼吸酶(乙醇脱氢酶、丙酮酸脱羧酶、乳酸脱氢酶)活性显著增加,且叶片与根系的乙醇脱氢酶活性均高于乳酸脱氢酶活性,中山杉的根系和叶片是通过加强以酒精发酵为主的无氧呼吸适应长期缺氧环境;不同水淹处理的叶片中3种无氧呼吸酶活性均高于根系,叶片对缺氧环境更加敏感;中山杉叶片和根系淀粉、可溶性糖含量均随水淹深度的增加显著增加,根系淀粉含量显著高于叶片,可溶性糖含量低于叶片;中山杉根系淀粉含量高是其能够长期忍受水淹的重要原因,且中山杉适应长期水淹的策略为忍耐型;经受长期水淹后中山杉根茎结合部长出气生根及茎基部膨大,同时根系外壁的木质化能将根系与外部水淹环境隔离,具有很强的耐淹性,可作为湿地生态修复、消落带生物治理的优良植物材料。     

发酵戊糖产酒精酵母菌株的选育

介绍了一种新的发酵戊糖产酒酵母菌种筛选方法并利用该方法筛选出一株性能优良的发酵戊糖酵母蓖株Z8,该菌株性能测试结果为：产酒精能力相当于发酵戊糖理论产量的60．0％,耐酒精能力14％（V／V）,在温度高达42℃的条件下仍能正常发酵,初步鉴定该菌株为管囊酵母（Pachysolen tannophilus)。     

pH对Themoanaerobacterium calidifontis Rx1乙醇发酵代谢的影响

Themoanaerobacterium calidifontis Rx1是一株能降解半纤维素发酵乙醇的高温厌氧菌。本研究以Rx1和其乙酸激酶基因(ack)敲除的突变菌株Rx1△ack作为研究对象,研究了不同pH值条件对两种菌株生长与代谢的影响,并通过测定碳中心代谢关键酶的表达量与酶活性分析了pH的代谢调控机制。结果表明,与自然pH相比,控制pH能够改变两种菌株的代谢产物分布;当控制pH值为7.0时,Rx1和Rx1△ack的乳酸产量分别下降了85.7%和89.9%,而乙醇产量分别提高了60%和69%;Rx1和Rx1△ack乙酸产量的变化趋势不同。与自然pH相比,控制p H时,Rx1和Rx1△ack的丙酮酸激酶(PK)基因表达量和酶活性都有所提高;野生型菌株的乙酸激酶(ACK)活性下降11%,而突变菌株基因表达量显著提高,酶活性只有野生型的25%,且与自然pH条件相比,有提高的的趋势;野生型菌株乙醇代谢途径中乙醇脱氢酶(ADH)活性显著下调;研究发现pH对Rx1和Rx1△ack乳酸代谢途径中乳酸脱氢酶(LDH)影响十分显著,在弱碱或中性条件下,其活性还不足酸性条件下的一半。Rx1和Rx1△ack在控制pH条件下乳酸产量下降,同时伴随乳酸生成途径的还原力(即NAD(P)H)积累,而乙醇生成途径可将积累的NAD(P)H氧化成NAD(P)+,这可能是控制pH条件下乙醇产量提高的原因。     

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.     

Saccharomyces cerevisiae genome shuffling through recursive population mating leads to improved tolerance to spent sulfite liquor

Spent sulfite liquor (SSL) is a waste effluent from sulfite pulping that contains monomeric sugars which can be fermented to ethanol. However, fermentative yeasts used for the fermentation of the sugars in SSL are adversely affected by the inhibitory substances in this complex feedstock. To overcome this limitation, evolutionary engineering of Saccharomyces cerevisiae was carried out using genome-shuffling technology based on large-scale population cross mating. Populations of UV-light-induced yeast mutants more tolerant than the wild type to hardwood spent sulfite liquor (HWSSL) were first isolated and then recursively mated and enriched for more-tolerant populations. After five rounds of genome shuffling, three strains were isolated that were able to grow on undiluted HWSSL and to support efficient ethanol production from the sugars therein for prolonged fermentation of HWSSL. Analyses showed that greater HWSSL tolerance is associated with improved viability in the presence of salt, sorbitol, peroxide, and acetic acid. Our results showed that evolutionary engineering through genome shuffling will yield robust yeasts capable of fermenting the sugars present in HWSSL, which is a complex substrate containing multiple sources of inhibitors. These strains may not be obtainable through classical evolutionary engineering and can serve as a model for further understanding of the mechanism behind simultaneous tolerance to multiple inhibitors.     

Construction of industrial xylose-fermenting Saccharomyces cerevisiae strains through combined approaches

Saccharomyces cerevisiae strain with excellent xylose-fermenting capacity and inhibitor tolerance is crucial for lignocellulosic ethanol production. In this study, a combined strategy including site-directed mutagenesis, mating, evolutionary engineering, and haploidization was applied to obtain strains with ideal xylose fermentabilities. Haploid industrial strain KFG4-6B was engineered to overexpress endogenous xylulokinase (XK) and heterologous native or mutated xylose reductase (XR) and xylitol dehydrogenase (XDH) from Scheffersomyces stipitis. The XR-mutated strain HX57D showed over 12% increase in both xylose consumption rate and ethanol yield compared with the XR-native strain. To improve the xylose uptake, the HX57D-derived diploids were subjected to evolutionary engineering. In comparison with HX57D, evolved diploid Z4X-21-18 achieved 4.5-fold increases in rates of xylose consumption and ethanol production when fermenting xylose. When fermenting mixed sugars, the glucose and xylose uptake rates were 1.4-fold and 8.3-fold, respectively, higher. H18s28, a haploid of Z4X-21-18, enabled a further 10% increase in xylose consumption rate when fermenting xylose only. However, it was inferior to its diploid parent when fermenting mixed sugars. In the presaccharification-simultaneous saccharification and fermentation (P-SSF) of the whole pretreated wheat straw slurry with high contents of multiple inhibitors, Z4X-21-18 produced approximately 42 g/L ethanol with a yield of 0.38 g/g total sugars.     

Towards industrial pentose-fermenting yeast strains

Production of bioethanol from forest and agricultural products requires a fermenting organism that converts all types of sugars in the raw material to ethanol in high yield and with a high rate. This review summarizes recent research aiming at developing industrial strains of Saccharomyces cerevisiae with the ability to ferment all lignocellulose-derived sugars. The properties required from the industrial yeast strains are discussed in relation to four benchmarks: (1) process water economy, (2) inhibitor tolerance, (3) ethanol yield, and (4) specific ethanol productivity. Of particular importance is the tolerance of the fermenting organism to fermentation inhibitors formed during fractionation/pretreatment and hydrolysis of the raw material, which necessitates the use of robust industrial strain background. While numerous metabolic engineering strategies have been developed in laboratory yeast strains, only a few approaches have been realized in industrial strains. The fermentation performance of the existing industrial pentose-fermenting S. cerevisiae strains in lignocellulose hydrolysate is reviewed. Ethanol yields of more than 0.4 g ethanol/g sugar have been achieved with several xylose-fermenting industrial strains such as TMB 3400, TMB 3006, and 424A(LNF-ST), carrying the heterologous xylose utilization pathway consisting of xylose reductase and xylitol dehydrogenase, which demonstrates the potential of pentose fermentation in improving lignocellulosic ethanol production.     

Effects of heat shock and ethanol stress on the viability of aSaccharomyces uvarum (carlsbergensis) brewing yeast strain during fermentation of high gravity wort

Summary The effects of heat shock and ethanol stress on the viability of a lager brewing yeast strain during fermentation of high gravity wort were studied. These stress effects resulted in reduced cell viability and inhibition of cell growth during fermentation. Cells were observed to be less tolerant to heat shock during the fermentation of 25°P (degree Plato) wort than cells fermenting 16°P wort. Degree Plato (^oP) is the weight of extract (sugar) equivalent to the weight of sucrose in a 100 g solution at 20°C. Relieving the stress effects of ethanol by washing the cells free of culture medium, improved their tolerance to heat shock. Cellular changes in yeast protein composition were observed after 24 h of fermentation at which time more than 2% (v/v) ethanol was present in the growth medium. The synthesis of these proteins was either induced by ethanol or was the result of the transition of cells from exponential phase to stationary phase of growth. No differences were observed in the protein composition of cells fermenting 16°P wort compared to those fermenting 25°P wort. Thus, the differences in the tolerance of these cells to heat shock may be due to the higher ethanol concentration produced in 25°P wort which enhanced their sensitivity to heat shock.     

Ethanol and biomass production of wild strains and respiratory deficient mutants of Saccharomyces cerevisiae under anaerobic and aerobic conditions

Summary In order to test the possibility of producing ethanol under aerobic conditions, 4 mitochondrial mutants of Saccharomyces cerevisiae lacking the capacity to respire were assayed for ethanol and biomass yield. As controls the corresponding wild strains were tested under anaerobic and aerobic conditions. In the latter case respiration was blocked by catabolite repression. The data show that the respiratory deficient mutants yield slightly less ethanol than the anaerobically grown wild strains, but more than those grown aerobically. Therefore, if for technical reasons aerobic fermentation is necessary, the use of mitochondrial mutants would be economically advantageous.     

Escherichia coli arcA mutants: metabolic profile characterization of microaerobic cultures using glycerol as a carbon source

ArcA is a global regulator that switches on the expression of fermentation genes and represses the aerobic pathways when Escherichia coli enters low oxygen growth conditions. The metabolic profile of E. coli CT1062 (DeltaarcA)and CT1061 (arcA2) grown in microaerobiosis with glycerol as carbon source were determined and compared with E. coli K1060, the arcA+ parent strain. Both arcA mutants achieved higher biomass yields than the wild-type strain. The production of acetate, formate, lactate, pyruvate, succinate and ethanol were determined in the supernatants of cultures grown on glycerol under microaerobic conditions for 48 h. The yield of extracellular metabolites on glycerol showed lower acid and higher ethanol values for the mutants. The ethanol/acetate ratio was 0.87 for the parent strain, 2.01 for CT1062, and 12.51 for CT1061. Accordingly, the NADH/NAD+ ratios were 0.18, 0.63, and 0.97, respectively. The extracellular succinate yield followed a different pattern, with yield values of 0.164 for K1060, 0.442 for CT1062 and 0.214 for CT1061. The dissimilarities observed can be attributed to the different effects exerted by the deletion and point mutations in a global regulator.     

Generation and characterisation of stable ethanol-tolerant mutants of Saccharomyces cerevisiae

Saccharomyces spp. are widely used for ethanologenic fermentations, however yeast metabolic rate and viability decrease as ethanol accumulates during fermentation, compromising ethanol yield. Improving ethanol tolerance in yeast should, therefore, reduce the impact of ethanol toxicity on fermentation performance. The purpose of the current work was to generate and characterise ethanol-tolerant yeast mutants by subjecting mutagenised and non-mutagenised populations of Saccharomyces cerevisiae W303-1A to adaptive evolution using ethanol stress as a selection pressure. Mutants CM1 (chemically mutagenised) and SM1 (spontaneous) had increased acclimation and growth rates when cultivated in sub-lethal ethanol concentrations, and their survivability in lethal ethanol concentrations was considerably improved compared with the parent strain. The mutants utilised glucose at a higher rate than the parent in the presence of ethanol and an initial glucose concentration of 20 g l⁻¹. At a glucose concentration of 100 g l⁻¹, SM1 had the highest glucose utilisation rate in the presence or absence of ethanol. The mutants produced substantially more glycerol than the parent and, although acetate was only detectable in ethanol-stressed cultures, both mutants produced more acetate than the parent. It is suggested that the increased ethanol tolerance of the mutants is due to their elevated glycerol production rates and the potential of this to increase the ratio of oxidised and reduced forms of nicotinamide adenine dinucleotide (NAD⁺/NADH) in an ethanol-compromised cell, stimulating glycolytic activity.     

Cloning and characterization of a gene complementing the mutation of an ethanol-sensitive mutant of sake yeast

Ethanol-sensitive mutants (esl to es10) were isolated from sake yeast, Saccharomyces cerevisiae SY-32. These mutants were unable to grow at 7% ethanol at which the wild type strain SY-32 does grow. The mutants had a variety of fermentation rates and viabilities in the presence of ethanol. The gene ERG6, complementing the ethanol-sensitive mutation of es5, was cloned from an SY-32 gene library. ERG6 encodes S-adenosylmethionine: delta 24-sterol-C-methyltransferase (EC 2.1.1.41) in the ergosterol synthetic pathway. Mutant es5 had a reduced ability to synthesize ergosterol. An erg6 disruptant was also ethanol-sensitive. These results suggested that ERG6 plays an important role in the ethanol tolerance of S. cerevisiae.