絮凝基因FLO1及FLO1c高表达提高工业酿酒酵母乙酸耐受性及发酵性能

乙酸是生物质乙醇发酵过程中酵母细胞面临的重要抑制剂之一,对细胞生长及发酵性能有强烈的抑制作用。增强酵母菌对乙酸胁迫的耐受性对提高乙醇产率具有重要意义。用分别带有完整絮凝基因FLO1及其重复序列单元C发生缺失的衍生基因FLO1c的重组表达质粒分别转化非絮凝型工业酿酒酵母CE6,获得絮凝型重组酵母菌株6-AF1和6-AF1c。同时以空载体p YCPGA1转化CE6的菌株CE6-V为对照菌株。与CE6-V相比,絮凝酵母明显提高了对乙酸胁迫的耐受性。在0.6%(V/V)乙酸胁迫下,6-AF1和6-AF1c的乙醇产率分别为对照菌株CE6-V的1.56倍和1.62倍;在1.0%(V/V)乙酸胁迫下,6-AF1和6-AF1c的乙醇产率分别为对照菌株CE6-V的1.21倍和1.78倍。可见絮凝能力改造能明显提高工业酿酒酵母的乙酸胁迫耐受性及发酵性能,而且FLO1内重复序列单元C缺失具有更加明显的效果。     

利用人工锌指文库选育高乙醇耐受性工业酵母菌株

选育高乙醇耐性的酿酒酵母菌株对提高燃料乙醇的发酵效率具有重要意义.锌指蛋白广泛存在于多种生物中,对基因的转录和翻译起重要的调节作用.利用人工设计的锌指蛋白可定向设计锌指序列及其排列顺序,实现对细胞内多个基因的全局调控.由于与环境胁迫反应相关的基因很多,因此可利用人工锌指蛋白技术获得耐受性提高的微生物重组菌.文中将人工锌指文库转入到酿酒酵母模式菌株S288c,选育了具有高乙醇耐受性的重组菌株M01,并分离了与乙醇耐受性提高相关的人工锌指蛋白表达载体pRS316ZFP-M01,转入工业酿酒酵母Sc4126,在含有不同浓度乙醇的平板上,工业酵母Sc4126的重组菌株表现出显著的耐受性提高.在高糖培养基(250 g/L)条件下进行乙醇发酵,发现重组菌的乙醇发酵效率明显快于野生型,发酵时间提前24 h,且发酵终点乙醇浓度提高6.3％.结果表明人工锌指文库能够提高酵母的乙醇耐受性,为构建发酵性能优良的酵母菌种奠定了基础.     

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。     

采用新型药物抗性选育高效黄酒酵母菌株

【目的】黄酒酵母菌种是影响大罐黄酒生产效率的关键因素之一,高发酵性能黄酒酵母的选育对于提升黄酒生产效率具有重要的实用意义。【方法】以一种药物抗性为基础设计快速初筛方法,对黄酒酿酒酵母XY进行诱变筛选得到克霉唑(CTZ)抗性的黄酒酵母突变株,进一步以酵母发酵性参数为指标筛选得到发酵速率提高的黄酒酵母新菌株XY-3。【结果】比较突变菌株与原始菌株酿造性能发现,XY-3菌株最大发酵速率较原始菌株提高5.21%。黄酒酿造小试结果显示发酵前4天XY-3菌株乙醇生成速率明显高于原始菌株,其最大乙醇生成速率从XY菌株的14.77 g/d提高到15.30 g/d。XY-3酵母菌株发酵速率的提高能够缩短黄酒发酵周期1-2天,有助于提高发酵设备的利用效率。进一步研究发现XY-3发酵速率的提升可能与XY-3菌株多药物抗性(PDR)有关。【结论】联合使用传统诱变和药物抗性筛选策略选育得到高效黄酒酵母新菌株,这种筛选策略也为发酵食品行业优良菌株的选育提供了一种新的思路。     

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。     

基因组改组技术选育耐高温、耐高乙醇酿酒酵母菌株的研究

当以f4、f5、f6作为出发菌株,用酵母菌原生质体紫外诱变的方法,在不同温度下,用含有不同浓度乙醇的平板筛选,分别获得了在耐高温和耐乙醇性状有较大提高的f4.2、f5.1、f6.2、f4.5等正突变菌株。以这些菌株作为出发菌株,进一步用硫酸二乙酯诱变,获得了f5.1.1、f4.2.1两个乙醇耐受性能较高的菌株。在建立了上述不同突变株后,通过基因组改组(genome shuffling)的方法,将上述不同特性的菌株经过两轮genome shuffling,获得了耐高温性能和耐乙醇性能都较好的酵母菌株。经过摇瓶发酵后证明,R24株在35℃发酵过程中,发酵液中的最高乙醇浓度12.93%(W/V),比原始出发菌株f4在35℃的发酵液中最高乙醇浓度8.11%提高了近5%。     

最新专利文摘

酵母菌混合发酵乳清生产燃料乙醇的方法;微生物处理工业废弃物的方法和发酵生产微生物油脂的方法及其专用菌株;重组表达载体和用其转化的宿主细胞发酵甘油高产1,3-丙二醇的方法;用基因工程菌发酵生产腺苷蛋氨酸的方法     

耐温性L-谷氨酸发酵菌种的选育

应用基因组改组技术提高,L-谷氨酸生产菌在高温发酵条件下的谷氨酸产量。以天津短杆菌T6—13变异株SW07-1为原始亲株,分别经紫外线（UV）-硫酸二乙酯（DES）和X射线诱变,获得5株耐温性能略有提高的突变菌株。经2轮基因组改组,获得耐高温（能在44℃生长）的L-谷氨酸菌株F2-50。F2—50在38℃下,摇瓶发酵40h,发酵液中L-谷氨酸浓度比原始出发菌株提高了近41％,在41℃高温下,摇瓶发酵40h,L-谷氨酸浓度比原始出发菌株提高了近2倍。     

响应面法优化重组工程菌的发酵工艺条件

目的：对基因改造运动发酵单胞菌的发酵工艺条件进行优化,提高重组菌发酵乙醇产量。方法：使用分子克隆实验操作技术构建重组运动发酵单胞菌,以单因素实验为基础,利用Box-Behnken中心组合实验和响应面分析法,确定了影响重组菌高产乙醇的三个重要因素。结果：成功构建含有YfdZ、MetB基因和Hsp基因的重组菌Zymomonas mobilis HYM,发酵主要影响因素的最佳条件分别为温度28℃,葡萄糖浓度24%（W/V）,pH7.4。在此优化条件下,Zymomonas mobilis HYM的乙醇产量可高达105.0735g/l,比原始菌株乙醇产量提高16.4%。结论：用中心组合设计和响应面分析法优化重组运动发酵单胞菌的发酵工艺条件,显著提高乙醇产量。     

紫外诱变结合驯化提高酿酒酵母对抑制物耐受性

[目的]提高工业酿酒酵母Sc4126对木质纤维素预处理过程中产生的发酵抑制物的耐受性。[方法]采用紫外诱变结合驯化对工业酿酒酵母Sc4126进行选育,对筛选出的三株菌株Sc4126-1、Sc4126-2、Sc4126-3在复合抑制剂和单一抑制剂存在下对比考查其发酵性能。[结果]相比原始菌株,3株菌株对抑制剂的耐受性均不同程度地提高。在复合抑制剂存在下,优势菌株Sc4126-1发酵时间为48 h,乙醇平均产率0.19 g/L·h,而原始菌株Sc4126发酵时间138 h,乙醇平均产率0.06 g/L·h,相比原始菌株,优势菌株发酵时间缩短65.22%,乙醇平均产率提高2.17倍。对于单一抑制剂,改造后的菌株对糠醛、香草醛的耐受性明显提高,而对乙酸的耐受性提高较少。[结论]通过紫外诱变结合驯化的方法,有效提高了工业酿酒酵母Sc4126对抑制剂的耐受性。     

Engineering of Saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of L-arabinose

For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as l-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of l-arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of l-arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the l-arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h(-1) g [dry weight](-1)) and ethanol production (0.29 g h(-1) g [dry weight](-1)) and a high ethanol yield (0.43 g g(-1)) during anaerobic growth on l-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.     

Identification of RCN1 and RSA3 as ethanol-tolerant genes in Saccharomyces cerevisiae using a high copy barcoded library

Saccharomyces cerevisiae (S.?cerevisiae) encounters a multitude of stresses during industrial processes such as wine fermentation including ethanol toxicity. High levels of ethanol reduce the viability of yeast and may prevent completion of fermentation. The identification of ethanol-tolerant genes is important for creating stress-resistant industrial yeast, and S.?cerevisiae genomic resources have been utilized for this purpose. We have employed a molecular barcoded yeast open reading frame (MoBY-ORF) high copy plasmid library to identify ethanol-tolerant genes in both the S.?cerevisiae S288C laboratory and M2 wine strains. We find that increased dosage of either RCN1 or RSA3 improves tolerance of S288C and M2 to toxic levels of ethanol. RCN1 is a regulator of calcineurin, whereas RSA3 has a role in ribosome maturation. Additional fitness advantages conferred upon overproduction of RCN1 and RSA3 include increased resistance to cell wall degradation, heat, osmotic and oxidative stress. We find that the M2 wine yeast strain is generally more tolerant of stress than S288C with the exception of translation inhibition, which affects M2 growth more severely than S288C. We conclude that regulation of ribosome biogenesis and ultimately translation is a critical factor for S.?cerevisiae survival during industrial-related environmental stress.     

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.     

固定化酵母乙醇发酵及固化小球微生态的研究

比较了SA-PVA-SiO₂固定化酿酒酵母和游离酿酒酵母的乙醇发酵能力,采用批次发酵试验研究固定化酿酒酵母的发酵稳定性。结果表明,SA-PVA-SiO₂固定化酿酒酵母的发酵速度比游离酿酒酵母快,发酵周期短;发酵稳定性很好,30℃,橡胶塞、90 r/min摇床培养24 h时,乙醇体积分数均在3%~3.5％之间,连续发酵14批次后,固化小球的形态依然完好,不发粘。通过扫描电镜对酿酒酵母包埋微生态环境进行了分析,图像表明固定化小球的内部环境非常有利于酵母细胞的厌氧发酵产乙醇,充分证明了SA-PVA-SiO₂固定化酿酒酵母乙醇发酵的优越性。     

High expression of XYL2 coding for xylitol dehydrogenase is necessary for efficient xylose fermentation by engineered Saccharomyces cerevisiae

The traditional ethanologenic yeast Saccharomyces cerevisiae cannot metabolize xylose, which is an abundant sugar in non-crop plants. Engineering this yeast for a practicable fermentation of xylose will therefore improve the economics of bioconversion for the production of fuels and chemicals such as ethanol. One of the most widely employed strategies is to express XYL1, XYL2, and XYL3 genes derived from Scheffersomyces stipitis (formerly Pichia stiptis) in S. cerevisiae. However, the resulting engineered strains have been reported to exhibit large variations in xylitol accumulation and ethanol yields, generating many hypotheses and arguments for elucidating these phenomena. Here we demonstrate that low expression levels of the XYL2 gene, coding for xylitol dehydrogenase (XDH), is a major bottleneck in efficient xylose fermentation. Through an inverse metabolic engineering approach using a genomic library of S. cerevisiae, XYL2 was identified as an overexpression target for improving xylose metabolism. Specifically, we performed serial subculture experiments after transforming a genomic library of wild type S. cerevisiae into an engineered strain harboring integrated copies of XYL1, XYL2 and XYL3. Interestingly, the isolated plasmids from efficient xylose-fermenting transformants contained XYL2. This suggests that the integrated XYL2 migrated into a multi-copy plasmid through homologous recombination. It was also found that additional overexpression of XYL2 under the control of strong constitutive promoters in a xylose-fermenting strain not only reduced xylitol accumulation, but also increased ethanol yields. As the expression levels of XYL2 increased, the ethanol yields gradually improved from 0.1 to 0.3g ethanol/g xylose, while the xylitol yields significantly decreased from 0.4 to 0.1g xylitol/g xylose. These results suggest that strong expression of XYL2 is a necessary condition for developing efficient xylose-fermenting strains.     

Tolerance and adaptation of ethanologenic yeasts to lignocellulosic inhibitory compounds

Synthetic mixtures of predominant lignocellulosic hexose sugars were supplemented with separate aliquots of three inhibitory compounds (furfural, hydroxymethylfurfural (HMF), and acetic acid) in a series of concentrations and fermented by the spent sulfite liquor (SSL)-adapted yeast strain Tembec T1 and the natural isolate Saccharomyces cerevisiae (S. cerevisiae) Y-1528 to compare tolerance and assess fermentative efficacy. The performance of Y-1528 exceeded that of Tembec T1 by a significant margin, with faster hexose sugar consumption, higher ethanol productivity, and in the case of furfural and HMF, faster inhibitor consumption. Nevertheless, furfural had a dose-proportionate effect on sugar consumption rate and ethanol productivity in both strains, but did not substantially affect ethanol yield. HMF had a similar effect on sugar consumption rate and ethanol productivity, and also lowered ethanol yield. Surprisingly, acetic acid had the least impact on sugar consumption rate and ethanol productivity, and stimulated ethanol yield at moderate concentrations. Sequential iterations of softwood (SW) and hardwood (HW) SSL were subsequently inoculated with the two yeast strains in order to compare adaptation to, and performance in lignocellulosic substrates in a cell recycle batch fermentation (CRBF) regime. Both strains were severely affected by the HW SSL, which was attributed to specific syringyl lignin-derived degradation products and synergistic interactions between inhibitors. Though ethanologenic capacity was preserved, a net loss of performance was evident from both strains, indicating the absence of adaptation to the substrates, regardless of the sequence in which the SSL types were employed.     

Fermentation performance of engineered and evolved xylose-fermenting Saccharomyces cerevisiae strains

Lignocellulose hydrolysate is an abundant substrate for bioethanol production. The ideal microorganism for such a fermentation process should combine rapid and efficient conversion of the available carbon sources to ethanol with high tolerance to ethanol and to inhibitory components in the hydrolysate. A particular biological problem are the pentoses, which are not naturally metabolized by the main industrial ethanol producer Saccharomyces cerevisiae. Several recombinant, mutated, and evolved xylose fermenting S. cerevisiae strains have been developed recently. We compare here the fermentation performance and robustness of eight recombinant strains and two evolved populations on glucose/xylose mixtures in defined and lignocellulose hydrolysate-containing medium. Generally, the polyploid industrial strains depleted xylose faster and were more resistant to the hydrolysate than the laboratory strains. The industrial strains accumulated, however, up to 30% more xylitol and therefore produced less ethanol than the haploid strains. The three most attractive strains were the mutated and selected, extremely rapid xylose consumer TMB3400, the evolved C5 strain with the highest achieved ethanol titer, and the engineered industrial F12 strain with by far the highest robustness to the lignocellulosic hydrolysate.     

Engineering Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: reflections and perspectives

Conversion of the abundant lignocellulosic biomass into ethanol is an environmentally sustainable solution to the energy crisis. Fermentation of lignocellulosic hydrolysates by Saccharomyces cerevisiae is not cost-effective yet as substantial amounts of xylose in the hydrolysates cannot be utilized by native S. cerevisiae strains. Extensive studies including both metabolic and evolutionary engineering have been carried out to develop an efficient xylose-fermenting S. cerevisiae strain, yet the ethanol yield and productivity from xylose fermentation of the best one are still far below expectation. This review compares the engineering approaches and resulted anaerobic xylose fermentation performance of recently reported xylose-utilizing S. cerevisiae strains, with the aim to understand the intrinsic reason for their low xylose fermentation capabilities. These comparative analyses revealed that some of the current engineering targets and the so-called "hot issues" might be overrated. Our opinions on the underrated parts and future efforts in this field are also presented. Overall, this review serves as a comprehensive reference to understanding xylose fermentation by S. cerevisiae.     

Ethanol-induced yeast flocculation directed by the promoter of TPS1 encoding trehalose-6-phosphate synthase 1 for efficient ethanol production

Yeast flocculation is an important trait in the brewing industry as well as in ethanol production, through which biomass can be recovered by cost-effective sedimentation. However, mass transfer limitation may affect yeast growth and ethanol fermentation if the flocculation occurs earlier before fermentation is completed. In this article, a novel type of cell-cell flocculation induced by trehalose-6-phosphate synthase 1 (TPS1) promoter was presented. The linear cassette HO-P(TPS1)-FLO1(SPSC01)-KanMX4-HO was constructed to transform the non-flocculating industrial yeast S. cerevisiae 4126 by chromosome integration to obtain a new flocculating yeast strain, ZLH01, whose flocculation was induced by ethanol produced during fermentation. The experimental results illustrated that flocculation of ZLH01 was triggered by 3% (v/v) ethanol and enhanced as ethanol concentration increased till complete flocculation was achieved at ethanol concentration of 8% (v/v). Real time PCR analysis confirmed that the expression of FLO1(SPSC01) was dependent on ethanol concentration. The growth and ethanol fermentation of ZLH01 were improved significantly, compared with the constitutive flocculating yeast BHL01 engineered with the same FLO gene but directed by the constitutive 3-phosphoglycerate kinase promoter PGK1, particularly under high temperature conditions. These characteristics make the engineered yeast more suitable for ethanol production from industrial substrates under high gravity and temperature conditions. In addition, this strategy offers advantage in inducing differential expression of other genes for metabolic engineering applications of S. cerevisiae.     

Comparative study on a series of recombinant flocculent Saccharomyces cerevisiae strains with different expression levels of xylose reductase and xylulokinase

Ethanol production from xylose is important for the utilization of lignocellulosic biomass as raw materials. Recently, we reported the development of an industrial xylose-fermenting Saccharomyces cerevisiae strain, MA-R4, which was engineered by chromosomal integration to express the genes encoding xylose reductase and xylitol dehydrogenase from Pichia stipitis along with S. cerevisiae xylulokinase gene constitutively using the alcohol-fermenting flocculent yeast strain, IR-2. IR-2 has the highest xylulose-fermenting ability of the industrial diploid strains, making it a useful host strain for genetically engineering xylose-utilizing S. cerevisiae. To optimize the activities of xylose metabolizing enzymes in the metabolic engineering of IR-2 for further improvement of ethanol production from xylose, we constructed a set of recombinant isogenic strains harboring different combinations of genetic modifications present in MA-R4, and investigated the effect of constitutive expression of xylulokinase and of different levels of xylulokinase and xylose reductase activity on xylose fermentation. This strain comparison showed that constitutive expression of xylulokinase increased ethanol production from xylose at the expense of xylitol excretion, and that high activity of xylose reductase resulted in an increased rate of xylose consumption and an increased glycerol yield. Moreover, strain MA-R6, which has moderate xylulokinase activity, grew slightly better but accumulated more xylitol than strain MA-R4. These results suggest that fine-tuning of introduced enzyme activity in S. cerevisiae is important for improving xylose fermentation to ethanol.