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。     

酿酒酵母木糖转运基因研究进展

由于对全球变暖等日益严重的环境问题的担忧,生产生物乙醇等清洁能源的技术正受到世界各国越来越多的关注。较之以粮食为原料生产乙醇,木质纤维素生产生物乙醇具有更大的发展潜力,因其来源广泛,廉价且可再生。以木质纤维素生产生物乙醇已经取得长足进步,但仍面临几个主要问题,比如天然酿酒酵母不能利用木糖发酵乙醇,木质纤维素酶成本过高,木质纤维素预处理环节成本高等。已经有基因改造的酵母菌株可以利用戊糖和己糖进行生物乙醇生产。然而,这些菌株对木糖的利用效率很低。这主要是因为酿酒酵母缺乏高效的特异性木糖转运基因,木糖运输依赖已糖转运基因。为了提高木糖利用速度,已有不少方法成功应用于构建重组酵母细胞。现对酵母木糖转运基因的最新研究进展进行简要概述。     

酿酒酵母异源表达纤维二糖转运蛋白LacY与纤维二糖利用菌株的构建北大核心

【目的】在酿酒酵母体内设计代谢通路,使酿酒酵母能利用纤维素水解产物纤维二糖生产乙醇。【方法】首先,用大肠杆菌DH5α总DNA为模板克隆编码大肠杆菌乳糖透过酶的LacY基因。为过表达LacY基因,以质粒YEplac181作为载体,将酿酒酵母PGK1p强启动子加到LacY基因之前,CYC1t终止子加到LacY基因之后,构建质粒YEplac181-PGK1p-LacY-CYC1t。之后,将纤维二糖转运蛋白LacY表达质粒和β-葡萄糖苷酶(β-glucosidase,BGL)表达质粒pRS316-PGK1p-gh1-1-CYC1t依次转入野生型酿酒酵母W303-1A中,使野生型酿酒酵母W303-1A异源表达可转运纤维二糖的LacY蛋白和β-葡萄糖苷酶GH1-1,构建可利用纤维二糖的酿酒酵母工程菌W303-1A GL。最后,通过发酵测定酿酒酵母工程菌W303-1A GL的纤维二糖利用情况和乙醇产量,并对纤维二糖代谢通路中纤维二糖酶活力进行测定。【结果】本研究构建了纤维二糖转运蛋白LacY和β-葡萄糖苷酶GH1-1协同表达的酿酒酵母工程菌W303-1AGL。W303-1AGL可以有效利用纤维二糖发酵生产乙醇,W303-1A GL发酵24 h时乙醇产量达到3.25 g/L,得率为0.325 g乙醇/g纤维二糖,利用葡萄糖产乙醇理论得率为0.511 g乙醇/g纤维二糖,达到葡萄糖产乙醇理论得率的64%,细胞密度最高在第54 h达到OD600=10.84,胞内β-葡萄糖苷酶的酶活在72 h最高,可达到0.51 U/mg。【结论】本研究成功构建了能有效利用纤维二糖的重组酿酒酵母工程菌W303-1A GL,为提高纤维素乙醇生产效率、降低纤维素乙醇生产成本提供了新思路。     

宿主遗传背景对重组酿酒酵母木糖共发酵影响的研究进展

木糖是纤维素原料水解液中最主要的五碳糖成分,由于野生的酿酒酵母缺乏有效的木糖利用途径,将外源木糖代谢途径整合至酿酒酵母中使其具有发酵木糖生产乙醇的能力是构建纤维素乙醇发酵菌株的关键。国内外学者的研究表明,同一木糖代谢途径导入不同酿酒酵母菌株中,所得到的重组菌发酵性能存在明显差异,表明宿主的遗传背景对菌株利用木糖能力和发酵性能具有重要的影响。就酿酒酵母宿主对重组菌株的木糖发酵性能的影响进行了综述,分析了产生宿主差异的内在机理,为进一步选育高效木糖共发酵菌种提供借鉴。     

CIT2基因对突变酿酒酵母木糖利用影响的研究

木糖利用困难是秸秆燃料乙醇产业化的制约因素,改造酿酒酵母使其能够代谢木糖生成乙醇是当前研究的热点之一。为了研究CIT2基因对遗传改良酿酒酵母C5D-P-M菌株中葡萄糖与木糖共发酵过程中木糖利用的影响,设计引物并应用重叠PCR技术获得敲除CIT2基因的敲除组件,通过同源重组的方法将C5D-P-M中的CIT2基因敲除得到突变菌株C5D-P-M-CIT2Δ。通过对出发菌株和突变菌株的生长速率、葡萄糖利用率、木糖利用率及乙醇产量进行研究,结果表明突变菌株C5D-P-M-CIT2Δ的上述指标比出发菌株C5D-P-M均有一定程度的提高。因此推测,酿酒酵母中CIT2基因是影响木糖利用的因素之一,CIT2基因的敲除可提高酿酒酵母的木糖利用率。     

酿酒酵母转化木糖生产化学品的研究进展

木糖的有效利用是木质纤维素生产生物燃料或化学品经济性转化的基础。30年来,通过理性代谢改造和适应性进化等工程策略,显著提高了传统乙醇发酵微生物——酿酒酵母Saccharomyces cerevisiae的木糖代谢能力。因此,近年来在酿酒酵母中利用木糖生产化学品的研究逐步展开。研究发现,酿酒酵母分别以木糖和葡萄糖为碳源时,其转录组和代谢组存在明显差异。与葡萄糖相比,木糖代谢过程中细胞整体呈现出Crabtree-negative代谢特征,如有限的糖酵解途径活性减少了丙酮酸到乙醇的代谢通量,以及增强的胞质乙酰辅酶A合成和呼吸能量代谢等,这都有利于以丙酮酸或乙酰辅酶A为前体的下游产物的有效合成。文中对酿酒酵母木糖代谢途径改造与优化、木糖代谢特征以及以木糖为碳源合成化学品的细胞工厂构建等方面进行了详细综述,并对木糖作为重要碳源在大宗化学品生物合成中存在的困难和挑战以及未来研究方向进行了总结与展望。     

代谢木糖产乙醇的酿酒酵母工程菌研究进展

随着能源价格的持续上涨, 使用木质纤维素生产燃料乙醇已具有重要的实践意义。木糖是多数木质纤维素水解产物中含量仅次于葡萄糖的一种单糖, 传统乙醇生产菌株酿酒酵母不能利用木糖, 这为使用以木质纤维素为原料发酵生产乙醇带来了困难。多年以来人们试图通过基因工程和细胞融合等方法对其进行改造使其能够代谢木糖生产乙醇。本文主要介绍这方面的研究进展。     

代谢木糖产乙醇的酿酒酵母工程菌研究进展

随着能源价格的持续上涨,使用木质纤维素生产燃料乙醇已具有重要的实践意义.木糖是多数木质纤维素水解产物中含量仅次于葡萄糖的一种单糖,传统乙醇生产菌株酿酒酵母不能利用木糖,这为使用以木质纤维素为原料发酵生产乙醇带来了困难.多年以来人们试图通过基因工程和细胞融合等方法对其进行改造使其能够代谢木糖生产乙醇.本文主要介绍这方面的研究进展.     

在酿酒酵母中共表达XYLA和XKS1基因后利用木糖的初步研究

为了使酿酒酵母较好地利用木糖产生乙醇,将来自Thermus thermophilus的木糖异构酶基因XYLA和酿酒酵母自身的木酮糖激酶基因XKS1,构建到酵母表达载体pESC-LEU中,导入酿酒酵母YPH499中,同时成功表达了两种酶基因。该菌以木糖为唯一碳源进行限氧发酵,木糖的利用率为9.64%,为宿主菌的4.17倍,产生2.22 mmol.L-1的乙醇。同时初步探讨了两种酶基因的表达量对酿酒酵母发酵木糖生成乙醇的影响。木糖异构酶对木糖的利用起关键性的作用,木酮糖激酶的过量表达不利于乙醇生成。     

可同化阿拉伯糖的木糖还原酿酒酵母菌株构建

[目的] 以秸秆等木质纤维素类生物质为原料生产液体生物燃料乙醇,目前生产成本高,大规模工业化生产尚有较大难度。构建能同化阿拉伯糖进行木糖还原生产木糖醇的重组酿酒酵母菌株,以实现原料中全糖利用、生产高附加值产品,实现产品多元化。[方法] 首先,利用CRISPR/Cas9基因编辑技术依次向出发菌株中导入阿拉伯糖代谢途径和木糖还原酶基因,使菌株获得代谢阿拉伯糖和将木糖转化为木糖醇的能力;其次,通过适应性驯化的进化工程手段,提高重组菌株对阿拉伯糖的利用效率;最后,通过混合糖发酵验证重组菌株利用阿拉伯糖和还原木糖产木糖醇的能力。[结果] 通过导入植物乳杆菌的阿拉伯糖代谢途径,酿酒酵母菌株获得了较好的利用阿拉伯糖生长繁殖的能力;进一步导入假丝酵母的木糖还原酶基因后,重组菌株在葡萄糖作为辅助碳源条件下可高效还原木糖产木糖醇,但阿拉伯糖的利用能力下降。利用以阿拉伯糖为唯一碳源的培养基进行反复批次驯化,阿拉伯糖的利用能力得以恢复和提升,得到表型较好的重组菌株KAX3-2。该菌株在木糖（50 g/L）和阿拉伯糖（20 g/L）混合糖发酵条件下发酵72 h时,对阿拉伯糖和木糖利用率分别达到42.1%和65.9%,木糖醇的收率为64%。[结论] 本研究成功构建了一株能有效利用阿拉伯糖并能将木糖转化为木糖醇的重组酿酒酵母菌株KAX3-2,为后续构建、获得阿拉伯糖代谢能力更强、木糖醇积累效率更高菌株的工作奠定了基础。     

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.     

Xylulokinase overexpression in two strains of Saccharomyces cerevisiae also expressing xylose reductase and xylitol dehydrogenase and its effect on fermentation of xylose and lignocellulosic hydrolysate

Fermentation of the pentose sugar xylose to ethanol in lignocellulosic biomass would make bioethanol production economically more competitive. Saccharomyces cerevisiae, an efficient ethanol producer, can utilize xylose only when expressing the heterologous genes XYL1 (xylose reductase) and XYL2 (xylitol dehydrogenase). Xylose reductase and xylitol dehydrogenase convert xylose to its isomer xylulose. The gene XKS1 encodes the xylulose-phosphorylating enzyme xylulokinase. In this study, we determined the effect of XKS1 overexpression on two different S. cerevisiae host strains, H158 and CEN.PK, also expressing XYL1 and XYL2. H158 has been previously used as a host strain for the construction of recombinant xylose-utilizing S. cerevisiae strains. CEN.PK is a new strain specifically developed to serve as a host strain for the development of metabolic engineering strategies. Fermentation was carried out in defined and complex media containing a hexose and pentose sugar mixture or a birch wood lignocellulosic hydrolysate. XKS1 overexpression increased the ethanol yield by a factor of 2 and reduced the xylitol yield by 70 to 100% and the final acetate concentrations by 50 to 100%. However, XKS1 overexpression reduced the total xylose consumption by half for CEN.PK and to as little as one-fifth for H158. Yeast extract and peptone partly restored sugar consumption in hydrolysate medium. CEN.PK consumed more xylose but produced more xylitol than H158 and thus gave lower ethanol yields on consumed xylose. The results demonstrate that strain background and modulation of XKS1 expression are important for generating an efficient xylose-fermenting recombinant strain of S. cerevisiae.     

酿酒酵母木糖发酵酒精途径工程的研究进展

途径工程(Pathway engineering),被称为第三代基因工程,改变代谢流向,开辟新的代谢途径是途径工程的主要目的。利用途径工程理念,对酿酒酵母（Saccharomyces cerevisiae）代谢途径进行理性设计,以拓展这一传统酒精生产菌的底物范围,使其充分利用可再生纤维质水解物中的各种糖分,是酿酒酵母酒精途径工程的研究热点之一。这里介绍了近年来酿酒酵母以木糖为底物的酒精途径工程的研究进展。     

产乙醇工程菌研究进展

伴随着21世纪的到来,低油价的时代也悄然落幕。简要概述了燃料乙醇产生菌代谢工程的研究进展,包括了利用淀粉、戊糖及纤维素的工程酵母构建,运动发酵单胞菌利用戊糖工程菌的构建,引入外源乙醇合成途径的大肠埃希氏菌和产酸克雷伯氏菌等。对燃料乙醇的重视将促进开发能利用廉价原料和要求粗放的工程菌株用于高产乙醇的生产过程,以降低成本和能耗,其中能利用生淀粉的工程酵母及利用木质纤维素水解物的运动发酵单胞菌工程菌有较大的工业化潜力。     

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.     

Expression of a heterologous xylose transporter in a <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain engineered to utilize xylose improves aerobic xylose consumption

The goal of this investigation was to determine the effect of a xylose transport system on glucose and xylose co-consumption as well as total xylose consumption in Saccharomyces cerevisiae. We expressed two heterologous transporters from Arabidopsis thaliana in recombinant xylose-utilizing S. cerevisiae cells. Strains expressing the heterologous transporters were grown on glucose and xylose mixtures. Sugar consumption rates and ethanol concentrations were determined and compared to an isogenic control strain lacking the A. thaliana transporters. Expression of the transporters increased xylose uptake and xylose consumption up to 46% and 40%, respectively. Xylose co-consumption rates (prior to glucose depletion) were also increased by up to 2.5-fold compared to the control strain. Increased xylose consumption correlated with increased ethanol concentration and productivity. During the xylose/glucose co-consumption phase, strains expressing the transporters had up to a 70% increase in ethanol production rate. It was concluded that in these strains, xylose transport was a limiting factor for xylose utilization and that increasing xylose/glucose co-consumption is a viable strategy for improving xylose fermentation.     

Simultaneous co-fermentation of mixed sugars: a promising strategy for producing cellulosic ethanol

The lack of microbial strains capable of fermenting all sugars prevalent in plant cell wall hydrolyzates to ethanol is a major challenge. Although naturally existing or engineered microorganisms can ferment mixed sugars (glucose, xylose and galactose) in these hydrolyzates sequentially, the preferential utilization of glucose to non-glucose sugars often results in lower overall yield and productivity of ethanol. Therefore, numerous metabolic engineering approaches have been attempted to construct optimal microorganisms capable of co-fermenting mixed sugars simultaneously. Here, we present recent findings and breakthroughs in engineering yeast for improved ethanol production from mixed sugars. In particular, this review discusses new sugar transporters, various strategies for simultaneous co-fermentation of mixed sugars, and potential applications of co-fermentation for producing fuels and chemicals.     

Metabolic engineering for improved fermentation of pentoses by yeasts

The fermentation of xylose is essential for the bioconversion of lignocellulose to fuels and chemicals, but wild-type strains of Saccharomyces cerevisiae do not metabolize xylose, so researchers have engineered xylose metabolism in this yeast. Glucose transporters mediate xylose uptake, but no transporter specific for xylose has yet been identified. Over-expressing genes for aldose (xylose) reductase, xylitol dehydrogenase and moderate levels of xylulokinase enable xylose assimilation and fermentation, but a balanced supply of NAD(P) and NAD(P)H must be maintained to avoid xylitol production. Reducing production of NADPH by blocking the oxidative pentose phosphate cycle can reduce xylitol formation, but this occurs at the expense of xylose assimilation. Respiration is critical for growth on xylose by both native xylose-fermenting yeasts and recombinant S, cerevisiae. Anaerobic growth by recombinant mutants has been reported. Reducing the respiration capacity of xylose-metabolizing yeasts increases ethanol production. Recently, two routes for arabinose metabolism have been engineered in S. cerevisiae and adapted strains of Pichia stipitis have been shown to ferment hydrolysates with ethanol yields of 0.45 g g^–1 sugar consumed, so commercialization seems feasible for some applications.