嗜热菌在纤维素乙醇中的研究进展及应用前景

利用纤维素生产乙醇的研究中,传统的中温微生物在基质范围、酶活性及热稳定性等方面存在不足,难以满足工业需求.嗜热菌具有独特的降解纤维素、半纤维素和生产乙醇的耐高温酶系和代谢途径,不但热稳定性高、而且底物范围广泛,在生物质能学领域有重要的研究价值和应用潜力.综述了在纤维素乙醇研究中具有潜力的嗜热菌的种类、特性、代谢机理和研究进展,并对嗜热菌的应用前景进行了分析与展望.     

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。     

酿酒酵母纤维素乙醇统合加工(CBP)的策略及研究进展

木质纤维素乙醇的统合生物加工过程(Consolidated bioprocessing,CBP)是将纤维素酶和半纤维素酶生产、纤维素水解和乙醇发酵过程组合或部分组合,通过一种微生物完成。统合生物加工过程有利于降低生物转化过程的成本,越来越受到研究者的普遍关注。酿酒酵母Saccharomyces cerevisiae是传统的乙醇发酵菌株。介绍了影响外源基因在酿酒酵母中表达水平的因素,纤维素酶和半纤维素酶在酿酒酵母中表达研究进展及利用酿酒酵母统合加工纤维素乙醇的策略。     

一株降解纤维素放线菌的产纤维素酶基因克隆与表达

【背景】纤维素在自然界中储量丰富,但天然纤维素的难降解性成为广泛应用纤维素资源的壁垒,近年来利用微生物来降解纤维素成为热点研究。【目的】筛选分离得到一株具有降解纤维素功能的放线菌菌株Lb1,通过全基因组测序确定其产纤维素酶关键基因5676,对基因5676进行克隆转化,使其在大肠杆菌中进行表达。【方法】通过基因工程技术将产纤维素基因连接到表达质粒上并导入表达菌株,对其降解纤维素生成葡萄糖的能力进行探究。【结果】将Lb1菌株的16S rRNA基因进行比对,确定菌株Lb1属于链霉菌属,命名为Streptomyces sp. Lb1。成功构建出纤维素酶表达载体,并且导入表达菌株大肠杆菌BL21(DE3),重组菌株的产纤维素酶能力大于空载菌株。【结论】通过基因工程技术成功克隆出产纤维素酶基因,从而表达纤维素酶,为今后利用微生物降解纤维素的大规模应用提供参考。     

产纤维素体菌厌氧降解纤维素制乙醇的研究进展

纤维素(植物细胞壁的主要成分)是自然界最丰富的一种可再生资源,但是极难降解利用。纤维素体是一种多酶复合体,能够高效降解纤维素,降解产物能够被某些厌氧微生物利用发酵产乙醇。综述了近年来产纤维素体菌厌氧降解纤维素制乙醇的研究进展,报道了纤维素体结构和功能、重组设计型纤维素体、代谢工程、混菌培养等研究方向的最新成果和思路,并对其前景作了展望。可以预期,随着研究的深入,生物质制乙醇必将日益显示出其强大的市场竞争力。     

瘤胃中木质纤维素降解菌及降解酶基因的研究进展

摘要：反刍动物瘤胃是公认的木质纤维素高效降解的天然反应器,对瘤胃微生物的研究成为开发生物能源的热点领域之一。其研究手段已经从传统的依赖分离培养从瘤胃中获得木质纤维素降解菌,并对降解菌中的木质纤维素降解酶逐一分析,发展到通过基因组/元基因组技术,直接从瘤胃中发现获得大量新的木质纤维素降解酶基因/基因簇,进而探讨其降解的分子机理。已有的研究结果表明,瘤胃微生物降解木质纤维素的过程非常复杂,其中涉及到大量不同种类的微生物、酶及基因/基因簇,随着新分析技术的建立和完善,对这些微生物、酶和基因的研究已取得了诸多进展。本论文综述报道了近期有关该方向的研究进展。     

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。     

酿酒酵母L-阿拉伯糖转化乙醇代谢工程的研究进展

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。     

瘤胃木质纤维素降解菌及降解酶基因的研究进展

反刍动物瘤胃是公认的木质纤维素高效降解的天然反应器,对瘤胃微生物的研究已成为开发生物能源的热点领域之一。目前的研究手段已经从传统的依赖分离培养从瘤胃中获得木质纤维素降解菌,并对降解菌中的木质纤维素降解酶逐一分析,发展到通过基因组/元基因组学技术,直接从瘤胃中发现并获得大量新的木质纤维素降解酶基因/基因簇,进而探讨其降解的分子机理。已有的研究结果表明,瘤胃微生物降解木质纤维素的过程非常复杂,涉及大量不同种类的微生物及基因/基因簇,随着新分析技术的建立和完善,对这些微生物和基因的研究已取得了诸多进展。本论文综述了近期有关该方向的研究进展。     

木质纤维素稀酸水解糖液乙醇发酵研究进展

以木质纤维素为原料生产燃料乙醇,首先要对原料进行预处理得到可发酵糖,在稀酸水解木质纤维素得到的糖液中,除含有葡萄糖、木糖等六碳糖和五碳糖外,根据水解温度、酸浓度和时间的不同,还含有不同浓度的发酵抑制剂。因此,在研究木质纤维素稀酸水解糖液的乙醇发酵中,对代谢木糖成乙醇的菌种的研究、对耐/代谢发酵抑制剂微生物的研究、对稀酸水解糖液的脱毒方法的研究以及对稀酸水解糖液不同发酵方式的乙醇发酵研究等非常重要。重点介绍了以上几个方面近几年研究的进展。     

Ethanol tolerance of yeast

The ethanol tolerance mechanism in yeasts is not very well understood. This may result from the complex inhibitory mechanisms of ethanol, the lack of a universally accepted definition and method to measure ethanol tolerance, and its complex polygenic characteristic. Recently, there has been some progress in understanding ethanol tolerance. Plasma membrane phospholipids have been shown to play an important role in the ethanol tolerance mechanism. Increases in membrane unsaturated fatty acids result in increased yeast ethanol tolerance. Supplementation of growth media with combinations of unsaturated fatty acids, vitamins and proteins also enhance ethanol tolerance. Physiological factors such as mode of substrate feeding, intracellular ethanol accumulation, temperature and osmotic pressure all contribute to the ethanol tolerance of yeast. The complex nature of ethanol toxicity suggests that a number of different genes are likely to be involved in the ethanol tolerance mechanism. Genetic approaches such as spheroplast or protoplast fusion, hybridization and continuous culture selection have been used to obtain ethanol tolerant yeasts. Isolation and characterization of such strains may provide a better understanding of ethanol tolerance.     

微生物木糖发酵产乙醇的代谢工程

利用木质纤维素发酵生产乙醇具有广泛的应用前景。而自然界中缺少有效转化木糖为乙醇的微生物是充分利用纤维素水解产物、提高乙醇产率、降低生产成本的关键因素。多年来研究者利用分子生物学技术对微生物菌株进行了代谢工程改造,使其能更有效地利用木糖生产乙醇。以下主要对运动发酵单胞菌、大肠杆菌和酵母等候选产乙醇微生物的木糖代谢工程研究进展进行了概述。     

Developing elite <Emphasis Type="Italic">Neurospora crassa</Emphasis> strains for cellulosic ethanol production using fungal breeding

The demand for renewable and sustainable energy has generated considerable interest in the conversion of cellulosic biomass into liquid fuels such as ethanol using a filamentous fungus. While attempts have been made to study cellulose metabolism through the use of knock-out mutants, there have been no systematic effort to characterize natural variation for cellulose metabolism in ecotypes adapted to different habitats. Here, we characterized natural variation in saccharification of cellulose and fermentation in 73 ecotypes and 89 laboratory strains of the model fungus Neurospora crassa. We observed significant variation in both traits among natural and laboratory generated populations, with some elite strains performing better than the reference strain. In the F1 population N345, 15% of the population outperformed both parents with the top performing strain having 10% improvement in ethanol production. These results suggest that natural alleles can be exploited through fungal breeding for developing elite industrial strains for bioethanol production.     

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。     

锌指蛋白及人工锌指蛋白对微生物代谢影响的研究进展

锌指蛋白由于锌指结构域序列相对保守,识别DNA序列具有高度特异性,所以成为研究较广泛的DNA结合蛋白,但目前对锌指蛋白的研究多集中在真核细胞,而对微生物锌指蛋白,尤其是原核微生物锌指蛋白的研究相对较少。本文综述了近年来微生物锌指蛋白,尤其是原核微生物锌指蛋白的发现及功能的最新研究进展,以及人工锌指蛋白技术在微生物菌株改造中的应用。特定人工锌指蛋白不仅可调控微生物细胞中多基因控制的复杂性状,例如耐热性、乙醇和丁醇耐性、渗透胁迫耐受性等,还可以利用锌指结构域构建DNA脚手架系统,进而构建复合酶系统,从而提高催化效率和代谢物产量。目前报道的用于微生物代谢调控的人工锌指蛋白利用的都是哺乳动物的基因,未来根据不同微生物中天然锌指蛋白的序列进行人工锌指的设计,将拓展人工转录因子技术在微生物全局基因表达调控中的应用。     

Metabolic engineering of Saccharomyces cerevisiae for production of carboxylic acids: current status and challenges

To meet the demands of future generations for chemicals and energy and to reduce the environmental footprint of the chemical industry, alternatives for petrochemistry are required. Microbial conversion of renewable feedstocks has a huge potential for cleaner, sustainable industrial production of fuels and chemicals. Microbial production of organic acids is a promising approach for production of chemical building blocks that can replace their petrochemically derived equivalents. Although Saccharomyces cerevisiae does not naturally produce organic acids in large quantities, its robustness, pH tolerance, simple nutrient requirements and long history as an industrial workhorse make it an excellent candidate biocatalyst for such processes. Genetic engineering, along with evolution and selection, has been successfully used to divert carbon from ethanol, the natural endproduct of S. cerevisiae , to pyruvate. Further engineering, which included expression of heterologous enzymes and transporters, yielded strains capable of producing lactate and malate from pyruvate. Besides these metabolic engineering strategies, this review discusses the impact of transport and energetics as well as the tolerance towards these organic acids. In addition to recent progress in engineering S. cerevisiae for organic acid production, the key limitations and challenges are discussed in the context of sustainable industrial production of organic acids from renewable feedstocks.     

酿酒酵母戊糖转运蛋白及C6/C5共代谢菌株的研究进展

充分利用木质纤维素中的糖分是提高以此类生物质为原料生产二代燃料乙醇经济盈利性的基本要求,也是实现其他生物基化学品规模化生产的基础。传统的乙醇生产微生物酿酒酵母Saccharomyces cerevisiae具有独特的生产性能及内在优势,是备受关注的底盘细胞,但其不能有效地利用戊糖。利用代谢工程、合成生物学策略,对二代燃料乙醇生产专用酿酒酵母的精准构制持续研究了30余年,已明显改善了其对木糖/葡萄糖的乙醇共发酵能力。近年来关注点集中在早期忽略的限速步骤即糖转运环节的研究上,以期实现不同糖分各行其道、高效专一性转运蛋白各行其责的二代燃料乙醇生产特种酿酒酵母所需的糖转运理想状态。文中主要综述了酿酒酵母戊糖转运蛋白的研究进展,及酿酒酵母的木糖和L-阿拉伯糖代谢工程的研究现状。     

能利用五碳糖和六碳糖生产乙醇的基因工程菌菌株的构建

燃料乙醇是一种极具前景的燃油代用品,近年来发展尤为迅速,为了推广这种能源和满足日益增长的需求,我们有必要开发更为高效的生产工艺和寻找更为廉价的原料。解决此问题的关键在于获得高效的工程菌,使其能利用木质纤维素水解液中的五碳糖和六碳糖发酵生产乙醇。通过代谢工程的研究和基因重组技术,几种重组细菌显示出良好的开发前景,它们是运动发酵单胞菌、大肠杆菌、产酸克雷伯氏菌和菊欧文氏菌。本文就这四种细菌的研究进展以及基因重组过程进行了介绍和评价。     

Isolation and characterization of ethanol tolerant yeast strains

Yeast strains are commonly associated with sugar rich environments. Various fruit samples were selected as source for isolating yeast cells. The isolated cultures were identified at Genus level by colony morphology, biochemical characteristics and cell morphological characters. An attempt has been made to check the viability of yeast cells under different concentrations of ethanol. Ethanol tolerance of each strain was studied by allowing the yeast to grow in liquid YEPD (Yeast Extract Peptone Dextrose) medium having different concentrations of ethanol. A total of fifteen yeast strains isolated from different samples were used for the study. Seven strains of Saccharomyces cerevisiae obtained from different fruit sources were screened for ethanol tolerance. The results obtained in this study show a range of tolerance levels between 7%-12% in all the stains. Further, the cluster analysis based on 22 RAPD (Random Amplified polymorphic DNA) bands revealed polymorphisms in these seven Saccharomyces strains.     

Antibodies to catecholamines and serotonin during alcoholic intoxication in animals with different predispositions to the development of experimental alcoholism

Experiments on C57Bl/6, CBA and DBA/2 mice characterized by different preferences for ethanol have shown that during chronic administration of alcohol to animals with natural ethanol motivation (strain C57Bl/6) the level of antibodies to catecholamines and serotonin was increased on the 3rd month of ethanol intoxication, with the voluntary alcohol consumption in mice decreased by this time. On the contrary in mice rejecting alcohol (strains DBA/2, CBA) no antibodies to catecholamines and serotonin have been found.