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。     

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。     

构建伴有β-葡萄糖苷酶表达的乙醇发酵大肠杆菌

研究构建能够分泌表达纤维素酶的产乙醇菌株,实现降解木质纤维素生产乙醇的整合生物加工过程。文中通过克隆来自运动发酵单胞菌Zymomonas mobilis ZM4的丙酮酸脱羧酶基因pdc和乙醇脱氢酶基因adhB,并通过Red重组将二者整合到大肠杆菌Escherichia coli JM109基因组中,首先构建了一株可以利用葡萄糖进行乙醇发酵的重组菌E. coli P81。随后将来源于多粘芽胞杆菌Bacillus polymyxa1.794的β-葡萄糖苷酶基因bglB在E. coli P81中进行了分泌表达,得到了一株可以进行纤维二糖降解和乙醇发酵双重功能的重组菌E. coli P81(pUC19-bglB)。该菌胞外分泌β-糖苷酶活达到84.78 mU/mL菌液,纤维二糖酶活达到了32.32 mU/mL菌液。该重组菌E. coli P81(pUC19-bglB) 以纤维二糖为碳源进行乙醇发酵,乙醇得率达到了理论产率55.8％,而在葡萄糖和纤维二糖的共发酵中,其乙醇产量达到了理论产率46.5％。构建得到的此株整合生物加工大肠杆菌能够利用β-葡萄糖苷酶生产乙醇,为构建能利用木质纤维素分解产物生产燃料乙醇的高效、稳定生产用工程菌奠定了良好的基础。     

代谢工程改造运动发酵单胞菌用于提高乙醇产量

目的:采用可以在运动发酵单胞菌中表达的操纵子构建重组运动发酵单胞菌,用于提高该细菌对高温高糖的耐受性和提高乙醇产量.方法:用外来的YfdZ、MetB和Hsp构建的多顺反子质粒,转化运动发酵单胞菌而使其获得新的代谢途径.在玉米水解液中,验证了该多顺反子质粒对运动发酵单胞菌产生乙醇的影响.结果:与对照菌相比,在37℃和糖浓度为28%的培养条件下,该基因工程菌的乙醇产量提高到183.2%.在37℃,糖浓度为28%并添加氮源的条件下,该基因工程菌的乙醇产量提高到148.0%.结论:YfdZ、MetB及Hsp三种基因的共同作用能显著提高运动发酵单胞菌的乙醇产量和发酵温度.     

运动发酵单胞菌在生物炼制中的研究进展

以木质纤维素生物质为原料的生物炼制技术已成为全球研发的热点和难点。欧盟国家和美国的中长期生物质能源发展路线图中均将木质纤维素生物炼制技术作为重要目标,但是目前整体水平尚处于中试阶段。我国的纤维素类生物质原料非常丰富,将其转化成燃料乙醇及生物基础化学品等具有较大的潜力,但当前要想实现商业化生产,还面临着很多瓶颈问题亟待解决。缺乏能够同时高效利用纤维素类水解物的发酵菌株,已成为纤维素生物质高效与高值转化的关键制约因素。运动发酵单胞菌是目前唯一一种通过ED途径兼性厌氧发酵葡萄糖的微生物,其独特的代谢途径使其成为构建产乙醇工程菌的优选宿主之一;同时由于该菌具有较高的糖利用效率等优点,也是其他生物基化学品生产的重要候选平台微生物,如山梨醇、葡萄糖酸、丁二酸和异丁醇等。本文从该菌的研究历程、分子生物学基础、菌种改良及该菌在生物能源及生物基化学品等生物炼制体系中的应用研究角度进行了综述,并提出该菌可作为纤维素生物质生物炼制系统的新的重要平台微生物。     

重组运动发酵单胞菌的构建及木糖利用特性研究

将大肠杆菌(Escherichia coli)木糖代谢的关键酶基因.引入到运动发酵单胞菌中,获得能利用木糖发酵生产乙醇的重组工程菌株PZM.混合糖发酵过程中,重组菌利用葡萄糖和木糖生成乙醇的效率分别达到理论值的81.2%和63.1%.     

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。     

木薯纤维素乙醇发酵的纤维素酶成本评价

木薯中的纤维素成分约占木薯干重的10％(W/W).文中以木薯燃料乙醇生产的木薯纤维素酒渣为原料,从纤维素酶成本角度评估了三种利用木薯纤维素组分发酵生产乙醇的方法,包括木薯纤维素酒渣的直接糖化和乙醇发酵、木薯纤维素酒渣预处理后的糖化与乙醇发酵、木薯乙醇发酵中同步淀粉与纤维素糖化以及乙醇发酵.结果表明,前两种方法的纤维素利用效率不高,酶成本分别达到13602、11659元/吨乙醇.第三种方法,即在木薯乙醇发酵过程同时加入糖化酶和纤维素酶,进行同步淀粉与纤维素糖化,进而进行乙醇发酵,木薯纤维素乙醇的收益最高.发酵结束时的乙醇浓度从101.5g/L提高到107.0g/L,纤维素酶成本为3 589元/吨乙醇.此方法利用木薯纤维素与木薯淀粉同时进行,不会带来额外的设备及操作投入,酶成本低于产品乙醇价格,可实现盈利,因此第三种方法为木薯纤维用于乙醇发酵的最适方法,本研究结果将为木薯乙醇产业深度利用木薯纤维提供依据.     

过表达INO1基因提高酿酒酵母乙醇耐受性

为获得燃料乙醇生产菌株,通过基因工程改造,构建能够利用能源甘蔗汁发酵、乙醇产率高的酿酒酵母工程菌株。即过表达肌醇-3-磷酸合成酶基因ino1,敲除kanMX抗性基因,获得重组菌。对过表达菌株的乙醇耐受性进行分析。利用甘蔗汁进行发酵培养,采用气相色谱(GC)对发酵产物乙醇进行检测。结果显示过表达菌株YI2-1能够耐19%(V/V)的乙醇,利用20oBx甘蔗汁厌氧发酵乙醇积累量为13.10%(V/V),较出发菌提高了8.55%。而过表达菌株YI2-1△KP的最大乙醇积累量为13.17%(V/V),较出发菌提高了9.16%。研究表明通过过表达酿酒酵母ino1基因能够有效提高菌株细胞活力、乙醇耐受性。构建的工程菌可利用甘蔗汁发酵,具有较高的乙醇产量。     

运动发酵单胞菌积累聚羟基丁酸提高乙醇产量

运动发酵单胞菌是一种很有潜力的酒精生产菌。PHB是生物合成的一种聚酯,有研究表明,该类物质在微生物体内的积累能够提高宿主菌的抗逆能力。本文对运动发酵单胞菌进行了如下改造：将PHB合成操纵子phbCAB与来源于运动发酵单胞菌的丙酮酸脱羧酶的启动子准确融合,插入广泛宿主载体pBBR1MCS-1中,并利用电转化的方法转入运动发酵单胞菌中。在重组菌中检测到了PhaA和PhaB的酶活;并首次在运动发酵单胞菌中实现了PHB的积累。摇瓶实验表明,前48小时重组菌的乙醇积累量提高了约10%,后续发酵中可能由于葡萄糖耗尽,重组菌与野生菌乙醇积累量差别不大。     

Fermentations with new recombinant organisms.

United States fuel ethanol production in 1998 exceeded the record production of 1.4 billion gallons set in 1995. Most of this ethanol was produced from over 550 million bushels of corn. Expanding fuel ethanol production will require developing lower-cost feedstocks, and only lignocellulosic feedstocks are available in sufficient quantities to substitute for corn starch. Major technical hurdles to converting lignocellulose to ethanol include the lack of low-cost efficient enzymes for saccharification of biomass to fermentable sugars and the development of microorganisms for the fermentation of these mixed sugars. To date, the most successful research approaches to develop novel biocatalysts that will efficiently ferment mixed sugar syrups include isolation of novel yeasts that ferment xylose, genetic engineering of Escherichia coli and other gram negative bacteria for ethanol production, and genetic engineering of Saccharoymces cerevisiae and Zymomonas mobilis for pentose utilization. We have evaluated the fermentation of corn fiber hydrolyzates by the various strains developed. E. coli K011, E. coli SL40, E. coli FBR3, Zymomonas CP4 (pZB5), and Saccharomyces 1400 (pLNH32) fermented corn fiber hydrolyzates to ethanol in the range of 21-34 g/L with yields ranging from 0.41 to 0.50 g of ethanol per gram of sugar consumed. Progress with new recombinant microorganisms has been rapid and will continue with the eventual development of organisms suitable for commercial ethanol production. Each research approach holds considerable promise, with the possibility existing that different "industrially hardened" strains may find separate applications in the fermentation of specific feedstocks.     

Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration

With industrial development growing rapidly, there is a need for environmentally sustainable energy sources. Bioethanol (ethanol from biomass) is an attractive, sustainable energy source to fuel transportation. Based on the premise that fuel bioethanol can contribute to a cleaner environment and with the implementation of environmental protection laws in many countries, demand for this fuel is increasing. Efficient ethanol production processes and cheap substrates are needed. Current ethanol production processes using crops such as sugar cane and corn are well-established; however, utilization of a cheaper substrate such as lignocellulose could make bioethanol more competitive with fossil fuel. The processing and utilization of this substrate is complex, differing in many aspects from crop-based ethanol production. One important requirement is an efficient microorganism able to ferment a variety of sugars (pentoses, and hexoses) as well as to tolerate stress conditions. Through metabolic engineering, bacterial and yeast strains have been constructed which feature traits that are advantageous for ethanol production using lignocellulose sugars. After several rounds of modification/evaluation/modification, three main microbial platforms, Saccharomyces cerevisiae, Zymomonas mobilis, and Escherichia coli, have emerged and they have performed well in pilot studies. While there are ongoing efforts to further enhance their properties, improvement of the fermentation process is just one of several factors-that needs to be fully optimized and integrated to generate a competitive lignocellulose ethanol plant.     

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 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.     

Ethanol production from wood hydrolysate using genetically engineered Zymomonas mobilis

An ethanologenic microorganism capable of fermenting all of the sugars released from lignocellulosic biomass through a saccharification process is essential for secondary bioethanol production. We therefore genetically engineered the ethanologenic bacterium Zymomonas mobilis such that it efficiently produced bioethanol from the hydrolysate of wood biomass containing glucose, mannose, and xylose as major sugar components. This was accomplished by introducing genes encoding mannose and xylose catabolic enzymes from Escherichia coli. Integration of E. coli manA into Z. mobilis chromosomal DNA conferred the ability to co-ferment mannose and glucose, producing 91 % of the theoretical yield of ethanol within 36 h. Then, by introducing a recombinant plasmid harboring the genes encoding E. coli xylA, xylB, tal, and tktA, we broadened the range of fermentable sugar substrates for Z. mobilis to include mannose and xylose as well as glucose. The resultant strain was able to ferment a mixture of 20 g/l glucose, 20 g/l mannose, and 20 g/l xylose as major sugar components of wood hydrolysate within 72 h, producing 89.8 % of the theoretical yield. The recombinant Z. mobilis also efficiently fermented actual acid hydrolysate prepared from cellulosic feedstock containing glucose, mannose, and xylose. Moreover, a reactor packed with the strain continuously produced ethanol from acid hydrolysate of wood biomass from coniferous trees for 10 days without accumulation of residual sugars. Ethanol productivity was at 10.27 g/l h at a dilution rate of 0.25 h(-1).     

Bacteria engineered for fuel ethanol production: current status

The lack of industrially suitable microorganisms for converting biomass into fuel ethanol has traditionally been cited as a major technical roadblock to developing a bioethanol industry. In the last two decades, numerous microorganisms have been engineered to selectively produce ethanol. Lignocellulosic biomass contains complex carbohydrates that necessitate utilizing microorganisms capable of fermenting sugars not fermentable by brewers' yeast. The most significant of these is xylose. The greatest successes have been in the engineering of Gram-negative bacteria: Escherichia coli, Klebsiella oxytoca, and Zymomonas mobilis. E. coli and K. oxytoca are naturally able to use a wide spectrum of sugars, and work has concentrated on engineering these strains to selectively produce ethanol. Z. mobilis produces ethanol at high yields, but ferments only glucose and fructose. Work on this organism has concentrated on introducing pathways for the fermentation of arabinose and xylose. The history of constructing these strains and current progress in refining them are detailed in this review.     

Genetic improvement of Saccharomyces cerevisiae for xylose fermentation

There is considerable interest in recent years in the bioconversion of forestry and agricultural residues into ethanol and value-added chemicals. High ethanol yields from lignocellulosic residues are dependent on efficient use of all the available sugars including glucose and xylose. The well-known fermentative yeast Saccharomyces cerevisiae is the preferred microorganism for ethanol production, but unfortunately, this yeast is unable to ferment xylose. Over the last 15 years, this yeast has been the subject of various research efforts aimed at improving its ability to utilize xylose and ferment it to ethanol. This review examines the research on S. cerevisiae strains that have been genetically modified or adapted to ferment xylose to ethanol. The current state of these efforts and areas where further research is required are identified and discussed.     

Engineering and Two-Stage Evolution of a Lignocellulosic Hydrolysate-Tolerant Saccharomyces cerevisiae Strain for Anaerobic Fermentation of Xylose from AFEX Pretreated Corn Stover

The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting S. cerevisiae strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent S. cerevisiae strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in GRE3, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH.     

Metabolic engineering of bacteria for ethanol production

Technologies are available which will allow the conversion of lignocellulose into fuel ethanol using genetically engineered bacteria. Assembling these into a cost-effective process remains a challenge. Our work has focused primarily on the genetic engineering of enteric bacteria using a portable ethanol production pathway. Genes encoding Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase have been integrated into the chromosome of Escherichia coli B to produce strain KO11 for the fermentation of hemicellulose-derived syrups. This organism can efficiently ferment all hexose and pentose sugars present in the polymers of hemicellulose. Klebsiella oxytoca M5A1 has been genetically engineered in a similar manner to produce strain P2 for ethanol production from cellulose. This organism has the native ability to ferment cellobiose and cellotriose, eliminating the need for one class of cellulase enzymes. The optimal pH for cellulose fermentation with this organism (pH 5.0-5.5) is near that of fungal cellulases. The general approach for the genetic engineering of new biocatalysts has been most successful with enteric bacteria thus far. However, this approach may also prove useful with Gram-positive bacteria which have other important traits for lignocellulose conversion. Many opportunities remain for further improvements in the biomass to ethanol processes. These include the development of enzyme-based systems which eliminate the need for dilute acid hydrolysis or other pretreatments, improvements in existing pretreatments for enzymatic hydrolysis, process improvements to increase the effective use of cellulase and hemicellulase enzymes, improvements in rates of ethanol production, decreased nutrient costs, increases in ethanol concentrations achieved in biomass beers, increased resistance of the biocatalysts to lignocellulosic-derived toxins, etc. To be useful, each of these improvements must result in a decrease in the cost for ethanol production. Copyright 1998 John Wiley & Sons, Inc.