Consolidated bioprocessing of cellulosic biomass: an update

Biologically mediated processes seem promising for energy conversion, in particular for the conversion of lignocellulosic biomass into fuels. Although processes featuring a step dedicated to the production of cellulase enzymes have been the focus of most research efforts to date, consolidated bioprocessing (CBP)--featuring cellulase production, cellulose hydrolysis and fermentation in one step--is an alternative approach with outstanding potential. Progress in developing CBP-enabling microorganisms is being made through two strategies: engineering naturally occurring cellulolytic microorganisms to improve product-related properties, such as yield and titer, and engineering non-cellulolytic organisms that exhibit high product yields and titers to express a heterologous cellulase system enabling cellulose utilization. Recent studies of the fundamental principles of microbial cellulose utilization support the feasibility of CBP.     

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。     

Recent progress in consolidated bioprocessing

Consolidated bioprocessing, or CBP, the conversion of lignocellulose into desired products in one step without added enzymes, has been a subject of increased research effort in recent years. In this review, the economic motivation for CBP is addressed, advances and remaining obstacles for CBP organism development are reviewed, and we comment briefly on fundamental aspects. For CBP organism development beginning with microbes that have native ability to utilize insoluble components of cellulosic biomass, key recent advances include the development of genetic systems for several cellulolytic bacteria, engineering a thermophilic bacterium to produce ethanol at commercially attractive yields and titers, and engineering a cellulolytic microbe to produce butanol. For CBP organism development, beginning with microbes that do not have this ability and thus requiring heterologous expression of a saccharolytic enzyme system, high-yield conversion of model cellulosic substrates and heterologous expression of CBH1 and CBH2 in yeast at levels believed to be sufficient for an industrial process have recently been demonstrated. For both strategies, increased emphasis on realizing high performance under industrial conditions is needed. Continued exploration of the underlying fundamentals of microbial cellulose utilization is likely to be useful in order to guide the choice and development of CBP systems.     

Consolidated bioprocessing and simultaneous saccharification and fermentation of lignocellulose to ethanol with thermotolerant yeast strains

Consolidated bioprocessing (CBP), which integrates enzyme production, saccharification and fermentation into a single process, is a promising strategy for effective ethanol production from lignocellulosic materials because of the resulting reduction in utilities, the substrate and other raw materials and simplification of operation. CBP requires a highly engineered microbial strain capable of hydrolyzing biomass with enzymes produced on its own and producing high-titer ethanol. Recently, heterologous production of cellulolytic enzymes has been pursued with yeast hosts, which has realized direct conversion of cellulose to ethanol. Specifically, the development of cell surface engineering, which provides a display of cellulolytic enzymes on the yeast cell surface, facilitates effective biomass hydrolysis concomitantly with ethanol production. On the other hand, the difference in optimum temperature between saccharification and fermentation is a drawback of efficient ethanol production in the simultaneous saccharification and fermentation (SSF). The application of thermotolerant yeast strains engineered to the SSF process would overcome the drawback by performing hydrolysis and fermentation at elevated temperature. In this review, we focus on the recent advances in the application of thermotolerant yeast to CBP and SSF of lignocellulosic material to ethanol. The development of thermotolerant and ethanologenic yeast strains with the ability to hydrolyze lignocellulosic materials is emphasized for high-temperature CBP.     

Engineering cellulolytic ability into bioprocessing organisms

Lignocellulosic biomass is an abundant renewable feedstock for sustainable production of commodities such as biofuels. The main technological barrier that prevents widespread utilization of this resource for production of commodity products is the lack of low-cost technologies to overcome the recalcitrance of lignocellulose. Organisms that hydrolyse the cellulose and hemicelluloses in biomass and produce a valuable product such as ethanol at a high rate and titre would significantly reduce the costs of current biomass conversion technologies. This would allow steps that are currently accomplished in different reactors, often by different organisms, to be combined in a consolidated bioprocess (CBP). The development of such organisms has focused on engineering naturally cellulolytic microorganisms to improve product-related properties or engineering non-cellulolytic organisms with high product yields to become cellulolytic. The latter is the focus of this review. While there is still no ideal organism to use in one-step biomass conversion, several candidates have been identified. These candidates are in various stages of development for establishment of a cellulolytic system or improvement of product-forming attributes. This review assesses the current state of the art for enabling non-cellulolytic organisms to grow on cellulosic substrates.     

Lessons from the cow: What the ruminant animal can teach us about consolidated bioprocessing of cellulosic biomass

Consolidated bioprocessing (CBP) of cellulosic biomass is a promising source of ethanol. This process uses anaerobic bacteria, their own cellulolytic enzymes and fermentation pathways that convert the products of cellulose hydrolysis to ethanol in a single reactor. However, the engineering and economics of the process remain questionable. The ruminal fermentation is a very highly developed natural cellulose-degrading system. We propose that breakthroughs developed by cattle and other ruminant animals in cellulosic biomass conversion can guide future improvements in engineered CBP systems. These breakthroughs include, among others, an elegant and effective physical pretreatment; operation at high solids loading under non-aseptic conditions; minimal nutrient requirements beyond the plant biomass itself; efficient fermentation of nearly all plant components; efficient recovery of primary fermentation end-products; and production of useful co-products. Ruminal fermentation does not produce significant amounts of ethanol, but it produces volatile fatty acids and methane at a rapid rate. Because these alternative products have a high energy content, efforts should be made to recover these products and convert them to other organic compounds, particularly transportation fuels.     

Engineering microbes for direct fermentation of cellulose to bioethanol

Consolidated bioprocessing (CBP) by micro-organisms is desired for efficient conversion of lignocellulosic biomass to bioethanol fuels. Potential candidates have been discovered, including cellulolytic bacteria and filamentous fungi. Genetic and metabolic manipulation of these organisms further promotes their fermentation capacities and the ethanol tolerance. In addition, Saccharomyces cerevisiae and several other yeasts were genetically modified to express recombinant cellulases in media or display them on the cell surface for CBP of cellulose. To compensate the insufficient capacity of a single strain, various microbial consortia have also been developed. In this article, we reviewed the recent advances in CBP microbes and focused on the efforts in strain improvement employing genetic engineering.     

Current progress on engineering microbial strains and consortia for production of cellulosic butanol through consolidated bioprocessing

In the last decades, fermentative production of n-butanol has regained substantial interest mainly owing to its use as drop-in-fuel. The use of lignocellulose as an alternative to traditional acetone–butanol–ethanol fermentation feedstocks (starchy biomass and molasses) can significantly increase the economic competitiveness of biobutanol over production from non-renewable sources (petroleum). However, the low cost of lignocellulose is offset by its high recalcitrance to biodegradation which generally requires chemical-physical pre-treatment and multiple bioreactor-based processes. The development of consolidated processing (i.e., single-pot fermentation) can dramatically reduce lignocellulose fermentation costs and promote its industrial application. Here, strategies for developing microbial strains and consortia that feature both efficient (hemi)cellulose depolymerization and butanol production will be depicted, that is, rational metabolic engineering of native (hemi)cellulolytic or native butanol-producing or other suitable microorganisms; protoplast fusion of (hemi)cellulolytic and butanol-producing strains; and co-culture of (hemi)cellulolytic and butanol-producing microbes. Irrespective of the fermentation feedstock, biobutanol production is inherently limited by the severe toxicity of this solvent that challenges process economic viability. Hence, an overview of strategies for developing butanol hypertolerant strains will be provided.     

费希尔曲霉脂肪酶在毕赤酵母中的优化表达及高密度发酵

【背景】脂肪酶广泛应用于纺织、食品、药品、皮革等工业领域,其在微生物中的异源表达研究进一步促进了脂肪酶产品的生产和应用。【目的】实现来源于费希尔曲霉的脂肪酶在毕赤酵母中的高效异源表达,探究其合适的表达及发酵条件,提高产量,降低成本。【方法】对费希尔曲霉的脂肪酶编码基因进行密码子优化后,应用pPIC9k质粒整合到毕赤酵母GS115基因组上,构建高产脂肪酶Lip605的毕赤酵母工程菌;并通过响应面发酵条件优化、筛选最适伴侣蛋白和高密度发酵相结合的方法,综合提高脂肪酶表达量。【结果】确定高产脂肪酶毕赤酵母工程菌的最优摇瓶发酵产酶条件为:甲醇3.103%(体积比),生物素0.4 mg/L,酵母粉11.5 g/L,酵母基础氮源培养基(yeast nitrogen base,YNB) 13.4 g/L,初始pH 6.4,装液量50 mL/250 mL,转速220 r/min,温度24°C,培养时间40 h。优化后的胞外脂肪酶酶活达到72.34 U/mL,较优化前提高了5.8倍;进一步选择12个伴侣蛋白分别与脂肪酶Lip605进行共表达,其中共表达伴侣蛋白Rpl10(pPICZA-RPL10)效果最佳,可使Lip605表达量进一步提高46.8%;在此基础上,经过10 L发酵罐分批补料的高密度发酵,工程菌株发酵142 h,胞外脂肪酶酶活最高达到680 U/mL,蛋白浓度为15.89 g/L。【结论】应用复合策略有效提高了脂肪酶Lip605在毕赤酵母中的发酵产量,为其进一步工业化生产奠定了良好的基础。     

Fungal Bioconversion of Lignocellulosic Residues; Opportunities & Perspectives

The development of alternative energy technology is critically important because of the rising prices of crude oil, security issues regarding the oil supply, and environmental issues such as global warming and air pollution. Bioconversion of biomass has significant advantages over other alternative energy strategies because biomass is the most abundant and also the most renewable biomaterial on our planet. Bioconversion of lignocellulosic residues is initiated primarily by microorganisms such as fungi and bacteria which are capable of degrading lignocellulolytic materials. Fungi such as Trichoderma reesei and Aspergillus niger produce large amounts of extracellular cellulolytic enzymes, whereas bacterial and a few anaerobic fungal strains mostly produce cellulolytic enzymes in a complex called cellulosome, which is associated with the cell wall. In filamentous fungi, cellulolytic enzymes including endoglucanases, cellobiohydrolases (exoglucanases) and β-glucosidases work efficiently on cellulolytic residues in a synergistic manner. In addition to cellulolytic/hemicellulolytic activities, higher fungi such as basidiomycetes (e.g. Phanerochaete chrysosporium) have unique oxidative systems which together with ligninolytic enzymes are responsible for lignocellulose degradation. This review gives an overview of different fungal lignocellulolytic enzymatic systems including extracellular and cellulosome-associated in aerobic and anaerobic fungi, respectively. In addition, oxidative lignocellulose-degradation mechanisms of higher fungi are discussed. Moreover, this paper reviews the current status of the technology for bioconversion of biomass by fungi, with focus on mutagenesis, co-culturing and heterologous gene expression attempts to improve fungal lignocellulolytic activities to create robust fungal strains.     

酿酒酵母细胞表面展示技术在燃料乙醇生产中的应用及研究进展

酿酒酵母Saccharomyces cerevisiae细胞表面展示表达系统是一种固定化表达异源蛋白质的真核展示系统,具有糖基化作用及蛋白翻译后折叠等优势,更利于基因工程操作。近年来,酵母细胞表面工程作为一种新兴策略来固定化淀粉水解酶、纤维素水解酶以及木聚糖降解酶,从而应用于燃料乙醇的生产。文中着重介绍了酵母细胞表面展示系统的基本原理、研究现状以及在生物乙醇生产中的应用前景及所面临的挑战。     

Consolidated bioprocessing for butanol production of cellulolytic Clostridia: development and optimization

Butanol is an important bulk chemical, as well as a promising renewable gasoline substitute, that is commonly produced by solventogenic Clostridia. The main cost of cellulosic butanol fermentation is caused by cellulases that are required to saccharify lignocellulose, since solventogenic Clostridia cannot efficiently secrete cellulases. However, cellulolytic Clostridia can natively degrade lignocellulose and produce ethanol, acetate, butyrate and even butanol. Therefore, cellulolytic Clostridia offer an alternative to develop consolidated bioprocessing (CBP), which combines cellulase production, lignocellulose hydrolysis and co-fermentation of hexose/pentose into butanol in one step. This review focuses on CBP advances for butanol production of cellulolytic Clostridia and various synthetic biotechnologies that drive these advances. Moreover, the efforts to optimize the CBP-enabling cellulolytic Clostridia chassis are also discussed. These include the development of genetic tools, pentose metabolic engineering and the improvement of butanol tolerance. Designer cellulolytic Clostridia or consortium provide a promising approach and resource to accelerate future CBP for butanol production.     

Cellobiohydrolase secretion by yeast: Current state and prospects for improvement

Lignocellulose is an abundant and renewable feedstock for the production of such commodities as fuels and chemicals, provided that a low-cost technology can be developed to overcome its recalcitrance. Organisms that hydrolyze the sugar polymers in lignocellulose to produce a valuable product at a high rate would significantly reduce the costs of current conversion technologies. To develop yeasts, such as Saccharomyces cerevisiae, for such consolidated bioprocessing (CBP), a secreted heterologous cellulolytic enzyme system must be engineered into it. While considerable progress has been made in this regard, the secretion of cellobiohydrolases (CBHs) at levels required for crystalline cellulose hydrolysis has remained elusive until recently. Recent results suggest the existence of a compatibility factor for the expression of foreign genes in a host and that expression of some genes or their products exerted varying degrees of stress on the cell. The secretion machinery of yeasts is a multi-step process and each step is directed and regulated by several proteins, providing a vast array of targets that can be manipulated to enhance heterologous protein secretion. This review assesses the current state of the field with respect to CBH secretion in yeast and the options for enhancing yeast secretion capacity through strain engineering.     

Merits of secretion of heterologous proteins from industrial microorganisms

The heterologous expression of proteins is without doubt one of the most fascinating applications of the recombinant DNA technique. Despite clear successes many attempts to produce a certain protein in a heterologous host cell have met with technical difficulties. Secretion from cells has been used as a solution to overcome the intracellular formation of inactive protein. Microorganisms with a history of use in the fermentation industry exhibit clear advantages over the frequently usedEscherichia coli as host cells for secreted products. Interleukin-3, chymosin and phytase are examples of commercial products that are produced efficiently with the aid of industrial microorganisms. Presented at the FEMS Symposium "Novel Methods and Standardization in Microbiology", Košice (Slovakia) 1996. Part of this study was conducted with sponsoring from theEU Commission (project CT9302540).     

巴斯德毕赤酵母表达系统的研究进展和前景展望

巴斯德毕赤酵母经过近二十来年的发展,已经成为表达外源基因的优秀表达系统之一,成功地表达了许多重组异源蛋白。从表达菌株,表达载体等方面详细综述了毕赤酵母表达系统的优点,如：营养要求低、可高密度发酵、遗传稳定性高等;分析了可能影响巴斯德毕赤酵母表达系统的相关因素,这些因素包括外源基因的特性、基因拷贝数、产物稳定性及发酵策略等,结合这些因素和具体实践经验,就如何提高外源基因在巴斯德毕赤酵母中表达量进行了阐述;讨论了该表达系统存在的不足之处并且展望了其发展前景。     

Elucidation of the metabolic fate of glucose in the filamentous fungus Trichoderma reesei using expressed sequence tag (EST) analysis and cDNA microarrays

Despite the intense interest in the metabolic regulation and evolution of the ATP-producing pathways, the long standing question of why most multicellular microorganisms metabolize glucose by respiration rather than fermentation remains unanswered. One such microorganism is the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). Using EST analysis and cDNA microarrays, we find that in T. reesei expression of the genes encoding the enzymes of the tricarboxylic acid cycle and the proteins of the electron transport chain is programmed in a way that favors the oxidation of pyruvate via the tricarboxylic acid cycle rather than its reduction to ethanol by fermentation. Moreover, the results indicate that acetaldehyde may be channeled into acetate rather than ethanol, thus preventing the regeneration of NAD(+), a pivotal product required for anaerobic metabolism. The studies also point out that the regulatory machinery controlled by glucose was most probably the target of evolutionary pressure that directed the flow of metabolites into respiratory metabolism rather than fermentation. This finding has significant implications for the development of metabolically engineered cellulolytic microorganisms for fuel production from cellulose biomass.     

Native xylose-inducible promoter expands the genetic tools for the biomass-degrading,extremely thermophilic bacterium Caldicellulosiruptor bescii

Regulated control of both homologous and heterologous gene expression is essential for precise genetic manipulation and metabolic engineering of target microorganisms. However, there are often no options available for inducible promoters when working with non-model microorganisms. These include extremely thermophilic, cellulolytic bacteria that are of interest for renewable lignocellulosic conversion to biofuels and chemicals. In fact, improvements to the genetic systems in these organisms often cease once transformation is achieved. This present study expands the tools available for genetically engineering Caldicellulosiruptor bescii, the most thermophilic cellulose-degrader known growing up to 90 °C on unpretreated plant biomass. A native xylose-inducible (P _xi ) promoter was utilized to control the expression of the reporter gene (ldh) encoding lactate dehydrogenase. The P _xi -ldh construct resulted in a both increased ldh expression (20-fold higher) and lactate dehydrogenase activity (32-fold higher) in the presence of xylose compared to when glucose was used as a substrate. Finally, lactate production during growth of the recombinant C. bescii strain was proportional to the initial xylose concentration, showing that tunable expression of genes is now possible using this xylose-inducible system. This study represents a major step in the use of C. bescii as a potential platform microorganism for biotechnological applications using renewable biomass.