General features of cellulosomal clostridial genomes compared with that of C. cellulovorans
1. Introduction
Lignocellulosic biomass such as agricultural, industrial, and forestry residues as well as dedicated crops constitute renewable and abundant resources with great potential for a low-cost and uniquely sustainable bioconversion to value-added bioproducts. Thus, many organic fuels and chemicals that can be obtained from lignocellulosic biomass can reduce greenhouse gas emissions, enhance energy security, improve the economy, dispose of problematic solid wastes, and improve air quality. In particular, liquid biofuels are attractive candidates, since little or no change is needed to the current petroleum-based fuel technologies. However, the biorefining process remains economically unfeasible due to a lack of biocatalysts that can overcome costly hurdles such as cooling from high temperature, pumping of oxygen/stirring, and, neutralization from acidic or basic pH. Therefore, bioconversion of the lignocellulosic components into fermentable sugars is an essential step in the biorefinery.
In nature, a variety of microorganisms including bacteria and fungi have the ability to degrade lignocellulosic biomass to C-5 and/or C-6 sugars. Moreover, new concepts have been proposed to enable the overall goal of cost reduction. These include genetically modifying the cell wall composition of energy crops in order to make their conversion easier, and combining the processes of glycoside hydrolases (GHs) and polysaccharide lyases (PLs) production, saccharification, and fermentation. Several clostridial species produce an extracellular enzyme complex called the cellulosomes and free extracellular enzymes called non-cellulosomes [1,2]. The cellulosomes are particularly designed for efficient degradation of plant cell wall polysaccharides such as cellulose, hemicellulose, and pectins. The component parts of the multi-component complex are integrated by virtue of a unique family of integrating modules, the cohesins and the dockerins, whose distribution and specificity dictate the overall cellulosome architecture. On the other hand, several clostridial species are able to ferment carbohydrates to acetone, butanol, and ethanol (ABE). Industrial application of this process, also known as ABE fermentation, has a long history, but the process economics after 1960 became unfavorable compared to the petrochemical process, and its commercial exploitation was gradually abandoned. The inefficiency of the fermentation still hampers commercial reintroduction of this renewable butanol production process. However, improving the yields and productivities of the solvent products is key to its successful reintroduction.
2. Solvent-producing clostridia
Biological production of butanol (
Several clostridial species such as
3. Metabolic engineering of mesophilic clostridia
Synthetic biology has recently been used to introduce biosynthetic capacity for butanol into non-natural hosts. The choice between using or engineering natural function versus importing biosynthetic function has been reviewed [16]. Commonly used host strains include
For successful metabolic engineering of
Several
Isobutanol is a more promising fermentation product because it is less toxic than 1-butanol. Unlike ethanol, isobutanol can also be blended at any ratio with gasoline or used directly in current engines without modification [30]. It is an attractive biofuel but cannot substitute for 1-butanol in the chemical market. One synthetic approach for isobutanol production involves the introduction of genes encoding enzymes that convert either acetyl-CoA or pyruvate to isobutanol. Alternatively, genes encoding enzymes that convert 2-keto acids intermediates (from amino acid synthesis) into isobutanol and branched-chain alcohols; 2-methyl-1-butanol and 3-methyl-1-butanol can be introduced [31,32,33]. Several companies are currently involved in scale-up and demonstration. Gevo Inc. (http://www.gevo.com) has engineered E. coli to produce isobutanol [34] and recently acquired a commercial-scale ethanol plant in Minnesota for retrofit to produce isobutanol. The company has also received Environmental Protection Agency certification to blend isobutanol in fossil fuels. DuPont has also engineered several biocatalysts for isobutanol [35] and assigned the technology to ButamaxTM Advanced Biofuels (http://www.butamax.com), a joint venture between BP and Dupont. ButamaxTM is collaborating with Kingston Research Limited, another BP–Dupont joint venture, to build a demonstration plant in the UK. Previously, the cellulosome-producing C. cellulolyticum has also been genetically engineered for improved ethanol production [36]. With this respect, most of the research concerning the construction of an organism for consolidated bioprocessing has focused on ethanol production. Despite this, it has been asserted that higher alcohols (i.e., alcohols with more than two carbons), such as isobutanol, are better candidates for gasoline replacement because they have energy density, octane value, and Reid vapor pressure that are more similar to those of gasoline [37].
4. Cellulosome-pruducing CLostridium cellulovorans
The anaerobic clostridia are found in the soil, on decaying plant materials, in rumens, in sewage sludge, in termite gut, in wood-chip piles, in compost piles, and at paper mills and wood processing plants (Table 1). Most of these bacteria occur in natural habitats such as soil and decaying plant materials, but some are enriched by human activities, such as in compost piles, in sewage plants, and at wood processing plants. Other natural habitats include the anaerobic rumen of various ruminants and the gut of termites, where they process plant materials for the host organism’s nutrition. The biotechnological potential of polysaccharolytic enzymes has resulted in the isolation and characterization of a large number of anaerobic, Gram-positive, spore-forming bacteria, the majority of which have been allocated to the genus Clostridium. Among some clostridia, the cellulosomes produced by Clostridium species are particularly designed for efficient degradation of plant cell wall polysaccharides. The component parts of the multicomponent complex are integrated by virtue of a unique family of integrating modules, the cohesins and the dockerins (Fig. 2A), whose distribution and specificity dictate the overall cellulosome architecture [38]. The cellulosomes are characterized by the presence of two general components: (1) the nonenzymatic scaffolding protein(s) with enzyme-binding sites called cohesins and (2) a variety of cellulosomal enzymes with dockerins, which interact with the cohesins in the scaffolding protein.
Since 2002, over 100 genome sequencing projects of Clostridium species have been done or are being done mainly by the United States Department of Energy Joint Genome Institute (DOE-JGI). The whole genome sequences of cellulosome-producing Clostridium species, i.e., thermophilic C. thermocellum ATCC27405 and mesophilic C. cellulolyticum H10 were sequenced by the JGI in 2007 and 2009, respectively. In 2009 the complete genome of C. cellulovorans was sequenced using the next-generation DNA sequencers to compare not only cellulosomal genes but also noncellulosomal ones among cellulosome-producing clostridia [39]. C. cellulovorans is able to degrade native substrates in soft biomass such as corn fiber and rice straw efficiently by producing the cellulosomes. The whole genome sequence of C. cellulovorans comprised 4,220 predicted genes in 5.10 Mbp. As a result, the genome size of C. cellulovorans was about 1 Mbp larger than that of other cellulosome-related clostridia, mesophilic C. acetobutylicum and C. cellulolyticum, and thermophilic C. thermocellum. A total of 57 cellulosomal genes were found in the C. cellulovorans genome (Table 2) and coded for not only CAZymes but also lipases, peptidases, and proteinase inhibitors [40,41]. Cellulosomal genes among clostridial genomes were identified and classified as cohesin-containing scaffolding proteins and dockerin-containing proteins. So far, the scaffolding proteins for constructing cellulosomes were found in C. acetobutylicum [42], C. cellulolyticum [43], C. cellulovorans [44], C. josui [45], and C. thermocellum [46].
Organism | GenBank Accession No. | Genome size (Mb) | No. of genes | No. of cellulosomal genes | % GC |
DF093537-DF093556 | 5.10 | 4220 | 57 | 31.1 | |
AE001437 | 3.94 | 3672 | 12 | 30.9 | |
CP001348 | 4.07 | 3390 | 65 | 37.4 | |
CP000568 | 3.84 | 3191 | 84 | 39.0 |
Among a total of 57 cellulosomal genes of the C. cellulovorans genome, 53 dockerin-containing proteins and four cohesin-containing scaffolding proteins were found, respectively [40]. More interestingly, two scaffolding proteins, CbpB and CbpC, consisting of a carbohydrate-binding module (CBM) of family 3, a surface–layer homology domain and a cohesin domain, were recently found and tandemly localized in the C. cellulovorans genome, while there were no such scaffolding proteins in other cellulosomal clostridia. Thus, by examining genome sequences from multiple Clostridium species, comparative genomics offers new insight into genome evolution and the way natural selection molds functional DNA sequence evolution. A recent model for the C. cellulovorans cellulosome reveals that the enzymatic subunits are bound to the scaffolding through the interaction of the cohesins and dockerins to form the cellulosome (Fig. 2B).
Carbohydrate-active enzymes (CAZymes) are categorized into different classes and families in the CAZy database (for more information please visit the CAZy web page; www.cazy.org). CAZymes that cleave, build, and rearrange oligo- and polysaccharides play a central role in the biology of bacteria and fungi and are key to optimizing biomass degradation by these species. Currently, more than 2,500 GHs have been identified and classified into 115 families [47]. Interestingly, the same enzyme family may contain members from bacteria, fungi, and plants with several different activities and substrate specifications [48]. However, fungal cellulases (hydrolysis of β-1,4-glycosidic bonds) have been mostly found within a few GH families including 5, 6, 7, 8, 9, 12, 44, 45, 48, 61, and 74 [47,49]. Cellulases have a small independently folded CBM that is connected to the catalytic domain by a flexible linker [48]. The CBMs are responsible for binding the enzyme to the crystalline cellulose, and thus enhance the enzyme activity [38]. Currently, many CBMs have been identified and classified into 54 families; however, only 20 families (1, 13, 14, 18, 19, 20, 21, 24, 29, 32, 35, 38, 39, 40, 42, 43, 47, 48, 50, and 52) have been found in fungi. Among 53 cellulosomal genes encoding dockerin containing proteins in the C. cellulovorans genome, a total of 29 genes coded for cellulolytic, hemicellulolytic and pectin-degrading enzymes [40]. Compared with the genome-sequenced species within cellulosomal clostridia, the proteome of C. cellulovorans focusing on dockerin-containing proteins showed representation of many proteins with known functions. In the C. cellulovorans cellulosome, there are 16 cellulase genes belonging to families GH5, GH9 and GH48, six mannanase genes belonging to families GH5 and GH26, three xylanase genes belonging to families GH8, GH10 and GH11, an endo-beta-galactosidase gene belonging to family GH98, and two pectate lyase genes belonging to families PL1 and PL9.
5. Cellulose metabolism of C. acetobutylicum
Cellulosomal gene clusters were conserved only in mesophilic clostridia (Fig. 3) [40]. Furthermore, these cellulosomal genes were randomly distributed in the C. cellulovorans genome except for the cellulosomal genes related to a large cellulosomal cluster, whereas two large cellulosomal gene clusters were found in the C. cellulolyticum genome. Even though the organization of genes encoding cellulosome subunits differs among mesophilic cellulolytic clostridia, there is nonetheless a clear similarity, particularly when looking at the cluster of genes following the main scaffoldin gene. Such a cluster is not found in C. thermocellum. This would suggest that the cellulosomes of the mesophilic clostridia, including the ‘ghost’ cellulosome of C. acetobutylicum, may have arisen from a common ancestral gene cluster. However, attempts have been made to develop a C. acetobutylicum strain that can utilize cellulose directly. There is evidence that C. acetobutylicum ATCC 824 can produce an active cellulosome. The celF gene, encoding a unique cellulase, was found to be up-regulated in C. acetobutylicum ATCC 824 during growth on xylose or lichenan [50]. However, C. acetobutylicum ATCC 824 had no cellulolytic activity suggesting that some element of the cellulosome is missing or not expressed. In an effort to make C. acetobutylicum utilize cellulose more directly, the engB gene from C. cellulovorans or the gene encoding the scaffold protein from C. cellulolyticum and C. thermocellum were introduced into C. acetobutylicum. However, the level of expressed heterologous cellulase was rather low [51,52]. On the other hand, the man5K gene encoding the mannanase Man5K from C. cellulolyticum was cloned alone or as an operon with the gene cipC1 encoding a truncated scaffoldin (miniCipC1) of the same origin in the solventogenic C. acetobutylicum [53]. The recombinant strains of the solventogenic bacterium were both found to secrete active Man5K in the range of milligrams per liter. In the case of the strain expressing only man5K, a large fraction of the recombinant enzyme was truncated and lost the N-terminal dockerin domain, but it remained active towards galactomannan. When man5K was coexpressed with cipC1 in C. acetobutylicum, the recombinant strain secreted almost exclusively full-length mannanase, which bound to the scaffoldin miniCipC1, thus showing that complexation to the scaffoldin stabilized the enzyme. Moreover, the secreted heterologous complex was found to be functional: it binds to crystalline cellulose via the carbohydrate-binding module of the miniscaffoldin, and the complexed mannanase is active towards galactomannan. Taken together, these data showed that C. acetobutylicum is a suitable host for the production, assembly, and secretion of heterologous minicellulosomes. More studies are needed to characterize the existing cellulosomal gene cluster in C. acetobutylicum before further metabolic engineering.
6. Consolidated bioprocessing by Clostridial species
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 [54]. Naturally occurring cellulolytic microorganisms are starting points for CBP organism development via the native strategy, with anaerobes being of particular interest [55]. The primary objective of such developments is to engineer product yields and titers to satisfy the requirements of an industrial process. Metabolic engineering of mixed-acid fermentations in relation to these objectives has been successful in the case of mesophilic, non-cellulolytic, enteric bacteria [56]. Far more limited work of this type has been undertaken with cellulolytic bacteria, primarily because of the absence of suitable gene-transfer techniques. Recent developments, however, appear to be removing this limitation for some organisms.
The lack of efficient genetic engineering tools including a gene knock-out system for C. acetobutylicum has hampered further strain improvement for a long time. As described earlier, much effort is exerted to develop genetic engineering tools for clostridia. In the mean time, Liao and collaborators recently reported metabolic engineering of E. coli for butanol production [57]. The mutant E. coli BW25113 (ΔadhE ΔldhA ΔfrdBC Δfnr Δpta) strain overexpressing the crt, bcd, etfAB, hbd and adhE2 genes of C. acetobutylicum, and atoB gene of E. coli was able to produce 552 mg/L butanol using 2% (w/v) glycerol as a carbon source. In another case, E. coli JM109 strain overexpressing the crt, bcd, etfAB, hbd, adhE and thiL genes of C. acetobutylicum was developed. This engineered E. coli strain was able to produce 16 mM butanol using 4% (w/v) glucose as a carbon source [58]. More recently, metabolic engineering has been used for the development of C. cellulolyticum H10 for isobutanol synthesis directly from cellulose [59] (Fig. 1B). In this study, by expressing enzymes that direct the conversion of pyruvate to isobutanol using an engineered valine biosynthesis pathway, the recombinant C. cellulolyticum was able to produce up to 660 mg/liter of isobutanol when grown on crystalline cellulose. To our knowledge, this was the first demonstration of isobutanol production directly from cellulose.
Butanol production from crystalline cellulose by co-cultures of the thermophilic and cellulosome-producing C. thermocellum and the mesophilic and butanol-producing C. saccharoperbutylacetonicum (strain N1-4) has been reported recently [60]. Butanol was produced from Avicel cellulose after it was incubated with C. thermocellum for at least 24 h at 60°C before the addition of the solventogenic strain N1-4. Butanol produced by strain N1-4 on 4% Avicel cellulose peaked (7.9 g/liter) after 9 days of incubation at 30°C, and acetone was undetectable in this coculture system. Less butanol was produced by C. acetobutylicum and C. beijerinckii in co-culture with C. thermocellum under the same conditions than by strain N1-4, indicating that strain N1-4 was the optimal strain for producing butanol from crystalline cellulose in this system.
7. Conclusion
It should be noted that one of the most critical factors not only for biofuel production but also for the whole biomass biorefinery concept is securing low price substrates for the processes. To compete with the conventional fossil resource-based chemical industry, the biotechnology industry needs a reliable, cost-effective raw materials infrastructure. The cost effectiveness of biomass production and the efficient storage and transport of harvested biomass resources will be critical elements for securing raw materials. Environmental impacts and sustainability are also important issues. There is a cautious prediction that agricultural crop production may not match future industrial demand. A significant amount of research has been dedicated to engineering organisms that are capable of consolidated bioprocessing (CBP). These CBP organisms are anticipated to have the ability to efficiently degrade lignocellulose, and to convert the resulting sugars to biofuels and chemical compounds at high productivities. Towards this goal, the production of biorefinery products from lignocellulose has been shown to be feasible using mesophilic clostridia. Both the successes and problems encountered in establishing new pathways in clostridial species will aid in the adaptation of the consolidated bioprocessing strategy in related mesophilic clostridial species such as C. acetobutylicum and C. cellulovorans.
References
- 1.
Tamaru Y. Miyake H. Kuroda K. Ueda M. Doi R. H. Comparative genomics of the mesophilic cellulosome-producing Clostridium cellulovorans and its application to biofuel production via consolidated bioprocessing. Environ Technol2010 31 889 903 - 2.
Chapter 20: Bacterial strategies for plant cell degradation and their genomic information. In Carbohydrate Modifying Biocatalysts (Ed. Peter Grunwald). Pan Stanford Publishing Pte. Ltd. (Singapore),Tamaru Y. Doi R. H. 2011 761 789 - 3.
Lee S. Y. Park J. H. Jang S. H. Nielsen L. K. Kim J. Jung K. S. Fermentative butanol production by Clostridia Biotechnol Bioeng2008 101 209 228 - 4.
Acetone-butanol fermentation revisited. Microbiol RevJones D. T. Woods D. R. 1986 50 484 524 - 5.
Zverlov V. V. Berezina O. Velikodvorskaya G. A. Schwarz W. H. Bacterial acetone and butanol production by industrial fermentation in the Soviet Union: Use of hydrolyzed agricultural waste for biorefinery. Appl Microbiol Biotechnol2006 71 587 597 - 6.
Jones D. T. Keis S. Origins and relationships of industrial solvent producing clostridial strains FEMS Microbiol Rev1995 17 223 232 - 7.
High-butanol ratio culturing method and its use. Chinese PatentZhang Y. Yang Y. Chen J. 1997 CN 1063483C. - 8.
Jiang Y. Xu C. Dong F. Yang Y. Jiang W. Yang S. Disruption of the acetoacetate decarboxylase gene in solvent-producing Clostridium acetobutylicum increases the butanol ratio Metab Eng2009 11 284 291 - 9.
Dürre P. Fermentative butanol production: bulk chemical and biofuel. Ann NY Acad Sci2008 1125 353 362 - 10.
Swana J. Yang Y. Behnam M. Thompson R. An analysis of net energy production and feedstock availability for biobutanol and bioethanol Bioresour Technol2011 102 2112 2117 - 11.
Ahn J. H. Sang B. I. Um Y. Butanol production from thin stillage using Clostridium pasteurianum Bioresour Technol2011 102 4934 4937 - 12.
Lee S. Y. Park J. H. Jang S. H. Nielsen L. K. Kim J. Jung K. S. Fermentative butanol production by Clostridia Biotechnol Bioeng2008 101 209 228 - 13.
Ezeji T. C. Qureshi N. Blaschek H. P. Bioproduction of butanol from biomass: from genes to bioreactors. Curr Opin Biotechnol2007 18 220 227 - 14.
Heap J. T. Pennington O. J. Cartman S. T. Carter G. P. Minton N. P. The ClosTron: A universal gene knock-out system for the genus Clostridium. J Microbiol Methods2007 70 452 464 - 15.
Bramono S. E. Lam Y. S. Ong S. L. He J. A mesophilic Clostridium species that produces butanol from monosaccharides and hydrogen from polysaccharides Bioresour Technol2011 102 9558 9563 - 16.
Engineering for biofuels: exploiting innate microbial capacity or importing biosynthetic potential? Nat Rev MicrobiolAlper H. Stephanopoulos G. 2009 7 715 723 - 17.
Knoshaug E. P. Zhang M. Butanol tolerance in a selection of microorganisms. Appl Biochem Biotechnol2009 153 13 20 - 18.
Nölling J. Breton G. Omelchenko M. V. Makarova K. S. Zeng Q. Gibson R. Lee H. M. Dubois J. Qiu D. Hitti J. Wolf Y. I. Tatusov R. L. Sabathe F. Doucette-stamm L. Soucaille P. Daly M. J. Bennett G. N. Koonin E. V. Smith D. R. Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. J Bacteriol2001 183 4823 4838 - 19.
Lee S. Y. Mermelstein L. D. Bennett G. N. Papoutsakis E. T. Vector construction, transformation, and gene amplification in Clostridium acetobutylicum ATCC 824. Ann NY Acad Sci1992 665 39 51 - 20.
J.D.,Minton N. P. Brehm J. K. Swinfield T. J. Whelan S. M. Mauchline M. L. Bodsworth N. Oultram 1993 Clostridial cloning vectors Woods DR, editor. The clostridia and biotechnology. Stoneham: Butterworth-Heinemann.119 150 - 21.
Mermelstein L. D. Welker N. E. Bennett G. N. Papoutsakis E. T. Expression of cloned homologous fermentative genes in Clostridium acetobutylicum ATCC 824. Bio/Technology1992 10 190 195 - 22.
methylation in Escherichia coli by the Bacillus subtilis phage w3TI methyl-transferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824. Appl Environ MicrobiolMermelstein L. D. Papoutsakis E. T. 1993 59 1077 1081 - 23.
Lee S. Y. Mermelstein L. D. Papoutsakis E. T. Determination of plasmid copy number and stability in Clostridium acetobutylicum ATCC 824. FEMS Microbiol Lett1993 108 319 324 - 24.
Green E. M. Fermentative production of butanol-the industrial perspective. Curr Opin Biotechnol2011 22 337 343 - 25.
Harris L. M. Blank L. Desai R. P. Welker N. E. Papoutsakis E. T. Fermentation characterization and flux analysis of recombinant strains of Clostridium acetobutylicum with an inactivated solR gene. J Ind Microbiol Biotechnol2001 27 322 328 - 26.
Characterization of recombinant strains of the butyrate kinase inactivation mutant: need for new phenomenological models for solventogenesis and butanol inhibition? Biotechnol BioengHarris L. M. Desai R. P. Welker N. E. Papoutsakis E. T. 2000 67 1 11 - 27.
Shao L. Hu S. Yang Y. Gu Y. Chen J. Yang Y. Jiang W. Yang S. 2007 Targeted gene disruption by use of a group II intron (targetron) vector in Clostridium acetobutylicum. Cell Res.17 963 965 - 28.
Heap J. T. Kuehne S. A. Ehsaan M. Cartman S. T. Cooksley C. M. Scott J. C. Minton N. P. The ClosTron: mutagenesis in Clostridium refined and streamlined J Microbiol Methods2010 80 49 55 - 29.
Methods. International Patent ApplicationHeap J. T. Minton N. P. 2009 PCT/GB2009/000380. - 30.
Biobutanol: an attractive biofuel. Biotechnol JDürre P. 2007 2 1525 1534 - 31.
Connor M. R. Liao J. C. Microbial production of advanced transportation fuels in non-natural hosts. Curr Opin Biotechnol2009 20 307 315 - 32.
Steen E. J. Chan R. Prasad N. Myers S. Petzold C. J. Redding A. Ouellet M. Keasling J. D. Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol. Microb Cell Fact2008 36 EOF - 33.
Nielsen D. R. Leonard E. Yoon S. H. Tseng H. C. Yuan C. Prather K. L. Engineering alternative butanol production platforms in heterologous bacteria Metab Eng2009 11 262 273 - 34.
Recovery of higher alcohols from dilute aqueous solutions. International Patent Application,Evanko W. A. Eyal A. M. Glassner D. A. Miao F. Aristidou A. Evans K. Gruber P. R. Hawkins A. C. 2009 PCT/US2008/088187. - 35.
Fermentative production of four carbon alcohols. US PatentDonaldson G. K. Eliot A. C. Flint D. Maggio-hall A. Nagarajan V. 2010 - 36.
Guedon E. Desvaux M. Petitdemange H. Improvement of cellulolytic properties of Clostridium cellulolyticum by metabolic engineering Appl Environ Microbiol2002 68 53 58 - 37.
Biobutanol-a replacement for bioethanol? Chem Eng ProgCascone R. 2008 S4 S9. - 38.
Bayer E. A. Lamed R. White B. A. Flint H. J. From cellulosomes to cellulosomics. Chem Rec2008 8 364 377 - 39.
Tamaru Y. Miyake H. Kuroda K. Nakanishi A. Kawade Y. Yamamoto K. Uemura M. Fujita Y. Doi R. H. Ueda M. Genome sequence of the cellulosome-producing mesophilic organism Clostridium cellulovorans 743B. J Bacteriol2010 192 901 902 - 40.
Tamaru Y. Miyake H. Kuroda K. Nakanishi A. Matsushima C. Doi R. H. Ueda M. Comparison of the mesophilic cellulosome-producing Clostridium cellulovorans genome with other cellulosome-related clostridial genomes Microb Biotechnol2011 4 64 73 - 41.
Meguro H. Morisaka H. Kuroda K. Miyake H. Tamaru Y. Ueda M. Putative role of cellulosomal protease inhibitors in Clostridium cellulovorans based on gene expression and measurement of activities. J Bacteriol2011 193 5527 5530 - 42.
Sabathe F. Bélaïch A. Soucaille P. Characterization of the cellulolytic complex (cellulosome) of Clostridium acetobutylicum FEMS Microbiol Lett2002 217 15 22 - 43.
Pagès S. Bélaïch A. Fierobe H. P. Tardif C. Gaudin C. Bélaïch J. P. Sequence analysis of scaffolding protein CipC and ORFXp, a new cohesin-containing protein in Clostridium cellulolyticum: comparison of various cohesin domains and subcellular localization of ORFXp. J Bacteriol1999 181 1801 1810 - 44.
Primary sequence analysis of cellulose binding protein A (CbpA). Proc Natl Acad Sci USAShoseyov O. Takagi M. Goldstein M. Doi R. H. 1992 89 3483 3487 - 45.
Cloning and DNA sequencing of the genes encoding scaffolding proteinCipA and cellulase CelD and identification of their gene products as major components of the cellulosome. J BacteriolKakiuchi M. Isui A. Suzuki K. Fujino T. Fujino E. Kimura T. Karita S. Sakka K. Ohmiya K. 1998 180 4303 4308 - 46.
Sequencing of a gene (CipA) encoding the cellulosomal SL-protein reveals an usual degree of internal homology. Mol MicrobiolGerngross U. T. Romaniec M. P. Kobayashi T. Huskisson N. S. Demain A. L. 1993 8 325 334 - 47.
Cantarel B. L. Coutinho P. M. Rancurel C. Bernard T. Lombard V. Henrissat B. The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res2009 D233 D238. - 48.
Dashtban M. Schraft H. Qin W. Fungal bioconversion of lignocellulosic residues; opportunities & perspectives Int J Biol Sci2009 5 578 595 - 49.
Sandgren M. Stahlberg J. Mitchinson C. Structural and biochemical studies of GH family 12 cellulases: improved thermal stability, and ligand complexes. Prog Biophys Mol Biol2005 89 246 291 - 50.
Substrate-induced production and secretion of cellulases by . Appl Environ MicrobiolLópez-contreras A. M. Gabor K. Martens A. A. Renckens B. A. Claassen P. A. Van Der Oost J. De Vos W. M. 2004 70 5238 5243 - 51.
Heterologous expression of endo-β-1,4-glucanase from in Clostridium acetobutylicum ATCC 824 following transformation of the engB gene. Appl Environ MicrobiolKim A. Y. Attwood G. T. Holt S. C. White B. A. Blaschek H. P. 1994 60 337 340 - 52.
Perret S. Casalot L. Fierobe H. P. Tardif C. Sabathe F. Bélaïch J. P. Bélaïch A. Production of heterologous and chimeric scaffoldins by Clostridium acetobutylicum ATCC 824 J Bacteriol2004 186 253 257 - 53.
Mingardon F. Perret S. Bélaïch A. Tardif C. Bélaïch J. P. Fierobe H. P. Heterologous production, assembly, and secretion of a minicellulosome by Clostridium acetobutylicum ATCC 824 Appl Environ Microbiol2005 71 1215 1222 - 54.
Joe Shaw A, Lynd LR.Olson D. G. Mcbride J. E. Recent progress in consolidated bioprocessing. Curr Opin Biotechnol2011 Dec 14. - 55.
Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol RevLynd L. R. Weimer P. J. Van Zyl W. H. Pretorius I. S. 2002 66 506 577 - 56.
Borges ACC, Causey TB, Martinez A, Morales F, Saleh A, Underwood SA, Yomano LP, York SW, Zaldivar J, Zhou S.Ingram L. O. Aldrich H. C. Enteric bacterial catalysts for fuel ethanol production. Biotechnol Prog1999 15 855 866 - 57.
Chou KJY, Hanai T, Liao JC.Atsumi S. Cann A. F. Connor M. R. Shen C. R. Smith K. M. Brynildsen M. P. Metab Eng of Escherichia coli for 1-butanol production2008 10 305 311 - 58.
Inui M. Suda M. Kimura S. Yasuda K. Suzuki H. Toda H. Yamamoto S. Okino S. Suzuki N. Yukawa H. Expression of Clostridium acetobutylicum butanol synthetic genes in Escherichia coli Appl Microbiol Biotechnol2008 77 1305 1316 - 59.
Higashide W. Li Y. Yang Y. Liao J. C. Metabolic engineering of Clostridium cellulolyticum for production of isobutanol from cellulose. Appl Environ Microbiol2011 77 2727 2733 - 60.
Nakayama S. Kiyoshi K. Kadokura T. Nakazato A. Butanol production from crystalline cellulose by cocultured Clostridium thermocellum and Clostridium saccharoperbutylacetonicum N1-4. Appl Environ Microbiol2011 77 6470 6475