The first evidence that a single cellulase can be essential for cellulose degradation in a cellulolytic microorganism

Cellulose is the most abundant carbon source in nature but it is very difficult to degrade because of its insolubility, quasi‐crystalline structure and its presence in plant cell walls in a matrix with other polymers that limit access to the cellulose surface. Most cellulose in soils is degraded by cellulolytic microorganisms that use a number of different approaches to overcome the recalcitrance of cellulose in plant cell walls. All of these approaches involve multiple cellulases and, since cellulose is insoluble and microorganisms cannot ingest particles, the cellulases are present outside of the cell although they can be attached to its outer surface. An impressive article by Tolonen et al. in this issue of Molecular Microbiology shows that deletion of the single family 9 cellulase gene in Clostridium phytofermentans prevents growth on cellulose although the mutant strain grows perfectly well on glucose and its other cellulase genes are transcribed normally. These results show for the first time that a single cellulase can be essential for cellulose degradation by an organism despite the presence of several other cellulases. It will be interesting to learn the detailed mechanism that C. phytofermentans uses to degrade cellulose.     

Synergistic effects of cellulosomal xylanase and cellulases from Clostridium cellulovorans on plant cell wall degradation

Plant cell walls are comprised of cellulose and hemicellulose and other polymers that are intertwined, and this complex structure presents a barrier to degradation by pure cellulases or hemicellulases. In this study, we determined the synergistic effects on corn cell wall degradation by the action of cellulosomal xylanase XynA and cellulosomal cellulases from Clostridium cellulovorans. XynA minicellulosomes and cellulase minicellulosomes were found to degrade corn cell walls synergistically but not purified substrates such as xylan and crystalline cellulose. The mixture of XynA and cellulases at a molar ratio of 1:2 showed the highest synergistic effect of 1.6 on corn cell wall degradation. The amounts both of xylooligosaccharides and cellooligosaccharides liberated from corn cell walls were increased by the synergistic action of XynA and cellulases. Although synergistic effects on corn cell wall degradation were found in simultaneous reactions with XynA and cellulases, no synergistic effects were observed in sequential reactions. The possible mechanism of synergism between XynA and cellulases is discussed.     

Estimate of the degradation potentials of cellulose,xylan, and chitin across global prokaryotic communities

Complex polysaccharides (e.g. cellulose, xylan, and chitin), the most abundant renewable biomass resources available on Earth, are mainly degraded by microorganisms in nature. However, little is known about the global distribution of the enzymes and microorganisms responsible for the degradation of cellulose, xylan, and chitin in natural environments. Through large-scale alignments between the sequences released by the Earth Microbiome Project and sequenced prokaryotic genomes, we determined that almost all prokaryotic communities have the functional potentials to degrade cellulose, xylan, and chitin. The median abundances of genes encoding putative cellulases, xylanases, and chitinases in global prokaryotic communities are 0.51 (0.17–1.01), 0.24 (0.05–0.57), and 0.33 (0.11–0.71) genes/cell, respectively, and the composition and abundance of these enzyme systems are environmentally varied. The taxonomic sources of the three enzymes are highly diverse within prokaryotic communities, and the main factor influencing the diversity is the community's alpha diversity index rather than gene abundance. Moreover, there are obvious differences in taxonomic sources among different communities, and most genera with degradation potentials are narrowly distributed. In conclusion, our analysis preliminarily depicts a panorama of cellulose-, xylan-, and chitin-degrading enzymatic systems across global prokaryotic communities.     

Genome analyses highlight the different biological roles of cellulases

Cellulolytic enzymes have been the subject of renewed interest owing to their potential role in the conversion of plant lignocellulose to sustainable biofuels. An analysis of ～1,500 complete bacterial genomes, presented here, reveals that ～40% of the genomes of sequenced bacteria encode at least one cellulase gene. Most of the bacteria that encode cellulases are soil and marine saprophytes, many of which encode a range of enzymes for cellulose hydrolysis and also for the breakdown of the other constituents of plant cell walls (hemicelluloses and pectins). Intriguingly, cellulases are present in organisms that are usually considered as non-saprophytic, such as Mycobacterium tuberculosis, Legionella pneumophila, Yersinia pestis and even Escherichia coli. We also discuss newly emerging roles of cellulases in such non-saprophytic organisms.     

Caldicellulosiruptor core and pangenomes reveal determinants for noncellulosomal thermophilic deconstruction of plant biomass

Extremely thermophilic bacteria of the genus Caldicellulosiruptor utilize carbohydrate components of plant cell walls, including cellulose and hemicellulose, facilitated by a diverse set of glycoside hydrolases (GHs). From a biofuel perspective, this capability is crucial for deconstruction of plant biomass into fermentable sugars. While all species from the genus grow on xylan and acid-pretreated switchgrass, growth on crystalline cellulose is variable. The basis for this variability was examined using microbiological, genomic, and proteomic analyses of eight globally diverse Caldicellulosiruptor species. The open Caldicellulosiruptor pangenome (4,009 open reading frames [ORFs]) encodes 106 GHs, representing 43 GH families, but only 26 GHs from 17 families are included in the core (noncellulosic) genome (1,543 ORFs). Differentiating the strongly cellulolytic Caldicellulosiruptor species from the others is a specific genomic locus that encodes multidomain cellulases from GH families 9 and 48, which are associated with cellulose-binding modules. This locus also encodes a novel adhesin associated with type IV pili, which was identified in the exoproteome bound to crystalline cellulose. Taking into account the core genomes, pangenomes, and individual genomes, the ancestral Caldicellulosiruptor was likely cellulolytic and evolved, in some cases, into species that lost the ability to degrade crystalline cellulose while maintaining the capacity to hydrolyze amorphous cellulose and hemicellulose.     

Microbial starch-binding domain

Glucosidic bonds from different non-soluble polysaccharides such as starch, cellulose and xylan are hydrolyzed by amylases, cellulases and xylanases, respectively. These enzymes are produced by microorganisms. They have a modular structure that is composed of a catalytic domain and at least one non-catalytic domain that is involved in polysaccharide binding. Starch-binding modules are present in microbial enzymes that are involved in starch metabolism; these are classified into several different families on the basis of their amino acid sequence similarities. Such binding domains promote attachment to the substrate and increase its concentration at the active site of the enzyme, which allows microorganisms to degrade non-soluble starch. Fold similarities are better conserved than sequences; nevertheless, it is possible to notice two evolutionary clusters of microbial starch-binding domains. These domains have enormous potential as tags for protein immobilization, as well as for the tailoring of enzymes that play a part in polysaccharide metabolism.     

Applications of computational science for understanding enzymatic deconstruction of cellulose

Understanding the molecular-level mechanisms that enzymes employ to deconstruct plant cell walls is a fundamental scientific challenge with significant ramifications for renewable fuel production from biomass. In nature, bacteria and fungi use enzyme cocktails that include processive and non-processive cellulases and hemicellulases to convert cellulose and hemicellulose to soluble sugars. Catalyzed by an accelerated biofuels R&D portfolio, there is now a wealth of new structural and experimental insights related to cellulases and the structure of plant cell walls. From this background, computational approaches commonly used in other fields are now poised to offer insights complementary to experiments designed to probe mechanisms of plant cell wall deconstruction. Here we outline the current status of computational approaches for a collection of critical problems in cellulose deconstruction. We discuss path sampling methods to measure rates of elementary steps of enzyme action, coarse-grained modeling for understanding macromolecular, cellulosomal complexes, methods to screen for enzyme improvements, and studies of cellulose at the molecular level. Overall, simulation is a complementary tool to understand carbohydrate-active enzymes and plant cell walls, which will enable industrial processes for the production of advanced, renewable fuels.     

Inability of Microorganisms To Degrade Cellulose Acetate Reverse-Osmosis Membranes

Operational cellulose acetate reverse-osmosis membranes were examined for evidence of biological degradation. Numerous fungi and bacteria were isolated by direct and enrichment techniques. When tested, most of the fungi were active cellulose degraders, but none of the bacteria were. Neither fungi nor bacteria were able to degrade cellulose acetate membrane in vitro, although many fungi were able to degrade cellulose acetate membrane after it had been deacetylated. Organisms did not significantly degrade powdered cellulose acetate in pure or mixed cultures as measured by reduction in acetyl content or intrinsic viscosity or production of reducing sugars. Organisms did not affect the performance of cellulose triacetate fibers when incubated with them. The inability of the organisms to degrade cellulose acetate was attributed to the high degree of acetate substitution of the cellulose polymer. The rate of salt rejection decline was strongly correlated with chlorination of feed water and inversely with densities of microorganisms. These data suggest that microbial degradation of operational cellulose acetate reverse-osmosis membranes is unlikely.     

Genetic modification of lignin biosynthesis for improved biofuel production

The energy in cellulosic biomass largely resides in plant cell walls. Cellulosic biomass is more difficult than starch to break down into sugars because of the presence of lignin and the complex structure of cell walls. Transgenic down-regulation of major lignin genes led to reduced lignin content, increased dry matter degradability, and improved accessibility of cellulases for cellulose degradation. This review provides background information on lignin biosynthesis and focuses on genetic manipulation of lignin genes in important monocot species as well as the dicot potential biofuel crop alfalfa. Reduction of lignin in biofuel crops by genetic engineering is likely one of the most effective ways of reducing costs associated with pretreatment and hydrolysis of cellulosic feedstocks, although some potential fitness issues should also be addressed.     

Reversible control of biofilm formation by Cellulomonas spp. in response to nitrogen availability

The microbial degradation of cellulose contributes greatly to the cycling of carbon in terrestrial environments and feedbacks to the atmosphere, a process that is highly responsive to nitrogen inputs. Yet how key groups of cellulolytic microorganisms adaptively respond to the global conditions of nitrogen limitation and/or anthropogenic or climate nitrogen inputs is poorly understood. The actinobacterial genus Cellulomonas is of special interest because it incorporates the only species known to degrade cellulose aerobically and anaerobically. Furthermore, despite their inability to fix nitrogen, they are active decomposers in nitrogen-limited environments. Here we show that nitrogen limitation induced biofilm formation in Cellulomonas spp., a process that was coupled to carbon sequestration and storage in a curdlan-type biofilm matrix. The response was reversible and the curdlan matrix was solubilized and used as a carbon and energy source for biofilm dispersal once nitrogen sources became available. The biofilms attached strongly to cellulosic surfaces and, despite the growth limitation, produced cellulases and degraded cellulose more efficiently. The results show that biofilm formation is a competitive strategy for carbon and nitrogen acquisition and provide valuable insights linking nitrogen inputs to carbon sequestration and remobilization in terrestrial environments.     

纤维素乙醇统合加工过程中的酵母多酶共展示体系研究进展

利用统合生物加工过程(Consolidated bioprocessing,CBP)生产纤维素乙醇是目前国内外的研究热点。CBP需要一种“集成化”微生物,既能生产水解木质纤维素的多种酶类又能利用水解木质纤维素产生的糖类发酵产乙醇。以酿酒酵母表面展示技术为依托,建立CBP菌株多酶共展示体系的研究主要分为以下两个方向：一是直接将纤维素酶展示在细胞表面,即非复合型纤维素酶体系;另一种是通过表面展示纤维小体(Cellulosome)将纤维素酶间接地锚定在细胞表面,即复合型纤维素酶体系,本文主要从以上两个方向阐述了近几年对于纤维素乙醇生物统合加工过程的研究进展。因纤维小体对纤维素的降解能力比非复合型纤维素酶体系更强,所以其在酿酒酵母细胞表面的组装研究受到越来越多的关注,为了更深入透彻地了解纤维小体的酵母展示技术,文中对纤维小体的结构与功能及其在纤维素乙醇发酵中的应用研究进行重点论述,并对该领域的发展方向进行展望。     

Biochemistry and genetics of actinomycete cellulases.

The order Actinomycetales includes a number of genera that contain species that actively degrade cellulose and these include both mesophilic and facultative thermophilic species. Cellulases produced by strains from two of the genera containing thermophilic organisms have been studied extensively: Microbispora bispora and Thermomonospora fusca. Fractionation of M. bispora cellulases has identified six different enzymes, all of which were purified to near homogeneity and partially characterized. Two of these enzymes appear to be exocellulases and gave synergism with each other and with the endocellulases. The structural genes of five M. bispora cellulases have been cloned and one was sequenced. Fractionation of T. fusca cellulases has identified five different enzymes, all of which were purified to near homogeneity and partially characterized. One of the T. fusca enzymes gives synergism in the hydrolysis of crystalline cellulose with several T. fusca endocellulases and with Trichoderma reesei CBHI but not with T. reesei CBHII. Each T. fusca cellulase contains distinct catalytic and cellulose binding domains. The structural genes of four of the T. fusca endoglucanases have been cloned and sequenced, while three cellulase genes have been cloned from "T. curvata". The T. fusca cellulase genes are expressed at a low level in Escherichia soli, but at a high level in Streptomyces lividans. Sequence comparisons have shown that there are no significant amino acid homologies between any of the catalytic domains of the four T. fusca cellulases, but each of them shows extensive homology to several other cellulases and fits in one of the five existing cellulase gene families. There have been extensive studies of the regulation of the synthesis of these cellulases and a number of regulatory mutants have been isolated. This work has shown that the different T. fusca cellulases are coordinately regulated over a 100-fold range by two independent controls; induction by cellobiose and repression by any good carbon source.     

Biological Pretreatment of Lignocellulosic Substrates for Enhanced Delignification and Enzymatic Digestibility

Sheer enormity of lignocellulosics makes them potential feedstock for biofuel production but, their conversion into fermentable sugars is a major hurdle. They have to be pretreated physically, chemically, or biologically to be used by fermenting organisms for production of ethanol. Each lignocellulosic substrate is a complex mix of cellulose, hemicellulose and lignin, bound in a matrix. While cellulose and hemicellulose yield fermentable sugars, lignin is the most recalcitrant polymer, consisting of phenyl-propanoid units. Many microorganisms in nature are able to attack and degrade lignin, thus making access to cellulose easy. Such organisms are abundantly found in forest leaf litter/composts and especially include the wood rotting fungi, actinomycetes and bacteria. These microorganisms possess enzyme systems to attack, depolymerize and degrade the polymers in lignocellulosic substrates. Current pretreatment research is targeted towards developing processes which are mild, economical and environment friendly facilitating subsequent saccharification of cellulose and its fermentation to ethanol. Besides being the critical step, pretreatment is also cost intensive. Biological treatments with white rot fungi and Streptomyces have been studied for delignification of pulp, increasing digestibility of lignocellulosics for animal feed and for bioremediation of paper mill effluents. Such lignocellulolytic organisms can prove extremely useful in production of bioethanol when used for removal of lignin from lignocellulosic substrate and also for cellulase production. Our studies on treatment of hardwood and softwood residues with Streptomyces griseus isolated from leaf litter showed that it enhanced the mild alkaline solubilisation of lignins and also produced high levels of the cellulase complex when growing on wood substrates. Lignin loss (Klason lignin) observed was 10.5 and 23.5% in case of soft wood and hard wood, respectively. Thus, biological pretreatment process for lignocellulosic substrate using lignolytic organisms such as actinomycetes and white rot fungi can be developed for facilitating efficient enzymatic digestibility of cellulose.     

The structure and function of an arabinan-specific alpha-1,2-arabinofuranosidase identified from screening the activities of bacterial GH43 glycoside hydrolases

Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo- and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific α-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave α-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific α-1,2-arabinofuranosidase, CjAbf43A, displays a five-bladed β-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for α-1,2-l-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.     

A new locus affects cell motility, cellulose binding, and degradation by Cytophaga hutchinsonii

Cytophaga hutchinsonii is a Gram-negative gliding bacterium, which can rapidly degrade crystalline cellulose via a novel strategy without any recognizable processive cellulases. Its mechanism of cellulose binding and degradation is still a mystery. In this study, the mutagenesis of C. hutchinsonii with the mariner-based transposon HimarEm3 and gene complementation with the oriC-based plasmid carrying the antibiotic resistance gene cfxA or tetQ were reported for the first time to provide valuable tools for mutagenesis and genetic manipulation of the bacterium. Mutant A-4 with a transposon mutation in gene CHU_0134, which encodes a putative thiol-disulfide isomerase exhibits defects in cell motility and cellulose degradation. The cellulose binding ability of A-4 was only half of that of the wild-type strain, while the endo-cellulase activity of the cell-free supernatants and on the intact cell surface of A-4 decreased by 40?%. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of proteins binding to cellulose in the outer membrane showed that most of them were significantly decreased or disappeared in A-4 including some Gld proteins and hypothetical proteins, indicating that these proteins might play an important role in cell motility and cellulose binding and degradation by the bacterium.     

Biotechnology report: single cell proteins from cellulosic wastes

Conventional sources of protein cannot meet the present or projected needs for human consumption. Single cell proteins from fermentation of petroleum and cellulosic wastes are likely sources of additional protein. The volume of cellulosic wastes is sufficient to supply all additional protein needs on a continuing basis for cellulose is a renewable resource. Both mesophilic and thermophilic microorganisms utilize cellulose at reasonable rates. Biodegradation of lignin and lignin–cellulose complexes constitutes a major obstacle to commercial utilization of cellulosic wastes. Thermophilic actinomyces appear to be the most effective organisms for single cell protein production from cellulosic wastes.     

Molecular engineering of the cellulosome complex for affinity and bioenergy applications

The cellulosome complex has evolved to degrade plant cell walls and, as such, combines tenacious binding to cellulose with diverse catalytic activities against amorphous and crystalline cellulose. Cellulolytic microorganisms provide an extensive selection of domains; those with affinity for cellulose, cohesins and their dockerin binding partners that define cellulosome stoichiometry and architecture, and a range of catalytic activities against carbohydrates. These robust domains provide the building blocks for molecular design. This review examines how protein modules derived from the cellulosome have been incorporated into chimaeric proteins to provide biosynthetic tools for research and industry. These applications include affinity tags for protein purification, and non-chemical methods for immobilisation and presentation of recombinant protein domains on cellulosic substrates. Cellulosomal architecture provides a paradigm for design of enzymatic complexes that synergistically combine multiple catalytic subunits to achieve higher specific activity than would be obtained using free enzymes. Multimeric enzymatic complexes may have industrial applications of relevance for an emerging carbon economy. Biocatalysis will lead to more efficient utilisation of renewable carbon-fixing energy sources with the added benefits of reducing chemical waste streams and reliance on petroleum.     

Symbiotic "Archaezoa" of the primitive termite Mastotermes darwiniensis still play a role in cellulase production

The relictual Mastotermes darwiniensis is one of the world's most destructive termites. Like all phylogenetically basal termites, it possesses protozoa in its hindgut, which are believed to help it digest wood. L. Li, J. Frohlich, P. Pfeiffer, and H. Konig (Eukaryot. Cell 2:1091-1098, 2003) recently cloned the genes encoding cellulases from the protozoa of M. darwiniensis; however, they claimed that these genes are essentially inactive, not contributing significantly to cellulose digestion. Instead, they suggested that the protozoa sequester enzymes produced by the termite in its salivary glands and use these to degrade cellulose in the hindgut. We tested this idea by performing gel filtration of enzymes in extracts of the hindgut, as well as in a combination of the salivary glands, foregut, and midgut. Three major cellulases were found in the hindgut, each of which had a larger molecular size than termite-derived salivary gland enzymes. N-terminal amino acid sequencing of one of the hindgut-derived enzymes showed that it was identical to the putative amino acid sequence of one mRNA sequence isolated by Li et al. (Eukaryot. Cell 2:1091-1098, 2003). The overall activity of the hindgut cellulases was found to be of approximately equal magnitude to the termite-derived cellulases detected in the mixture of salivary gland, foregut, and midguts. Based on these results, we conclude that, contrary to Li et al. (Eukaryot. Cell 2:1091-1098, 2003), the hindgut protozoan fauna of M. darwiniensis actively produce cellulases, which play an important role in cellulose digestion of the host termite.