Proteogenomic Analysis of a Thermophilic Bacterial Consortium Adapted to Deconstruct Switchgrass

Thermophilic bacteria are a potential source of enzymes for the deconstruction of lignocellulosic biomass. However, the complement of proteins used to deconstruct biomass and the specific roles of different microbial groups in thermophilic biomass deconstruction are not well-explored. Here we report on the metagenomic and proteogenomic analyses of a compost-derived bacterial consortium adapted to switchgrass at elevated temperature with high levels of glycoside hydrolase activities. Near-complete genomes were reconstructed for the most abundant populations, which included composite genomes for populations closely related to sequenced strains of Thermus thermophilus and Rhodothermus marinus, and for novel populations that are related to thermophilic Paenibacilli and an uncultivated subdivision of the little-studied Gemmatimonadetes phylum. Partial genomes were also reconstructed for a number of lower abundance thermophilic Chloroflexi populations. Identification of genes for lignocellulose processing and metabolic reconstructions suggested Rhodothermus, Paenibacillus and Gemmatimonadetes as key groups for deconstructing biomass, and Thermus as a group that may primarily metabolize low molecular weight compounds. Mass spectrometry-based proteomic analysis of the consortium was used to identify >3000 proteins in fractionated samples from the cultures, and confirmed the importance of Paenibacillus and Gemmatimonadetes to biomass deconstruction. These studies also indicate that there are unexplored proteins with important roles in bacterial lignocellulose deconstruction.     

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.     

Isolation and characterization of lignin‐degrading bacteria from rainforest soils

The deconstruction of lignin to enhance the release of fermentable sugars from plant cell walls presents a challenge for biofuels production from lignocellulosic biomass. The discovery of novel lignin‐degrading enzymes from bacteria could provide advantages over fungal enzymes in terms of their production and relative ease of protein engineering. In this study, 140 bacterial strains isolated from soils of a biodiversity‐rich rainforest in Peru were screened based on their oxidative activity on ABTS, a laccase substrate. Strain C6 (Bacillus pumilus) and strain B7 (Bacillus atrophaeus) were selected for their high laccase activity and identified by 16S rDNA analysis. Strains B7 and C6 degraded fragments of Kraft lignin and the lignin model dimer guaiacylglycerol‐β‐guaiacyl ether, the most abundant linkage in lignin. Finally, LC–MS analysis of incubations of strains B7 and C6 with poplar biomass in rich and minimal media revealed that a higher number of compounds were released in the minimal medium than in the rich one. These findings provide important evidence that bacterial enzymes can degrade and/or modify lignin and contribute to the release of fermentable sugars from lignocellulose. Biotechnol. Bioeng. 2013; 110: 1616–1626. © 2013 Wiley Periodicals, Inc.     

Efficient screening of potential cellulases and hemicellulases produced by <Emphasis Type="Italic">Bosea</Emphasis> sp. FBZP-16 using the combination of enzyme assays and genome analysis

Identification of bacteria that produce carbohydrolytic enzymes is extremely important given the increased demand for these enzymes in many industries. Twenty lignocellulose-degrading bacterial isolates from Algerian compost and different soils were screened for their potential to produce different enzymes involved in biomass deconstruction. Based on 16S rRNA gene sequencing, the isolates belonged to Proteobacteria and Actinobacteria. Differences among species were reflected both as the presence/absence of enzymes or at the level of enzyme activity. Among the most active species, Bosea sp. FBZP-16 demonstrated cellulolytic activity on both amorphous cellulose (CMC) and complex lignocellulose (wheat straw) and was selected for whole-genomic sequencing. The genome sequencing revealed the presence of a complex enzymatic machinery required for organic matter decomposition. Analysis of the enzyme-encoding genes indicated that multiple genes for endoglucanase, xylanase, β-glucosidase and β-mannosidase are present in the genome with enzyme activities displayed by the bacterium, while other enzymes, such as certain cellobiohydrolases, were not detected at the genomic level. This indicates that a combination of functional screening of bacterial cultures with the use of genome-derived information is important for the prediction of potential enzyme production. These results provide insight into their possible exploitation for the production of fuels and chemicals derived from plant biomass.     

Rumen Cellulosomics: Divergent Fiber-Degrading Strategies Revealed by Comparative Genome-Wide Analysis of Six Ruminococcal Strains

Background

A complex community of microorganisms is responsible for efficient plant cell wall digestion by many herbivores, notably the ruminants. Understanding the different fibrolytic mechanisms utilized by these bacteria has been of great interest in agricultural and technological fields, reinforced more recently by current efforts to convert cellulosic biomass to biofuels.

Methodology/Principal Findings

Here, we have used a bioinformatics-based approach to explore the cellulosome-related components of six genomes from two of the primary fiber-degrading bacteria in the rumen: Ruminococcus flavefaciens (strains FD-1, 007c and 17) and Ruminococcus albus (strains 7, 8 and SY3). The genomes of two of these strains are reported for the first time herein. The data reveal that the three R. flavefaciens strains encode for an elaborate reservoir of cohesin- and dockerin-containing proteins, whereas the three R. albus strains are cohesin-deficient and encode mainly dockerins and a unique family of cell-anchoring carbohydrate-binding modules (family 37).

Conclusions/Significance

Our comparative genome-wide analysis pinpoints rare and novel strain-specific protein architectures and provides an exhaustive profile of their numerous lignocellulose-degrading enzymes. This work provides blueprints of the divergent cellulolytic systems in these two prominent fibrolytic rumen bacterial species, each of which reflects a distinct mechanistic model for efficient degradation of cellulosic biomass.     

Bacterial enzymes for lignin depolymerisation: new biocatalysts for generation of renewable chemicals from biomass

The conversion of polymeric lignin from plant biomass into renewable chemicals is an important unsolved problem in the biorefinery concept. This article summarises recent developments in the discovery of bacterial enzymes for lignin degradation, our current understanding of their molecular mechanism of action, and their use to convert lignin or lignocellulose into aromatic chemicals. The review also discusses the recent developments in screening of metagenomic libraries for new biocatalysts, and the use of protein engineering to enhance lignin degradation activity.     

Mining diversity of the natural biorefinery housed within Tipula abdominalis larvae for use in an industrial biorefinery for production of lignocellulosic ethanol

Abstract Although they are the largest taxonomic group of animals, relatively few insects have been examined for symbiotic relationships with micro-organisms. However, this is rapidly changing because of the potential for examination of the natural insect–microbe–lignocellulose interactions to provide insights for biofuel technology. Micro-organisms associated with lignocellulose-consuming insects often facilitate the digestion of the recalcitrant plant diet; therefore these microbial communities may be mined for novel lignocellulose-degrading microbes, or for robust and inexpensive biocatalysts necessary for economically feasible biofuel production from lignocellulose. These insect–microbe interactions are influenced by the ecosystem and specific lignocellulose diet, and appreciating the whole ecosystem–insect–microbiota–lignocellulose as a natural biorefinery provides a rich and diverse framework from which to design novel industrial processes. One such natural biorefinery, the Tipula abdominalis larvae in riparian ecosystems, is reviewed herein with applications for biochemical processes and overcoming challenges involved in conversion of lignocellulosic biomass to fuel ethanol. From the dense and diverse T. abdominalis larval hindgut microbial community, a cellulolytic bacterial isolate, 27C64, demonstrated enzymatic activity toward many model plant polymers and also produced a bacterial antibiotic. 27C64 was co-cultured with yeast in fermentation of pine to ethanol, which allowed for a 20% reduction of commercial enzyme. In this study, a micro-organism from a lignocellulose-consuming insect was successfully applied for improvement of biomass-to-biofuel technology.     

An Efficient Proteomic Approach to Analyze Agriculture Crop Biomass

While a plant cell wall is formed by a complex of various components, including polysaccharides and structural proteins, its composition and representation may vary during cell growth. Currently, plant research targets the proteins participating in wall loosening. Multiple classes of enzymes, including various hemicellulases and cellulases, are required for plant material degradation to achieve the maximum decomposition. Identifying the set of proteins involved in the breakdown of cell-wall polymers is important to understand plant material conversion into suitable products. The objective of this study was to describe a method which can be used to carry out proteomics analysis of complex plant samples and identify enzymes degrading biomass. For this purpose we used proteomic techniques including gel electrophoresis, high pressure liquid chromatography combinated with mass spectrometry followed by data evaluation using databases searching. Results show that more than 50 % of these activities correspond to enzymes with proteolytic function. This study was focused primarily on enzymes able to breakdown the lignocellulosic and hemicellulosic parts that are very important for the material conversion into required products of degradation.     

Recombinant Bacillus subtilis That Grows on Untreated Plant Biomass

Lignocellulosic biomass is a promising feedstock to produce biofuels and other valuable biocommodities. A major obstacle to its commercialization is the high cost of degrading biomass into fermentable sugars, which is typically achieved using cellulolytic enzymes from Trichoderma reesei. Here, we explore the use of microbes to break down biomass. Bacillus subtilis was engineered to display a multicellulase-containing minicellulosome. The complex contains a miniscaffoldin protein that is covalently attached to the cell wall and three noncovalently associated cellulase enzymes derived from Clostridium cellulolyticum (Cel48F, Cel9E, and Cel5A). The minicellulosome spontaneously assembles, thus increasing the practicality of the cells. The recombinant bacteria are highly cellulolytic and grew in minimal medium containing industrially relevant forms of biomass as the primary nutrient source (corn stover, hatched straw, and switch grass). Notably, growth did not require dilute acid pretreatment of the biomass and the cells achieved densities approaching those of cells cultured with glucose. An analysis of the sugars released from acid-pretreated corn stover indicates that the cells have stable cellulolytic activity that enables them to break down 62.3% ± 2.6% of the biomass. When supplemented with beta-glucosidase, the cells liberated 21% and 33% of the total available glucose and xylose in the biomass, respectively. As the cells display only three types of enzymes, increasing the number of displayed enzymes should lead to even more potent cellulolytic microbes. This work has important implications for the efficient conversion of lignocellulose to value-added biocommodities.     

Discrete and Structurally Unique Proteins (Tāpirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose

A variety of catalytic and noncatalytic protein domains are deployed by select microorganisms to deconstruct lignocellulose. These extracellular proteins are used to attach to, modify, and hydrolyze the complex polysaccharides present in plant cell walls. Cellulolytic enzymes, often containing carbohydrate-binding modules, are key to this process; however, these enzymes are not solely responsible for attachment. Few mechanisms of attachment have been discovered among bacteria that do not form large polypeptide structures, called cellulosomes, to deconstruct biomass. In this study, bioinformatics and proteomics analyses identified unique, discrete, hypothetical proteins (“tāpirins,” origin from Māori: to join), not directly associated with cellulases, that mediate attachment to cellulose by species in the noncellulosomal, extremely thermophilic bacterial genus Caldicellulosiruptor. Two tāpirin genes are located directly downstream of a type IV pilus operon in strongly cellulolytic members of the genus, whereas homologs are absent from the weakly cellulolytic Caldicellulosiruptor species. Based on their amino acid sequence, tāpirins are specific to these extreme thermophiles. Tāpirins are also unusual in that they share no detectable protein domain signatures with known polysaccharide-binding proteins. Adsorption isotherm and trans vivo analyses demonstrated the carbohydrate-binding module-like affinity of the tāpirins for cellulose. Crystallization of a cellulose-binding truncation from one tāpirin indicated that these proteins form a long β-helix core with a shielded hydrophobic face. Furthermore, they are structurally unique and define a new class of polysaccharide adhesins. Strongly cellulolytic Caldicellulosiruptor species employ tāpirins to complement substrate-binding proteins from the ATP-binding cassette transporters and multidomain extracellular and S-layer-associated glycoside hydrolases to process the carbohydrate content of lignocellulose.     

Plant genetic engineering to improve biomass characteristics for biofuels

Currently, most ethanol produced in the United States is derived from maize kernel, at levels in excess of four billion gallons per year. Plant lignocellulosic biomass is renewable, cheap and globally available at 10-50 billion tons per year. At present, plant biomass is converted to fermentable sugars for the production of biofuels using pretreatment processes that disrupt the lignocellulose and remove the lignin, thus allowing the access of microbial enzymes for cellulose deconstruction. Both the pretreatments and the production of enzymes in microbial tanks are expensive. Recent advances in plant genetic engineering could reduce biomass conversion costs by developing crop varieties with less lignin, crops that self-produce cellulase enzymes for cellulose degradation and ligninase enzymes for lignin degradation, or plants that have increased cellulose or an overall biomass yield.     

Drought-Tolerance of Wheat Improved by Rhizosphere Bacteria from Harsh Environments: Enhanced Biomass Production and Reduced Emissions of Stress Volatiles

Water is the key resource limiting world agricultural production. Although an impressive number of research reports have been published on plant drought tolerance enhancement via genetic modifications during the last few years, progress has been slower than expected. We suggest a feasible alternative strategy by application of rhizospheric bacteria coevolved with plant roots in harsh environments over millions of years, and harboring adaptive traits improving plant fitness under biotic and abiotic stresses. We show the effect of bacterial priming on wheat drought stress tolerance enhancement, resulting in up to 78% greater plant biomass and five-fold higher survivorship under severe drought. We monitored emissions of seven stress-related volatiles from bacterially-primed drought-stressed wheat seedlings, and demonstrated that three of these volatiles are likely promising candidates for a rapid non-invasive technique to assess crop drought stress and its mitigation in early phases of stress development. We conclude that gauging stress by elicited volatiles provides an effectual platform for rapid screening of potent bacterial strains and that priming with isolates of rhizospheric bacteria from harsh environments is a promising, novel way to improve plant water use efficiency. These new advancements importantly contribute towards solving food security issues in changing climates.     

植物基因工程改善生物质能利用的研究进展

利用植物木质纤维资源发酵生产乙醇越来越受到人们的重视,但是要实现工业化生产仍然存在很多难题。最近,利用植物基因工程技术,改善植物自身性状,包括减少植物自身细胞壁中木质素含量、细胞中积累表达纤维素酶和木聚糖酶等方法,使自生产生的生物质更利于降解利用。目前,对这种新的能源转基因植物的研究取得了一定进展。     

基因工程改善植物生物质能利用的研究进展

利用植物木质纤维资源发酵产乙醇越来越受到人们的重视,但是要达到工业生产仍然存在很多难题。最近在利用植物基因工程技术改善植物自身性状,以利于能源植物的研究方面取得了一定的进展,这些研究包括减少植物自身细胞壁中的木质素含量、细胞中积累表达纤维素酶和木聚耱酶等的方法,使产生的生物质更利于降解利用。     

Research advances in expansins and expansion-like proteins involved in lignocellulose degradation

Biofuel derived from lignocellulosic biomass has attracted considerable attention as a renewable energy source. Nevertheless, the conversion of lignocellulose into fermentable sugars is inherently difficult because of the complex structures of lignocelluloses. Accessory proteins, like expansins, have a non-hydrolytic disruptive effect on crystalline cellulose and can synergistically cooperate with cellulase to improve hydrolysis efficiency. This review summarizes recent studies on expansins and expansin-like proteins, in terms of their expression and purification, synergism in lignocellulose hydrolysis, structure–function studies and binding characteristics. Future research prospects are also presented. This review provides a discussion of expansins in the context of lignocellulose hydrolysis.     

Characterization of extracellular lignocellulolytic enzymes of Coniochaeta sp. during corn stover bioconversion

Biorefinery of renewable lignocellulosic biomass to biochemical and biofuel is a promising technology to mitigate global warming and fuel shortage but hydrolysis of recalcitrant lignocellulose to its constitutive components is the bottleneck of the process. This work isolated and characterized a new lignocellulose degrading filamentous fungus from decomposing wood in mangrove area. The strain was identified as Coniochaeta sp. according to ITS rRNA sequences and its phylogenic analysis. The extracellular lignocellulolytic enzymes of this fungal strain, when grown on corn stover, were profiled by LC–MS/MS and exponentially modified protein abundance index (emPAI) based label-free quantitative proteomics approach. We identified 107 potential lignocellulolytic enzymes and their functional classification revealed unique extracellular enzyme system constituting multienzyme complexes of cellulases (29%), hemicellulases (17%), glycoside hydrolases (10%), proteases and peptidases (24%), lignin degrading enzymes (7%) and hypothetical proteins (13%). The growth behavior, biochemical assay and LC–MS/MS analysis of secretome by isolated fungal strain revealed its lignocellulose degradation potential when cultivated with corn stover as a major carbon source.