Fungal lysis by a soil bacterium fermenting cellulose

Recycling of plant biomass by a community of bacteria and fungi is fundamental to carbon flow in terrestrial ecosystems. Here we report how the plant fermenting, soil bacterium Clostridium phytofermentans enhances growth on cellulose by simultaneously lysing and consuming model fungi from soil. We investigate the mechanism of fungal lysis to show that among the dozens of different glycoside hydrolases C. phytofermentans secretes on cellulose, the most highly expressed enzymes degrade fungi rather than plant substrates. These enzymes, the GH18 Cphy1799 and Cphy1800, synergize to hydrolyse chitin, a main component of the fungal cell wall. Purified enzymes inhibit fungal growth and mutants lacking either GH18 grow normally on cellulose and other plant substrates, but have a reduced ability to hydrolyse chitinous substrates and fungal hyphae. Thus, C. phytofermentans boosts growth on cellulose by lysing fungi with its most highly expressed hydrolases, highlighting the importance of fungal interactions to the ecology of cellulolytic bacteria.     

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.     

Enzyme diversity of the cellulolytic system produced by Clostridium cellulolyticum explored by two-dimensional analysis: identification of seven genes encoding new dockerin-containing proteins

The enzyme diversity of the cellulolytic system produced by Clostridium cellulolyticum grown on crystalline cellulose as a sole carbon and energy source was explored by two-dimensional electrophoresis. The cellulolytic system of C. cellulolyticum is composed of at least 30 dockerin-containing proteins (designated cellulosomal proteins) and 30 noncellulosomal components. Most of the known cellulosomal proteins, including CipC, Cel48F, Cel8C, Cel9G, Cel9E, Man5K, Cel9M, and Cel5A, were identified by using two-dimensional Western blot analysis with specific antibodies, whereas Cel5N, Cel9J, and Cel44O were identified by using N-terminal sequencing. Unknown enzymes having carboxymethyl cellulase or xylanase activities were detected by zymogram analysis of two-dimensional gels. Some of these enzymes were identified by N-terminal sequencing as homologs of proteins listed in the NCBI database. Using Trap-Dock PCR and DNA walking, seven genes encoding new dockerin-containing proteins were cloned and sequenced. Some of these genes are clustered. Enzymes encoded by these genes belong to glycoside hydrolase families GH2, GH9, GH10, GH26, GH27, and GH59. Except for members of family GH9, which contains only cellulases, the new modular glycoside hydrolases discovered in this work could be involved in the degradation of different hemicellulosic substrates, such as xylan or galactomannan.     

Function and Structure Studies of GH Family 31 and 97 α-Glycosidases

A huge number of glycoside hydrolases are classified into the glycoside hydrolase family (GH family) based on their amino-acid sequence similarity. The glycoside hydrolases acting on α-glucosidic linkage are in GH family 4, 13, 15, 31, 63, 97, and 122. This review deals mainly with findings on GH family 31 and 97 enzymes. Research on two GH family 31 enzymes is described: clarification of the substrate recognition of Escherichia coli α-xylosidase, and glycosynthase derived from Schizosaccharomyces pombe α-glucosidase. GH family 97 is an aberrant GH family, containing inverting and retaining glycoside hydrolases. The inverting enzyme in GH family 97 displays significant similarity to retaining α-glycosidases, including GH family 97 retaining α-glycosidase, but the inverting enzyme has no catalytic nucleophile residue. It appears that a catalytic nucleophile has been eliminated during the molecular evolution in the same way as a man-made nucleophile mutant enzyme, which catalyzes the inverting reaction, as in glycosynthase and chemical rescue.     

Composition and Expression of Genes Encoding Carbohydrate-Active Enzymes in the Straw-Degrading Mushroom Volvariella volvacea

Volvariella volvacea is one of a few commercial cultivated mushrooms mainly using straw as carbon source. In this study, the genome of V. volcacea was sequenced and assembled. A total of 285 genes encoding carbohydrate-active enzymes (CAZymes) in V. volvacea were identified and annotated. Among 15 fungi with sequenced genomes, V. volvacea ranks seventh in the number of genes encoding CAZymes. In addition, the composition of glycoside hydrolases in V. volcacea is dramatically different from other basidiomycetes: it is particularly rich in members of the glycoside hydrolase families GH10 (hemicellulose degradation) and GH43 (hemicellulose and pectin degradation), and the lyase families PL1, PL3 and PL4 (pectin degradation) but lacks families GH5b, GH11, GH26, GH62, GH93, GH115, GH105, GH9, GH53, GH32, GH74 and CE12. Analysis of genome-wide gene expression profiles of 3 strains using 3′-tag digital gene expression (DGE) reveals that 239 CAZyme genes were expressed even in potato destrose broth medium. Our data also showed that the formation of a heterokaryotic strain could dramatically increase the expression of a number of genes which were poorly expressed in its parental homokaryotic strains.     

Several Genes Encoding Enzymes with the Same Activity Are Necessary for Aerobic Fungal Degradation of Cellulose in Nature

The cellulose-degrading fungal enzymes are glycoside hydrolases of the GH families and lytic polysaccharide monooxygenases. The entanglement of glycoside hydrolase families and functions makes it difficult to predict the enzymatic activity of glycoside hydrolases based on their sequence. In the present study we further developed the method Peptide Pattern Recognition to an automatic approach not only to find all genes encoding glycoside hydrolases and lytic polysaccharide monooxygenases in fungal genomes but also to predict the function of the genes. The functional annotation is an important feature as it provides a direct route to predict function from primary sequence. Furthermore, we used Peptide Pattern Recognition to compare the cellulose-degrading enzyme activities encoded by 39 fungal genomes. The results indicated that cellobiohydrolases and AA9 lytic polysaccharide monooxygenases are hallmarks of cellulose-degrading fungi except brown rot fungi. Furthermore, a high number of AA9, endocellulase and β-glucosidase genes were identified, not in what are known to be the strongest, specialized lignocellulose degraders but in saprophytic fungi that can use a wide variety of substrates whereas only few of these genes were found in fungi that have a limited number of natural, lignocellulotic substrates. This correlation suggests that enzymes with different properties are necessary for degradation of cellulose in different complex substrates. Interestingly, clustering of the fungi based on their predicted enzymes indicated that Ascomycota and Basidiomycota use the same enzymatic activities to degrade plant cell walls.     

Structural analysis of CPF_2247, a novel α-amylase from Clostridium perfringens

CPF_2247 from Clostridium perfringens ATCC 13124 was identified as a putative carbohydrate‐active enzyme by its low sequence identity to endo‐β‐1,4‐glucanases belonging to family 8 of the glycoside hydrolase classification. The X‐ray crystal structure of CPF_2247 determined to 2.0 Å resolution by single‐wavelength anomalous dispersion using seleno‐methionine‐substituted protein revealed an (α/α)₆ barrel fold. A large cleft on the surface of the protein contains residues that are structurally conserved with key elements of the catalytic machinery in clan GH‐M glycoside hydrolases. Assessment of CPF_2247 as a carbohydrate‐active enzyme disclosed α‐glucanase activity on amylose, glycogen, and malto‐oligosaccharides. Proteins 2011;. © 2011 Wiley‐Liss, Inc.     

Oxidoreductive cellulose depolymerization by the enzymes cellobiose dehydrogenase and glycoside hydrolase 61

Several members of the glycoside hydrolase 61 (GH61) family of proteins have recently been shown to dramatically increase the breakdown of lignocellulosic biomass by microbial hydrolytic cellulases. However, purified GH61 proteins have neither demonstrable direct hydrolase activity on various polysaccharide or lignacious components of biomass nor an apparent hydrolase active site. Cellobiose dehydrogenase (CDH) is a secreted flavocytochrome produced by many cellulose-degrading fungi with no well-understood biological function. Here we demonstrate that the binary combination of Thermoascus aurantiacus GH61A (TaGH61A) and Humicola insolens CDH (HiCDH) cleaves cellulose into soluble, oxidized oligosaccharides. TaGH61A-HiCDH activity on cellulose is shown to be nonredundant with the activities of canonical endocellulase and exocellulase enzymes in microcrystalline cellulose cleavage, and while the combination of TaGH61A and HiCDH cleaves highly crystalline bacterial cellulose, it does not cleave soluble cellodextrins. GH61 and CDH proteins are coexpressed and secreted by the thermophilic ascomycete Thielavia terrestris in response to environmental cellulose, and the combined activities of T. terrestris GH61 and T. terrestris CDH are shown to synergize with T. terrestris cellulose hydrolases in the breakdown of cellulose. The action of GH61 and CDH on cellulose may constitute an important, but overlooked, biological oxidoreductive system that functions in microbial lignocellulose degradation and has applications in industrial biomass utilization.     

The noncellulosomal family 48 cellobiohydrolase from Clostridium phytofermentans ISDg: heterologous expression, characterization, and processivity

Family 48 glycoside hydrolases (cellobiohydrolases) are among the most important cellulase components for crystalline cellulose hydrolysis mediated by cellulolytic bacteria. Open reading frame (Cphy_3368) of Clostridium phytofermentans ISDg encodes a putative family 48 glycoside hydrolase (CpCel48) with a family 3 cellulose-binding module. CpCel48 was successfully expressed as two soluble intracellular forms with or without a C-terminal His-tag in Escherichia coli and as a secretory active form in Bacillus subtilis. It was found that calcium ion enhanced activity and thermostability of the enzyme. CpCel48 had high activities of 15.1 U μmol⁻¹ on Avicel and 35.9 U μmol⁻¹ on regenerated amorphous cellulose (RAC) with cellobiose as a main product and cellotriose and cellotetraose as by-products. By contrast, it had very weak activities on soluble cellulose derivatives (e.g., carboxymethyl cellulose (CMC)) and did not significantly decrease the viscosity of the CMC solution. Cellotetraose was the smallest oligosaccharide substrate for CpCel48. Since processivity is a key characteristic for cellobiohydrolases, the new initial false/right attack model was developed for estimation of processivity by considering the enzyme's substrate specificity, the crystalline structure of homologous Cel48 enzymes, and the configuration of cellulose chains. The processivities of CpCel48 on Avicel and RAC were estimated to be ∼3.5 and 6.0, respectively. Heterologous expression of secretory active cellobiohydrolase in B. subtilis is an important step for developing recombinant cellulolytic B. subtilis strains for low-cost production of advanced biofuels from cellulosic materials in a single step.     

Glycoside hydrolases as components of putative carbohydrate biosensor proteins in <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

The composition of the cellulase system in the cellulosome-producing bacterium, Clostridium thermocellum, has been reported to change in response to growth on different carbon sources. Recently, an extensive carbohydrate-sensing mechanism, purported to regulate the activation of genes coding for polysaccharide-degrading enzymes, was suggested. In this system, CBM modules, comprising extracellular components of RsgI-like anti-σ factors, were proposed to function as carbohydrate sensors, through which a set of cellulose utilization genes are activated by the associated σ^I-like factors. An extracellular module of one of these RsgI-like proteins (Cthe_2119) was annotated as a family 10 glycoside hydrolase, RsgI6-GH10, and a second putative anti-σ factor (Cthe_1471), related in sequence to Rsi24, was found to contain a module that resembles a family 5 glycoside hydrolase (termed herein Rsi24C-GH5). The present study examines the relevance of these two glycoside hydrolases as sensors in this signal-transmission system. The RsgI6-GH10 was found to bind xylan matrices but exhibited low enzymatic activity on this substrate. In addition, this glycoside hydrolase module was shown to interact with crystalline cellulose although no hydrolytic activity was detected on cellulosic substrates. Bioinformatic analysis of the Rsi24C-GH5 showed a glutamate-to-glutamine substitution that would presumably preclude catalytic activity. Indeed, the recombinant module was shown to bind to cellulose, but showed no hydrolytic activity. These observations suggest that these two glycoside hydrolases underwent an evolutionary adaptation to function as polysaccharide binding agents rather than enzymatic components and thus serve in the capacity of extracellular carbohydrate sensors.     

Cloning, expression and characterization of an alkaline thermostable GH9 endoglucanase from Thermobifida halotolerans YIM 90462 T

The endoglucanase gene, thcel9A, from Thermobifida halotolerans YIM 90462^T was cloned and expressed in Escherichia coli BL 21(DE). The 2895-bp full-length gene encodes a 964-residue polypeptide (Thcel9A) containing a catalytic domain belonging to glycosyl hydrolases (GH) family 9. Phylogenetic analysis indicated that Thcel9A is closely related to Cel9A of Thermobifidafusca YX. Thcel9A was purified from the culture supernatant by Ni²⁺-affinity chromatography and the purified enzyme exhibited optimal activity at 55 °C and pH 8.0. Substrate specificity assays showed that it not only had CMCase activity, but also hydrolase activity on microcrystalline cellulose and filter paper. These properties suggested that Thcel9A is a classical GH9 group A endoglucanase.     

The crystal structure of a family GH25 lysozyme from Bacillus anthracis implies a neighboring-group catalytic mechanism with retention of anomeric configuration

Lysozymes are found in many of the sequence-based families of glycoside hydrolases (www.cazy.org) where they show considerable structural and mechanistic diversity. Lysozymes from glycoside hydrolase family GH25 adopt a (α/β)₅(β)₃-barrel-like fold with a proposal in the literature that these enzymes act with inversion of anomeric configuration; the lack of a suitable substrate, however, means that no group has successfully demonstrated the configuration of the product. Here we report the 3-D structure of the GH25 enzyme from Bacillus anthracis at 1.4 Å resolution. We show that the active center is extremely similar to those from glycoside hydrolase families GH18, GH20, GH56, GH84, and GH85 implying that, in the absence of evidence to the contrary, GH25 enzymes also act with net retention of anomeric configuration using the neighboring-group catalytic mechanism that is common to this ‘super-family’ of enzymes.     

A novel GH6 cellobiohydrolase from Paenibacillus curdlanolyticus B-6 and its synergistic action on cellulose degradation

We recently discovered a novel glycoside hydrolase family 6 (GH6) cellobiohydrolase from Paenibacillus curdlanolyticus B-6 (PcCel6A), which is rarely found in bacteria. This enzyme is a true exo-type cellobiohydrolase which exhibits high substrate specificity on amorphous cellulose and low substrate specificity on crystalline cellulose, while this showed no activity on substitution substrates, carboxymethyl cellulose and xylan, distinct from all other known GH6 cellobiohydrolases. Product profiles, HPLC analysis of the hydrolysis products and a schematic drawing of the substrate-binding subsites catalysing cellooligosaccharides can explain the new mode of action of this enzyme which prefers to hydrolyse cellopentaose. PcCel6A was not inhibited by glucose or cellobiose at concentrations up to 300 and 100 mM, respectively. A good synergistic effect for glucose production was found when PcCel6A acted together with processive endoglucanase Cel9R from Clostridium thermocellum and β-glucosidase CglT from Thermoanaerobacter brockii. These properties of PcCel6A make it a suitable candidate for industrial application in the cellulose degradation process.

     

里氏木霉不同糖苷水解酶基因转录水平比较分析

以前期里氏木霉RNA-seq中发现的7个糖苷水解酶基因为对象,分析其不同条件下的表达特性,以期为寻找新的纤维素降解功能酶提供证据。运用生物信息学方法,分析了7个基因可能的编码产物和结构特征。以不同的产纤维素酶菌株（QM 9414、RUT C30）为材料,采用实时荧光定量PCR,对7个糖苷水解酶基因（编号4–10）在各种碳源条件下转录情况与主要的3个纤维素酶基因cbh1,cbh2,egl1（编号1–3）进行了比较分析。信息学分析表明,7个基因编码蛋白分属于GH47（4号、5号）,GH92（6–8号）,GH16（9号）,GH31（10号）糖苷水解酶家族,具有典型的信号肽序列。cbh1,cbh2,egl1基因在纤维素酶诱导条件下,转录水平均表现显著的增加,上调倍数以QM 9414菌株表现的最高。QM 9414菌株中,cbh1,cbh2,egl1基因在纤维素条件下的上调倍数显著高于乳糖,3个基因在RUT C30菌株中的转录水平则显示乳糖条件下上调幅度更大。7个糖苷水解酶基因也存在类似的情况,而且编码α-甘露糖苷酶和内切β-葡聚糖酶的8号、9号基因上调倍数在纤维素酶诱导条件下仅次于纤维素酶基因,而以甘油为碳源条件下,8号、9号基因上调倍数高于纤维素酶基因。4号基因在上述碳源条件下,转录水平变化不大。结果表明：4号基因可能是组成型表达。基因5、6、7、8、9、10的表达呈现明显的菌株和碳源依赖性,且在纤维素酶诱导条件下基本上是和3个纤维素酶基因共转录的。     

Temporal Alterations in the Secretome of the Selective Ligninolytic Fungus Ceriporiopsis subvermispora during Growth on Aspen Wood Reveal This Organism's Strategy for Degrading Lignocellulose

The white-rot basidiomycetes efficiently degrade all wood cell wall polymers. Generally, these fungi simultaneously degrade cellulose and lignin, but certain organisms, such as Ceriporiopsis subvermispora, selectively remove lignin in advance of cellulose degradation. However, relatively little is known about the mechanism of selective ligninolysis. To address this issue, C. subvermispora was grown in liquid medium containing ball-milled aspen, and nano-liquid chromatography-tandem mass spectrometry was used to identify and estimate extracellular protein abundance over time. Several manganese peroxidases and an aryl alcohol oxidase, both associated with lignin degradation, were identified after 3 days of incubation. A glycoside hydrolase (GH) family 51 arabinofuranosidase was also identified after 3 days but then successively decreased in later samples. Several enzymes related to cellulose and xylan degradation, such as GH10 endoxylanase, GH5_5 endoglucanase, and GH7 cellobiohydrolase, were detected after 5 days. Peptides corresponding to potential cellulose-degrading enzymes GH12, GH45, lytic polysaccharide monooxygenase, and cellobiose dehydrogenase were most abundant after 7 days. This sequential production of enzymes provides a mechanism consistent with selective ligninolysis by C. subvermispora.     

Proteomic characterization of lignocellulose-degrading enzymes secreted by Phanerochaete carnosa grown on spruce and microcrystalline cellulose

Proteins secreted by the white-rot, softwood-degrading fungus Phanerochaete carnosa during growth on cellulose and spruce were analyzed using tandem mass spectrometry and de novo sequencing. Homology-driven proteomics was applied to compare P. carnosa peptide sequences to proteins in Phanerochaete chrysosporium using MS BLAST and non-gapped alignment. In this way, 665 and 365 peptides from cellulose and spruce cultivations, respectively, were annotated. Predicted activities included endoglucanases from glycoside hydrolase (GH) families 5, 16, and 61, cellobiohydrolases from GH6 and GH7, GH3 β-glucosidases, xylanases from GH10 and GH11, GH2 β-mannosidases, and debranching hemicellulases from GH43 and CE15. Peptides corresponding to glyoxal oxidases, peroxidases, and glycopeptides that could participate in lignin degradation were also detected. Overall, predicted activities detected in extracellular filtrates of cellulose and spruce cultures were similar, suggesting that the adaptation of P. carnosa to growth on lignocellulose might result from fine tuning the expression of similar enzyme families.     

Characterization of a family 5 glycoside hydrolase isolated from the outer membrane of cellulolytic Cytophaga hutchinsonii

Cytophaga hutchinsonii is an abundant aerobic cellulolytic bacterium that rapidly digests crystalline cellulose in a contact-dependent manner. The different roles of various predicted glycoside hydrolases and the detailed mechanism used by C. hutchinsonii in cellulose digestion are, however, not known. In this study, an endoglucanase belonging to glycoside hydrolase family 5 (GH5) named as ChCel5A was isolated from the outer membrane of C. hutchinsonii. The catalytic domain of ChCel5A exhibited typical endoglucanase activity and was capable of hydrolyzing insoluble cellulose with cellobiose and cellotriose as the predominant digestion products. Site-directed mutagenesis identified two aromatic amino acids in ChCle5A, W61 and W308, that dramatically decreased its hydrolytic activity toward filter paper while causing only a slight decrease in carboxymethylcellulase (CMCase) activity. Disruption of chu_1107 encoding ChCel5A caused no drastic effect on the growth parameters on cellulose for the resulting mutant strain with negligible reduction in the specific CMCase activities for intact cells. The demonstration of targeted gene inactivation capability for C. hutchinsonii has provided an opportunity to improve understanding of the details of the mechanism underlying its efficient utilization of cellulose.     

Structural basis for cellulose binding by the type A carbohydrate‐binding module 64 of Spirochaeta thermophila

Spirochaeta thermophila secretes seven glycoside hydrolases for plant biomass degradation that carry a carbohydrate‐binding module 64 (CBM64) appended at the C‐terminus. CBM64 adsorbs to various β1‐4‐linked pyranose substrates and shows high affinity for cellulose. We present the first crystal structure of a CBM64 at 1.2 Å resolution, which reveals a jelly‐roll‐like fold corresponding to a surface‐binding type A CBM. Modeling of its interaction with cellulose indicates that CBM64 achieves association with the hydrophobic face of β‐linked pyranose chains via a unique coplanar arrangement of four exposed tryptophan side chains. Proteins 2016; 84:855–858. © 2016 Wiley Periodicals, Inc.