Random forest machine-learning algorithm classifies white- and brown-rot fungi according to the number of the genes encoding Carbohydrate-Active enZyme families.

Wood-rotting fungi play an important role in the global carbon cycle because they are the only known organisms that digest wood, the largest carbon stock in nature. In the present study, we used linear discriminant analysis and random forest (RF) machine learning algorithms to predict white- or brown-rot decay modes from the numbers of genes encoding Carbohydrate-Active enZymes with over 98% accuracy. Unlike other algorithms, RF identified specific genes involved in cellulose and lignin degradation, including auxiliary activities (AAs) family 9 lytic polysaccharide monooxygenases, glycoside hydrolase family 7 cellobiohydrolases, and AA family 2 peroxidases, as critical factors. This study sheds light on the complex interplay between genetic information and decay modes and underscores the potential of RF for comparative genomics studies of wood-rotting fungi. IMPORTANCE: Wood-rotting fungi are categorized as either white- or brown-rot modes based on the coloration of decomposed wood. The process of classification can be influenced by human biases. The random forest machine learning algorithm effectively distinguishes between white- and brown-rot fungi based on the presence of Carbohydrate-Active enZyme genes. These findings not only aid in the classification of wood-rotting fungi but also facilitate the identification of the enzymes responsible for degrading woody biomass.

Effect of Free Cysteine Residues to Serine Mutation on Cellodextrin Phosphorylase.

Cellodextrin phosphorylase (CDP) plays a key role in energy-efficient cellulose metabolism of anaerobic bacteria by catalyzing phosphorolysis of cellodextrin to produce cellobiose and glucose 1-phosphate, which can be utilized for glycolysis without consumption of additional ATP. As the enzymatic phosphorolysis reaction is reversible, CDP is also employed to produce cellulosic materials in vitro. However, the enzyme is rapidly inactivated by oxidation, which hinders in vitro utilization in aerobic environments. It has been suggested that the cysteine residues of CDP, which do not form disulfide bonds, are responsible for the loss of activity, and the aim of the present work was to test this idea. For this purpose, we replaced all 11 free cysteine residues of CDP from Acetivibrio thermocellus (formerly known as Clostridium thermocellum) with serine, which structurally resembles cysteine in our previous work. Herein, we show that the resulting CDP variant, named CDP-CS, has comparable activity to the wild-type enzyme, but shows increased stability to oxidation during long-term storage. X-Ray crystallography indicated that the mutations did not markedly alter the overall structure of the enzyme. Ensemble refinement of the crystal structures of CDP and CDP-CS indicated that the C372S and C625S mutations reduce structural fluctuations in the protein main chain, which may contribute to the increased stability of CDP-CS to oxidation.

Thermotolerance Mechanism of Fungal GH6 Cellobiohydrolase. Part II. Structural Analysis of Thermotolerant Mutant from the Basidiomycete Phanerochaete chrysosporium.

Glycoside hydrolase family 6 cellobiohydrolase (GH6 CBH) is a group of cellulases capable of hydrolyzing crystalline cellulose. However, the synergistic reaction of GH6 CBH with other cellulases is hindered by its relatively low thermotolerance. We previously obtained a thermotolerant double mutant, C240S/C393S, of GH6 CBH from the basidiomycete Phanerochaete chrysosporium (PcCel6A) by replacing the two free cysteine (Cys) residues, C240 and C393, with serine (Yamaguchi et al., J Appl Glycosci. 2020; 67;79-86). In the accompanying paper (Part I; Yamaguchi et al., J Appl Glycosci. 2024; 71: 55-62), we measured the temperature dependence of the activity and folding of C240S/C393S and its single mutants, C240S and C393S, and found that replacement of C393 was the major contributor to the increased thermotolerance of C240S/C393S. Here, in order to investigate the mechanism involved, we crystallized the wild-type and the mutant enzymes and compared their X-ray crystal structures. The overall structures of the wild-type and the three mutant enzymes were similar. However, C240S/C393S had the lowest relative B-factor at both the N-terminal loop (residues 172-177) and the C-terminal loop (residues 390-425). This result suggests that reduced structural fluctuation of the substrate-enclosing loops, possibly due to stronger hydrogen bonding involving C393, could account for the increased thermotolerance of C240S/C393S.

Thermotolerance Mechanism of Fungal GH6 Cellobiohydrolase. Part I. Characterization of Thermotolerant Mutant from the Basidiomycete Phanerochaete chrysosporium.

Cellobiohydrolase (CBH), belonging to glycoside hydrolase family 6 (GH6), plays an essential role in cellulose saccharification, but its low thermotolerance presents a challenge in improving the reaction efficiency. Based on a report that chimeric CBH II (GH6) engineered to remove non-disulfide-bonded free Cys shows increased thermotolerance, we previously mutated the two free Cys residues to Ser in GH6 CBH from the basidiomycete Phanerochaete chrysosporium (PcCel6A) and obtained a thermotolerant double mutant, C240S/C393S (Yamaguchi et al., J. Appl. Glycosci. 2020; 67: 79-86). Here, characterization of the double mutant revealed that its activity towards both amorphous and crystalline cellulose was higher than that of the wild-type enzyme at elevated temperature, suggesting that the catalytic domain is the major contributor to the increased thermotolerance. To investigate the role of each free Cys residue, we prepared both single mutants, C240S and C393S, of the catalytic domain of PcCel6A and examined their residual activity at high temperature and the temperature-dependent changes of folding by means of circular dichroism measurements and thermal shift assay. The results indicate that the C393S mutation is the main contributor to both the increased thermotolerance of C240S/C393S and the increased activity of the catalytic domain at high temperature.

Effect of degree of substitution on the microphase separation and mechanical properties of cellooligosaccharide acetate-based elastomers.

Thermoplastic elastomers (TPEs) have long been used in a wide range of industries. However, most existing TPEs are petroleum-derived polymers. To realize environmentally benign alternatives to conventional TPEs, cellulose acetate is a promising TPE hard segment because of its sufficient mechanical properties, availability from renewable sources, and biodegradability in natural environments. Because the degree of substitution (DS) of cellulose acetate governs a range of physical properties, it is a useful parameter for designing novel cellulose acetate-based TPEs. In this study, we synthesized cellulose acetate-based ABA-type triblock copolymers (AcCelx-b-PDL-b-AcCelx) containing a celloologosaccharide acetate hard A segment (AcCelx, where x is the DS; x = 3.0, 2.6, and 2.3) and a poly(δ-decanolactone) (PDL) soft B segment. Small-angle X-ray scattering showed that decreasing the DS of AcCelx-b-PDL-b-AcCelx resulted in the formation of a more ordered microphase-separated structure. Owing to the microphase separation of the hard cellulosic and soft PDL segments, all the AcCelx-b-PDL-b-AcCelx samples exhibited elastomer-like properties. Moreover, the decrease in DS improved toughness and suppressed stress relaxation. Furthermore, preliminary biodegradation tests in an aqueous environment revealed that the decrease in DS endowed AcCelx-b-PDL-b-AcCelx with greater biodegradability potential. This work demonstrates the usefulness of cellulose acetate-based TPEs as next-generation sustainable materials.

Lytic polysaccharide monooxygenase increases cellobiohydrolases activity by promoting decrystallization of cellulose surface.

Efficient depolymerization of crystalline cellulose requires cooperation between multiple cellulolytic enzymes. Through biochemical approaches, molecular dynamics (MD) simulation, and single-molecule observations using high-speed atomic force microscopy (HS-AFM), we quantify and track synergistic activity for cellobiohydrolases (CBHs) with a lytic polysaccharide monooxygenase (LPMO) from Phanerochaete chrysosporium. Increasing concentrations of LPMO (AA9D) increased the activity of a glycoside hydrolase family 6 CBH, Cel6A, whereas the activity of a family 7 CBH (Cel7D) was enhanced only at lower concentrations of AA9D. MD simulation suggests that the result of AA9D action to produce chain breaks in crystalline cellulose can oxidatively disturb the crystalline surface by disrupting hydrogen bonds. HS-AFM observations showed that AA9D increased the number of Cel7D molecules moving on the substrate surface and increased the processivity of Cel7D, thereby increasing the depolymerization performance, suggesting that AA9D not only generates chain ends but also amorphizes the crystalline surface, thereby increasing the activity of CBHs.

Acetylated Xylan Degradation by Glycoside Hydrolase Family 10 and 11 Xylanases from the White-rot Fungus Phanerochaete chrysosporium.

Endo-type xylanases are key enzymes in microbial xylanolytic systems, and xylanases belonging to glycoside hydrolase (GH) families 10 or 11 are the major enzymes degrading xylan in nature. These enzymes have typically been characterized using xylan prepared by alkaline extraction, which removes acetyl sidechains from the substrate, and thus the effect of acetyl groups on xylan degradation remains unclear. Here, we compare the ability of GH10 and 11 xylanases, PcXyn10A and PcXyn11B, from the white-rot basidiomycete Phanerochaete chrysosporium to degrade acetylated and deacetylated xylan from various plants. Product quantification revealed that PcXyn10A effectively degraded both acetylated xylan extracted from Arabidopsis thaliana and the deacetylated xylan obtained by alkaline treatment, generating xylooligosaccharides. In contrast, PcXyn11B showed limited activity towards acetyl xylan, but showed significantly increased activity after deacetylation of the xylan. Polysaccharide analysis using carbohydrate gel electrophoresis showed that PcXyn11B generated a broad range of products from native acetylated xylans extracted from birch wood and rice straw, including large residual xylooligosaccharides, while non-acetylated xylan from Japanese cedar was readily degraded into xylooligosaccharides. These results suggest that the degradability of native xylan by GH11 xylanases is highly dependent on the extent of acetyl group substitution. Analysis of 31 fungal genomes in the Carbohydrate-Active enZymes database indicated that the presence of GH11 xylanases is correlated to that of carbohydrate esterase (CE) family 1 acetyl xylan esterases (AXEs), while this is not the case for GH10 xylanases. These findings may imply co-evolution of GH11 xylanases and CE1 AXEs.

Enzymatic synthesis of cellulose in space: gravity is a crucial factor for building cellulose II gel structure.

ABSTRACT: We previously reported in vitro synthesis of highly ordered crystalline cellulose II by reverse reaction of cellodextrin phosphorylase from the cellulolytic bacterium Clostridium (Hungateiclostridium) thermocellum (CtCDP), but the formation mechanism of the cellulose crystals and highly ordered structure has long been unclear. Considering the specific density of cellulose versus water, the formation of crystalline and highly ordered structure in an aqueous solution should be affected by gravity. Thus, we synthesized cellulose with CtCDP stable variant at the International Space Station, where sedimentation and convection due to gravity are negligible. Optical microscopic observation suggested that cellulose in space has a gel-like appearance without apparent aggregation, in contrast to cellulose synthesized on the ground. Small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS) indicated that cellulose synthesized in space has a more uniform particle distribution in the ~ 100 nm scale region than cellulose synthesized on the ground. Scanning electron microscopy (SEM) showed that both celluloses have a micrometer scale network structure, whereas a fine fiber network was constructed only under microgravity. These results indicate that gravity plays a role in cellulose II crystal sedimentation and the building of network structure, and synthesis in space could play a role in designing unique materials. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10570-021-04399-0.

Comparison of glycoside hydrolase family 3 ß-xylosidases from basidiomycetes and ascomycetes reveals evolutionarily distinct xylan degradation systems.

Xylan is the most common hemicellulose in plant cell walls, though the structure of xylan polymers differs between plant species. Here, to gain a better understanding of fungal xylan degradation systems, which can enhance enzymatic saccharification of plant cell walls in industrial processes, we conducted a comparative study of two glycoside hydrolase family 3 (GH3) ß-xylosidases (Bxls), one from the basidiomycete Phanerochaete chrysosporium (PcBxl3), and the other from the ascomycete Trichoderma reesei (TrXyl3A). A comparison of the crystal structures of the two enzymes, both with saccharide bound at the catalytic center, provided insight into the basis of substrate binding at each subsite. PcBxl3 has a substrate-binding pocket at subsite -1, while TrXyl3A has an extra loop that contains additional binding subsites. Furthermore, kinetic experiments revealed that PcBxl3 degraded xylooligosaccharides faster than TrXyl3A, while the KM values of TrXyl3A were lower than those of PcBxl3. The relationship between substrate specificity and degree of polymerization of substrates suggested that PcBxl3 preferentially degrades xylobiose (X2), while TrXyl3A degrades longer xylooligosaccharides. Moreover, docking simulation supported the existence of extended positive subsites of TrXyl3A in the extra loop located at the N-terminus of the protein. Finally, phylogenetic analysis suggests that wood-decaying basidiomycetes use Bxls such as PcBxl3 that act efficiently on xylan structures from woody plants, whereas molds use instead Bxls that efficiently degrade xylan from grass. Our results provide added insights into fungal efficient xylan degradation systems.

Glucomannan and beta-glucan degradation by Mytilus edulis Cel45A: Crystal structure and activity comparison with GH45 subfamily A, B and C.

The enzymatic hydrolysis of barley beta-glucan, konjac glucomannan and carboxymethyl cellulose by a ß-1,4-D-endoglucanase MeCel45A from blue mussel, Mytilus edulis, which belongs to subfamily B of glycoside hydrolase family 45 (GH45), was compared with GH45 members of subfamilies A (Humicola insolens HiCel45A), B (Trichoderma reesei TrCel45A) and C (Phanerochaete chrysosporium PcCel45A). Furthermore, the crystal structure of MeCel45A is reported. Initial rates and hydrolysis yields were determined by reducing sugar assays and product formation was characterized using NMR spectroscopy. The subfamily B and C enzymes exhibited mannanase activity, whereas the subfamily A member was uniquely able to produce monomeric glucose. All enzymes were confirmed to be inverting glycoside hydrolases. MeCel45A appears to be cold adapted by evolution, as it maintained 70% activity on cellohexaose at 4 °C relative to 30 °C, compared to 35% for TrCel45A. Both enzymes produced cellobiose and cellotetraose from cellohexaose, but TrCel45A additionally produced cellotriose.

Role of Tryptophan 38 in Loading Substrate Chain into the Active-site Tunnel of Cellobiohydrolase I from Trichoderma reesei.

Cellobiohydrolase I from Trichoderma reesei ( Tr Cel7A) is one of the best-studied cellulases, exhibiting high activity towards crystalline cellulose. Tryptophan residues at subsites -7 and -4 (Trp40 and Trp38 respectively) are located at the entrance and middle of the tunnel-like active site of Tr Cel7A, and are conserved among the GH family 7 cellobiohydrolases. Trp40 of Tr Cel7A is important for the recruitment of cellulose chain ends on the substrate surface, but the role of Trp38 is less clear. Comparison of the effects of W38A and W40A mutations on the binding energies of sugar units at the two subsites indicated that the contribution of Trp38 to the binding was greater than that of Trp40. In addition, the smooth gradient of binding energy was broken in W38A mutant. To clarify the importance of Trp38, the activities of Tr Cel7A WT and W38A towards crystalline cellulose and amorphous cellulose were compared. W38A was more active than WT towards amorphous cellulose, whereas its activity towards crystalline cellulose was only one-tenth of that of WT. To quantify the effect of mutation at subsite -4, we measured kinetic parameters of Tr Cel7A WT, W40A and W38A towards cello-oligosaccharides. All combinations of enzymes and substrates showed substrate inhibition, and comparison of the inhibition constants showed that the Trp38 residue increases the velocity of substrate intake ( k on for forming productive complex) from the minus side of the subsites. These results indicate a key role of Trp38 residue in processively loading the reducing-end of cellulose chain into the catalytic tunnel.

An amperometric biosensor of L-fucose in urine for the first screening test of cancer.

Quantitative routine detection of fucose, which is a cancer marker, in urine is effective for the preliminary screening of cancer. Amperometric biosensing methods have the advantage of being simple, rapid, and precise for urinalysis. However, coexisting electroactive interferences such as ascorbic acid (AA), dopamine (DA), and uric acid (UA) prevent accurate measurements. In this work, an amperometric l-fucose biosensor unaffected by interferences was developed and utilizes direct electron transfer type bioelectrocatalysis of pyrroloquinoline quinone (PQQ)-dependent pyranose dehydrogenase from Coprinopsis cinerea (CcPDH). The isolated PQQ domain from CcPDH was immobilized on gold nanoparticle (AuNP)-modified electrodes, which obtained a catalytic current at a lower potential than the oxidation potential of the interfering compounds. Applying an operating potential of -0.1 V vs. Ag|AgCl (3 M NaCl) enabled the detection of l-fucose while completely eliminating the oxidation of AA, DA, and UA on the electrodes. The increase in the specific area of the electrodes by increasing the AuNP drop-casting time resulted in an improvement in the sensor performance. The biosensor exhibited a linear range for l-fucose detection between 0.1 mM and 1 mM (R2 = 0.9996), including a cut-off value, the sensitivity was 3.12 ± 0.05 µA mM-1 cm-2, and the detection limit was 13.6 µM at a signal-to-noise ratio of three. The biosensor can be used to quantify the concentration of l-fucose at physiological levels and does not require urine preprocessing, making it applicable to practical use for point-of-care testing with urine.

Molecular analysis of cyclic α-maltosyl-(1→6)-maltose binding protein in the bacterial metabolic pathway.

Cyclic α-maltosyl-(1→6)-maltose (CMM) is a cyclic glucotetrasaccharide with alternating α-1,4 and α-1,6 linkages. Here, we report functional and structural analyses on CMM-binding protein (CMMBP), which is a substrate-binding protein (SBP) of an ABC importer system of the bacteria Arthrobacter globiformis. Isothermal titration calorimetry analysis revealed that CMMBP specifically bound to CMM with a Kd value of 9.6 nM. The crystal structure of CMMBP was determined at a resolution of 1.47 Å, and a panose molecule was bound in a cleft between two domains. To delineate its structural features, the crystal structure of CMMBP was compared with other SBPs specific for carbohydrates, such as cyclic α-nigerosyl-(1→6)-nigerose and cyclodextrins. These results indicate that A. globiformis has a unique metabolic pathway specialized for CMM.

Unique active-site and subsite features in the arabinogalactan-degrading GH43 exo-ß-1,3-galactanase from Phanerochaete chrysosporium.

Arabinogalactan proteins (AGPs) are plant proteoglycans with functions in growth and development. However, these functions are largely unexplored, mainly because of the complexity of the sugar moieties. These carbohydrate sequences are generally analyzed with the aid of glycoside hydrolases. The exo-ß-1,3-galactanase is a glycoside hydrolase from the basidiomycete Phanerochaete chrysosporium (Pc1,3Gal43A), which specifically cleaves AGPs. However, its structure is not known in relation to its mechanism bypassing side chains. In this study, we solved the apo and liganded structures of Pc1,3Gal43A, which reveal a glycoside hydrolase family 43 subfamily 24 (GH43_sub24) catalytic domain together with a carbohydrate-binding module family 35 (CBM35) binding domain. GH43_sub24 is known to lack the catalytic base Asp conserved among other GH43 subfamilies. Our structure in combination with kinetic analyses reveals that the tautomerized imidic acid group of Gln263 serves as the catalytic base residue instead. Pc1,3Gal43A has three subsites that continue from the bottom of the catalytic pocket to the solvent. Subsite -1 contains a space that can accommodate the C-6 methylol of Gal, enabling the enzyme to bypass the ß-1,6-linked galactan side chains of AGPs. Furthermore, the galactan-binding domain in CBM35 has a different ligand interaction mechanism from other sugar-binding CBM35s, including those that bind galactomannan. Specifically, we noted a Gly → Trp substitution, which affects pyranose stacking, and an Asp → Asn substitution in the binding pocket, which recognizes ß-linked rather than α-linked Gal residues. These findings should facilitate further structural analysis of AGPs and may also be helpful in engineering designer enzymes for efficient biomass utilization.

Substrate-recognition mechanism of tomato ß-galactosidase 4 using X-ray crystallography and docking simulation.

MAIN CONCLUSION: TBG4 recognize multiple linkage types substrates due to having a spatially wide subsite + 1. This feature allows the degradation of AGI, AGII, and AGP leading to the fruit ripening. ß-galactosidase (EC 3. 2. 1. 23) catalyzes the hydrolysis of ß-galactan and release of D-galactose. Tomato has at least 17 ß-galactosidases (TBGs), of which, TBG 4 is responsible for fruit ripening. TBG4 hydrolyzes not only ß-1,4-bound galactans, but also ß-1,3- and ß-1,6-galactans. In this study, we compared each enzyme-substrate complex using X-ray crystallography, ensemble refinement, and docking simulation to understand the broad substrate-specificity of TBG4. In subsite - 1, most interactions were conserved across each linkage type of galactobioses; however, some differences were seen in subsite + 1, owing to the huge volume of catalytic pocket. In addition to this, docking simulation indicated TBG4 to possibly have more positive subsites to recognize and hydrolyze longer galactans. Taken together, our results indicated that during tomato fruit ripening, TBG4 plays an important role by degrading arabinogalactan I (AGI), arabinogalactan II (AGII), and the carbohydrate moiety of arabinogalactan protein (AGP).

In Vitro Synthesis and Self-Assembly of Cellulose II Nanofibrils Catalyzed by the Reverse Reaction of Clostridium thermocellum Cellodextrin Phosphorylase.

In nature, various organisms produce cellulose as microfibrils, which are processed into their nano- and microfibrillar and/or crystalline components by humans in order to obtain desired material properties. Interestingly, the natural synthesis machinery can be circumvented by enzymatically synthesizing cellulose from precursor molecules in vitro. This approach is appealing for producing tailor-made cellulosic particles and materials because it enables optimization of the reaction conditions for cellulose synthesis in order to generate particles with a desired morphology in their pure form. Here, we present enzymatic cellulose synthesis catalyzed by the reverse reaction of Clostridium thermocellum cellodextrin phosphorylase in vitro. We were able to produce cellulose II nanofibril networks in all conditions tested, using varying concentrations of the glycosyl acceptors d-glucose or d-cellobiose (0.5, 5, and 50 mM). We show that shorter cellulose chains assemble into flat ribbon-like fibrils with greater diameter, while longer chains assemble into cylindrical fibrils with smaller diameter.

Domain architecture divergence leads to functional divergence in binding and catalytic domains of bacterial and fungal cellobiohydrolases.

Cellobiohydrolases directly convert crystalline cellulose into cellobiose and are of biotechnological interest to achieve efficient biomass utilization. As a result, much research in the field has focused on identifying cellobiohydrolases that are very fast. Cellobiohydrolase A from the bacterium Cellulomonas fimi (CfCel6B) and cellobiohydrolase II from the fungus Trichoderma reesei (TrCel6A) have similar catalytic domains (CDs) and show similar hydrolytic activity. However, TrCel6A and CfCel6B have different cellulose-binding domains (CBDs) and linkers: TrCel6A has a glycosylated peptide linker, whereas CfCel6B's linker consists of three fibronectin type 3 domains. We previously found that TrCel6A's linker plays an important role in increasing the binding rate constant to crystalline cellulose. However, it was not clear whether CfCel6B's linker has similar function. Here we analyze kinetic parameters of CfCel6B using single-molecule fluorescence imaging to compare CfCel6B and TrCel6A. We find that CBD is important for initial binding of CfCel6B, but the contribution of the linker to the binding rate constant or to the dissociation rate constant is minor. The crystal structure of the CfCel6B CD showed longer loops at the entrance and exit of the substrate-binding tunnel compared with TrCel6A CD, which results in higher processivity. Furthermore, CfCel6B CD showed not only fast surface diffusion but also slow processive movement, which is not observed in TrCel6A CD. Combined with the results of a phylogenetic tree analysis, we propose that bacterial cellobiohydrolases are designed to degrade crystalline cellulose using high-affinity CBD and high-processivity CD.

Convergent evolution of processivity in bacterial and fungal cellulases.

Cellulose is the most abundant biomass on Earth, and many microorganisms depend on it as a source of energy. It consists mainly of crystalline and amorphous regions, and natural degradation of the crystalline part is highly dependent on the degree of processivity of the degrading enzymes (i.e., the extent of continuous hydrolysis without detachment from the substrate cellulose). Here, we report high-speed atomic force microscopic (HS-AFM) observations of the movement of four types of cellulases derived from the cellulolytic bacteria Cellulomonas fimi on various insoluble cellulose substrates. The HS-AFM images clearly demonstrated that two of them (CfCel6B and CfCel48A) slide on crystalline cellulose. The direction of processive movement of CfCel6B is from the nonreducing to the reducing end of the substrate, which is opposite that of processive cellulase Cel7A of the fungus Trichoderma reesei (TrCel7A), whose movement was first observed by this technique, while CfCel48A moves in the same direction as TrCel7A. When CfCel6B and TrCel7A were mixed on the same substrate, "traffic accidents" were observed, in which the two cellulases blocked each other's progress. The processivity of CfCel6B was similar to those of fungal family 7 cellulases but considerably higher than those of fungal family 6 cellulases. The results indicate that bacteria utilize family 6 cellulases as high-processivity enzymes for efficient degradation of crystalline cellulose, whereas family 7 enzymes have the same function in fungi. This is consistent with the idea of convergent evolution of processive cellulases in fungi and bacteria to achieve similar functionality using different protein foldings.

Structural analysis of ß-L-arabinobiose-binding protein in the metabolic pathway of hydroxyproline-rich glycoproteins in Bifidobacterium longum.

Bifidobacterium longum is a symbiotic human gut bacterium that has a degradation system for ß-arabinooligosaccharides, which are present in the hydroxyproline-rich glycoproteins of edible plants. Whereas microbial degradation systems for α-linked arabinofuranosyl carbohydrates have been extensively studied, little is understood about the degradation systems targeting ß-linked arabinofuranosyl carbohydrates. We functionally and structurally analyzed a substrate-binding protein (SBP) of a putative ABC transporter (BLLJ_0208) in the ß-arabinooligosaccharide degradation system. Thermal shift assays and isothermal titration calorimetry revealed that the SBP specifically bound Araf-ß1,2-Araf (ß-Ara2 ) with a Kd of 0.150 µm, but did not bind L-arabinose or methyl-ß-Ara2 . Therefore, the SBP was termed ß-arabinobiose-binding protein (BABP). Crystal structures of BABP complexed with ß-Ara2 were determined at resolutions of up to 1.78 Å. The findings showed that ß-Ara2 was bound to BABP within a short tunnel between two lobes as an α-anomeric form at its reducing end. BABP forms extensive interactions with ß-Ara2 , and its binding mode was unique among SBPs. A molecular dynamics simulation revealed that the closed conformation of substrate-bound BABP is stable, whereas substrate-free form can adopt a fully open and two distinct semi-open states. The importer system specific for ß-Ara2 may contribute to microbial survival in biological niches with limited amounts of digestible carbohydrates. DATABASE: Atomic coordinates and structure factors (codes 6LCE and 6LCF) have been deposited in the Protein Data Bank (http://wwpdb.org/).

The Lipomyces starkeyi gene Ls120451 encodes a cellobiose transporter that enables cellobiose fermentation in Saccharomyces cerevisiae.

Processed lignocellulosic biomass is a source of mixed sugars that can be used for microbial fermentation into fuels or higher value products, like chemicals. Previously, the yeast Saccharomyces cerevisiae was engineered to utilize its cellodextrins through the heterologous expression of sugar transporters together with an intracellular expressed ß-glucosidase. In this study, we screened a selection of eight (putative) cellodextrin transporters from different yeast and fungal hosts in order to extend the catalogue of available cellobiose transporters for cellobiose fermentation in S. cerevisiae. We confirmed that several in silico predicted cellodextrin transporters from Aspergillus niger were capable of transporting cellobiose with low affinity. In addition, we found a novel cellobiose transporter from the yeast Lipomyces starkeyi, encoded by the gene Ls120451. This transporter allowed efficient growth on cellobiose, while it also grew on glucose and lactose, but not cellotriose nor cellotetraose. We characterized the transporter more in-depth together with the transporter CdtG from Penicillium oxalicum. CdtG showed to be slightly more efficient in cellobiose consumption than Ls120451 at concentrations below 1.0 g/L. Ls120451 was more efficient in cellobiose consumption at higher concentrations and strains expressing this transporter grew slightly slower, but produced up to 30% more ethanol than CdtG.