Efficient production of polyhydroxybutyrate using lignocellulosic biomass derived from oil palm trunks by the inhibitor-tolerant strain Burkholderia ambifaria E5-3.

Lignocellulosic biomass is a valuable, renewable substrate for the synthesis of polyhydroxybutyrate (PHB), an ecofriendly biopolymer. In this study, bacterial strain E5-3 was isolated from soil in Japan; it was identified as Burkholderia ambifaria strain E5-3 by 16 S rRNA gene sequencing. The strain showed optimal growth at 37 °C with an initial pH of 9. It demonstrated diverse metabolic ability, processing a broad range of carbon substrates, including xylose, glucose, sucrose, glycerol, cellobiose, and, notably, palm oil. Palm oil induced the highest cellular growth, with a PHB content of 65% wt. The strain exhibited inherent tolerance to potential fermentation inhibitors derived from lignocellulosic hydrolysate, withstanding 3 g/L 5-hydroxymethylfurfural and 1.25 g/L acetic acid. Employing a fed-batch fermentation strategy with a combination of glucose, xylose, and cellobiose resulted in PHB production 2.7-times that in traditional batch fermentation. The use of oil palm trunk hydrolysate, without inhibitor pretreatment, in a fed-batch fermentation setup led to significant cell growth with a PHB content of 45% wt, equivalent to 10 g/L. The physicochemical attributes of xylose-derived PHB produced by strain E5-3 included a molecular weight of 722 kDa, a number-average molecular weight of 191 kDa, and a polydispersity index of 3.78. The amorphous structure of this PHB displayed a glass transition temperature of 4.59 °C, while its crystalline counterpart had a melting point of 171.03 °C. This research highlights the potential of lignocellulosic feedstocks, especially oil palm trunk hydrolysate, for PHB production through fed-batch fermentation by B. ambifaria strain E5-3, which has high inhibitor tolerance.

A chemo-enzymatic pathway to expand cellooligosaccharide chemical space through amine bond introduction.

Enzymatic functionalization of oligosaccharides is a useful and environmentally friendly way to expand their structural chemical space and access to a wider range of applications in the health, food, feed, cosmetics and other sectors. In this work, we first tested the laccase/TEMPO system to generate oxidized forms of cellobiose and methyl ß-D-cellobiose, and obtained high yields of novel anionic disaccharides (>60 %) at pH 6.0. Laccase/TEMPO system was then applied to a mix of cellooligosaccharides and to pure D-cellopentaose. The occurrence of carbonyl and carboxyl groups in the oxidation products was shown by LC-HRMS, MALDI-TOF and reductive amination of the carbonyl groups was attempted with p-toluidine a low molar mass amine to form the Schiff base, then reduced by 2-picoline borane to generate a more stable amine bond. The new grafted products were characterized by LC-HRMS, LC-UV-MS/MS and covalent grafting was evidenced. Next, the same procedure was adopted to successfully graft a dye, the rhodamine 123, larger in size than toluidine. This two-step chemo-enzymatic approach, never reported before, for functionalization of oligosaccharides, offers attractive opportunities to anionic cellooligosaccharides and derived glucoconjugates of interest for biomedical or neutraceutical applications. It also paves the way for more environmentally-friendly cellulose fabric staining procedures.

Kinetics and products of Thermotoga maritima ß-glucosidase with lactose and cellobiose.

Galacto-oligosaccharides (GOS) are prebiotic compounds that are mainly used in infant formula to mimic bifidogenic effects of mother's milk. They are synthesized by ß-galactosidase enzymes in a trans-glycosylation reaction with lactose. Many ß-galactosidase enzymes from different sources have been studied, resulting in varying GOS product compositions and yields. The in vivo role of these enzymes is in lactose hydrolysis. Therefore, the best GOS yields were achieved at high lactose concentrations up to 60%wt, which require a relatively high temperature to dissolve. Some thermostable ß-glucosidase enzymes from thermophilic bacteria are also capable of using lactose or para nitrophenyl-galactose as a substrate. Here, we describe the use of the ß-glucosidase BglA from Thermotoga maritima for synthesis of oligosaccharides derived from lactose and cellobiose and their detailed structural characterization. Also, the BglA enzyme kinetics and yields were determined, showing highest productivity at higher lactose and cellobiose concentrations. The BglA trans-glycosylation/hydrolysis ratio was higher with 57%wt lactose than with a nearly saturated cellobiose (20%wt) solution. The yield of GOS was very high, reaching 72.1%wt GOS from lactose. Structural elucidation of the products showed mainly ß(1 → 3) and ß(1 → 6) elongating activity, but also some ß(1 → 4) elongation was observed. The ß-glucosidase BglA from T. maritima was shown to be a very versatile enzyme, producing high yields of oligosaccharides, particularly GOS from lactose. KEY POINTS: • ß-Glucosidase of Thermotoga maritima synthesizes GOS from lactose at very high yield. • Thermotoga maritima ß-glucosidase has high activity and high thermostability. • Thermotoga maritima ß-glucosidase GOS contains mainly (ß1-3) and (ß1-6) linkages.

Recombinant cellobiose dehydrogenase from Thermothelomyces thermophilus: Its functional characterization and applicability in cellobionic acid production.

The fungus Thermothelomyces thermophilus is a thermotolerant microorganism that has been explored as a reservoir for enzymes (hydrolytic enzymes and oxidoreductases). The functional analysis of a recombinant cellobiose dehydrogenase (MtCDHB) from T. thermophilus demonstrated a thermophilic behavior, an optimal pH in alkaline conditions for inter-domain electron transfer, and catalytic activity on cellooligosaccharides with different degree of polymerization. Its applicability was evaluated to the sustainable production of cellobionic acid (CBA), a potential pharmaceutical and cosmetic ingredient rarely commercialized. Dissolving pulp was used as a disaccharide source for MtCDHB. Initially, recombinant exoglucanases (MtCBHI and MtCBHII) from T. thermophilus hydrolyzed the dissolving pulp, resulting in 87% cellobiose yield, which was subsequently converted into CBA by MtCDHB, achieving a 66% CBA yield after 24 h. These findings highlight the potential of MtCDHB as a novel approach to obtaining CBA through the bioconversion of a plant-based source.

Single-molecule tracking reveals dual front door/back door inhibition of Cel7A cellulase by its product cellobiose.

Degrading cellulose is a key step in the processing of lignocellulosic biomass into bioethanol. Cellobiose, the disaccharide product of cellulose degradation, has been shown to inhibit cellulase activity, but the mechanisms underlying product inhibition are not clear. We combined single-molecule imaging and biochemical investigations with the goal of revealing the mechanism by which cellobiose inhibits the activity of Trichoderma reesei Cel7A, a well-characterized exo-cellulase. We find that cellobiose slows the processive velocity of Cel7A and shortens the distance moved per encounter; effects that can be explained by cellobiose binding to the product release site of the enzyme. Cellobiose also strongly inhibits the binding of Cel7A to immobilized cellulose, with a Ki of 2.1 mM. The isolated catalytic domain (CD) of Cel7A was also inhibited to a similar degree by cellobiose, and binding of an isolated carbohydrate-binding module to cellulose was not inhibited by cellobiose, suggesting that cellobiose acts on the CD alone. Finally, cellopentaose inhibited Cel7A binding at micromolar concentrations without affecting the enzyme's velocity of movement along cellulose. Together, these results suggest that cellobiose inhibits Cel7A activity both by binding to the "back door" product release site to slow activity and to the "front door" substrate-binding tunnel to inhibit interaction with cellulose. These findings point to strategies for engineering cellulases to reduce product inhibition and enhance cellulose degradation, supporting the growth of a sustainable bioeconomy.

Structural analysis of Tris binding in ß-glucosidases.

ß-glucosidases (Bgls) are glycosyl hydrolases that catalyze the conversion of cellobiose or glucosyl-polysaccharide into glucose. Bgls are widely used in industry to produce bioethanol, wine and juice, and feed. Tris (tris(hydroxymethyl)aminomethane) is an organic compound that can inhibit the hydrolase activity of some Bgls, but the inhibition state and selectivity have not been fully elucidated. Here, three crystal structures of Thermoanaerobacterium saccharolyticum Bgl complexed with the Tris molecule were determined at 1.55-1.95 Å. The configuration of Tris binding to TsaBgl remained consistent across three crystal structures, and the amino acids interacting with the Tris molecule were conserved across Bgl enzymes. The positions O1 and O3 atoms of Tris exhibit the same binding moiety as the hydroxyl group of the glucose molecule. Tris molecules are stably positioned at the glycone site and coordinate with surrounding water molecules. The Tris-binding configuration of TsaBgl is similar to that of HjeBgl, HgaBgl, ManBgl, and KflBgl, but the arrangement of the water molecule coordinating Tris at the aglycone site differs. Meanwhile, both the arrangement of Tris and the water molecules in ubBgl, NkoBgl, and SfrBgl differ from those in TsaBgl. The binding configuration and affinity of the Tris molecule for Bgl may be affected by the residues on the aglycone and gatekeeper regions. This result will extend our knowledge of the inhibitory effect of Tris molecules on TsaBgl.

Engineering of Ogataea polymorpha strains with ability for high-temperature alcoholic fermentation of cellobiose.

Successful conversion of cellulosic biomass into biofuels requires organisms capable of efficiently utilizing xylose as well as cellodextrins and glucose. Ogataea (Hansenula) polymorpha is the natural xylose-metabolizing organism and is one of the most thermotolerant yeasts known, with a maximum growth temperature above 50°C. Cellobiose-fermenting strains, derivatives of an improved ethanol producer from xylose O. polymorpha BEP/cat8∆, were constructed in this work by the introduction of heterologous genes encoding cellodextrin transporters (CDTs) and intracellular enzymes (ß-glucosidase or cellobiose phosphorylase) that hydrolyze cellobiose. For this purpose, the genes gh1-1 of ß-glucosidase, CDT-1m and CDT-2m of cellodextrin transporters from Neurospora crassa and the CBP gene coding for cellobiose phosphorylase from Saccharophagus degradans, were successfully expressed in O. polymorpha. Through metabolic engineering and mutagenesis, strains BEP/cat8∆/gh1-1/CDT-1m and BEP/cat8∆/CBP-1/CDT-2mAM were developed, showing improved parameters for high-temperature alcoholic fermentation of cellobiose. The study highlights the need for further optimization to enhance ethanol yields and elucidate cellobiose metabolism intricacies in O. polymorpha yeast. This is the first report of the successful development of stable methylotrophic thermotolerant strains of O. polymorpha capable of coutilizing cellobiose, glucose, and xylose under high-temperature alcoholic fermentation conditions at 45°C.

Conversion of Phytochemicals by Lactobacilli: (Phospho)-ß-glucosidases Are Specific for Glucosylated Phytochemicals Rather than Disaccharides.

Food-fermenting lactobacilli convert glycosylated phytochemicals to glycosyl hydrolases and thereby alter their biological activity. This study aimed to investigate the microbial transformation of ß-glucosides of phytochemicals in comparison with utilization of cellobiose. Four homofermentative and four heterofermentative lactobacilli were selected to represent the metabolic diversity of Lactobacillaceae. The genomes of Lactobacillus crispatus, Companilactobacillus paralimentarius, Lacticaseibacillus paracasei, and Lactiplantibacillus plantarum encoded for 8 to 22 enzymes, predominantly phospho-ß-glucosidases, with predicted activity on ß-glucosides. Levilactobacillus hammesii and Furfurilactobacillus milii encoded for 3 ß-glucosidases, Furfurilactobacillus rossiae for one, and Fructilactobacillus sanfranciscensis for none. The hydrolysis of amygdalin, esculin, salicin, glucosides of quercetin and genistein, and ginsenosides demonstrated that several strains hydrolyzed ß-glucosides of phytochemicals but not cellobiose. Taken together, several of the carbohydrate-active enzymes of food-fermenting lactobacilli are specific for glycosides of phytochemicals.

Characterization of a novel acidophilic, ethanol tolerant and halophilic GH12 ß-1,4-endoglucanase from Trichoderma asperellum ND-1 and its synergistic hydrolysis of lignocellulosic biomass.

A novel acidophilic GH5 ß-1,4-endoglucanase (TaCel12) from Trichoderma asperellum ND-1 was efficiently expressed in Pichia pastoris (a 1.5-fold increase). Deglycosylated TaCel12 migrated as a single band (26.5 kDa) in SDS-PAGE. TaCel12 was acidophilic with a pH optimum of 4.0 and displayed great pH stability (>80 % activity over pH 3.0-5.0). TaCel12 exhibited considerable activity towards sodium carboxymethyl cellulose and sodium alginate with Vmax values of 197.97 µmol/min/mg and 119.06 µmol/min/mg, respectively. Moreover, TaCel12 maintained >80 % activity in the presence of 20 % ethanol and 4.28 M NaCl. Additionally, Mn2+, Pb2+ and Cu2+ negatively affected TaCel12 activity, while the presence of 5 mM Co2+ significantly increased the enzyme activity. Analysis of action mode revealed that TaCel12 required at least four glucose (cellotetraose) residues for hydrolysis to yield cellobiose and cellotriose. Site-directed mutagenesis results suggested that Glu133 and Glu217 of TaCel12 are crucial catalytic residues, with Asp116 displaying an auxiliary function. Production of soluble sugars from lignocellulose is a crucial step in bioethanol development, and it is noteworthy that TaCel12 could synergistically yield fermentable sugars from corn stover and bagasse, respectively. Thus TaCel12 with excellent properties will be considered a potential biocatalyst for applications in various industries, especially for lignocellulosic biomass conversion.

Production of cello-oligosaccharides from corncob residue by degradation-synthesis reactions.

The cellulose-rich corncob residue (CCR) is an abundant and renewable agricultural biomass that has been under-exploited. In this study, two strategies were compared for their ability to transform CCR into cello-oligosaccharides (COS). The first strategy employed the use of endo-glucanases. Although selected endo-glucanases from GH9, GH12, GH45, and GH131 could release COS with degrees of polymerization from 2 to 4, the degrading efficiency was low. For the second strategy, first, CCR was efficiently depolymerized to glucose and cellobiose using the cellulase from Trichoderma reesei. Then, using these simple sugars and sucrose as the starting materials, phosphorylases from different microorganisms were combined to generate COS to a level up to 100.3 g/L with different patterns and degrees of polymerization. Using tomato as a model plant, the representative COS obtained from BaSP (a sucrose phosphorylase from Bifidobacterium adolescens), CuCbP (a cellobiose phosphorylase from Cellulomonas uda), and CcCdP (a cellodextrin phosphorylase from Clostridium cellulosi) were shown to be able to promote plant growth. The current study pointed to an approach to make use of CCR for production of the value-added COS. KEY POINTS: • Sequential use of cellulase and phosphorylases effectively generated cello-oligosaccharides from corncob residue. • Cello-oligosaccharides patterns varied in accordance to cellobiose/cellodextrin phosphorylases. • Spraying cello-oligosaccharides promoted tomato growth.

Proteome profiling of enriched membrane-associated proteins unraveled a novel sophorose and cello-oligosaccharide transporter in Trichoderma reesei.

BACKGROUND: Trichoderma reesei is an organism extensively used in the bioethanol industry, owing to its capability to produce enzymes capable of breaking down holocellulose into simple sugars. The uptake of carbohydrates generated from cellulose breakdown is crucial to induce the signaling cascade that triggers cellulase production. However, the sugar transporters involved in this process in T. reesei remain poorly identified and characterized. RESULTS: To address this gap, this study used temporal membrane proteomics analysis to identify five known and nine putative sugar transporters that may be involved in cellulose degradation by T. reesei. Docking analysis pointed out potential ligands for the putative sugar transporter Tr44175. Further functional validation of this transporter was carried out in Saccharomyces cerevisiae. The results showed that Tr44175 transports a variety of sugar molecules, including cellobiose, cellotriose, cellotetraose, and sophorose. CONCLUSION: This study has unveiled a transporter Tr44175 capable of transporting cellobiose, cellotriose, cellotetraose, and sophorose. Our study represents the first inventory of T. reesei sugar transportome once exposed to cellulose, offering promising potential targets for strain engineering in the context of bioethanol production.

Self-Assembly of Unusually Stable Thermotropic Network Phases by Cellobiose-Based Guerbet Glycolipids.

Bicontinuous thermotropic liquid crystal (LC) materials, e.g., double gyroid (DG) phases, have garnered significant attention due to the potential utility of their 3D network structures in wide-ranging applications. However, the utility of these materials is significantly constrained by the lack of robust molecular design rules for shape-filling amphiphiles that spontaneously adopt the saddle curvatures required to access these useful supramolecular assemblies. Toward this aim, we synthesized anomerically pure Guerbet-type glycolipids bearing cellobiose head groups and branched alkyl tails and studied their thermotropic LC self-assembly. Using a combination of differential scanning calorimetry, polarized optical microscopy, and small-angle X-ray scattering, our studies demonstrate that Guerbet cellobiosides exhibit a strong propensity to self-assemble into DG morphologies over wide thermotropic phase windows. The stabilities of these assemblies sensitively depend on the branched alkyl tail structure and the anomeric configuration of the glycolipid in a previously unrecognized manner. Complementary molecular simulations furnish detailed insights into the observed self-assembly characteristics, thus unveiling molecular motifs that foster network phase self-assembly that will enable future designs and applications of network LC materials.

Pushing the boundaries of phosphorylase cascade reaction for cellobiose production I: Kinetic model development.

One-pot cascade reactions of coupled disaccharide phosphorylases enable an efficient transglycosylation via intermediary α-d-glucose 1-phosphate (G1P). Such transformations have promising applications in the production of carbohydrate commodities, including the disaccharide cellobiose for food and feed use. Several studies have shown sucrose and cellobiose phosphorylase for cellobiose synthesis from sucrose, but the boundaries on transformation efficiency that result from kinetic and thermodynamic characteristics of the individual enzyme reactions are not known. Here, we assessed in a step-by-step systematic fashion the practical requirements of a kinetic model to describe cellobiose production at industrially relevant substrate concentrations of up to 600 mM sucrose and glucose each. Mechanistic initial-rate models of the two-substrate reactions of sucrose phosphorylase (sucrose + phosphate → G1P + fructose) and cellobiose phosphorylase (G1P + glucose → cellobiose + phosphate) were needed and additionally required expansion by terms of glucose inhibition, in particular a distinctive two-site glucose substrate inhibition of the cellobiose phosphorylase (from Cellulumonas uda). Combined with mass action terms accounting for the approach to equilibrium, the kinetic model gave an excellent fit and a robust prediction of the full reaction time courses for a wide range of enzyme activities as well as substrate concentrations, including the variable substoichiometric concentration of phosphate. The model thus provides the essential engineering tool to disentangle the highly interrelated factors of conversion efficiency in the coupled enzyme reaction; and it establishes the necessary basis of window of operation calculations for targeted optimizations toward different process tasks.

An extracellular glucose sensor for substrate-dependent secretion and display of cellulose-degrading enzymes.

The efficient hydrolysis of lignocellulosic biomass into fermentable sugars is key for viable economic production of biofuels and biorenewable chemicals from second-generation feedstocks. Consolidated bioprocessing (CBP) combines lignocellulose saccharification and chemical production in a single step. To avoid wasting valuable resources during CBP, the selective secretion of enzymes (independent or attached to the surface) based on the carbon source available is advantageous. To enable enzyme expression and secretion based on extracellular glucose levels, we implemented a G-protein-coupled receptor (GPCR)-based extracellular glucose sensor; this allows the secretion and display of cellulases in the presence of the cellulosic fraction of lignocellulose by leveraging cellobiose-dependent signal amplification. We focused on the glucose-responsiveness of the HXT1 promoter and engineered PHXT1 by changing its core to that of the strong promoter PTHD3 , increasing extracellular enzyme activity by 81%. We then demonstrated glucose-mediated expression and cell-surface display of the ß-glucosidase BglI on the surface of Saccharomyces cerevisiae. The display system was further optimized by re-directing fatty acid pools from lipid droplet synthesis toward formation of membrane precursors via knock-out of PAH1. This resulted in an up to 4.2-fold improvement with respect to the baseline strain. Finally, we observed cellobiose-dependent signal amplification of the system with an increase in enzymatic activity of up to 3.1-fold when cellobiose was added.

Pushing the boundaries of phosphorylase cascade reaction for cellobiose production II: Model-based multiobjective optimization.

The inherent complexity of coupled biocatalytic reactions presents a major challenge for process development with one-pot multienzyme cascade transformations. Kinetic models are powerful engineering tools to guide the optimization of cascade reactions towards a performance suitable for scale up to an actual production. Here, we report kinetic model-based window of operation analysis for cellobiose production (≥100 g/L) from sucrose and glucose by indirect transglycosylation via glucose 1-phosphate as intermediate. The two-step cascade transformation is catalyzed by sucrose and cellobiose phosphorylase in the presence of substoichiometric amounts of phosphate (≤27 mol% of substrate). Kinetic modeling was instrumental to uncover the hidden effect of bulk microviscosity due to high sugar concentrations on decreasing the rate of cellobiose phosphorylase specifically. The mechanistic-empirical hybrid model thus developed gives a comprehensive description of the cascade reaction at industrially relevant substrate conditions. Model simulations serve to unravel opposed relationships between efficient utilization of the enzymes and maximized concentration (or yield) of the product within a given process time, in dependence of the initial concentrations of substrate and phosphate used. Optimum balance of these competing key metrics of process performance is suggested from the model-calculated window of operation and is verified experimentally. The evidence shown highlights the important use of kinetic modeling for the characterization and optimization of cascade reactions in ways that appear to be inaccessible to purely data-driven approaches.

Discovery of a Novel Cellobiose Dehydrogenase from Cellulomonas palmilytica EW123 and Its Sugar Acids Production.

Cellobiose dehydrogenases (CDHs) are a group of enzymes belonging to the hemoflavoenzyme group, which are mostly found in fungi. They play an important role in the production of acid sugar. In this research, CDH annotated from the actinobacterium Cellulomonas palmilytica EW123 (CpCDH) was cloned and characterized. The CpCDH exhibited a domain architecture resembling class-I CDH found in Basidiomycota. The cytochrome c and flavin-containing dehydrogenase domains in CpCDH showed an extra-long evolutionary distance compared to fungal CDH. The amino acid sequence of CpCDH revealed conservative catalytic amino acids and a distinct flavin adenine dinucleotide region specific to CDH, setting it apart from closely related sequences. The physicochemical properties of CpCDH displayed optimal pH conditions similar to those of CDHs but differed in terms of optimal temperature. The CpCDH displayed excellent enzymatic activity at low temperatures (below 30°C), unlike other CDHs. Moreover, CpCDH showed the highest substrate specificity for disaccharides such as cellobiose and lactose, which contain a glucose molecule at the non-reducing end. The catalytic efficiency of CpCDH for cellobiose and lactose were 2.05 x 105 and 9.06 x 104 (M-1 s-1), respectively. The result from the Fourier-transform infrared spectroscopy (FT-IR) spectra confirmed the presence of cellobionic and lactobionic acids as the oxidative products of CpCDH. This study establishes CpCDH as a novel and attractive bacterial CDH, representing the first report of its kind in the Cellulomonas genus.

Efficient and Economic Utilization of Cellobiose for Glycosylation Modification by Regulating Carbon Source Supply and Metabolic Pathway In Vivo.

Glycosylation, one of the most common and significant modifications in nature, has prompted the development of a cellobiose phosphorolysis route for glycosylation in vivo. However, the process of glycosylation is hampered by the notably low conversion rate of cellobiose. In this work, regulation of the carbon source supply by changing the ratio of glucose to cellobiose improved the conversion rate of cellobiose, resulting in enhancing the efficiency of glycosylation and the production of vitexin. Moreover, three genes (pgm, agp, and ushA) involved in the degradation of UDP-glucose were knocked out to relieve the degradation and diversion of the cellobiose phosphorolysis route. Finally, through the optimization of conversion conditions, we observed a continuous enhancement in cellobiose conversion rate and vitexin production in BL21ΔushAΔagp-TcCGT-CepA, corresponding to an increased concentration of added glucose. The maximum production of vitexin reached 2228 mg/L with the addition of 2 g/L cellobiose and 6 g/L glucose, which was 312% of that in BL21-TcCGT-CepA with the addition of 2 g/L cellobiose. The conversion rate of cellobiose in BL21ΔushAΔagp-TcCGT-CepA reached 88%, which was the highest conversion rate of cellobiose to date. Therefore, this study presents a cost-effective and efficient method to enhance the conversion rate of cellobiose during the glycosylation process.

The role of intracellular ß-glucosidase in cellulolytic response induction in filamentous fungus Penicillium verruculosum.

In this study, CRISPR/Cas9 genome editing was used to knockout the bgl2 gene encoding intracellular ß-glucosidase filamentous fungus Penicillium verruculosum. This resulted in a dramatic reduction of secretion of cellulolytic enzymes. The study of P. verruculosum Δbgl2 found that the transcription of the cbh1 gene, which encodes cellobiohydrolase 1, was impaired when induced by cellobiose and cellotriose. However, the transcription of the cbh1 gene remains at level of the host strain when induced by gentiobiose. This implies that gentiobiose is the true inducer of the cellulolytic response in P. verruculosum, in contrast to Neurospora crassa where cellobiose acts as an inducer.

The Conformational Change of the L3 Loop Affects the Structural Changes in the Substrate Binding Pocket Entrance of ß-Glucosidase.

ß-glucosidase (Bgl) hydrolyzes cellobiose to glucose, thereby releasing non-reducing terminal glucosyl residues. Bgl is an essential enzyme belonging to the biomass-degrading enzyme family, which plays a vital role in enzymatic saccharification during biofuel production. The four loops above the Bgl substrate-binding pocket undergo a conformational change upon substrate recognition. However, the structural dynamism of this loop and how it is conserved among Bgl family members remain unknown. Herein, to better understand the four loops above the substrate-binding pocket of Bgl, four Bgl crystal structures in Thermoanaerobacterium saccharolyticum (TsaBgl) were determined at 1.5-2.1 Å. The L1, L2, and L4 loops of TsaBgl showed a rigid conformation stabilized by their neighboring residues via hydrogen bonds and hydrophobic interactions. The TsaBgl L3 loop showed relatively high flexibility and two different N-terminal region conformations. The conformational change in the TsaBgl L3 loop induced a change in charge and shaped at the substrate-binding pocket entrance. The amino acid sequences and structures of the TsaBgl L1-4 loops were compared with other 45 Bgl proteins, and a diversity of the L2 and L3 loops was observed. Differences in amino acids and lengths of Bgls L2-L3 loop induced differences in the conformation and structure of the Bgls substrate-binding pocket entrance. These findings expand our knowledge on the molecular function of the loops in the Bgl enzyme family.

Heterologously Expressed Cellobiose Dehydrogenase Acts as Efficient Electron-Donor of Lytic Polysaccharide Monooxygenase for Cellulose Degradation in Trichoderma reesei.

The conversion of lignocellulosic biomass to second-generation biofuels through enzymes is achieved at a high cost. Filamentous fungi through a combination of oxidative enzymes can easily disintegrate the glycosidic bonds of cellulose. The combination of cellobiose dehydrogenase (CDH) with lytic polysaccharide monooxygenases (LPMOs) enhances cellulose degradation in many folds. CDH increases cellulose deconstruction via coupling the oxidation of cellobiose to the reductive activation of LPMOs by catalyzing the addition of oxygen to C-H bonds of the glycosidic linkages. Fungal LPMOs show different regio-selectivity (C1 or C4) and result in oxidized products through modifications at reducing as well as nonreducing ends of the respective glucan chain. T. reesei LPMOs have shown great potential for oxidative cleavage of cellobiose at C1 and C4 glucan bonds, therefore, the incorporation of heterologous CDH further increases its potential for biofuel production for industrial purposes at a reduced cost. We introduced CDH of Phanerochaete chrysosporium (PcCDH) in Trichoderma reesei (which originally lacked CDH). We purified CDH through affinity chromatography and analyzed its enzymatic activity, electron-donating ability to LPMO, and the synergistic effect of LPMO and CDH on cellulose deconstruction. The optimum temperature of the recombinant PcCDH was found to be 45 °C and the optimum pH of PcCDH was observed as 4.5. PcCDH has high cello-oligosaccharide kcat, Km, and kcat/Km values. The synergistic effect of LPMO and cellulase significantly improved the degradation efficiency of phosphoric acid swollen cellulose (PASC) when CDH was used as the electron donor. We also found that LPMO undergoes auto-oxidative inactivation, and when PcCDH is used an electron donor has the function of a C1-type LPMO electron donor without additional substrate increments. This work provides novel insights into finding stable electron donors for LPMOs and paves the way forward in discovering efficient CDHs for enhanced cellulose degradation.