Revealing nutrient limitation status of microorganisms in the soil of Robinia pseudoacacia plantation through soil stoichiometry and enzyme metrology.

Exploring nutrient limitation in forest soil holds significant implications for forest tending and management. However, current research on nutrient limitation status of microorganisms in Robinia pseudoacacia plantations within the Loess Plateau remains insufficient. To investigate soil microbial nutrient limitation of R. pseu-doacacia plantations on the Loess Plateau, we selected R. pseudoacacia plantations with different afforestation time series (15, 25, 35, and 45 years) and a pile of barren slope cropland (control) in Yongshou County, Shaanxi Province as the research objects. We analyzed the contents of soil organic matter, total nitrogen, and total phosphorus, and the activities of ß-1,4-glucosidase (BG), cellobiose hydrolase (CBH), leucine aminopeptidase (LAP), ß-1,4-N-acetylglucoside (NAG) and phosphatase (AP). We analyzed the soil nutrient limitation by stoichiometry and enzyme metrology. The results showed a shift in soil pH from alkaline to acidic during vegetation restoration process, and that total phosphorus exhibited a gradual decrease over the course of 0 to 25 years. Soil orga-nic matter, total nitrogen and enzyme activities exhibited an increasing trend during the same time frame. However, between 25 and 45 years of age, soil total phosphorus, soil organic matter, total nitrogen, AP and LAP gradually declined while NAG, BG, and CBH initially increased and then decreased. Notably, the values of (BG+CBH)/(LAP+NAG), (BG+CBH)/AP and (LAP+NAG)/AP in R. pseudoacacia plantations were higher than the global average throughout the process of vegetation restoration. In the study area, the vector length was less than 1 and gradually increased, indicating that a progressive increase in microbial carbon limitation during the process of vegetation restoration. The vector angle exceeded 45° and exhibited an overall decreasing trend, suggesting that soil microorganisms were constrained by phosphorus (P) with a gradual deceleration of P limitation, without any nitrogen (N) limitation. The restoration of R. pseudoacacia plantation resulted in significant change in soil physical and chemical properties, while the time series of afforestation also influenced nutrient limitation of soil microorganisms.

Upgraded cellulose and xylan digestions for synergistic enhancements of biomass enzymatic saccharification and bioethanol conversion using engineered Trichoderma reesei strains overproducing mushroom LeGH7 enzyme.

Crop straws provide enormous lignocellulose resources transformable for sustainable biofuels and valuable bioproducts. However, lignocellulose recalcitrance basically restricts essential biomass enzymatic saccharification at large scale. In this study, the mushroom-derived cellobiohydrolase (LeGH7) was introduced into Trichoderma reesei (Rut-C30) to generate two desirable strains, namely GH7-5 and GH7-6. Compared to the Rut-C30 strain, both engineered strains exhibited significantly enhanced enzymatic activities, with ß-glucosidases, endocellulases, cellobiohydrolases, and xylanase activities increasing by 113 %, 140 %, 241 %, and 196 %, respectively. By performing steam explosion and mild alkali pretreatments with mature straws of five bioenergy crops, diverse lignocellulose substrates were effectively digested by the crude enzymes secreted from the engineered strains, leading to the high-yield hexoses released for bioethanol production. Notably, the LeGH7 enzyme purified from engineered strain enabled to act as multiple cellulases and xylanase at higher activities, interpreting how synergistic enhancement of enzymatic saccharification was achieved for distinct lignocellulose substrates in major bioenergy crops. Therefore, this study has identified a novel enzyme that is active for simultaneous hydrolyses of cellulose and xylan, providing an applicable strategy for high biomass enzymatic saccharification and bioethanol conversion in bioenergy crops.

Lipid production from corn straw by cellobiohydrolase and delta-6 desaturase engineered Mucor circinelloides strains under solid state fermentation.

Previously, we constructed engineered M. circinelloides strains that can not only utilize cellulose, but also increase the yield of γ-linolenic acid (GLA). In the present study, an in-depth analysis of lipid accumulation by engineered M. circinelloides strains using corn straw was to be explored. When a two-stage temperature control strategy was adopted with adding 1.5% cellulase and 15% inoculum, the engineered strains led to increases in the lipid yield (up to 1.56 g per 100 g dry medium) and GLA yield (up to 274 mg per 100 g dry medium) of 1.8- and 2.3-fold, respectively, compared with the control strain. This study proved the engineered M. circinelloides strains, especially for Mc-C2PD6, possess advantages in using corn straw to produce GLA. This work provided a reference for transformation from agricultural cellulosic waste to functional lipid in one step, which might play a positive role in promoting the sustainable development of biological industry.

Effects of Exocellobiohydrolase CBHA on Fermentation of Tobacco Leaves.

The quality of tobacco is directly affected by macromolecular content, fermentation is an effective method to improve biochemical properties. In this study, we utilized CBHA (cellobiohydrolase A) glycosylase, which was expressed by Pichia pastoris, as an additive for fermentation. The contents of main chemical components of tobacco leaves after fermentation were determined, and the changes of microbial community structure and abundance in tobacco leaves during fermentation were analyzed. The relationship between chemical composition and changes in microbial composition was investigated, and the function of bacteria and fungi in fermentation was predicted to identify possible metabolic pathways. After 48 h of CBHA fermentation, the contents of starch, cellulose and total nitrogen in tobacco leaf decreased by 17.60%, 28.91% and 16.05%, respectively. The microbial community structure changed significantly, with Aspergillus abundance decreasing significantly, while Filobasidum, Cladosporium, Bullera, Komagataella, etc., increased in CBHA treated group. Soluble sugar was most affected by microbial community in tobacco leaves, which was negatively correlated with starch, cellulose and total nitrogen. During the fermentation process, the relative abundance of metabolism-related functional genes increased, and the expressions of cellulase and endopeptidase also increased. The results showed that the changes of bacterial community and dominant microbial community on tobacco leaves affected the content of chemical components in tobacco leaves, and adding CBHA for fermentation had a positive effect on improving the quality of tobacco leaves.

Study on the mechanism of lignin non-productive adsorption on cellobiohydrolase.

Enzymatic hydrolysis is important for lignocellulosic biomass conversion into fermentable sugars. However, the nonproductive adsorption of enzyme on lignin was major hinderance for the enzymatic hydrolysis efficiency. In this study, non-productive adsorption mechanism of cellulase component cellobiohydrolase (CBH) onto lignin was specific investigated. Research revealed that the adsorption behavior of CBH on eucalyptus alkali lignin (EuA) was affected by reaction conditions. As study on the adsorption kinetic, it was indicated that the adsorption cellulose binding domain (CBD) of CBH onto EuA well fitted with Langmuir adsorption model and pseudo second-order adsorption kinetics model. And the tyrosine site related to the adsorption of CBD onto lignin was proved by the fluorescence and UV spectra analysis. The results of this work provide a theoretical guidance to understanding the nonproductive adsorption mechanism and building method to reduce the adsorption of cellulase on the lignin.

Cloning, expression and purification of cellobiohydrolase gene from Caldicellulosiruptor bescii for efficient saccharification of plant biomass.

Anthropogenic activities have led to a drastic shift from natural fuels to alternative renewable energy reserves that demand heat-stable cellulases. Cellobiohydrolase is an indispensable member of cellulases that play a critical role in the degradation of cellulosic biomass. This article details the process of cloning the cellobiohydrolase gene from the thermophilic bacterium Caldicellulosiruptor bescii and expressing it in Escherichia coli (BL21) CondonPlus DE3-(RIPL) using the pET-21a(+) expression vector. Multi-alignments and structural modeling studies reveal that recombinant CbCBH contained a conserved cellulose binding domain III. The enzyme's catalytic site included Asp-372 and Glu-620, which are either involved in substrate or metal binding. The purified CbCBH, with a molecular weight of 91.8 kDa, displayed peak activity against pNPC (167.93 U/mg) at 65°C and pH 6.0. Moreover, it demonstrated remarkable stability across a broad temperature range (60-80°C) for 8 h. Additionally, the Plackett-Burman experimental model was employed to assess the saccharification of pretreated sugarcane bagasse with CbCBH, aiming to evaluate the cultivation conditions. The optimized parameters, including a pH of 6.0, a temperature of 55°C, a 24-hour incubation period, a substrate concentration of 1.5% (w/v), and enzyme activity of 120 U, resulted in an observed saccharification efficiency of 28.45%. This discovery indicates that the recombinant CbCBH holds promising potential for biofuel sector.

Short-term responses of soil enzyme activities and stoichiometry to litter input in Castanopsis carlesii and Cunninghamia lanceolata plantations.

Litter input triggers the secretion of soil extracellular enzymes and facilitates the release of carbon (C), nitrogen (N), and phosphorus (P) from decomposing litter. However, how soil extracellular enzyme activities were controlled by litter input with various substrates is not fully understood. We examined the activities and stoichiometry of five enzymes including ß-1,4-glucosidase, ß-D-cellobiosidase, ß-1,4-N-acetyl-glucosaminidase, leucine aminopeptidase and acidic phosphatase (AP) with and without litter input in 10-year-old Castanopsis carlesii and Cunninghamia lanceolata plantations monthly during April to August, in October, and in December 2021 by using an in situ microcosm experiment. The results showed that: 1) There was no significant effect of short-term litter input on soil enzyme activity, stoichiometry, and vector properties in C. carlesii plantation. In contrast, short-term litter input significantly increased the AP activity by 1.7% in May and decreased the enzymatic C/N ratio by 3.8% in August, and decreased enzymatic C/P and N/P ratios by 11.7% and 10.3%, respectively, in October in C. lanceolata plantation. Meanwhile, litter input increased the soil enzymatic vector angle to 53.8° in October in C. lanceolata plantations, suggesting a significant P limitation for soil microorganisms. 2) Results from partial least squares regression analyses showed that soil dissolved organic matter and microbial biomass C and N were the primary factors in explaining the responses of soil enzymatic activity to short-term litter input in both plantations. Overall, input of low-quality (high C/N) litter stimulates the secretion of soil extracellular enzymes and accelerates litter decomposition. There is a P limitation for soil microorganisms in the study area.

The S-S bridge mutation between the A2 and A4 loops (T416C-I432C) of Cel7A of Aspergillus fumigatus enhances catalytic activity and thermostability.

Disulfide bonds are important for maintaining the structural conformation and stability of the protein. The introduction of the disulfide bond is a promising strategy to increase the thermostability of the protein. In this report, cysteine residues are introduced to form disulfide bonds in the Glycoside Hydrolase family GH 7 cellobiohydrolase (GH7 CBHs) or Cel7A of Aspergillus fumigatus. Disulfide by Design 2.0 (DbD2), an online tool is used for the detection of the mutation sites. Mutations are created (D276C-G279C; DSB1, D322C-G327C; DSB2, T416C-I432C; DSB3, G460C-S465C; DSB4) inside and outside of the peripheral loops but, not in the catalytic region. The introduction of cysteine in the A2 and A4 loop of DSB3 mutant showed higher thermostability (70% activity at 70°C), higher substrate affinity (Km = 0.081 mM) and higher catalytic activity (Kcat = 9.75 min-1; Kcat/Km = 120.37 mM min-1) compared to wild-type AfCel7A (50% activity at 70°C; Km = 0.128 mM; Kcat = 4.833 min-1; Kcat/Km = 37.75 mM min-1). The other three mutants with high B factor showed loss of thermostability and catalytic activity. Molecular dynamic simulations revealed that the mutation T416C-I432C makes the tunnel wider (DSB3: 13.6 Å; Wt: 5.3 Å) at the product exit site, giving flexibility in the entrance region or mobility of the substrate in the exit region. It may facilitate substrate entry into the catalytic tunnel and release the product faster than the wild type, whereas in other mutants, the tunnel is not prominent (DSB4), the exit is lost (DSB1), and the ligand binding site is absent (DSB2). This is the first report of the gain of function of both thermostability and enzyme activity of cellobiohydrolase Cel7A by disulfide bond engineering in the loop.IMPORTANCEBioethanol is one of the cleanest renewable energy and alternatives to fossil fuels. Cost efficient bioethanol production can be achieved through simultaneous saccharification and co-fermentation that needs active polysaccharide degrading enzymes. Cellulase enzyme complex is a crucial enzyme for second-generation bioethanol production from lignocellulosic biomass. Cellobiohydrolase (Cel7A) is an important member of this complex. In this work, we engineered (disulfide bond engineering) the Cel7A to increase its thermostability and catalytic activity which is required for its industrial application.

Specific activity and utility of recombinant cellobiohydrolase II (Cel6A) produced in maize endosperm.

Cellobiohydrolase II (CBH II) is an exo-glucanase that is part of a fungal mixture of enzymes from a wood-rot fungus, Trichoderma reesei. It is therefore difficult to purify and to establish a specific activity assay. The gene for this enzyme, driven by the rice Os glutelin promoter, was transformed into High II tissue culture competent corn, and the enzyme accumulated in the endosperm of the seed. The transgenic line recovered from tissue culture was bred into male and female elite Stine inbred corn lines, stiff stalk 16083-025 (female) and Lancaster MSO411 (male), for future production in their hybrid. The enzyme increases its accumulation throughout its 6 generations of back crosses, 27-266-fold between T1 and T2, and 2-10-fold between T2 and T3 generations with lesser increases in T4-T6. The germplasm of the inbred lines replaces the tissue culture corn variety germplasm with each generation, with the ultimate goal of producing a high-yielding hybrid with the transgene. The CBH II enzyme was purified from T5 inbred male grain 10-fold to homogeneity with 47.5% recovery. The specific activity was determined to be 1.544 units per µg protein. The corn-derived CBH II works in biopolishing of cotton by removing surface fibers to improve dyeability and increasing glucose from corn flour for increasing ethanol yield from starch-based first-generation processes.

Methionine oxidation of carbohydrate-active enzymes during white-rot wood decay.

White-rot fungi employ secreted carbohydrate-active enzymes (CAZymes) along with reactive oxygen species (ROS), like hydrogen peroxide (H2O2), to degrade lignocellulose in wood. H2O2 serves as a co-substrate for key oxidoreductases during the initial decay phase. While the degradation of lignocellulose by CAZymes is well documented, the impact of ROS on the oxidation of the secreted proteins remains unclear, and the identity of the oxidized proteins is unknown. Methionine (Met) can be oxidized to Met sulfoxide (MetO) or Met sulfone (MetO2) with potential deleterious, antioxidant, or regulatory effects. Other residues, like proline (Pro), can undergo carbonylation. Using the white-rot Pycnoporus cinnabarinus grown on aspen wood, we analyzed the Met content of the secreted proteins and their susceptibility to oxidation combining H218O2 with deep shotgun proteomics. Strikingly, their overall Met content was significantly lower (1.4%) compared to intracellular proteins (2.1%), a feature conserved in fungi but not in metazoans or plants. We evidenced that a catalase, widespread in white-rot fungi, protects the secreted proteins from oxidation. Our redox proteomics approach allowed the identification of 49 oxidizable Met and 40 oxidizable Pro residues within few secreted proteins, mostly CAZymes. Interestingly, many of them had several oxidized residues localized in hotspots. Some Met, including those in GH7 cellobiohydrolases, were oxidized up to 47%, with a substantial percentage of sulfone (13%). These Met are conserved in fungal homologs, suggesting important functional roles. Our findings reveal that white-rot fungi safeguard their secreted proteins by minimizing their Met content and by scavenging ROS and pinpoint redox-active residues in CAZymes.IMPORTANCEThe study of lignocellulose degradation by fungi is critical for understanding the ecological and industrial implications of wood decay. While carbohydrate-active enzymes (CAZymes) play a well-established role in lignocellulose degradation, the impact of hydrogen peroxide (H2O2) on secreted proteins remains unclear. This study aims at evaluating the effect of H2O2 on secreted proteins, focusing on the oxidation of methionine (Met). Using the model white-rot fungi Pycnoporus cinnabarinus grown on aspen wood, we showed that fungi protect their secreted proteins from oxidation by reducing their Met content and utilizing a secreted catalase to scavenge exogenous H2O2. The research identified key oxidizable Met within secreted CAZymes. Importantly, some Met, like those of GH7 cellobiohydrolases, undergone substantial oxidation levels suggesting important roles in lignocellulose degradation. These findings highlight the adaptive mechanisms employed by white-rot fungi to safeguard their secreted proteins during wood decay and emphasize the importance of these processes in lignocellulose breakdown.

Functional characterisation of a new halotolerant seawater active glycoside hydrolase family 6 cellobiohydrolase from a salt marsh.

Realising a fully circular bioeconomy requires the valorisation of lignocellulosic biomass. Cellulose is the most attractive component of lignocellulose but depolymerisation is inefficient, expensive and resource intensive requiring substantial volumes of potable water. Seawater is an attractive prospective replacement, however seawater tolerant enzymes are required for the development of seawater-based biorefineries. Here, we report a halophilic cellobiohydrolase SMECel6A, identified and isolated from a salt marsh meta-exo-proteome dataset with high sequence divergence to previously characterised cellobiohydrolases. SMECel6A contains a glycoside hydrolase family 6 (GH6) domain and a carbohydrate binding module family 2 (CBM2) domain. Characterisation of recombinant SMECel6A revealed SMECel6A to be active upon crystalline and amorphous cellulose. Mono- and oligosaccharide product profiles revealed cellobiose as the major hydrolysis product confirming SMECel6A as a cellobiohydrolase. We show SMECel6A to be halophilic with optimal activity achieved in 0.5X seawater displaying 80.6 ± 6.93% activity in 1 × seawater. Structural predictions revealed similarity to a characterised halophilic cellobiohydrolase despite sharing only 57% sequence identity. Sequential thermocycling revealed SMECel6A had the ability to partially reversibly denature exclusively in seawater retaining significant activity. Our study confirms that salt marsh ecosystems harbour enzymes with attractive traits with biotechnological potential for implementation in ionic solution based bioprocessing systems.

Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening.

Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, glycoside hydrolase family 7 (GH7) CBHs are critically important for the bioeconomy and typically difficult to engineer. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity. Using experimentally assayed activities of genome mined CBHs, we applied sequence and structural alignments to top performers to identify key point mutations linked to improved activity. From ∼1500 known GH7 sequences, an evolutionarily diverse subset of 57 GH7 CBH genes was expressed in Trichoderma reesei and screened using a multiplexed activity screening assay. Ten catalytically enhanced natural variants were identified, produced, purified, and tested for efficacy using industrially relevant conditions and substrates. Three key amino acids in CBHs with performance comparable or superior to Penicillium funiculosum Cel7A were identified and combinatorially engineered into P. funiculosum cel7a, expressed in T. reesei, and assayed on lignocellulosic biomass. The top performer generated using this combined approach of natural diversity genome mining, experimental assays, and computational modeling produced a 41% increase in conversion extent over native P. funiculosum Cel7A, a 55% increase over the current industrial standard T. reesei Cel7A, and 10% improvement over Aspergillus oryzae Cel7C, the best natural GH7 CBH previously identified in our laboratory.

Impact of Synergy Partner Cel7B on Cel7A Binding Rates: Insights from Single-Molecule Data.

Enzymatic degradation of cellulosic biomass is a well-established route for the sustainable production of biofuels, chemicals, and materials. A strategy employed by nature and industry to achieve an efficient degradation of cellulose is that cellobiohydrolases (or exocellulases), such as Cel7A, work synergistically with endoglucanases, such as Cel7B, to achieve the complete degradation of cellulose. However, a complete mechanistic understanding of this exo-endo synergy is still lacking. Here, we used single-molecule fluorescence microscopy to quantify the binding kinetics of Cel7A on cellulose when it is acting alone on the cellulose fibrils and in the presence of its synergy partner, the endoglucanase Cel7B. To this end, we used a fluorescently tagged Cel7A and studied its binding in the presence of the unlabeled Cel7B. This provided the single-molecule data necessary for the estimation of the rate constants of association kON and dissociation kOFF of Cel7A for the substrate. We show that the presence of Cel7B does not impact the dissociation rate constant, kOFF. But, the association rate of Cel7A decreases by a factor of 2 when Cel7B is present at a molar proportion of 10:1. This ratio has previously been shown to lead to synergy. This decrease in association rate is observed in a wide range of total enzyme concentrations, from sub nM to µM concentrations. This decrease in kON is consistent with the formation of cellulase clusters recently observed by others using atomic force microscopy.

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.

Assay of cellulose 1,4-ß-cellobiosidase activity in soil.

As the most abundant biopolymer on earth, cellulose undergoes degradation by a diverse set of enzymes with varying specificities that act in synergism. An assay protocol was developed to detect and quantify activity of cellulose 1,4-ß-cellobiosidase (EC 3.2.1.91) in soil. The optimum pH and temperature for ß-cellobiosidase activity were approximately pH 5.5 and 60 °C, respectively. In the tested six soils, the Michaelis constants (Km) ranged from 0.08 to 0.51 mM, and maximum velocity (Vmax) ranged from 71.5 to 318.1 µmol kg soil-1 h-1. The temperature coefficient (Q10) ranged from 1.72 to 1.99 at non-denaturing temperatures from 10 to 50 °C, and the activation energy (Ea) ranged from 42.5 to 53.7 kJ mol-1. The assay procedure provided reproducible results with a coefficient of variance ≤4.7% and demonstrated a limit of quantification (LOQ) of 50.9 µmol p-nitrophenol release kg-1 soil h-1 for ß-cellobiosidase activity in soil. Notably, the developed assay protocol offers reproducibility and precision comparable to bench-scale assays while reducing costs associated with reagents, supplies, and labor.

First evidence of a horizontally-acquired GH-7 cellobiohydrolase from a longhorned beetle genome.

Xylophagous larvae of longhorned beetles (Coleoptera; Cerambycidae) efficiently break down polysaccharides of the plant cell wall, which make the bulk of their food, using a range of carbohydrate-active enzymes (CAZymes). In this study, we investigated the function and evolutionary history of the first identified example of insect-encoded members of glycoside hydrolase family 7 (GH7) derived from the Lamiinae Exocentrus adspersus. The genome of this beetle contained two genes encoding GH7 proteins located in tandem and flanked by transposable elements. Phylogenetic analysis revealed that the GH7 sequences of E. adspersus were closely related to those of Ascomycete fungi, suggesting that they were acquired through horizontal gene transfer (HGT) from fungi. However, they were more distantly related to those encoded by genomes of Crustacea and of protist symbionts of termites and cockroaches, supporting that the same enzyme family was recruited several times independently in Metazoa during the course of their evolution. The recombinant E. adspersus GH7 was found to primarily break down cellulose polysaccharides into cellobiose, indicating that it is a cellobiohydrolase, and could also use smaller cellulose oligomers as substrates. Additionally, the cellobiohydrolase activity was boosted by the presence of calcium chloride. Our findings suggest that the combination of GH7 cellobiohydrolases with other previously characterized endo-ß-1,4-glucanases and ß-glucosidases allows longhorned beetles like E. adspersus to efficiently break down cellulose into monomeric glucose.

ß-1,4-Cellobiohydrolase is involved in full expression of phcA, contributing to the feedback loop in quorum sensing of Ralstonia pseudosolanacearum strain OE1-1.

After infecting roots of tomato plants, the gram-negative bacterium Ralstonia pseudosolanacearum strain OE1-1 activates quorum sensing (QS) to induce production of plant cell wall-degrading enzymes, such as ß-1,4-endoglucanase (Egl) and ß-1,4-cellobiohydrolase (CbhA), via the LysR family transcriptional regulator PhcA and then invades xylem vessels to exhibit virulence. The phcA-deletion mutant (ΔphcA) exhibits neither the ability to infect xylem vessels nor virulence. Compared with strain OE1-1, the egl-deletion mutant (Δegl) exhibits lower cellulose degradation activity, lower infectivity in xylem vessels, and reduced virulence. In this study, we analysed functions of CbhA other than cell wall degradation activity that are involved in the virulence of strain OE1-1. The cbhA-deletion mutant (ΔcbhA) lacked the ability to infect xylem vessels and displayed loss of virulence, similar to ΔphcA, but exhibited less reduced cellulose degradation activity compared with Δegl. Transcriptome analysis revealed that the phcA expression levels in ΔcbhA were significantly lower than in OE1-1, with significantly altered expression of more than 50% of PhcA-regulated genes. Deletion of cbhA led to a significant change in QS-dependent phenotypes, similar to the effects of phcA deletion. Complementation of ΔcbhA with native cbhA or transformation of this mutant with phcA controlled by a constitutive promoter recovered its QS-dependent phenotypes. The expression level of phcA in ΔcbhA-inoculated tomato plants was significantly lower than in strain OE1-1-inoculated plants. Our results collectively suggest that CbhA is involved in the full expression of phcA, thereby contributing to the QS feedback loop and virulence of strain OE1-1.

Enhancing the production of a heterologous Trametes laccase (LacA) by replacement of the major cellulase CBH1 in Trichoderma reesei.

The laccases from white-rot fungi exhibit high redox potential in treating phenolic compounds. However, their application in commercial purposes has been limited because of the relatively low productivity of the native hosts. Here, the laccase A-encoding gene lacA of Trametes sp. AH28-2 was overexpressed under the control of the strong promoter of cbh1 (Pcbh1), the gene encoding the endogenous cellobiohydrolase 1 (CBH1), in the industrial workhorse fungus Trichoderma reesei. Firstly, the lacA expression cassette was randomly integrated into the T. reesei chromosome by genetic transformation. The lacA gene was successfully transcribed, but the laccase couldn't be detected in the liquid fermentation condition. Meanwhile, it was found that the endoplasmic reticulum-associated degradation (ERAD) was strongly activated, indicating that the expression of LacA probably triggered intense endoplasmic reticulum (ER) stress. Subsequently, the lacA expression cassette was added with the downstream region of cbh1 (Tcbh1) to construct the new expression cassette lacA::Δcbh1, which could replace the cbh1 locus in the genome via homologous recombination. After genetic transformation, the lacA gene was integrated into the cbh1 locus and transcribed. And the unfolded protein response (UPR) and ERAD were only slightly induced, for which the loss of endogenous cellulase CBH1 released the pressure of secretion. Finally, the maximum laccase activity of 168.3 U/l was obtained in the fermentation broth. These results demonstrated that the reduction of secretion pressure by deletion of endogenous protein-encoding genes would be an efficient strategy for the secretion of heterologous target proteins in industrial fungi. ONE-SENTENCE SUMMARY: The reduction of the secretion pressure by deletion of the endogenous cbh1 gene can contribute to heterologous expression of the laccase (LacA) from Trametes sp. AH28-2 in Trichoderma reesei.

Molecular cloning and characterization of a novel bifunctional cellobiohydrolase/ß-xylosidase from a metagenomic library of mangrove soil.

A metagenomic library of mangrove soil samples consisting of approximately 11,000 clones was constructed, and a rare bifunctional cellobiohydrolase/ß-xylosidase Cbh2124 was identified by functional screening. Cbh2124 displayed the highest homology (56.43%) with a protein of the glycoside hydrolase 10 (GH10) family from Proteobacteria. Phylogenetic analysis confirmed that Cbh2124 belongs to the GH10 family. The recombinant enzyme showed a strong cellobiohydrolase activity and a relatively high ß-xylosidase activity, and its catalytic efficiency to the cellobiose substrate was as high as 1.27 × 105 s-1·mM-1, the highest efficiency among reported cellobiohydrolases. Of particular interest, some enzymatic properties of the ß-xylosidase activity of Cbh2124 were significantly different from those of the cellobiohydrolase activity. The optimal pH and temperature of the cellobiohydrolase activity of Cbh2124 was 6.4 and 36 °C, and the activity was essentially lost after treatment at 45 °C for 1 h. The optimal pH and temperature of the ß-xylosidase activity of Cbh2124 was 8.0 and 60 °C, and the residual activity was still over 90% after treatment at 80 °C for 6 h. The molecular docking results of the ß-xylosidase activity of Cbh2124 revealed the additional presence of catalytic amino acids Ser175 and Lys420, thus increasing the number of hydrogen bonds involved in the catalytic process, which possibly let to the improved thermostability compared with that of the cellobiohydrolase activity.

Increasing extracellular cellulase activity of the recombinant Saccharomyces cerevisiae by engineering cell wall-related proteins for improved consolidated processing of carbon neutral lignocellulosic biomass.

Sustainable bioproduction usingcarbon neutral feedstocks, especially lignocellulosic biomass, has attracted increasing attention due to concern over climate change and carbon reduction. Consolidated bioprocessing (CBP) of lignocellulosic biomass using recombinantyeast of Saccharomyces cerevisiaeis a promising strategy forlignocellulosic biorefinery. However, the economic viability is restricted by low enzyme secretion levels.For more efficient CBP, MIG1spsc01isolated from the industrial yeast which encodes the glucose repression regulator derivative was overexpressed. Increased extracellular cellobiohydrolase (CBH) activity was observed with unexpectedly decreased cell wall integrity. Further studies revealed that disruption ofCWP2, YGP1, andUTH1,which are functionally related toMIG1spsc01, also enhanced CBH secretion. Subsequently, improved cellulase production was achieved by simultaneous disruption ofYGP1and overexpression ofSED5, which remarkably increased extracellular CBH activity of 2.2-fold over the control strain. These results provide a novel strategy to improve the CBP yeast for bioconversion of carbon neutral biomass.