Enzymatic and biophysical characterization of a novel modular cellulosomal GH5 endoglucanase multifunctional from the anaerobic gut fungus Piromyces finnis.

Cellulases from anaerobic fungi are enzymes less-studied biochemically and structurally than cellulases from bacteria and aerobic fungi. Currently, only thirteen GH5 cellulases from anaerobic fungi were biochemically characterized and two crystal structures were reported. In this context, here, we report the functional and biophysical characterization of a novel multi-modular cellulosomal GH5 endoglucanase from the anaerobic gut fungus Piromyces finnis (named here PfGH5). Multiple sequences alignments indicate that PfGH5 is composed of a GH5 catalytic domain and a CBM1 carbohydrate-binding module connected through a CBM10 dockerin module. Our results showed that PfGH5 is an endoglucanase from anaerobic fungus with a large spectrum of activity. PfGH5 exhibited preference for hydrolysis of oat ß-glucan, followed by galactomannan, carboxymethyl cellulose, mannan, lichenan and barley ß-glucan, therefore displaying multi-functionality. For oat ß-glucan, PfGH5 reaches its optimum enzymatic activity at 40 °C and pH 5.5, with Km of 7.1 µM. Ion exchange chromatography analyzes revealed the production of oligosaccharides with a wide degree of polymerization indicated that PfGH5 has endoglucanase activity. The ability to bind and cleave different types of carbohydrates evidence the potential of PfGH5 for use in biotechnology and provide a useful basis for future investigation and application of new anaerobic fungi enzymes.

Metabolic engineering of Saccharomyces cerevisiae for second-generation ethanol production from xylo-oligosaccharides and acetate.

Simultaneous intracellular depolymerization of xylo-oligosaccharides (XOS) and acetate fermentation by engineered Saccharomyces cerevisiae offers significant potential for more cost-effective second-generation (2G) ethanol production. In the present work, the previously engineered S. cerevisiae strain, SR8A6S3, expressing enzymes for xylose assimilation along with an optimized route for acetate reduction, was used as the host for expressing two ß-xylosidases, GH43-2 and GH43-7, and a xylodextrin transporter, CDT-2, from Neurospora crassa, yielding the engineered SR8A6S3-CDT-2-GH34-2/7 strain. Both ß-xylosidases and the transporter were introduced by replacing two endogenous genes, GRE3 and SOR1, that encode aldose reductase and sorbitol (xylitol) dehydrogenase, respectively, and catalyse steps in xylitol production. The engineered strain, SR8A6S3-CDT-2-GH34-2/7 (sor1Δ gre3Δ), produced ethanol through simultaneous XOS, xylose, and acetate co-utilization. The mutant strain produced 60% more ethanol and 12% less xylitol than the control strain when a hemicellulosic hydrolysate was used as a mono- and oligosaccharide source. Similarly, the ethanol yield was 84% higher for the engineered strain using hydrolysed xylan, compared with the parental strain. Xylan, a common polysaccharide in lignocellulosic residues, enables recombinant strains to outcompete contaminants in fermentation tanks, as XOS transport and breakdown occur intracellularly. Furthermore, acetic acid is a ubiquitous toxic component in lignocellulosic hydrolysates, deriving from hemicellulose and lignin breakdown. Therefore, the consumption of XOS, xylose, and acetate expands the capabilities of S. cerevisiae for utilization of all of the carbohydrate in lignocellulose, potentially increasing the efficiency of 2G biofuel production.

Sustainable biosynthetic pathways to value-added bioproducts from hydroxycinnamic acids.

The biorefinery concept, in which biomass is utilized for the production of fuels and chemicals, emerges as an eco-friendly, cost-effective, and renewable alternative to petrochemical-based production. The hydroxycinnamic acid fraction of lignocellulosic biomass represents an untapped source of aromatic molecules that can be converted to numerous high-value products with industrial applications, including in the flavor and fragrance sector and pharmaceuticals. This review describes several biochemical pathways useful in the development of a biorefinery concept based on the biocatalytic conversion of the hydroxycinnamic acids ferulic, caffeic, and p-coumaric acid into high-value molecules. KEY POINTS: • The phenylpropanoids bioconversion pathways in the context of biorefineries • Description of pathways from hydroxycinnamic acids to high-value compounds • Metabolic engineering and synthetic biology advance hydroxycinnamic acid-based biorefineries.

Oxidative cleavage of polysaccharides by a termite-derived superoxide dismutase boosts the degradation of biomass by glycoside hydrolases.

Wood-feeding termites effectively degrade plant biomass through enzymatic degradation. Despite their high efficiencies, however, individual glycoside hydrolases isolated from termites and their symbionts exhibit anomalously low effectiveness in lignocellulose degradation, suggesting hereto unknown enzymatic activities in their digestome. Herein, we demonstrate that an ancient redox-active enzyme encoded by the lower termite Coptotermes gestroi, a Cu/Zn superoxide dismutase (CgSOD-1), plays a previously unknown role in plant biomass degradation. We show that CgSOD-1 transcripts and peptides are up-regulated in response to an increased level of lignocellulose recalcitrance and that CgSOD-1 localizes in the lumen of the fore- and midguts of C. gestroi together with termite main cellulase, CgEG-1-GH9. CgSOD-1 boosts the saccharification of polysaccharides by CgEG-1-GH9. We show that the boosting effect of CgSOD-1 involves an oxidative mechanism of action in which CgSOD-1 generates reactive oxygen species that subsequently cleave the polysaccharide. SOD-type enzymes constitute a new addition to the growing family of oxidases, ones which are up-regulated when exposed to recalcitrant polysaccharides, and that are used by Nature for biomass degradation.

Comparison of Spathaspora passalidarum and recombinant Saccharomyces cerevisiae for integration of first- and second-generation ethanol production.

First-generation ethanol (E1G) is based on the fermentation of sugars released from saccharine or starch sources, while second-generation ethanol (E2G) is focused on the fermentation of sugars released from lignocellulosic feedstocks. During the fractionation process to release sugars from hemicelluloses (mainly xylose), some inhibitor compounds are released hindering fermentation. Thus, the biggest challenge of using hemicellulosic hydrolysate is selecting strains and processes able to efficiently ferment xylose and tolerate inhibitors. With the aim of diluting inhibitors, sugarcane molasses (80% of sucrose content) can be mixed to hemicellulosic hydrolysate in an integrated E1G-E2G process. Cofermentations of xylose and sucrose were evaluated for the native xylose consumer Spathaspora passalidarum and a recombinant Saccharomyces cerevisiae strain. The industrial S. cerevisiae strain CAT-1 was modified to overexpress the XYL1, XYL2 and XKS1 genes and a mutant ([4-59Δ]HXT1) version of the low-affinity HXT1 permease, generating strain MP-C5H1. Although S. passalidarum showed better results for xylose fermentation, this yeast showed intracellular sucrose hydrolysis and low sucrose consumption in microaerobic conditions. Recombinant S. cerevisiae showed the best performance for cofermentation, and a batch strategy at high cell density in bioreactor achieved unprecedented results of ethanol yield, titer and volumetric productivity in E1G-E2G production process.

On the roles of AA15 lytic polysaccharide monooxygenases derived from the termite Coptotermes gestroi.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes which catalyze the oxidative cleavage of polysaccharides. LPMOs belonging to family 15 in the Auxiliary Activity (AA) class from the Carbohydrate-Active Enzyme database are found widespread across the Tree of Life, including viruses, algae, oomycetes and animals. Recently, two AA15s from the firebrat Thermobia domestica were reported to have oxidative activity, one towards cellulose or chitin and the other towards chitin, signalling that AA15 LPMOs from insects potentially have different biochemical functions. Herein, we report the identification and characterization of two family AA15 members from the lower termite Coptotermes gestroi. Addition of Cu(II) to CgAA15a or CgAA15b had a thermostabilizing effect on both. Using ascorbate and O2 as co-substrates, CgAA15a and CgAA15b were able to oxidize chitin, but showed no activity on celluloses, xylan, xyloglucan and starch. Structural models indicate that the LPMOs from C. gestroi (CgAA15a/CgAA15b) have a similar fold but exhibit key differences in the catalytic site residues when compared to the cellulose/chitin-active LPMO from T. domestica (TdAA15a), especially the presence of a non-coordinating phenylalanine nearby the Cu ion in CgAA15a/b, which appears as a tyrosine in the active site of TdAA15a. Despite the overall similarity in protein folds, however, mutation of the active site phenylalanine in CgAA15a to a tyrosine did not expanded the enzymatic specificity from chitin to cellulose. Our data show that CgAA15a/b enzymes are likely not involved in lignocellulose digestion but might play a role in termite developmental processes as well as on chitin and nitrogen metabolisms.

Enzymatic removal of inhibitory compounds from lignocellulosic hydrolysates for biomass to bioproducts applications.

The physicochemical pretreatment is an important step to reduce biomass recalcitrance and facilitate further processing of plant lignocellulose into bioproducts. This process results in soluble and insoluble biomass fractions, and both may contain by-products that inhibit enzymatic biocatalysts and microbial fermentation. These fermentation inhibitory compounds (ICs) are produced during the degradation of lignin and sugars, resulting in phenolic and furanic compounds, and carboxylic acids. Therefore, detoxification steps may be required to improve lignocellulose conversion by microoganisms. Several physical and chemical methods, such as neutralization, use of activated charcoal and organic solvents, have been developed and recommended for removal of ICs. However, biological processes, especially enzyme-based, have been shown to efficiently remove ICs with the advantage of minimizing environmental issues since they are biogenic catalysts and used in low quantities. This review focuses on describing several enzymatic approaches to promote detoxification of lignocellulosic hydrolysates and improve the performance of microbial fermentation for the generation of bioproducts. Novel strategies using classical carbohydrate active enzymes (CAZymes), such as laccases (AA1) and peroxidases (AA2), as well as more advanced strategies using prooxidant, antioxidant and detoxification enzymes (dubbed as PADs), i.e. superoxide dismutases, are discussed as perspectives in the field.

Redox potential as a key parameter for monitoring and optimization of xylose fermentation with yeast Spathaspora passalidarum under limited-oxygen conditions.

The determination of optimum values of volumetric oxygen transfer coefficient (kLa) for Spathaspora passalidarum is an important aspect for the optimization of ethanol production from pentoses since oxygen plays a key role on yeast metabolism. By studying the fermentation of a xylose and glucose mixture, the highest ethanol volumetric productivity was achieved at a kLa of 45 h-1 (1.12 gethanol L-1 h-1), reducing the fermentation time to half when compared to other oxygen-limiting conditions that were considered optimum for other native strains, besides increasing xylose consumption rates. The high cell density fermentation showed to be a good strategy to be applied in industrial processes with S. passalidarum, enabling the complete exhaustion of a high initial substrate concentration (90 g L-1) in less than 24 h, with a final ethanol titer of 28.61 (± 0.42) g L-1. By performing a detailed investigation on oxidation-reduction potential (ORP), it was possible to conclude that the highest ethanol formation rates were registered at oxireduction potential values around - 100 mV, becoming an important parameter to be controlled when oxygen-limiting conditions are applied in industrial fermentations. The oxygen availability also affected the activity of enzyme XR and its preference for NADH or NADPH, directly affecting the activity of enzyme XDH and the redox imbalance on the xylose pathway. In addition, respirometric parameters were determined for the yeast S. passalidarum under an aerobic growth condition.

Designing a cocktail containing redox enzymes to improve hemicellulosic hydrolysate fermentability by microorganisms.

Bioproducts production using monomeric sugars derived from lignocellulosic biomass presents several challenges, such as to require a physicochemical pretreatment to improve its conversion yields. Hydrothermal lignocellulose pretreatment has several advantages and results in solid and liquid streams. The former is called hemicellulosic hydrolysate (HH), which contains inhibitory phenolic compounds and sugar degradation products that hinder microbial fermentation products from pentose sugars. Here, we developed and applied a novel enzyme process to detoxify HH. Initially, the design of experiments with different redox activities enzymes was carried out. The enzyme mixture containing the peroxidase (from Armoracia rusticana) together with superoxide dismutase (from Coptotermes gestroi) are the most effective to detoxify HH derived from sugarcane bagasse. Butanol fermentation by the bacteria Clostridium saccharoperbutylacetonicum and ethanol production by the yeast Scheffersomyces stipitis increased by 24.0× and 2.4×, respectively, relative to the untreated hemicellulosic hydrolysates. Detoxified HH was analyzed by chromatographic and spectrometric methods elucidating the mechanisms of phenolic compound modifications by enzymatic treatment. The enzyme mixture degraded and reduced the hydroxyphenyl- and feruloyl-derived units and polymerized the lignin fragments. This strategy uses biocatalysts under environmentally friendly conditions and could be applied in the fuel, food, and chemical industries.

Exopolysaccharides from Aspergillus terreus: Production, chemical elucidation and immunoactivity.

Aspergillus terreus, a fungus commonly used in pharmaceutical industry to produce lovastatin and other secondary metabolites, has been reported to have beneficial biological properties. In this study the exopolysaccharides (AT-EPS) produced by A. terreus were evaluated as potential modulators of certain functions of macrophages. The production parameters for EPS obtained from the liquid culture broth of the studied fungus were optimized using response surface methodology (RSM) and indicated good correlation between the experimental and predicted values. The optimum conditions for AT-EPS extraction included fermentation at 28 °C, pH 8.79, under 98 rpm of agitation, using 2.39% glucose (carbon source) and 0.957% ammonium nitrate (nitrogen source). Under these optimized conditions, AT-EPS production was 1.34 g/L medium. The chemical analyses showed that AT-EPS was composed by mannose (Man; 40.5 mol%), galactose (Gal; 35.2 mol%), and glucose (Glc; 24.3 mol%), and the spectroscopic (FTIR; NMR) and methylation analyses indicated the presence of galactomannans, ß-1,3-glucans, and glycogen-like glucans. AT-EPS was tested on murine macrophages to verify its immunoactivity and the treated cells were able to produce nitric oxide, superoxide anion, TNF-α and interleukin 6 similarly to the positive control cells. Furthermore, the macrophages treated with AT-EPS showed activated-like morphological alterations.

Suppression of a single BAHD gene in Setaria viridis causes large, stable decreases in cell wall feruloylation and increases biomass digestibility.

Feruloylation of arabinoxylan (AX) in grass cell walls is a key determinant of recalcitrance to enzyme attack, making it a target for improvement of grass crops, and of interest in grass evolution. Definitive evidence on the genes responsible is lacking so we studied a candidate gene that we identified within the BAHD acyl-CoA transferase family. We used RNA interference (RNAi) silencing of orthologs in the model grasses Setaria viridis (SvBAHD01) and Brachypodium distachyon (BdBAHD01) and determined effects on AX feruloylation. Silencing of SvBAHD01 in Setaria resulted in a c. 60% decrease in AX feruloylation in stems consistently across four generations. Silencing of BdBAHD01 in Brachypodium stems decreased feruloylation much less, possibly due to higher expression of functionally redundant genes. Setaria SvBAHD01 RNAi plants showed: no decrease in total lignin, approximately doubled arabinose acylated by p-coumarate, changes in two-dimensional NMR spectra of unfractionated cell walls consistent with biochemical estimates, no effect on total biomass production and an increase in biomass saccharification efficiency of 40-60%. We provide the first strong evidence for a key role of the BAHD01 gene in AX feruloylation and demonstrate that it is a promising target for improvement of grass crops for biofuel, biorefining and animal nutrition applications.

Biochemical and biophysical properties of a metagenome-derived GH5 endoglucanase displaying an unconventional domain architecture.

Endoglucanases are key enzymes in the degradation of cellulose, the most abundant polymer on Earth. The aim of this work was to perform the biochemical and biophysical characterization of CelE2, a soil metagenome derived endoglucanase. CelE2 harbors a conserved domain from glycoside hydrolase family 5 (GH5) and a C-terminal domain with identity to Calx-beta domains. The recombinant CelE2 displayed preference for hydrolysis of oat beta-glucan, followed by lichenan and carboxymethyl cellulose. Optimum values of enzymatic activity were observed at 45°C and pH 5.3, and CelE2 exhibited considerable thermal stability at 40°C for up to 360min. Regarding the cleavage pattern on polysaccharides, the release of oligosaccharides with a wide degree of polymerization indicated a characteristic of endoglucanase activity. Furthermore, the analysis of products generated from the cleavage of cellooligosaccharides suggested that CelE2 exhibited transglycosylation activity. Interestingly, the presence of CaCl2 positively affect CelE2, including in the presence of surfactants. SAXS experiments provided key information on the effect of CaCl2 on the stability of CelE2 and dummy atom and rigid-body models were generated. To the best of our knowledge this is the first biochemical and biophysical characterization of an endoglucanase from family GH5 displaying this unconventional modular organization.

The Coptotermes gestroi aldo-keto reductase: a multipurpose enzyme for biorefinery applications.

BACKGROUND: In nature, termites can be considered as a model biological system for biofuel research based on their remarkable efficiency for lignocellulosic biomass conversion. Redox enzymes are of interest in second-generation ethanol production because they promote synergic enzymatic activity with classical hydrolases for lignocellulose saccharification and inactivate fermentation inhibitory compounds produced after lignocellulose pretreatment steps. RESULTS: In the present study, the biochemical and structural characteristics of the Coptotermes gestroi aldo-keto reductase (CgAKR-1) were comprehensively investigated. CgAKR-1 displayed major structural differences compared with others AKRs, including the differences in the amino acid composition of the substrate-binding site, providing basis for classification as a founding member of a new AKR subfamily (family AKR1 I). Immunolocalization assays with anti-CgAKR-1 antibodies resulted in strong fluorescence in the salivary gland, proventriculus, and foregut. CgAKR-1 supplementation caused a 32% reduction in phenolic aldehydes, such as furfural, which act as fermentation inhibitors of hemicellulosic hydrolysates, and improved ethanol fermentation by the xylose-fermenting yeast Scheffersomyces stipitis by 45%. We observed synergistic enzymatic interactions between CgAKR-1 and commercial cellulosic cocktail for sugarcane bagasse saccharification, with a maximum synergism degree of 2.17 for sugar release. Our data indicated that additive enzymatic activity could be mediated by reactive oxygen species because CgAKR-1 could produce hydrogen peroxide. CONCLUSION: In summary, we identified the founding member of an AKRI subfamily with a potential role in the termite digestome. CgAKR-1 was found to be a multipurpose enzyme with potential biotechnological applications. The present work provided a basis for the development and application of integrative and multipurpose enzymes in the bioethanol production chain.

Expanding the Knowledge on Lignocellulolytic and Redox Enzymes of Worker and Soldier Castes from the Lower Termite Coptotermes gestroi.

Termites are considered one of the most efficient decomposers of lignocelluloses on Earth due to their ability to produce, along with its microbial symbionts, a repertoire of carbohydrate-active enzymes (CAZymes). Recently, a set of Pro-oxidant, Antioxidant, and Detoxification enzymes (PAD) were also correlated with the metabolism of carbohydrates and lignin in termites. The lower termite Coptotermes gestroi is considered the main urban pest in Brazil, causing damage to wood constructions. Recently, analysis of the enzymatic repertoire of C. gestroi unveiled the presence of different CAZymes. Because the gene profile of CAZy/PAD enzymes endogenously synthesized by C. gestroi and also by their symbiotic protists remains unclear, the aim of this study was to explore the eukaryotic repertoire of these enzymes in worker and soldier castes of C. gestroi. Our findings showed that worker and soldier castes present similar repertoires of CAZy/PAD enzymes, and also confirmed that endo-glucanases (GH9) and beta-glucosidases (GH1) were the most important glycoside hydrolase families related to lignocellulose degradation in both castes. Classical cellulases such as exo-glucanases (GH7) and endo-glucanases (GH5 and GH45), as well as classical xylanases (GH10 and GH11), were found in both castes only taxonomically related to protists, highlighting the importance of symbiosis in C. gestroi. Moreover, our analysis revealed the presence of Auxiliary Activity enzyme families (AAs), which could be related to lignin modifications in termite digestomes. In conclusion, this report expanded the knowledge on genes and proteins related to CAZy/PAD enzymes from worker and soldier castes of lower termites, revealing new potential enzyme candidates for second-generation biofuel processes.

Bioethanol production by recycled Scheffersomyces stipitis in sequential batch fermentations with high cell density using xylose and glucose mixture.

Here, it is shown three-step investigative procedures aiming to improve pentose-rich fermentations performance, involving a simple system for elevated mass production by Scheffersomyces stipitis (I), cellular recycle batch fermentations (CRBFs) at high cell density using two temperature strategies (fixed at 30°C; decreasing from 30 to 26°C) (II), and a short-term adaptation action seeking to acclimatize the microorganism in xylose rich-media (III). Cellular propagation provided 0.52gdrycellweightgRS(-1), resulting in an expressive value of 45.9gdrycellweightL(-1). The yeast robustness in CRBF was proven by effective ethanol production, reaching high xylose consumption (81%) and EtOH productivity (1.53gL(-1)h(-1)). Regarding the short-term adaptation, S. stipitis strengthened its robustness, as shown by a 6-fold increase in xylose reductase (XR) activity. The short fermentation time (20h for each batch) and the fermentation kinetics for ethanol production from xylose are quite promising.

Cooperation of Aspergillus nidulans enzymes increases plant polysaccharide saccharification.

Efficient polysaccharide degradation depends on interaction between enzymes acting on the main chain and the side chains. Previous studies demonstrated cooperation between several enzymes, but not all enzyme combinations have been explored. A better understanding of enzyme cooperation would enable the design of better enzyme mixtures, optimally profiting from synergistic effects. In this study, we analyzed the cooperation of several enzymes involved in the degradation of xylan, glucan, xyloglucan and crude plant biomass from Aspergillus nidulans by single and combined incubations with their polymeric substrate. Positive effects were observed between most enzymes, although not always to the same extent. Moreover, the tailor made cocktails formulated in this study resulted in efficient release of glucose from plant biomass. This study also serves as an example for the complex cooperation that occurs between enzymes in plant biomass saccharification and how expression in easily-accessible hosts, such as Pichia pastoris, can help in revealing these effects.