Functional characterization and structural insights of three cutinases from the ascomycete Fusarium verticillioides.

Cutinases are serine esterases that belong to the α/ß hydrolases superfamily. The natural substrates for these enzymes are cutin and suberin, components of the plant cuticle, the first barrier in the defense system against pathogen invasion. It is well-reported that plant pathogens produce cutinases to facilitate infection. Fusarium verticillioides, one important corn pathogens, is an ascomycete upon which its cutinases are poorly explored. Consequently, the objective of this study was to perform the biochemical characterization of three precursor cutinases (FvCut1, FvCut2, and FvCut3) from F. verticillioides and to obtain structural insights about them. The cutinases were produced in Escherichia coli and purified. FvCut1, FvCut2, and FvCut3 presented optimal temperatures of 20, 40, and 35 °C, and optimal pH of 9, 7, and 8, respectively. Some chemicals stimulated the enzymatic activity. The kinetic parameters revealed that FvCut1 has higher catalytic efficiency (Kcat/Km) in the p-nitrophenyl-butyrate (p-NPB) substrate. Nevertheless, the enzymes were not able to hydrolyze polyethylene terephthalate (PET). Furthermore, the three-dimensional models of these enzymes showed structural differences among them, mainly FvCut1, which presented a narrower opening cleft to access the catalytic site. Therefore, our study contributes to exploring the diversity of fungal cutinases and their potential biotechnological applications.

Insights into the dual cleavage activity of the GH16 laminarinase enzyme class on ß-1,3 and ß-1,4 glycosidic bonds.

Glycoside hydrolases (GHs) are involved in the degradation of a wide diversity of carbohydrates and present several biotechnological applications. Many GH families are composed of enzymes with a single well-defined specificity. In contrast, enzymes from the GH16 family can act on a range of different polysaccharides, including ß-glucans and galactans. SCLam, a GH16 member derived from a soil metagenome, an endo-ß-1,3(4)-glucanase (EC 3.2.1.6), can cleave both ß-1,3 and ß-1,4 glycosidic bonds in glucans, such as laminarin, barley ß-glucan, and cello-oligosaccharides. A similar cleavage pattern was previously reported for other GH16 family members. However, the molecular mechanisms for this dual cleavage activity on (1,3)- and (1,4)-ß-D-glycosidic bonds by laminarinases have not been elucidated. In this sense, we determined the X-ray structure of a presumably inactive form of SCLam cocrystallized with different oligosaccharides. The solved structures revealed general bound products that are formed owing to residual activities of hydrolysis and transglycosylation. Biochemical and biophysical analyses and molecular dynamics simulations help to rationalize differences in activity toward different substrates. Our results depicted a bulky aromatic residue near the catalytic site critical to select the preferable configuration of glycosidic bonds in the binding cleft. Altogether, these data contribute to understanding the structural basis of recognition and hydrolysis of ß-1,3 and ß-1,4 glycosidic linkages of the laminarinase enzyme class, which is valuable for future studies on the GH16 family members and applications related to biomass conversion into feedstocks and bioproducts.

The periplasmic expression and purification of AA15 lytic polysaccharide monooxygenases from insect species in Escherichia coli.

Lytic polysaccharide monooxygenases (LPMOs) are metalloenzymes that cleave structural polysaccharides through an oxidative mechanism. The enzymatic activity of LPMOs relies on the presence of a Cu2+ histidine-brace motif in their flat catalytic surface. Upon reduction by an external electron donor and in the presence of its co-substrates, O2 or H2O2, LPMOs can generate reactive oxygen species to oxidize the substrates. Fungal and bacterial LPMOs are involved in the catabolism of polysaccharides, such as chitin, cellulose, and hemicelluloses, and virulence mechanisms. Based on the reports on the discovery of LPMOs from the family AA15 in termites, firebrats, and flies, the functional role of the LPMO in the biosphere could expand, as these enzymes may be correlated with chitin remodeling and molting in insects. However, there is limited knowledge of AA15 LPMOs due to difficulties in recombinant expression of soluble proteins and purification protocols. In this study, we describe a protocol for the cloning, expression, and purification of insect AA15 LPMOs from Arthropoda, mainly from termites, followed by the expression and purification of an AA15 LPMO from the silkworm Bombyx mori, which contains a relatively high number of disulfide bonds. We also report the recombinant expression and purification of a protein with homology to AA15 family from the western European honeybee Apis mellifera, an LPMO-like enzyme lacking the canonical histidine brace. Therefore, this work can support future studies concerning the role of LPMOs in the biology of insects and inspire molecular entomologists and insect biochemists in conducting activities in this field.

Applying biochemical and structural characterization of hydroxycinnamate catabolic enzymes from soil metagenome for lignin valorization strategies.

The biocatalytic production of fuels and chemicals from plant biomass represents an attractive alternative to fossil fuel-based refineries. In this context, the mining and characterization of novel biocatalysts can promote disruptive innovation opportunities in the field of lignocellulose conversion and valorization. In the present work, we conducted the biochemical and structural characterization of two novel hydroxycinnamic acid catabolic enzymes, isolated from a lignin-degrading microbial consortium, a feruloyl-CoA synthetase, and a feruloyl-CoA hydratase-lyase, named LM-FCS2 and LM-FCHL2, respectively. Besides establishing the homology model structures for novel FCS and FCHL members with unique characteristics, the enzymes presented interesting biochemical features: LM-FCS2 showed stability in alkaline pHs and was able to convert a wide array of p-hydroxycinnamic acids to their respective CoA-thioesters, including sinapic acid; LM-FCHL2 efficiently converted feruloyl-CoA and p-coumaroyl-CoA into vanillin and 4-hydroxybenzaldehyde, respectively, and could produce vanillin directly from ferulic acid. The coupled reaction of LM-FCS2 and LM-FCHL2 produced vanillin, not only from commercial ferulic acid but also from a crude lignocellulosic hydrolysate. Collectively, this work illuminates the structure and function of two critical enzymes involved in converting ferulic acid into high-value molecules, thus providing valuable concepts applied to the development of plant biomass biorefineries. KEY POINTS: • Comprehensive characterization of feruloyl-CoA synthetase from metagenomic origin. • Novel low-resolution structures of hydroxycinnamate catabolic enzymes. • Production of vanillin via enzymatic reaction using lignocellulosic hydrolysates.

Cloning, heterologous expression and biochemical characterization of a non-specific endoglucanase family 12 from Aspergillus terreus NIH2624.

The cellulases from Glycoside Hydrolyses family 12 (GH12) play an important role in cellulose degradation and plant cell wall deconstruction being widely used in a number of bioindustrial processes. Aiming to contribute toward better comprehension of these class of the enzymes, here we describe a high-yield secretion of a endoglucanase GH12 from Aspegillus terreus (AtGH12), which was cloned and expressed in Aspergillus nidulans strain A773. The purified protein was used for complete biochemical and functional characterization. The optimal temperature and pH of the enzyme were 55°C and 5.0 respectively, which has high activity against ß-glucan and xyloglucan and also is active toward glucomannan and CMC. The enzyme retained activity up to 60°C. AtGH12 is strongly inhibited by Cu2+, Fe2+, Cd2+, Mn2+, Ca2+, Zn2+ and EDTA, whereas K+, Tween, Cs+, DMSO, Triton X-100 and Mg2+ enhanced the enzyme activity. Furthermore, SAXS data reveal that the enzyme has a globular shape and CD analysis demonstrated a prevalence of a ß-strand structure corroborating with typical ß-sheets fold commonly found for other endoglucanases from GH12 family.

Structural and functional characterization of a highly secreted α-l-arabinofuranosidase (GH62) from Aspergillus nidulans grown on sugarcane bagasse.

Carbohydrate-Active Enzymes are key enzymes for biomass-to-bioproducts conversion. α-l-Arabinofuranosidases that belong to the Glycoside Hydrolase family 62 (GH62) have important applications in biofuel production from plant biomass by hydrolyzing arabinoxylans, found in both the primary and secondary cell walls of plants. In this work, we identified a GH62 α-l-arabinofuranosidase (AnAbf62Awt) that was highly secreted when Aspergillus nidulans was cultivated on sugarcane bagasse. The gene AN7908 was cloned and transformed in A. nidulans for homologous production of AnAbf62Awt, and we confirmed that the enzyme is N-glycosylated at asparagine 83 by mass spectrometry analysis. The enzyme was also expressed in Escherichia coli and the studies of circular dichroism showed that the melting temperature and structural profile of AnAbf62Awt and the non-glycosylated enzyme from E. coli (AnAbf62Adeglyc) were highly similar. In addition, the designed glycomutant AnAbf62AN83Q presented similar patterns of secretion and activity to the AnAbf62Awt, indicating that the N-glycan does not influence the properties of this enzyme. The crystallographic structure of AnAbf62Adeglyc was obtained and the 1.7Å resolution model showed a five-bladed ß-propeller fold, which is conserved in family GH62. Mutants AnAbf62AY312F and AnAbf62AY312S showed that Y312 was an important substrate-binding residue. Molecular dynamics simulations indicated that the loop containing Y312 could access different conformations separated by moderately low energy barriers. One of these conformations, comprising a local minimum, is responsible for placing Y312 in the vicinity of the arabinose glycosidic bond, and thus, may be important for catalytic efficiency.

Xyloglucan breakdown by endo-xyloglucanase family 74 from Aspergillus fumigatus.

Xyloglucan is the most abundant hemicellulose in primary walls of spermatophytes except for grasses. Xyloglucan-degrading enzymes are important in lignocellulosic biomass hydrolysis because they remove xyloglucan, which is abundant in monocot-derived biomass. Fungal genomes encode numerous xyloglucanase genes, belonging to at least six glycoside hydrolase (GH) families. GH74 endo-xyloglucanases cleave xyloglucan backbones with unsubstituted glucose at the -1 subsite or prefer xylosyl-substituted residues in the -1 subsite. In this work, 137 GH74-related genes were detected by examining 293 Eurotiomycete genomes and Ascomycete fungi contained one or no GH74 xyloglucanase gene per genome. Another interesting feature is that the triad of tryptophan residues along the catalytic cleft was found to be widely conserved among Ascomycetes. The GH74 from Aspergillus fumigatus (AfXEG74) was chosen as an example to conduct comprehensive biochemical studies to determine the catalytic mechanism. AfXEG74 has no CBM and cleaves the xyloglucan backbone between the unsubstituted glucose and xylose-substituted glucose at specific positions, along the XX motif when linked to regions deprived of galactosyl branches. It resembles an endo-processive activity, which after initial random hydrolysis releases xyloglucan-oligosaccharides as major reaction products. This work provides insights on phylogenetic diversity and catalytic mechanism of GH74 xyloglucanases from Ascomycete fungi.

Insights into the plant polysaccharide degradation potential of the xylanolytic yeast Pseudozyma brasiliensis.

In second-generation (2G) bioethanol production, plant cell-wall polysaccharides are broken down to release fermentable sugars. The enzymes of this process are classified as carbohydrate-active enzymes (CAZymes) and contribute substantially to the cost of biofuel production. A novel basidiomycete yeast species, Pseudozyma brasiliensis, was recently discovered. It produces an endo-ß-1,4-xylanase with a higher specific activity than other xylanases. This enzyme is essential for the hydrolysis of biomass-derived xylan and has an important role in 2G bioethanol production. In spite of the P. brasiliensis biotechnological potential, there is no information about how it breaks down polysaccharides. For the first time, we characterized the secretome of P. brasiliensis grown on different carbon sources (xylose, xylan, cellobiose and glucose) and also under starvation conditions. The growth and consumption of each carbohydrate and the activity of the CAZymes of culture supernatants were analyzed. The CAZymes found in its secretomes, validated by enzymatic assays, have the potential to hydrolyze xylan, mannan, cellobiose and other polysaccharides. The data show that this yeast is a potential source of hydrolases, which can be used for biomass saccharification.

Molecular basis of substrate recognition and specificity revealed in family 12 glycoside hydrolases.

Fungal GH12 enzymes are classified as xyloglucanases when they specifically target xyloglucans, or promiscuous endoglucanases when they exhibit catalytic activity against xyloglucan and ß-glucan chains. Several structural and functional studies involving GH12 enzymes tried to explain the main patterns of xyloglucan activity, but what really determines xyloglucanase specificity remains elusive. Here, three fungal GH12 enzymes from Aspergillus clavatus (AclaXegA), A. zonatus (AspzoGH12), and A. terreus (AtEglD) were studied to unveil the molecular basis for substrate specificity. Using functional assays, site-directed mutagenesis, and molecular dynamics simulations, we demonstrated that three main regions are responsible for substrate selectivity: (i) the YSG group in loop 1; (ii) the SST group in loop 2; and (iii) loop A3-B3 and neighboring residues. Functional assays and sequence alignment showed that while AclaXegA is specific to xyloglucan, AtEglD cleaves ß-glucan, and xyloglucan. However, AspzoGH12 was also shown to be promiscuous contrarily to a sequence alignment-based prediction. We find that residues Y111 and R93 in AtEglD harbor the substrate in an adequate orientation for hydrolysis in the catalytic cleft entrance and that residues Y19 in AclaXegA and Y30 in AspzoGH12 partially compensate the absence of the YSG segment, typically found in promiscuous enzymes. The results point out the multiple structural factors underlying the substrate specificity of GH12 enzymes. Biotechnol. Bioeng. 2016;113: 2577-2586. © 2016 Wiley Periodicals, Inc.

The functional properties of a xyloglucanase (GH12) of Aspergillus terreus expressed in Aspergillus nidulans may increase performance of biomass degradation.

Filamentous fungi are attractive hosts for heterologous protein expression due to their capacity to secrete large amounts of enzymes into the extracellular medium. Xyloglucanases, which specifically hydrolyze xyloglucan, have been recently applied in lignocellulosic biomass degradation and conversion in many other industrial processes. In this context, this work aimed to clone, express, and determine the functional properties of a recombinant xyloglucanase (AtXEG12) from Aspergillus terreus, and also its solid-state (SSF) and submerged (SmF) fermentation in bioreactors. The purified AtXEG12 showed optimum pH and temperature of 5.5 and 65 °C, respectively, demonstrating to be 90 % stable after 24 h of incubation at 50 °C. AtXEG12 activity increased in the presence of 2-mercaptoethanol (65 %) and Zn+2 (45 %), while Cu+2 and Ag+ ions drastically decreased its activity. A substrate assay showed, for the first time for this enzyme's family, xylanase activity. The enzyme exhibited high specificity for tamarind xyloglucan (K M 1.2 mg mL-1) and V max of 17.4 µmol min-1 mg-1 of protein. The capillary zone electrophoresis analysis revealed that AtXEG12 is an endo-xyloglucanase. The heterologous xyloglucanase secretion was greater than the production by wild-type A. terreus cultivated in SmF. On the other hand, AtXEG12 activity reached by SSF was sevenfold higher than values achieved by SmF, showing that the expression of recombinant enzymes can be significantly improved by cultivation under SSF.

Enhanced xyloglucan-specific endo-ß-1,4-glucanase efficiency in an engineered CBM44-XegA chimera.

Xyloglucan-specific endo-ß-1,4-glucanases (Xegs, EC 3.2.1.151) exhibit high catalytic specificity for ß-1,4 linkages of xyloglucan, a branched hemicellulosic polysaccharide abundant in dicot primary cell walls and present in many monocot species. In nature, GH12 Xegs are not associated with carbohydrate-binding modules (CBMs), and here, we have investigated the effect of the fusion of the xyloglucan-specific CBM44 on the structure and function of a GH12 Xeg from Aspergillus niveus (XegA). This fusion presented enhanced catalytic properties and conferred superior thermal stability on the XegA. An increased k cat (chimera, 177.03 s(-1); XegA, 144.31 s(-1)) and reduced KM (chimera, 1.30 mg mL(-1); XegA, 1.50 mg mL(-1)) resulted in a 1.3-fold increase in catalytic efficiency of the chimera over the parental XegA. Although both parental and chimeric enzymes presented catalytic optima at pH 5.5 and 60 °C, the thermostabilitiy of the chimera at 60 °C was greater than the parental XegA. Moreover, the crystallographic structure of XegA together with small-angle X-ray scattering (SAXS) and molecular dynamics simulations revealed that the spatial arrangement of the domains in the chimeric enzyme resulted in the formation of an extended binding cleft that may explain the improved kinetic properties of the CBM44-XegA chimera.

Assembling a xylanase-lichenase chimera through all-atom molecular dynamics simulations.

Multifunctional enzyme engineering can improve enzyme cocktails for emerging biofuel technology. Molecular dynamics through structure-based models (SB) is an effective tool for assessing the tridimensional arrangement of chimeric enzymes as well as for inferring the functional practicability before experimental validation. This study describes the computational design of a bifunctional xylanase-lichenase chimera (XylLich) using the xynA and bglS genes from Bacillus subtilis. In silico analysis of the average solvent accessible surface area (SAS) and the root mean square fluctuation (RMSF) predicted a fully functional chimera, with minor fluctuations and variations along the polypeptide chains. Afterwards, the chimeric enzyme was built by fusing the xynA and bglS genes. XylLich was evaluated through small-angle X-ray scattering (SAXS) experiments, resulting in scattering curves with a very accurate fit to the theoretical protein model. The chimera preserved the biochemical characteristics of the parental enzymes, with the exception of a slight variation in the temperature of operation and the catalytic efficiency (kcat/Km). The absence of substantial shifts in the catalytic mode of operation was also verified. Furthermore, the production of chimeric enzymes could be more profitable than producing a single enzyme separately, based on comparing the recombinant protein production yield and the hydrolytic activity achieved for XylLich with that of the parental enzymes.

α-(1,4)-Amylase, but not α- and ß-(1,3)-glucanases, may be responsible for the impaired growth and morphogenesis of Paracoccidioides brasiliensis induced by N-glycosylation inhibition.

The cell wall of Paracoccidioides brasiliensis, which consists of a network of polysaccharides and glycoproteins, is essential for fungal pathogenesis. We have previously reported that N-glycosylation of proteins such as N-acetyl-ß-D-glucosaminidase is required for the growth and morphogenesis of P. brasiliensis. In the present study, we investigated the influence of tunycamicin (TM)-mediated inhibition of N-linked glycosylation on α- and ß-(1,3)-glucanases and on α-(1,4)-amylase in P. brasiliensis yeast and mycelium cells. The addition of 15 µg/ml TM to the fungal cultures did not interfere with either α- or ß-(1,3)-glucanase production and secretion. Moreover, incubation with TM did not alter α- and ß-(1,3)-glucanase activity in yeast and mycelium cell extracts. In contrast, α-(1,4)-amylase activity was significantly reduced in underglycosylated yeast and mycelium extracts after exposure to TM. In spite of its importance for fungal growth and morphogenesis, N-glycosylation was not required for glucanase activities. This is surprising because these activities are directed to wall components that are crucial for fungal morphogenesis. On the other hand, N-glycans were essential for α-(1,4)-amylase activity involved in the production of malto-oligosaccharides that act as primer molecules for the biosynthesis of α-(1,3)-glucan. Our results suggest that reduced fungal α-(1,4)-amylase activity affects cell wall composition and may account for the impaired growth of underglycosylated yeast and mycelium cells.

Understanding the function of conserved variations in the catalytic loops of fungal glycoside hydrolase family 12.

Enzymes that cleave the xyloglucan backbone at unbranched glucose residues have been identified in GH families 5, 7, 12, 16, 44, and 74. Fungi produce enzymes that populate 20 of 22 families that are considered critical for plant biomass deconstruction. We searched for GH12-encoding genes in 27 Eurotiomycetes genomes. After analyzing 50 GH12-related sequences, the conserved variations of the amino acid sequences were examined. Compared to the endoglucanases, the endo-xyloglucanase-associated YSG deletion at the negative subsites of the catalytic cleft with a SST insertion at the reducing end of the substrate-binding crevice is highly conserved. In addition, a highly conserved alanine residue was identified in all xyloglucan-specific enzymes, and this residue is substituted by arginine in more promiscuous glucanases. To understand the basis for the xyloglucan specificity displayed by certain GH12 enzymes, two fungal GH12 endoglucanases were chosen for mutagenesis and functional studies: an endo-xyloglucanase from Aspergillus clavatus (AclaXegA) and an endoglucanase from A. terreus (AtEglD). Comprehensive molecular docking studies and biochemical analyses were performed, revealing that mutations at the entrance of the catalytic cleft in AtEglD result in a wider binding cleft and the alteration of the substrate-cleavage pattern, implying that a trio of residues coordinates the interactions and binding to linear glycans. The loop insertion at the crevice-reducing end of AclaXegA is critical for catalytic efficiency to hydrolyze xyloglucan. The understanding of the structural elements governing endo-xyloglucanase activity on linear and branched glucans will facilitate future enzyme modifications with potential applications in industrial biotechnology.

Development of hemicellulolytic enzyme mixtures for plant biomass deconstruction on target biotechnological applications.

An essential step in the conversion of lignocellulosic biomass to ethanol and other biorefinery products is conversion of cell wall polysaccharides into fermentable sugars by enzymatic hydrolysis. The objective of the present study was to understand the mode of action of hemicellulolytic enzyme mixtures for pretreated sugarcane bagasse (PSB) deconstruction and wheat arabinoxylan (WA) hydrolysis on target biotechnological applications. In this study, five hemicellulolytic enzymes-two endo-1,4-xylanases (GH10 and GH11), two α-L-arabinofuranosidases (GH51 and GH54), and one ß-xylosidase (GH43)-were submitted to combinatorial assays using the experimental design strategy, in order to analyze synergistic and antagonistic effects of enzyme interactions on biomass degradation. The xylooligosaccharides (XOSs) released from hydrolysis were analyzed by capillary electrophoresis and quantified by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Based on this analysis, it was possible to define which enzymatic combinations favor xylose (X1) or XOS production and thus enable the development of target biotechnological applications. Our results demonstrate that if the objective is X1 production from WA, the best enzymatic combination is GH11 + GH54 + GH43, and for xylobiose (X2) production from WA, it is best to combine GH11 + GH51. However, if the goal is to produce XOS, the five enzymes used in WA hydrolysis are important, but for PSB hydrolysis, only GH11 is sufficient. If the final objective is bioethanol production, GH11 is responsible for hydrolyzing 64.3 % of hemicellulose from PSB. This work provides a basis for further studies on enzymatic mechanisms for XOS production, and the development of more efficient and less expensive enzymatic mixtures, targeting commercially viable lignocellulosic ethanol production and other biorefinery products.

A Timeline of Biosynthetic Gene Cluster Discovery in Aspergillus fumigatus: From Characterization to Future Perspectives.

In 1999, the first biosynthetic gene cluster (BGC), synthesizing the virulence factor DHN melanin, was characterized in Aspergillus fumigatus. Since then, 19 additional BGCs have been linked to specific secondary metabolites (SMs) in this species. Here, we provide a comprehensive timeline of A. fumigatus BGC discovery and find that initial advances centered around the commonly expressed SMs where chemical structure informed rationale identification of the producing BGC (e.g., gliotoxin, fumigaclavine, fumitremorgin, pseurotin A, helvolic acid, fumiquinazoline). Further advances followed the transcriptional profiling of a ΔlaeA mutant, which aided in the identification of endocrocin, fumagillin, hexadehydroastechrome, trypacidin, and fumisoquin BGCs. These SMs and their precursors are the commonly produced metabolites in most A. fumigatus studies. Characterization of other BGC/SM pairs required additional efforts, such as induction treatments, including co-culture with bacteria (fumicycline/neosartoricin, fumigermin) or growth under copper starvation (fumivaline, fumicicolin). Finally, four BGC/SM pairs were discovered via overexpression technologies, including the use of heterologous hosts (fumicycline/neosartoricin, fumihopaside, sphingofungin, and sartorypyrone). Initial analysis of the two most studied A. fumigatus isolates, Af293 and A1160, suggested that both harbored ca. 34-36 BGCs. However, an examination of 264 available genomes of A. fumigatus shows up to 20 additional BGCs, with some strains showing considerable variations in BGC number and composition. These new BGCs present a new frontier in the future of secondary metabolism characterization in this important species.

Unveiling the crystal structure of thermostable dienelactone hydrolase exhibiting activity on terephthalate esters.

Dienelactone hydrolase (DLH) is one of numerous hydrolytic enzymes with an α/ß-hydrolase fold, which catalyze the hydrolysis of dienelactone to maleylacetate. The DLHs share remarkably similar tertiary structures and a conserved arrangement of catalytic residues. This study presents the crystal structure and comprehensive functional characterization of a novel thermostable DLH from the bacterium Hydrogenobacter thermophilus (HtDLH). The crystal structure of the HtDLH, solved at a resolution of about 1.67 Å, exhibits a canonical α/ß-hydrolase fold formed by eight ß-sheet strands in the core, with one buried α-helix and six others exposed to the solvent. The structure also confirmed the conserved catalytic triad of DHLs formed by Cys121, Asp170, and His202 residues. The HtDLH forms stable homodimers in solution. Functional studies showed that HtDLH has the expected esterase activity over esters with short carbon chains, such as p-nitrophenyl acetate, reaching optimal activity at pH 7.5 and 70 °C. Furthermore, HtDLH maintains more than 50 % of its activity even after incubation at 90 °C for 16 h. Interestingly, HtDLH exhibits catalytic activity towards polyethylene terephthalate (PET) monomers, including bis-1,2-hydroxyethyl terephthalate (BHET) and 1-(2-hydroxyethyl) 4-methyl terephthalate, as well as other aliphatic and aromatic esters. These findings associated with the lack of activity on amorphous PET indicate that HtDLH has characteristic of a BHET-degrading enzyme. This work expands our understanding of enzyme families involved in PET degradation, providing novel insights for plastic biorecycling through protein engineering, which could lead to eco-friendly solutions to reduce the accumulation of plastic in landfills and natural environments.

A Multiomics Perspective on Plant Cell Wall-Degrading Enzyme Production: Insights from the Unexploited Fungus Trichoderma erinaceum.

Trichoderma erinaceum is a filamentous fungus that was isolated from decaying sugarcane straw at a Brazilian ethanol biorefinery. This fungus shows potential as a source of plant cell wall-degrading enzymes (PCWDEs). In this study, we conducted a comprehensive multiomics investigation of T. erinaceum to gain insights into its enzymatic capabilities and genetic makeup. Firstly, we performed genome sequencing and assembly, which resulted in the identification of 10,942 genes in the T. erinaceum genome. We then conducted transcriptomics and secretome analyses to map the gene expression patterns and identify the enzymes produced by T. erinaceum in the presence of different substrates such as glucose, microcrystalline cellulose, pretreated sugarcane straw, and pretreated energy cane bagasse. Our analyses revealed that T. erinaceum highly expresses genes directly related to lignocellulose degradation when grown on pretreated energy cane and sugarcane substrates. Furthermore, our secretome analysis identified 35 carbohydrate-active enzymes, primarily PCWDEs. To further explore the enzymatic capabilities of T. erinaceum, we selected a ß-glucosidase from the secretome data for recombinant production in a fungal strain. The recombinant enzyme demonstrated superior performance in degrading cellobiose and laminaribiose compared to a well-known enzyme derived from Trichoderma reesei. Overall, this comprehensive study provides valuable insights into both the genetic patterns of T. erinaceum and its potential for lignocellulose degradation and enzyme production. The obtained genomic data can serve as an important resource for future genetic engineering efforts aimed at optimizing enzyme production from this fungus.

Plastic-degrading microbial communities reveal novel microorganisms, pathways, and biocatalysts for polymer degradation and bioplastic production.

Plastics derived from fossil fuels are used ubiquitously owing to their exceptional physicochemical characteristics. However, the extensive and short-term use of plastics has caused environmental challenges. The biotechnological plastic conversion can help address the challenges related to plastic pollution, offering sustainable alternatives that can operate using bioeconomic concepts and promote socioeconomic benefits. In this context, using soil from a plastic-contaminated landfill, two consortia were established (ConsPlastic-A and -B) displaying versatility in developing and consuming polyethylene or polyethylene terephthalate as the carbon source of nutrition. The ConsPlastic-A and -B metagenomic sequencing, taxonomic profiling, and the reconstruction of 79 draft bacterial genomes significantly expanded the knowledge of plastic-degrading microorganisms and enzymes, disclosing novel taxonomic groups associated with polymer degradation. The microbial consortium was utilized to obtain a novel Pseudomonas putida strain (BR4), presenting a striking metabolic arsenal for aromatic compound degradation and assimilation, confirmed by genomic analyses. The BR4 displays the inherent capacity to degrade polyethylene terephthalate (PET) and produce polyhydroxybutyrate (PHB) containing hydroxyvalerate (HV) units that contribute to enhanced copolymer properties, such as increased flexibility and resistance to breakage, compared with pure PHB. Therefore, BR4 is a promising strain for developing a bioconsolidated plastic depolymerization and upcycling process. Collectively, our study provides insights that may extend beyond the artificial ecosystems established during our experiments and supports future strategies for effectively decomposing and valorizing plastic waste. Furthermore, the functional genomic analysis described herein serves as a valuable guide for elucidating the genetic potential of microbial communities and microorganisms in plastic deconstruction and upcycling.

Sirtuin E deacetylase is required for full virulence of Aspergillus fumigatus.

Aspergillus fumigatus represents a public health problem due to the high mortality rate in immunosuppressed patients and the emergence of antifungal-resistant isolates. Protein acetylation is a crucial post-translational modification that controls gene expression and biological processes. The strategic manipulation of enzymes involved in protein acetylation has emerged as a promising therapeutic approach for addressing fungal infections. Sirtuins, NAD+-dependent lysine deacetylases, regulate protein acetylation and gene expression in eukaryotes. However, their role in the human pathogenic fungus A. fumigatus remains unclear. This study constructs six single knockout strains of A. fumigatus and a strain lacking all predicted sirtuins (SIRTKO). The mutant strains are viable under laboratory conditions, indicating that sirtuins are not essential genes. Phenotypic assays suggest sirtuins' involvement in cell wall integrity, secondary metabolite production, thermotolerance, and virulence. Deletion of sirE attenuates virulence in murine and Galleria mellonella infection models. The absence of SirE alters the acetylation status of proteins, including histones and non-histones, and triggers significant changes in the expression of genes associated with secondary metabolism, cell wall biosynthesis, and virulence factors. These findings encourage testing sirtuin inhibitors as potential therapeutic strategies to combat A. fumigatus infections or in combination therapy with available antifungals.