Vanillyl alcohol oxidase from Diplodia corticola: Residues Ala420 and Glu466 allow for efficient catalysis of syringyl derivatives.

Vanillyl alcohol oxidases (VAOs) belong to the 4-phenol oxidases family and are found predominantly in lignin-degrading ascomycetes. Systematical investigation of the enzyme family at the sequence level resulted in discovery and characterization of the second recombinantly produced VAO member, DcVAO, from Diplodia corticola. Remarkably high activities for 2,6-substituted substrates like 4-allyl-2,6-dimethoxy-phenol (3.5 ± 0.02 U mg-1) or 4-(hydroxymethyl)-2,6-dimethoxyphenol (6.3 ± 0.5 U mg-1) were observed, which could be attributed to a Phe to Ala exchange in the catalytic center. In order to rationalize this rare substrate preference among VAOs, we resurrected and characterized three ancestral enzymes and performed mutagenesis analyses. The results indicate that a Cys/Glu exchange was required to retain activity for É£-hydroxylations and shifted the acceptance towards benzyl ethers (up to 4.0 ± 0.1 U mg-1). Our findings contribute to the understanding of the functionality of VAO enzyme group, and with DcVAO, we add a new enzyme to the repertoire of ether cleaving biocatalysts.

Discovery of non-squalene triterpenes.

All known triterpenes are generated by triterpene synthases (TrTSs) from squalene or oxidosqualene1. This approach is fundamentally different from the biosynthesis of short-chain (C10-C25) terpenes that are formed from polyisoprenyl diphosphates2-4. In this study, two fungal chimeric class I TrTSs, Talaromyces verruculosus talaropentaene synthase (TvTS) and Macrophomina phaseolina macrophomene synthase (MpMS), were characterized. Both enzymes use dimethylallyl diphosphate and isopentenyl diphosphate or hexaprenyl diphosphate as substrates, representing the first examples, to our knowledge, of non-squalene-dependent triterpene biosynthesis. The cyclization mechanisms of TvTS and MpMS and the absolute configurations of their products were investigated in isotopic labelling experiments. Structural analyses of the terpene cyclase domain of TvTS and full-length MpMS provide detailed insights into their catalytic mechanisms. An AlphaFold2-based screening platform was developed to mine a third TrTS, Colletotrichum gloeosporioides colleterpenol synthase (CgCS). Our findings identify a new enzymatic mechanism for the biosynthesis of triterpenes and enhance understanding of terpene biosynthesis in nature.

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

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

Cloning and Characterization of Three Novel Enzymes Responsible for the Detoxification of Zearalenone.

Zearalenone is a common mycotoxin contaminant in cereals that causes severe economic losses and serious risks to health of human and animals. Many strategies have been devised to degrade ZEN and keep food safe. The hydrolase ZHD101 from Clonostachys rosea, which catalyzes the hydrolytic degradation of ZEN, has been studied widely. In the current research, three new enzymes that have the capacity to detoxify ZEN were identified, namely CLA, EXO, and TRI, showing 61%, 63%, and 97% amino acids identities with ZHD101, respectively. Three coding genes was expressed as heterologous in Escherichia coli BL21. Through biochemical analysis, the purified recombinant CLA, EXO, TRI, and ZHD101 exhibited high activities of degrading ZEN with the specific activity of 114.8 U/mg, 459.0 U/mg, 239.8 U/mg, and 242.8 U/mg. The optimal temperatures of CLA, EXO, TRI, and ZHD101 were 40 °C, 40 °C, 40 °C, and 45 °C, and their optimum pH were 7.0, 9.0, 9.5, and 9.0, respectively. Our study demonstrated that the novel enzymes CLA, EXO, and TRI possessed high ability to degrade ZEN from the model solutions and could be the promising candidates for ZEN detoxification in practical application.

Phenolic degradation by catechol dioxygenases is associated with pathogenic fungi with a necrotrophic lifestyle in the Ceratocystidaceae.

Fungal species of the Ceratocystidaceae grow on their host plants using a variety of different lifestyles, from saprophytic to highly pathogenic. Although many genomes of fungi in the Ceratocystidaceae are publicly available, it is not known how the genes that encode catechol dioxygenases (CDOs), enzymes involved in the degradation of phenolic plant defense compounds, differ among members of the Ceratocystidaceae. The aim of this study was therefore to identify and characterize the genes encoding CDOs in the genomes of Ceratocystidaceae representatives. We found that genes encoding CDOs are more abundant in pathogenic necrotrophic species of the Ceratocystidaceae and less abundant in saprophytic species. The loss of the CDO genes and the associated 3-oxoadipate catabolic pathway appears to have occurred in a lineage-specific manner. Taken together, this study revealed a positive association between CDO gene copy number and fungal lifestyle in Ceratocystidaceae representatives.

Gene deletion and constitutive expression of the pectate lyase gene 1 (MoPL1) lead to diminished virulence of Magnaporthe oryzae.

Phytopathogenic fungi are known to secrete specific proteins which act as virulence factors and promote host colonization. Some of them are enzymes with plant cell wall degradation capability, like pectate lyases (Pls). In this work, we examined the involvement of Pls in the infection process of Magnaporthe oryzae, the causal agent of rice blast disease. From three Plgenes annotated in the M. oryzae genome, only transcripts of MoPL1 considerably accumulated during the infection process with a peak at 72 h post inoculation. Both, gene deletion and a constitutive expression of MoPL1 in M. oryzae led to a significant reduction in virulence. By contrast, mutants that constitutively expressed an enzymatic inactive version of MoPl1 did not differ in virulence compared to the wild type isolate. This indicates that the enzymatic activity of MoPl1 is responsible for diminished virulence, which is presumably due to degradation products recognized as danger associated molecular patterns (DAMPs), which strengthen the plant immune response. Microscopic analysis of infection sites pointed to an increased plant defense response. Additionally, MoPl1 tagged with mRFP, and not the enzymatic inactive version, focally accumulated in attacked plant cells beneath appressoria and at sites where fungal hyphae transverse from one to another cell. These findings shed new light on the role of pectate lyases during tissue colonization in the necrotrophic stage of M. oryzae's life cycle.

Phospholipase C (AoPLC2) regulates mycelial development, trap morphogenesis, and pathogenicity of the nematode-trapping fungus Arthrobotrys oligospora.

AIMS: Phospholipase C (PLC) is a hydrolase involved in signal transduction in eukaryotic cells. This study aimed to understand the function of PLC in the nematode-trapping fungus Arthrobotrys oligospora. METHODS AND RESULTS: Orthologous PLC (AoPLC2) of A. oligospora was functionally analysed using gene disruption and multi-phenotypic analysis. Disrupting Aoplc2 caused a deformation of partial hyphal cells (about 10%) and conidia (about 50%), decreased the number of nuclei in both conidia and hyphal cells, and increased the accumulation of lipid droplets. Meanwhile, the sporulation-related genes fluG and abaA were downregulated in ΔAoplc2 mutants than in the wild-type strain. Moreover, ΔAoplc2 mutants were more sensitive to osmotic stressors. Importantly, the number of traps, electron-dense bodies in traps, and nematicidal activity of ΔAoplc2 mutants were reduced, and the shape of the traps was deformed. In addition, AoPLC2 was involved in the biosynthesis of secondary metabolites in A. oligospora. CONCLUSIONS: AoPLC2 plays an important role in the development of hyphae, spores, and cell nuclei, responses to stress, formation of traps, and predation of nematodes in A. oligospora. SIGNIFICANCE AND IMPACT OF STUDY: This study reveals the various functions of phospholipase C and elucidates the regulation of trap morphogenesis in nematode-trapping fungi.

Heterologous Expression of Macrollins from Phytopathogenic Macrophomina phaseolina Revealed a Cytochrome P450 Mono-oxygenase in the Biosynthesis of ß-Hydroxyl Tetramic Acid.

Macrophomina phaseolina (M. phaseolina) is a crucial pathogenic fungus that can cause severe charcoal rot in economic crops and other plants. In this study, four new natural products, macrollins A-D, were discovered from M. phaseolina by the strategy of heterologous expression. To our knowledge, macrollins are the first reported polyketide-amino acid hybrids from the plant pathogen. Heterologous expression and in vitro reactions revealed a cytochrome P450 mono-oxygenase (MacC) catalyzing the hydroxylation at the ß-carbon of tetramic acid molecules, which is different from P450s leading to the ring expansion in the biosynthesis of fungal 2-pyridones. Phylogenetic analysis of P450s involved in the fungal polyketide-amino acid hybrids showed that MacC was not classified in any known clades. The putative oxidative mechanisms of the P450s and the biosynthetic pathway of macrollins were also proposed.

Molecular characterization and overexpression of the difenoconazole resistance gene CYP51 in Lasiodiplodia theobromae field isolates.

Stem-end rot (SER) caused by Lasiodiplodia theobromae is an important disease of mango in China. Demethylation inhibitor (DMI) fungicides are widely used for disease control in mango orchards. The baseline sensitivity to difenoconazole of 138 L. theobromae isolates collected from mango in the field in 2019 was established by the mycelial growth rate method. The cross-resistance to six site-specific fungicides with different modes of action were investigated using 20 isolates randomly selected. The possible mechanism for L. theobromae resistance to difenoconazole was preliminarily determined through gene sequence alignment and quantitative real-time PCR analysis. The results showed that the EC50 values of 138 L. theobromae isolates to difenoconazole ranged from 0.01 to 13.72 µg/mL. The frequency of difenoconazole sensitivity formed a normal distribution curve when the outliers were excluded. Difenoconazole showed positive cross-resistance only with the DMI tebuconazole but not with non-DMI fungicides carbendazim, pyraclostrobin, fludioxonil, bromothalonil, or iprodione. Some multifungicide-resistant isolates of L. theobromae were found. Two amino acid substitutions (E209k and G207A) were found in the CYP51 protein, but they were unlikely to be related to the resistance phenotype. There was no alteration in the promoter region of the CYP51 gene. However, difenoconazole significantly increased the expression of the CYP51 gene in the resistant isolates compared to the susceptible isolates. These results are vital to develop effective mango disease management strategies to avoid the development of further resistance.

Secretome analysis identified extracellular superoxide dismutase and catalase of Macrophomina phaseolina.

Macrophomina phaseolina, a necrotrophic fungal pathogen is known to cause charcoal rot disease in food crops, pulse crops, oil crops and cotton and fibre crops. Necrotrophic fungi survive on dead plant tissue. It is well known that reactive oxygen species (ROS) are produced by the host plant during plant-pathogen interaction. However, it is still unclear how M. phaseolina can overcome the ROS-induced cellular damage. To mimic the invasion of M. phaseolina inside the plant cell wall, we developed solid substrate fermentation where M. phaseolina spore suspension was inoculated on a wheat bran bed and incubated for vegetative growth. To analyse the secretome of M. phaseolina after different day interval, its secretory material was collected and concentrated. Both superoxide dismutase (SOD) and catalase were detected in the secretome by zymogram. The presence of SOD and catalase was further confirmed by liquid chromatography based mass spectrometry. The physicochemical properties of M. phaseolina catalase in terms of stability towards pH, temperature, metal ions and chaotropic agent and inhibitors indicated its fitness at different environmental conditions. Apart from the production of catalase in SSF, the studies on this particular microorganism may also have significance in necrotrophic fungal pathogen and their susceptible host plant interaction.

Kinetic and Structural Characterization of Sialidases (Kdnases) from Ascomycete Fungal Pathogens.

Sialidases catalyze the release of sialic acid from the terminus of glycan chains. We previously characterized the sialidase from the opportunistic fungal pathogen, Aspergillus fumigatus, and showed that it is a Kdnase. That is, this enzyme prefers 3-deoxy-d-glycero-d-galacto-non-2-ulosonates (Kdn glycosides) as the substrate compared to N-acetylneuraminides (Neu5Ac). Here, we report characterization and crystal structures of putative sialidases from two other ascomycete fungal pathogens, Aspergillus terreus (AtS) and Trichophyton rubrum (TrS). Unlike A. fumigatus Kdnase (AfS), hydrolysis with the Neu5Ac substrates was negligible for TrS and AtS; thus, TrS and AtS are selective Kdnases. The second-order rate constant for hydrolysis of aryl Kdn glycosides by AtS is similar to that by AfS but 30-fold higher by TrS. The structures of these glycoside hydrolase family 33 (GH33) enzymes in complex with a range of ligands for both AtS and TrS show subtle changes in ring conformation that mimic the Michaelis complex, transition state, and covalent intermediate formed during catalysis. In addition, they can aid identification of important residues for distinguishing between Kdn and Neu5Ac substrates. When A. fumigatus, A. terreus, and T. rubrum were grown in chemically defined media, Kdn was detected in mycelial extracts, but Neu5Ac was only observed in A. terreus or T. rubrum extracts. The C8 monosaccharide 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) was also identified in A. fumigatus and T. rubrum samples. A fluorescent Kdn probe was synthesized and revealed the localization of AfS in vesicles at the cell surface.

A Histone Deacetylase, Magnaporthe oryzae RPD3, Regulates Reproduction and Pathogenic Development in the Rice Blast Fungus.

Acetylation and deacetylation of histones are key epigenetic mechanisms for gene regulation in response to environmental stimuli. RPD3 is a well-conserved class I histone deacetylase (HDAC) that is involved in diverse biological processes. Here, we investigated the roles of the Magnaporthe oryzae RPD3 (MoRPD3) gene, an ortholog of Saccharomyces cerevisiae Rpd3, during development and pathogenesis in the model plant-pathogenic fungus Magnaporthe oryzae. We demonstrated that the MoRPD3 gene is able to functionally complement the yeast Rpd3 deletion mutant despite the C-terminal extension of the MoRPD3 protein. MoRPD3 localizes primarily to the nuclei of vegetative hyphae, asexual spores, and invasive hyphae. Deletion of MoRPD3 appears to be lethal. Depletion of MoRPD3 transcripts via gene silencing (MoRPD3kd, where "kd" stands for "knockdown") has opposing effects on asexual and sexual reproduction. Although conidial germination and appressorium formation rates of the mutants were almost comparable to those of the wild type, in-depth analysis revealed that the appressoria of mutants are smaller than those of the wild type. Furthermore, the MoRPD3kd strain shows a significant reduction in pathogenicity, which can be attributed to the delay in appressorium-mediated penetration and impaired invasive growth. Interestingly, MoRPD3 does not regulate potassium transporters, as shown for Rpd3 of S. cerevisiae. However, it functioned in association with the target of rapamycin (TOR) kinase pathway, resulting in the dependency of appressorium formation on hydrophilic surfaces and on TOR's inhibition by MoRPD3. Taken together, our results uncovered distinct and evolutionarily conserved roles of MoRPD3 in regulating fungal reproduction, infection-specific development, and virulence. IMPORTANCE RPD3 is an evolutionarily conserved class I histone deacetylase (HDAC) that plays a pivotal role in diverse cellular processes. In filamentous fungal pathogens, abrogation of the gene encoding RPD3 results in either lethality or severe growth impairment, making subsequent genetic analyses challenging. Magnaporthe oryzae is a causal agent of rice blast disease, which is responsible for significant annual yield losses in rice production. Here, we characterized the RPD3 gene of M. oryzae (MoRPD3) in unprecedented detail using a gene-silencing approach. We provide evidence that MoRPD3 is a bona fide HDAC regulating fungal reproduction and pathogenic development by potentially being involved in the TOR-mediated signaling pathway. To the best of our knowledge, this work is the most comprehensive genetic dissection of RPD3 in filamentous fungal pathogens. Our work extends and deepens our understanding of how an epigenetic factor is implicated in the development and virulence of fungal pathogens of plants.

Green Oxidation of Amines by a Novel Cold-Adapted Monoamine Oxidase MAO P3 from Psychrophilic Fungi Pseudogymnoascus sp. P3.

The use of monoamine oxidases (MAOs) in amine oxidation is a great example of how biocatalysis can be applied in the agricultural or pharmaceutical industry and manufacturing of fine chemicals to make a shift from traditional chemical synthesis towards more sustainable green chemistry. This article reports the screening of fourteen Antarctic fungi strains for MAO activity and the discovery of a novel psychrozyme MAOP3 isolated from the Pseudogymnoascus sp. P3. The activity of the native enzyme was 1350 ± 10.5 U/L towards a primary (n-butylamine) amine, and 1470 ± 10.6 U/L towards a secondary (6,6-dimethyl-3-azabicyclohexane) amine. MAO P3 has the potential for applications in biotransformations due to its wide substrate specificity (aliphatic and cyclic amines, pyrrolidine derivatives). The psychrozyme operates at an optimal temperature of 30 °C, retains 75% of activity at 20 °C, and is rather thermolabile, which is beneficial for a reduction in the overall costs of a bioprocess and offers a convenient way of heat inactivation. The reported biocatalyst is the first psychrophilic MAO; its unique biochemical properties, substrate specificity, and effectiveness predispose MAO P3 for use in environmentally friendly, low-emission biotransformations.

Cytotoxic function of xylanase VdXyn4 in the plant vascular wilt pathogen Verticillium dahliae.

Phytopathogen xylanases play critical roles in pathogenesis, likely due to their ability to degrade plant structural barriers and manipulate host immunity. As an invader of plant xylem vessels, the fungus Verticillium dahliae is thought to deploy complex cell wall degrading enzymes. Comparative genomics analyses revealed that the V. dahliae genome encodes a family of six xylanases, each possessing a glycosyl hydrolase 11 domain, but the functions of these enzymes are undetermined. Characterizing gene deletion mutants revealed that only V. dahliae xylanase 4 (VdXyn4) degraded the plant cell wall and contributed to the virulence of V. dahliae. VdXyn4 displayed cytotoxic activity and induced a necrosis phenotype during the late stages of infection, leading to vein and petiole collapse that depended on the enzyme simultaneously localizing to nuclei and chloroplasts. The internalization of VdXyn4 was in conjunction with that of the plasma membrane complexLeucine-rich repeat (LRR)-receptor-like kinase suppressor of BIR1-1 (SOBIR1)/LRR-RLK BRI1-associated kinase-1 (BAK1), but we could not rule out the possibility that VdXyn4 may also act as an apoplastic effector. Immune signaling (in the SA-JA pathways) induced by VdXyn4 relative to that induced by known immunity effectors was substantially delayed. While cytotoxic activity could be partially suppressed by known effectors, they failed to impede necrosis in Nicotiana benthamiana. Thus, unlike typical effectors, cytotoxicity of VdXyn4 plays a crucial intracellular role at the late stages of V. dahliae infection and colonization, especially following pathogen entry into the xylem; this cytotoxic activity is likely conserved in the corresponding enzyme families in plant vascular pathogens.

Appressorium-mediated plant infection by Magnaporthe oryzae is regulated by a Pmk1-dependent hierarchical transcriptional network.

Rice blast is a devastating disease caused by the fungal pathogen Magnaporthe oryzae that threatens rice production around the world. The fungus produces a specialized infection cell, called the appressorium, that enables penetration through the plant cell wall in response to surface signals from the rice leaf. The underlying biology of plant infection, including the regulation of appressorium formation, is not completely understood. Here we report the identification of a network of temporally coregulated transcription factors that act downstream of the Pmk1 mitogen-activated protein kinase pathway to regulate gene expression during appressorium-mediated plant infection. We show that this tiered regulatory mechanism involves Pmk1-dependent phosphorylation of the Hox7 homeobox transcription factor, which regulates genes associated with induction of major physiological changes required for appressorium development-including cell-cycle control, autophagic cell death, turgor generation and melanin biosynthesis-as well as controlling a additional set of virulence-associated transcription factor-encoding genes. Pmk1-dependent phosphorylation of Mst12 then regulates gene functions involved in septin-dependent cytoskeletal re-organization, polarized exocytosis and effector gene expression, which are necessary for plant tissue invasion. Identification of this regulatory cascade provides new potential targets for disease intervention.

First Dye-Decolorizing Peroxidase from an Ascomycetous Fungus Secreted by Xylaria grammica.

BACKGROUND: Fungal DyP-type peroxidases have so far been described exclusively for basidiomycetes. Moreover, peroxidases from ascomycetes that oxidize Mn2+ ions are yet not known. METHODS: We describe here the physicochemical, biocatalytic, and molecular characterization of a DyP-type peroxidase (DyP, EC 1.11.1.19) from an ascomycetous fungus. RESULTS: The enzyme oxidizes classic peroxidase substrates such as 2,6-DMP but also veratryl alcohol and notably Mn2+ to Mn3+ ions, suggesting a physiological function of this DyP in lignin modification. The KM value (49 µM) indicates that Mn2+ ions bind with high affinity to the XgrDyP protein but their subsequent oxidation into reactive Mn3+ proceeds with moderate efficiency compared to MnPs and VPs. Mn2+ oxidation was most effective at an acidic pH (between 4.0 and 5.0) and a hypothetical surface exposed an Mn2+ binding site comprising three acidic amino acids (two aspartates and one glutamate) could be localized within the hypothetical XgrDyP structure. The oxidation of Mn2+ ions is seemingly supported by four aromatic amino acids that mediate an electron transfer from the surface to the heme center. CONCLUSIONS: Our findings shed new light on the possible involvement of DyP-type peroxidases in lignocellulose degradation, especially by fungi that lack prototypical ligninolytic class II peroxidases.

Insights into the Lignocellulose-Degrading Enzyme System of Humicola grisea var. thermoidea Based on Genome and Transcriptome Analysis.

Humicola grisea var. thermoidea is a thermophilic ascomycete and important enzyme producer that has an efficient enzymatic system with a broad spectrum of thermostable carbohydrate-active (CAZy) enzymes. These enzymes can be employed in lignocellulose biomass deconstruction and other industrial applications. In this work, the genome of H. grisea var. thermoidea was sequenced. The acquired sequence reads were assembled into a total length of 28.75 Mbp. Genome features correlate with what was expected for thermophilic Sordariomycetes. The transcriptomic data showed that sugarcane bagasse significantly upregulated genes related to primary metabolism and polysaccharide deconstruction, especially hydrolases, at both pH 5 and pH 8. However, a number of exclusive and shared genes between the pH values were found, especially at pH 8. H. grisea expresses an average of 211 CAZy enzymes (CAZymes), which are capable of acting in different substrates. The top upregulated genes at both pH values represent CAZyme-encoding genes from different classes, including acetylxylan esterase, endo-1,4-ß-mannosidase, exoglucanase, and endoglucanase genes. For the first time, the arsenal that the thermophilic fungus H. grisea var. thermoidea possesses to degrade the lignocellulosic biomass is shown. Carbon source and pH are of pivotal importance in regulating gene expression in this organism, and alkaline pH is a key regulatory factor for sugarcane bagasse hydrolysis. This work paves the way for the genetic manipulation and robust biotechnological applications of this fungus. IMPORTANCE Most studies regarding the use of fungi as enzyme producers for biomass deconstruction have focused on mesophile species, whereas the potential of thermophiles has been evaluated less. This study revealed, through genome and transcriptome analyses, the genetic repertoire of the biotechnological relevant thermophile fungus Humicola grisea. Comparative genomics helped us to further understand the biology and biotechnological potential of H. grisea. The results demonstrate that this fungus possesses an arsenal of carbohydrate-active (CAZy) enzymes to degrade the lignocellulosic biomass. Indeed, it expresses more than 200 genes encoding CAZy enzymes when cultivated in sugarcane bagasse. Carbon source and pH are key factors for regulating the gene expression in this organism. This work shows, for the first time, the great potential of H. grisea as an enzyme producer and a gene donor for biotechnological applications and provides the base for the genetic manipulation and robust biotechnological applications of this fungus.

Comparative molecular evolution of chitinases in ascomycota with emphasis on mycoparasitism lifestyle.

Chitinases are involved in multiple aspects of fungal life cycle, such as cell wall remodelling, chitin degradation and mycoparasitism lifestyle. To improve our knowledge of the chitinase molecular evolution of Ascomycota, the gene family of 72 representatives of this phylum was identified and subjected to phylogenetic, evolution trajectory and selective pressure analyses. Phylogenetic analysis showed that the chitinase gene family size and enzyme types varied significantly, along with species evolution, especially for groups B and C. In addition, two new subgroups, C3 and C4, are recognized in group C chitinases. Random birth and death testing indicated that gene expansion and contraction occurred in most of the taxa, particularly for species in the order Hypocreales (class Sordariomycetes). From an enzyme function point of view, we speculate that group A chitinases are mainly involved in species growth and development, while the expansion of genes in group B chitinases is related to fungal mycoparasitic and entomopathogenic abilities, and, to a certain extent, the expansion of genes in group C chitinases seems to be correlated with the host range broadening of some plant-pathogenic fungi in Sordariomycetes. Further selection pressure testing revealed that chitinases and the related amino acid sites were under positive selection in the evolutionary history, especially at the nodes sharing common ancestors and the terminal branches of Hypocreales. These results give a reasonable explanation for the size and function differences of chitinase genes among ascomycetes, and provide a scientific basis for understanding the evolutionary trajectories of chitinases, particularly that towards a mycoparasitic lifestyle.

Novel Cold-Adapted Recombinant Laccase KbLcc1 from Kabatiella bupleuri G3 IBMiP as a Green Catalyst in Biotransformation.

Cold-adapted enzymes are useful tools in the organic syntheses conducted in mixed aqueous-organic or non-aqueous solvents due to their molecular flexibility that stabilizes the proteins in low water activity environments. A novel psychrophilic laccase gene from Kabatiella bupleuri, G3 IBMiP, was spliced by Overlap-Extension PCR (OE-PCR) and expressed in Pichia pastoris. Purified recombinant KbLcc1 laccase has an optimal temperature of 30 °C and pH of 3.5, 5.5, 6.0, and 7.0 in the reaction with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), guaiacol, sinapic acid, and syringaldazine, respectively. Moreover, laccase KbLcc1 is highly thermolabile, as it loses 40% of activity after 30 min at 40 °C and is inactivated at 50 °C after the same period of incubation. The new enzyme remained active with 1 mM of Ni2+, Cu2+, Mn2+, and Zn2+ and with 2 mM of Co2+, Ca2+, and Mg2+, but Fe2+ greatly inhibited the laccase activity. Moreover, 1% ethanol had no impact on KbLcc1, although acetone and ethyl acetate decreased the laccase activity. The presence of hexane (40%, v/v) caused a 58% increase in activity. Laccase KbLcc1 could be applied in the decolorization of synthetic dyes and in the biotransformation of ferulic acid to vanillin. After 5 days of reaction at 20 °C, pH 3.5, with 1 mM ABTS as a mediator, the vanillin concentration was 21.9 mg/L and the molar yield of transformation reached 14.39%.

Hydroxybenzoate hydroxylase genes underlying protocatechuic acid production in Valsa mali are required for full pathogenicity in apple trees.

Valsa mali is the causative agent of apple tree valsa canker, which causes significant losses in apple production. It produces various toxic compounds that kill plant cells, facilitating infection. Among these, protocatechuic acid exhibits the highest phytotoxic activity. However, those genes involved in toxin production have not been studied. In this study we identified four hydroxybenzoate hydroxylase genes (VmHbh1, VmHbh2, VmHbh3, and VmHbh4) from the transcriptome of V. mali. The VmHbh protein had high enzymatic activities of hydroxybenzoate hydroxylase, which could convert 4-hydroxybenzoate to protocatechuic acid. These four VmHbh genes all had highly elevated transcript levels during the V. mali infection process, especially VmHbh1 and VmHbh4, with 26.0- and 53.4-fold increases, respectively. Mutants of the four genes were generated to study whether VmHbhs are required for V. mali pathogenicity. Of the four genes, the VmHbh1 and VmHbh4 deletion mutants considerably attenuated V. mali virulence in apple leaves and in twigs, coupled with much reduced toxin levels. The VmHbh2 and VmHbh3 deletion mutants promoted the transcript levels of the other VmHbhs, suggesting functional redundancies of VmHbhs in V. mali virulence. The results provide insights into the functions of VmHbhs in the production of protocatechuic acid by V. mali during its infection of apple trees.