Construction of an enzyme-constrained metabolic network model for Myceliophthora thermophila using machine learning-based kcat data.

BACKGROUND: Genome-scale metabolic models (GEMs) serve as effective tools for understanding cellular phenotypes and predicting engineering targets in the development of industrial strain. Enzyme-constrained genome-scale metabolic models (ecGEMs) have emerged as a valuable advancement, providing more accurate predictions and unveiling new engineering targets compared to models lacking enzyme constraints. In 2022, a stoichiometric GEM, iDL1450, was reconstructed for the industrially significant fungus Myceliophthora thermophila. To enhance the GEM's performance, an ecGEM was developed for M. thermophila in this study. RESULTS: Initially, the model iDL1450 underwent refinement and updates, resulting in a new version named iYW1475. These updates included adjustments to biomass components, correction of gene-protein-reaction (GPR) rules, and a consensus on metabolites. Subsequently, the first ecGEM for M. thermophila was constructed using machine learning-based kcat data predicted by TurNuP within the ECMpy framework. During the construction, three versions of ecGEMs were developed based on three distinct kcat collection methods, namely AutoPACMEN, DLKcat and TurNuP. After comparison, the ecGEM constructed using TurNuP-predicted kcat values performed better in several aspects and was selected as the definitive version of ecGEM for M. thermophila (ecMTM). Comparing ecMTM to iYW1475, the solution space was reduced and the growth simulation results more closely resembled realistic cellular phenotypes. Metabolic adjustment simulated by ecMTM revealed a trade-off between biomass yield and enzyme usage efficiency at varying glucose uptake rates. Notably, hierarchical utilization of five carbon sources derived from plant biomass hydrolysis was accurately captured and explained by ecMTM. Furthermore, based on enzyme cost considerations, ecMTM successfully predicted reported targets for metabolic engineering modification and introduced some new potential targets for chemicals produced in M. thermophila. CONCLUSIONS: In this study, the incorporation of enzyme constraint to iYW1475 not only improved prediction accuracy but also broadened the model's applicability. This research demonstrates the effectiveness of integrating of machine learning-based kcat data in the construction of ecGEMs especially in situations where there is limited measured enzyme kinetic parameters for a specific organism.

Structural and molecular insights into a bifunctional glycoside hydrolase 30 xylanase specific to glucuronoxylan.

Glycoside hydrolase (GH) 30 family xylanases are enzymes of biotechnological interest due to their capacity to degrade recalcitrant hemicelluloses, such as glucuronoxylan (GX). This study focuses on a subfamily 7 GH30, TtXyn30A from Thermothelomyces thermophilus, which acts on GX in an "endo" and "exo" mode, releasing methyl-glucuronic acid branched xylooligosaccharides (XOs) and xylobiose, respectively. The crystal structure of inactive TtXyn30A in complex with 23-(4-O-methyl-α-D-glucuronosyl)-xylotriose (UXX), along with biochemical analyses, corroborate the implication of E233, previously identified as alternative catalytic residue, in the hydrolysis of decorated xylan. At the -1 subsite, the xylose adopts a distorted conformation, indicative of the Michaelis complex of TtXyn30AEE with UXX trapped in the semi-functional active site. The most significant structural rearrangements upon substrate binding are observed at residues W127 and E233. The structures with neutral XOs, representing the "exo" function, clearly show the nonspecific binding at aglycon subsites, contrary to glycon sites, where the xylose molecules are accommodated via multiple interactions. Last, an unproductive ligand binding site is found at the interface between the catalytic and the secondary ß-domain which is present in all GH30 enzymes. These findings improve current understanding of the mechanism of bifunctional GH30s, with potential applications in the field of enzyme engineering.

Genome and secretome insights: unravelling the lignocellulolytic potential of Myceliophthora verrucosa for enhanced hydrolysis of lignocellulosic biomass.

Lignocellulolytic enzymes from a novel Myceliophthora verrucosa (5DR) strain was found to potentiate the efficacy of benchmark cellulase during saccharification of acid/alkali treated bagasse by ~ 2.24 fold, indicating it to be an important source of auxiliary enzymes. The De-novo sequencing and analysis of M. verrucosa genome (31.7 Mb) revealed to encode for 7989 putative genes, representing a wide array of CAZymes (366) with a high proportions of auxiliary activity (AA) genes (76). The LC/MS QTOF based secretome analysis of M. verrucosa showed high abundance of glycosyl hydrolases and AA proteins with cellobiose dehydrogenase (CDH) (AA8), being the most prominent auxiliary protein. A gene coding for lytic polysaccharide monooxygenase (LPMO) was expressed in Pichia pastoris and CDH produced by M. verrucosa culture on rice straw based solidified medium were purified and characterized. The mass spectrometry of LPMO catalyzed hydrolytic products of avicel showed the release of both C1/C4 oxidized products, indicating it to be type-3. The lignocellulolytic cocktail comprising of in-house cellulase produced by Aspergillus allahabadii strain spiked with LPMO & CDH exhibited enhanced and better hydrolysis of mild alkali deacetylated (MAD) and unwashed acid pretreated rice straw slurry (UWAP), when compared to Cellic CTec3 at high substrate loading rate.

Histone binding of ASF1 is required for fruiting body development but not for genome stability in the filamentous fungus Sordaria macrospora.

IMPORTANCE: Histone chaperones are proteins that are involved in nucleosome assembly and disassembly and can therefore influence all DNA-dependent processes including transcription, DNA replication, and repair. ASF1 is a histone chaperone that is conserved throughout eukaryotes. In contrast to most other multicellular organisms, a deletion mutant of asf1 in the fungus Sordaria macrospora is viable; however, the mutant is sterile. In this study, we could show that the histone-binding ability of ASF1 is required for fertility in S. macrospora, whereas the function of ASF1 in maintenance of genome stability does not require histone binding. We also showed that the histone modifications H3K27me3 and H3K56ac are misregulated in the Δasf1 mutant. Furthermore, we identified a large duplication on chromosome 2 of the mutant strain that is genetically linked to the Δasf1 allele present on chromosome 6, suggesting that viability of the mutant might depend on the presence of the duplicated region.

Development of an efficient protein expression system in the thermophilic fungus Myceliophthora thermophila.

BACKGROUND: Thermophilic fungus Myceliophthora thermophila has been widely used in industrial applications due to its ability to produce various enzymes. However, the lack of an efficient protein expression system has limited its biotechnological applications. RESULTS: In this study, using a laccase gene reporting system, we developed an efficient protein expression system in M. thermophila through the selection of strong constitutive promoters, 5'UTRs and signal peptides. The expression of the laccase was confirmed by enzyme activity assays. The results showed that the Mtpdc promoter (Ppdc) was able to drive high-level expression of the target protein in M. thermophila. Manipulation of the 5'UTR also has significant effects on protein expression and secretion. The best 5'UTR (NCA-7d) was identified. The transformant containing the laccase gene under the Mtpdc promoter, NCA-7d 5'UTR and its own signal peptide with the highest laccase activity (1708 U/L) was obtained. In addition, the expression system was stable and could be used for the production of various proteins, including homologous proteins like MtCbh-1, MtGh5-1, MtLPMO9B, and MtEpl1, as well as a glucoamylase from Trichoderma reesei. CONCLUSIONS: An efficient protein expression system was established in M. thermophila for the production of various proteins. This study provides a valuable tool for protein production in M. thermophila and expands its potential for biotechnological applications.

Genome-scale phylogeny and comparative genomics of the fungal order Sordariales.

The order Sordariales is taxonomically diverse, and harbours many species with different lifestyles and large economic importance. Despite its importance, a robust genome-scale phylogeny, and associated comparative genomic analysis of the order is lacking. In this study, we examined whole-genome data from 99 Sordariales, including 52 newly sequenced genomes, and seven outgroup taxa. We inferred a comprehensive phylogeny that resolved several contentious relationships amongst families in the order, and cleared-up intrafamily relationships within the Podosporaceae. Extensive comparative genomics showed that genomes from the three largest families in the dataset (Chaetomiaceae, Podosporaceae and Sordariaceae) differ greatly in GC content, genome size, gene number, repeat percentage, evolutionary rate, and genome content affected by repeat-induced point mutations (RIP). All genomic traits showed phylogenetic signal, and ancestral state reconstruction revealed that the variation of the properties stems primarily from within-family evolution. Together, the results provide a thorough framework for understanding genome evolution in this important group of fungi.

Construction and characterization of a Myceliophthora thermophila lytic polysaccharide monooxygenase mutant S174C/A93C with improved thermostability.

Lytic polysaccharide monooxygenases (LPMOs) can oxidatively cleave the glycosidic bonds of crystalline polysaccharides, providing more accessible sites for polysaccharide hydrolases and promoting efficient conversion of biomass. In order to promote industrial applications of LPMOs, the stability of an LPMO of Myceliophthora thermophila C1 (MtC1LPMO) was improved by adding disulfide bonds in this study. Firstly, the structural changes of wild-type (WT) MtC1LPMO at different temperatures were explored using molecular dynamics simulations, and eight mutants were selected by combining the predicted results from Disulfide by Design (DBD), Multi agent stability prediction upon point mutations (Maestro) and Bridge disulfide (BridgeD) websites. Then, the enzymatic properties of the different mutants were determined after their expression and purification, and the mutant S174C/A93C with the highest thermal stability was obtained. The specific activities of unheated S174C/A93C and WT were 160.6 ± 1.7 U/g and 174.8 ± 7.5 U/g, respectively, while those of S174C/A93C and WT treated at 70 °C for 4 h were 77.7 ± 3.4 U/g and 46.1 ± 0.4 U/g, respectively. The transition midpoint temperature of S174C/A93C was 2.7 °C higher than that of WT. The conversion efficiency of S174C/A93C for both microcrystalline cellulose and corn straw was about 1.5 times higher than that of WT. Finally, molecular dynamics simulations revealed that the introduction of disulfide bonds increased the ß-sheet content of the H1-E34 region, thus improving the rigidity of the protein. Therefore, the overall structural stability of S174C/A93C was improved, which in turn improved its thermal stability.

Stepwise recombination suppression around the mating-type locus in an ascomycete fungus with self-fertile spores.

Recombination is often suppressed at sex-determining loci in plants and animals, and at self-incompatibility or mating-type loci in plants and fungi. In fungal ascomycetes, recombination suppression around the mating-type locus is associated with pseudo-homothallism, i.e. the production of self-fertile dikaryotic sexual spores carrying the two opposite mating types. This has been well studied in two species complexes from different families of Sordariales: Podospora anserina and Neurospora tetrasperma. However, it is unclear whether this intriguing association holds in other species. We show here that Schizothecium tetrasporum, a fungus from a third family in the order Sordariales, also produces mostly self-fertile dikaryotic spores carrying the two opposite mating types. This was due to a high frequency of second meiotic division segregation at the mating-type locus, indicating the occurrence of a single and systematic crossing-over event between the mating-type locus and the centromere, as in P. anserina. The mating-type locus has the typical Sordariales organization, plus a MAT1-1-1 pseudogene in the MAT1-2 haplotype. High-quality genome assemblies of opposite mating types and segregation analyses revealed a suppression of recombination in a region of 1.47 Mb around the mating-type locus. We detected three evolutionary strata, indicating a stepwise extension of recombination suppression. The three strata displayed no rearrangement or transposable element accumulation but gene losses and gene disruptions were present, and precisely at the strata margins. Our findings indicate a convergent evolution of self-fertile dikaryotic sexual spores across multiple ascomycete fungi. The particular pattern of meiotic segregation at the mating-type locus was associated with recombination suppression around this locus, that had extended stepwise. This association between pseudo-homothallism and recombination suppression across lineages and the presence of gene disruption at the strata limits are consistent with a recently proposed mechanism of sheltering deleterious alleles to explain stepwise recombination suppression.

Protease production and molecular characterization of a protease dipeptidyl-aminopeptidase gene from different strains of Sordaria fimicola.

The current research was designed to reach extracellular protease production potential in different strains of Sordaria fimicola which were previously obtained from Dr. Lamb (Imperial College, London) from North Facing Slope and South Facing Slope of Evolution Canyon. After initial and secondary screening, two hyper-producers strains S2 and N6 were selected for submerged fermentation and cultural conditions including temperature, pH, incubation period, inoculum size, substrate concentration, and different carbon and nitrogen sources were optimized for enzyme production. S2 strain showed maximum protease production of 3.291 U/mL after 14 days of incubation at 30 °C with 7 pH, 1% substrate concentration and 1 mL inoculum, While N6 strain showed maximum protease production of 1.929 U/mL under fermentation optimized conditions. Another aim of the present research was to underpin the biodiversity of genetics and post-translational modifications (PTMs) of protease DPAP (peptidyl-aminopeptidase) in Sordaria fimicola. Five polymorphic sites were observed in amino acid sequence of S. fimicola strains with reference to Neurospora crassa. PTMs prediction from bioinformatics tools predicted 38 phosphorylation sites on serine residues for protease peptidyl-aminopeptidase in S1 strain of S. fimicola while 45 phosphorylation sites on serine in N7 strain and 47 serine phosphorylation modifications were predicted in N. crassa. Current research gave an insight that change in genetic makeup effected PTMs which ultimately affected the production of protease enzyme in different strains of same organism (S. fimicola). The production and molecular data of the research revealed that environmental stress has strong effects on the specific genes through mutations which may cause genetic diversity. S. fimicola is non- pathogenic fungus and has a short life cycle. This fungus can be chosen to produce protease enzyme on a commercial scale.

Reconstruction and analysis of genome-scale metabolic model for thermophilic fungus Myceliophthora thermophila.

Myceliophthora thermophila, a thermophilic fungus that can degrade and utilize all major polysaccharides in plant biomass, has great potential in biotechnological industries. Here, the first manually curated genome-scale metabolic model iDL1450 for M. thermophila was reconstructed using an autogenerating pipeline with thorough manual curation. The model contains 1450 genes, 2592 reactions, and 1784 unique metabolites. High accuracy was shown in predictions related to carbon and nitrogen source utilization based on data obtained from Biolog experiments. Besides, metabolism profiles were analyzed using iDL1450 integrated with transcriptomics data of M. thermophila at various growth temperatures. The refined model provides new insights into thermophilic fungi metabolism and sheds light on model-driven strain design to improve biotechnological applications of this thermophilic lignocellulosic fungus.

Functional characterization of the developmental genes asm2, asm3, and spt3 required for fruiting body formation in the filamentous ascomycete Sordaria macrospora.

The formation of fruiting bodies is one of the most complex developmental processes in filamentous ascomycetes. It requires the development of sexual structures that give rise to meiosporangia (asci) and meiotic spores (ascospores) as well as surrounding structures for protection and dispersal of the spores. Previous studies have shown that these developmental processes are accompanied by significant changes of the transcriptome, and comparative transcriptomics of different fungi as well as the analysis of transcriptome changes in developmental mutants have aided in the identification of differentially regulated genes that are themselves involved in regulating fruiting body development. In previous analyses, we used transcriptomics to identify the genes asm2 and spt3, which result in developmental phenotypes when deleted in Sordaria macrospora. In this study, we identified another gene, asm3, required for fruiting body formation, and performed transcriptomics analyses of Δasm2, Δasm3, and Δspt3. Deletion of spt3, which encodes a subunit of the SAGA complex, results in a block at an early stage of development and drastic changes in the transcriptome. Deletion mutants of asm2 and asm3 are able to form fruiting bodies, but have defects in ascospore maturation. Transcriptomics analysis of fruiting bodies revealed a large overlap in differentially regulated genes in Δasm2 and Δasm3 compared to the wild type. Analysis of nuclear distribution during ascus development showed that both mutants undergo meiosis and postmeiotic divisions, suggesting that the transcriptomic and morphological changes might be related to defects in the morphogenesis of structural features of the developing asci and ascospores.

Improving the Heterologous Production of Fungal Peroxygenases through an Episomal Pichia pastoris Promoter and Signal Peptide Shuffling System.

Fungal peroxygenases (UPOs) have emerged as oxyfunctionalization catalysts of tremendous interest in recent years. However, their widespread use in the field of biocatalysis is still hampered by their challenging heterologous production, substantially limiting the panel of accessible enzymes for investigation and enzyme engineering. Building upon previous work on UPO production in yeast, we have developed a combined promoter and signal peptide shuffling system for episomal high throughput UPO production in the industrially relevant, methylotrophic yeast Pichia pastoris. Eleven endogenous and orthologous promoters were shuffled with a diverse set of 17 signal peptides. Three previously described UPOs were selected as first test set, leading to the identification of beneficial promoter/signal peptide combinations for protein production. We applied the system then successfully to produce two novel UPOs: MfeUPO from Myceliophthora fergusii and MhiUPO from Myceliophthora hinnulea. To demonstrate the feasibility of the developed system to other enzyme classes, it was applied for the industrially relevant lipase CalB and the laccase Mrl2. In total, approximately 3200 transformants of eight diverse enzymes were screened and the best promoter/signal peptide combinations studied at various cofeeding, derepression, and induction conditions. High volumetric production titers were achieved by subsequent creation of stable integration lines and harnessing orthologous promoters from Hansenula polymorpha. In most cases promising yields were also achieved without the addition of methanol under derepressed conditions. To foster the use of the episomal high throughput promoter/signal peptide Pichia pastoris system, we made all plasmids available through Addgene.

Targeted Quantification of Phosphorylation Sites Identifies STRIPAK-Dependent Phosphorylation of the Hippo Pathway-Related Kinase SmKIN3.

We showed recently that the germinal center kinase III (GCKIII) SmKIN3 from the fungus Sordaria macrospora is involved in sexual development and hyphal septation. Our recent extensive global proteome and phosphoproteome analysis revealed that SmKIN3 is a target of the striatin-interacting phosphatase and kinase (STRIPAK) multisubunit complex. Here, using protein samples from the wild type and three STRIPAK mutants, we applied absolute quantification by parallel-reaction monitoring (PRM) to analyze phosphorylation site occupancy in SmKIN3 and other septation initiation network (SIN) components, such as CDC7 and DBF2, as well as BUD4, acting downstream of SIN. For SmKIN3, we show that phosphorylation of S668 and S686 is decreased in mutants lacking distinct subunits of STRIPAK, while a third phosphorylation site, S589, was not affected. We constructed SmKIN3 mutants carrying phospho-mimetic and phospho-deficient codons for phosphorylation sites S589, S668, and S686. Investigation of hyphae in a ΔSmkin3 strain complemented by the S668 and S686 mutants showed a hyper-septation phenotype, which was absent in the wild type, the ΔSmkin3 strain complemented with the wild-type gene, and the S589 mutant. Furthermore, localization studies with SmKIN3 phosphorylation variants and STRIPAK mutants showed that SmKIN3 preferentially localizes at the terminal septa, which is distinctly different from the localization of the wild-type strains. We conclude that STRIPAK-dependent phosphorylation of SmKIN3 has an impact on controlled septum formation and on the time-dependent localization of SmKIN3 on septa at the hyphal tip. Thus, STRIPAK seems to regulate SmKIN3, as well as DBF2 and BUD4 phosphorylation, affecting septum formation.IMPORTANCE Phosphorylation and dephosphorylation of proteins are fundamental posttranslational modifications that determine the fine-tuning of their biological activity. Involved in this modification process is the recently identified striatin-interacting phosphatase and kinase (STRIPAK) multisubunit complex, which is evolutionarily conserved from fungi to humans. STRIPAK functions as a macromolecular assembly communicating through physical interactions with other conserved signaling protein complexes to constitute larger dynamic protein networks. Its function is implied in many cellular processes, such as signal transduction pathways, growth, and cellular differentiation. We applied absolute quantification of protein phosphorylation by parallel-reaction monitoring (PRM) to analyze phosphorylation site occupancy in signaling components that are linked to the STRIPAK complex. Using the filamentous fungus Sordaria macrospora, we provide evidence for the phosphorylation-dependent role of the Hippo-like germinal center kinase SmKIN3, which controls septum formation, and localize it in a time-dependent manner on septa at the hyphal tip.

Isolation and modification of nano-scale cellulose from organosolv-treated birch through the synergistic activity of LPMO and endoglucanases.

Nanocellulose isolation from lignocellulose is a tedious and expensive process with high energy and harsh chemical requirements, primarily due to the recalcitrance of the substrate, which otherwise would have been cost-effective due to its abundance. Replacing the chemical steps with biocatalytic processes offers opportunities to solve this bottleneck to a certain extent due to the enzymes substrate specificity and mild reaction chemistry. In this work, we demonstrate the isolation of sulphate-free nanocellulose from organosolv pretreated birch biomass using different glycosyl-hydrolases, along with accessory oxidative enzymes including a lytic polysaccharide monooxygenase (LPMO). The suggested process produced colloidal nanocellulose suspensions (ζ-potential -19.4 mV) with particles of 7-20 nm diameter, high carboxylate content and improved thermostability (To = 301 °C, Tmax = 337 °C). Nanocelluloses were subjected to post-modification using LPMOs of different regioselectivity. The sample from chemical route was the least favorable for LPMO to enhance the carboxylate content, while that from the C1-specific LPMO treatment showed the highest increase in carboxylate content.

Probing the role of Val228 on the catalytic activity of Scytalidium catalase.

Scytalidium catalase is a homotetramer including heme d in each subunit. Its primary function is the dismutation of H2O2 to water and oxygen, but it is also able to oxidase various small organic compounds including catechol and phenol. The crystal structure of Scytalidium catalase reveals the presence of three linked channels providing access to the exterior like other catalases reported so far. The function of these channels has been extensively studied, revealing the possible routes for substrate flow and product release. In this report, we have focussed on the semi-conserved residue Val228, located near to the vinyl groups of the heme at the opening of the lateral channel. Its replacement with Ala, Ser, Gly, Cys, Phe and Ile were tested. We observed a significant decrease in catalytic efficiency in all mutants with the exception of a remarkable increase in oxidase activity when Val228 was mutated to either Ala, Gly or Ser. The reduced catalytic efficiencies are characterized in terms of the restriction of hydrogen peroxide as electron acceptor in the active centre resulting from the opening of lateral channel inlet by introducing the smaller side chain residues. On the other hand, the increased oxidase activity is explained by allowing the suitable electron donor to approach more closely to the heme. The crystal structures of V228C and V228I were determined at 1.41 and 1.47 Å resolution, respectively. The lateral channels of the V228C and V228I presented a broadly identical chain of arranged waters to that observed for wild-type enzyme.

Mollicellins S-U, three new depsidones from Chaetomium brasiliense SD-596 with anti-MRSA activities.

Fungi are important resources for drug development, as they have a diversity of genes, that can produce novel secondary metabolites with effective bioactivities. Here, five depsidone-based analogs were isolated from the rice media of Chaetomium brasiliense SD-596. Their structures were elucidated using NMR and mass spectrometry analysis. Five compounds, including three new depsidone analogs, mollicellin S (1), mollicellin T (2), and mollicellin U (3), and two known compounds, mollicellin D (4) and mollicellin H (5), exhibited significant inhibition against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), with MIC values ranging from 6.25 to 12.5 µg ml-1. Herein, we identified the predicted plausible biosynthetic cluster of the compounds and discussed the structure-activity relationship. Finally, we found that the introduction of aldehyde and methoxyl groups provide marked improvement for the inhibition against MRSA.

Golden Gate vectors for efficient gene fusion and gene deletion in diverse filamentous fungi.

The cloning of plasmids can be time-consuming or expensive. Yet, cloning is a prerequisite for many standard experiments for the functional analysis of genes, including the generation of deletion mutants and the localization of gene products. Here, we provide Golden Gate vectors for fast and easy cloning of gene fusion as well as gene deletion vectors applicable to diverse fungi. In Golden Gate cloning, restriction and ligation occur simultaneously in a one-pot reaction. Our vector set contains recognition sites for the commonly used type IIS restriction endonuclease BsaI. We generated plasmids for C- as well as N-terminal tagging with GFP, mRFP and 3xFLAG. For gene deletion, we provide five different donor vectors for selection marker cassettes. These include standard cassettes for hygromycin B, nourseothricin and phleomycin resistance genes as well as FLP/FRT-based marker recycling cassettes for hygromycin B and nourseothricin resistance genes. To make cloning most feasible, we provide robust protocols, namely (1) an overview of cloning procedures described in this paper, (2) specific Golden Gate reaction protocols and (3) standard primers for cloning and sequencing of plasmids and generation of deletion cassettes by PCR and split-marker PCR. We show that our vector set is applicable for the biotechnologically relevant Penicillium chrysogenum and the developmental model system Sordaria macrospora. We thus expect these vectors to be beneficial for other fungi as well. Finally, the vectors can easily be adapted to organisms beyond the kingdom fungi.

Safety evaluation of a ß-mannanase enzyme preparation produced with Thermothelomyces thermophilus expressing a protein-engineered ß-mannanase gene.

Mannanase 19287 enzyme is an engineered ß-mannanase that can be added to diets for animals raised for human consumption to hydrolyze ß-mannans. Established toxicological analyses were conducted with the enzyme preparation to ensure the safety of this product for the intended use. The mannanase 19287 preparation was produced with Thermothelomyces thermophilus strain DSM 33149. In vitro toxicity studies presented here used dosages of the mannanase 19287 test articles up to 5000 µg/plate. For in vivo toxicity studies in Wistar rats, test articles were administered at 5.1 mg/L for inhalation toxicity and up to 15,000 mg/kg rat feed for oral toxicity, based on the Total Organic Solids (TOS) content in each test article. No treatment related adverse effects were reported in any study. The No Observed Adverse Effect Levels in the high dose group of the subchronic oral toxicity study were calculated as 1117-1298 mg TOS/kg bw/day in rats. Comparing these values to an Estimated Daily Intake for poultry demonstrated safety factors larger than 5000. Our results confirm that T. thermophilus fulfills the recognized safety criteria for the manufacture of food enzyme preparations and represent the first peer-reviewed safety evaluation of an enzyme preparation by T. thermophilus. The results of the toxicity studies presented herein attest to the safety of the mannanase 19287 enzyme for its intended use.

Molecular dynamics simulations of two GH74 endo-processive xyloglucanases and the mutated variants to understand better the mechanism of the enzyme action.

BACKGROUND: GH74 xyloglucanases are composed of two separate domains connected by two unstructured peptides. Previously, a hypothesis was made that the movement of domains may affect the enzyme mechanism of catalysis. METHODS: The molecular dynamics (MD) simulations of endo-processive xyloglucanases from Paenibacillus odorifer (PoGH74cat) and Myceliophthora thermophila (MtXeg74A) were carried out. RESULTS: MD simulations for both enzymes in complex with XXLG and XGXXLG oligosaccharides confirmed the possibility of domain movement. In the case of MtXeg74A, changes in the distances between Cα atoms of aromatic residues involved in xyloglucan binding in -3 and +3 subsites of the active site cleft and those of selected residues on the opposite side of the cleft reached values up to 10-12 Å. For PoGH74cat the conformational changes were less pronounced. In MtXeg74A variants, the deletion of loop 1, which partially closes the entrance to the cleft, and the additional double mutation of two Trp residues in +3 and +5 subsites caused the enhanced mobility of the XGXXLG and also induced changes in topography of the cleft. CONCLUSIONS: These findings demonstrate the possibility of existence of GH74 xyloglucanases in a more open and more closed enzyme conformation. The enzyme in an open conformation may more easily accommodate the branched polysaccharide, while its transition to the closed conformation, together with loop 1 function, should aid processivity. GENERAL SIGNIFICANCE: Our results provide an insight into a mechanism of action of GH74 xyloglucanases and may be useful for discussing the catalytic mechanisms of glycoside hydrolases from other families.

The STRIPAK signaling complex regulates dephosphorylation of GUL1, an RNA-binding protein that shuttles on endosomes.

The striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit signaling complex is highly conserved within eukaryotes. In fungi, STRIPAK controls multicellular development, morphogenesis, pathogenicity, and cell-cell recognition, while in humans, certain diseases are related to this signaling complex. To date, phosphorylation and dephosphorylation targets of STRIPAK are still widely unknown in microbial as well as animal systems. Here, we provide an extended global proteome and phosphoproteome study using the wild type as well as STRIPAK single and double deletion mutants (Δpro11, Δpro11Δpro22, Δpp2Ac1Δpro22) from the filamentous fungus Sordaria macrospora. Notably, in the deletion mutants, we identified the differential phosphorylation of 129 proteins, of which 70 phosphorylation sites were previously unknown. Included in the list of STRIPAK targets are eight proteins with RNA recognition motifs (RRMs) including GUL1. Knockout mutants and complemented transformants clearly show that GUL1 affects hyphal growth and sexual development. To assess the role of GUL1 phosphorylation on fungal development, we constructed phospho-mimetic and -deficient mutants of GUL1 residues. While S180 was dephosphorylated in a STRIPAK-dependent manner, S216, and S1343 served as non-regulated phosphorylation sites. While the S1343 mutants were indistinguishable from wild type, phospho-deficiency of S180 and S216 resulted in a drastic reduction in hyphal growth, and phospho-deficiency of S216 also affects sexual fertility. These results thus suggest that differential phosphorylation of GUL1 regulates developmental processes such as fruiting body maturation and hyphal morphogenesis. Moreover, genetic interaction studies provide strong evidence that GUL1 is not an integral subunit of STRIPAK. Finally, fluorescence microscopy revealed that GUL1 co-localizes with endosomal marker proteins and shuttles on endosomes. Here, we provide a new mechanistic model that explains how STRIPAK-dependent and -independent phosphorylation of GUL1 regulates sexual development and asexual growth.