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The GDS(L)-like lipase from the Basidiomycota Pleurotus sapidus (PSA_Lip) was heterologously expressed using Trichoderma reesei with an activity of 350 U L -1 . The isoelectric point of 5.0 was determined by isoelectric focusing. The novel PSA_Lip showed only 23.8-25.1%, 25.5%, 26.6% and 28.4% identity to the previously characterized GDSL-like enzymes phospholipase, plant lipase, acetylcholinesterase and acetylxylan esterase, from the carbohydrate esterase family 16, respectively. Therefore, the enzyme was purified from the culture supernatant and the catalytic properties and the substrate specificity of the enzyme were investigated using different assays to reveal its potential function. While no phospholipase, acetylcholinesterase and acetylxylan esterase activities were detected, studies on the hydrolysis of ferulic acid methyl ester (~ 8.3%) and feruloylated carbohydrate 5-O-transferuloyl-arabino-furanose (~ 0.8%) showed low conversions of these substrates. By investigating the hydrolytic activity towards p-nitrophenyl-(pNP)-esters with various chain-lengths, the highest activity was determined for medium chain-length pNP-octanoate at 65 °C and a pH value of 8, while almost no activity was detected for pNP-hexanoate. The enzyme is highly stable when stored at pH 10 and 4 °C for at least 7 days. Moreover, using consensus sequence analysis and homology modeling, we could demonstrate that the PSA_Lip does not contain the usual SGNH residues in the actives site, which are usually present in GDS(L)-like enzymes., (© 2024. The Author(s).)

Due to the increasing demand for natural food ingredients, including taste-active compounds, enzyme-catalyzed conversions of natural substrates, such as flavonoids, are promising tools to align with the principles of Green Chemistry. In this study, a novel O -methyltransferase activity was identified in the mycelium of Lentinula edodes , which was successfully applied to generate the taste-active flavonoids hesperetin, hesperetin dihydrochalcone, homoeriodictyol, and homoeriodictyol dihydrochalcone. Furthermore, the mycelium-mediated OMT activity allowed for the conversion of various catecholic substrates, yielding their respective (iso-)vanilloids, while monohydroxylated compounds were not converted. By means of a bottom-up proteomics approach, three putative O -methyltransferases were identified, and subsequently, synthetic, codon-optimized genes were heterologously expressed in Escherichia coli . The purified enzymes confirmed the biocatalytic O -methylation activity against targeted flavonoids containing catechol motifs.

Metagenomic sequencing is a promising method that has the potential to revolutionize the world of pathogen detection and antimicrobial resistance (AMR) surveillance in food-producing environments. However, the analysis of the huge amount of data obtained requires performant bioinformatics tools and databases, with intuitive and straightforward interpretation. In this study, based on long-read metagenomics data of chicken fecal samples with a spike-in mock community, we proposed confidence levels for taxonomic identification and AMR gene detection, with interpretation guidelines, to help with the analysis of the output data generated by KMA, a popular k- mer read alignment tool. Additionally, we demonstrated that the completeness and diversity of the genomes present in the reference databases are key parameters for accurate and easy interpretation of the sequencing data. Finally, we explored whether KMA, in a two-step procedure, can be used to link the detected AMR genes to their bacterial host chromosome, both detected within the same long-reads. The confidence levels were successfully tested on 28 metagenomics datasets which were obtained with sequencing of real and spiked samples from fecal (chicken, pig, and buffalo) or food (minced beef and food enzyme products) origin. The methodology proposed in this study will facilitate the analysis of metagenomics sequencing datasets for KMA users. Ultimately, this will contribute to improvements in the rapid diagnosis and surveillance of pathogens and AMR genes in food-producing environments, as prioritized by the EU., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Gand, Navickaite, Bartsch, Grützke, Overballe-Petersen, Rasmussen, Otani, Michelacci, Matamoros, González-Zorn, Brouwer, Di Marcantonio, Bloemen, Vanneste, Roosens, AbuOun and De Keersmaecker.)

Rapid, accurate and comprehensive diagnostics are essential for outbreak prevention and pathogen surveillance. Real-time, on-site metagenomics on miniaturized devices, such as Oxford Nanopore Technologies MinION sequencing, could provide a promising approach. However, current sample preparation protocols often require substantial equipment and dedicated laboratories, limiting their use. In this study, we developed a rapid on-site applicable DNA extraction and library preparation approach for nanopore sequencing, using portable devices. The optimized method consists of a portable mechanical lysis approach followed by magnetic bead-based DNA purification and automated sequencing library preparation, and resulted in a throughput comparable to a current optimal, laboratory-based protocol using enzymatic digestion to lyse cells. By using spike-in reference communities, we compared the on-site method with other workflows, and demonstrated reliable taxonomic profiling, despite method-specific biases. We also demonstrated the added value of long-read sequencing by recovering reads containing full-length antimicrobial resistance genes, and attributing them to a host species based on the additional genomic information they contain. Our method may provide a rapid, widely-applicable approach for microbial detection and surveillance in a variety of on-site settings., (© 2023. The Author(s).)

Macrophage migration inhibitory factor (MIF) is a pleiotropic protein with chemotactic, pro-inflammatory, and growth-promoting activities first discovered in mammals. In parasites, MIF homologs are involved in immune evasion and pathogenesis. Here, we present the first comprehensive analysis of an MIF protein from the devastating plant pathogen Magnaporthe oryzae ( Mo ). The fungal genome encodes a single MIF protein ( Mo MIF1) that, unlike the human homolog, harbors multiple low-complexity regions (LCRs) and is unique to Ascomycota. Following infection, MoMIF1 is expressed in the biotrophic phase of the fungus, and is strongly down-regulated during subsequent necrotrophic growth in leaves and roots. We show that Mo MIF1 is secreted during plant infection, affects the production of the mycotoxin tenuazonic acid and inhibits plant cell death. Our results suggest that Mo MIF1 is a novel key regulator of fungal virulence that maintains the balance between biotrophy and necrotrophy during the different phases of fungal infection., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Background: Oxford Nanopore Technologies (ONT) offers an accessible platform for long-read sequencing, which improves the reconstruction of genomes and helps to resolve complex genomic contexts, especially in the case of metagenome analysis. To take the best advantage of long-read sequencing, DNA extraction methods must be able to isolate pure high molecular weight (HMW) DNA from complex metagenomics samples, without introducing any bias. New methods released on the market, and protocols developed at the research level, were specifically designed for this application and need to be assessed., Results: In this study, with different bacterial cocktail mixes, analyzed as pure or spiked in a synthetic fecal matrix, we evaluated the performances of 6 DNA extraction methods using various cells lysis and purification techniques, from quick and easy, to more time-consuming and gentle protocols, including a portable method for on-site application. In addition to the comparison of the quality, quantity and purity of the extracted DNA, the performance obtained when doing Nanopore sequencing on a MinION flow cell was also tested. From the obtained results, the Quick-DNA HMW MagBead Kit (Zymo Research) was selected as producing the best yield of pure HMW DNA. Furthermore, this kit allowed an accurate detection, by Nanopore sequencing, of almost all the bacterial species present in a complex mock community., Conclusion: Amongst the 6 tested methods, the Quick-DNA HMW MagBead Kit (Zymo Research) was considered as the most suitable for Nanopore sequencing and would be recommended for bacterial metagenomics studies using this technology., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The comprehensive identification of the proteome content from a white wine (cv. Silvaner) is described here for the first time. The wine protein composition isolated from a representative wine sample (250 L) was identified via mass spectrometry (MS)-based proteomics following in-solution and in-gel digestion methods after being submitted to size exclusion chromatographic (SEC) fractionation to gain a comprehensive insight into proteins that survive the vinification processes. In total, we identified 154 characterized (with described functional information) or so far uncharacterized proteins, mainly from Vitis vinifera L. and Saccharomyces cerevisiae . With the complementarity of the two-step purification, the digestion techniques and the high-resolution (HR)-MS analyses provided a high-score identification of proteins from low to high abundance. These proteins can be valuable for future authentication of wines by tracing proteins derived from a specific cultivar or winemaking process. The proteomics approach presented herein may also be generally helpful to understand which proteins are important for the organoleptic properties and stability of wines.

Thermolabile grape berry proteins such as thaumatin-like proteins (TLPs) and chitinases (CHIs) promote haze formation in bottled wines if not properly fined. As a natural grapevine pest, the spotted-wing fly Drosophila suzukii is a promising source of peptidases that break down grape berry proteins because the larvae develop and feed inside mature berries. Therefore, we produced recombinant TLP and CHI as model thermolabile wine haze proteins and applied a peptidomics strategy to investigate whether D. suzukii larval peptidases were able to digest them under acidic conditions (pH 3.5), which are typically found in winemaking practices. The activity of the novel peptidases was confirmed by mass spectrometry, and cleavage sites within the wine haze proteins were visualized in 3D protein models. The combination of recombinant haze proteins and peptidomics provides a valuable screening tool to identify optimal peptidases suitable for clarification processes in the winemaking industry.

The rapidly evolving Coronavirus Disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide with thousands of deaths and infected cases. For the identification of effective treatments against this disease, the main protease (M pro ) of SARS‑CoV‑2 was found to be an attractive drug target, as it played a central role in viral replication and transcription. Here, we report the results of high-throughput molecular docking with 1,045,468 ligands' structures from 116 kinds of traditional Chinese medicine (TCM). Subsequently, 465 promising candidates were obtained, showing high binding affinities. The dynamic simulation, ADMET (absorption, distribution, metabolism, excretion and toxicity) and drug-likeness properties were further analyzed the screened docking results. Basing on these simulation results, 23 kinds of Chinese herbal extracts were employed to study their inhibitory activity for M pro of SARS‑CoV‑2. Plants extracts from Forsythiae Fructus, Radix Puerariae, Radix astragali, Anemarrhenae Rhizoma showed acceptable inhibitory efficiencies, which were over 70%. The best candidate was Anemarrhenae Rhizoma , reaching 78.9%., Competing Interests: The authors declare that they have no competing interests., (© 2022 Elsevier B.V. All rights reserved.)

In this proof-of-concept study on food contaminated with norovirus, we investigated the feasibility of metagenomics as a new method to obtain the whole genome sequence of the virus and perform strain level characterization but also relate to human cases in order to resolve foodborne outbreaks. We tested several preparation methods to determine if a more open sequencing approach, i.e., shotgun metagenomics, or a more targeted approach, including hybrid capture, was the most appropriate. The genetic material was sequenced using Oxford Nanopore technologies with or without adaptive sampling, and the data were analyzed with an in-house bioinformatics workflow. We showed that a viral genome sequence could be obtained for phylogenetic analysis with shotgun metagenomics if the contamination load was sufficiently high or after hybrid capture for lower contamination. Relatedness to human cases goes well beyond the results obtained with the current qPCR methods. This workflow was also tested on a publicly available dataset of food spiked with norovirus and hepatitis A virus. This allowed us to prove that we could detect even fewer genome copies and two viruses present in a sample using shotgun metagenomics. We share the lessons learnt on the satisfactory and unsatisfactory results in an attempt to advance the field.

Cross-linking net aggregates of thermolabile thaumatin-like proteins (TLPs) and chitinases (CHIs) are the primary source of haze in white wines. Although bentonite fining is still routinely used in winemaking, alternative methods to selectively remove haze proteins without affecting wine organoleptic properties are needed. The availability of pure TLPs and CHIs would facilitate the research for the identification of such technological advances. Therefore, we proposed the usage of recombinant TLP (rTLP) and CHI (rCHI), expressed by Komagataella phaffii , as haze-protein models, since they showed similar characteristics (aggregation potential, melting point, functionality, glycosylation levels and bentonite adsorption) to the native-haze proteins from Vitis vinifera . Hence, rTLP and rCHI can be applied to study haze formation mechanisms on a molecular level and to explore alternative fining methods by screening proteolytic enzymes and ideal adsorptive resins.

In traditional cheese making, pregastric lipolytic enzymes of animal origin are used for the acceleration of ripening and the formation of spicy flavor compounds. Especially for cheese specialities, such as Pecorino, Provolone, or Feta, pregastric esterases (PGE) play an important role. A lipase from Pleurotus citrinopileatus could serve as a substitute for these animal-derived enzymes, thus offering vegetarian, kosher, and halal alternatives. However, the hydrolytic activity of this enzyme towards long-chain fatty acids is slightly too high, which may lead to off-flavors during long-term ripening. Therefore, an optimization via protein engineering (PE) was performed by changing the specificity towards medium-chain fatty acids. With a semi-rational design, possible mutants at eight different positions were created and analyzed in silico. Heterologous expression was performed for 24 predicted mutants, of which 18 caused a change in the hydrolysis profile. Three mutants (F91L, L302G, and L305A) were used in application tests to produce Feta-type brine cheese. The sensory analyses showed promising results for cheeses prepared with the L305A mutant, and SPME-GC-MS analysis of volatile free fatty acids supported these findings. Therefore, altering the chain length specificity via PE becomes a powerful tool for the replacement of PGEs in cheese making.

Air pollution exposure can lead to exacerbation of respiratory disorders in children. Using sensitive biomarkers helps to assess the impact of air pollution on children's respiratory health and combining protein, genetic and epigenetic biomarkers gives insights on their interrelatedness. Most studies do not contain such an integrated approach and investigate these biomarkers individually in blood, although its collection in children is challenging. Our study aimed at assessing the feasibility of conducting future integrated larger-scale studies evaluating respiratory health risks of air pollution episodes in children, based on a qualitative analysis of the technical and logistic aspects of a small-scale field study involving 42 children. This included the preparation, collection and storage of non-invasive samples (urine, saliva), the measurement of general and respiratory health parameters and the measurement of specific biomarkers (genetic, protein, epigenetic) of respiratory health and air pollution exposure. Bottlenecks were identified and modifications were proposed to expand this integrated study to a higher number of children, time points and locations. This would allow for non-invasive assessment of the impact of air pollution exposure on the respiratory health of children in future larger-scale studies, which is critical for the development of policies or measures at the population level.

The azinothricin family comprises several cyclic hexadepsipeptides with diverse pharmacological bioactivities, including antimicrobial, antitumoral, and apoptosis induction. In this work, using a genome mining approach, a biosynthetic gene cluster encoding an azinothricin-like compound was identified from the Streptomyces sp. s120 genome sequence ( pop BGC). Comparative MS analysis of extracts from the native producer and a knockout mutant led to the identification of metabolites corresponding to the pop BGC. Furthermore, regulatory elements of the BGC were identified. By overexpression of an LmbU-like transcriptional activator, the production yield of 1 and 2 was increased, enabling isolation and structure elucidation of polyoxyperuin A seco acid ( 1 ) and polyoxyperuin A ( 2 ) using high-resolution mass spectrometry and NMR spectroscopy. Compound 1 exhibited a low antibiotic effect against Micrococcus luteus , while 2 showed a strong Gram-positive antibiotic effect in a micro-broth-dilution assay.

Traditionally produced piquant cheeses such as Feta or Provolone rely on pregastric lipolytic enzymes of animal origin to intensify flavor formation during ripening. Herein, we report a novel fungal lipase, derived from the phylum Basidiomycota to replace animal-derived products. A screening of 31 strains for the desired hydrolytic activities was performed, which revealed a promising fungal species. The secretome of an edible golden oyster mushroom, Pleurotus citrinopileatus , provided suitable enzymatic activity, and the coding sequence of the corresponding enzyme was identified by combining transcriptome and liquid chromatography high-resolution electrospray tandem mass spectroscopy (LC-HR-ESI-MS/MS) data. Recombinant expression in Escherichia coli BL21 (DE3) using chaperones GroES-GroEL and DnaK-DnaJ-GrpE was established. The recombinant lipolytic enzyme was purified and biochemically characterized in terms of thermal and pH stability, optimal reaction conditions, and kinetic data toward p -nitrophenyl esters. An application in the microscale production of Feta-type brine cheese revealed promising sensory properties, which were confirmed by headspace solid-phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS) analyses in comparison with the reference enzyme opti-zym z10uc from goat origin. Supplementation with 2.3 U of the heterologously expressed fungal lipase produced the most comparable free fatty acid profile after 30 days of ripening. The flavor and texture formed during the application of the new lipase from P. citrinopileatus proved to be competitive to the use of pregastric lipases and could therefore replace the products of animal origin.

To meet consumer expectations, white wines must be clear and stable against haze formation. Temperature variations during transport and storage may induce protein aggregation, mainly caused by thaumatin like-proteins (TLPs) and chitinases (CHIs), which thus need to be fined before bottling of the wine. Currently, bentonite clay is employed to inhibit or minimize haze formation in wines. Alternatively, peptidases have emerged as an option for the removal of these thermolabile proteins, although their efficacy under winemaking conditions has not yet been fully demonstrated. The simultaneous understanding of the chemistry behind the cleavage of haze proteins and the haze formation may orchestrate alternative methods of technological and economic importance in winemaking. Therefore, we provide an overview of wine fining by peptidases, and new perspectives are developed to reopen discussions on the aforementioned challenges.

Prevention of haze formation in wines is challenging for winemakers. Thermolabile proteins in wines, notably thaumatin-like proteins (TLPs) and chitinases (CHIs), undergo structural changes under varying physicochemical conditions, resulting in protein aggregation and visible haze in bottled products. Peptidases are an alternative fining method, although an effective proteolysis under typical winemaking conditions (acidic pH and low temperature) is difficult to achieve. In this study, tryptic peptides from TLPs and CHIs were identified by MS-based peptidomics (top-down proteomics) after exposure of scissile bonds on the protein surface. As proposed by the theory of limited proteolysis, protein conformational changes following temperature and pH variations allowed the detection of enzyme-accessible regions. Protein structure visualization and molecular dynamics simulations were used to highlight cleavage spots and provide the scientific basis for haze formation mechanisms. The described method offers a tool to the search for ideal enzymes to prevent wine haze., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The worldwide emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since 2019 has highlighted the importance of rapid and reliable diagnostic testing to prevent and control the viral transmission. However, inaccurate results may occur due to false negatives (FN) caused by polymorphisms or point mutations related to the virus evolution and compromise the accuracy of the diagnostic tests. Therefore, PCR-based SARS-CoV-2 diagnostics should be evaluated and evolve together with the rapidly increasing number of new variants appearing around the world. However, even by using a large collection of samples, laboratories are not able to test a representative collection of samples that deals with the same level of diversity that is continuously evolving worldwide. In the present study, we proposed a methodology based on an in silico and in vitro analysis. First, we used all information offered by available whole-genome sequencing data for SARS-CoV-2 for the selection of the two PCR assays targeting two different regions in the genome, and to monitor the possible impact of virus evolution on the specificity of the primers and probes of the PCR assays during and after the development of the assays. Besides this first essential in silico evaluation, a minimal set of testing was proposed to generate experimental evidence on the method performance, such as specificity, sensitivity and applicability. Therefore, a duplex reverse-transcription droplet digital PCR (RT-ddPCR) method was evaluated in silico by using 154 489 whole-genome sequences of SARS-CoV-2 strains that were representative for the circulating strains around the world. The RT-ddPCR platform was selected as it presented several advantages to detect and quantify SARS-CoV-2 RNA in clinical samples and wastewater. Next, the assays were successfully experimentally evaluated for their sensitivity and specificity. A preliminary evaluation of the applicability of the developed method was performed using both clinical and wastewater samples.

Staphylotrichum longicolleum FW57 (DSM105789) is a prolific chitinolytic fungus isolated from wood, with a chitinase activity of 0.11 ± 0.01 U/mg. We selected this strain for genome sequencing and annotation, and compiled its growth characteristics on four different chitinous substrates as well as two agro-industrial waste products. We found that the enzymatic mixture secreted by FW57 was not only able to digest pre-treated sugarcane bagasse, but also untreated sugarcane bagasse and maize leaves. The efficiency was comparable to a commercial enzymatic cocktail, highlighting the potential of the S. longicolleum enzyme mixture as an alternative pretreatment method. To further characterize the enzymes, which efficiently digested polymers such as cellulose, hemicellulose, pectin, starch, and lignin, we performed in-depth mass spectrometry-based secretome analysis using tryptic peptides from in-gel and in-solution digestions. Depending on the growth conditions, we were able to detect from 442 to 1092 proteins, which were annotated to identify from 134 to 224 putative carbohydrate-active enzymes (CAZymes) in five different families: glycoside hydrolases, auxiliary activities, carbohydrate esterases, polysaccharide lyases, glycosyl transferases, and proteins containing a carbohydrate-binding module, as well as combinations thereof. The FW57 enzyme mixture could be used to replace commercial enzyme cocktails for the digestion of agro-residual substrates.

For 1 year now, the world is undergoing a coronavirus disease-2019 (COVID-19) pandemic due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The most widely used method for COVID-19 diagnosis is the detection of viral RNA by RT-qPCR with a specific set of primers and probe. It is important to frequently evaluate the performance of these tests and this can be done first by an in silico approach. Previously, we reported some mismatches between the oligonucleotides of publicly available RT-qPCR assays and SARS-CoV-2 genomes collected from GISAID and NCBI, potentially impacting proper detection of the virus. In the present study, 11 primers and probe sets investigated during the first study were evaluated again with 84,305 new SARS-CoV-2 unique genomes collected between June 2020 and January 2021. The lower inclusivity of the China CDC assay targeting the gene N has continued to decrease with new mismatches detected, whereas the other evaluated assays kept their inclusivity above 99%. Additionally, some mutations specific to new SARS-CoV-2 variants of concern were found to be located in oligonucleotide annealing sites. This might impact the strategy to be considered for future SARS-CoV-2 testing. Given the potential threat of the new variants, it is crucial to assess if they can still be correctly targeted by the primers and probes of the RT-qPCR assays. Our study highlights that considering the evolution of the virus and the emergence of new variants, an in silico (re-)evaluation should be performed on a regular basis. Ideally, this should be done for all the RT-qPCR assays employed for SARS-CoV-2 detection, including also commercial tests, although the primer and probe sequences used in these kits are rarely disclosed, which impedes independent performance evaluation.

Background: The transition to a biobased economy involving the depolymerization and fermentation of renewable agro-industrial sources is a challenge that can only be met by achieving the efficient hydrolysis of biomass to monosaccharides. In nature, lignocellulosic biomass is mainly decomposed by fungi. We recently identified six efficient cellulose degraders by screening fungi from Vietnam., Results: We characterized a high-performance cellulase-producing strain, with an activity of 0.06 U/mg, which was identified as a member of the Fusarium solani species complex linkage 6 (Fusarium metavorans), isolated from mangrove wood (FW16.1, deposited as DSM105788). The genome, representing nine potential chromosomes, was sequenced using PacBio and Illumina technology. In-depth secretome analysis using six different synthetic and artificial cellulose substrates and two agro-industrial waste products identified 500 proteins, including 135 enzymes assigned to five different carbohydrate-active enzyme (CAZyme) classes. The F. metavorans enzyme cocktail was tested for saccharification activity on pre-treated sugarcane bagasse, as well as untreated sugarcane bagasse and maize leaves, where it was complemented with the commercial enzyme mixture Accellerase 1500. In the untreated sugarcane bagasse and maize leaves, initial cell wall degradation was observed in the presence of at least 196 µg/mL of the in-house cocktail. Increasing the dose to 336 µg/mL facilitated the saccharification of untreated sugarcane biomass, but had no further effect on the pre-treated biomass., Conclusion: Our results show that F. metavorans DSM105788 is a promising alternative pre-treatment for the degradation of agro-industrial lignocellulosic materials. The enzyme cocktail promotes the debranching of biopolymers surrounding the cellulose fibers and releases reduced sugars without process disadvantages or loss of carbohydrates.

The basidiomycetous lipoxygenase Lox1 from Cyclocybe aegerita catalyzes the oxygenation of polyunsaturated fatty acids (PUFAs) with a high preference towards the C18-PUFA linoleic acid (C18:2 (ω-6)). In contrast, longer PUFAs, generally not present in the fungal cell such as eicosatrienoic acid (C20:3(ω-3)) and docosatrienoic acid (C22:3 (ω-3)), are converted with drastically lower activities. With site-directed mutagenesis, we were able to create two variants with enhanced activities towards longer chain PUFAs. The W330L variant showed a ~ 20 % increased specific activity towards C20:3(ω-3), while a ~ 2.5-fold increased activity against C22:3 (ω-3) was accomplished by the V581 variant.

Humans are mostly contaminated by Salmonella through the consumption of pork- and poultry-derived food products. Therefore, a strict monitoring of Salmonella serotypes in food-producing animals is needed to limit the transmission of the pathogen to humans. Additionally, Salmonella can lead to economic loss in the food sector. Previously, a genoserotyping method using the MOL-PCR and Luminex technology was developed for the identification of the 6 Salmonella serotypes, and their variants, subjected to an official control in the Belgian food sector. In this study, 3 additional assays using the same technology were developed for the rapid and cost-effective detection of 13 dangerous highly invasive serotypes or other serotypes frequently isolated from the Belgian poultry and pork sector, i.e. Agona, Anatum, Brandenburg, Choleraesuis, Derby, Enteritidis vaccine strains, Gallinarum var. Gallinarum/Pullorum, Livingstone, Mbandaka, Minnesota, Ohio, Rissen and Senftenberg. Moreover, the previously developed first MOL-PCR assay was improved for S. Paratyphi B and serogroup O:3 detection. Finally, a Decision Support System hosted by a web application was created for an automatic and objective interpretation of the Luminex raw data. The 3 new assays and the modifications of the first assay were validated with a 100% accuracy, using 553 Salmonella and non-Salmonella strains in total., Competing Interests: Declaration of competing interest All authors declare that they have no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A prerequisite for the transition toward a biobased economy is the identification and development of efficient enzymes for the usage of renewable resources as raw material. Therefore, different xylanolytic enzymes are important for efficient enzymatic hydrolysis of xylan-heteropolymers. A powerful tool to overcome the limited enzymatic toolbox lies in exhausting the potential of unexplored habitats. By screening a Vietnamese fungal culture collection of 295 undiscovered fungal isolates, 12 highly active xylan degraders were identified. One of the best xylanase producing strains proved to be an Aspergillus sydowii strain from shrimp shell (Fsh102), showing a specific activity of 0.6 U/mg. Illumina dye sequencing was used to identify our Fsh102 strain and determine differences to the A. sydowii CBS 593.65 reference strain. With activity based in-gel zymography and subsequent mass spectrometric identification, three potential proteins responsible for xylan degradation were identified. Two of these proteins were cloned from the cDNA and, furthermore, expressed heterologously in Escherichia coli and characterized. Both xylanases, were entirely different from each other, including glycoside hydrolases (GH) families, folds, substrate specificity, and inhibition patterns. The specific enzyme activity applying 0.1% birch xylan of both purified enzymes were determined with 181.1 ± 37.8 or 121.5 ± 10.9 U/mg for xylanase I and xylanase II, respectively. Xylanase I belongs to the GH11 family, while xylanase II is member of the GH10 family. Both enzymes showed typical endo-xylanase activity, the main products of xylanase I are xylobiose, xylotriose, and xylohexose, while xylobiose, xylotriose, and xylopentose are formed by xylanase II. Additionally, xylanase II showed remarkable activity toward xylotriose. Xylanase I is stable when stored up to 30°C and pH value of 9, while xylanase II started to lose significant activity stored at pH 9 after exceeding 3 days of storage. Xylanase II displayed about 40% activity when stored at 50°C for 24 h. The enzymes are tolerant toward mesophilic temperatures, while acting in a broad pH range. With site directed mutagenesis, the active site residues in both enzymes were confirmed. The presented activity and stability justify the classification of both xylanases as highly interesting for further development., (Copyright © 2020 Brandt, Ellinger, van Nguyen, Harder, Schlüter, Hahnke, Rühl, Schäfer and Gand.)

The current COronaVIrus Disease 2019 (COVID-19) pandemic started in December 2019. COVID-19 cases are confirmed by the detection of SARS-CoV-2 RNA in biological samples by RT-qPCR. However, limited numbers of SARS-CoV-2 genomes were available when the first RT-qPCR methods were developed in January 2020 for initial in silico specificity evaluation and to verify whether the targeted loci are highly conserved. Now that more whole genome data have become available, we used the bioinformatics tool SCREENED and a total of 4755 publicly available SARS-CoV-2 genomes, downloaded at two different time points, to evaluate the specificity of 12 RT-qPCR tests (consisting of a total of 30 primers and probe sets) used for SARS-CoV-2 detection and the impact of the virus' genetic evolution on four of them. The exclusivity of these methods was also assessed using the human reference genome and 2624 closely related other respiratory viral genomes. The specificity of the assays was generally good and stable over time. An exception is the first method developed by the China Center for Disease Control and prevention (CDC), which exhibits three primer mismatches present in 358 SARS-CoV-2 genomes sequenced mainly in Europe from February 2020 onwards. The best results were obtained for the assay of Chan et al. (2020) targeting the gene coding for the spiking protein (S). This demonstrates that our user-friendly strategy can be used for a first in silico specificity evaluation of future RT-qPCR tests, as well as verifying that the former methods are still capable of detecting circulating SARS-CoV-2 variants.

Salmonella is a major pathogen having a public health and economic impact in both humans and animals. Six serotypes of the Salmonella genus are mentioned in the Belgian and European regulation as to be rapidly excluded from the food chain (EU regulation N°2160/2003, Belgian royal decree 27/04/2017). The reference method for Salmonella serotyping, including slide-agglutination and biochemical tests, is time-consuming, expensive, not always objective, and therefore does not match the fast identification criteria required by the legislation. In this study, a molecular method, using genetic markers detected by Multiplex Oligonucleotide Ligation - PCR and Luminex technology, was developed for the identification of the 6 Salmonella serotypes and their variants subjected to an official control. The resulting method was validated with the analysis of 971 Salmonella isolated from different matrixes (human, animal, food or environment) and 33 non-Salmonella strains. The results were compared with the reference identifications, achieving an accuracy of 99.7%. The cost-effective high-throughput genoserotyping assay is performed in 1 day and generates objective results, thanks to the automatic interpretation of raw data using a barcode system. In conclusion, it is fully adapted to the implementation in first line laboratories and meets the requirements of the regulation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Natural products (NPs) from microorganisms have been important sources for discovering new therapeutic and chemical entities. While their corresponding biosynthetic gene clusters (BGCs) can be easily identified by gene-sequence-similarity-based bioinformatics strategies, the actual access to these NPs for structure elucidation and bioactivity testing remains difficult. Deletion of the gene encoding the RNA chaperone, Hfq, results in strains losing the production of most NPs. By exchanging the native promoter of a desired BGC against an inducible promoter in Δhfq mutants, almost exclusive production of the corresponding NP from the targeted BGC in Photorhabdus, Xenorhabdus and Pseudomonas was observed including the production of several new NPs derived from previously uncharacterized non-ribosomal peptide synthetases (NRPS). This easyPACId approach (easy Promoter Activated Compound Identification) facilitates NP identification due to low interference from other NPs. Moreover, it allows direct bioactivity testing of supernatants containing secreted NPs, without laborious purification., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Discriminating between D-tartrate fermenting and non-fermenting strains of Salmonella enterica subsp. enterica serotype Paratyphi B is of major importance as these two variants have different pathogenic profiles. While D-tartrate non-fermenting S. Paratyphi B isolates are the causative agent of typhoid-like fever, D-tartrate fermenting isolates (also called variant Java) of the same serotype trigger the less dangerous gastroenteritis. The determination of S. Paratyphi B variants requires a time-consuming process and complex biochemical tests. Therefore, a quadruplex real-time PCR method, based on the allelic discrimination of molecular markers selected from the scientific literature and from whole genome sequencing data produced in-house, was developed in this study, to be applied to Salmonella isolates. This method was validated with the analysis of 178 S. Paratyphi B (D-tartrate fermenting and non-fermenting) and other serotypes reaching an accuracy, compared with the classical methods, of 98% for serotyping by slide agglutination and 100% for replacement of the biochemical test. The developed real-time PCR permits to save time and to obtain an accurate identification of a S. Paratyphi B serotype and its D-tartrate fermenting profile, which is needed in routine laboratories for fast and efficient diagnostics.

Fungal strains are abundantly used throughout all areas of biotechnology and many of them are adapted to degrade complex biopolymers like chitin or lignocellulose. We therefore assembled a collection of 295 fungi from nine different habitats in Vietnam, known for its rich biodiversity, and investigated their cellulase, chitinase, xylanase and lipase activity. The collection consists of 70 isolates from wood, 55 from soil, 44 from rice straw, 3 found on fruits, 24 from oil environments (butchery), 12 from hot springs, 47 from insects as well as 27 from shrimp shells and 13 strains from crab shells. These strains were cultivated and selected by growth differences to enrich phenotypes, resulting in 211 visually different fungi. DNA isolation of 183 isolates and phylogenetic analysis was performed and 164 species were identified. All were subjected to enzyme activity assays, yielding high activities for every investigated enzyme set. In general, enzyme activity corresponded with the environment of which the strain was isolated from. Therefore, highest cellulase activity strains were isolated from wood substrates, rice straw and soil and similar substrate effects were observed for chitinase and lipase activity. Xylanase activity was similarly distributed as cellulase activity, but substantial activity was also found from fungi isolated from insects and shrimp shells. Seven strains displayed significant activities against three of the four tested substrates, while three degraded all four investigated carbon sources. The collection will be an important source for further studies. Therefore representative strains were made available to the scientific community and deposited in the public collection of the Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig., Competing Interests: The authors have declared that no competing interests exist.

In order to control food losses and wastage, monitoring the microbial diversity of food products, during processing and storage is important, as studies have highlighted the metabolic activities of some microorganisms which can lead to spoilage. Knowledge of this diversity can be greatly improved by using a metagenetic approach based on high throughput 16S rRNA gene sequencing, which enables a much higher resolution than culture-based methods. Moreover, the Jameson effect, a phenomenon described by Jameson in 1962, is often used to classify bacterial strains within an ecosystem. According to this, we have studied the bacterial microbiota of Belgian white pudding during storage at different temperatures using culture-dependent and independent methods. The product was inoculated with a mix of dominant strains previously isolated from this foodstuff at the end of its shelf life (Carnobacterium maltaromaticum, Lactobacillus fuchuensis, Lactobacillus graminis, Lactobacillus oligofermentans, Lactococcus lactis, Leuconostoc mesenteroides, Raoultella terrigena and Serratia sp.). Daily during 16days, the absolute abundance of inoculated strain was monitored by combining total count on plate agar and metagenetic analysis. The results were confirmed by qPCR analysis. The growth of each species was modelled for each temperature conditions, representative of good or bad storage practices. These data allowed the bacterial strains subdivision into three classes based on criteria of growth parameters for the studied temperature: the "dominant", the "subdominant" and the "inhibited" bacterial species, according to their maximal concentration (Nmax, log CFU/g), growth rate (μmax, 1/h) and time to reach the stationary phase (TRSP, days). Thereby, depending on the storage conditions, these data have permitted to follow intrinsically the evolution of each strain on the bacterial ecosystem of Belgian white pudding. Interestingly, it has shown that the reliability of the Jameson effect can be discussed. For example, at 4°C when Lactococcus lactis and Serratia sp. stopped growth at day 12, at the same time Carnobacterium maltaromaticum reached its maximal concentration and entered its stationary phase. In opposition to this, it can be noticed that in the same condition, the "sub-dominant" organisms continued their growth independently of the "dominant" species behaviour. In this case, the Jameson effect was not illustrated. This pattern is described for all storage conditions with the same strain classifications. These results highlighted the importance of combining metagenetic analysis and classical methods, with modelling, to offer a new tool for studying the evolution of microorganisms present in perishable food within different environmental conditions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Engineering cofactor specificity of enzymes is a promising approach that can expand the application of enzymes for biocatalytic production of industrially relevant chemicals. Until now, only NADPH-dependent imine reductases (IREDs) are known. This limits their applications to reactions employing whole cells as a cost-efficient cofactor regeneration system. For applications of IREDs as cell-free catalysts, (i) we created an IRED variant showing an improved activity for NADH. With rational design we were able to identify four residues in the (R)-selective IRED from Streptomyces GF3587 (IR-Sgf3587), which coordinate the 2'-phosphate moiety of the NADPH cofactor. From a set of 15 variants, the highest NADH activity was caused by the single amino acid exchange K40A resulting in a 3-fold increased acceptance of NADH. (ii) We showed its applicability using an immobilisate obtained either from purified enzyme or from lysate using the EziG(™) carriers. Applying the variant and NADH, we reached 88% conversion in a preparative scale biotransformation when employing 4% (w/v) 2-methylpyrroline. (iii) We demonstrated a one-enzyme cofactor regeneration approach using the achiral amine N-methyl-3-aminopentanone as a hydrogen donor co-substrate., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Among small GTPases from the Rho family, Cdc42, RAC, and Rho are well known to mediate a large variety of cellular processes linked with cancer biology through their ability to cycle between an inactive (GDP-bound) and an active (GTP-bound) state. Guanine nucleotide exchange factors (GEFs) stimulate the exchange of GDP for GTP to generate the activated form, whereas the GTPase-activating proteins (GAPs) catalyze GTP hydrolysis, leading to the inactivated form. Modulation of Rho GTPase activity following altered expression of RHO-GEFs and/or RHO-GAPs has already been reported in various human tumors. However, nothing is known about the Rho GTPase activity or the expression of their regulators in human pheochromocytomas, a neuroendocrine tumor (NET) arising from chromaffin cells of the adrenal medulla. In this study, we demonstrate, through an ELISA-based activity assay, that Rac1 and Cdc42 activities decrease in human pheochromocytomas (PCCs) compared with the matched adjacent non-tumor tissue. Furthermore, through quantitative mass spectrometry (MS) approaches, we show that the expression of two RHO-GEF proteins, namely ARHGEF1 and FARP1, is significantly reduced in tumors compared with matched non-tumor tissue, whereas ARHGAP36 expression is increased. Moreover, siRNA-based knockdown of ARHGEF1 and FARP1 in PC12 cells leads to a significant inhibition of Rac1 and Cdc42 activities, respectively. Finally, a principal component analysis (PCA) of our dataset was able to discriminate PCC from non-tumor tissue and indicates a close correlation between Cdc42/Rac1 activity and FARP1/ARHGEF1 expression. Altogether, our findings reveal for the first time the importance of modulation of Rho GTPase activities and expression of their regulators in human PCCs., (© 2016 Society for Endocrinology.)