Bi-directionalized promoter systems allow methanol-free production of hard-to-express peroxygenases with Komagataella Phaffii.

BACKGROUND: Heme-incorporating peroxygenases are responsible for electron transport in a multitude of organisms. Yet their application in biocatalysis is hindered due to their challenging recombinant production. Previous studies suggest Komagataella phaffi to be a suitable production host for heme-containing enzymes. In addition, co-expression of helper proteins has been shown to aid protein folding in yeast. In order to facilitate recombinant protein expression for an unspecific peroxygenase (AnoUPO), we aimed to apply a bi-directionalized expression strategy with Komagataella phaffii. RESULTS: In initial screenings, co-expression of protein disulfide isomerase was found to aid the correct folding of the expressed unspecific peroxygenase in K. phaffi. A multitude of different bi-directionalized promoter combinations was screened. The clone with the most promising promoter combination was scaled up to bioreactor cultivations and compared to a mono-directional construct (expressing only the peroxygenase). The strains were screened for the target enzyme productivity in a dynamic matter, investigating both derepression and mixed feeding (methanol-glycerol) for induction. Set-points from bioreactor screenings, resulting in the highest peroxygenase productivity, for derepressed and methanol-based induction were chosen to conduct dedicated peroxygenase production runs and were analyzed with RT-qPCR. Results demonstrated that methanol-free cultivation is superior over mixed feeding in regard to cell-specific enzyme productivity. RT-qPCR analysis confirmed that mixed feeding resulted in high stress for the host cells, impeding high productivity. Moreover, the bi-directionalized construct resulted in a much higher specific enzymatic activity over the mono-directional expression system. CONCLUSIONS: In this study, we demonstrate a methanol-free bioreactor production strategy for an unspecific peroxygenase, yet not shown in literature. Hence, bi-directionalized assisted protein expression in K. phaffii, cultivated under derepressed conditions, is indicated to be an effective production strategy for heme-containing oxidoreductases. This very production strategy might be opening up further opportunities for biocatalysis.

Applicability of non-invasive and live-cell holotomographic imaging on fungi.

The ability to acquire three-dimensional (3D) information of cellular structures without the need for fluorescent tags or staining makes holotomographic imaging a powerful tool in cellular biology. It provides valuable insights by measuring the refractive index (RI), an optical parameter describing the phase delay of light that passes through the living cell. Here, we demonstrate holotomographic imaging on industrial relevant ascomycete fungi and study their development and morphogenesis. This includes conidial germination, subcellular dynamics, and cytoplasmic flow during hyphal growth in Aspergillus niger. In addition, growth and budding of Aureobasidium pullulans cells are captured using holotomographic microscopy. Coupled to fluorescence imaging, lipid droplets, vacuoles, the mitochondrial network, and nuclei are targeted and analyzed in the 3D RI reconstructed images. While lipid droplets and vacuoles can be assigned to a specific RI pattern, mitochondria and nuclei were not pronounced. We show, that the lower sensitivity of RI measurements derives from the fungal cell wall that acts as an additional barrier for the illumination light of the microscope. After cell wall digest of hyphae and protoplast formation of A. niger expressing GFP-tagged histone H2A, location of nuclei could be determined by non-invasive RI measurements. Furthermore, we used coupled fluorescence microscopy to observe migration of nuclei in unperturbed hyphal segments and duplication during growth on a single-cell level. Detailed micromorphological studies in Saccharomyces cerevisiae and Trichoderma reesei are challenging due to cell size restrictions. Overall, holotomography opens up new avenues for exploring dynamic cellular processes in real time and enables the visualization of fungi from a new perspective.

Genomic deletions in Aureobasidium pullulans by an AMA1 plasmid for gRNA and CRISPR/Cas9 expression.

BACKGROUND: Aureobasidium pullulans is a generalist polyextremotolerant black yeast fungus. It tolerates temperatures below 0 °C or salt concentrations up to 18%, among other stresses. A. pullulans genome sequencing revealed a high potential for producing bioactive metabolites. Only few molecular tools exist to edit the genome of A. pullulans, hence it is important to make full use of its potential. Two CRISPR/Cas9 methods have been proposed for the protoplast-based transformation of A. pullulans. These methods require the integration of a marker gene into the locus of the gene to be deleted, when the deletion of this gene does not yield a selectable phenotype. We present the adaptation of a plasmid-based CRISPR/Cas9 system developed in Aspergillus niger for A. pullulans to create deletion strains. RESULTS: The A. niger CRISPR/Cas9 plasmid led to efficient genomic deletions in A. pullulans. In this study, strains with deletions ranging from 30 to 862 bp were obtained by using an AMA1 plasmid-based genome editing strategy. CONCLUSION: The CRISPR/Cas9 transformation system presented in this study provides new opportunities for strain engineering of A. pullulans. This system allows expression of Cas9 and antibiotic resistance while being easy to adapt. This strategy could open the path to intensive genomic engineering in A. pullulans.

Exploration of Trichoderma reesei as an alternative host for erythritol production.

BACKGROUND: Erythritol, a natural polyol, is a low-calorie sweetener synthesized by a number of microorganisms, such as Moniliella pollinis. Yet, a widespread use of erythritol is limited by high production costs due to the need for cultivation on glucose-rich substrates. This study explores the potential of using Trichoderma reesei as an alternative host for erythritol production, as this saprotrophic fungus can be cultivated on lignocellulosic biomass residues. The objective of this study was to evaluate whether such an alternative host would lead to a more sustainable and economically viable production of erythritol by identifying suitable carbon sources for erythritol biosynthesis, the main parameters influencing erythritol biosynthesis and evaluating the feasibility of scaling up the defined process. RESULTS: Our investigation revealed that T. reesei can synthesize erythritol from glucose but not from other carbon sources like xylose and lactose. T. reesei is able to consume erythritol, but it does not in the presence of glucose. Among nitrogen sources, urea and yeast extract were more effective than ammonium and nitrate. A significant impact on erythritol synthesis was observed with variations in pH and temperature. Despite successful shake flask experiments, the transition to bioreactors faced challenges, indicating a need for further scale-up optimization. CONCLUSIONS: While T. reesei shows potential for erythritol production, reaching a maximum concentration of 1 g/L over an extended period, its productivity could be improved by optimizing the parameters that affect erythritol production. In any case, this research contributes valuable insights into the polyol metabolism of T. reesei, offering potential implications for future research on glycerol or mannitol production. Moreover, it suggests a potential metabolic association between erythritol production and glycolysis over the pentose phosphate pathway.

Complete genome of Microbacterium plantarum CoE-159-22, isolated from traditionally produced Montenegrin cheese.

Microbacterium plantarum (M. plantarum) was recently described as a new species isolated from copper globemallow (Sphaeralcea angustifolia). Here, we report the complete genome of M. plantarum CoE-159-22, which was obtained from traditionally produced Montenegrin cheese.

Radiation dose fractionation and its potential hormetic effects on male Glossina palpalis gambiensis (Diptera: Glossinidae): a comparative study of reproductive and flight quality parameters.

One of the most critical factors for implementing the sterile insect technique for the management of tsetse is the production of large quantities of highly competitive sterile males in the field. Several factors may influence the biological quality of sterile males, but optimizing the irradiation protocols to limit unwanted somatic cell damage could improve male performance. This study evaluated the effect of fractionation of gamma radiation doses on the fertility and flight quality of male Glossina palpalis gambiensis. Induced sterility was assessed by mating irradiated males with virgin fertile females. Flight quality was assessed using a standard protocol. The male flies were irradiated as pupae on day 23-27 post larviposition with 110 Gy, either in a single dose or in fractionations of 10 + 100 Gy and 50 + 60 Gy separated by 1-, 2- and 3-day intervals or 55 + 55 Gy separated by 4-, 8-, and 24-hour intervals. All treatments induced more than 90% sterility in females mated with irradiated males, as compared with untreated males. No significant differences were found in emergence rate or flight propensity between fractionated and single radiation doses, nor between the types of fractionations. Overall, the 50(D0) + 60(D1) Gy dose showed slightly higher induced sterility, flight propensity, and survival of males under feeding regime. Dose fractionation resulted in only small improvements with respect to flight propensity and survival, and this should be traded off with the required increase in labor that dose fractionation entails, especially in larger control programs.

Title: Fractionnement de la dose de rayonnement et ses effets hormétiques potentiels sur les Glossina palpalis gambiensis mâles (Diptera : Glossinidae) : une étude comparative des paramètres de reproduction et de qualité de vol. Abstract: L'un des facteurs les plus critiques pour la mise en œuvre de la technique de l'insecte stérile pour la gestion des glossines est la production de grandes quantités de mâles stériles hautement compétitifs sur le terrain. Plusieurs facteurs peuvent influencer la qualité biologique des mâles stériles, mais l'optimisation des protocoles d'irradiation pour limiter les dommages indésirables aux cellules somatiques pourrait améliorer les performances des mâles. Cette étude a évalué l'effet du fractionnement de la dose d'irradiation gamma sur la fertilité et la qualité de vol des mâles de Glossina palpalis gambiensis. La stérilité induite a été évaluée en accouplant des mâles irradiés avec des femelles vierges et fertiles. La qualité du vol a été évaluée à l'aide d'un protocole standard. Les mouches mâles ont été irradiées sous forme de pupes agées de 23 à 27 jours après la larviposition avec 110 Gy, soit en dose unique, soit en fractions de 10 + 100 Gy et 50 + 60 Gy séparées par 1, 2 et 3 jours ou 55 + 55 Gy séparés par des intervalles de 4, 8 et 24 heures. Tous les traitements ont induit plus de 90 % de stérilité chez les femelles accouplées avec des mâles irradiés par rapport aux mâles non traités. Aucune différence significative n'a été trouvée dans le taux d'émergence ou la propension au vol entre les doses d'irradiation fractionnées et uniques ni entre les types de fractionnements. Dans l'ensemble, la dose de 50 (J0) + 60 (J1) Gy a montré une stérilité induite, une propension à voler et une survie légèrement plus élevées chez les mâles sous régime alimentaire. Le fractionnement de dose n'a entraîné que de légères améliorations en ce qui concerne la propension à voler et la survie, et cela devrait être compensé par l'augmentation nécessaire du travail qu'implique le fractionnement de dose, en particulier dans les programmes de contrôle de grande envergure.

Protein secretion and associated stress in industrially employed filamentous fungi.

Application of filamentous fungi for the production of commercial enzymes such as amylase, cellulase, or xylanase is on the rise due to the increasing demand to degrade several complex carbohydrates as raw material for biotechnological processes. Also, protein production by fungi for food and feed gains importance. In any case, the protein production involves both cellular synthesis and secretion outside of the cell. Unfortunately, the secretion of proteins or enzymes can be hampered due to accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) as a result of too high synthesis of enzymes or (heterologous) protein expression. To cope with this ER stress, the cell generates a response known as unfolded protein response (UPR). Even though this mechanism should re-establish the protein homeostasis equivalent to a cell under non-stress conditions, the enzyme expression might still suffer from repression under secretory stress (RESS). Among eukaryotes, Saccharomyces cerevisiae is the only fungus, which is studied quite extensively to unravel the UPR pathway. Several homologs of the proteins involved in this signal transduction cascade are also found in filamentous fungi. Since RESS seems to be absent in S. cerevisiae and was only reported in Trichoderma reesei in the presence of folding and glycosylation inhibitors such as dithiothreitol and tunicamycin, more in-depth study about this mechanism, specifically in filamentous fungi, is the need of the hour. Hence, this review article gives an overview on both, protein secretion and associated stress responses in fungi. KEY POINTS: • Enzymes produced by filamentous fungi are crucial in industrial processes • UPR mechanism is conserved among many fungi, but mediated by different proteins • RESS is not fully understood or studied in industrially relevant filamentous fungi.

Enterococcus montenegrensis sp. nov., isolated from artisanal Montenegrin dry sausage.

A novel, Gram-positive, facultative anaerobe, coccoid and non-motile bacterium, designated as CoE-012-22T was isolated from dried beef sausage (the original name in Montenegro is Govedji Kulen) manufactured in the municipality of Rozaje (Montenegro) in 2021. Cells of this strain were oxidase- and catalase-negative. Growth occurred at 4-50 °C, at pH 5.0-8.0 and with 0-6.5 % (w/v) NaCl in diverse growth media. MALDI-TOF analysis identified the strain as Enterococcus canintestini (log score 2). Phylogenetic analysis of the 16S rRNA gene and whole genome sequences assigned the strain to the genus Enterococcus. The closest relatives were E. canintestini DSM 21207T and E. dispar ATCC 51266T with 16S rRNA gene sequence pairwise similarities of 99.34 and 98.59 %, respectively. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between isolate CoE-012-22T and other enterococci species were below the thresholds for species delineation thresholds (95.0 % ANI; 70.0 % dDDH) with maximum identities of 84.13 % (ANIb), 86.43 % (ANIm) and 28.4 % (dDDH) to E. saigonensis JCM 31193T and 70.97 % (ANIb), 88.99 % (ANIm) and 32.4 % (dDDH) to E. malodoratus ATCC 43197T. Two unknown Enterococcus isolates, Enterococcus sp. MJM12 and Enterococcus SMC-9, showed identities of 99.87 and 99.94 % (16S rRNA), 98.57 and 98.65 % (ANIb), 98.93 and 99.02 % (ANIm), and 89.8 and 90.0 % (dDDH) to strain CoE-012-22T and can therefore be regarded as the same species. Based on the characterization results, strain CoE-012-22T was considered to represent a novel species, for which the name Enterococcus montenegrensis sp. nov. is proposed. The type strain is CoE-012-22T (=DSM 115843T=NCIMB 15468T).

Prevalence of Spiroplasma and interaction with wild Glossina tachinoides microbiota.

Tsetse flies (Diptera: Glossinidae) are vectors of the tropical neglected diseases sleeping sickness in humans and nagana in animals. The elimination of these diseases is linked to control of the vector. The sterile insect technique (SIT) is an environment-friendly method that has been shown to be effective when applied in an area-wide integrated pest management approach. However, as irradiated males conserve their vectorial competence, there is the potential risk of trypanosome transmission with their release in the field. Analyzing the interaction between the tsetse fly and its microbiota, and between different microbiota and the trypanosome, might provide important information to enhance the fly's resistance to trypanosome infection. This study on the prevalence of Spiroplasma in wild populations of seven tsetse species from East, West, Central and Southern Africa showed that Spiroplasma is present only in Glossina fuscipes fuscipes and Glossina tachinoides. In G. tachinoides, a significant deviation from independence in co-infection with Spiroplasma and Trypanosoma spp. was observed. Moreover, Spiroplasma infections seem to significantly reduce the density of the trypanosomes, suggesting that Spiroplasma might enhance tsetse fly's refractoriness to the trypanosome infections. This finding might be useful to reduce risks associated with the release of sterile males during SIT implementation in trypanosome endemic areas.

Title: Prévalence de Spiroplasma et interaction avec le microbiote des Glossina tachinoides sauvages. Abstract: Les mouches tsé-tsé (Diptera : Glossinidae) sont les vecteurs de maladies tropicales négligées, la maladie du sommeil chez l'homme et la nagana chez les animaux. L'élimination de ces maladies est liée à la lutte contre le vecteur. La technique de l'insecte stérile (TIS) est une méthode respectueuse de l'environnement qui s'est révélée efficace lorsqu'elle est appliquée dans le cadre d'une approche de lutte antiparasitaire intégrée à l'échelle d'une zone. Cependant, comme les mâles irradiés conservent leur compétence vectorielle, il existe un risque potentiel de transmission des trypanosomes lors de la libération des mâles sur le terrain. L'analyse de l'interaction entre la mouche tsé-tsé et son microbiote, et entre différents microbiotes et le trypanosome, pourrait fournir des informations importantes pour améliorer la résistance de la mouche à l'infection trypanosomienne. Cette étude sur la prévalence de Spiroplasma dans les populations sauvages de sept espèces de glossines d'Afrique de l'Est, de l'Ouest, centrale et australe a montré que Spiroplasma est présent uniquement chez Glossina fuscipes fuscipes et Glossina tachinoides. Chez G. tachinoides, un écart significatif par rapport à l'indépendance dans la co-infection par Spiroplasma et Trypanosoma spp. a été observé. De plus, les infections à Spiroplasma semblent réduire considérablement la densité des trypanosomes, ce qui suggère que Spiroplasma pourrait renforcer le caractère réfractaire de la mouche tsé-tsé aux infections trypanosomiennes. Cette découverte pourrait être utile pour réduire le risque associé à la libération de mâles stériles lors de la mise en œuvre de la TIS dans les zones d'endémie trypanosomienne.

X-rays are as effective as gamma-rays for the sterilization of Glossina palpalis gambiensis Vanderplank, 1911 (Diptera: Glossinidae) for use in the sterile insect technique.

An area-wide integrated pest management strategy with a sterile insect technique (SIT) component requires a radiation source for the sterilisation of male insects. Self-contained gamma irradiators, which were exclusively used in past SIT programmes, are now facing increasing constraints and challenges due to stringent regulations. As a potential alternative, new generation high output X-ray irradiators have been proposed. The feasibility of using X-ray irradiators was assessed by comparing the effects of both gamma- and X-ray irradiators on biological parameters of Glossina palpalis gambiensis (Vanderplank, 1911), that are important for SIT applications. The gamma irradiator Foss Model 812 and two X-ray irradiators, the Rad Source 2400 and the blood irradiator Raycell Mk2 were used. Glossina palpalis gambiensis males were exposed to radiation as pupae. A radiation dose of 110 Gy or above induced more than 97% sterility in females that mated with the irradiated males for all the irradiators. Adult emergence rate, flight propensity, survival and mating performance did not differ between gamma- and X-rays irradiators. These results suggest that irradiating pupae with a dose of 110 Gy is optimal for both gamma-and X-ray irradiators used in this study, to achieve a sterility of approximately 99%. Similar research on other tsetse species could gradually phase out the use of gamma-ray irradiators in favour of X-rays irradiators, especially for smaller SIT programmes.

Development and characterization of microsatellite markers for the tsetse species Glossina brevipalpis and preliminary population genetics analyses.

Tsetse flies, the vectors of African trypanosomes are of key medical and economic importance and one of the constraints for the development of Africa. Tsetse fly control is one of the most effective and sustainable strategies used for controlling the disease. Knowledge about population structure and level of gene flow between neighbouring populations of the target vector is of high importance to develop appropriate strategies for implementing effective management programmes. Microsatellites are commonly used to identify population structure and assess dispersal of the target populations and have been developed for several tsetse species but were lacking for Glossina brevipalpis. In this study, we screened the genome of G. brevipalpis to search for suitable microsatellite markers and nine were found to be efficient enough to distinguish between different tsetse populations. The availability of these novel microsatellite loci will help to better understand the population biology of G. brevipalpis and to assess the level of gene flow between different populations. Such information will help with the development of appropriate strategies to implement the sterile insect technique (SIT) in the framework of an area-wide integrated pest management (AW-IPM) approach to manage tsetse populations and ultimately address the trypanosomoses problem in these targeted areas.

Title: Développement et caractérisation de marqueurs microsatellites pour l'espèce de mouche tsé-tsé Glossina brevipalpis et analyses génétiques préliminaires des populations. Abstract: Les mouches tsé-tsé, vecteurs des trypanosomes africains, sont d'une importance médicale et économique majeure et l'une des contraintes pour le développement de l'Afrique. La lutte contre la mouche tsé-tsé est l'une des stratégies les plus efficaces et durables utilisées pour contrôler la maladie. La connaissance de la structure de la population et du niveau de flux de gènes entre les populations voisines du vecteur cible est d'une grande importance pour développer des stratégies appropriées pour la mise en œuvre de programmes de gestion efficaces. Les microsatellites sont couramment utilisés pour identifier la structure de la population et évaluer la dispersion des populations cibles et ont été développés pour plusieurs espèces de glossines mais manquaient pour Glossina brevipalpis. Dans cette étude, nous avons criblé le génome de G. brevipalpis pour rechercher des marqueurs microsatellites appropriés et neuf ont été trouvés suffisamment efficaces pour faire la distinction entre différentes populations de glossines. La disponibilité de ces nouveaux locus microsatellites aidera à mieux comprendre la biologie des populations de G. brevipalpis et à évaluer le niveau de flux de gènes entre différentes populations. Ces informations aideront à l'élaboration de stratégies appropriées pour mettre en œuvre la technique de l'insecte stérile dans le cadre d'une approche de lutte antiparasitaire intégrée à l'échelle de la zone pour gérer les populations de glossines et, en fin de compte, résoudre le problème des trypanosomoses dans les zones concernées.

The phenomenon of strain degeneration in biotechnologically relevant fungi.

Fungi are widely exploited for large-scale production in the biotechnological industry to produce a diverse range of substances due to their versatility and relative ease of growing on various substrates. The occurrence of a phenomenon-the so-called fungal strain degeneration-leads to the spontaneous loss or decline of production capacity and results in an economic loss on a tremendous scale. Some of the most commonly applied genera of fungi in the biotechnical industry, such as Aspergillus, Trichoderma, and Penicillium, are threatened by this phenomenon. Although fungal degeneration has been known for almost a century, the phenomenon and its underlying mechanisms still need to be understood. The proposed mechanisms causing fungi to degenerate can be of genetic or epigenetic origin. Other factors, such as culture conditions, stress, or aging, were also reported to have an influence. This mini-review addresses the topic of fungal degeneration by describing examples of productivity losses in biotechnical processes using Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, and Penicillium chrysogenum. Further, potential reasons, circumvention, and prevention methods are discussed. This is the first mini-review which provides a comprehensive overview on this phenomenon in biotechnologically used fungi, and it also includes a collection of strategies that can be useful to minimize economic losses which can arise from strain degeneration. KEY POINTS: • Spontaneous loss of productivity is evident in many fungi used in biotechnology. • The properties and mechanisms underlying this phenomenon are very versatile. • Only studying these underlying mechanisms enables the design of a tailored solution.

Evaluation of reference genes for transcript analyses in Komagataella phaffii (Pichia pastoris).

BACKGROUND: The yeast Komagataella phaffii (Pichia pastoris) is routinely used for heterologous protein expression and is suggested as a model organism for yeast. Despite its importance and application potential, no reference gene for transcript analysis via RT-qPCR assays has been evaluated to date. In this study, we searched publicly available RNASeq data for stably expressed genes to find potential reference genes for relative transcript analysis by RT-qPCR in K. phaffii. To evaluate the applicability of these genes, we used a diverse set of samples from three different strains and a broad range of cultivation conditions. The transcript levels of 9 genes were measured and compared using commonly applied bioinformatic tools. RESULTS: We could demonstrate that the often-used reference gene ACT1 is not very stably expressed and could identify two genes with outstandingly low transcript level fluctuations. Consequently, we suggest the two genes, RSC1, and TAF10 to be simultaneously used as reference genes in transcript analyses by RT-qPCR in K. phaffii in future RT-qPCR assays. CONCLUSION: The usage of ACT1 as a reference gene in RT-qPCR analysis might lead to distorted results due to the instability of its transcript levels. In this study, we evaluated the transcript levels of several genes and found RSC1 and TAF10 to be extremely stable. Using these genes holds the promise for reliable RT-qPCR results.

Suitability of Raycell MK2 Blood X-ray Irradiator for the Use in the Sterile Insect Technique: Dose Response in Fruit Flies, Tsetse Flies and Mosquitoes.

The sterile insect technique (SIT) is based on the inundatory field release of a target pest following their reproductive sterilization via exposure to radiation. Until recently, gamma irradiation from isotopic sources has been the most widely used in SIT programs. As isotopic sources are becoming increasingly expensive, especially for small programs, and regulations surrounding their procurement and shipment increasingly strict, irradiation capacity is one of the limiting factors in smaller or newly developing SIT projects. For this reason, the possibility of using X-ray irradiators has been evaluated in the recent decade. The availability of "off-the-shelf" blood X-ray irradiators that meet the technical requirements for insect irradiation can provide irradiation capacity for those SIT projects in which the acquisition of gamma ray irradiators is not feasible. Following the recent technical characterization of a Raycell MK2 X-ray blood irradiator, it was found in this study, that MK2 instruments were suitable for the sterilization of fruit flies, tsetse flies and mosquitoes, inducing comparable, even slightly higher, sterility levels compared to those achieved by gamma ray irradiation. This, together with its estimated processing efficiency, shows that MK2 irradiators are suitable for small- to mid-sized SIT programs.

Draft Genome Sequence of Enterococcus dispar CoE-457-22, Isolated from Traditionally Produced Montenegrin Dry Sausage.

Enterococcus dispar was isolated for the first time from synovial fluid and stool cultures and described as a new species in 1991. Here, we report the genome of E. dispar CoE-457-22, which was obtained from traditionally produced Montenegrin dry sausage (sudzuk).

In Vitro Characterization of a Nuclear Receptor-like Domain of the Xylanase Regulator 1 from Trichoderma reesei.

Engineering transcription factors is an interesting research target gaining increasing attention, such as in the case of industrially used organisms. With respect to sustainability, biomass-degrading saprophytic fungi, such as Trichoderma reesei, are promising industrial work horses because they exhibit a high secretory capacity of native and heterologously expressed enzymes and compounds. A single-point mutation in the main transactivator of xylanase and cellulase expressions in T. reesei Xyr1 led to a strongly deregulated and enhanced xylanase expression. Circular dichroism spectroscopy revealed a change in secondary structure caused by this mutation. According to electrophoretic mobility shift assays and determination of the equilibrium-binding constants, the DNA-binding affinity of the mutated Xyr1 was considerably reduced compared to the wild-type Xyr1. Both techniques were also used to investigate the allosteric response to carbohydrates (D-glucose-6-phosphate, D-xylose, and sophorose) signalling the repression or induction of Xyr1 target genes. The mutated Xyr1 no longer exhibited a conformational change in response to these carbohydrates, indicating that the observed deregulation is not a simple matter of a change in DNA-binding of the transactivator. Altogether, we postulate that the part of Xyr1 where the mutation is located functions as a nuclear receptor-like domain that mediates carbohydrate signals and modulates the Xyr1 transactivating activity.

Interactions between Glossina pallidipes salivary gland hypertrophy virus and tsetse endosymbionts in wild tsetse populations.

BACKGROUND: Tsetse control is considered an effective and sustainable tactic for the control of cyclically transmitted trypanosomosis in the absence of effective vaccines and inexpensive, effective drugs. The sterile insect technique (SIT) is currently used to eliminate tsetse fly populations in an area-wide integrated pest management (AW-IPM) context in Senegal. For SIT, tsetse mass rearing is a major milestone that associated microbes can influence. Tsetse flies can be infected with microorganisms, including the primary and obligate Wigglesworthia glossinidia, the commensal Sodalis glossinidius, and Wolbachia pipientis. In addition, tsetse populations often carry a pathogenic DNA virus, the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) that hinders tsetse fertility and fecundity. Interactions between symbionts and pathogens might affect the performance of the insect host. METHODS: In the present study, we assessed associations of GpSGHV and tsetse endosymbionts under field conditions to decipher the possible bidirectional interactions in different Glossina species. We determined the co-infection pattern of GpSGHV and Wolbachia in natural tsetse populations. We further analyzed the interaction of both Wolbachia and GpSGHV infections with Sodalis and Wigglesworthia density using qPCR. RESULTS: The results indicated that the co-infection of GpSGHV and Wolbachia was most prevalent in Glossina austeni and Glossina morsitans morsitans, with an explicit significant negative correlation between GpSGHV and Wigglesworthia density. GpSGHV infection levels > 103.31 seem to be absent when Wolbachia infection is present at high density (> 107.36), suggesting a potential protective role of Wolbachia against GpSGHV. CONCLUSION: The result indicates that Wolbachia infection might interact (with an undefined mechanism) antagonistically with SGHV infection protecting tsetse fly against GpSGHV, and the interactions between the tsetse host and its associated microbes are dynamic and likely species specific; significant differences may exist between laboratory and field conditions.

A bioinformatic-assisted workflow for genome-wide identification of ncRNAs.

With the upcoming of affordable Next-Generation Sequencing technologies, the number of known non-protein coding RNAs increased drastically in recent years. Different types of non-coding RNAs (ncRNAs) emerged as key players in the regulation of gene expression on the RNA-RNA, RNA-DNA as well as RNA-protein level, ranging from involvement in chromatin remodeling and transcription regulation to post-transcriptional modifications. Prediction of ncRNAs involves the use of several bioinformatics tools and can be a daunting task for researchers. This led to the development of analysis pipelines such as UClncR and lncpipe. However, these pipelines are limited to datasets from human, mouse, zebrafish or fruit fly and are not able to analyze RNA sequencing data from other organisms. In this study, we developed the analysis pipeline Pinc (Pipeline for prediction of ncRNA) as an enhanced tool to predict ncRNAs based on sequencing data by removing transcripts that show protein-coding potential. Additionally, a feature for differential expression analysis of annotated genes as well as for identification of novel ncRNAs is implemented. Pinc uses Nextflow as a framework and is built with robust and well-established analysis tools. This will allow researchers to utilize sequencing data from every organism in order to reliably identify ncRNAs.

Fast and efficient CRISPR-mediated genome editing in Aureobasidium using Cas9 ribonucleoproteins.

Species of the genus Aureobasidium are ubiquitous, polyextremotolerant, "yeast-like" ascomycetes used for the industrial production of pullulan and other products and as biocontrol agents in agriculture. Their application potential and wide-spread occurrence make Aureobasidium spp. interesting study objects. The availability of a fast and efficient genome editing method is an obvious advantage for future basic and applied research on Aureobasidium. In this study, we describe the development of a CRISPR/Cas9-based genome editing method using ribonucleoproteins (RNPs) in A. pullulans and A. melanogenum. We demonstrate that this method can be used for single and multiplex genome editing using only RNPs by targeting URA3 (encoding for orotidine-5'-phosphate decarboxylase), ADE2 (encoding for phosphoribosylaminoimidazole carboxylase) and ARG4 (encoding for argininosuccinate lyase). We demonstrate the applicability of Trichoderma reesei pyr4 and Aspergillus fumigatus pyrG to complement the URA3 deficiency. Further, we show that using RNPs improves the homologous recombination rate and 20 bp long homologous flanks are sufficient. Therefore, the repair cassettes can be constructed by a single PCR, abolishing the need for laborious and time-consuming cloning, which is necessary for previously described methods for CRISPR-mediated genome editing in these fungi. The here presented method allows fast and efficient genome editing for gene deletions, modifications, and insertions in Auresobasidium with a minimized risk of off-target effects.