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Background Biocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET). Microbial whole-cell biocatalysts for simultaneous PET degradation and growth on PET monomers would offer a one-step solution toward PET recycling or upcycling. We set out to engineer the industry-proven bacterium Pseudomonas putida for (i) metabolism of PET monomers as sole carbon sources, and (ii) efficient extracellular expression of PET hydrolases. We pursued this approach for both PET and the related polyester polybutylene adipate co-terephthalate (PBAT), aiming to learn about the determinants and potential applications of bacterial polyester-degrading biocatalysts. Results P. putida was engineered to metabolize the PET and PBAT monomer terephthalic acid (TA) through genomic integration of four tphII operon genes from Comamonas sp. E6. Efficient cellular TA uptake was enabled by a point mutation in the native P. putida membrane transporter MhpT. Metabolism of the PET and PBAT monomers ethylene glycol and 1,4-butanediol was achieved through adaptive laboratory evolution. We then used fast design-build-test-learn cycles to engineer extracellular PET hydrolase expression, including tests of (i) the three PET hydrolases LCC, HiC, and IsPETase; (ii) genomic versus plasmid-based expression, using expression plasmids with high, medium, and low cellular copy number; (iii) three different promoter systems; (iv) three membrane anchor proteins for PET hydrolase cell surface display; and (v) a 30-mer signal peptide library for PET hydrolase secretion. PET hydrolase surface display and secretion was successfully engineered but often resulted in host cell fitness costs, which could be mitigated by promoter choice and altering construct copy number. Plastic biodegradation assays with the best PET hydrolase expression constructs genomically integrated into our monomer-metabolizing P. putida strains resulted in various degrees of plastic depolymerization, although self-sustaining bacterial growth remained elusive. Conclusion Our results show that balancing extracellular PET hydrolase expression with cellular fitness under nutrient-limiting conditions is a challenge. The precise knowledge of such bottlenecks, together with the vast array of PET hydrolase expression tools generated and tested here, may serve as a baseline for future efforts to engineer P. putida or other bacterial hosts towards becoming efficient whole-cell polyester-degrading biocatalysts., Microbial Cell Factories, 21 (1), ISSN:1475-2859

This review deals with the mechanism of antagonistic action of bacterial and fungal biocontrol agents such as the production of antibiotics, siderophores, enzyme secretion, competition for nutritio...

Background Fungal saccharification of lignocellulosic biomass occurs concurrently with the secretion of a diverse collection of proteins, together functioning as a catalytic system to liberate soluble sugars from insoluble composite biomaterials. How different fungi respond to different substrates is of fundamental interest to the developing biomass saccharification industry. Among the cornerstones of fungal enzyme systems are the highly expressed cellulases (endo-β-glucanases and cellobiohydrolases). Recently, a cyclophellitol-derived activity-based probe (ABP-Cel) was shown to be a highly sensitive tool for the detection and identification of cellulases. Results Here we show that ABP-Cel enables endo-β-glucanase profiling in diverse fungal secretomes. In combination with established ABPs for β-xylanases and β-d-glucosidases, we collected multiplexed in-gel fluorescence activity-based protein profiles of 240 secretomes collected over ten days from biological replicates of ten different basidiomycete fungi grown on maltose, wheat straw, or aspen pulp. Our results reveal the remarkable dynamics and unique enzyme fingerprints associated with each species substrate combination. Chemical proteomic analysis identifies significant arsenals of cellulases secreted by each fungal species during growth on lignocellulosic biomass. Recombinant production and characterization of a collection of probe-reactive enzymes from GH5, GH10, and GH12 confirm that ABP-Cel shows broad selectivity towards enzymes with endo-β-glucanase activity. Conclusion Using small-volume samples with minimal sample preparation, the results presented here demonstrate the ready accessibility of sensitive direct evidence for fungal enzyme secretion during early stages of growth on complex lignocellulosic substrates.

Many challenges are faced in the conversion of biomass into advanced biofuels, one of which is finding the correct organism for the job. The filamentous fungus Aspergillus niger has been chosen as a biocatalyst for cellulose, hemicellulose, and lignin degradation because it can secrete numerous hydrolytic enzymes, such as lignin-degrading enzymes and, in particular, laccase enzymes (1). However, low transformation efficiency has hindered efforts to unlock the full potential of this organism. This thesis presents a novel method to efficiently edit the genome of A. niger that overcomes several of the current challenges encountered when using CRISPR/Cas9 (2). We designed a genetic construct that is efficient and precise concerning knockout efficiency and phenotype control and demonstrated its utility for genetic knockouts, integrations, and alterations. Using the new CRISPR/Cas9 toolbox, we developed a high throughput platform to transform A. niger using robotics. We then transformed a library of 81 laccases that could be potentially secreted by A. niger in two different strain backgrounds and obtained six new laccases secreted by A. niger. We engineered a strain that increased total protein secretion fourfold and extracellular laccase activity threefold. We are currently applying for a patent for this discovery. We characterized each secreted enzyme by downscaling in micro-culture to study their optimum efficiency (e.g., media, pH, and fermentation time). These discoveries bring us significantly closer to generating strains that can produce a cocktail of laccases to unlock recalcitrant biomass for downstream processing. Additionally, the methods we developed will enable the rapid building and testing of genetic variants in A. niger for metabolic engineering, synthetic biology, and many other applications.

Trypanosoma brucei are protozoan parasites infecting mammals in Sub-Saharan Africa causing the disease known as Sleeping sickness. The parasites are transmitted between the mammal hosts through bites of an insect vector. The transmission from insect to mammal is one out of many steps in a complex life cycle causing numbers of metabolic and physiological changes. While the majority of a population is preparing to reenter the insect vector, a fraction of the population continuously adapt to the immune system of the host, showing a heterogeneous pattern within the population. Though numerous analytical methods for studying single cells are continuously developed, little is still know of T. bruceion a single-cell level. Droplet-based microfluidicsis a high-throughput tool for single cell analysis emerging within the last decades. We here developed an analytical method for quantitative measurements at the single cell level based on encapsulation and cultivation of T. brucei in microcompartment droplets. We described a microfluidic system for single-cell encapsulation and analysis based on fluorescence measurements. We first showed the survival of T. brucei for several hours to days in droplets with influence on both survival and growth by the droplet size. We unraveled various growth patterns within a population, and found that droplet cultivation of trypanosomes revealed highly dividing cell variants with10-fold higher cell densities compared to standard cultivation techniques. We further combined the encapsulation of T. brucei with an enzymatic detection assay to study protease secretion. Using a fluorescence based substrate allowed detection of protease activity secreted from single cells. Lastly, we tested the setup in two additional applications both used on single-cell T. brucei. Conclusively, droplet-based microfluidics have shown promising as an analytical tool for trypanosome cultivation and analysis with a further potential for high-throughput single-cell analysis., Trypanosoma brucei est un parasite protozoaire infectant les mammifères en Afrique subsaharienne et provoquant la maladie connue sous le nom de maladie du sommeil. Les parasites sont transmis entre les hôtes mammifères par les piqûres d’un insecte vecteur. La transmission de l’insecte au mammifère est l’une des nombreuses étapes d’un cycle de vie complexe provoquant de nombreux changements métaboliques et physiologiques. Alors que la majorité d’une population se prépare à réintégrer l’insecte vecteur, une fraction de la population s’adapte en permanence au système immunitaire de l’hôte, montrant un schéma hétérogène au sein de la population. Bien que de nombreuses méthodes analytiques soient continuellement développées pour étudier les cellules individuelles, on sait encore peu de choses sur T. brucei au niveau cellulaire. La microfluidique en gouttes est un outil à haut débit pour l’analyse de cellules uniques qui a émergé au cours des dernières décennies. Nous avons développé ici une méthode analytique pour les mesures quantitatives au niveau de la cellule unique basées sur l’encapsulation et la culture de T. brucei dans des gouttes agissant comme des microcompartiments. Nous avons décrit un système microfluidique pour l’encapsulation et l’analyse de cellules individuelles basées sur des mesures de fluorescence. Nous avons d’abord montré la survie de T. brucei pendant plusieurs heures à plusieurs jours dans des gouttelettes avec une influence à la fois sur la survie et la croissance par la taille des gouttelettes. Nous avons observé divers modèles de croissance au sein d’une population et avons découvert que la culture par gouttes de trypanosomes révélait des variants cellulaires à division élevée avec des densités cellulaires 10 fois plus élevées que les techniques de culture standard. Nous avons en outre combiné l’encapsulation de T.brucei avec un test de détection enzymatique pour étudier la sécrétion de protéase. L’utilisation d’un substrat basé sur la fluorescence a permis la détection de l’activité protéase sécrétée par des cellules individuelles. Enfin, nous avons testé la configuration dans deux applications supplémentaires, toutes deux utilisées sur T. brucei unicellulaire. En conclusion, la microfluidique en gouttes s’est révélée prometteuse en tant qu’outil analytique pour la culture et l’analyse des trypanosomes avec un potentiel supplémentaire pour l’analyse unicellulaire à haut débit.

To fight against antibiotic‐resistant bacteria adhering and developing on medical devices, which is a growing problem worldwide, researchers are currently developing new “smart” materials and coatings. They consist in delivery of antimicrobial agents in an intelligent way, i.e., only when bacteria are present. This requires the use of new and sophisticated tools combining antimicrobial agents with lipids or polymers, synthetic and/or natural. In this review, three classes of innovative materials are described: hydrogels, nanomaterials, and thin films. Moreover, smart antibacterial materials can be classified into two groups depending on the origin of the stimulus used: those that respond to a nonbiological stimulus (light, temperature, electric and magnetic fields) and those that respond to a biological stimulus related to the presence of bacteria, such as changes in pH or bacterial enzyme secretion. The bacteria presence can induce a pH change that constitutes a first potential biological trigger allowing the system to become active. A second biological trigger signal consists in enzymes produced by bacteria themselves. A complete panel of recent studies will be given focusing on the design of such innovative smart materials that are sensitive to biological triggers.

A major hurdle in the production of bioethanol with second-generation feedstocks is the high cost of the enzymes for saccharification of the lignocellulosic biomass into fermentable sugars. Simultaneous saccharification and fermentation with Saccharomyces cerevisiae yeast that secretes a range of lignocellulolytic enzymes might address this problem, ideally leading to consolidated bioprocessing. However, it has been unclear how many enzymes can be secreted simultaneously and what the consequences would be on the C6 and C5 sugar fermentation performance and robustness of the second-generation yeast strain. We have successfully expressed seven secreted lignocellulolytic enzymes, namely endoglucanase, β-glucosidase, cellobiohydrolase I and II, xylanase, β-xylosidase and acetylxylan esterase, in a single second-generation industrial S. cerevisiae strain, reaching 94.5 FPU/g CDW and enabling direct conversion of lignocellulosic substrates into ethanol without preceding enzyme treatment. Neither glucose nor the engineered xylose fermentation were significantly affected by the heterologous enzyme secretion. This strain can therefore serve as a promising industrial platform strain for development of yeast cell factories that can significantly reduce the enzyme cost for saccharification of lignocellulosic feedstocks. ispartof: METABOLIC ENGINEERING vol:59 pages:131-141 ispartof: location:Belgium status: published

The pancreas is a glandular organ responsible for diverse homeostatic functions, including hormone production from the endocrine islet cells to regulate blood sugar levels and enzyme secretion from the exocrine acinar cells to facilitate food digestion. These pancreatic functions are essential for life; therefore, preserving pancreatic function is of utmost importance. Pancreas dysfunction can arise either from developmental disorders or adult onset disease, both of which are caused by defects in shared molecular pathways. In this chapter, we discuss what is known about the molecular mechanisms controlling pancreas development, how disruption of these mechanisms can lead to developmental defects and disease, and how essential pancreas functions can be modeled using human pluripotent stem cells. At the core of understanding of these molecular processes are animal model studies that continue to be essential for elucidating the mechanisms underlying human pancreatic functions and diseases.

Dissertation, RWTH Aachen University, 2020; Aachen : RWTH Aachen University 1 Online-Ressource : Illustrationen, Diagramme (2021). = Dissertation, RWTH Aachen University, 2020, Enzymes are biological catalysts that are produced by living organisms. Their properties make them indispensable in different fields of industry and research. Obtaining enzymes in sufficient quantities is important for their application, as well as for their scientific analysis. Heterologous enzyme production is a common method to achieve high enzyme titers and employing secretion can improve enzyme yield as well as reduce downstream processing. The goal of this thesis was to establish a platform for enzyme production that can be used for simple and straightforward optimization of enzyme expression and secretion. The main objective was the generation of a signal peptide library fusion platform that can be used to screen for suitable signal peptides. To this end, a cloning strategy was established for simple and fast library generation. Subsequently, a signal peptide library was built with signal peptides from B. subtilis. This library served as starting point for subcloning into different vectors and for generating fusion libraries of different target genes. Two different vectors were used for subcloning: a C. glutamicum vector to enable enzyme production in the gram positive bacterium and a vector containing the fluorescent reporter protein Pp1FbFP to enable monitoring the target protein secretion during the expression process. A cutinase, a phytase and a diguanylate cyclase were cloned into the library to test the library’s applicability. For cutinase, the signal peptide screening resulted in a threefold increase in activity, which was achieved by the signal peptide YobB. The best signal peptide for the phytase AppA showed 3.5-fold improvement compared to the starting variant. For the diguanylate cyclase tDGCsh, secretion could not be observed. Fusion with signal peptides does not only enable enzyme secretion, it can also be used for the secretion of peptides. Secretory production of antimicrobial peptides in C. glutamicum was tested for the four antimicrobial peptides AFP1, LCI, Psoriasin and VarvF. With the signal peptide NprE, LCI and Psoriasin were secreted at high levels. Titers of up to 130 mg/L have been reached for LCI while Psoriasin concentration was estimated to be 54 mg/L. Directed evolution was performed to improve different signal peptides towards more efficient secretion. For this purpose, the signal peptides PhrF and YbdN were chosen. Two rounds of directed evolution for PhrF resulted in a 2.4-fold increase in activity. YbdN was evolved in one round of mutagenesis, resulting in a 1.36-fold improvement. Coexpression of enzymes with additional unfused signal peptides resulted in significantly improved enzyme secretion. The effect of coexpressed signal peptides on secretion was strongly depended on the expression conditions and the used signal peptide. To further improve enzyme production, vector backbone evolution with the epMEGAWHOP method was performed for the compact E. coli vector pALXtreme-1a and the E. coli-C. glutamicum shuttle vector pEKEx2. The activity of the model enzyme cutinase was increased up to 2.5-fold after two evolution rounds for pEKEx2 and up to ninefold after three rounds of directed evolution for pALXtreme-1a., Published by RWTH Aachen University, Aachen

Yeasts occur in all environments and have been described as potent antagonists of various plant pathogens. Due to their antagonistic ability, undemanding cultivation requirements, and limited biosafety concerns, many of these unicellular fungi have been considered for biocontrol applications. Here, we review the fundamental research on the mechanisms (e.g., competition, enzyme secretion, toxin production, volatiles, mycoparasitism, induction of resistance) by which biocontrol yeasts exert their activity as plant protection agents. In a second part, we focus on five yeast species (Candida oleophila, Aureobasidium pullulans, Metschnikowia fructicola, Cryptococcus albidus, Saccharomyces cerevisiae) that are or have been registered for the application as biocontrol products. These examples demonstrate the potential of yeasts for commercial biocontrol usage, but this review also highlights the scarcity of fundamental studies on yeast biocontrol mechanisms and of registered yeast-based biocontrol products. Yeast biocontrol mechanisms thus represent a largely unexplored field of research and plentiful opportunities for the development of commercial, yeast-based applications for plant protection exist. Electronic supplementary material The online version of this article (10.1007/s11274-019-2728-4) contains supplementary material, which is available to authorized users.

SummaryDuring the degradation of biological materials such as biopolymers, extracellular enzymes liberate oligosaccharides that act as common goods and become available for all cells in the local neighborhood. This phenomenon can lead to cooperative growth, whereby cell-cell aggregation increases both the per-capita availability of resources and the per cell growth rate. However, aggregation can also have detrimental consequences for growth, as gradients form within aggregates limiting the resource accessibility. We used a computational model to show that high bacterial densities and high enzyme secretion rates restrict cooperation in aggregates larger than 10μm, due to the emergence of polymer and oligomer counter-gradients. We compared these predictions against experiments performed with two well-studied alginate degrading Vibrios, one of which displayed a strong density dependent growth. We observed that both strains can form large aggregates (

Made available in DSpace on 2020-12-12T02:23:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-11-14 Background: β-Xylosidases are glycoside hydrolases (GHs) that cleave xylooligosaccharides and/or xylobiose into shorter oligosaccharides and xylose. Aspergillus nidulans is an established genetic model and good source of carbohydrate-active enzymes (CAZymes). Most fungal enzymes are N-glycosylated, which influences their secretion, stability, activity, signalization, and protease protection. A greater understanding of the N-glycosylation process would contribute to better address the current bottlenecks in obtaining high secretion yields of fungal proteins for industrial applications. Results: In this study, BxlB-a highly secreted GH3 β-xylosidase from A. nidulans, presenting high activity and several N-glycosylation sites-was selected for N-glycosylation engineering. Several glycomutants were designed to investigate the influence of N-glycans on BxlB secretion and function. The non-glycosylated mutant (BxlBnon-glyc) showed similar levels of enzyme secretion and activity compared to the wild-type (BxlBwt), while a partially glycosylated mutant (BxlBN1;5;7) exhibited increased activity. Additionally, there was no enzyme secretion in the mutant in which the N-glycosylation context was changed by the introduction of four new N-glycosylation sites (BxlBCC), despite the high transcript levels. BxlBwt, BxlBnon-glyc, and BxlBN1;5;7 formed similar secondary structures, though the mutants had lower melting temperatures compared to the wild type. Six additional glycomutants were designed based on BxlBN1;5;7, to better understand its increased activity. Among them, the two glycomutants which maintained only two N-glycosylation sites each (BxlBN1;5 and BxlBN5;7) showed improved catalytic efficiency, whereas the other four mutants' catalytic efficiencies were reduced. The N-glycosylation site N5 is important for improved BxlB catalytic efficiency, but needs to be complemented by N1 and/or N7. Molecular dynamics simulations of BxlBnon-glyc and BxlBN1;5 reveals that the mobility pattern of structural elements in the vicinity of the catalytic pocket changes upon N1 and N5 N-glycosylation sites, enhancing substrate binding properties which may underlie the observed differences in catalytic efficiency between BxlBnon-glyc and BxlBN1;5. Conclusions: This study demonstrates the influence of N-glycosylation on A. nidulans BxlB production and function, reinforcing that protein glycoengineering is a promising tool for enhancing thermal stability, secretion, and enzymatic activity. Our report may also support biotechnological applications for N-glycosylation modification of other CAZymes. Department of Biochemistry and Tissue Biology Institute of Biology University of Campinas (UNICAMP), Rua Monteiro Lobato, 255 Cidade Universitária Zeferino Vaz Department of Physics Institute of Biosciences Humanities and Exact Sciences São Paulo State University (UNESP) Department of Medical Science Faculty of Medicine University of Campinas (UNICAMP) Department of Biochemistry and Immunology Ribeirão Preto Medical School University of São Paulo (USP) Institute of Chemistry and Center for Computing in Engineering and Sciences University of Campinas (UNICAMP) Department of Physics Institute of Biosciences Humanities and Exact Sciences São Paulo State University (UNESP)

Agradecimentos: We thank the LNBio Mass Spectrometry staff for their assistance with LC–MS/MS and Professor Hernandes Faustino de Carvalho Laboratory for his assistance with the immunohistochemistry methodology. We would like to thank Paul Whitford for the simulation and analysis program codes. The simulations were performed at CENAPAD-SP (Centro Nacional de Processamento de Alto Desempenho em São Paulo), under the UNICAMP/FINEP-MCT project. Research was supported by LNBR-Brazilian Biorenewables National Laboratory (CNPEM/MCTIC) using the Characterization of Macromolecules (MAC) open-access facility. The authors thank Espaço da Escrita-Pró-Reitoria de Pesquisa, UNICAMP for the language services provided Abstract: ß-Xylosidases are glycoside hydrolases (GHs) that cleave xylooligosaccharides and/or xylobiose into shorter oligosaccharides and xylose. Aspergillus nidulans is an established genetic model and good source of carbohydrate-active enzymes (CAZymes). Most fungal enzymes are N-glycosylated, which influences their secretion, stability, activity, signalization, and protease protection. A greater understanding of the N-glycosylation process would contribute to better address the current bottlenecks in obtaining high secretion yields of fungal proteins for industrial applications. In this study, BxlB - a highly secreted GH3 ß-xylosidase from A. nidulans, presenting high activity and several N-glycosylation sites - was selected for N-glycosylation engineering. Several glycomutants were designed to investigate the influence of N-glycans on BxlB secretion and function. The non-glycosylated mutant (BxlBnon-glyc) showed similar levels of enzyme secretion and activity compared to the wild-type (BxlBwt), while a partially glycosylated mutant (BxlBN1;5;7) exhibited increased activity. Additionally, there was no enzyme secretion in the mutant in which the N-glycosylation context was changed by the introduction of four new N-glycosylation sites (BxlBCC), despite the high transcript levels. BxlBwt, BxlBnon-glyc, and BxlBN1;5;7 formed similar secondary structures, though the mutants had lower melting temperatures compared to the wild type. Six additional glycomutants were designed based on BxlBN1;5;7, to better understand its increased activity. Among them, the two glycomutants which maintained only two N-glycosylation sites each (BxlBN1;5 and BxlBN5;7) showed improved catalytic efficiency, whereas the other four mutants’ catalytic efficiencies were reduced. The N-glycosylation site N5 is important for improved BxlB catalytic efficiency, but needs to be complemented by N1 and/or N7. Molecular dynamics simulations of BxlBnon-glyc and BxlBN1;5 reveals that the mobility pattern of structural elements in the vicinity of the catalytic pocket changes upon N1 and N5 N-glycosylation sites, enhancing substrate binding properties which may underlie the observed differences in catalytic efficiency between BxlBnon-glyc and BxlBN1;5. This study demonstrates the influence of N-glycosylation on A. nidulans BxlB production and function, reinforcing that protein glycoengineering is a promising tool for enhancing thermal stability, secretion, and enzymatic activity. Our report may also support biotechnological applications for N-glycosylation modification of other CAZymes FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ Fechado

The aim of the present study was to assess the ability of different white-rot fungi to tolerate polychlorinated biphenyls (PCBs) using predictive mycology, by relating fungal growth inhibition to ligninolityc enzyme secretion. Fungal strains were grown in the presence of PCBs in solid media and their radial growth values were modelled through the Dantigny-logistic like function in order to estimate the time required by the fungal colonies to attain half their maximum diameter. The principal component analysis (PCA) revealed an inverse correlation between strain tolerance to PCBs and the laccase secretion over time, being laccase production closely associated with fungal growth capacity. Finally, a PCA was run to regroup and split between resistant and sensitive fungi. Simultaneously, a function associated with a model predicting the tolerance to PCBs was developed. Some of the assayed isolates showed a promising capacity to be applied in PCB bioremediation. Abbreviations: Polychlorinated biphenyls (PCBs), white-rot fungi (WRF). Fil: Sadañoski, Marcela Alejandra. Universidad Nacional de Misiones. Facultad de Ciencias Exactas Químicas y Naturales. Departamento de Bioquímica Clínica. Laboratorio de Biotecnología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina Fil: Velázquez, Juan Ernesto. Universidad Nacional de Misiones. Facultad de Ciencias Exactas Químicas y Naturales. Departamento de Bioquímica Clínica. Laboratorio de Biotecnología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina Fil: Fonseca, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional de Misiones. Facultad de Ciencias Exactas Químicas y Naturales. Departamento de Bioquímica Clínica. Laboratorio de Biotecnología Molecular; Argentina Fil: Zapata, Pedro Dario. Universidad Nacional de Misiones. Facultad de Ciencias Exactas Químicas y Naturales. Departamento de Bioquímica Clínica. Laboratorio de Biotecnología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina Fil: Levin, Laura Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina Fil: Villalba, Laura. Universidad Nacional de Misiones. Facultad de Ciencias Exactas Químicas y Naturales. Departamento de Bioquímica Clínica. Laboratorio de Biotecnología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina

Deinococcus radiodurans is one of the most yet discovered extremophilic microbe, the isolation of which from the various habitats of Kotumsar cave is always a matter of enticement to discover its ecological economics. In the present work we studied the intra versus extracellular alkaline protease and glucose isomerase secretion capabilities of Deinococcus radiodurans; KCB21, KCB50, KCB93 isolated from three distinct subterranean niches of Kotumsar cave. The selected niches/zones were the entrance zone, transient zone and the deep inner zone from where the soil sediments were collected to isolate the bacterial strains. The results revealed high extracellular alkaline protease activity from the Deinococcus radiodurans strain which was isolated from the deeper zones of the cave, whereas no such phenomenon was revealed for glucose isomerase. The possible reason for the obtained results has been discussed.

Melatonin is an indoleamine produced from the amino acid l-tryptophan, whereas metabolites of melatonin are known as kynuramines. One of the best-known kynuramines is N1-acetyl-N1-formyl-5-methoxykynuramine (AFMK). Melatonin has attracted scientific attention as a potent antioxidant and protector of tissue against oxidative stress. l-Tryptophan and kynuramines share common beneficial features with melatonin. Melatonin was originally discovered as a pineal product, has been detected in the gastrointestinal tract, and its receptors have been identified in the pancreas. The role of melatonin in the pancreatic gland is not explained, however several arguments support the opinion that melatonin is probably implicated in the physiology and pathophysiology of the pancreas. (1) Melatonin stimulates pancreatic enzyme secretion through the activation of entero-pancreatic reflex and cholecystokinin (CCK) release. l-Tryptophan and AFMK are less effective than melatonin in the stimulation of pancreatic exocrine function; (2) Melatonin is a successful pancreatic protector, which prevents the pancreas from developing of acute pancreatitis and reduces pancreatic damage. This effect is related to its direct and indirect antioxidant action, to the strengthening of immune defense, and to the modulation of apoptosis. Like melatonin, its precursor and AFMK are able to mimic its protective effect, and it is commonly accepted that all these substances create an antioxidant cascade to intensify the pancreatic protection and acinar cells viability; (3) In pancreatic cancer cells, melatonin and AFMK activated a signal transduction pathway for apoptosis and stimulated heat shock proteins. The role of melatonin and AFMK in pancreatic tumorigenesis remains to be elucidated.

In fungi, ambient pH sensing involves the activation of the Pal/PacC signalling pathway. In the dermatophyte Trichophyton rubrum, pH-dependent secretion of keratinases, which are major virulence determinants, is affected by disruption of the pacC gene. Here, the transcription profiling of the genes coding for N- and O-linked mannosyltransferases, enzymes involved in protein glycosylation, was evaluated in T. rubrum in response to disruption of the pacC gene and growth in keratin, glucose, and glucose plus glycine. We show that transcription of these mannosyltransferase genes is affected by nutrients at acidic pH and by PacC., Highlights ▸ The PacC/Pal transduction pathway mediates the metabolic response to pH sensing. ▸ pH-dependent secretion of keratinases is modulated by the pacC gene in T. rubrum. ▸ The pacC gene modulates transcription of O- and N-mannosyltransferase genes at acidic pH. ▸ An O-mannosyltransferase gene is preferentially transcribed in keratin at pH 5.0. ▸ An N-mannosyltransferase gene is preferentially transcribed in non-buffered glucose at pH 5.0.

Low back pain is a common disorder. Pathological innervation and intervertebral disc degeneration are two major factors associated with this disease. Semaphorin 3A, originally known for its potent inhibiting effect on axonal outgrowth, is recently found to correlate with disease activity and histological features in some skeletal disorders. Based on its effects on innervation and vascularization, as well as enzyme secretion, we presume that semaphorin 3A may act as a potential target for low back pain.

In fungi a genetic system ensures that enzymes are secreted mainly at ambient pH values corresponding to their optima of activity. Although a great deal of information has been obtained concerning this environmental response, there is a lack of studies involving phytopathogenic, endophytic and entomopathogenic fungi as well as different aspects of fungus-host interactions. This study compares in a plate-clearing assays, the effect of ambient pH in the secretion of amylase, cellulase, lipase, pectinase and protease by endophytic, phytopathogenic, and entomopathogenic isolates belonging to several species of Colletotrichum. All enzymes were secreted in a pH-dependent manner by all isolates. Endophytes and pathogens showed distinct patterns of protease secretion, with optima at alkaline and acid growth conditions, respectively. In liquid medium, a Pi-repressible acid phosphatase of an endophytic isolate responded to ambient pH, having a 14-fold increase in secreted specific activity at acid pH, as compared to alkaline pH. Furthermore, part of a Colletotrichum pacC homologue gene, coding for a transcriptional factor responsible for pH-regulated gene expression, was cloned. Ambient pH seems to be a general factor controlling enzyme secretion in fungus-host interactions through a conserved genetic circuit. Em fungos, um sistema de regulação gênica garante que enzimas sejam secretadas predominantemente em valores de pH do ambiente próximos aos pH ótimos de atividade correspondentes. Embora muita informação tenha sido acumulada sobre essa resposta adaptativa, não existem estudos envolvendo fungos fitopatogênicos, endofíticos e entomopatogênicos, bem como sobre outros aspectos relacionados às interações fungo-hospedeiro. No presente trabalho foi comparado, em meio sólido, o efeito do pH do ambiente na secreção das enzimas amilase, celulase, lipase, pectinase e protease por isolados endofíticos, fitopatogênico e entomopatogênicos pertencentes a diferentes espécies de Colletotrichum. Para todas as enzimas e em todos os isolados, observou-se um padrão de secreção dependente dos valores do pH do ambiente. Isolados endofíticos e patogênicos apresentaram padrões distintos de secreção de protease, com ótimos em pH de crescimento alcalino e ácido, respectivamente. Em meio líquido, uma fosfatase ácida Pi-repressível, secretada por um isolado endofítico, respondeu ao pH do ambiente, apresentando um aumento de 14 vezes na sua atividade específica durante o crescimento do fungo em meio ácido, quando comparado a meio alcalino. Além disso, foi clonada parte do gene pacC de Colletotrichum, o qual codifica um fator de transcrição responsável pela regulação dependente do pH do ambiente. É plausível a hipótese de que o pH ambiente é um fator de amplo espectro controlando a secreção enzimática durante as interações fungo-hospedeiro por meio de um circuito genético conservado.

In this communication, we show that the growth of isolate H6 of the dermatophyte Trichophyton rubrum on non-buffered medium and under saturating phosphate conditions is dependent on the initial growth pH, with an apparent optimum at pH 4.0. In addition, irrespective of the initial growth pH, the pH of the medium alteredduring cultivation reaching values that ranged from 8.3 to 8.9. Furthermore, this isolate synthesized and secreted almost the same levels of an alkaline phosphatase with an apparent optimum pH ranging from 9.0 to 10.0 when grown on both low- and high-phosphate medium. Also, this alkaline phosphatase is activated by Mg2+ and is EDTA-sensitive. On the other hand, the very low levels of the enzyme retained by the mycelium grown on buffered medium at pH 5.0-5.2 suggest that this enzyme is encoded by an alkaline gene, i.e., a gene responsive to ambient pH signaling.Nesta comunicação nós mostramos que o crescimento do isolado H6 do dermatófito T. rubrum em meio não tamponado e sob condição saturante de fosfato, é dependente do pH inicial de cultivo, com um ótimo aparente em pH 4,0. Além disto, independente do pH inicial, o pH do meio se altera durante o cultivo alcançando valores que variam de 8,3 a 8,9. Verificou-se também que este isolado sintetiza e secreta quase os mesmos níveis de fosfatase alcalina, com um ótimo de atividade aparente entre os valores de pH 9,0 e 10,0, independentemente da concentração de fosfato no meio. Também mostramos que essa fosfatase alcalina é inibida por EDTA e ativada por Mg2+. Por outro lado, o nível dessa enzima retida no micélio cultivado em meio tamponado em pH 5,0-5,2 é baixo, sugerindo que ela seja codificada por um gene alcalino, isto é, um gene responsivo à sinalização pelo pH ambiente.

In this communication, we show that the growth of isolate H6 of the dermatophyte Trichophyton rubrum on non-buffered medium and under saturating phosphate conditions is dependent on the initial growth pH, with an apparent optimum at pH 4.0. In addition, irrespective of the initial growth pH, the pH of the medium alteredduring cultivation reaching values that ranged from 8.3 to 8.9. Furthermore, this isolate synthesized and secreted almost the same levels of an alkaline phosphatase with an apparent optimum pH ranging from 9.0 to 10.0 when grown on both low- and high-phosphate medium. Also, this alkaline phosphatase is activated by Mg2+ and is EDTA-sensitive. On the other hand, the very low levels of the enzyme retained by the mycelium grown on buffered medium at pH 5.0-5.2 suggest that this enzyme is encoded by an alkaline gene, i.e., a gene responsive to ambient pH signaling. Nesta comunicação nós mostramos que o crescimento do isolado H6 do dermatófito T. rubrum em meio não tamponado e sob condição saturante de fosfato, é dependente do pH inicial de cultivo, com um ótimo aparente em pH 4,0. Além disto, independente do pH inicial, o pH do meio se altera durante o cultivo alcançando valores que variam de 8,3 a 8,9. Verificou-se também que este isolado sintetiza e secreta quase os mesmos níveis de fosfatase alcalina, com um ótimo de atividade aparente entre os valores de pH 9,0 e 10,0, independentemente da concentração de fosfato no meio. Também mostramos que essa fosfatase alcalina é inibida por EDTA e ativada por Mg2+. Por outro lado, o nível dessa enzima retida no micélio cultivado em meio tamponado em pH 5,0-5,2 é baixo, sugerindo que ela seja codificada por um gene alcalino, isto é, um gene responsivo à sinalização pelo pH ambiente.

In order to investigate further the adaptive response of moulds to ambient pH, we have measured by ELISA the pho-2-encoded Pi-repressible alkaline phosphatase synthesised by Neurospora crassa. We showed that the 74A and pho-2A strains of this mould secrete similar amounts of the pho-2-encoded enzyme irrespective of ambient pH, when both the preg and pgov genes are not functional, i.e., in strains nuc-2+ growing under Pi-starvation. This suggests that pho-2, which is responsive to Pi starvation via the action of genes nuc-2, preg, pgov and nuc-1, is not a gene responsive to ambient pH and that the differential glycosylation observed for the Pi-repressible alkaline phosphatase retained by the mycelium at pH 5.6 or secreted into the growth medium at pH 8.0 is the genetic response to ambient pH sensing in N. crassa.

Approximately a tenth of all described fungal species can cause diseases in plants. A common feature of this process is the necessity to pass through the plant cell wall, an important barrier against pathogen attack. To this end, fungi possess a diverse array of secreted enzymes to depolymerize the main structural polysaccharide components of the plant cell wall, i.e., cellulose, hemicellulose, and pectin. Recent advances in genomic and systems-level studies have begun to unravel this diversity and have pinpointed cell wall-degrading enzyme (CWDE) families that are specifically present or enhanced in plant-pathogenic fungi. In this review, we discuss differences between the CWDE arsenal of plant-pathogenic and non-plant-pathogenic fungi, highlight the importance of individual enzyme families for pathogenesis, illustrate the secretory pathway that transports CWDEs out of the fungal cell, and report the transcriptional regulation of expression of CWDE genes in both saprophytic and phytopathogenic fungi. ©2014 by Annual Reviews. All rights reserved.

Solid-state culture encourages high-level enzyme secretion by Aspergillus oryzae. Using the real-time quantitative reverse transcriptase-polymerase chain reaction, we confirmed that expression of the glucoamylase-encoding gene in A. oryzae cultured in solid-state culture depends on the water content of the culture.

Acid phosphatase (ACP) activity was demonstrated in blood stream form of Trypanosome brucei brucei harvested from infected Wister rats by Ion Exchange DEAE Cellulose 52 chromatography. Whole parasite extract (WPE) and Excretory Secretory Extract (ESE) were prepared and analyzed for acid phosphatase activity. A higher ACP activity (85.5 μmol/min) was recorded in WPE compared to ESE (36.8 μmol/min). ACP activity in ESE is suggestive of the presence of a cell rich enzyme. Phase separation of the extracts using the detergent Triton X-114 (TX-114), resulted in protein partitioning into aqueous and detergent phases. ACP activity was higher in the detergent phases (56.2 μmol/min and 28.8 μmol/min) of WPE and ESE respectively. ACP activity recorded in the aqueous phases of WPE and EPE was 27.8 and 7.6 μmol/min respectively. On a Size Exclusion chromatography column using Sephacryl-300, ESE emerged as five distinct protein peaks. ACP activity of the eluted fractions showed two peaks of relative molecular weights 195 and 325 KD. This study shows that T. brucei releases acid phosphatase extracellularly via a yet to be determined mechanism. Acid phosphatase activity in ESE is indicative of a soluble enzyme within the cell matrix which may also play an important role in the pathology of African Trypanosomiasis.Keywords: Trypanosoma brucei; acid phosphatase; enzyme secretion.

An epithelial duct is a well-defined multicellular structure composed of tightly packed cells separating and protecting body compartments that are used for enzyme secretion and its transport across the internal. The structural and functional integrity (homeostasis) of such ducts is vital in carrying many life functions (breathing, lactation, production of hormones). However, the processes involved in maintaining the homeostatic balance are not yet fully understood. On the other hand, the loss of epithelial tissue architecture, such as filled lumens or ductal disorganization, are among the first symptoms of the emerging epithelial tumors (carcinomas). Using the previously developed biomechanical model of epithelial ducts:IBCell, we investigated how different signals and mechanical stimuli imposed on individual epithelial cells can impact the homeostatic (im)balance and integrity of the whole epithelial tissue. We provide a link between erroneous responses of individual epithelial cells to specific signals and the emerging ductal morphologies characteristic for preinvasive cancers observed in pathology specimens, or characteristic for multicellular structures arising from mutated cells culturedin vitro. We summarize our finding in terms of altered properties of epithelial cell polarization, and discuss the relative importance of various polarization signals on the formation of tumor-like multicellular structures.

Arthrobacter ureafaciensのinulin fructotoransferse(IFT)の誘導生産と菌体外への分泌機構に対する温度の影響を見た。本酵素の生産はイヌリンを炭素源として40℃で培養すると,20℃培養時に比べ約1.5倍増加するが,菌体外への遊離は明らかに抑制された。このことは,本酵素をdi-D-fructofuranose1,2':2,3'dianhydride(DFAIII)で誘導させたとき,培養温度を20℃から40℃へ変化させると菌体内酵素レベルが上昇し,逆に40℃から20℃へ下げると急速な菌体内酵素レベルの低下と菌体外酵素レベルの上昇が認められることからも確かめられた。さらに,40℃で酵素を誘導中,15分間20℃で低温処理すると,処理直後から約30分間,菌体内酵素レベルが停滞し以後約1時間で急速に回復,低温未処理の場合と同レベルに達した。これらのことは高温下では主としてIFTの菌体外への遊離が抑制されることを示している。, Experiments were performed to determine how temperature affects inulin fructotransferase(IFT)production in Arthrobacter ureafaciens.At 40℃,IFT was produced roughly 1.5-fold compared with those at 20℃.This increase was apparently reflected the accumulation of cell-bound form.Culturing A.ureafaciens(inducible)at 20℃ for 5 hr and shift-up to 40℃ leads preferentially to an increase in cell-bound IFT activity,but the extracellular one keeps the same amount of the control.Contraversely, culturing at 40℃ and shift-down to 20℃ leads rapidly to the release of enzyme.In the way of the 40℃-induction,the cold shock(15min)leads the cessation of production of cell-bound form for about 30 min and then rapidly recovers to the level of untreated one.The level of extracellular one is increased by this treatment.The heat shock causes the increase of the level of cell-bound and extracellular forms.These indicates that the higher temperature repress the release of IFT.

As atividades amilolítica e pectinolítica de 45 isolados de Alternaria solani, provenientes de diferentes hospedeiros, foram estimadas por meio da difusão enzimática em meio sólido específico e mensuração do halo de degradação do substrato. Todos os isolados degradaram pectina. Apenas 17 isolados apresentaram atividade amilolítica, sendo nove isolados provenientes de batateira. Somente o isolado AS18 se destacou como bom produtor de ambas as enzimas. Uma vez que a atividade pectinolítica foi mais evidente, avaliou-se a influência de pectinases na agressividade de A. solani ao tomateiro. Para isso, cinco isolados (2 de berinjela, 2 de tomateiro e 1 de batateira) contrastantes quanto à produção de pectinases foram selecionados para testes em folíolos destacados e plantas inteiras. Quatro isolados foram utilizados no teste em folíolos destacados (AS6, AS7, AS12 e AS26), e constatou-se haver variabilidade patogênica. A correlação obtida entre o tamanho das lesões e a atividade pectinolítica foi de r = 0,963 (P = 0,087). Cinco isolados (AS6, AS7, AS12, AS25 e AS26) foram inoculados em plantas inteiras de tomate. Os isolados não diferiram quanto ao número de lesões/cm² de área foliar, porém variaram em agressividade. Houve correlação (r = 0,916; P = 0,042) entre a atividade de pectinases e o índice de doença, sugerindo possível papel para as enzimas pécticas durante a infecção de A. solani em tomateiro. É provável que as diferenças no perfil enzimático dos isolados estejam associadas ao hospedeiro original de onde os mesmos foram obtidos. Os resultados reforçaram evidências de especificidade por hospedeiro em populações de A. solani. Amilolytic and pectinolytic activities of 45 isolates of Alternaria solani from distinct hosts were investigated. Enzyme secretion was evaluated in glucose-free medium amended with pectin or starch as carbon source. All isolates degraded pectin. Only 17 isolates had amilolytic activity, and nine of these isolates were isolated from potato. Only AS18 had good amilolytic and pectinolytic activities. The role of pectinases produced by A. solani in its aggressiveness on tomato was examined. Five isolates showing substantial differences regarding the ability to produce pectic enzymes were selected, two from eggplant, two from tomato plants, and one from potato plant. Four isolates (AS6, AS7, AS12, and AS26) were inoculated on detached leaflets of tomato and pectinolytic activity was correlated with lesion size (r = 0.963; P = 0.087). Pathogenicity test with five isolates (AS6, AS7, AS12, AS25, and AS26) was conducted in tomato plants. Variation in the number of lesions per cm² of leaf area was not detected, but isolates varied in aggressiveness. Pectinolytic activity and aggressiveness were correlated (r = 0.916; P = 0.042). In general, pectinases-producing isolates caused larger lesions on tomato plants. It is possible that enzymatic profile differences are associated with the host origin. The results contribute to cumulative evidences of host specificity in populations of A. solani.