Academic literature on the topic 'Fengycine'

The mycotoxigenic pathogen Fusarium verticillioides threatens the quality and utility of maize across industrial and agricultural purposes. Chemical control is complicated by the intimate endophytic lifestyle of the pathogen with its host. Bacillus mojavensis RRC101, a maize-endophytic bacterium, has been observed to reduce F. verticillioides disease severity and fumonisin accumulation when coinoculated to maize. Genome sequencing and annotation identified a number of biocontrol-relevant pathways in RRC101. Biochemical assays confirmed the presence and activity of surfactin- and fengycin-type lipopeptides, with fengycins responsible for antifungal activity against F. verticillioides. This antagonism manifests as inhibition of filamentous growth, with microscopy revealing hyphal distortions, vacuolization, and lysis. F. verticillioides secondary metabolism also responds to antagonism, with lipopeptide challenge inducing greater fumonisin production and, in the case of fengycins, eliciting pigment accumulation at sites of inhibition. Together, these data suggest that antibiotic and toxin production are components of a complex biochemical interaction among maize endophytes, one pathogenic and one beneficial.

Endophytic microorganisms (EMs) have recently attracted interest for applications in plant protection, mainly due to their bioactive compound-producing capacity. Therefore, we underwent the task of isolating olive tree EMs and investigating their bioactivity against the devastating pathogen Colletotrichum acutatum. Several EMs were isolated; however, the Bacillus sp. PTA13 isolate exhibited the highest toxicity to the phytopathogen. Bacteria of the genus Bacillus exhibit superior bioactive metabolite-producing capacity, with the lipopeptides (LPs) of surfactin, iturin, and fengycin groups being the most studied. A total LP extract and several fractions were obtained, and their bioactivity was assessed against C. acutatum strains. LPs of the major surfactin, iturin, and fengycin groups and the minor gageotetrin and bacilotetrin groups were annotated. The results confirmed the bioactivity of the major LPs, with fengycins being the most fungitoxic. Interestingly, the minor LP fraction exhibited selective toxicity to the fungicide-resistant C. acutatum isolate, an observation that highlights the significance of our approach to comprehensively mine the total LP extract. This work represents a proof of concept of the exploitation of EMs in customized olive tree plant protection and aligns well with strategies that focus on the sustainability and safety of food production via the development of next-generation plant protection products.

A lot of crops are destroyed by the phytopathogens such as fungi, bacteria, and yeast leading to economic losses to the farmers. Members of theBacillusgenus are considered as the factories for the production of biologically active molecules that are potential inhibitors of growth of phytopathogens. Plant diseases constitute an emerging threat to global food security. Many of the currently available antimicrobial agents for agriculture are highly toxic and nonbiodegradable and thus cause extended environmental pollution. Moreover, an increasing number of phytopathogens have developed resistance to antimicrobial agents. The lipopeptides have been tried as potent versatile weapons to deal with a variety of phytopathogens. All the three families ofBacilluslipopeptides, namely, Surfactins, Iturins and Fengycins, have been explored for their antagonistic activities towards a wide range of phytopathogens including bacteria, fungi, and oomycetes. Iturin and Fengycin have antifungal activities, while Surfactin has broad range of potent antibacterial activities and this has also been used as larvicidal agent. Interestingly, lipopeptides being the molecules of biological origin are environmentally acceptable.

Probiotics may offer an attractive alternative for standard antibiotic therapy to treat Clostridium difficile infections (CDI). In this study, the antibacterial mechanism in vitro of newly isolated B. amyloliquefaciens C-1 against C. difficile was investigated. The lipopeptides surfactin, iturin, and fengycin produced by C-1 strongly inhibited C. difficile growth and viability. Systematic research of the bacteriostatic mechanism showed that the C-1 lipopeptides damage the integrity of the C. difficile cell wall and cell membrane. In addition, the lipopeptide binds to C. difficile genomic DNA, leading to cell death. Genome resequencing revealed many important antimicrobial compound-encoding clusters, including six nonribosomal peptides (surfactins (srfABCD), iturins (ituABCD), fengycins (fenABCDE), bacillibactin (bmyABC), teichuronic, and bacilysin) and three polyketides (bacillaene (baeEDLMNJRS), difficidin (difABCDEFGHIJ), and macrolactin (mlnABCDEFGHI)). In addition, there were other beneficial genes, such as phospholipase and seven siderophore biosynthesis gene clusters, which may contribute synergistically to the antibacterial activity of B. amyloliquefaciens C-1. We suggest that proper application of antimicrobial peptides may be effective in C. difficile control.

The antibacterial potential of four strains of Bacillus subtilis, UMAF6614, UMAF6619, UMAF6639, and UMAF8561, previously selected on the basis of their antifungal activity and efficacy against cucurbit powdery mildew, was examined. Among these strains, UMAF6614 and UMAF6639 showed the highest antibacterial activity in vitro, especially against Xanthomonas campestris pv. cucurbitae and Pectobacterium carotovorum subsp. carotovorum. These strains produced the three families of lipopeptide antibiotics known in Bacillus spp.: surfactins, iturins, and fengycins. Using thin-layer chromatography analysis and direct bioautography, the antibacterial activity could be associated with iturin lipopeptides. This result was confirmed by mutagenesis analysis using lipopeptide-defective mutants. The antibacterial activity was practically abolished in iturin-deficient mutants, whereas the fengycin mutants retained certain inhibitory capabilities. Analyses by fluorescence and transmission electron microscopy revealed the cytotoxic effect of these compounds at the bacterial plasma membrane level. Finally, biological control assays on detached melon leaves demonstrated the ability of UMAF6614 and UMAF6639 to suppress bacterial leaf spot and soft rot; accordingly, the biocontrol activity was practically abolished in mutants deficient in iturin biosynthesis. Taken together, our results highlight the potential of these B. subtilis strains as biocontrol agents against fungal and bacterial diseases of cucurbits and the versatility of iturins as antifungal and antibacterial compounds.

This study investigate the inhibiting effect of fengycin on respiration and nutrient utilization of Penicillium expansum. The respiratory inhibition rate of the P. expansum was determined by the test of dissolved oxygen fengycin, The effect of fengycin treatment on the activity of P. expansum mitochondrial complex enzyme was detected by mitochondrial enzyme activity assay. The ability of fengycin treatment to P. expansum the utilization of total sugar and total protein was determined by DNS colorimetric method and biuret method. After fengycin treatments, the TCA pathway of respiratory metabolism in P. expansum was inhibited. Besides, fengycin could block the gene expression in P. expansum by binding P. expansum mitochondrial complex enzyme II and III related genes. Therefore, the activity of mitochondrial enzymes was affected. With the increasement of fengycin concentration, the absorption and utilization capacity of P. expansum to total sugar and total protein decreased significantly. Fengycin could inhibit the respiratory metabolism and reduce the biochemical metabolism level in P. expansum and finally caused the growth inhibition.

Fengycin is an antimicrobial cyclic lipopeptide produced by various Bacillus subtilis strains, including strain CU12. Direct effects of fengycin include membrane pore formation and efflux of cellular contents leading to cell death in sensitive microorganisms. In this study, four plant pathogens were studied in order to elucidate the role of membrane lipids in their relative sensitivity to fengycin. Inhibition of mycelial growth in these pathogens varied considerably. Analysis of membrane lipids in these microorganisms indicated that sensitivity correlated with low ergosterol content and shorter phospholipid fatty acyl chains. Sensitivity to fengycin also correlated with a lower anionic/zwitterionic phospholipid ratio. Our data suggest that decreased fluidity buffering capacity, as a result of low ergosterol content, and higher intrinsic fluidity afforded by short fatty acyl chain length may increase the sensitivity of microbial membranes to fengycin. Our results also suggest that lower content in anionic phospholipids may increase fengycin insertion into the membrane through reduced electrostatic repulsion with the negatively charged fengycin. The intrinsic membrane lipid composition may contribute, in part, to the observed level of antimicrobial activity of fengycin in various plant pathogens.

Fengycin is a lipopeptide produced by Bacillus that has a strong inhibitory effect on filamentous fungi; however, its use is restricted due to poor production and low yield. Previous studies have shown that fengycin biosynthesis in B. amyloliquefaciens was found to be significantly increased after fructose addition. This study investigated the effect of fructose on fengycin production and its regulation mechanism in B. amyloliquefaciens by transcriptome sequencing. According to the RNA sequencing data, 458 genes were upregulated and 879 genes were downregulated. Transcriptome analysis results showed that fructose changed the transcription of amino acid synthesis, fatty acid metabolism, and energy metabolism; alterations in these metabolic pathways contribute to the synthesis of fengycin. In an MLF medium (modified Landy medium with fructose), the expression level of the fengycin operon was two-times higher than in an ML medium (modified Landy medium). After fructose was added to B. amyloliquefaciens, the fengycin-synthesis-associated genes were activated in the process of fengycin synthesis.

Aim. To study the metabolomic profile of bacterial strains of the genus Bacillus promising for the development of biofungicides using thin layer chromatography (TLC), bioautography and high performance liquid chromatography with mass spectrometry (HPLC‐MS).Materials and Methods. The objects of study are strains of bacteria of the genus Bacillus. Exometabolites were isolated from the liquid culture and their metabolomic profile was analyzed by TLC, bioautography, and HPLC‐MS.Results. By the method of bioautography with a test culture of the fungus F. oxysporum var. orthoceras BZR F6 metabolites of bacterial strains of the genus Bacillus surfactin, iturin and fengycin were identified. Quantitative analysis using HPLC‐MS analysis allows us to state that the B. velenzensis BZR 336g and B. amyloliquefaciens BZR 277 strains produce more surfactin than the others. An increased content of ituric lipopeptides was found in strains B. velezensis BZR 517 and B. velenzensis BZR 336g. According to the ability to produce fengycins, strains of B. velezensis BZR 517 and B. velenzensis BZR 336g are ahead of other strains.Conclusion. Studies using two analytical methods reveal that the strains produce all three antifungal lipopeptides. This is important, since metabolites are able not only to suppress phytopathogenic fungi, but also to enhance the antifungal effect due to synergism. The results obtained allow us to state the possibility of using all four strains as producers of effective biofungicides.

Abstract Penicillium expansum is the main cause of apple rot. Besides, it can also produce mycotoxin patulin (PAT). Therefore, the search for substances that can inhibit the activity and toxigenicity of P. expansum has become a hot research topic. This study investigates the inhibitory effects of fengycin on patulin production in P. expansum. P. expansum was cultured under different environments with different concentrations of fengycin. The patulin content produced per unit weight of P. expansum mycelium was detected and determined by high pressure liquid chromatography (HPLC). Synergy brands (SYBR) GreenI Real-time PCR was used to detect the expression levels of 6-methylsalicylic acid synthase (6-MSAS) and isoepoxydon dehydrogenase (IDH), which were the key genes of producing patulin of P. expansum mycelium, in the conditions treated by fengycin and untreated. After fengycin treatments, not only the patulin content in every unit weight of P. expansum mycelium but also the expression level of 6-MSAS decreased significantly. The expression level of 6-MSAS of treatment was 0.11 folds of control. However, the expression level of IDH treated by fengycin decreased slightly. Fengycin could inhibit the P. expansum from producing patulin by downregulating the expression of key synthetic genes 6-MSAS.

Ce travail a pour objectif d’analyser la surproduction de fengycine par la souche de B. subtilis BBG 21, en comparant la synthèse à celle d’autres souches de Bacillus. BBG21 produisant également des surfactines, nous avons aussi analysé la co-production des lipopeptides dans cette souche. Le travail montre le rôle des promoteurs Pfen et Ppps dans la synthèse des fengycines. L’analyse de la séquence met en évidence 10 nucléotides manquants entre Ppps168 et PfenBBG21 et la modification d’un nucléotide de « l’UP element » entre BBG 21 et ATCC 21332. Dans un second temps, certaines conditions biotiques et abiotiques influençant l’expression du promoteur Pfen et la synthèse de fengycines et de surfactines ont été testées dans le dérivé BBG 208. Les sources de carbone orientent la synthèse vers une famille de lipopeptides alors que la plupart des sources d’azote permettent la cosynthèse à haut niveau des 2 molécules. Une forte expression du promoteur Pfen couplée à une synthèse importante de fengycines a été mise en évidence lorsque l’urée ou le mélange urée ammonium sont utilisés comme source d’azote et le mannitol comme source de carbone. Les conditions de température, de pH et d’oxygénation sont importantes pour la synthèse de fengycine et ces conditions sont spécifiques aux sources de carbone ou d’azote présentes dans le milieu. Enfin, nous avons étudié la synthèse des molécules chez des mutants surfactine - et pnp ase – issus de la souche Bs 168. Les résultats montrent que le régulateur srfAA affecte significativement la production de fengycines alors qu’il n’y pas action de srfAC. Chez les mutants pnpase - il y a une forte diminution des 2 lipopeptides
This work aimed at analyzing the overproduction of fengycin in Bacillus subtilis BBG21 and then was compared to a set of Bacilli strains. As BBG21 also produces surfactin, we also studied coproduction of lipopeptides in this strain. The work highlighted the role the promotor Pfen and Ppps in the synthesis of fengicins. The analysis of sequences was unveiled 10 missing nucleotides between Ppps168 and PfenBBG21 and modification of one nucleotide of the UP element between strains BBG21 and 21332, respectively. Secondly, environmental conditions that might influence the promotor Pfen expression and synthesis of both lipopeptides were also tested in B. subtilis BBG 208. Thus, carbon sources appeared to direct synthesis of one family of lipopeptides, whilst most of nitrogen sources allowed high level of both lipopeptides co-synthesis. A strong expression of promotor Pfen and an important synthesis of fengycins were obtained with urea or urea ammonium mixture used as nitrogen source and with mannitol as carbon source. Temperature, pH and filling volume are important for fengycins synthesis and these conditions are carbon and nitrogen sources dependent. Finally we studied fengycins synthesis in surfactin and PNPase mutants derived from Bs 168. The result showed that srfAA regulator decreased fengycin synthesis whereas srfAC has not any affect. Notably, an important decrease in surfactin and fengycin was observed for PNPase mutant strains

La souche de Bacillus subtilis ATCC 21332 produit deux familles de lipopeptides (les surfactines et les fengycines/plipastatines) qui présentent des propriétés biologiques d’intérêt. La productivité des procédés fermentaires en mode continu ou discontinu et leur extrapolation à grande échelle est limitée par des problèmes cinétiques et technologiques notamment par le transfert d’oxygène et la formation massive de mousse. Le travail réalisé porte sur la mise en œuvre d’un procédé innovant permettant la production de lipopeptides sans mousse et sur l’extraction des molécules à partir du milieu de fermentation. Après des travaux concernant l’amélioration de la production des lipopeptides par des cellules de B. subtilis immobilisées sur des supports solides, un bioréacteur à disques tournants permettant la production importante et sélective de lipopeptides a été mis en œuvre. Des concentrations de plus de 1 g L-1 de lipopeptides ont été obtenues lors des fermentations. Le transfert d’oxygène, facteur limitant primordial de ce métabolisme chez B. subtilis, a été étudié. Il a été fortement affecté par l’agitation pour toutes les configurations du bioréacteur étudiées. Pour des KLa se situant entre 0,001 et 0,003 s-1, le métabolisme est orienté vers la synthèse de fengycines qui représentent jusqu’à 80 % des lipopeptides synthétisés. La productivité en fengycine particulièrement intéressante et la simplicité de la mise en œuvre du bioréacteur permet d’envisager une extrapolation de la production à l’échelle industrielle. Simultanément à la production, des études concernant l’extraction des lipopeptides par pertraction dans un contacteur à disques tournants ont été entreprises. Les résultats obtenus sur l’extraction de la surfactine montrent le potentiel de cette technique séparative pour la récupération des lipopeptides à partir de leur milieu de fermentation
Bacillus subtilis ATCC 21332 produces two families of lipopeptides (surfactins and fengycins/plipastatins) with different biological properties of interest. The productivity of fermentation process in batch or continuous conditions and bioreactors scale-up are limited by the problems of oxygen transfer limitation and foaming. This work presents a novel process of non-foaming production of lipopeptides in a rotating discs bioreactor and pertraction studies on the recovery of lipopeptides from fermentation broth. The improving production of lipopeptides by B. subtilis immobilized on solid supports enabled the implementation of an original bubbleless bioreactor for a selective production of lipopeptides. More than 1 g L-1 of lipopeptides was produced in this new rotating discs bioreactor. The oxygen transfer, a key factor for the metabolism of B. subtilis, was studied, also. In every studied configuration of bioreactor, the transfer of oxygen was strongly affected by the agitation conditions. At KLa in the range of 0.001-0.003 s-1, mainly fengycin was produced (up to 80% of total lipopeptides). The obtained high fengycin selectivity and the simplicity of the implementation of the rotating discs bioreactor suggest its potential scale-up. The extraction of lipopeptides by pertraction in a rotating discs contactor was studied, also. The obtained results on surfactin recovery by pertraction allow to consider this technique as suitable for lipopeptides extraction from fermentation broth

Dans cette étude, 36 génomes de souches de Bacillus publiés ont été analysés ; neuf d’entre eux ne portent pas de gènes ou opérons codant pour une synthèse de peptides par la voie non ribosomiale (NRPS). Chez les 18 souches restantes ont été détectées l’existence de gènes/opérons codant pour des molécules de type NRP nouvelles et des molécules issues d’une synthèse hybride NRPS/PKS (Polycétide synthases). L’analyse des opérons impliqués dans la synthèse des fengycines et plipastatines chez la souche de laboratoire B. subtilis 168, B. subtilis F29-3 et B. amyloliquefaciens FZB42, a montré des homologies élevées entre ces opérons et des grandes similitudes entre ces molécules de type NRP. De plus, l’importance du séquençage du génome de la souche de B. subtilis S499 a été soulevée. L’obtention d’un mutant mono-producteur de plipastatine, dérivé de B. subtilis 168, par le remplacement du promoteur natif Ppps par un promoteur constitutif PrepU associé à un gène de résistance à la néomycine (neo), n’a pas abouti. Par contre, l’inactivation de l’expression de l’opéron plipastatine chez le mutant BBG111, constitutif pour la synthèse de surfactine, par cette cassette PrepU-neo située en sens inverse de la transcription de l’opéron pps, a conduit à l’isolement du dérivé BBG140 montrant une production accrue de surfactine par rapport à la souche parentale. Les promoteurs Ppps (B. subtilis 168) et Pfen (B. subtilis BBG21) ont été clonés dans le vecteur d’expression pDG1661, portant la cassette lacZ, et intégrés dans le chromosome de la souche 168. L’expression et la régulation en différentes conditions de croissance de ces promoteurs Ppps et Pfen, comparées à celles du promoteur PsrfA de B. subtilis 168, ont été étudiées dans les mutants dérivés respectifs BBG142, BBG139 et BBG127
In this study, 36 Bacillus strain genomes were analyzed; nine of them have no NRPS molecules while the detection of one NRPS molecule ‘Bacillibactin siderophore’ was showed for another nine strains. The other 18 strains showed the presence of 17 other new NRPS and NRPS-PKS hybrid molecules. The analysis of plipastatin or fengycin operons sequences of Bs F29-3, Bs 168 and B. amyloliqufaciens FZB42 were studied, which refer to the similarity of these molecules and clarified the importance of sequencing the Bs S499 fengycin operon which have a fengycin molecule cannot be correlated with the structure of the synthetases described in other fengycin- or plipastatin-producing strains interesting to come over the difficulty of the differentiation between fengycin and plipastatin. The obtaining of plipastatin mono-producer derivative from Bs 168 by the replacement of the plipastatin native promoter by a constitutive one Prepu was failed. The constructed plasmid with Prepu-neo gene have two types; pBG180 which couldn’t be transformed and pBG180* which has inverted Prepu-neo gene and transformed into BBG111 to obtain BBG140 which showed no plipastatin production and high surfactin production than the mother strain BBG111. The expression and the regulation of Ppps, Pfen and Psrf were studied in Bs 168 derivatives: BBG142, BBG139 and BBG127 respectively

La lutte contre la tavelure du pommier due à Venturia inaequalis, une maladie préjudiciable, nécessite des alternatives aux fongicides chimiques conventionnels plus respectueuses de l'environnement. Une possibilité prometteuse est l'utilisation de lipopeptides produits par différentes espèces de Bacillus. Un des objectifs de ce travail a été d'évaluer l'efficacité de trois familles de lipopeptides (fengycines, iturines et surfactines) et de leurs mélanges pour lutter contre V. inaequalis, tant in vitro qu'in vivo. Il s'est basé sur une souche de V. inaequalis sensible au tébuconazole (S755) et une souche ayant une sensibilité réduite au tébuconazole (Rs552) qui présentent également une résistance différenciée à la fengycine avec pour objectif d'approfondir la compréhension des mécanismes responsables de cette différence de sensibilité à ces deux molécules antifongiques. Contrairement à la fengycine, les lipopeptides de la famille des iturines ont une activité similaire sur les deux souches, alors que ceux de la famille des surfactines ne sont pas actifs, sauf en mélange binaire avec la fengycine. Les mélanges de fengycine/surfactine 50-50% et de mycosubtiline/surfactine 80-20% sont aussi efficaces que les lipopeptides seuls in vitro. A partir de ces mélanges, des essais en vergers ont montré une réduction significative de l'incidence de la tavelure pouvant aller jusqu'à 70%. Les différences de sensibilités ont été investiguées en utilisant des techniques de biologie moléculaire, de microscopie et de lipidomique. Les différences de sensibilités aux triazoles sont liées à des mécanismes moléculaires distincts, incluant la surexpression du gène Cyp51A et un efflux accru par des pompes membranaires chez la souche Rs552. Alors que ces mécanismes ne semblent pas impliqués dans la sensibilité réduite à la fengycine. Les observations microscopiques indiquent des altérations cellulaires induites par celle-ci, suggérant une interaction avec les membranes plasmiques. Les analyses lipidiques révèlent quelques variations dans la composition en stérols et acides gras totaux ainsi qu'en phospholipides entre les souches, pouvant influencer leur sensibilité à la fengycine. En conclusion, l'efficacité potentielle des lipopeptides produits par Bacillus comme alternative aux fongicides chimiques dans la lutte contre la tavelure du pommier a été mis en lumière
The fight against apple scab caused by Venturia inaequalis, a harmful disease, requires more environmentally friendlier alternatives to conventional chemical fungicides. One promising possibility is the use of lipopeptides produced by various Bacillus species. One of the objectives of this work was to evaluate the efficacy of three families of lipopeptides (fengycins, iturins and surfactins) and their mixtures in controlling V. inaequalis, both in vitro and in vivo. The project was based on a V. inaequalis strain sensitive to tebuconazole (S755) and a strain with reduced sensitivity to tebuconazole (Rs552), which also exhibit differential resistance to fengycin, with the aim of deepening the understanding of the mechanisms responsible for this difference in sensitivity to these two antifungal treatments. Unlike fengycin, lipopeptides from the iturin family have a similar activity on both strains, while those from the surfactin family are not active, except in binary mixtures with fengycin. Mixtures of fengycin/surfactin 50-50% and mycosubtilin/surfactin 80-20% are as effective as individual lipopeptides in vitro. From these mixtures, orchard trials have shown a significant reduction in scab incidence of up to 70%. Sensitivity differences were investigated using molecular biology, microscopy and lipidomic techniques. Sensitivity differences to triazole are related to distinct molecular mechanisms, including overexpression of the Cyp51A gene and increased efflux by membrane pumps in the Rs552 strain. Whereas these mechanisms do not seem to be involved in reduced sensitivity to fengycin. Microscopic observations indicate cellular alterations induced by fengycin, suggesting an interaction with plasma membranes. Lipid analyses reveal some variations in sterol and total fatty acid composition as well as phospholipids between strains, which could influence their sensitivity to fengycin. In conclusion, the potential effectiveness of lipopeptides produced by Bacillus as an alternative to chemical fungicides in the biocontrol of apple scab has been highlighted

Orientador: Lúcia Regina Durrant
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Os biossurfactantes são compostos amplamente estudados em todo o mundo. Suas características o tornam muito atrativo em relação aos surfactantes sintéticos. Dentre essas características podemos citar, a baixa toxicidade, boa compatibilidade com a pele e olhos, biodegradabilidade e produção a partir de substratos renováveis. No entanto, o custo de produção dos biossurfactantes ainda inviabiliza sua produção em escala industrial. Tradicionalmente, os hidrocarbonetos têm sido os substratos escolhidos para a produção de biossurfactantes e bioemulsicadores. É assumido que a produção de biossurfactantes é induzida para tornar o substrato hidrofóbico acessível a célula. Contudo, substratos solúveis em água também podem ser utilizados para a produção dos surfactantes biológicos. Esses últimos são mais baratos do que os hidrocarbonetos e preferidos em processos fermentativos, devido ao fato de que as fermentações monofásicas são mais simples do que as fermentações bifásicas. Além disso, o uso de hidrocarbonetos são inaceitáveis para a produção de biossurfactantes destinados a aplicações em alimentos, cosméticos e produtos farmacêuticos. Uma grande variedade de matérias-primas estão atualmente disponíveis como substrato orgânico para fermentações industriais, dentre as quais podemos destacar os resíduos agroindustriais. Essas matérias-primas possuem vantagens de serem encontradas em excesso e produzidas em regiões de clima tropical ou temperado. Atualmente, com o aumento da demanda de produção de biodiesel, o glicerol tem ganhado atenção no cenário de bioprocessos, demonstrando ser uma matéria-prima de grande disponibilidade para produção de biomoléculas de interesse industrial. Segundo a Agência Nacional de Petróleo (ANP-Brasil), o Brasil é ranqueado como o maior produtor e consumidor de biodiesel do mundo: 1,2 bilhões de litros/ano em 2008, gerando de 120.000 toneladas de glicerina residual nesse processo. Logo, o presente trabalho teve como objetivo selecionar um substrato orgânico de baixo custo, de forma a tornar viável a produção e comercialização dos biossurfactantes em larga escala. Dentre as 19 linhagens microbianas avaliadas, o microrganismo Bacillus subtilis LSFM_05 foi selecionado para a produção de biossurfactantes, utilizando glicerina residual bruta como substrato orgânico. A temperatura de 32oC e concentração de glicerina de 5% v/v foram designadas como condições ótimas para a produção de biossurfactantes, através do planejamento experimental. A produção dos biossurfactantes foi realizada em fermentador um litro nas rotações de 150, 250 e 350 rev.min -1. A fermentação conduzida à 250 rev.min-1 apresentou melhor desempenho na formação de espuma e produção de biossurfactante. Em seguida, essa condição foi reproduzida em fermentador de 10 litros, que apresentou rendimento de 0,69 g.L-1. Após a execu-ção dos processos fermentativos, os biossurfactantes foram recuperados por precipitação ácida, puri_cados em coluna de adsorção e caracterizados utilizando Espectrometria no Infravermelho com Transformada de Fourier (IV-FT), Espectrometria de Ressonância Magnética (1H e 13C RMN) e Espectrometria de Massa (ESI-MS/MS). O biossurfactante foi caracterizado como uma isoforma do lipopeptídio surfactina, contendo 14 átomos de carbono na cadeia lipídica e sete aminoácidos do anel peptíco. A espectrometria de massa foi capaz de elucidar a composição e sequência de aminoácidos do peptídio cíclico GluOMe(1)/Leu(2)/Leu(3)/Val(4)/Asp(5)/Leu(6)/Leu(7) e os espectros de 1H e 13C - RMN foram de extrema importância para comprovar a esteri_cação do aminoácido ácido glutâmico. O microrganismo Bacillus subtilis LSFM_05 também foi caracterizado como co-produtor de fengicina. A espectrometria de massa apresentou ser uma técnica sensível e rápida para a caracterização dos homólogos A e B de fengicina. A surfactina C14/Leu7 foi avaliada com relação a toxicidade ambiental sobDaphnia similis, apresentando EC50 em 1500 mg.L-1. A surfactina apresentou atividade antiviral contra o vírus envelopado Herpervirus bovino (BoHV-1), inibindo 100% da infectividade do vírus na concentração de 0,25 _M, porém, não apresentou atividade antimicrobiana para os microrganismos avaliados até a concentração de 1mg.mL-1. A surfactina também foi avaliada com relação a citotoxicidade sob _broblastos c3T3 de camundongos, e o valor de EC50 não foi determinado na faixa de concentração estudada, indicando níveis de toxicidade para esse tipo de célula. No âmbito na nanotecnologia, o extrato bruto contendo os biossurfactantes mostrou-se uma ferramenta útil na dispersão de nanotubos de carbono, em água de cultivo de Daphnia, tornando possível o estudo de ecotoxicologia desses nanomateriais
Abstract: In recent years biosurfactants have attracted considerable attention because they o_er several advantages in comparison with synthetic surfactants: low toxicity, greater biodegradability, better environmental compatibility, greater foaming, speci_c activity at extreme temperatures, pH and salinity, and the possibility of being produced from renewable sources and industrial wastes However, biosurfactants have not yet been employed extensively in industry because of their relatively high production and recovery costs. The cost can be reduced by strain improvement, optimizing medium composition by statistical methods or by using alternative inexpensive substrates. Traditionally, hydrocarbons have been the substrates of choice to produce biosurfactants and bioemulsi_ers. It is assumed that surfactant production is induced to render hydrophobic substrates accessible to the cell, but water-soluble substrates such as molasses, cassava waste-water and potato substrates have also been used for biosurfactant production. The latter are cheaper than hydrocarbons and are the preferred substrates, because single-phase fermentation is simpler than biphasic fermentation and in addition the hydrocarbon substrates are unacceptable for many applications, such as in foods, cosmetics and pharmaceutical products. One alternative substrate aimed at decreasing costs in the production of biosurfactants could be the waste glycerol obtained from the biodiesel industry. Brazil is ranked amongst the greatest producers and consumers of biodiesel in the world: 1.2 billion liters /year in 2008, according to the National Petroleum Agency-Brazil (ANP) generating 120,000 tons of waste glycerol in this process. The data described above may support the idea of applying the raw glycerol in the production of biosurfactant on a large-scale. This present study aimed to select an low cost organic substrate, in order to become viable the biosurfactant production on a large scale. Among the 19 microbial strains evaluated, the microorganism Bacillus subtilis LSFM-05 was selected for the biosurfactant production using raw glycerol as organic substrate. The temperature of 32oC and glycerol concentration of 5% v/v were designated as optimal conditions for the production of biosurfactants, by response surface methodology. The production of biosurfactants was carried out by one liter fermentor at 150, 250 and 350 rev.min-1. The 250 rev.min-1 fermentation was the best performance in foaming and biosurfactant production. Then, this condition was reproduced in 10 liter fermentor, with yield of 0.69 g.L-1. After the fermentation processes, the biosurfactants were recovered by acid precipitation, puri_ed by adsorption column and characterized using Infrared Spectroscopy Fourier Transform (FT-IR), Magnetic Resonance Spectroscopy ( 1H and 13C NMR) and Mass Spectrometry (ESI-MS/MS). The biosurfactant was characterized as a surfactin isoform, containing14 carbon atoms in the lipid chain and 7 aminoacids in the peptide portion. The mass spectrometer was able to elucidate the composition and aminoacids sequence in the cyclic peptide GluOMe(1)/Leu(2)/Leu(3)/Val(4)/Asp(5)/Leu(6)/Leu(7). The microorganism Bacillus subtilis LSFM-05 was characterized as a fengycin co-producer. The mass spectrometry have been a sensitive and rapid technique for fengycin homologues characterization. The C14/Leu7 surfactin presented EC50 value of the 1500 mg.L-1 against Daphnia similis in ecotoxicological studies. The surfactin showed antiviral activity against enveloped bovine herpesvirus (BoHV-1), inhibiting 100 % of the infectivity at concentration of 0.25 _M, however, it didn't show antimicrobial activity for microorganisms evaluated until concentration of 1mg.mL-1. The surfactin cytotoxicity in mice _broblasts c3T3 was evaluated as well, and the EC50 value was not determined in the studied concentration range, indicating low levels of toxicity for this cell type. Concerning to applications in nanotechnology, the biosurfactants proved to be a useful tool in the dispersion of carbon nanotubes in standard water cultivation of Daphnia, enabling future studies about the environmental toxicology of these nanomaterials
Doutorado
Doutor em Ciência de Alimentos

Les rhamnolipides (RLs) et les fengycines (FGs), des composés sécrétés par des bactéries, ont des propriétés antifongiques contre les champignons phytopathogènes Sclerotinia sclerotiorum et Botrytis cinerea. Cependant, les effets biocides induits et les mécanismes impliqués sont peu étudiés chez les champignons. De par leur caractère amphiphile, un mode d’action membranotrope est proposé pour ces composés intéréssants pour le biocontrôle. Les travaux présentés ici démontrent que les deux Sclérotiniacées ont des sensibilités opposées aux RLs et aux FGs. Une étude en microscopie montre que les RLs peuvent induire une mort cellulaire programmée (PCD) ou nécrotique chez les deux champignons selon la concentration alors que les FGs induisent systématiquement une PCD, probablement par un mécanisme d’autophagie. Des analyses lipidomiques (contenus en acides gras, phospholipides et ergostérol) de souches de S. sclerotiorum et B. cinerea plus ou moins sensibles aux RLs et aux FGs permettent de rapprocher les contenus lipidiques des champignons à leurs sensibilités. Ces données ont été utilisées pour étudier les interactions entre les RLs ou les FGs et des modèles de membranes plasmiques biomimétiques des deux champignons. Les études de dynamique moléculaire montrent que les RLs s’insèrent, sous forme de monomères, dans les différents modèles étudiés sans les fluidifier et que les FGs s’agrègent puis s’insèrent dans certains modèles en induisant une fluidification. L’ergostérol et les acides phosphatidiques défavoriseraient cette insertion tandis que les phosphatidylcholines et les phosphatidyléthanolamines les favoriseraient. Ces travaux permettent de mieux appréhender le mode d’action antifongique des RLs et des FGs, et contribueront, à terme, au développement de produits de biocontrôle plus efficaces pour la protection des cultures vis-à-vis de pathogènes spécifiques
Rhamnolipids (RLs) and fengycins (FGs), are compounds produced by bacteria displaying antifungal properties against the phytopathogenic fungi Sclerotinia sclerotiorum and Botrytis cinerea. However, the induced biocidal effects, and the involved mechanisms are poorly understood in fungi. Due to their amphiphilic properties, a membranotropic mode of action is proposed for these interesting compounds for biocontrol. The present work demonstrates that the two Sclerotiniaceae have opposite sensitivities to RLs and FGs. A microscopy study shows that RLs can induce programmed cell death (PCD) or necrotic cell death in both fungi depending on the concentration whereas FGs systematically induce PCD, probably by triggering autophagy. Lipidomic analyses (fatty acid, phospholipid and ergosterol contents) of S. sclerotiorum and B. cinerea strains differently sensitive to RLs and FGs allow to correlate the lipid contents of the fungi to their sensitivities. These data are used to study the interactions of RLs or FGs on biomimetic plasma membrane models of the two fungi. The dynamics show that the RLs monomers insert into the models without fluidizing them and that the FGs auto-aggregate themselves and insert into some models, inducing fluidization. Ergosterol and phosphatidic acids seems to disfavour this insertion while phosphatidylcholine and phosphatidylethanolamine seem to favour it.This work allows to better understand the antifungal mode of action of RLs and FGs, with a view to develop more effective biocontrol products for crop protection targeting specific pathogens

This thesis is focused on the development of synthetic approaches to obtain new bioactive peptides. The first part deals with the design of new antimicrobial peptide/cell-penetrating peptide conjugates as anticancer agents. Their conjugation enhanced the activity of the antimicrobial peptides against cancer cells while maintained their low toxicity. These compounds are interesting for the design of new anticancer agents. On the second part, a new versatile methodology for the synthesis of natural fengycin derivatives is described. Our strategy represents the first synthetic approach for the total solid-phase synthesis of these cyclic lipodepsipeptides and can be easily adapted to obtain a wide range of analogues.
Aquesta tesi doctoral s’ha centrat en el desenvolupament d’estratègies sintètiques útils per a l’obtenció de nous pèptids bioactius. Primerament, s’han dissenyat nous pèptids conjugats antitumorals a través de la unió d’un pèptid antimicrobià i un cell-pentrating peptide. Aquesta conjugació augmenta l’activitat antitumoral del pèptid mantenint la toxicitat baixa. Aquests conjugats són interessants pel desenvolupament de nous agents antitumorals. A continuació, s’ha desenvolupat una metodologia per a la preparació de pèptids cíclics derivats de les fengicines. Aquesta metodologia representa la primera estratègia sintètica descrita per a l’obtenció en fase sòlida d’aquesta família de ciclolipodepsipèptids i pot ser fàcilment adaptada per a l’obtenció d’una àmplia varietat d’anàlegs.

Nas células cancerosas, a via apoptótica é danificada pela supressão ou mutação de genes importantes, como genes supressores de tumor p53 ou Check2. Isto faz com que as células cancerígenas percam a habilidade de executar a morte celular controlada, resultando na desobstrução da divisão celular e crescimento do tumor. Muitos tumores também mostram resistências aos tratamentos tradicionais como quimioterapia ou radioterapia. Tratamentos de câncer baseados na apoptose induzida ou em aumento na inibição das células cancerosas estão em desenvolvimento. O objetivo deste trabalho é investigar nas células cancerígenas humanas o efeito anti-proliferativo dos lipopeptídeos iturin A e fengycin obtidos das estirpes de Bacillus spp. bem como dos da proteina VP3 de Avian gyrovirus II (AGVII). A proteína VP3 do AGV II foi descoberta em 2011 e sua seqüência de aminoácido mostra 32.2% de homologia e domínios funcionas similares à proteina apoptina do vírus da anemia infecciosa das galinhas (CAV - apoptin), uma proteína que induz apoptose nas células cancerígenas mas que não afeta células normais. Inicialmente, para obter a proteína VP3 em uma análise adicional, a sequência PTD4, conhecida como uma sequência de transmissão dentro da célula, foi adicionada à seqüência VP3 através de PCR. Após a determinação da sequencia de nucleotideos, o produto de PCR foi clonado dentro do vetor de expressão PET-SUMO e a construção final foi transformada em E. coli BL21 (DE3) pLyS. Após a super expressão da proteína e a purificação subsequente, a proteína PTD4-VP3 foi incubada com culturas de células de câncer humano. Os lipopeptídeos iturin A e fengycin foram produzidos pelo Bacillus amyloliquefaciens LBM 5006 e pelo Bacillus sp. P34, respectivamente. Os lipopeptídeos foram purificados e adicionados às culturas de células de câncer humano. A linhagem de célula nãocancerígena humana AS405 (fibroblasto) foi escolhida como o controle. O efeito da proteína na viabilidade celular foi determinado através de testes de MTT. Os resultados mostraram que o efeito antiproliferativo dos lipopeptídeos utilizados iturin e fengycin, bem como os peptídeos virais PTD4-VP3 (T) e PTD4-VP3 (SM) dependem da dose. Para os lipopeptídeos o efeito de inibição do crescimento dependente do tempo, também pode ser detectada. Um efeito anti-proliferativo em células humanas normais não pode ser excluído, embora não tenha sido claramente demonstrado. Este é o primeiro estudo validando o 24 potencial anti-proliferativo dos lipopeptideos, iturin e fengycin e das proteínas de fusão viral PTD4-VP3 (T) e PTD4-VP3 (SM) em inibir o crescimento de células, principalmente células cancerígenas humanas.
In cancer cells the apoptotic pathway is damaged by deletion or mutation of important genes, e.g. tumor-suppressor gene p53 or Check2. This causes a loss in cancer cells to undergo controlled cell death resulting in unstopped cell division and tumor growth. Also, many tumors show resistances against the traditional applied treatments like chemo or radiation therapy. Therefore, accompanied or independent cancer treatments based on induced apoptosis or strengthened growth inhibition in cancer cells are under development. The objective of this work was to investigate the anti-proliferative effect on human cancer cells of the lipopeptides iturin A and fengycin obtained from strains of Bacillus spp. as well as of the Avian Gyrovirus II (AGVII) protein -VP3. The VP3 protein of the AGV II was discovered in 2011 and its amino acid sequence showed 32.2% homology and similar functional domains to the chicken anemia virus apoptin (CAV-apoptin), a protein that was proven to induce apoptosis in cancer cells but not in normal cells. Initially, to obtain the VP3 protein for further analysis, the PTD4 sequence, known as a transmission sequence into the cell, was N-terminally added to the VP3 sequence via PCR. After sequencing, the PCR product was cloned into the expression vector pET-SUMO and the final construct transformed into E. coli BL21(DE3)pLyS. After induced protein overexpression and subsequent purification, the PTD4-VP3 protein was quantified and incubated with human cancer-cell cultures. The lipopeptides iturin A and fengycin were produced by Bacillus amyloliquefaciens LBM 5006 and Bacillus sp. P34, respectively. The lipopeptides were purified and added to the cell cultures of human tumor cells. The human non-cancer cell line AS405 (skin fibroblasts) was chosen as control. The protein effect on cell viability was determined via MTT assays. The results showed that the lipopeptides iturin and fengycin as well as the viral peptides PTD4-VP3 (T) and PTD4- VP3 (SM) demonstrated dose-dependent anti-proliferative activity on cancer cells. For the lipopeptides also time-dependent growth-inhibition effect could be detected. A anti-proliferation effect on normal human cells was not excludable but could not clearly be demonstrated. 22 This is the first study validating the anti-proliferative potential of the lipopeptides iturin and fengycin and the viral fusion proteins PTD4-VP3 (T) and PTD4-VP3 (SM) to inhibit cell growth mainly in human cancer cells.

碩士
長庚大學
基礎醫學研究所
91
Fengycin, an antifugal antibiotic, is a cyclic lipopeptidic antibiotics produced by Bacillus subtilis F29-3. Fengycin contains 10 amino acids and is synthesized nonribosomally by peptide synthetases encoded by an operon containing fenC, fenD, fenE, fenA and fenB. In this study, I analyze the functions of fengycin synthetase modules, FenA1, FenA2 and FenA3. FenA1, with a predicted molecular mass of 126 kDa, was expressed in Escherichia coli BL21(DE3)pLysS and was purified by Ni-affinity chromatography. ATP-PPi exchange assay revealed that FenA1 activates proline with an optimun temperature between 25。 to 37。, an optimun pH of 4.5, a Km value of 1 mM and a Kcat value of 0.58 s-1. FenA3, with a predicted molecular mass of 169 kDa, activates tyrosine. FenA2, with a predicted molecular mass of 132 kDa, was expressed as inclusion body in E. coli BL21(DE3) and E. coli BL21(DE3)pLysS. Results presented herein suggest that fengycin synthetase genes and amino acids in fengycin are collinear.

Fengycin is a cyclic lipopeptide produced mainly by the Bacillus genus, which is structurally composed of a β-hydroxy fatty acid and 10 amino acids. The biosynthesis of fengycin is catalyzed by large non-ribosomal peptide synthetases. Fengycin is an amphiphilic molecule with strong surface activity and displays strong antimicrobial activity. In this chapter, the molecular structure and biological properties of fengycin, and the function and catalyzing mechanism of fengycin multienzyme were summarized. Multiple antimicrobial mechanisms of fengycin and the strategies for increasing the production of fengycin were introduced. Fengycin has the advantages of low toxicity, biodegradation and high stability. Its applications, including biological control of plant pathogens, bioremediation of a contaminated environment, postharvest disease control of fruit and vegetables, food processing and preservation, etc., were reviewed finally.

This study was designed to assess the suppressive effects of various anaerobically digested slurries (ADSs), and the microorganisms inhabiting them, against Fusarium wilt in spinach. We used five different ADSs from a range of source materials (dairy cow manure, sewage sludge, food garbage, pig manure, night soil sludge), combined in different proportions. All five raw ADSs suppressed the growth of Fusarium oxysporum f. sp. spinaciae (Fos) on agar plates using a co-culture test. In contrast, filtrate ADSs did not suppress the growth of Fos. In total, 32 bacterial strains were isolated from five ADSs, and eight isolates showed antagonistic activities against Fos. Based on 16S rDNA sequences, the strain AD-3 isolated from ADS from dairy cow manure belonged to Bacillus velezensis. Genome analysis revealed that AD-3 had two kinds of genes related to the production of the non-ribosomal lipopeptides, fengycin/plipastatin (pps genes), and surfactin (srf genes). In pot assays, inoculation of AD-3 (1.0 × 106 CFU·g −1 dry soil) into Fos-infected soil (1.0 × 105 bud-cells·g −1 dry soil) significantly reduced the severity of Fusarium wilt disease at 28 d after seedling. The percentage reductions in disease severity in two replicates were 64.3% and 44.3%, respectively. Thus, bacterial strain AD-3 could be applied to reduce Fusarium wilt in spinach.