Dissertations / Theses on the topic 'Fengycine'

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.

碩士
長庚醫學暨工程學院
基礎醫學研究所
85
abstractFengycin is a cyclic lipopeptidic antibiotic produced by Bacillus subtilis F29-3. This antibiotic consists of ten amino acids and exhibits a broad spectrum of antifungal activity. Results obtained from our laboratory have shown that fengycin is synthesized nonribosomally by peptide synthetases. Our laboratory has previously isolated a 46-kb cosmid clone, containing a large numbers of genes involved in fengycin biosynthesis. Despite of the size of the cosmid, this clone does not contain all the fengycin biosynthesis genes. To take one step further in understanding the mechanism of fengycin biosynthesis, I have carried out studies to identify the fengycin biosynthesis genes that are not located in pFC660. I have identified two cosmid clones, pFC3-1 and pFC768, overlapping with pFC660. Sequencing results showed that pFC3-1 contained a complete peptide synthetase gene, fenE and 3'' region of a peptide synthetase gene, ''fenD. Gene fenE is 7,665 bp long, encoding a 2,554-amino acid peptide synthetase. The protein encoded by this gene, FenE consists two amino acid activation modules, FenEA and FenEB. I also found that FenEB contain an epimerase domain at C-terminal region of the module. In all the peptide synthetase genes identified so far, an epimerase domain is always present at the C-terminal region of the last amino acid activation module of a peptide synthetase. Therefore, FenEB should be the last module of the FenE peptide synthetase. Gene ''fenD is 2,877 bp long. This segment of DNA encodes the last module of a peptide synthetase, ''FenD. This module also consists of an epimerase domain. Sequence comparison also revealed that FenEA, FenEB, and ''FenD consist of conserved elongation, ATP- binding, ATPase, and pantethein-binding motifs, typical of a peptide synthetase. Phylogenetic analysis suggested that FenEA, FenEB, and ''FenD may activate glutamic acid, valine, and leucine, respectively. However, the biochemical properties of these modules has to be confirmed. Since nonribosomal peptide synthesis is potentially useful for the production of industrially important peptides, the understanding of fenE and ''fneD will be useful for us to achieve this goal.

碩士
長庚大學
基礎醫學研究所
93
Fengycin is an antifungal antibiotic produced by Bacillus subtilis F29-3, which is composed of a fatty acid and a peptide chain. Fengycin is synthesized nonribosomally by fengycin synthetases, including FenC, FenD, FenE, FenA, and FenB. It is known that fengycin peptide elongates in an orderly fashion during peptide synthesis on FenC, FenD, FenE, FenA, and FenB. Thus, it is hypothesized that specific interactions among fengycin synthetases are critical to the synthesis process. Our earlier studies demonstrated that these five peptide synthetases form a complex in vivo. However, whether these enzymes interact specifically is still unknown. To understand how fengycin is synthesized, this study investigates the interaction between FenA and FenB to elucidate the domains in these two enzymes involved in the interaction. This study demonstrates that the N-terminal region of FenB interacts specifically with the C-terminal region of FenA. Deletion analysis delineates the interaction regions to the N-terminal 538 amino acids of FenB and the C-terminal 500 amino acids of FenA. Because this study finds that FenB interacts only with FenA but not the other fengycin synthesis; the finding suggests that nonribosomal peptide synthesis involves specific interactions among peptide synthetases.

碩士
長庚醫學暨工程學院
基礎醫學研究所
85
AbstractFengycin is a lipopeptide which exhibits a broad spectrum of antifungal activity. Previous studies have shown that fengycin peptide consists of 10 amino acids and is synthesized nonribosomally by peptide synthetases. These peptide synthetases can bind to and activate specific amino acids. These activated amino acids are then linked together by the action of the enzymes to form the fengycin peptide. The purpose of this study was to characterize the gene, fenA, which encodes the peptide synthetase, FenA. Sequencing analysis revealed that fenA has a length of 10,513 bp and encodes a peptide synthetase, FenA which can activate three amino acids. Each of the three amino acid activation modules (FenAA, FenAB, FenAC) of FenA contains conserved spacer, ATP-binding, ATPase, and pantetheinin-binding domains. The sequence similarities between FenAA and the SrfAC module of surfactin synthetase, FenAB and the TyrA module of tyrocidine synthetase, FenAC and the GrsA module of gramicidin S synthetase are 32.8%, 33.7%, and 34.9%, respectively. I have also cloned and overexpressed the fenAC module of fenA in Escherichia coli. The expressed protein was purified by His-tag affinity chromatography. The purified protein was incubated with one of the twenty amino acids in a reaction mixture containing [32P]sodium pyrophosphate and ATP. Results showed that only alanine was able to promote the pyrophosphate exchange between sodium pyrophosphate and ATP, indicating that the function of FenAC is responsible for the activation of alanine. Studies reported by other laboratory showed that the last amino acid activation module of all the peptide synthetases studied so far consists of a racemase domain, indicating that this domain is required not only for the conversion of L-amino acid to D-amino acid but also for the translocation of the peptide translocation from one enzyme to the other enzyme. I found that a conserved racemase region is also present at the end of FenAC, showing that FenAC is the last module of FenA synthetase. I have also determined the locations of the seven Tn917lux insertions in fenA and analyzed the expression of fenA by monitoring the luciferase activity exhibited by two of the Tn917lux mutants, FS22 and FS24. Results showed that the luciferase was expressed during the stationary phase, suggesting that the promoter which transcribes fenA is activated during the stationary phase. Since it is possible to generate artificial peptide synthetases consisting of different amino acid modules for the synthesis of a specific peptide, the understanding of the structure and the function of fenA will help us to achieve this goal.

博士
國立臺灣大學
植物學研究所
87
Abstract Fengycin, an antibiotic produced by Bacillus subtilis F29-3, is a cyclic lipopeptide containing one each of D-ornithine, L-tyrosine, D-allo-theronine, L-proline, D-tyrosine, L-isoleucine, D-alanine or D-valine and three L-glutamic acids. In the past, our laboratory isolated three overlapping cosmid clones which contain at least two gene clusters that are involved in fengycin synthesis. One of the gene cluster contains five fengycin synthetase genes, namely fenC, fenD, fenE, fenA and fenB. These genes are transcribed form a promoter located immediately upstream from fenC. FenB, which activates the last amino acid of fengycin, was overexpressed and purified. This enzyme activates L-isoleucine. The enzyme has a Km value of 922 mM and a turnover number of 236S-1. The optimal reaction conditions are 25 ℃ and pH 4.5. I have also overexpressed the three amino acid-activation modules of FenA in E. coli. However, these recombinant proteins form inclusion bodies in the cell and was difficult to refold. Therefore, intact FenA was purified from B. subtilis F29-3 directly. The FenA protein has a molecular mass of 406 kDa. This protein was purified by gel filtration, ion exchange chromatography, and FPLC. Results revealed that FenA activates L-proline, L-glutamic acid and L-tyrosine. The results presented in this dissertation will lead us to a better understanding on the functions of fengycin synthetases and the mechanism invloved in nonribosomal peptide synthesis.

碩士
元智大學
生物科技暨生物資訊研究所
95
Fengycin, a cyclic lipopeptidic antibiotic produced by B. subtilis F29-3, is composed of a peptide chain of 10 amino acids and a long-chain fatty acid. Five fengycin synthetases, including FenC, FenD, FenE, FenA and FenB, catalyze the synthesis of peptide moiety. Until now, the mechanism about the covalent linkage of a long-chain fatty acid moiety to peptide moiety is not well studied. Early studies on the synthesis of the fatty acid chain in fengycin showed that, instead of a binding of long-chain acyl-group, malonyl-group that bound to FenC (the initial fengycin synthetases) and FenB (the last fengycin synthetases), catalyzed by two transacylases, YvsB and YdjL. Furthermore, a set of enzymes related to fatty acid metabolism has also been identified to be essential for fengycin synthesis. Only the thiolation domain 1 of FenC can bind the malonyl-group. In this study, the FenD and FenA1 was not linkage with malonyl-group, and the malonyl-group binding site of FenC was investigated. Based on our studies, the model for the synthesis of fatty acid moiety of fengycin is also proposed.

碩士
元智大學
生物科技暨生物資訊研究所
95
Bacillus subtilis F29-3 was identified as a producer of fengycin, a potent anti-fungal cyclic lipopeptide antibiotic composed of a circular decapeptide and a ß-hydroxy fatty acid linked to the N-terminal of the decapeptide. The formation of fatty acid moiety and its linkage to the peptide chain are yet to be elucidated. The purpose of this study was to explore the relationship between the expression of transacylases genes, yvsB and ydjL, and the fatty acid synthesis of fengycin. According to the in vitro assays of transacylases activity, two putative transacylases, YvsB and YdjL, from B. subtilis could transfer the malonyl group from malonyl-CoA to FenC. Furthermore, the promoter analysis of yvsB and ydjL displayed that the expression of yvsB and ydjL were related to fengycin production. Therefore, YvsB and YdjL were involved in the fatty acid synthesis of fengycin.

博士
長庚大學
生物醫學研究所
104
Fengycin is a lipopeptide antibiotic synthesized nonribosomally by five fengycin synthetases. These enzymes are linked in a specific order via interactions between the C-terminal region of an upstream enzyme and the N-terminal region of its downstream partner enzyme, to form a fengycin synthetase complex. This study shows that the communication-mediating donor (COM-D) domains of fengycin synthetases are required for enzyme interaction during complex formation, and can be specifically recognized by downstream partner enzymes. In addition, although communication-mediating acceptor (COM-A) domains are also required for binding between two partner enzymes, non-partner enzymes can non-specifically recognize these domains as well. We have confirmed that a large N-terminal region in fengycin synthetases is crucial to achieving specific recognition of upstream partner enzymes during complex formation. This region includes the COM-A domain, the entire condensation domain, and a portion of the adenylation domain. When the COM-A domain of BN657 or FenB is replaced with the corresponding region in FenA, the resulting construct, BN657(COM-AA) or His-FenB(COM-AA), lost the ability to specifically recognize FenA and was able to interact with all fengycin synthetases, indicating that the COM-A domain and the protein structure of N-terminal region in fengycin synthetases are critically involved in the specific recognition of partner synthetases. These findings shed light on key domains involved in the specific recognition of upstream and downstream partner enzymes, and serve to advance the current understanding of fengycin synthetase enzyme complex formation.

碩士
嘉南藥理科技大學
生物科技系暨研究所
92
Fengycin is a lipopeptidic antifungal antibiotic produced by Bacillus subtilis F29-3. This antibiotic contains ten amino acids and a long-chain fatty acid residue. This peptide is synthesized nonribosomally by five fengycin synthetases, including FenC, FenD, FenE, FenA and FenC. Earlier studies show that FenD contains two amino acid modules, FenD1 and FenD2, each of which contains a condensation, adenylation and thiolation domain, that are typically present in peptide synthetases. In addition, FenD2 also contains an epimerization domain in C-terminal region. A protein containing the condensation, adenylation and thiolation domains of FenD1 (CAT-FenD1) and adenylation domain of FenD2 (AD-FenD2) was expressed in Escherchia coli and purified by Ni-chelate affinity chromatography. Biochemical analysis revealed that FenD1 and FenD2 activate the third and the fourth amino acid in fengycin, L-Tyr and L-Thr, respectively. In the case of iturin synthesis, initiation of peptide synthesis involves fatty acid synthesis and the functiosn of acyl-CoA synthetase and acyl carrier protein. Our earlier Tn917 mutagenesis studies had identified a gene, fenL , which encodes a protein with a sequence similar to acyl-CoA synthetase. Therefore, this investigation mutagenizes fenL and demonstrates the importance of the gene. Meanwhile, the fenL was expressed in Escherchia coli and purified by affinity chromatography with a His resin. The overexpressed recombinant protein has an estimated molecular mass of 70 kDa.

碩士
元智大學
生物科技暨生物資訊研究所
93
Abstract Fengycin, an antifungal antibiotic produced by Bacillus subtilis F29-3, is a cyclic lipopeptidic antibiotic. This compound is produced by a series of enzymes, i.e., FenC, FenD, FenE, FenA, and FenB, underlying a nonribosomal mechanism of peptide synthesis. The structure of fengycin includes a cyclic polypeptide, consisted of one each of D-ornithine, L-tyrosine, D-allo-theronine, L-proline, D-tyrosine, L-isoleucine, D-alanine or D-valine, and three L-glutamic acids, and a fatty acid moiety linking to the N terminal of the peptide backbone. Although fengycin synthesis has been studied for about forty years, lots of details are not yet elucidated, including mechanisms underlying the initialization, elongation, and termination of peptide chain as well as the covalent conjugation of fatty acid moiety to the peptide backbone. Previous studies have showed that a malonyl-group, the precursor of fatty acid synthesis, could bind to the fengycin synthetase C (FenC), indicating that this phenomenon might be associated with the formation of fatty acid moiety in the nonribosomal peptide synthesis. In this thesis, three models were proposed to explain how the fatty acid moiety of fengycin might be synthesized. In order to determine which model is correct, the domain of FenC responsible for malonyl-CoA binding was clarified. The result showed that malonyl-CoA bound to the first thiolation domain of FenC, supporting the assumption of Model 3, that malonyl-CoA binds to the thiolation domain of each peptide synthetase. Both fatty acid and peptide chain are elongated simultaneously but independently until liking to the terminal enzyme, which catalyzes the linkage of peptide chain and fatty acid, and completes fengycin synthesis by releasing the product, an entire molecule of lipopeptidic antibiotic.

博士
長庚大學
生物醫學研究所
97
Bacillus subtilis F29-3 produces an anti-fungal peptidic antibiotic that is synthesized nonribosomally by fengycin synthetases. Our earlier work established that the promoter of the fengycin synthetase operon (fenp) is located 86 nucleotides upstream of the translational initiation codon of fenC. This investigation generated transcriptional fusions with a DNA fragments that contains the region between -105 and +80 in fenp. Analyzing the fusion plasmids revealed that deleting the region between -55 and -42 reduces the promoter activity by 64.5%. Transcriptional fusions generated on the B. subtilis DB2 chromosome also indicate that this region is important to the transcription. In vitro transcription analysis confirms that the transcription is inefficient when the sequence in this region is mutated. The electrophoretic mobility shift and footprinting analyses demonstrate that the C-terminal domain of the RNA polymerase α subunit binds to the region between -55 and -39. These results indicated that the sequence is an UP element. Finally, this UP element is critical to the production of fengycin, since mutating the UP sequence on the chromosome of B. subtilis F29-3 reduces the transcription of the fen operon by 85% and prevents the cells from producing enough fengycin to suppress the germination of Paecilomyces variotii spores on agar plates. Moreover, 8 predicted PerR boxes were found in fenp. In a perR mutant, the transcription activity of fenp was decreases by 86%. However, integrating perR into the chromosome of the perR mutant restores the promoter activity, showing that transcription from fenp is regulated by PerR. Chromatin immunoprecipitation and eletrophoretic mobility shift assay revealed that PerR binds to the region bewteen -20 and +80 in fenp. These results suggested that PerR binds to fenp and positively regulated the transcription activity of fenp. This study demonstrated that fenp has an UP element to activate the transcription activity and PerR may be involved in the transcription regulation of fenp. These results reveal the mechanism by which B. subtilis F29-3 regulates the transcription of fengycin synthetase operon.

碩士
國立陽明大學
微生物暨免疫學研究所
87
Fengycin is an antifungal lipopeptidic antibiotics produced by Bacillus subtilis F29-3. Ealier studies demostrated that fengycin peptide contains 10 amino acids, which is synthesized nonribosomally by peptide synthetases. The purpose of this study is to analyze a 9.6-kb gene cluster in cosmid pFC660, which is potentially involved in the synthesis of the fatty acid in fengycin. DNA sequencing revealed that the region contains nine genes, fenH, fenI, fenJ, fenK, fenL, fenM, fenN, fenO, fenP. Among these genes, fenJ belongs to TetR/AcrR family regulator; fenK, acyl-CoA dehydrogenase; fenL, long-chain fatty acid CoA-ligase; fenM, acetyl-CoA carboxylase; fenN, hydroxymethylglutaryl-CoA lyase; fenO, enoyl-CoA hydratase; fenP, methylmalonyl-CoA decarboxylase. The upstream region of the two genes, fenH, fenJ , were cloned to a promoter-probe vector, pGHL6. Afetr transforming the plasmid into B. subtilis F29-3 and assaying the luciferase activity, only the fenJ upstream region is active. Gene fenJ was overexpressed in E. coli, The overexpression resulted in the formation of insoluble inclusion body. The function of FenJ is now under investigation.

碩士
元智大學
生物科技暨生物資訊研究所
95
Fengycin is an antifungal lipopeptide produced by Bacillus subtilis F29-3. It inhibits filamentous fungi but is ineffective against yeast and bacteria. Fengycin was isolated and purified from the broth and was characterized by FAB-MS, FT-IR, UV-Vis, 13C-NMR and MALDI-TOF /MASS. The results of characteristic analysis show that the molecular weight and molecular structure of the fermentative metabolite resembled those of fengycin . Fengycin consists of two main components differing by one amino acid exchange. Fengycin A is composed of 1 D-Ala, 1 L-Ile, 1 L-Pro, 1 D-allo-Thr, 3 L-Glx, 1 D-Tyr, 1 L-Tyr, 1 D-Orn, whereas in fengyicn B the D-Ala is replaced by D-Val. Fengycin production from the B. subtilis F29-3 was affected by temperature and agitation rate, as 30 ℃ and 200 rpm agitation were favorable for fengycin production. Furthermore, statistical experimental design (Response surface methodology, RSM)) was applied for the purpose of identifying optimal formulation in the medium. For five-level four-factor central composite design, the production was significantly affected by Soybean meal, NaNO3, MnSO4, mannitol-mannitol, soybean meal-mannitol, soybean meal-soybean meal, NaNO3-NaNO3 and MnSO4-MnSO4. The model had a coefficient of determination (R2) of 0.9043, which can explain 90.43 % variability of the data. The results of steepest ascent and central composite design revealed that 26.2 g/L mannitol, 21.9 g/L soybean meal, 3.1 g/L NaNO3 and 0.2 g/L MnSO4 were optimum for the production of fengycin. The optimization strategy led to the enhancement of fengycin from 1.45 g/L to 3.5 g/L. The results of repeated fed-batch cultures in fermentor revealed that the optimum feeding nutrient is 2.6 % mannitol and 2.2 % soybean meal as carbon and nitrogen source, and the optimum feeding timing is 12, 24, 36 and 48 hr. The fengycin production was boosted to 4.7 g/L. Meanwhile, the results of repeated fed-batch cultures in shaking flask revealed that obtain the supernatant, add 1 % inoculum and culture for 96 hr, fengycin production could not inhibit by fengycin or toxic in the supernatant. This strategy led to the enhancement of fengycin from 3.0 g/L to 5.2 g/L.

碩士
元智大學
生物科技暨生物資訊研究所
94
Fengycin, a cyclic lipopeptidic antibiotic produced by B. subtilis F29-3, is composed of a peptide chain of 10 amino acids and a long-chain fatty acid. The synthesis of peptide moiety of fengycin is catalyzed nonribosomally by five fengycin synthetases -- FenC, FenD, FenE, FenA and FenB. So far, it is unknown how the fatty acid moiety is synthesized and linked to the peptide. Earlier investigations revealed that the synthesis of surfactin involves attachment of a prefabricated long-chain fatty acid to the initiating peptide synthetase (SrfAA). Our current studies show that fengycin is synthesized by a different mechanism. Investigation displayed that a set of genes from B3 region, in the 16-kb BamHI fragment downstream to the fengycin synthetase operon, are involved in fengycin synthesis. Sequence analysis shows that the genes in B3 region are closely related to fatty acid metabolism. HPLC and MALDI-TOF mass spectrometry analysis confirmed the compound accumulated in cultured medium of B. subtilis F29-3. The results showed that B. subtilis F29-3 produces fengycin and surfactin. fenI ~ fenO, the genes of B3 region in B. subtilis F29-3, were knockedout by double-crossover recombination. Four mutants were obtained and named as B. subtilis FB3-1, FB3-2, FB3-3 and FB3-4. Elimination of fenI ~ fenO disservice that cell density after the stationary phase decrease and less of fengycin productivity. When the media containing acetic acid and malonic acid, the cell density after the stationary phase and fengycin productivity of FB3-1 and FB3-4 were recovered. These results indicate that the genes of B3 region are involved in the synthesis of fatty acid moiety of fengycin indirectly. There are probably involved in the fatty acid metabolism after the stationary phase. Furthermore, B. subtilis F29-3 was found to have the ability to produce the second nonribosomal lipopeptide, surfactin. When fenF, another gene in the B3 region, was eliminated, B. subtilis F29-3 lost the ability to produce surfactin. The fenF knock-out mutant still produce fengycin. This observation suggests the fatty acid moieties of surfactin and fengycin are perhaps synthesized by different mechanisms.

碩士
國立陽明大學
微生物暨免疫學研究所
89
Abstract Fengycin, a ten amino-acid lipopeptide, is an antibiotic synthesized via nonribosomal mechanism by Bacillus subtilis F29-3. To understand the regulation mechanism for the synthesis of fengycin, a 1.2-kb DNA fragment which contains the putative promoter involved in the synthesis of fengycin was characterized in this thesis. We determined the location of this putative promoter and identified the transcriptional initiation site by primer extension method. The results demonstrated that this putative promoter, designated as PfenK, was responsible for the transcription of fenK. The deletion of the -35 sequence in PfenK resulted in the constitutive and small-amount expression of PfenK-drived luciferase gene. To investigate whether sigma factors of sporulation of Bacillus subtilis affect the expression of PfenK , the sporulation mutant strains were used to detect the expression of PfenK-drived luciferase gene. The results revealed that PfenK did not express in the spo0B mutant strain Bacillus subtilis 174 and the sigma F mutant strain Bacillus subtilis 176. PfenK would express in the sigma G mutant strain Bacillus subtilis 178, although the degree of expression was less than that of wild type. In addition, we also investigated the effect of variable medium on the PfenK expression. The results showed that in the DSM medium PfenK has a small-amount expression in the middle log phase but a large-amount expression in the stationary phase of the bacterial culture. In the nHA medium, the expression of PfenK started in the begining of log phase and reached to the peak in the stationary phase. As to the role of PfenK involved in the fengycin synthesis remains to be studied.

博士
國立中興大學
植物學系
83
Bacillus subtilis F29-3 is a gram-positve, rod-shaped,aerobic, spore-forming, motile bacterium. This organism is known to produce an antifungal antibiotic, fengycin. A total of twenty B. subtilis F29-3 mutants defective in fengycin bio- synthesis was obtained by Tn917 mutagenesis. Those sequence flanking the Tn917 insertion site in these twenty mutants were cloned, mapped and sequenced. Results showed that Tn917 was inserted in eleven different locations on the chromosome. One of the clones, pBTS7, was selected and used as a probe to screen for genes encoding fengycin biosynthesis in a cosmid library established with pHC79. A clone, pFC660 which hybridized to the probe, was selected. Mapping study revealed that eight mutants had insertion within the DNA fragment cloned in pFC660. Among them, four had Tn917 inserted in regions which encoded peptide sequences similar to part of peptide synthetases. Another four mutants had Tn917 inserted in the regions encoding the peptide sequences that were similar to part of the enzymes required for lipid synthesis. The 12-kb BamHI fragment of pFC660 was sequences. The length of this fragment was 11,459 bp long. This fragment contains a 3,825-bp peptide synthetase gene, fenB which encodes a protein of 143.6-kDa with 21.0-46.2% similarity to the amino acid-binding domains of different peptide synthetases. The 12-kb BamHI fragment also contains an incomplete open reading frame, ''fenA, which encodes a peptide synthetase of 227.1 kDa. Protein analysis revealed that fenB indeed encodes a 143-kDa protein and intact fenA may encode a protein of approximately 500 kDa. The results obtained from this dissertation suggest that a large number of genes in B. subtilis F29-3 is involved in fengycin biosynthesis.

博士
國立陽明大學
微生物暨免疫學研究所
90
Abstract Fengycin, an antifugal antibiotic, is a cyclic lipopeptidic antibiotic produced by Bacillus subtilis F29-3. Fengycin is composed of a peptide and a fatty acid residue of 15-17 carbons with a structure of a single side chain, saturated, or unsaturated residue. The fengycin peptide contains 10 amino acids in the order of L-Glu‧D-Orn‧L-Tyr‧D-allo-Thr‧L-Glu‧D-Ala (D-Val)‧L-Pro‧L-Glu‧D-Tyr‧L-Ile. Earlier studies demonstrated that fengycin is synthesized nonribosomally by fegycin synthetases encoded by an operon containing fenC, fenD, fenE, fenA and fenB. The current study analyzes the enzymatic functions of FenE which contains two amino acid activation modules (FenE1 and FenE2); each of these contains an amino acid activation domain activating a specific amino acid. Proteins containing the activation domains of FenE were expressed in Escherichia coli and purified by Ni-affinity chromatography. ATP-PPi exchange assay reveled that recombinant protein containing the FenE1 activation domain (AD-FenE1) activates L-Glu; protein containing the FenE2 activation domain (ATED-FenE2) activates L-Ala, L-Val, and 2-aminobutyric acid. Optimum temperature for both of these two enzymes is 25oC. Both of these two enzymes display higher ATP-PPi exchange activity in the range of pH of 6.5 to 8.0. AD-FenE1 has a Km value of 0.1 mM and Kcat value 0.07 s-1 toward L-Glu; ATED-FenE2, Km values of 0.36 mM, 0.61 mM, and 0.46 mM and Kcat values 0.11 s-1, 0.21 s-1, and 0.15 s-1 toward L-Ala, L-Val, and L-2-aminobutyric acid, respectivity. The function of phosphopantetheinyl transferase (PPTase) is required for fengycins synthesis. Promoter activity was analyzed by transcriptional fusion assay. The regions from 81 to 90 and from 40 to +20 are required for the fgp promoter activity. Gel retardation assay also showed that Bacillus subtilis F29-3 proteins could bind to the 81 to 90 region. Fengycin synthetase FenC is the initiating enzyme for fengycin synthesis. This enzyme contains an N-terminal condensation domain, which is commonly present in the initiating peptide synthetases involved in lipopeptide antibiotic synthesis, i.e. SrfAA, but is lacking in those involved in the initiation of the synthesis of peptide antibiotics such as GrsA. Presence of this N-terminal condensation domain likely prevents fengycin peptide synthesis unless a fatty acyl-CoA triggers the initiation process. This study deleted the N-terminal condensation domain (FenC-dC) from FenC. Radioactively labeling the peptide synthesized by FenC-dC reveled that this truncated protein catalyzes the formation of L-Glu-L-Orn peptide. On the other hand, this peptide is not synthesized by FenC, demonstrating the role of the condensation domain in fengycin synthesis.