Publications of Philippe Jacques
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See detailLimiting factors of mycopesticide development
Zaki, Omran ULiege; Weekers, F; Thonart, Philippe ULiege et al

in Biological Control (2020)

Phytosanitary crop protection products have shown their impact on the environment. They may not be very selective and their excessive use in agriculture causes pollution of soil and groundwater ... [more ▼]

Phytosanitary crop protection products have shown their impact on the environment. They may not be very selective and their excessive use in agriculture causes pollution of soil and groundwater, destruction of many beneficial insects and the emergence of resistant pests. Hence there is strong public and political pressure driven by consumers to implement phytosanitary alternatives that are less aggressive to the environment, such of biopesticides. Biopesticides and, in particular, mycopesticides are frequently used as an environmentally friendly tools to reduce plant diseases by inhibiting the growth of pathogens and inducing resistance in plants. <br /> <br />In this review, we will first analyze the current evolution of the global market of biopesticides. Boosted by different political initiatives all over the world, this market has increased 6-fold between 2005 and 2016. In 2016, it represented 6% of the global pesticide market with a compound annual growth rate (CGAR) of 14.1%. The mycopesticides accounted for only 10% of the global biopesticide market of 2016. <br /> <br />We then focused on two main factors which should contribute to the future development of mycopesticides: the large panel of their modes of action and the physiological state of the active product. Indeed, several modes of action could, in some instances, increase the efficacy of a biopesticide and postpone the emergence of resistance mechanism. The broad set of mechanisms use by Trichoderma harzianum and T. atroviride to reduce plant diseases: nutrient competition, direct antagonism, mycoparasitism and induction of resistance mechanism in plants were developed as an example. Stability of the active substance is another essential factor for creating competitive mycopesticides. For this, the choice of propagule types could be a major factor in enhancing their stability. This choice was discussed highlighting the advantages of conidia or sporidia from solid-state fermentation. <br /> <br />To end with, the registration process in European Union was described revealing another limiting factor that delays the development of mycopesticides in this zone. [less ▲]

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See detailOptimization of biosurfactant production in a trickle-bed biofilm reactor with genetically improved bacteria
Brück, Hannah ULiege; Coutte, François; Delvigne, Frank ULiege et al

Poster (2020)

The Gram-positive soil bacterium Bacillus subtilis is a potential producer of the very powerful biosurfactant surfactin [1]. In the phytosanitary field, surfactin gains increasing attention for the use as ... [more ▼]

The Gram-positive soil bacterium Bacillus subtilis is a potential producer of the very powerful biosurfactant surfactin [1]. In the phytosanitary field, surfactin gains increasing attention for the use as biocontrol agent since it is able to induce systemic resistance in plants [1]. Furthermore, surfactin has many applications in different industrial sectors due to the exceptional foaming and emulsifying properties [1]. Biofilm bioreactors are efficient alternative production systems for bacterial biosurfactants since excessive foam formation can be avoided while a high air/liquid mass transfer can be obtained [2-5]. The widely used B. subtilis 168 strain has very low cell adhesion capacities and thus a reduced growth in biofilm-based systems. In this work, genetically modified B. subtilis 168 mutants have been designed to improve the natural cell immobilization via biofilm formation. The mutants were first cultivated in a drip-flow biofilm reactor to assess their biofilm formation capacities [6]. Then, the strains with the best performances were selected for the cultivation in a trickle-bed biofilm reactor. The mutants with functional biofilm matrix production showed a significantly improved adhesion capacity compared to the control strain as well as an improved surfactin productivity. The introduced cell filamentation seemed to improve cell cohesion and to decrease cell detachment. [less ▲]

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See detailGrowth Dynamics of Bacterial Populations in a Two-Compartment Biofilm Bioreactor Designed for Continuous Surfactin Biosynthesis
Brück, Hannah ULiege; Coutte, François; Dhulster, Pascal et al

in Microorganisms (2020)

Biofilm bioreactors are promising systems for continuous biosurfactant production since they provide process stability through cell immobilization and avoid foam formation. In this work, a two-compartment ... [more ▼]

Biofilm bioreactors are promising systems for continuous biosurfactant production since they provide process stability through cell immobilization and avoid foam formation. In this work, a two-compartment biofilm bioreactor was designed consisting of a stirred tank reactor and a trickle-bed reactor containing a structured metal packing for biofilm formation. A strong and poor biofilm forming B. subtilis 168 strain due to restored exopolysaccharides (EPS) production or not were cultivated in the system to study the growth behavior of the planktonic and biofilm population for the establishment of a growth model. A high dilution rate was used in order to promote biofilm formation on the packing and wash out unwanted planktonic cells. Biofilm development kinetics on the packing were assessed through a total organic carbon mass balance. The EPS+ strain showed a significantly improved performance in terms of adhesion capacity and surfactin production. The mean surfactin productivity of the EPS+ strain was about 37% higher during the continuous cultivation compared to the EPS- strain. The substrate consumption together with the planktonic cell and biofilm development were properly predicted by the model (α = 0.05). The results show the efficiency of the biofilm bioreactor for continuous surfactin production using an EPS producing strain. [less ▲]

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See detailPalantir: a springboard for the analysis of secondary metabolite gene clusters in large-scale genome mining projects.
Meunier, Loïc ULiege; Tocquin, Pierre ULiege; Cornet, Luc ULiege et al

in Bioinformatics (Oxford, England) (2020)

SUMMARY: To support small and large-scale genome mining projects, we present Palantir (Post-processing Analysis tooLbox for ANTIsmash Reports), a dedicated software suite for handling and refining ... [more ▼]

SUMMARY: To support small and large-scale genome mining projects, we present Palantir (Post-processing Analysis tooLbox for ANTIsmash Reports), a dedicated software suite for handling and refining secondary metabolite biosynthetic gene cluster (BGC) data annotated with the popular antiSMASH pipeline. Palantir provides new functionalities building on NRPS/PKS predictions from antiSMASH, such as improved BGC annotation, module delineation and easy access to sub-sequences at different levels (cluster, gene, module, domain). Moreover, it can parse user-provided antiSMASH reports and reformat them for direct use or storage in a relational database. AVAILABILITY: Palantir is released both as a Perl API available on CPAN (https://metacpan.org/release/Bio-Palantir) and as a web application (http://palantir.uliege.be). As a practical use case, the web interface also features a database built from the mining of 1616 cyanobacterial genomes, of which 1488 were predicted to encode at least one BGC. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. [less ▲]

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See detailAgrobacterium tumefaciens C58 presence affects Bacillus velezensis 32a ecological fitness in the tomato rhizosphere
Abdallah, D. B.; Krier, F.; Jacques, Philippe ULiege et al

in Environmental Science and Pollution Research (2020), 27(22), 28429-28437

The persistence of pathogenic Agrobacterium strains as soil-associated saprophytes may cause an inconsistency in the efficacy of the biocontrol inoculants under field condition. The study of the ... [more ▼]

The persistence of pathogenic Agrobacterium strains as soil-associated saprophytes may cause an inconsistency in the efficacy of the biocontrol inoculants under field condition. The study of the interaction occurring in the rhizosphere between the beneficial and the pathogenic microbes is thus interesting for the development of effective biopesticides for the management of crown gall disease. However, very little is still known about the influence of these complex interactions on the biocontrol determinants of beneficial bacteria, especially Bacillus strains. This study aimed to evaluate the effect of the soil borne pathogen Agrobacterium tumefaciens C58 on root colonization and lipopeptide production by Bacillus velezensis strain 32a during interaction with tomato plants. Results show that the presence of A. tumefaciens C58 positively impacted the root colonization level of the Bacillus strain. However, negative impact on surfactin production was observed in Agrobacterium-treated seedling, compared with control. Further investigation suggests that these modulations are due to a modified tomato root exudate composition during the tripartite interaction. Thus, this work contributes to enhance the knowledge on the impact of interspecies interaction on the ecological fitness of Bacillus cells living in the rhizosphere. © 2020, Springer-Verlag GmbH Germany, part of Springer Nature. [less ▲]

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See detailNorine: update of the nonribosomal peptide resource
Flissi, A.; Ricart, E.; Campart, C. et al

in Nucleic Acids Research (2020), 48(D1), 465-469

Norine, the unique resource dedicated to nonribosomal peptides (NRPs), is now updated with a new pipeline to automate massive sourcing and enhance annotation. External databases are mined to extract NRPs ... [more ▼]

Norine, the unique resource dedicated to nonribosomal peptides (NRPs), is now updated with a new pipeline to automate massive sourcing and enhance annotation. External databases are mined to extract NRPs that are not yet in Norine. To maintain a high data quality, successive filters are applied to automatically validate the NRP annotations and only validated data is inserted in the database. External databases were also used to complete annotations of NRPs already in Norine. Besides, annotation consistency inside Norine and between Norine and external sources have reported annotation errors. Some can be corrected automatically, while others need manual curation. This new approach led to the insertion of 539 new NRPs and the addition or correction of annotations of nearly all Norine entries. Two new tools to analyse the chemical structures of NRPs (rBAN) and to infer a molecular formula from the mass-to-charge ratio of an NRP (Kendrick Formula Predictor) were also integrated. Norine is freely accessible from the following URL: https://bioinfo.cristal.univ-lille.fr/norine/. © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. [less ▲]

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See detailAntarctic cyanobacteria sources of biosignatures
Lara, Yannick ULiege; Demoulin, Catherine ULiege; Lambion, Alexandre ULiege et al

Conference (2019, September 05)

The high UV intensities and extreme seasonality make some of Antarctic habitats interesting to the study life adaptive strategies in extreme conditions, and the biosignatures that can be preserved. In ... [more ▼]

The high UV intensities and extreme seasonality make some of Antarctic habitats interesting to the study life adaptive strategies in extreme conditions, and the biosignatures that can be preserved. In Antarctica, most of the surface, lacustrine and endolithic photosynthetic niches are occupied by cyanobacteria, which are well equipped to survive cold, desiccation or UV exposure. To provide a better understanding of the cyanobacteria survival strategies to extreme conditions, we used transmitted light and TEM microscopy as well as high-throughput sequencing technologies on the Antarctic lineage Phormidesmis priestleyi. We observed and characterized the production of a gloeocapsin-like UV-screening pigment and compared it to the pigment produced by Gloeocapsa alpina. Cyanobacteria are considered to be the inventors of oxygenic photosynthesis and therefore played a pivotal role in early Life and Earth evolution during the Precambrian. However, to perform photosynthesis in the UV exposure of the Early Earth unprotected by an ozone layer, their ancestors must have developed multiple molecular strategies. The presence of a gloeocapsin-like pigment in different cyanobacterial lineages may suggest its early production by their common ancestor, potentially present before the oxidation of the atmosphere. In Polar regions, low temperatures lead to the success of particular organisms featuring adaptations to molecular and cellular disturbances such as rigidity of membranes, reduction of enzyme-catalyzed reactions, and solute transport. Our results underline the importance of functional categories of genes involved in the production of key molecules for the survival of polar P. priestleyi (e.g. exopolysaccharides, chaperone proteins, fatty acids and phospholipids). The study of Antarctic cyanobacteria is promising to find new analog biosignatures for Life in rocky habitable planets. This project is supported by the mini-ARC PUMA (ULiège, Belgium). [less ▲]

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See detailAstin C production by the endophytic fungus Cyanodermella asteris in planktonic and immobilized culture conditions
Vassaux, Antoine ULiege; Tarayre, Cédric; Arguelles Arias, Anthony ULiege et al

in Biotechnology Journal (2019)

The fungal endophyte Cyanodermella asteris was recently isolated from the medicinal plant Aster tataricus. This fungus produces astin C, a cyclic pentapeptide with anticancer and anti-inflammatory ... [more ▼]

The fungal endophyte Cyanodermella asteris was recently isolated from the medicinal plant Aster tataricus. This fungus produces astin C, a cyclic pentapeptide with anticancer and anti-inflammatory properties. The production of this secondary metabolite was compared in immobilized and planktonic conditions. For immobilized cultures, a stainless steel packing immersed in the culture broth was used as a support. In these conditions, the fungus exclusively grew on the packing, which provides a considerable advantage for astin C recovery and purification. C. asteris metabolism was different according to the culture conditions in terms of substrate consumption rate, cell-growth, and astin C production. Immobilized-cell cultures yielded a 30% increase of astin C production associate to a 39% increase in biomass. The inoculum type as spores rather than hyphae, and a pre-inoculation washing procedure with sodium hydroxide, turned out to be beneficial both for astin C production and fungus development onto the support. Finally, influence of culture parameters such as pH and medium composition, on astin C production was evaluated. With optimized culture conditions, astin C yield was further improved reaching a five times higher final specific yield compared to the value reported with astin C extraction from Aster tataricus (0.89 and 0.16 mg/g respectively). [less ▲]

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See detailOptimization of scalable biofilm bioreactors with genetically improved bacteria for microbial biosurfactant production
Brück, Hannah ULiege; Coutte, François; Delvigne, Frank ULiege et al

Poster (2019, May)

The Gram-positive soil bacterium Bacillus subtilis is a potential producer of a very powerful biosurfactant called surfactin. It offers many environmental advantages because surfactin is biodegradable and ... [more ▼]

The Gram-positive soil bacterium Bacillus subtilis is a potential producer of a very powerful biosurfactant called surfactin. It offers many environmental advantages because surfactin is biodegradable and less toxic than chemical compounds. In the phytosanitary field, surfactin represents a very promising biocontrol agent as it is able to induce systemic resistance in plants. Furthermore, due to its exceptional foaming and emulsifying properties, surfactin has many applications in different industrial sectors. Industrial surfactin production in conventional stirred tank reactors necessitates the control of excessive foam formation. Biofilm bioreactors are more efficient alternative bubbleless systems for biosurfactant production since foam formation can be avoided while a high air/liquid mass transfer can be obtained. This is a very important parameter because a high aeration rate is necessary for a good surfactin productivity. However, the control of the cell colonization on the reactor support is challenging as biofilm formation is a heterogeneous phenomenon. The laboratory strain B. subtilis 168 is very easy to cultivate and manipulate genetically. Yet, this strain has very low cell adhesion capacities making it difficult to fix the cells by natural cell immobilization through biofilm formation on the bioreactor support. Here, genetically modified B. subtilis 168 mutants have been tested for improved colonization capacities on the bioreactor support to achieve a more robust and stable process. The cell adhesion capacities of the selected B. subtilis 168 mutants have been improved through the restoration of the biofilm matrix production and the induction of cell filamentation by genetic engineering. Through the additional change of cell shape, the aim was to promote the initial cell adhesion step followed by the support colonization and to reduce the cell detachment under stress conditions. The biofilm adhesion capacities were analyzed by means of a drip flow biofilm reactor. The mutants with the best performance were then selected for the cultivation in a lab-scale trickle-bed biofilm bioreactor. This device contains a structured metal packing providing a very high specific surface area for cell immobilization. For the mutant with functional biofilm matrix production the cell attachment capacities were significantly improved compared to the control strain resultant in an improved surfactin productivity. Cell filamentation seemed to decrease cell detachment under stress conditions, leading to a more stable colonization and process. [less ▲]

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See detailNatural products: what is the biosynthetic potential of unicellular eukaryotes?
Meunier, Loïc ULiege; Jacques, Philippe ULiege; Baurain, Denis ULiege

Poster (2019, March 14)

Microbial organisms are a precious source of natural products for medical and agricultural fields. These natural products are produced by different enzymes classes from the secondary metabolism, resulting ... [more ▼]

Microbial organisms are a precious source of natural products for medical and agricultural fields. These natural products are produced by different enzymes classes from the secondary metabolism, resulting in a large diversity of molecular structures and bioactivities. Currently, the main known microbial producers of bioactive compounds are bacteria and fungi, which have already been extensively exploited for the pharmaceutical and phytosanitary drugs. However, other unicellular eukaryotes seem to also have potential for the production of bioactive compounds, which present somehow a new continent for the discovery of natural products. A preliminary large-scale in silico analysis through unicellular eukaryotic lineages, for which genomic data are available, indicates that a part of these organisms might possess diverse classes of secondary metabolism pathways and thus be potential candidates for the discovery of novel compounds. [less ▲]

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See detailOptimisation de la production de biosurfactants dans des bioréacteurs à biofilm en utilisant des bactéries génétiquement modifiées ayant des capacités d’adhérence améliorées
Brück, Hannah ULiege; Coutte, François; Delvigne, Frank ULiege et al

Poster (2019)

La bactérie du sol à Gram positif Bacillus subtilis est un producteur potentiel d'un biosurfactant très puissant appelé « surfactine » [1]. Ce biosurfactant présente des nombreux avantages ... [more ▼]

La bactérie du sol à Gram positif Bacillus subtilis est un producteur potentiel d'un biosurfactant très puissant appelé « surfactine » [1]. Ce biosurfactant présente des nombreux avantages environnementaux puisque la surfactine est biodégradable et moins toxique que les composés chimiques [2]. Dans le domaine des produits phytosanitaires, la surfactine est un agent de biocontrôle très prometteur car elle est capable d'induire une résistance systémique chez les plantes [1]. En outre, en raison de ses propriétés moussantes et émulsionnantes exceptionnelles, la surfactine trouve de nombreuses applications dans différents secteurs industriels [1]. La production industrielle de surfactine dans des réacteurs à cuve agitée classique implique le pénible contrôle de la formation excessive de mousse [3]. Les bioréacteurs à biofilm sont des systèmes alternatifs sans bulle et ainsi mieux adaptés et plus efficaces pour la production de biosurfactants. La formation de mousse peut être évitée tout en permettant un transfert de masse air/liquide élevé [2], [4]–[6]. Ceci est un paramètre très important car un taux d’aération élevé est nécessaire pour une bonne productivité de surfactine par B. subtilis [7]. Cependant, vu que la formation de biofilm est un phénomène très hétérogène, le contrôle de la colonisation cellulaire sur le support du réacteur reste un défi. La souche de laboratoire B. subtilis 168 a comme avantage d’être très facile à cultiver et à manipuler génétiquement. Toutefois, cette souche possède des très faibles capacités d'adhérence cellulaire, ce qui rend la fixation des cellules par immobilisation naturelle, c’est-à-dire par la formation d’un biofilm sur le support du bioréacteur, difficile. Ici, des souches de B. subtilis 168 génétiquement modifiées ont été testées afin d’identifier les souches avec les meilleures capacités de colonisation pour obtenir un procédé plus robuste et stable. Les capacités d'adhésion cellulaire des mutants de B. subtilis 168 ont été améliorées grâce à la restauration de la production des exopolysaccharides pour la matrice du biofilm et l'induction d’une filamentation cellulaire par génie génétique. Le but de ce changement supplémentaire de la forme cellulaire est de promouvoir l’étape initiale d’adhérence cellulaire suivie par la colonisation du support et de réduire par la suite le détachement cellulaire dans des conditions de stress. Les capacités d’adhérence des cellules ont été analysées au moyen d’un réacteur à biofilm à écoulement goutte à goutte. Les mutants avec les meilleures performances ont ensuite été sélectionnés pour la culture dans un bioréacteur à biofilm à film tombant. Ce dispositif contient une garniture métallique structurée offrant une très grande surface spécifique pour l'immobilisation des cellules. Les mutants produisant une matrice de biofilm ont montré une capacité d'adhérence cellulaire considérablement améliorée par rapport à la souche de contrôle. Par conséquent, il en résulte une productivité augmentée en surfactine. La filamentation cellulaire semblait diminuer le détachement dans des conditions de stress ce qui mène à une colonisation et un procédé plus stables. [less ▲]

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See detailSurfactin production optimization in biofilm bioreactors using genetically modified Bacillus subtilis 168 strains with improved adhesion capacities
Brück, Hannah ULiege; Coutte, François; Delvigne, Frank ULiege et al

Conference (2019)

1. Introduction Biofilm bioreactors have shown to be efficient cultivation systems for the production of bacterial biosurfactants [1]-[3]. They provide improved productivity and process stability through ... [more ▼]

1. Introduction Biofilm bioreactors have shown to be efficient cultivation systems for the production of bacterial biosurfactants [1]-[3]. They provide improved productivity and process stability through cell immobilization while avoiding foam formation. The widely known Gram-positive bacterium B. subtilis 168 is a potential producer of a very powerful biosurfactant, surfactin, with many applications in different industrial sectors [4]. The genome of B. subtilis 168 is completely sequenced and the strain can easily be genetically modified [5]. However, as a result of its domestication process, this laboratory strain is not able to produce surfactin anymore due to genetic mutations in the sfp gene coding for a co-factor required for the synthesis [6]. Furthermore, B. subtilis 168 possesses only poor biofilm formation capacities. This is mostly due to a deficiency in exopolysaccharide production [7]. In this work, different surfactin producing mutants of B. subtilis 168 with increased adhesion and biofilm formation capacities have been investigated for the cultivation in a trickle bed biofilm reactor [2]. 2. Methods The used B. subtilis 168 mutants contained all a functional sfp gene necessary for surfactin production. Firstly, a mutant with restored exopolysaccharide production (epsC+) has been selected to optimize the natural immobilization of the bacterial cells on the bioreactor support. Secondly, cell filamentation has been additionally provoked through the deletion of the sepF gene which is involved in the cell division process [8]. The idea was to promote further the initial cell adhesion step as well as the support colonization through this change of cell shape. The surfactin productivity and biofilm formation capacity of these mutant strains have been studied under batch and continuous process conditions in a trickle-bed biofilm reactor containing a structured metal packing with a high specific surface area for the cell colonization [1], [2]. Moreover, the effect of the dilution rate on biofilm formation has been examined. 3. Results and discussion As expected, the epsC+ mutants showed significant improved attachment capacities on the biofilm bioreactor support resulting in an increased surfactin productivity compared to the control strain. The restoration of the exopolysaccharides permitted the cells to produce a biofilm matrix which helps to stick the cells together for their immobilization on the reactor support. The surfactin productivity could be further increased through the transition from a batch to a continuous production mode. An increased dilution rate (D=0.5 h-1) permitted to enhance the biofilm formation and thus higher cell densities on the reactor support could be achieved. By choosing a dilution rate higher than the maximum specific growth rate, the number of suspended cells could be reduced by washing out the cells. Microscope images of biofilm samples of the epsC+ ΔsepF mutant revealed strongly filamentous cells. The deletion of sepF did not affect the cell metabolism and had a minor impact on the support colonization in comparison to the presence of exopolysaccharides. 4. Conclusions A continuous production mode is more favorable for surfactin production than the production in a batch reactor. A dilution rate that is higher than the maximum specific growth rate permits to reduce suspended cells and increase the biofilm formation in the trickle-bed biofilm reactor and thus improve the surfactin productivity. Exopolysaccharide production is important for an increased support colonization by B. subtilis 168 whereas induced morphological changes seem to have a lower impact on the final biofilm formation but may facilitate initial adhesion on the reactor support. References [1] Q. Zune et al., Chem. Eng. Sci., vol. 170, pp. 628–638, 2017. [2] Q. Zune et al., J. Chem. Technol. Biotechnol., vol. 89, no. 3, pp. 382–390, 2013. [3] F. Coutte et al., Biotechnol. J., vol. 12, no. 1600566, pp. 1–10, 2017. [4] P. Jacques, in: G. Soberon-Chavez (Ed.), Biosurfactants, Microbiology Monographs, Vol. 20, Springer Verlag Berlin Heidelberg, 2011, pp.57–91. [5] R. Gallegos-Monterrosa et al., Microbiology, pp. 1–11, 2016. [6] F. Coutte et al., J. Appl. Microbiol., vol. 109, no. 2, pp. 480–491, 2010. [7] A. L. McLoon et al., J. Bacteriol., vol. 193, no. 8, pp. 2027–2034, 2011. [8] L. W. Hamoen et al., Mol. Microbiol., vol. 59, no. 3, pp. 989–999, 2006. [less ▲]

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See detailMolecular strategies for adapting Bacillus subtilis 168 biosurfactant production to biofilm cultivation mode
Brück, Hannah ULiege; Delvigne, Frank ULiege; Dhulster, Pascal et al

in Bioresource Technology (2019), 293

Biofilm bioreactors have already been proven to be efficient systems for microbial lipopeptide production since they avoid foam formation. However, the cell adhesion capacities of the laboratory strain B ... [more ▼]

Biofilm bioreactors have already been proven to be efficient systems for microbial lipopeptide production since they avoid foam formation. However, the cell adhesion capacities of the laboratory strain B. subtilis 168 to the biofilm bioreactor support are limited. In this work, we present a novel approach for increasing cell adhesion through the generation of filamentous and/or exopolysaccharide producing B. subtilis 168 mutants by genetic engineering. The single cell growth behavior was analyzed using time-lapse microscopy and the colonization capacities were investigated under continuous flow conditions in a drip-flow reactor. Cell adhesion could be increased three times through filamentous growth in lipopeptide producing B. subtilis 168 derivatives strains. Further restored exopolysaccharide production increased up to 50 times the cell adhesion capacities. Enhanced cell immobilization resulted in 10 times increased surfactin production. These findings will be of particular interest regarding the design of more efficient microbial cell factories for biofilm cultivation. © 2019 Elsevier Ltd [less ▲]

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See detailNonribosomal peptides in fungal cell factories: from genome mining to optimized heterologous production
Vassaux, A.; Meunier, Loïc ULiege; Vandenbol, Micheline ULiege et al

in Biotechnology Advances (2019)

Fungi are notoriously prolific producers of secondary metabolites including nonribosomal peptides (NRPs). The structural complexity of NRPs grants them interesting activities such as antibiotic, anti ... [more ▼]

Fungi are notoriously prolific producers of secondary metabolites including nonribosomal peptides (NRPs). The structural complexity of NRPs grants them interesting activities such as antibiotic, anti-cancer, and anti-inflammatory properties. The discovery of these compounds with attractive activities can be achieved by using two approaches: either by screening samples originating from various environments for their biological activities, or by identifying the related clusters in genomic sequences thanks to bioinformatics tools. This genome mining approach has grown tremendously due to recent advances in genome sequencing, which have provided an incredible amount of genomic data from hundreds of microbial species. Regarding fungal organisms, the genomic data have revealed the presence of an unexpected number of putative NRP-related gene clusters. This highlights fungi as a goldmine for the discovery of putative novel bioactive compounds. Recent development of NRP dedicated bioinformatics tools have increased the capacity to identify these gene clusters and to deduce NRPs structures, speeding-up the screening process for novel metabolites discovery. Unfortunately, the newly identified compound is frequently not or poorly produced by native producers due to a lack of expression of the related genes cluster. A frequently employed strategy to increase production rates consists in transferring the related biosynthetic pathway in heterologous hosts. This review aims to provide a comprehensive overview about the topic of NRPs discovery, from gene cluster identification by genome mining to the heterologous production in fungal hosts. The main computational tools and methods for genome mining are herein presented with an emphasis on the particularities of the fungal systems. The different steps of the reconstitution of NRP biosynthetic pathway in heterologous fungal cell factories will be discussed, as well as the key factors to consider for maximizing productivity. Several examples will be developed to illustrate the potential of heterologous production to both discover uncharacterized novel compounds predicted in silico by genome mining, and to enhance the productivity of interesting bio-active natural products. © 2019 Elsevier Inc. [less ▲]

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See detailMOLECULAR STRATEGIES FOR ADAPTING BACILLUS CELL TO OVERPRODUCE SURFACTIN IN BIOFILM BASED PROCESSES
Coutte, François; Brück, Hannah ULiege; Dhali, Debarun et al

Poster (2019)

The scope of this work was to develop molecular strategies to get genetically engineered strains designed to overproduce surfactin in biofilm based bioprocesses. Surfactin is a well-known lipopeptide ... [more ▼]

The scope of this work was to develop molecular strategies to get genetically engineered strains designed to overproduce surfactin in biofilm based bioprocesses. Surfactin is a well-known lipopeptide biosurfactant synthesized by the non-ribosomal pathway [1]. It is composed of a ring of seven amino acid residues connected to a β-hydroxylated fatty acid chain of different length and isomery. The peptide moiety contains four leucines. The limiting factors of surfactin synthesis were identified and solved step by step in view to overproduce this compound but also to facilitate its recovery in continuous bioprocesses. Bacillus subtilis 168 or BSB1 were used as the main microbial chassis for this optimization process. Four molecular/differentiation steps were specifically addressed, i.e. gene transcription, post-translational modification, non-ribosomal synthetase activity and biofilm formation. Gene transcription and post-translational modifications were challenged by promoter replacement and through the insertion of an efficient sfp gene from B.subtilis ATCC 21332 [2]. This later is responsible for post-translational modification of surfactin synthetase from apo-form to holo-form by the addition of a phosphopantetheinate group. Regarding the non-ribosomal synthetase activity the main limiting factor is the intracellular concentration of surfactin precursor. A hybrid modelling approach involving metabolic pathways and the main transcriptional regulation steps was thus implemented for designing genetic engineering strategies in order to increase these intracellular concentrations. This hybrid model was more specifically focused on the metabolic pathways related to branched chain amino acids (BCAA: isoleucine, leucine and valine) for predicting the effects of gene disruption on the intracellular concentration of leucine and on surfactin biosynthesis [3,4]. The last optimization step was focused on strain engineering for increasing the ability of cells to grow in biofilm-based bioprocesses [5]. To this end, a set of Bacillus filamentous strains with restored exopolysaccharide (EPS) synthesis was designed and further checked for surfactin overproduction. [less ▲]

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See detailBioprocédés innovants pour la production de biosurfactants microbiens de nature lipopeptidique
Brück, Hannah ULiege; Coutte, François; Lecouturier, Didier et al

Conference (2019)

PORTEE DU TRAVAIL Les lipopeptides sont des biomolécules amphiphiles présentant des applications intéressantes dans de nombreux domaines tels que l'environnement, la détergence, l'agriculture et la ... [more ▼]

PORTEE DU TRAVAIL Les lipopeptides sont des biomolécules amphiphiles présentant des applications intéressantes dans de nombreux domaines tels que l'environnement, la détergence, l'agriculture et la médecine [1]. Ils sont composés d'une partie peptidique liée à une chaîne d'acide gras de longueur et d'isomérie variables. Bacillus sp. produit principalement trois familles de lipopeptides: les surfactines, les fengycines et les iturines. Ces molécules diffèrent par leur structure, leurs propriétés physico-chimiques et leurs activités biologiques. L’un des principaux verrous technologiques liés à la production de ces molécules est la gestion de la mousse qu’ils génèrent en cours de production. Par ailleurs, la synthèse de ces molécules dépend d’un mécanisme de régulation, appelé le quorum sensing, qui est activé dans des conditions de haute densité cellulaire et, en particulier, dans les cultures en biofilms. OBJECTIFS Différents procédés ont été développés prenant en compte deux critères : d’une part favoriser, contrôler ou empêcher la formation de mousse et d’autre part cultiver les cellules sous forme planctoniques ou sous forme de biofilm. RESULTATS Deux grandes approches ont été développées. Dans la première, un bioréacteur à débordement favorisant la formation de mousse a été mis au point. Dans ce type de procédé, le biosurfactant produit est extrait en continu par moussage (Fig. 1) [2]. Dans la seconde approche, l’objectif a été de réduire ou d’éviter la formation de mousse et de favoriser la formation de biofilm. Pour ce faire, quatre dispositifs différents on été élaborés : un réacteur à lit fluidisé inversé [3], un réacteur à disque [4], un réacteur à biofilm [5] et un contacteur membranaire air-liquide (Fig. 2) [6]. En comparant ces différents bioprocédés, deux paramètres principaux influençant clairement la production de lipopeptides ont été identifiés: la croissance en biofilm et le taux de transfert d'oxygène. Ce dernier influence fortement la synthèse des lipopeptides. A titre d’exemple, le tableau 1 montre que pour une souche capable de produire les deux familles de lipopeptides, fengycine et surfactine, le niveau de transfert d’oxygène permet d’orienter le métabolisme vers l’un de ces lipopeptides. CONCLUSIONS ET PERSPECTIVES Les avantages et inconvénients des différents procédés sont résumés dans le tableau 2. Un procédé permettant un taux de transfert d’oxygène plus élevé sera, par exemple, utilisé pour la production de surfactine. Une approche moléculaire innovante a été récemment développée au laboratoire pour favoriser la croissance en biofilm de Bacillus sp. Cette approche qui se base sur l’obtention de bactéries filamenteuses et productrices d’exopolymères (EPS), devrait permettre une meilleure adhésion des cellules au support et faciliter la purification des lipopeptides. [less ▲]

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See detailAntifungal activities of bacillus subtilis lipopeptides to two venturia inaequalis strains possessing different tebuconazole sensitivity
Desmyttere, H.; Deweer, C.; Muchembled, J. et al

in Frontiers in Microbiology (2019), 10(OCT),

Within the framework of biocontrol development, three natural substances produced by Bacillus subtilis, called lipopeptides, have been studied: fengycin (F), surfactin (S), and mycosubtilin (M). Their ... [more ▼]

Within the framework of biocontrol development, three natural substances produced by Bacillus subtilis, called lipopeptides, have been studied: fengycin (F), surfactin (S), and mycosubtilin (M). Their antifungal properties were tested in vitro, in liquid medium, on two strains of Venturia inaequalis, ascomycete fungi causing apple scab. These two strains were, respectively sensitive and less sensitive to tebuconazole, an active substance of the triazole family. These three molecules were tested on their own, in binary (FS, FM, SM) and ternary mixtures (FSM). The antifungal activities of lipopeptides were estimated by calculating an IC50, compared to tebuconazole chemical substance. In tests involving the sensitive strain, all lipopeptide modalities exhibited antifungal activity. However, modalities involving fengycin and its mixtures exhibited the best antifungal activities; the activity of fengycin alone being very similar to that of tebuconazole. Interestingly, regarding the strain with reduced sensitivity to tebuconazole, surfactin and fengycin alone were not efficient while mycosubtilin and the different mixtures showed interesting antifungal activities. Specifically, the antifungal activity of FS and FSM mixture were equivalent to that of tebuconazole. For both fungal strains, microscopic observations revealed important morphological modifications in the presence of fengycin and in a less important proportion in the presence of surfactin but not in the presence of mycosubtilin. Overall, this study highlights the diversity in mode of action of lipopeptides on apple scab strains. © 2019 Desmyttere, Deweer, Muchembled, Sahmer, Jacquin, Coutte and Jacques. [less ▲]

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See detailBio-emulsifying and biodegradation activities of syringafactin producing Pseudomonas spp. strains isolated from oil contaminated soils
Zouari, O.; Lecouturier, D.; Rochex, A. et al

in Biodegradation (2019), 30(4), 259-272

Pseudomonas strains isolated from oil contaminated soils were screened for biosurfactant production. Three out of eleven Pseudomonas isolates were selected for their high emulsifying activity (E24 value ... [more ▼]

Pseudomonas strains isolated from oil contaminated soils were screened for biosurfactant production. Three out of eleven Pseudomonas isolates were selected for their high emulsifying activity (E24 value on n-hexadecane ~ 78%). These isolates (E39, E311 and E313) were identified as members of the P. putida group using phenotypical methods and a molecular approach. To identify the chemical nature of produced biosurfactants, thin layer chromatography and MALDI-ToF mass spectrometry analysis were carried out and revealed lipopeptides belonging to the syringafactin family. The activity of the produced biosurfactants was stable over a pH range of 6–12, at high salinity (10%) and after heating at 80 °C. Tests in contaminated sand micro-bioreactors showed that the three strains were able to degrade diesel. These results suggest the potential of these syringafactin producing strains for application in hydrocarbon bioremediation. © 2018, Springer Nature B.V. [less ▲]

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