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See detailHigh-level biosynthesis of the anteiso-C(17) isoform of the antibiotic mycosubtilin in Bacillus subtilis and characterization of its candidacidal activity.
Fickers, Patrick ULg; Guez, Jean-Sebastien; Damblon, Christian ULg et al

in Applied and Environmental Microbiology (2009), 75(13), 4636-40

High-level production (880 mg liter(-1)) and isolation of the anteiso-C(17) isoform of the lipopeptide mycosubtilin produced by a genetically engineered Bacillus subtilis strain are reported. Antifungal ... [more ▼]

High-level production (880 mg liter(-1)) and isolation of the anteiso-C(17) isoform of the lipopeptide mycosubtilin produced by a genetically engineered Bacillus subtilis strain are reported. Antifungal activity of this isoform, as determined via culture and fluorometric and cell leakage assays, suggests its potential therapeutic use as an antifungal agent, in particular against Candida spp. [less ▲]

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See detailBacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens.
Arguelles-Arias, A.; Ongena, MARC ULg; Halimi, B. et al

in Microbial Cell Factories (2009), 8(1), 63

ABSTRACT: BACKGROUND: Phytopathogenic fungi affecting crop and post-harvested vegetables are a major threat to food production and food storage. To face these drawbacks, producers have become increasingly ... [more ▼]

ABSTRACT: BACKGROUND: Phytopathogenic fungi affecting crop and post-harvested vegetables are a major threat to food production and food storage. To face these drawbacks, producers have become increasingly dependent on agrochemicals. However, intensive use of these compounds has led to the emergence of pathogen resistance and severe negative environmental impacts. There are also a number of plant diseases for which chemical solutions are ineffective or non-existent as well as an increasing demand by consumers for pesticide-free food. Thus, biological control through the use of natural antagonistic microorganisms has emerged as a promising alternative to chemical pesticides for more rational and safe crop management. RESULTS: The genome of the plant-associated B. amyloliquefaciens GA1 was sample sequenced. Several gene clusters involved in the synthesis of biocontrol agents were detected. Four gene clusters were shown to direct the synthesis of the cyclic lipopeptides surfactin, iturin A and fengycin as well as the iron-siderophore bacillibactin. Beside these non-ribosomaly synthetised peptides, three additional gene clusters directing the synthesis of the antibacterial polyketides macrolactin, bacillaene and difficidin were identified. Mass spectrometry analysis of culture supernatants led to the identification of these secondary metabolites, hence demonstrating that the corresponding biosynthetic gene clusters are functional in strain GA1. In addition, genes encoding enzymes involved in synthesis and export of the dipeptide antibiotic bacilysin were highlighted. However, only its chlorinated derivative, chlorotetaine, could be detected in culture supernatants. On the contrary, genes involved in ribosome-dependent synthesis of bacteriocin and other antibiotic peptides were not detected as compared to the reference strain B. amyloliquefaciens FZB42. CONCLUSION: The production of all of these antibiotic compounds highlights B. amyloliquefaciens GA1 as a good candidate for the development of biocontrol agents. [less ▲]

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See detailEcological fitness of Bacillus subtilis BGS3 regarding production of the surfactin lipopeptide in the rhizosphere
Nihorimbere, Venant ULg; Fickers, Patrick ULg; Thonart, Philippe ULg et al

in Environmental Microbiology Reports (2009), 1(2), 124-130

Cyclic lipopeptides and particularly surfactins produced by Bacillus species retain antibacterial, antiviral, biofilm-forming and plant resistance-inducing activities. In most cases, their role in ... [more ▼]

Cyclic lipopeptides and particularly surfactins produced by Bacillus species retain antibacterial, antiviral, biofilm-forming and plant resistance-inducing activities. In most cases, their role in biological control of plant diseases was evoked on the basis of in vitro assays or by using non-producing/ overproducing mutants but there is a need for more direct evidence of an efficient lipopeptide biosynthesis in the rhizosphere. In this work, we coupled LC-MS quantification of the lipopeptides secreted by cells colonizing tomato plants with the use of psrfA– lacZ reporter system integrated within the BGS3 chromosome to study the expression of the surfactin operon in planta. Results showed that a higher level of psrfA induction was observed upon the establishment of a stable BGS3 population on roots and surfactins extracted from the rhizosphere were produced in biologically significant quantities. Our results also demonstrate that BGS3 efficiently utilizes the main substrates from plant exudates to produce surfactins. This synthesis is also efficient in cells forming colonies and the production may be favoured in bacteria developing slowly in the rhizosphere. This provides a first understanding of how environmental factors may influence lipopeptide production by beneficial Bacillus strains. [less ▲]

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See detailInfluence of rhizosphere-specific parameters on surfactin production by Bacillus subtilis.
Ongena, MARC ULg; Nihorimbere, Venant; Fickers, Patrick ULg et al

in Bulletin OILB/SROP = IOBC/WPRS Bulletin (2009), (43), 317-320

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See detailTemperature dependence of mycosubtilin homologue production in Bacillus subtilis ATCC6633.
Fickers, Patrick ULg; Leclere, Valerie; Guez, Jean*-Sebastien et al

in Research in Microbiology (2008), 159(6), 449-57

Bacillus subtilis ATCC6633 produces mycosubtilin, a non-ribosomally synthesized lipopeptide of the iturin family which presents antagonistic activities toward various phytopathogens. Different homologues ... [more ▼]

Bacillus subtilis ATCC6633 produces mycosubtilin, a non-ribosomally synthesized lipopeptide of the iturin family which presents antagonistic activities toward various phytopathogens. Different homologues with fatty acid moiety varying from C15 to C17 are usually co-produced, with their biological activities increasing with the number of carbons in the fatty acid chain. In the present report, we highlight that growth temperature modulates both the extent of mycosubtilin production and the relative abundance of the different homologues. A 30-fold increase in mycosubtilin production was observed when the temperature was decreased from 37 degrees C to 25 degrees C for both strain ATCC6633 and its derivative BBG100, a constitutive mycosubtilin overproducer. However, no significant difference in either the expression of the mycosubtilin synthetase encoding genes or in the intracellular synthetase concentration could be found, suggesting that the observed phenotype originated from a higher mycosubtilin synthetase turnover at lower temperature. We also point out that lower growth temperature leads to an increased proportion of odd-numbered fatty acid homologues as a consequence of de novo synthesis of C17 anteiso fatty acid following cell adaptation to low temperatures. [less ▲]

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See detailLes lipases sont des hydrolases atypiques : principales caractéristiques et applications
Fickers, Patrick ULg; Destain, Jacqueline ULg; Thonart, Philippe ULg

in Biotechnologie, Agronomie, Société et Environnement = Biotechnology, Agronomy, Society and Environment [=BASE] (2008), 12(2), 119-130

Due to their kinetic and substrate specificities, triacylglycerol acyl-hydrolases or lipases are atypical enzymes. In function of their microenvironment, lipases are able to act as hydrolases in aqueous ... [more ▼]

Due to their kinetic and substrate specificities, triacylglycerol acyl-hydrolases or lipases are atypical enzymes. In function of their microenvironment, lipases are able to act as hydrolases in aqueous solution or as biocatalysts in organic synthesis. As hydrolases, they are responsible of the triglycerids catabolism into fatty acids and glycerol. In many organisms, this reaction plays a major role in the fat and lipid metabolism. In addition, lipases are also able to hydrolyse phospholipids and cholesterol esters. In organic solvent, lipases could catalyse reactions such as esterifications, acidolysis or alcoolysis with enantio-, regio- and chimioselectivity. Lipases form a mixed class of enzyme due to their animal, vegetal or microbial origins. All those properties led to the development of many applications in the food and chemical industries but also in the medical and therapeutic field. [less ▲]

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See detailProduction and down-stream processing of an extracellular lipase from the yeast Yarrowia lipolytica
Fickers, Patrick ULg; Ongena, MARC ULg; Destain, Jacqueline ULg et al

in Enzyme & Microbial Technology (2006), 38(6), 756-759

Lipase constitutes an interesting class of enzyme with many biotechnological applications. However, the development of a fruitful process must be set up to obtained a product compatible with the ... [more ▼]

Lipase constitutes an interesting class of enzyme with many biotechnological applications. However, the development of a fruitful process must be set up to obtained a product compatible with the industrial and commercial needs. Here, we report the development of such a process for the extracellular lipase secreted by the yeast Yarrowia lipolytica. The enzyme production, carried out in a 2000 L bioreactor, led to a lipase activity of approximately 1100 U mL(-1) after 53 h of fermentation. The post-culture treatment, consisting of a centrifugation, a filtration and an ultra-filtration steps, led to 15-fold volume reduction and a 8-fold increased of the lipase activity. Finally, addition of 12% of milk powder and 3% of gum arabic before spray-drying dehydration led to a stable powder with a lipolytic activity of 37,500 U g D.W.(-1). (c) 2005 Elsevier Inc. All rights reserved. [less ▲]

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See detailInvolvement of hexokinase HXK1 in glucose catabolite repression of LIP2 encoding extracellular lipase in the yeast Yarrowia lipolytica
Fickers, Patrick ULg; Nicaud, J. M.; Destain, Jacqueline ULg et al

in Current Microbiology (2005), 50(3), 133-137

The yeast Yarrowia lipolytica produces an extracellular lipase encoded by the LIP2 gene. However, very little is known about the mechanisms controlling its expression, especially on glucose media. In this ... [more ▼]

The yeast Yarrowia lipolytica produces an extracellular lipase encoded by the LIP2 gene. However, very little is known about the mechanisms controlling its expression, especially on glucose media. In this work, the involvement of hexokinase Hxk1 in the glucose catabolite repression of LIP2 was investigated in a lipase overproducing mutant less sensitive to glucose repression. This mutant has a reduced capacity to phosphorylate hexose compared with the wild-type strain, but no differences could be observed between the HXK1 sequences in the two isolates. This suggested that the reduced phosphorylating activity of the mutant strain probably resulted from a modification in the level of HXK1 expression. However, overexpression of the HXK1 gene in this mutant led to a decrease of both LIP2 induction and extracellular lipase activity, suggesting that the hexokinase is involved in the glucose catabolite repression of LIP2 in Y lipolytica. [less ▲]

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See detailIdentification and characterisation of LIP7 and LIP8 genes encoding two extracellular triacylglycerol lipases in the yeast Yarrowia lipolytiea
Fickers, Patrick ULg; Fudalej, F.; Le Dall, M. T. et al

in Fungal Genetics and Biology (2005), 42(3), 264-274

In the lipolytic yeast Yarrowia lipolytica, the LIP2 gene was previously reported to encode all extracellular lipase. The growth of a Deltalip2 strain on triglycerides as sole carbon source suggest all ... [more ▼]

In the lipolytic yeast Yarrowia lipolytica, the LIP2 gene was previously reported to encode all extracellular lipase. The growth of a Deltalip2 strain on triglycerides as sole carbon source suggest all alternative pathway for triglycerides utilisation ill this yeast. Here, we describe the isolation and the characterisation of the LIP7 and LIP8 genes which were found to encode a 366 and a 371-amino acid precursor protein, respectively. These proteins which belong to the triacylglycerol hydrolase family (EC 3.1.1.3) presented a high homology with the extracellular lipase CdLIP2 and CdLIP3 from Candida deformans. The physiological function of the lipase isoenzymes was investigated by creating single and multi-disrupted strains. Lip7p and Lip8p were found to correspond to active secreted lipases. The lack of lipase production in a Deltalip2 Deltalip7 Deltalip8 strain suggest that no additional extracellular lipase remains to be discovered in Y. lipolytica. The substrate specificity towards synthetic ester molecules indicates that Lip7p presented a maximum activity centred on caproate (C-6) while that of Lip8p is in caprate (C-10). (C) 2004 Elsevier Inc. All rights reserved. [less ▲]

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See detailSelection of new over-producing derivatives for the improvement of extracellular lipase production by the non-conventional yeast Yarrowia lipolytica
Fickers, Patrick ULg; Fudalej, F.; Nicaud, J. M. et al

in Journal of Biotechnology (2005), 115(4), 379-386

The non-conventional yeast Yarrowia lipolytica produces an extracellular lipase encoded by the LIP2 gene. Mutant strains with enhanced productivity were previously obtained either by chemical mutagenesis ... [more ▼]

The non-conventional yeast Yarrowia lipolytica produces an extracellular lipase encoded by the LIP2 gene. Mutant strains with enhanced productivity were previously obtained either by chemical mutagenesis or genetic engineering. In this work, we used one of these mutants, named LgX64.81 to select new overproducing strains following by amplification of the LIP2 gene. We also developed a process for lipase production in bioreactors and compared lipase production levels in batch and fed-batch cultures. Batch culture led to a lipase production of 26 450 U ml(-1) in a media containing olive oil and tryptone as carbon and nitrogen sources. Feeding of a combination of tryptone and olive oil at the end of the exponential growth phase yielded to lipase activity of 158 246 U ml(-1) after 80 h of cultivation. In addition this production system developed for the extracellular lipase could also be applied for other heterologous protein production since we have demonstrated that LgX64.81 is an interesting alternative host strain. (C) 2004 Elsevier B.V. All rights reserved. [less ▲]

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See detailUtilization of methyloleate in production of microbial lipase
Destain, Jacqueline ULg; Fickers, Patrick ULg; Weekers, F. et al

in Applied Biochemistry and Biotechnology (2005), 121(Spring), 269-277

In this article, we report the development and optimization of an industrial culture medium for the production of extracellular lipase in the yeast Yarrowia lipolytica. Until now olive oil in combination ... [more ▼]

In this article, we report the development and optimization of an industrial culture medium for the production of extracellular lipase in the yeast Yarrowia lipolytica. Until now olive oil in combination with glucose was used as the carbon source and inducer for the production of lipase. Our results demonstrate that methyloleate, a cheap hydrophobic compound, could efficiently substitute olive oil as the inducer and carbon source for lipase production. A new process of lipase production was developed yielding a twofold increase in the level of production compared with the levels in previous reports. [less ▲]

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See detailCarbon and nitrogen sources modulate lipase production in the yeast Yarrowia lipolytica
Fickers, Patrick ULg; Nicaud, J. M.; Gaillardin, C. et al

in Journal of Applied Microbiology (2004), 96(4), 742-749

AIMS: To analyse the influence of nitrogen and carbon sources on extracellular lipase production by Yarrowia lipolytica-overproducing mutant in order to optimize its production in large-scale bioreactors ... [more ▼]

AIMS: To analyse the influence of nitrogen and carbon sources on extracellular lipase production by Yarrowia lipolytica-overproducing mutant in order to optimize its production in large-scale bioreactors. METHODS AND RESULTS: The level of lipase production and LIP2 induction, measured using an LIP2-LacZ reporter gene, were compared for different carbon and nitrogen sources and for different concentrations. The localization of the enzyme during growth was also determined by Western blotting analysis using a six-histidine-tagged lipase. SIGNIFICANCE AND IMPACT OF THE STUDY: Tryptone N1 and oleic acid are the most suitable nitrogen and carbon sources for the production of the extracellular lipase by the Y. lipolytica mutant. Higher levels of lipase production were obtained as the tryptone concentration increased in the culture medium. Such a positive correlation was not observed with oleic acid media where the highest lipolytic productivities were obtained in the presence of low concentration. We also demonstrate that in the presence of oleic acid, lipase is cell-bound during the growth phase before being released in the media. CONCLUSIONS: This work provides a better understanding of the mechanism controlling LIP2 expression and, thus, extracellular lipase production in the yeast Y. lipolytica. [less ▲]

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See detailOverproduction of lipase by Yarrowia lipolytica mutants
Fickers, Patrick ULg; Nicaud, J. M.; Destain, Jacqueline ULg et al

in Applied Microbiology & Biotechnology (2003), 63(2), 136-142

Non-genetically modified mutants with increased capacities of extracellular lipase production were obtained from Yarrowia lipolytica strain CBS6303 by chemical mutagenesis. Of the 400 mutants isolated ... [more ▼]

Non-genetically modified mutants with increased capacities of extracellular lipase production were obtained from Yarrowia lipolytica strain CBS6303 by chemical mutagenesis. Of the 400 mutants isolated, LgX64.81 had the highest potential for the development of an industrial lipase production process. This mutant exhibits lipase production uncoupled from catabolite repression by glucose, and a 10-fold increased productivity upon addition of oleic acid. Using a LIP2-LacZ reporter gene, we demonstrate that the mutant phenotype originates from a trans-acting mutation. The glucose uptake capacity of LgX64.81 is reduced 2.5-fold compared to the wild-type-strain, and it exhibits high lipase production on glucose medium. A trans-acting mutation in a gene involved in glucose transport could thus explain this mutant phenotype. [less ▲]

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See detailNew disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica
Fickers, Patrick ULg; Le Dall, M. T.; Gaillardin, C. et al

in Journal of Microbiological Methods (2003), 55(3), 727-737

Yarrowia lipolytica is one of the most extensively studied nonconventional yeasts. Unfortunately, few methods for gene disruption have been reported for this yeast, and all of them are time-consuming and ... [more ▼]

Yarrowia lipolytica is one of the most extensively studied nonconventional yeasts. Unfortunately, few methods for gene disruption have been reported for this yeast, and all of them are time-consuming and laborious. The functional analysis of unknown genes requires powerful disruption methods. Here, we describe such a new method for rapid gene disruption in Y lipolytica. This knockout system combines SEP method and the Cre-lox recombination system, facilitating efficient marker rescue. Versatility was increased by using both auxotrophic markers like ylURA3 and ylLEU2, as well as the antibiotic resistance marker hph. The hph marker, which confers resistance to hygromycin-B, allows gene disruption in a strain lacking any conventional auxothrophic marker. The disruption cassette was shown to integrate at the correct locus at an average frequency of 45%. Upon expression of Cre recombinase, the marker was excised at a frequency of 98%, by recombination between the two lox sites. This new method for gene disruption is an ideal tool for the functional analysis of gene families, or for creating large-scale mutant collections in general. (C) 2003 Elsevier B.V. All rights reserved. [less ▲]

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