Bioinformatics Modelling and Metabolic Engineering of the Branched Chain Amino Acid Pathway for Specific Production of Mycosubtilin Isoforms in Bacillus subtilis.

Mycosubtilin belongs to the family of lipopeptides. Different isoforms with various antifungal activities can be obtained according to the length and the isomery of the fatty acid. In this work, the activities of the mycosubtilin isoforms were first studied against the pathogen Aspergillus niger, revealing the high activity of the anteiso-C17 isoform. Modification of the mycosubtilin isoform patterns during cultures of the natural strain Bacillus subtilis ATCC 6633 was then investigated through amino acid feeding experiments. In parallel, single-gene knockouts and single-gene overexpression, leading to the overproduction of the anteiso-C15 fatty acid chains, were predicted using informatics tools which provide logical reasoning with formal models of reaction networks. In this way, it was in silico predicted that the single overexpression of the ilvA gene as well as the single knockout of the codY gene may lead to the overproduction of anteiso-C15 fatty acid chains. For the first time, it has been demonstrated that overexpression of ilvA helps to enhance the furniture of odd anteiso fatty acids leading to a favored mycosubtilin anteiso-C17 production pattern (+41%). Alternatively, a knock-out codY mutant led to a higher furniture of even iso fatty acids, leading to a favored mycosubtilin iso-C16 production pattern (+180%). These results showed that increased selective synthesis of particular isoforms of mycosubtilin through metabolic engineering is feasible, disclosing the interest of these approaches for future development of lipopeptide-producing strains.

Lipopeptide biodiversity in antifungal Bacillus strains isolated from Algeria.

Several Bacillus strains have been well studied for their ability to control soil-borne plant diseases. This property is linked to the production of several families of lipopeptides. Depending of their structure, these compounds show antifungal and/or plant systemic resistance inducing activities. In this work, the biodiversity of lipopeptides produced by different antifungal Bacillus strains isolated from seeds, rhizospheric, and non-rhizospheric soils in Algeria was analyzed. Sixteen active strains were characterized by PCR for their content in genes involved in lipopeptide biosynthesis and by MALDI-ToF for their lipopeptide production, revealing a high biodiversity of products. The difficulty to detect kurstakin genes led us to design two new sets of specific primers. An interesting potential of antifungal activity and the synthesis of two forms of fengycins differing in the eighth amino acid (Gln/Glu) were found from the strain 8. Investigation of its genome led to the finding of an adenylation domain of the fengycin synthetase predicted to activate the glutamate residue instead of the glutamine one. According to the comparison of both the results of MALDI-ToF-MS and genome analysis, it was concluded that this adenylation domain could activate both residues at the same time. This study highlighted that the richness of the Algerian ecosystems in Bacillus strains is able to produce: surfactin, pumilacidin, lichenysin, kurstakin, and different types of fengycins.

Polynucleotide phosphorylase is involved in the control of lipopeptide fengycin production in Bacillus subtilis.

Bacillus subtilis is a wealth source of lipopeptide molecules such as iturins, surfactins and fengycins or plipastatins endowed with a range of biological activities. These molecules, designated secondary metabolites, are synthesized via non-ribosomal peptides synthesis (NRPS) machinery and are most often subjected to a complex regulation with involvement of several regulatory factors. To gain novel insights on mechanism regulating fengycin production, we investigated the effect of the fascinating polynucleotide phosphorylase (PNPase), as well as the effect of lipopeptide surfactin. Compared to the wild type, the production of fengycin in the mutant strains B. subtilis BBG235 and BBG236 altered for PNPase has not only decreased to about 70 and 40%, respectively, but also hampered its antifungal activity towards the plant pathogen Botrytis cinerea. On the other hand, mutant strains BBG231 (srfAA-) and BBG232 (srfAC-) displayed different levels of fengycin production. BBG231 had registered an important decrease in fengycin production, comparable to that observed for BBG235 or BBG236. This study permitted to establish that the products of pnpA gene (PNPase), and srfAA- (surfactin synthetase) are involved in fengycin production.

Biofilm formation is determinant in tomato rhizosphere colonization by Bacillus velezensis FZB42.

In this work, the behavior in tomato rhizosphere of Bacillus velezensis FZB42 was analyzed taking into account the surfactin production, the use of tomato roots exudate as substrates, and the biofilm formation. B. velezensis FZB42 and B. amyloliquefaciens S499 have a similar capability to colonize tomato rhizosphere. Little difference in this colonization was observed with surfactin non producing B. velezensis FZB42 mutant strains. B. velezensis is able to grow in the presence of root exudate and used preferentially sucrose, maltose, glutamic, and malic acids as carbon sources. A mutant enable to produce exopolysaccharide (EPS-) was constructed to demonstrate the main importance of biofilm formation on rhizosphere colonization. This mutant had completely lost its ability to form biofilm whatever the substrate present in the culture medium and was unable to efficiently colonize tomato rhizosphere.

Study of the correlation between fengycin promoter expression and its production by Bacillus subtilis under different culture conditions and the impact on surfactin production.

This work aimed to rely expression of the fengycin promoter to fengycin production under different culture conditions. To this end, Bacillus subtilis BBG208, derived from BBG21, which is a fengycin overproducing strain carrying the green fluorescent protein (GFP) under the control of fengycin promoter, was used to assess the effects of different carbon and nitrogen sources on surfactin and fengycin production and the fengycin promoter expression. The data showed that some carbon sources oriented synthesis of one family of lipopeptides, while most of the nitrogen sources allowed high co-production of fengycin and surfactin. High expressions of promoter Pfen and fengycin synthesis were obtained with urea or urea + ammonium mixture as nitrogen source and mannitol as carbon source. Moreover, temperature, pH and oxygenation influenced their biosynthesis based on the nutrition conditions. Optimization of the production medium increased the fengycin production to 768 mg L-1, which is the highest level reported for this strain. This study defines the suitable nutrient conditions allowing as well the highest expression of the fengycin promoter and portrays the conditions relying on the fengycin and surfactin production.

Identification and natural functions of cyclic lipopeptides from Bacillus amyloliquefaciens An6.

Lipopeptides constitute a structurally diverse group of metabolites produced by various bacterial and fungal genera. In the past decades, research on lipopeptides has been fueled by their surfactant activities. However, natural functions of lipopeptides compounds have received considerably less attention. The aim of this study was to isolate and identify the lipopeptides from Bacillus amyloliquefaciens An6, and further evaluate their biological activities. An6 lipopeptides were detected by PCR using degenerated primers and MALDI-TOF-MS. An6 strain was found to produce surfactin, fengycin, and bacillomycin. Following their purification, the in vitro antioxidant activity of An6 lipopeptides was studied through different assays. The scavenging effect on 1,1-diphenyl-2-picrylhydrazyl radicals at a dosage of 0.75 mg/mL was 81%. Its reducing power was concentration-dependant and reached a maximum of 1.07 at 2.5 mg/mL. Moreover, they showed a strong inhibition of ß-carotene bleaching. An6 lipopeptides mixture was also found to display significant antimicrobial activity against several Gram-positive, Gram-negative bacteria, and fungal strains. An6 lipopeptides were insensitive to proteolytic enzymes, stable between pH 4.0 and 12.0, and resistant to high temperature. Our results provided enough evidence proving that An6 lipopeptides could be used as functional-food components.

Production of Bacillus amyloliquefaciens OG and its metabolites in renewable media: valorisation for biodiesel production and p-xylene decontamination.

Biosurfactants are important in many areas; however, costs impede large-scale production. This work aimed to develop a global sustainable strategy for the production of biosurfactants by a novel strain of Bacillus amyloliquefaciens. Initially, Bacillus sp. strain 0G was renamed B. amyloliquefaciens subsp. plantarum (syn. Bacillus velezensis) after analysis of the gyrA and gyrB DNA sequences. Growth in modified Landy's medium produced 3 main recoverable metabolites: surfactin, fengycin, and acetoin, which promote plant growth. Cultivation was studied in the presence of renewable carbon (as glycerol) and nitrogen (as arginine) sources. While diverse kinetics of acetoin production were observed in different media, similar yields (6-8 g·L-1) were obtained after 72 h of growth. Glycerol increased surfactin-specific production, while arginine increased the yields of surfactin and fengycin and increased biomass significantly. The specific production of fengycin increased ∼10 times, possibly due to a connecting pathway involving arginine and ornithine. Adding value to crude extracts and biomass, both were shown to be useful, respectively, for the removal of p-xylene from contaminated water and for biodiesel production, yielding ∼70 mg·g-1 cells and glycerol, which could be recycled in novel media. This is the first study considering circular bioeconomy to lower the production costs of biosurfactants by valorisation of both microbial cells and their primary and secondary metabolites.

High-throughput strategies for the discovery and engineering of enzymes for biocatalysis.

Innovations in novel enzyme discoveries impact upon a wide range of industries for which biocatalysis and biotransformations represent a great challenge, i.e., food industry, polymers and chemical industry. Key tools and technologies, such as bioinformatics tools to guide mutant library design, molecular biology tools to create mutants library, microfluidics/microplates, parallel miniscale bioreactors and mass spectrometry technologies to create high-throughput screening methods and experimental design tools for screening and optimization, allow to evolve the discovery, development and implementation of enzymes and whole cells in (bio)processes. These technological innovations are also accompanied by the development and implementation of clean and sustainable integrated processes to meet the growing needs of chemical, pharmaceutical, environmental and biorefinery industries. This review gives an overview of the benefits of high-throughput screening approach from the discovery and engineering of biocatalysts to cell culture for optimizing their production in integrated processes and their extraction/purification.

Burkholderia genome mining for nonribosomal peptide synthetases reveals a great potential for novel siderophores and lipopeptides synthesis.

Burkholderia is an important genus encompassing a variety of species, including pathogenic strains as well as strains that promote plant growth. We have carried out a global strategy, which combined two complementary approaches. The first one is genome guided with deep analysis of genome sequences and the second one is assay guided with experiments to support the predictions obtained in silico. This efficient screening for new secondary metabolites, performed on 48 gapless genomes of Burkholderia species, revealed a total of 161 clusters containing nonribosomal peptide synthetases (NRPSs), with the potential to synthesize at least 11 novel products. Most of them are siderophores or lipopeptides, two classes of products with potential application in biocontrol. The strategy led to the identification, for the first time, of the cluster for cepaciachelin biosynthesis in the genome of Burkholderia ambifaria AMMD and a cluster corresponding to a new malleobactin-like siderophore, called phymabactin, was identified in Burkholderia phymatum STM815 genome. In both cases, the siderophore was produced when the strain was grown in iron-limited conditions. Elsewhere, the cluster for the antifungal burkholdin was detected in the genome of B. ambifaria AMMD and also Burkholderia sp. KJ006. Burkholderia pseudomallei strains harbor the genetic potential to produce a novel lipopeptide called burkhomycin, containing a peptidyl moiety of 12 monomers. A mixture of lipopeptides produced by Burkholderia rhizoxinica lowered the surface tension of the supernatant from 70 to 27 mN·m(-1) . The production of nonribosomal secondary metabolites seems related to the three phylogenetic groups obtained from 16S rRNA sequences. Moreover, the genome-mining approach gave new insights into the nonribosomal synthesis exemplified by the identification of dual C/E domains in lipopeptide NRPSs, up to now essentially found in Pseudomonas strains.

Influence of promoters on the production of fengycin in Bacillus spp.

Fengycin is a promising antifungal lipopeptide from Bacillus spp. synthesized by non-ribosomal peptide synthetases (NRPS). In this work, fengycin production of a spontaneous fengycin overproducing strain, Bacillus subtilis BBG21, was first compared to those of B. subtilis BBG111 (a 168 derivative), B. subtilis ATCC 21332 and Bacillus amyloliquefaciens FZB42 under two different experimental conditions. In both conditions, very high fengycin yields were obtained from strain BBG21 (480 mg/L) in comparison to its counterparts. The high efficiency of the fengycin promoter (Pfen) of BBG21 compared to the promoter of BBG111 and FZB42 was confirmed using a GFP reporter gene. Under all tested conditions, this promoter showed highest expression in comparison to the other strains. The highest fluorescence rate was obtained with mannitol as carbon source. In addition, when the Ppps promoter from B. subtilis BBG111 was replaced by promoter Pfen from BBG21, fengycin production increased about 10-fold, while no fengycin overproduction was observed when replacement was performed with Pfen from ATCC 21332. Comparative sequence analysis of these different promoters revealed one nucleotide modification in the UP element known for its importance in the regulation process. This point mutation is thus responsible for overproduction of fengycin in BBG21.

The Tunisian oasis ecosystem is a source of antagonistic Bacillus spp. producing diverse antifungal lipopeptides.

The use of microbial products has become a promising alternative approach to controlling plant diseases caused by phytopathogenic fungi. Bacteria isolated from the date palm tree rhizosphere of the Tunisian oasis ecosystem could provide new biocontrol microorganisms adapted to extreme conditions, such as drought, salinity and high temperature. The aim of this study was to screen bacteria isolated from the rhizosphere of the date palm tree for their ability to inhibit phytopathogenic fungi, and to identify molecules responsible for their antifungal activity. Screening for antifungal activity was performed on twenty-eight isolates. Five antagonistic isolates were selected and identified as different species of Bacillus using phenotypical methods and a molecular approach. The five antagonistic Bacillus isolated showed tolerance to abiotic stresses (high temperature, salinity, drought). Their ability to produce lipopeptides was investigated using a combination of two techniques: PCR amplification and MALDI-ToF mass spectrometry. Analyses revealed that the antagonistic isolates produced a high diversity of lipopeptides that belonged to surfactin, fengycin, iturin and kurstakin families. Their antagonistic activity, related to their capacity for producing diverse antifungal lipopeptides and their tolerance to abiotic stresses, highlighted Bacillus strains isolated from the rhizosphere of the date palm tree as potential biocontrol agents for combatting plant diseases in extreme environments.

Mycosubtilin and surfactin are efficient, low ecotoxicity molecules for the biocontrol of lettuce downy mildew.

The use of surfactin and mycosubtilin as an eco-friendly alternative to control lettuce downy mildew caused by the obligate pathogen Bremia lactucae was investigated. Preliminary ecotoxicity evaluations obtained from three different tests revealed the rather low toxicity of these lipopeptides separately or in combination. The EC50 (concentration estimated to cause a 50 % response by the exposed test organisms) was about 100 mg L(-1) in Microtox assays and 6 mg L(-1) in Daphnia magna immobilization tests for mycosubtilin and 125 mg L(-1) and 25 mg L(-1) for surfactin, respectively. The toxicity of the mixture mycosubtilin/surfactin (1:1, w/w) was close to that obtained with mycosubtilin alone. In addition, the very low phytotoxic effect of these lipopeptides has been observed on germination and root growth of garden cress Lepidium sativum L. While a surfactin treatment did not influence the development of B. lactucae on lettuce plantlets, treatment with 100 mg L(-1) of mycosubtilin produced about seven times more healthy plantlets than the control samples, indicating that mycosubtilin strongly reduced the development of B. lactucae. The mixture mycosubtilin/surfactin (50:50 mg L(-1)) gave the same result on B. lactucae development as 100 mg L(-1) of mycosubtilin. The results of ecotoxicity as well as those obtained in biocontrol experiments indicated that the presence of surfactin enhances the biological activities of mycosubtilin. Mycosubtilin and surfactin were thus found to be efficient compounds against lettuce downy mildew, with low toxicity compared to the toxicity values of chemical pesticides. This is the first time that Bacillus lipopeptides have been tested in vivo against an obligate pathogen and that ecotoxic values have been given for surfactin and mycosubtilin.

Production of a novel mixture of mycosubtilins by mutants of Bacillus subtilis.

Using promoter exchange and gene knock-out strategies, two mutant strains, the so-called BBG116 and BBG125, were constructed from Bacillus subtilis wild-type strain ATCC 6633, a surfactin and mycosubtilin producer. Compared to the parental strain, both mutants overproduced constitutively mycosubtilin, while BBG125 had lost the ability to synthesize surfactin. Surprisingly, BBG125 was found to produce about 2-fold less mycosubtilin than BBG116 despite an expected higher availability of the cytoplasmic precursors and cofactors pool for biosynthesis. Further physiological characterization of BBG125 also highlighted: (i) a strong influence of temperature on mycosubtilin biosynthesis in BBG125 with a maximal productivity observed at 22°C, compared to 15 and 30°C; (ii) substantial changes in fatty acid profiles and thereby in mycosubtilin isoforms, compared to the wild-type strain; and (iii) the presence of five novel mycosubtilin isoforms. The antifungal activities of the new mix were higher than or equal to those of purified isoforms.

Structure, biosynthesis, and properties of kurstakins, nonribosomal lipopeptides from Bacillus spp.

A new family of lipopeptides produced by Bacillus thuringiensis, the kurstakins, was discovered in 2000 and considered as a biomarker of this species. Kurstakins are lipoheptapeptides displaying antifungal activities against Stachybotrys charatum. Recently, the biosynthesis mechanism, the regulation of this biosynthesis and the potential new properties of kurstakins were described in the literature. In addition, kurstakins were also detected in other species belonging to Bacillus genus such as Bacillus cereus. This mini-review gathers all the information about these promising bioactive molecules.

Production of surfactin and fengycin by Bacillus subtilis in a bubbleless membrane bioreactor.

Surfactin and fengycin are lipopeptide biosurfactants produced by Bacillus subtilis. This work describes for the first time the use of bubbleless bioreactors for the production of these lipopeptides by B. subtilis ATCC 21332 with aeration by a hollow fiber membrane air-liquid contactor to prevent foam formation. Three different configurations were tested: external aeration module made from either polyethersulfone (reactor BB1) or polypropylene (reactor BB2) and a submerged module in polypropylene (reactor BB3). Bacterial growth, glucose consumption, lipopeptide production, and oxygen uptake rate were monitored during the culture in the bioreactors. For all the tested membranes, the bioreactors were of satisfactory bacterial growth and lipopeptide production. In the three configurations, surfactin production related to the culture volume was in the same range: 242, 230, and 188 mg l(-1) for BB1, BB2, and BB3, respectively. Interestingly, high differences were observed for fengycin production: 47 mg l(-1) for BB1, 207 mg l(-1) for BB2, and 393 mg l(-1) for BB3. A significant proportion of surfactin was adsorbed on the membranes and reduced the volumetric oxygen mass transfer coefficient. The degree of adsorption depended on both the material and the structure of the membrane and was higher with the submerged polypropylene membrane.

High-level biosynthesis of the anteiso-C(17) isoform of the antibiotic mycosubtilin in Bacillus subtilis and characterization of its candidacidal activity.

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.

Temperature dependence of mycosubtilin homologue production in Bacillus subtilis ATCC6633.

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.

The lipopeptides mycosubtilin and surfactin enhance spreading of Bacillus subtilis strains by their surface-active properties.

The colonizing behaviour and the pellicle formation of Bacillus subtilis strains producing different families of lipopeptides were evaluated under several cultural conditions. The pattern of lipopeptides produced determined the architecture of the colony on a swarming medium as well as the flotation and the thickness of the pellicle formed at the air/liquid interface. The overproduction of mycosubtilin, a lipopeptide of the iturin family, led to increased spreading but had no effect on pellicle formation. A physico-chemical approach was developed to gain an insight into the mode of action of the biosurfactants facilitating the colonization. A relationship between surface tension of the culture medium and spreading of a lipopeptide non-producing strain, B. subtilis 168, was established. Goniometry was used to highlight the modification of the in situ wettability in the area where spreading was enhanced. On a solid medium, co-cultures of a surfactin producing with other strains showed a diffusion ring of the surfactin around the colony. This ring characterized by a higher wettability favoured the propagation of other colonies.

Mycosubtilin overproduction by Bacillus subtilis BBG100 enhances the organism's antagonistic and biocontrol activities.

A Bacillus subtilis derivative was obtained from strain ATCC 6633 by replacement of the native promoter of the mycosubtilin operon by a constitutive promoter originating from the replication gene repU of the Staphylococcus aureus plasmid pUB110. The recombinant strain, designated BBG100, produced up to 15-fold more mycosubtilin than the wild type produced. The overproducing phenotype was related to enhancement of the antagonistic activities against several yeasts and pathogenic fungi. Hemolytic activities were also clearly increased in the modified strain. Mass spectrometry analyses of enriched mycosubtilin extracts showed similar patterns of lipopeptides for BBG100 and the wild type. Interestingly, these analyses also revealed a new form of mycosubtilin which was more easily detected in the BBG100 sample. When tested for its biocontrol potential, wild-type strain ATCC 6633 was almost ineffective for reducing a Pythium infection of tomato seedlings. However, treatment of seeds with the BBG100 overproducing strain resulted in a marked increase in the germination rate of seeds. This protective effect afforded by mycosubtilin overproduction was also visualized by the significantly greater fresh weight of emerging seedlings treated with BBG100 compared to controls or seedlings inoculated with the wild-type strain.