NEW STRAINS OF BACILLUS SUBTILIS PRODUCING NEW MYCOSUBTILIN ISOFORMS

Abstract

The invention relates to the field of antifungal biosurfactant molecules of bacterial origin. More particularly, the invention concerns novel strains of Bacillus subtilis producing new isoforms of mycosubtilin, their preparation process, and compositions containing them. These new isoforms of mycosubtilin exhibit improved antifungal activity and reduced cytotoxic properties compared to prior art mycosubtilins.

Description

Title of the invention: NEW DEBACILLUS SUBTILIS STRAINS PRODUCING NEW MYCOSUBTILIN ISOFORMS

[0001] The invention relates to the field of antifungal biosurfactant molecules of bacterial origin. More particularly, the invention relates to new strains of Bacillus subtilis producing new isoforms of mycosubtilin, their preparation process, and compositions containing them. These new isoforms of mycosubtilin exhibit improved antifungal activity and reduced cytotoxic properties compared to prior art mycosubtilins. Field of the invention

[0002] The world's population is growing and is expected to reach 8.3 billion in 2025 and nearly 10 billion in 2050. Depending on the various assumptions used, global food demand could increase by 40% to 68% by 2050 (Hérault, 2011). This increase in demand could be met by even more intensive agriculture, leading to increased consumption of water, fossil fuels, fertilizers, and pesticides to ensure high yields. To meet these challenges, the scientific community and industry must develop innovative research approaches. In the field of agriculture, this relates to the issue of securing yields and farm profitability while limiting the use of inputs derived from chemical synthesis.These substances are the subject of much debate, and their impact on the health of farmers, consumers, the environment, and biodiversity has been demonstrated in numerous cases. Around the world, authorities have implemented regulations aimed at limiting the use of toxic compounds in the agricultural, food, and other sectors.

[0003] The demand for bio-derived molecules in agriculture is therefore very high. A recent report indicates that the biopesticide market is expected to reach USD 8.5 billion by 2025. Other sectors are also heavily impacted by increased regulatory pressure on synthetic molecules in Europe since the implementation of the REACH directive in 2007. Manufacturers are particularly seeking preservatives, biosurfactants, and new antimicrobial molecules. Microbial secondary metabolites produced by many microorganisms, such as B. subtilis, possess all these properties.

[0004] In the agricultural sector, there are many plant pathogens (approximately 7000 species), and among them, Botrytis cinerea (responsible for grey mold) and Zymoseptoria tritici (responsible for septoria leaf blotch of wheat) are classified in the top 10 plant pathogens that have the most impact on crops (Dean et al. 2012).

[0005] Lipopeptides are bioactive molecules produced by various bacterial strains, particularly Bacillus and Pseudomonas (Jacques, 2011). Bacillus strains are capable of producing three different families of molecules, namely surfactins (surfactin, pumillacidin); fengycins (fengycin, pli-pastatin, agrastatin); and iturines (iturine, mycosubtiline, bacilomycin, mo-javensin). Among the iturine family, mycosubtiline is the compound exhibiting the highest antifungal activity (Besson et al., 1979). Mycosubtiline is a lipopeptide whose heptapeptide ring is linked to a fatty acid chain by a [3-amino] bond. The fatty acid chain length can vary from C15 to C18, and the isomerism can be linear, iso, or anteiso (Stein, 2005). The classic formula of the peptide chain is Asn / D-Tyr / D-Asn / Gln / Pro / D-Ser / Asn, as described in [Fig. 1].

[0006] The composition of the peptide ring, as well as the length and isomerism of the fatty acid chain, are factors that influence the activity of the molecule, and in particular its antifungal activity. In 2009, a study showed that the anteiso-C17 isoform of mycosubtilin was the most active against the yeast C. albicans (Fickers et al., 2009). In 2013, another study investigated the effect of different alkyl chains of mycosubtilin against B. cinerea. The authors showed that the anteiso-C17 isoform was the most active against B. cinerea with a MIC of 8 µM, followed by the n-C16 and iso-C17 isoforms (MIC = 16 µM) and iso-C16 (MIC = 32 µM). Furthermore, the presence (even at low concentrations) of the Cl8 chain has been shown to significantly enhance the activity of the mixture, suggesting very potent activity for this isoform (Béchet et al., 2013). More recently, the antifungal activities of mycosubtilin isoforms have been measured against A.niger, the results showed that the anteiso-C17 isoform was the most active against A. niger with an MIC of 8 qM, followed by the n-C16 and iso-C17 isoforms (MIC = 16 qM) and iso-C16 (MIC = 32 qM). (J.-S. Guez et al., 2022).

[0007] Numerous scientific studies have shown the broad spectrum of antifungal activity of mycosubtiline. This is particularly the case against Candida albicans, Bremia lactucae, Zymoseptoria tritici, Botrytis cinera, Fusarium oxysporum, Fusarium gra-minearum and Fusarium verticillioides Candida krusei, Paecilomyces variotii, Bys-socchlamys fulva, Venturia inaequalis, Verticillium dahlia or Aspergilus niger (Béchet et al., 2013; Chen et al., 2021; Deravel et al., 2014; Desmyttere et al., 2019; Farace et al., 2015; Fickers et al., 2009; Guez et al., 2022; Kourmentza et al., 2021; Mejri et al., 2017; Mihalache et al., 2018; Yu et al., 2021)

[0008] In 2011, a new isoform of mycosubtiline was discovered in a modified strain of B. subtilis and patented. This molecule with a fatty acid chain in C17 contains Glutamine at position 3 [Gln3] instead of Asparagine (W02013 / 050700). In this patent, the inventors showed that a mixture of mycosubtilin isoforms containing this new isoform [Gln3-C17] (even in small quantities, on the order of 1%) was as active as a mixture not containing it, demonstrating the superior antifungal potency of this isoform compared to others.

[0009] There are very few studies on the toxicity and ecotoxicity of the lipopeptide molecules produced by B. subtilis, and in particular of each lipopeptide isoform. Ecotoxicity has been studied on mixtures of surfactin and mycosubtilin isoforms alone or in binary mixtures. The results of this study showed that the EC50 of mycosubtilines in the microtox test (on Aliivibrio fis chéri) was at least 4.5 times lower than that of surfactins.But also that the EC50 on Daphnia magna for mycosubtilins (8 mg / L) is 3 times lower than that of surfactins (26 mg / L) (Deravel et al. 2014). In another study on iturine A, its acute toxicity was investigated in rats over 28 days. Iturine A showed no toxic effects on the lungs, heart, or kidneys after the 28 days of treatment, as well as after a recovery period (14 days) (Dey et al., 2016). More recently, a cytotoxicity study was conducted with a mixture of three lipopeptide families (surfactins, fengycins, and mycosubtilins) on two different cell lines: Caco-2 and Vero cells. The results showed that the mixture of mycosubtilin isoforms has an IC50 between 10 and 20 mg / L on these two cell lines (Kourmentza et al., 2021).

[0010] To date, there is a need to have antifungal molecules available in large quantities and with improved properties in order to make these biological alternatives economically viable on an industrial scale. Description of the invention

[0011] The inventors have developed new strains of Bacillus subtilis capable of producing new isoforms of mycosubtiline that are both more effective as an antifungal agent and less cytotoxic than the mycosubtilines described to date.

[0012] Thus, the invention relates to two new strains of Bacillus subtilis genetically modified by 7 point mutations so that they produce the new isoforms of mycosubtilin. These two strains are registered with the CNCM under numbers CNCM 1-5565 and CNCM 1-5679.

[0013] The invention relates to 3 new isoforms of mycosubtiline namely Gln3-C16, Glnl-C16 and Glnl-C17 as well as compositions containing them.

[0014] The invention also relates to the use of these 3 new isoforms as an antifungal agent, in particular against strains of B. cinerea, Aspergillus sp and Z. tritici.

[0015] Finally, the invention relates to a method for producing at least one of these new isoforms by culturing a strain of Bacillus subtilis genetically modified according to the invention. Advantages of the invention

[0016] The present invention provides access to surfactants suitable for use in agriculture, namely compounds that are not toxic and are available in large quantities.

[0017] Indeed, the new mycosubtiline isoforms presented here exhibit superior antifungal activity compared to previously described mycosutilins. In particular, the GlnlCl₂ isoform exhibits superior antifungal activity compared to the Cl₆ isoform. Furthermore, these new isoforms exhibit lower cytotoxicity compared to these known mycosubtilines.

[0018] In agriculture, these new isoforms can be used in the production of biopesticides or biosurfactants for the plant protection industry for the biocontrol of plant diseases or post-harvest treatment, as well as for stimulating plant growth.

[0019] Such surfactants are of interest in other fields due to their properties, particularly in the food, cosmetic, chemical, medical, pharmaceutical, detergent, petroleum, and environmental industries. DETAILED DESCRIPTION OF THE INVENTION

[0020] A first object of the invention relates to a genetically modified strain of Bacillus sp. characterized in that it comprises at least the following point mutations: - replacement of a serine by an asparagine due to a mutation at position 1118 of the resE_l gene - replacement of aspartic acid by asparagine due to a mutation at position 3466 of the rpoC gene - replacement of alanine by valine due to a mutation at position 886 of the addB gene - replacement of glutamic acid by lysine due to a mutation at position 121 of the araA gene - replacement of a proline by a serine due to a mutation at position 154 of the cotY gene - replacement of a serine by a phenylalanine due to a mutation at position 32 of gene 03427 - replacement of a serine by a phenylalanine due to a mutation at position 92 of gene 03457

[0021] The mutations carried out in these genes are in particular point nucleotide mutations.

[0022] An example of each of the mutated genes is shown in the sequence listing, namely: - The mutated resE_l gene is represented by the sequence SEQ ID NO. 1 - The mutated rpoC gene is represented by the sequence SEQ ID NO. 2 - The mutated addB gene is represented by the sequence SEQ ID NO. 3 - The mutated araA gene is represented by the sequence SEQ ID NO. 4 - The mutated cotY gene is represented by the sequence SEQ ID NO. 5 - The mutated gene 03427 is represented by the sequence SEQ ID NO. 6 - The mutated gene 03457 is represented by the sequence SEQ ID NO. 7

[0023] Other mutations are possible because the genetic code is degenerated. A person skilled in the art will be able to determine the alternative mutations to those described in these sequences.

[0024] The inventors have shown that the combination of these 7 mutations allows the production of new isoforms of mycosubtilin, in particular the Gln3-C16, Glnl-C16 and Glnl-C17 isoforms.

[0025] The seven genes modified in the Bacillus sp. strains according to the invention are as follows:

[0026] - resE is a gene that codes for a protein similar to that of the systems of ResE is involved in two-component signal transduction and plays a regulatory role in respiration. It is implicated in the overall regulation of aerobic and anaerobic respiration in B. subtilis. ResE (as well as ResD) are two-component regulatory proteins required for the transcriptional activation of Fnr under oxygen limitation in B. subtilis (Sun et al., 1996). Mycosubtilin production is strongly influenced by oxygenation conditions (Guez et al., 2008).

[0027] - rpoC is a gene responsible for the beta subunit of the RNA polymerase directed by DNA. A point mutation in rpoC leads to high expression of stress-sensitive regulators, including those of extracytoplasmic function (ECF) factors (oM, oW, and oX) and general stress factor (oB). SigB (oB) plays an important role in the adaptive response, including the pathogen presence response, leading to a positive impact on antifungal molecules produced by Bacillus species. (Lee et al., 2013; Rodriguez Ayala et al., 2020).

[0028] - araA is a gene encoding L-arabinose isomerase. This gene is repressed by a high glucose concentration (Sâ-Nogueira et al., 1997).

[0029] - addB is a gene that contributes to DNA repair and recombination. The enzyme is functional as a heterodimer of the AddA and AddB subunits, is a fast and processive DNA helicase, and catalyzes unwinding. of DNA (Yeeles et al., 2009). A point mutation in this gene can have an impact on the activity of the enzyme (Haijema et al., 1996).

[0030] - cotY is the gene that codes for the Y protein of the spore envelope. This gene is part of the sigE regulator and there is a SigE binding site in the mycosubtilin operon promoter region. (Wu et al., 2015).

[0031] - Gene 03427 codes for a protein whose function is not precisely described in the literature, but which could be related to the Rap protein. Rap ​​proteins in B. subtilis regulate the phosphorylation level or DNA-binding activity of response regulators such as SpoOF, involved in sporulation initiation, or ComA, regulating competence development (Diaz et al., 2012). Rap ​​proteins are also involved in the expression of genes encoding lipopeptide biosynthesis.

[0032] - Gene 03457 codes for a protein whose function is unknown but which could be related to the transport of secondary metabolites.

[0033] The Bacillus strain may be selected from among the strains Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Paenibacillus polymixa, Bacillus pumilus, Bacillus thuringiensis, Bacillus sphaericus, Bacillus coagulans, Bacillus mycoides, Bacillus velenzensis, Bacillus firmus, Bacillus methylotrophicus, Bacillus megaterium, and Bacillus vallismortis. Advantageously, the Bacillus strains are selected from among the strains of Bacillus subtilis and Bacillus amyloliquefaciens (recently recognized as Bacillus velenzensis). In a preferred embodiment, the Bacillus strain is a Bacillus subtilis strain.

[0034] A first particular strain according to the invention is the Bacillus subtilis strain deposited on July 30, 2020 under the number CNCM 1-5565 at the National Collection of Microorganism Cultures (CNCM) of the Pasteur Institute (Paris, France).

[0035] A second particular strain according to the invention is the Bacillus subtilis strain deposited on May 5, 2021 under the number CNCM 1-5679 in the National Collection of Microorganism Cultures (CNCM) of the Pasteur Institute (Paris, France).

[0036] Strains 1-5565 and 1-5679 both have the 7 point mutations and differ in the nature of the mycosubtiline promoter: strain 1-5565 includes the native promoter whereas strain 1-5679 has been modified by replacing the endogenous promoter with the constitutive PrepU promoter.

[0037] The introduction of the seven mutations led to an increase in mycosubtilin production and the appearance of new isoforms. In this context, the promoter change led to an increase in the overall production of mycosubtilin: of the classical form of mycosubtilin and of the new isoforms.

[0038] A second object of the invention relates to a new isoform of mycosubtiline

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047] chosen from the isoforms Gln3-C16, Glnl-C16 and Glnl-C17. These molecules are represented in Figures 3, 4 and 5 respectively. It is to be noted that strains 1-5565 and 1-5679 produce 14 other novel minor mycosubtilins (in terms of production level) namely: Gln3-C16; Gln7-C16; Glnl, Gln3-C16; Gln3,Gln7-C16; Glnl-C16; Glnl,Gln3,Gln7-C16; Gln7-C17; Glnl,Gln3-C17; Gln3,Gln7-C17; Glnl-C17; Gln3-C18; Gln7-C18; Glnl-C18 and A-C19. A third object of the invention relates to a composition comprising at least one mycosubtiline selected from the isoforms Gln3-C16, Glnl-C16 and Glnl-C17. A composition according to the invention comprises one, two or three of these isoforms, namely: - the Gln3-C16 isoform - the Glnl-Cl6 isoform - the Glnl-Cl7 isoform - the Gln3-C16 and Glnl-Cl6 isoforms - the Gln3-C16 and Glnl-C17 isoforms - the Glnl-C16 and Glnl-C17 isoforms the isoforms Gln3-C16, Glnl-C16 and Glnl-C17. Furthermore, a composition according to the invention may contain at least one of the novel myco-subtilines selected from Gln3-C16; Gln7-C16; Gln1, Gln3-C16; Gln3,Gln7-C16; Glnl-C16; Glnl,Gln3,Gln7-C16; Gln7-C17; Glnl,Gln3-C17; Gln3,Gln7-C17; Glnl-C17; Gln3-C18; Gln7-C18; Glnl-C18 and A-C19, as well as any other known mycosubtiline. Such a composition may also include other lipopeptides besides mycosubtiline (which is an iturin), notably: - molecules from the iturine family, such as iturine A, mojavensin, and bacillomycins A, B, C, D, F and L; - molecules from the surfactin family, such as surfactins A, B or C, lichenysin and pumilacidine; - molecules from the fengycin family, such as fengycins A and B, plipastatins A and B and agrastatins A and B. Such a composition may also include other components such as other surfactant molecules, preservatives, and adjuvants. Surfactant molecules include amphiphilic molecules, surfactants, lipopeptides, such as surfactin, fengycin, chemical surfactants and biological surfactants, such as rhamnolipids, polysaccharides, etc. Examples of surfactants include heparin, hyaluronic acid, and... dextran, amylose, chitosan, anionic surfactants derived from amino acids, non-ionic surfactants derived from polyglycosides, hydrotropic surfactants, lipopeptides such as surfactin isomers and / or fengycin (or plipastatin) isomers, rhamnolipids and vegetable oils.

[0048] Surfactants from the family of non-ionic surfactants are chosen for example from: fatty alcohol axalkylate, pentylene glycol and its derivatives, hydrotropic molecules of the alkylpolyglycoside type (alkypolyglycoside and alkylethoxypolyglycoside), molecules of the polyglycoside texturizing agent type (xanthan gum, gum arabic, tragacanth gum, guar gum, locust bean gum, tamarind gum, pectin, gellan gum, carrageenans, agar-agar, alginates.)

[0049] Surfactants from the family of anionic surfactants are chosen for example from surfactins, fengycins, sodium laureth sulfate and its derivatives or amino acid derivatives.

[0050] Surfactants from the oil family are chosen for example from modified oils and oil extracts (acidified, methylated, esterified) in particular from: almond, peanut, argan, avocado, rapeseed, ricin, lorenzo, neem, hazelnut, cashew nut, macadamia nut, olive, pistachio, rice, high oleic sunflower, camelina, flax, borage, safflower, hemp, cotton, wheat germ, maize, walnut, poppy, evening primrose, barley, pumpkin seed, grape seed, pea, sesame, soy, sunflower.

[0051] Among the adjuvants, some can facilitate the penetration of the composition into the plant (such as oils), others increase the contact surface between the plant leaf and the composition (wetting agents), others can absorb moisture from the air and thus combat desiccation (salts), and still others can fix the composition to the leaves so as to limit leaching and volatilization. Adjuvants must therefore be adapted to the modes of action of the compositions (root, contact, systemic, or penetrant), the types of product formulations, and the types of plants targeted (glabrous or hairy leaves, cuticle thickness, plant growth stages, stomatal positions, etc.).

[0052] A third object of the invention relates to the use of at least one mycosubtiline chosen from the isoforms Gln3-C16, Glnl-C16 and Glnl-C17 or of a composition comprising such a mycosubtiline (as described above) as an antifungal agent.

[0053] These three isoforms are effective against an ascomycete fungus, in particular against strains B. cinera, Aspergillus sp. and Z. tritici.

[0054] In a particular embodiment, this use consists of using at least one mycosubtiline corresponding to the Gin 1-Cl6 isoform as an antifungal agent against the B. Cinera strain.

[0055] In another particular embodiment, this use consists of using at less a mycosubtiline corresponding to the Glnl-C17 isoform as an antifungal agent against the Aspergillus sp. strain.

[0056] In yet another particular embodiment, this use consists of using at least one mycosubtiline corresponding to one of the Gin 1-Cl6 or Gln3-C16 isoforms as an antifungal agent against the Z. tritici strain.

[0057] A fourth object of the invention relates to a method for producing at least one new isoform of mycosubtilin chosen from the isoforms Gln3-C16, Gin 1-Cl6 and Gin 1-Cl7 consisting of cultivating a genetically modified Bacillus strain as defined above.

[0058] Sequentially, this process consists of:

[0059] -to place a strain of Bacillus sp. according to the invention in culture

[0060] - incubate this strain in a suitable culture medium

[0061] - harvest the culture strain and / or supernatant.

[0062] This process can be continued by one or more of the following steps: - Purification of mycosubtilins (global or differentiated by isoform) - Concentration of the solution containing mycosubtilin or the isoforms of interest of mycosubtilin - dehydration of a solution comprising mycosubtiline or isoforms of interest of mycosubtiline

[0063] The new isoforms of mycosubtiline described above, as well as the compositions containing them, can be used in many applications due to their surfactant and / or antifungal properties and their absence or low level of toxicity: - In agriculture: production of biopesticides or biosurfactants intended for the plant protection industry for the biocontrol of plant diseases or post-harvest treatment or as a plant growth stimulant - In the food industry: preservative - In cosmetics: as a preservative and as a formulation agent - In chemistry - In the medical and pharmaceutical fields: antifungal action or prevention, preservative - As a detergent - In the oil industry - In the field of environmental protection: biological pest control, non-toxic formulation agent.

[0064] The present invention will be better understood upon reading the following examples, which are provided by way of illustration and shall in no way be considered as limiting the Scope of the present invention. DESCRIPTION OF THE FIGURES

[0065] [Fig. 1] [Fig. 1]: Classical formula of mycosubtiline A-C17 (n=13)

[0066] [Fig.2] [Fig.2]: Schematic representation of the HPLC chromatogram preparative. Peaks P3, P5, P6, P7, and P8 are classical isoforms of mycosubtilin, while peaks P1, P2, and P4 are novel isoforms of mycosubtilin. The modified amino acid in the peptide ring has been labeled.

[0067] [Fig.3] [Fig.3]: Formula of the new isoform of mycosubtiline Gln3-C16

[0068] [Fig.4] [Fig.4]: Formula of the new isoform of mycosubtiline Glnl-C16

[0069] [Fig.5] [Fig.5]: Formula of the new isoform of mycosubtiline Glnl-C17

[0070] [Fig.6] [Fig.6]: Quantitative analysis of mycosubtilin produced by the new modified strains of Bacillus subtilis.

[0071] [Fig.7] [Fig.7]: Percentage of the cell viability test of the treated vero cells with different isoforms of mycosubtiline at different concentrations of each isoform. EXAMPLES

[0072] EXAMPLE 1: Construction of Bacillus subtilis strains 1-5565 and 1-5679

[0073] The Bacillus subtilis strain 1-5679 is derived from strain 1-5565; the constructs of these strains are described below

[0074] A first strain deposit was made on July 30, 2020, under number CNCM 1-5565, at the National Collection of Microorganism Cultures (CNCM) of the Pasteur Institute (Paris, France). A second strain deposit was made on May 5, 2021, under number CNCM 1-5679, at the National Collection of Microorganism Cultures (CNCM) of the Pasteur Institute (Paris, France).

[0075] 1.1 Procedure for obtaining the B. subtilis 1-5565 strain

[0076] Strain 1-5565 was constructed from a B. subtilis strain deposited on February 6, 2020 under the number CNCM 1-5487 at the National Collection of Microorganism Cultures (CNCM) of the Pasteur Institute (Paris, France).

[0077] In this strain 1-5487, seven genes (resE, rpoC, addB, araA, cotY, 03427, and 03457) were modified by a point mutation, meaning that a single nucleotide is changed. The deletion or modification of a single nucleotide (point mutation) can change the amino acid sequence, resulting in a new phenotype in the mutant strain. Various genetic engineering techniques can be used to induce this type of genetic mutation: - The CRISPR-Cas9-mediated genome engineering method has surpassed previous methods in terms of accuracy and has made it possible to create reliable mutants without foreign DNA in many wild-type bacterium strains terians, including in B. subtilis, with a point mutation efficiency of up to ~68%. This method can create a new phenotype without introducing any foreign DNA into the genome (So et al., 2017). Site-directed mutagenesis is one of the cornerstones of modern molecular biology, allowing for precise control of protein sequences. Several strategies have been developed, with the QuikChange™ site-directed mutagenesis system developed by Stratagene (La Jolla, CA) probably being the most widely used. QuikChange™ works by using a pair of complementary primers containing a mutation. In a series of PCR cycles, these primers hybridize to the template DNA, replicating the plasmid DNA with the mutation. The mutant DNA product exhibits a strand break (nick). The resulting DNA pool (mutant and parental) is then treated with Dpnl to destroy the methylated parental DNA in the newly synthesized unmethylated mutant DNA and transformed into E. coli cells, where the break is ligated by host repair enzymes. (Liu & Naismith, 2008). - Targeted mutagenesis. This method relies on the use of chemical mutagens (ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS)) or physical agents such as strong exposure to ultraviolet (UV) radiation. A genetic modification occurs, creating a change in phenotype. EMS is chosen to target GC-rich regions, while UV radiation is chosen to target AT-rich regions.

[0078] Following genetic modification, using the point mutation procedure described above, interesting mutants were obtained. The method used for screening the mutant strains involves diluting one unit volume of stock from the mutant library in one unit volume of physiological saline. The cell suspension is spread onto nutrient agar plates (supplied by Condalab) to obtain single colonies. Several single colonies are then picked using the Qpix 460 (from Molecular Devices) and inoculated into a nutrient culture medium. After incubation at 30°C for 24 h, the cells are harvested, and the supernatant is used for mycosubtilin analysis by reversed-phase high-performance liquid chromatography as described in Example 2. In a different embodiment, other culture media can be used, such as LB medium, Landy medium, or Cooper medium.

[0079] Using this methodology, after several series of point mutation procedures, strain B. subtilis 1-5565 was selected. Genome sequencing using Illumina Paired end 2*150 bp (Library prepared following Nextera XT) of this strain showed changes in the sequence of these seven genes as described below.

[0080] 1.2 Genetic characterization of strain B. subtilis 1-5565

[0081] Genome sequencing of strain B. subtilis 1-5565 was performed using Illumina Paired End 2*150 bp (Nextera XT fowling library). The genome sequencing results confirm changes in the nucleotide sequence of seven genes, namely: resE, rpoC, addB, araA, cotY, 03427 and 03457.

[0082] The impact of these nucleotide changes was then analyzed and confirmed on the primary structure of the corresponding proteins, and is described below:

[0083] - change in the amino acid sequence by replacing a serine with an asparagine due to a mutation in the RseE_l gene at position 1118.

[0084] - change in the amino acid sequence by replacement of the amino acid practiced by an asparagine in the RpoC gene at position 3466.

[0085] - change in the amino acid sequence by replacing an alanine with valine due to a mutation in the AddB gene at position 886.

[0086] - change in the amino acid sequence by replacement of an acid glutamic acid by a lysine due to a mutation in the AraA gene at position 121.

[0087] - change in the amino acid sequence by replacing a proline with a serine due to a mutation in the CotY gene at position 154.

[0088] - change in the amino acid sequence by replacing a serine with phenylalanine due to a mutation in gene 03427 at position 32.

[0089] - change in the amino acid sequence by replacing a serine with phenylalanine due to a mutation in gene 03457 at position 92.

[0090] Compared with B. subtilis LBS0, the Bacillus subtilis 1-5565 strain produces more mycosubtilin as shown in Example 2 and several novel isoforms. 1.3 pLIPl plasmid construction protocol

[0091] In order to replace the native promoter of the mycosubtilin operon in the B. subtilis strain I-5565, a hybrid plasmid containing epbp-P^y-neo-efenF and rep(R6K) was first constructed as follows. The bLIPl plasmid was digested with the restriction enzymes Xmil (Thermo Scientific; reference FD1484) and Pael (Thermo Scientific; reference FD0604) to extract and purify a 4.5 kilobase pair (Kb) fragment following the protocol of the GeneJET gel extraction kit (Thermo Scientific; reference K0692). The pbp gene fragment (950 base pairs) was amplified from B. subtilis 1-5565 using primers containing similar restriction sites. The amplified fragment was extracted and purified. The 4.5 kb fragment and the 950 bp PCR fragment were ligated according to the protocol of the rapid DNA ligation kit (Thermo Scientific; reference K1422). The ligation product was processed in E cells.coli JM 109 competent ready-to-use (Promega, reference . L2005). Clone selection is performed on Luria-Bertani medium containing agar and 50p / ml neomycin / kanamycin. The final plasmid from the positive clones is extracted following the protocol of the GeneJET plasmid minipreparation kit (Thermo Scientific; reference K0503). The resulting final plasmid is called pLIPl (5.5 kb). 1.4 Protocol for obtaining B. subtilis 1-5679

[0092] The B. subtilis 1-5565 strain is then transformed with the pLIPl plasmid using the natural transformation protocol. Clone selection is performed on Luria-Bertani medium containing agar and 20p,g / ml neomycin / kanamycin. The positive clone is verified by colony PCR. The final construct is known as B. subtilis 1-5679. Compared to the B. subtilis 1-5565 strain, the Bacillus subtilis 1-5679 strain produces a higher concentration of mycosubtilin and a greater number of the different novel isoforms shown in Example 2.

[0093] EXAMPLE 2: Production and purification of mycosubtiline from new modified strains of B. subtilis 2.1 Composition of the modified Landy medium

[0094] The composition of the modified Landy medium is as follows: glucose, 60 g / L; ammonium sulfate, 8 g / L; yeast extract, 4 g / L; MgSO4, 0.25 g / L; K2HPO4, 1 g / L; KCl, 0.5 g / L; CuSO4·5H2O, 1.6 mg / L; FeSO4·7H2O, 1.2 mg / L; MnSO4·H2O, 0.4 mg / L. 2.2 Stock Solutions

[0095] To ensure the reproducibility of the medium's composition, sterile concentrated solutions are prepared. A glucose solution (400 g / L) is sterilized by autoclaving at 121°C for 10 minutes. Other solutions include an ammonium sulfate solution (80 g / L); a yeast extract solution (40 g / L); and a mineral salt solution (KHPO₄ 10 g / L; MgSO₄ 2.5 g / L; KCl 5 g / L). a solution of mineral salts l0OOx n°2 (CuSO4 .5H2 O, 1.6 g / l; FeSO4 .7H2O, 1.2 g / l; MnSO4 .H2 O, 0.4 g / l) was acidified with concentrated sulfuric acid for the total dissolution of the salts, all these stock solutions are sterilized by autoclaving at 121°C, for 20 minutes.

[0096] 2.3 Production of one liter of modified Landy medium with MOPS

[0097] A 150 ml glucose solution is taken under sterile conditions and poured into A 1 L bottle of reagent is used. Each solution is added successively under sterile conditions: 100 ml of yeast extract solution, 100 ml of ammonium sulfate solution, 100 ml of mineral solution #1, and finally 1 ml of mineral solution #2. A 20xMOPS (2M) buffer is prepared by dissolving 3-N-Morpholinopropanesulfonic acid (MOPS) (Sigma, reference M3183) in water; the pH is adjusted to 7.0 with NaOH. The solution is then sterilized using a filter. porosity of 0.2 pm. To produce 1 liter of modified Landy medium buffered at 100 mM with MOPS, 50 ml of 20xMOPS were added to the mixture.

[0098] The final pH was adjusted to 7.0 using a sterile 6M NaOH solution. The volume was made up to 1 litre with sterile water. 2.4 Preparation of an inoculum

[0099] The inoculum was prepared from a stock of strains stored at -80°C in 25% glycerol. The glycerol stock was inoculated into Luria-Bertani (LB) broth for 7 to 8 hours. The culture was then spread onto Luria-Bertani (LB) agar plates containing 20 pg / ml of kanamycin (kanamycin sulfate, Sigma, reference 60615) to obtain a single colony. The plate was incubated at 37°C overnight. A preculture PI was then produced with a single colony from the overnight-cultured plate inoculated into a final volume of 10 ml of Luria-Bertani (LB) broth containing 20 pg / ml of kanamycin in a 100 ml Erlenmeyer flask. The culture is incubated at 30°C with shaking at 200 rpm for 10 to 14 hours. After the PI incubation period of the preculture, the culture is centrifuged at 2000 g for 10 minutes at 25°C. The cells are washed with physiological saline and finally resuspended in sterile physiological saline.The suspension is then ready for inoculation. A preculture P2 is produced with an inoculation OD of 600 0.2 in a final volume of 100 ml of modified Landy medium contained in a 1 L Erlenmeyer flask. The culture is incubated at 30°C with shaking at 200 rpm for 7 to 8 hours. 2.5 Cultures in Erlenmeyer flasks

[0100] Before inoculation, the cells from the P2 preculture are harvested and washed with physiological saline and then suspended in sterile physiological saline. This suspension is used for inoculation. The OD of inoculation is between 0.1 and 0.2. The volume of the Erlenmeyer flasks is 11, each containing 100 ml of modified Landy's medium. Similarly, ten 11 Erlenmeyer flasks containing 100 ml of modified Landy's medium are prepared to obtain a high concentration of mycosubtilin. The culture is incubated at 30°C with shaking at 200 rpm for a maximum of 72 hours. After the incubation period, the culture supernatant is used for lipopeptide analysis.

[0101] 2.6 Concentration and purification of mycosubtiline

[0102] The cultures are mixed with acetonitrile (VWR Chemical, reference 83639.320) to a final acetonitrile concentration of 50%. The mixture is centrifuged at 8500 g for 10 minutes at 25°C. The cells are separated, and the supernatant is used to concentrate the mycosubtilin using a laboratory rotary evaporator (Buchi, Rotavapor R300). The sample was concentrated to a concentration final of 3 g / L.

[0103] 2.7 Quantitative analysis of mycosubtilin by UPLC

[0104] The samples were analyzed using a Waters brand complete UPLC system (Aqcuity H class plus) (Waters SAS, Guyancourt, France) using a C18 column (2.1 X150mm, CORTECS. UPLC. C18.1.6pm). To analyze mycosubtilin: 3 pL of purified sample were injected and compared to a The standard for iturine A at 500 mg / L (11774, Sigma-Aldrich, St. Louis, MO, USA) was eluted at a flow rate of 0.4 mL / min. Elution was performed isocratically using a 60 / 40 / 0.1 (v / v / v) water / acetonitrile / formic acid solvent. The retention time and second derivative of the spectrum between 200 and 400 nm for each peak were automatically analyzed using Empower 3 software for identification of the eluted molecules.

[0105] As shown in [Fig.6], cultures of each strain were analyzed in triplicate and the mycosubtilin production of the three strains (LBS0,1-5565 and 1-5679) was compared.

[0106] As shown in [Fig.6], mycosubtilin production was multiplied by 6 in strain 1-5565 compared to its parent strain LBS0, while mycosubtilin production in strain 1-5679 (where the native mycosubtilin promoter was replaced by the continuative prepU promoter in 1-5565) is respectively 28.5 and 2.5 times greater than that of LBS0 and 1-5565 respectively. Comparing these results with those previously obtained with the B. subtilis strain BBG125 by suppressing surfactin production and inserting the PrepU promoter upstream of the mycosubtilin gene (WO2013 / 050700), strain 1-5565 produced 5 times more than strain BBG125, and strain 1-5679 produced 12 times more than strain BBG125. In conclusion, the disruption of the resE, rpoC, addB, araA, cotY, 03427, and 03457 genes in strain 1-5565 unexpectedly led to a significantly greater increase in production than previously obtained.Most importantly, the combination of these seven gene interruptions and the insertion of the PrepU promoter in strain 1-5679 further increased mycosubtilin production by a factor of 2.5 compared to strain 1-5565.

[0107] 2.8 Preparatory analysis of mycosubtiline by HPLC

[0108] The PuriFlash preparative HPLC column - interchim is used to separate the isoforms. Purification was carried out on the interchim US5C18HQ-250 / 300 column (UPTISPHERE STRATEGY C18-HQ 5µm 250X30mm HPLC column).

[0109] The parameters used for the preparative HPLC are as follows: - Injection volume: 10ml - Mobile phase: water / acetonitrile with 0.1 TFA - Flow rate: 35 ml / min - Volume of the fraction: 14 ml and the threshold was 3 mAU. - Detector: 214 nm

[0110] The program used for the separation of isoforms is presented in Table 1 below: [YES] [Table 1] Time (minutes) Acenitrile (%) Water (%) 0 20 80 2 20 80 3 43 57 43 43 57 44 90 10 46 90 10 47 20 80 49 20 80

[0112] Table 1: Size of acetonitrile used for the separation of my-cosubtiline isoforms by preparative HPLC.

[0113] The different peaks representing the different isoforms of mycosubtilin are shown in [Fig. 2]. Each peak was collected in several 14 mL tubes. The number of tubes depends on the peak volume. A total of 5 runs were performed (10 mL of sample per run). Each isoform sample was then analyzed by mass spectrometry (Q-tof analysis) as described in Example 3. Antifungal analysis was performed on the different isoforms as described in Example 4. Cytotoxicity analysis was also performed on the different isoforms as described in Example 5.

[0114] EXAMPLE 3: Qualitative analysis of mycosubtilin produced by new modified strains of B. subtilis

[0115] The parameters used for the LC / MS-Qtof analysis are as follows:

[0116] The UPLC experiments were performed on Agilent 1290 Infinity II 2D-LC technology. A C18 Acquity UPLC BEH column (2.1 x 50 mm x 1.7 pm; Waters) was used at a flow rate of 0.3 mL / min and a temperature of 40 °C. The injection volume was 20 pL, and the diode array detector (DAD) scanned a wavelength spectrum between 190 and 600 nm. Agilent OpenLab CDS ChemStation software and Agilent 1290 Infinity 2D-LC acquisition software were used for the LC analysis.

[0117] The parameters used for the UPLC are as follows: - Injection volume: 1 Opl - Column temperature: 40°C - Mobile phase: Water / Acetonitrile with 0.1% formic acid.

[0118] The program used for the separation of isoforms is presented in Table 2 below:

[0119] [Tables2] Time (minutes) Acenitrile (%) Water (%) 0 37 63 5 37 63 7.5 45 55 8 100 0 10 100 0 10.5 37 63

[0120] Table 2: Acetonitrile content used for the analysis of mycosubtiline isoforms by LC / MS-Qtof

[0121] LC / MS-Qtof analyses were performed using the 1290 Infinity II coupled to the Jet Stream ESLQ-TOF 6530 (Agilent Technologies) in negative mode with the following parameters: capillary voltage: 3.5 kV; nebulizer pressure: 35 psig; drying gas: 8 L / min; drying gas temperature: 300°C; sheath gas flow rate: 11 L / min; sheath gas temperature: 350°C; fragmenter voltage: 175 V; skimmer voltage: 65 V; CTOPOLE RF: 750 V. Accurate mass spectra were recorded in the range of m / z = 100-1200. In brief, 10 pL of sample were injected and separated using a Cl8 Acquity UPLC BEH (2.1 x 50 mm x 1.7 pm; Waters) with 0.1% formic acid (FA) as solvent A and ACN + 0.1% FA as solvent B. The gradient started with 0% solvent B and increased to 30% over 5 min. The data were processed using MassHunter Qualitative Analysis software (Agilent Technologies).Table 3 shows the new mycosubtilin isoforms C16 and C17 compared with the classical C16 and C17 obtained from LC / MS-Qtof analysis as described above.

[0122] [Tables3] Peak number Variant s Fatty acid AAI AA2 AA3 AA4 AA5 AA6 AA7 m / z 1 Gln3-Cl 6 C16 Asn Tyr Gin Gin Pro Ser Asn 1085 2 Glnl-Cl 6 C16 Gin Tyr Asn Gin Pro Ser Asn 1085 3 A-C16 C16 Asn Tyr Asn Gin Pro Ser Asn 1071 4 Glnl-Cl 7 C17 Gin Tyr Asn Gin Pro Ser Asn 1099 5 A-C17 C17 Asn Tyr Gin Gin Pro Ser Asn 1085 6 A-C17 C17 Asn Tyr Asn Gin Pro Ser Asn 1085 7 A-C17 C17 Asn Tyr Asn Gin Pro Ser Asn 1085 8 A-C18 C18 Asn Tyr Asn Gin Pro Ser Asn 1099

[0123] Table 3: Analysis of mycosubilin isoforms by LC / MS-Qtof of the different fractions purified by preparative HPLC (new isoforms are in bold)

[0124] In addition to the classical C16 isoform of mycosubtiline (C16-myco), the C17 isoform of mycosubtiline (C17-myco), and the C18 isoform of mycosubtiline (peaks 3, 5, 6, 7, and 8), three new isoforms were detected. Peaks 1, 2, and 4, which correspond to C16-Gln3, C16-Glnl, and C17-Glnl, respectively, are new isoforms of mycosubtiline. Asparagine is replaced by glutamine in the third position for peak 1, while asparagine is replaced by glutamine in the third position for peaks 2 and 4.

[0125] [Tables4] Variantes Acide gras AAI AA2 AA3 AA4 AA5 AA6 AA m / z Gln3-C16 C16 Asn Tyr Gin Gin Pro Ser Asn 1085 Gln7-C16 C16 Asn Tyr Asn Gin Pro Ser Gin 1085 Glnl,Gln3-Cl 6 C16 Gin Tyr Gin Gin Pro Ser Asn 1099 Gln3,Gln7-Cl 6 C16 Asn Tyr Gin Gin Pro Ser Gin 1099 Glnl-C16 C16 Gin Tyr Asn Gin Pro Ser Asn 1085 A-C16 C16 Asn Tyr Asn Gin Pro Ser Asn 1071 Glnl,Gln3, Gln7-C16 C16 Gin Tyr Gin Gin Pro Ser Gin 1113 GLN3-C17 C17 Asn Tyr Gin Gin Pro Ser Asn 1099 GLN7-C17 C17 Asn Tyr Asn Gin Pro Ser Gin 1099 GLN3-CL 7 C17 Gin Tyr Gin Gin Pro Ser Asn 1113 GLN3,GLN7-CL 7 C17 Asn Tyr Gin Gin Pro Ser Gin 1113 GLN1-C17 C17 Gin Tyr Asn Gin Pro Ser Asn 1099 A-C17 C17 Asn Tyr Asn Gin Pro Ser Asn 1085 A-C17 C17 Asn Tyr Asn Gin Pro Ser Asn 1085 A-C17 C17 Asn Tyr Asn Gin Pro Ser Asn 1085 A-C18 C18 Asn Tyr Asn Gin Pro Ser Asn 1099 Gln3-C18 C18 Asn Tyr Asn Gin Gin Pro Ser Asn 1113 Gln7-C18 C18 Asn Tyr Asn Gin Pro Ser Gin 1113 Glnl-C18 C18 Gin Tyr Asn Gin Pro Ser Asn 1113 A-C19 C19 Asn Tyr Asn Gin Pro Ser Asn 1113

[0126] Table 4: Analysis of mycosubilin isoforms performed by LC / MS-Qtof on the complete supernatant of B. subtilis strains 1-5565 and 1-5679 (new isoforms are in bold) (AA = amino acid).

[0127] In addition to the eight main isoforms purified by preparative HPLC (shown (in Table 3), several other minor isoforms were separated by UPLC from the supernatants of strains 1-5565 and 1-5679. These minor isoforms were then identified and characterized using the highly sensitive MS-Qtof method described above. As shown in Table 4, a total of 20 isoforms were identified in the supernatants of the new B. subtilis strains. Among these, 14 new minor isoforms were discovered, listed here: Gln3-C16; Gln7-C16; Glnl,Gln3-C16; Gln3,Gln7-C16; Glnl-C16; Glnl,Gln3,Gln7-C16; Gln7-C17; Glnl,Gln3-C17; Gln3,Gln7-C17; Glnl-C17; Gln3-C18; Gln7-C18; Glnl-C18 and A-C19. In addition to all the new isoforms discovered by this work, it also turns out that in our overproducing mutants, the adenylation domains of my-cosubtiline synthetase can activate both a Gin and an Asn at positions 1, 3, and 7 of the peptide ring. The new isoforms are being used for activity and cytotoxicity experiments to determine their efficacy. The isoforms are dissolved in DMSO and used to study the activity. The results are described in examples 4 and 5.

[0128] EXAMPLE 4: Antifungal activities of the different isoforms of mycosubtiline

[0129] Antifungal activity was demonstrated on three different fungal strains, namely B. cinerea, Aspergillus sp., and Z. tritici. In addition to the two conventional isoforms, three new isoforms were tested for their antifungal properties. A range of concentrations (pg ma / ml or ppm) of 0, 0.0037, 0.0146, 0.0586, 0.2344, 0.9375, 3.75, and 15 was used to determine the activity coefficient. Based on this concentration range, the EC50 (concentration that reduces the growth of the fungal strain by 50%) and EC95 (concentration that reduces the growth of the pathogen by 95%) were calculated and are presented as described in Table 5. 4.1 Antifungal activity against B. cinerea

[0130] The EC50 value of A-C16 is 0.933 ppm, while that of Gin 1-Cl6 is 1.048 ppm, indicating a 10% lower concentration of the former required to achieve the same activity as the latter. The EC95 value of TA-C16 is 1.457 ppm, while that of Gin 1-Cl6 is 1.282 ppm, indicating a 17% higher concentration of the former required to achieve the same activity as the latter. This new isoform (Gin 1-Cl6) proves effective against this fungal strain.

[0131] The two other new isoforms Gln3-C16 and Glnl-C17 proved effective against this fungal strain but at a higher concentration.

[0132] 4.2 Antifungal activity on Aspergillus sp.

[0133] The EC50 value of A-C17 is 1.191 ppm, while that of Glnl-C17 is 1.019 ppm, indicating a 14% higher concentration of the former required to achieve the same activity as the latter. The EC95 value of A-C17 is 1.905 ppm, while that of Gin 1-Cl7 is 1.290 ppm, indicating a 32% higher concentration of the former is required to achieve the same activity as the latter. This new isoform proves effective against this fungal strain.

[0134] The two other new isoforms Gin 1-Cl6 and Gln3-C16 proved effective against this fungal strain but at a higher concentration. 4.3 Antifungal activity on Z. tritici

[0135] The EC50 value of A-C16 is 4.258 ppm, while that of Gin 1-Cl6 is 1.906 ppm, indicating a 55% higher concentration of the former is required to achieve the same activity as the latter. The EC95 value of A-C16 is 26.106 ppm, while that of Gln3-C16 is 17.874 ppm, indicating a 32% higher concentration of the former is required to achieve the same activity as the latter. These new isoforms have proven effective against this fungal strain.

[0136] The other new isoform Glnl-C17 proves effective against this fungal strain but at a higher concentration.

[0137] [Tables5] CES0 (pg / ml) Gln3-C16 Glnl-C16 A-C16 Gln3-C17 Glnl-C17 A-C17 B. cinerea 9.918 1.048 0.933 1.646 0.920 0.467 As parsley lus sp. 13.148 1.691 1.062 6.335 1.019 1.191 Z. tritici 10.077 1.906 4.258 4.314 2.156 0.943 CE9S(pg / ml) Gln3-C16 Glnl-C16 A-C16 Gln3-C17 Glnl-C17 A-C17 B. cinerea 14.232 1.282 1.457 3.437 1.117 0.677 As parsley lus sp. 14,772 3,040 1,374 13,659 1,290 1,905 Z. tritici 17,874 24,286 26,106 16,078 17,040 18,010

[0138] Table 5: Antifungal activity of 6 different isoforms of mycosubtiline on three phytopathogenic fungi. EXAMPLE 5: Cytotoxicity Tests

[0139] The cytotoxicity test is performed using Vero cells and the CyQUANT™ XTT Cell Viability assay kit (supplied by Invitrogen™).

[0140] The samples are dissolved in 100% DMSO at a concentration of 20-50 g / L. The final concentration in the DMEM medium is 40 pg / L. The cells were prepared in flasks containing DMEM cell culture medium with 10% FBS as follows:

[0141] A 175 ml vial containing 20-30 million cells was prepared with 25 ml of medium and 2 ml of trypsin. The medium was then discarded because the cells were attached to the vial wall. The vial was rinsed twice with PBS, and then trypsin was added to detach the cells from the vial wall. The mixture was incubated for 5 minutes at 37°C. To inactivate the trypsin, 6 ml of trypsin was added to the medium (twice the volume of the original trypsin in the medium).

[0142] In a 50 mL bottle, the medium was centrifuged for 5 minutes at 300 g. The medium was discarded, and the cells were resuspended. 100 µL of cells were added to 100 µL of dye, and then 12 µL were used for counting. The percentage of viability was compared to a control of untreated cells.

[0143] The test is carried out in a 96-well plate. A range of concentrations (mg / 1) 0.14; 0.28; 0.56; 1.16; 2.25; 4.5 is used to determine the toxicity limit of each isoform.

[0144] The results presented in [Fig. 7] show that the percentage of Vero cells The viable concentration is significantly higher in the presence of the novel C16 and C17 isoforms compared to the conventional A-C16 and A-C17 isoforms. From these data, the EC50 can be determined: the EC50 of Gln3-C16 is greater than 4.5 mg / L, the EC50 of Gin 1-Cl6 is 4.5 mg / L, and the EC50 of A-C16 is 1.7 mg / L, which is at least 2.64 times lower than that of the novel C16 isoforms of mycosubtilin. The cytotoxicity profile of the C16 isoforms is as follows: Gln3-C16 < Gln1-C16 < A-C16. The novel C16 isoforms, namely Gln3-C16 and Gln1-C16, are significantly less toxic than the conventional isoform, A-C16. The same profile is observed for the C17 isoforms based on mycosubtiline. The EC50 of Gln3-C17 is greater than 4.5 mg / L, the EC50 of Glnl-C17 is about 3.4 mg / L, the EC50 of A-C17 is about 1.7 mg / L, which is at least 2 times lower than the new C17 isoforms of mycosubtiline.For C17 isoforms, the toxicity profile is as follows: Gln3-C17 < Glnl-C17 < A-C17.

Claims

Demands

1. Genetically modified Bacillus sp strain characterized as the Bacillus subtilis strain deposited on July 30, 2020 under CNCM number 1-5565 at the National Collection of Microorganism Cultures (CNCM) of the Pasteur Institute (Paris, France).

2. Strain as defined in claim 1 modified by replacing the endogenous promoter with the constitutive promoter PrepU characterized in that it is the Bacillus subtilis strain deposited on May 5, 2021 under number CNCM 1-5679 in the National Collection of Microorganism Cultures (CNCM) of the Pasteur Institute (Paris, France).

3. Novel isoform of mycosubtiline selected from the Glnl-C16 and Glnl-C17 isoforms.

4. Composition comprising at least one mycosubtiline selected from the isoforms Glnl-C16 and Glnl-C17.

5. Use of at least one mycosubtiline selected from the Glnl-C16 and Glnl-C17 isoforms as an antifungal agent in the fields of agriculture and the food, chemical, detergent, petroleum and environmental industries

6. Mycosubtiline selected from the Glnl-C16 and Glnl-C17 isoforms for use as an antifungal agent in the cosmetic, medical and pharmaceutical industries.

7. Use according to claim 5 wherein at least one mycosubtiline corresponding to the Glnl-Cl6 isoform is used as an antifungal agent against the B. cinera strain.

8. Use according to claim 5 wherein at least one mycosubtiline corresponding to the Glnl-C17 isoform is used as an antifungal agent against the Aspergillus sp. strain.

9. Use according to claim 5 wherein at least one mycosubtiline corresponding to the Glnl-Cl6 isoform is used as an antifungal agent against the Z. tritici strain.

10. Use of at least one mycosubtiline corresponding to the Gln3-C16 isoform as an antifungal agent against the Z. tritici strain in the fields of agriculture and the food, chemical, detergent, petroleum and environmental industries.

11. A method for producing at least one new isoform of mycosubtilin selected from the isoforms Gln3-C16, Glnl-C16 and Glnl-C17, consisting of cultivating a strain as defined in one of the claims indications 1 or 2.

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