A novel Bacillus subtilis strain producing a novel mycosubtyrin isoform

Abstract

The present invention relates to the field of antifungal biosurfactant molecules of bacterial origin. More specifically, the present invention relates to novel Bacillus subtilis strains producing novel isoforms of mycosubtyrin, methods for their preparation, and compositions containing them. These novel mycosubtyrin isoforms exhibit improved antifungal activity and reduced cytotoxicity compared to prior art mycosubtyrins.

Description

Detailed Description of the Invention

[0001] [Technical field] The present invention relates to the field of antifungal biosurfactant molecules of bacterial origin. More specifically, the present invention relates to novel Bacillus subtilis strains producing novel isoforms of mycosubtyrin, methods for their preparation, and compositions containing them. These novel mycosubtyrin isoforms exhibit improved antifungal activity and reduced cytotoxicity compared to prior art mycosubtyrins.

[0002] [Background technology] FIELD OF THE INVENTION The world's population is growing and is expected to reach 8.3 billion by 2025 and nearly 10 billion by 2050. Depending on the various assumptions used, global food demand could increase by 40% to 68% by 2050 (Herault, 2011). This increase in demand can be met by even more intensive agriculture, which involves greater consumption of water, fossil fuels, fertilizers, and plant protection products to ensure high yields. To address these challenges, the scientific community and industry must develop innovative research approaches. In the agricultural sector, this raises issues regarding ensuring yields and farm profitability, while limiting the use of inputs derived from chemical synthesis. The latter is the subject of much debate, and their impacts on farmers, consumer health, the environment, and biodiversity have been documented in numerous examples. Authorities around the world are introducing regulations to limit the use of toxic compounds in agriculture, food, and other sectors.

[0003] Therefore, the demand for organic molecules in agriculture is very high. Recent reports indicate that the biopesticides market is set to reach US$8.5 billion by 2025. Since the implementation of the REACH regulation in 2007, increased regulatory pressure on synthetic molecules in Europe has also had a major impact on other business sectors. In particular, manufacturers are exploring preservatives, biosurfactants, and novel antimicrobial molecules. Microbial secondary metabolites, produced by many microorganisms such as Bacillus subtilis, possess all of these properties.

[0004] In the agricultural sector, there are many plant pathogens (approximately 7,000 species), among which Botrytis cinerea (causing gray mold) and Zymoseptoria tritici (causing wheat leaf blight) rank among the top 10 plant pathogens with the greatest 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 can produce three distinct families of molecules: surfactins (surfactins, pumillacidins); fengycins (fengycins, plipastatins, agrastatins); and iturins (iturins, mycosubtilins, bacilomycins, mojavensins). Within the iturin family, mycosubtilins are the compounds with the highest antifungal activity (Besson et al., 1979). Mycosubtilins are lipopeptides in which a heptapeptide ring is linked to a fatty acid chain by a β-amino bond. The length of the fatty acid chain can vary from C15 to C18, and the isomerism can be linear, iso, or anteiso. (Stein, 2005). The classical peptide chain formula is Asn / D-Tyr / D-Asn / Gln / Pro / / D-Ser / Asn, as disclosed in Figure 1.

[0006] The composition of the peptide ring and the length and isomerism of the fatty acid chain are factors that affect the activity of a molecule, particularly its antifungal activity. In 2009, a study showed that the anteiso-C17 isoform of mycosubtilin was most active against the yeast C. albicans (Fickers et al., 2009). In 2013, another study tested the effects of different alkyl chains of mycosubtilin against B. cinerea. The authors showed that the anteiso-C17 isoform had the highest activity against B. cinerea, with an MIC of 8 μM, followed by the n-C16 and iso-C17 isoforms (MIC = 16 μM), and the iso-C16 isoform (MIC = 32 μM). Furthermore, the presence of the C18 chain (even at low concentrations) significantly enhanced the activity of the mixture, suggesting very potent activity for this isoform (Bechet et al., 2013). More recently, the antifungal activity of mycosubtyrin isoforms against A. niger was measured, and the results showed that the anteiso-C17 isoform had the highest activity against A. niger, with an MIC of 8 μM, followed by the n-C16 and iso-C17 isoforms (MIC = 16 μM), and iso-C16 (MIC = 32 μM) (J.-S. Guez et al., 2022).

[0007] Numerous scientific studies have demonstrated the broad spectrum of antifungal activity of mycosubtyrin. This includes Candida albicans, Bremia lactucae, Zymoseptoria tritici, Botrytis cinera, Fusarium oxysporum, Fusarium graminearum, and Fusarium verticillioides, Candida krusei, Paecilomyces variotii, Byssocchlamys fulva, Venturia inaequalis, Verticillium dahlia, or Aspergillus niger. niger) (Bechet 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 novel mycosubtyrin isoform was discovered in an engineered strain of Bacillus subtilis and patented. This molecule, with a C17 fatty acid chain, contains glutamine [Gln3] at position 3 instead of asparagine (WO 2013 / 050700). In this patent, the inventors showed that a mixture of mycosubtyrin isoforms containing this novel isoform [Gln3-C17] (even in small amounts, approximately 1%) was as active as a mixture without it, demonstrating better antifungal activity of this isoform relative to the others.

[0009] Very few studies have been conducted on the toxicity and ecotoxicity of lipopeptide molecules produced by Bacillus subtilis, especially on the individual lipopeptide isoforms. The ecotoxicity of surfactin and mycosubtilin isoforms alone or in binary mixtures has been investigated. The results of this study show that the EC of mycosubtilin in the microtox test (against Aliivibrio fischeri) is 50 However, the EC of mycosubtilin (8 mg / L) on Daphnia magna was at least 4.5 times lower than that of surfactin. 50 It was also shown to have an IC50 activity three times lower than that of surfactin (26 mg / L) (Deravel et al., 2014). Another study on iturin A investigated its acute toxicity in rats over a 28-day period. Iturin A showed no toxic effects on the lungs, heart, and kidneys after 28 days of treatment and a 14-day recovery period (Dey et al., 2016). More recently, a cytotoxicity study was conducted on two different cell lines: Caco-2 and Vero cells, using a mixture of three lipopeptide families (surfactin, fengycin, and mycosubtilin). The results showed that a mixture of mycosubtilin isoforms had an IC50 activity of 10–20 mg / L against these two cell lines. 50 (Kourmentza et al., 2021).

[0010] Today, there is a large need for antifungal molecules with improved properties to make these biological alternatives economically viable on an industrial scale.

[0011] [Summary of the Invention] The present inventors have developed a new Bacillus subtilis strain capable of producing a novel mycosubtyrin isoform that is more effective as an antifungal agent and less cytotoxic than mycosubtyrins reported to date.

[0012] The present invention relates to two new Bacillus subtilis strains that have been genetically modified with seven mutations to produce novel mycosubtyrin isoforms, and these two strains have been registered with the CNCM under numbers CNCM I-5565 and CNCM I-5679.

[0013] The present invention relates to two novel mycosubtyrin isoforms, Gln1-C16 and Gln1-C17, and compositions containing them.

[0014] The present invention also relates to the use of the novel isoforms as antifungal agents, particularly against B. cinerea, Aspergillus spp., and Z. tritici strains.

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

[0016] Effect of the invention The present invention provides access to surfactants suitable for agricultural use, compounds that are non-toxic and available in large quantities.

[0017] Indeed, the novel mycosubtilin isoforms presented herein exhibit superior antifungal activity to mycosubtilins disclosed to date. In particular, the GlnlC16 isoform exhibits greater antifungal activity than the C16 isoform. Furthermore, these novel isoforms exhibit lower cytotoxicity than the same known mycosubtilins.

[0018] In agriculture, these novel isoforms can be used for the plant health industry for the biological control of plant diseases or post-harvest treatments, as well as in the production of biopesticides or biosurfactants for plant growth stimulation.

[0019] These surfactant properties make them particularly useful in other fields such as the food, cosmetic, chemical, medical, pharmaceutical, detergent, oil, and environmental industries.

[0020] [Mode for Carrying Out the Invention] A first object of the present invention relates to the Bacillus subtilis strain deposited on July 30, 2020 at the Collection nationale de cultures de micro-organismes (CNCM) of the Institut Pasteur (Paris, France) under the number CNCM I-5565.

[0021] A second particular strain according to the invention is the Bacillus subtilis strain deposited on May 05, 2021 at the Collection National des Cultures de Microorganisms (CNCM) of the Pasteur Institute (Paris, France) under the number CNCM I-5679.

[0022] Strain I-5565 contains seven mutations that are responsible for metabolic changes, leading to increased mycosubtilin production and the appearance of novel isoforms, particularly Gln1-C16 and Gln1-C17.

[0023] Strain I-5679 differs from strain I-5565 in the nature of the promoter that allows mycosubtilin expression: while strain I-5565 contains the native promoter, strain I-5679 has been modified by replacing the endogenous promoter with the constitutive promoter PrepU. The promoter change increased overall production of mycosubtilin: both the conventional form of mycosubtilin and the novel isoform produced by strain I-5565.

[0024] The present invention also relates to a strain as defined above (strain I-5565) modified by replacing the endogenous promoter with a constitutive PrepU promoter, characterized in that it is the Bacillus subtilis strain deposited at the Collection National des Cultures de Microorganisms (CNCM) of the Pasteur Institute (Paris, France) under number CNCM I-5679 on May 05, 2021.

[0025] The strain according to the invention is a genetically modified Bacillus strain characterized in that it contains at least the following mutations: - substitution of serine by asparagine due to a mutation at position 1118 of the resE_1 gene; - substitution of aspartic acid with asparagine due to a mutation at position 3466 of the rpoC gene; - substitution of alanine with valine due to a mutation at position 886 of the addB gene; - substitution of glutamic acid by lysine due to a mutation at position 121 of the araA gene; - substitution of proline by serine due to a mutation at position 154 of the cotY gene; -03427 gene, resulting from a mutation at position 32, resulting in a substitution of serine with phenylalanine; - substitution of serine with phenylalanine due to a mutation at position 92 of the 03457 gene; The mutations in these genes are primarily nucleotide variations.

[0026] Examples of each of the mutant genes are shown in the sequence listing: - the mutant resE_1 gene is represented by the sequence SEQ ID NO: 1; - the mutant 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 mutant araA gene is represented by the sequence SEQ ID NO: 4; - the mutant cotY gene is represented by the sequence SEQ ID NO: 5; - the mutant 03427 gene is represented by the sequence SEQ ID NO: 6; The mutant 03457 gene is represented by the sequence SEQ ID NO:7.

[0027] Given the degeneracy of the genetic code, other variations are possible, and one skilled in the art will be able to identify variations other than those disclosed in these sequences.

[0028] The inventors have shown that the combination of these seven mutations results in an overall increase in mycosubtyrin production, in particular the production of novel mycosubtyrin isoforms such as Gln1-C16 and Gln1-C17.

[0029] The seven genes modified in the Bacillus strain according to the present invention are as follows: -resE is a gene encoding a protein similar to those found in two-component signal transduction systems and plays a regulatory role in respiration. ResE is involved in the overall regulation of aerobic and anaerobic respiration in B. subtilis. ResE (as well as ResD) is a two-component regulatory protein required for transcriptional activation of Fnr under oxygen limitation in B. subtilis (Sun et al., 1996). Mycosubtilin production is significantly affected by oxygenation conditions (Guez et al., 2008); -rpoC is a gene involved in the β subunit of DNA-dependent RNA polymerase. Mutations in rpoC increase the expression of stress-sensitive regulatory factors, including extracytoplasmic function (ECF) σ factors (σM, σW, and σX) and general stress σ factor (σB). SigB (σB) plays an important role in adaptive responses, including pathogen presence responses, which positively influence the antifungal molecules produced by Bacillus species (Lee et al., 2013; Rodriguez Ayala et al., 2020); -araA is the gene encoding L-arabinose isomerase. This gene is repressed by high glucose concentrations (Sa-Nogueira et al., 1997); -addB is a gene that contributes to DNA repair and recombination; This enzyme functions as a heterodimer of AddA and AddB subunits and is a rapid processive DNA helicase, catalyzing DNA unwinding (Yeeles et al., 2009). Mutations in this gene can affect enzyme activity (Haijema et al., 1996); -cotY is a gene encoding the spore coat protein Y. This gene is part of the SigE regulatory element, and a SigE binding site exists in the promoter region of the mycosubtilin operon (Wu et al., 2015); The -03427 gene encodes a protein that may be related to Rap proteins, although its function has not been precisely described in the literature. In B. subtilis, it regulates the phosphorylation level or DNA binding activity of response regulators such as Spo0F, which is involved in sporulation initiation, or ComA, which regulates the expression of transformation competence (Diaz et al., 2012). Rap ​​proteins are also involved in the expression of genes encoding lipopeptide biosynthesis; The -03457 gene encodes a protein whose function is unknown but which may be involved in the transport of secondary metabolites.

[0030] A second object of the present invention relates to novel mycosubtyrin isoforms selected from the Gln1-C16 and Gln1-C17 isoforms.

[0031] These molecules are shown in Figures 4 and 5, respectively.

[0032] Strains I-5565 and I-5679 produced 13 other novel minor mycosubtilins (in terms of production levels), namely, Gln7-C16; Gln1, Gln3-C16; Gln3, Gln7-C16; Gln1-C16; Gln1, Gln3, Gln7-C16; Gln7-C17; It should be noted that the enzyme produces Gln1, Gln3-C17; Gln3, Gln7-C17; Gln1-C17; Gln3-C18; Gln7-C18; Gln1-C18 and A-C19.

[0033] A second object of the invention relates to a composition comprising at least one mycosubtilin selected from the Gln1-C16 and Gln1-C17 isoforms.

[0034] The composition according to the invention may contain one, two or three isoforms of mycosubtilin, namely: -Gln1-C16 isoform -Gln1-C17 isoform -Gln1-C16 and Gln3-C16 isoforms -Gln1-C17 isoform and Gln3-C16 isoform -Gln1-C16 and Gln1-C17 isoforms -Gln1-C16 isoform, Gln1-C17 isoform, and Gln3-C16 isoform.

[0035] The Gln3-C16 isoform is shown in Figure 3.

[0036] In a preferred embodiment of the invention, the composition comprises at least the Gln1-C16 isoform.

[0037] Furthermore, the composition according to the present invention may contain at least one of the novel mycosubtilins selected from Gln7-C16; Gln1, Gln3-C16; Gln3, Gln7-C16; Gln1-C16; Gln1, Gln3, Gln7-C16; Gln7-C17; Gln1, Gln3-C17; Gln3, Gln7-C17; Gln1-C17; Gln3-C18; Gln7-C18; Gln1-C18 and A-C19, and any other known mycosubtilins.

[0038] Such compositions also include - molecules of the iturin family, such as iturin A, mojavencin, and bacillomycins A, B, C, D, F, and L; - molecules of the surfactin family, such as surfactin A, B, or C, lichenisin, and pumilacidin; - molecules of the fengycin family, such as fengycin A and B, plipastatin A and B, and agrastatin A and B Lipopeptides other than mycosubtilin (which is iturin) may also be included, including:

[0039] Such compositions may also contain other ingredients such as other surfactant molecules, preservatives, and adjuvants.

[0040] Surfactant molecules include amphiphilic molecules, surfactants, lipopeptides (eg, surfactin, fengycin), chemical surfactants, and biological surfactants (eg, rhamnolipids, polysaccharides).

[0041] Surfactants include heparin, hyaluronic acid, dextran, amylose, chitosan, anionic surfactants derived from amino acids, nonionic surfactants derived from polyglycosides, hydrotropic surfactants, lipopeptides such as surfactin isomers and / or fengycin (or plipastatin) isomers, rhamnolipids, and vegetable oils.

[0042] Surfactants from the nonionic surfactant family are chosen, for example, from fatty alcohol axalkylates, pentylene glycol and its derivatives, hydrotropes of the alkyl polyglycoside type (alkyl polyglycosides and alkylethoxy polyglycosides), texturizing molecules of the polyglycoside type (xanthan gum, gum arabic, tragacanth gum, guar gum, locust bean gum, tamarind gum, pectin, gellan gum, carrageenates, agar, alginates).

[0043] The surfactants from the anionic surfactant family are chosen, for example, from surfactin, fengycin, sodium laureth sulfate and its derivatives, or amino acid derivatives.

[0044] Surfactants from the oil family are chosen, for example, from modified (acidified, methylated, esterified) oils and oil extracts, in particular from almond, peanut, argan, avocado, rapeseed, ricin, Lorenzo, neem, hazelnut, cashew, macadamia nut, olive, pistachio, rice, oleic sunflower, camelina, linseed, borage, safflower, hemp, cotton, wheat germ, corn, walnut, poppy, evening primrose, barley, pumpkin seed, grape seed, pea, sesame, soybean, sunflower.

[0045] Among adjuvants, some can facilitate penetration of the composition into the plant (e.g., oils), others increase the contact surface between the plant leaf and the composition (humectants), others can counteract drying by absorbing moisture from the air (salts), and still others can fix the composition to the leaf so as to limit leaching and volatilization. Thus, adjuvants must be adapted to the composition's mode of action (root, contact, systemic translocation, or penetration), the type of product formulation, and the type of target plant (hairy or glabrous leaves, cuticle thickness, plant stage, stomata position, etc.).

[0046] A third object of the present invention relates to the use of at least one mycosubtilin selected from the Gln1-C16 and Gln1-C17 isoforms or a composition comprising such a mycosubtilin (as disclosed above) as an antifungal agent.

[0047] Both isoforms are effective against ascomycetes, particularly B. cinera, Aspergillus, and Z. tritici.

[0048] In a particular embodiment, the use consists in using at least one mycosubtilin corresponding to the Gln1-C16 isoform as an antifungal agent against B. cinerea strains.

[0049] In another particular embodiment, the use consists in using at least one mycosubtilin corresponding to the Gln1-C17 isoform as an antifungal agent against Aspergillus strains.

[0050] In yet another particular embodiment, the use comprises using at least one mycosubtilin corresponding to one of the Gln1-C16 or Gln3-C16 isoforms as an antifungal agent against Z. tritici strains. One particular embodiment comprises using Gln1-C16 or Gln1-C17 in combination with Gln3-C16 as an antifungal agent against Z. tritici strains.

[0051] A fourth object of the present invention relates to a method for producing an antifungal composition, which comprises cultivating a genetically modified Bacillus strain as defined above.

[0052] In a particular embodiment, the production method produces at least one novel mycosubtilin isoform selected from the Gln1-C16 isoform and the Gln1-C17 isoform by culturing a genetically modified Bacillus strain as defined above.

[0053] As a result, this method - culturing a Bacillus strain according to the invention, - incubating the strain in a suitable culture medium, - Harvesting the strain and / or culture supernatant, It consists of:

[0054] The method may continue with one or more of the following steps: - purifying mycosubtilins (total or differentiated by isoforms) - concentrating the solution containing mycosubtilin or the desired isoform of mycosubtilin - dehydrating the solution containing mycosubtyrin or the desired isoform of mycosubtyrin.

[0055] The novel mycosubtyrin isoforms disclosed above and compositions containing them may be used in many applications due to their surfactant and / or antifungal properties as well as their lack or low level of toxicity; - in agriculture, the manufacture of biopesticides or biosurfactants for the plant health industry for the biological control of plant diseases or for post-harvest treatment, or as plant growth stimulants; -In the food industry: preservatives; - in cosmetics: preservatives and formulation agents; - in chemistry; - in the medical and pharmaceutical sector: antifungal or preventive, antiseptic; - as a detergent; -In the oil industry; -In the field of environmental protection: biological pest control, non-toxic formulations.

[0056] They can therefore be used in non-therapeutic applications such as agricultural, food, chemical, detergent, oil and environmental industries, or in medical applications such as cosmetics, medicine and pharmaceuticals.

[0057] The invention will be better understood on reading the following examples, which are provided by way of illustration and are not to be considered in any way as limiting the scope of the invention.

[0058] [Brief description of the drawing] [Figure 1] Classical formula of mycosubtilin A-C17 (n=13).

[0059] [Figure 2] Schematic diagram of preparative HPLC chromatogram. Peaks P3, P5, P6, P7, and P8 are classical mycosubtyrin isoforms, while peaks P1, P2, and P4 are novel mycosubtyrin isoforms. Modified amino acids in the peptide ring are labeled.

[0060] [Figure 3] Expression of mycosubtilin isoform Gln3-C16.

[0061] [Figure 4] Expression of the novel mycosubtilin isoform Gln1-C16.

[0062] [Figure 5] Expression of the novel mycosubtilin isoform Gln1-C17.

[0063] [Figure 6] Quantitative analysis of mycosubtilin produced by the newly engineered Bacillus subtilis strain.

[0064] [Figure 7] Cell viability test of Vero cells treated with different mycosubtyrin isoforms at different concentrations of each isoform.

[0065] [Example] Example 1: Construction of Bacillus subtilis strains I-5565 and I-5679 B. subtilis strain I-5679 was derived from strain I-5565; the construction of these strains is described below; The first strain was deposited at the Collection National des Cultures de Microbiologie (CNCM) of the Institut Pasteur (Paris, France) under the number CNCM I-5565 on July 30, 2020. The second strain was deposited at the Collection National des Cultures de Microbiologie (CNCM) of the Institut Pasteur (Paris, France) under the number CNCM I-5679 on May 5, 2021.

[0066] 1.1 Procedure for obtaining Bacillus subtilis strain I-5565 Strain I-5565 was constructed from Bacillus subtilis deposited at the Collection National des Cultures de Microbiologie (CNCM) of the Pasteur Institute (Paris, France) on February 6, 2020, under CNCM number I-5487.

[0067] In this strain I-5487, seven genes were modified by point modifications, i.e., single nucleotide changes (resE, rpoC, addB, araA, cotY03427, and 03457). Deletion or modification of a single nucleotide (point mutation) can alter the amino acid sequence and result in a new phenotype in the mutant strain. To achieve this type of genetic modification, various genetic engineering techniques can be used: -CRISPR-Cas9-mediated genome engineering surpasses previous methods in terms of precision, enabling reliable generation of mutants without foreign DNA in many wild-type bacterial strains, including Bacillus subtilis, with point mutation efficiencies of up to approximately 68%. This method can generate novel phenotypes 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 complete control over protein sequence. Several strategies have been developed, with the QuikChange™ site-directed mutagenesis system developed by Stratagene (La Jolla, CA) being perhaps the most widely used. QuikChange™ works by using a pair of complementary primers carrying the mutation. In a series of PCR cycles, these primers hybridize to the template DNA, replicating the plasmid DNA carrying the mutation. The mutant DNA product exhibits a strand break (nick). The resulting DNA pool (mutant and parent) is then treated with DpnI to disrupt the methylated parent DNA from the newly synthesized unmethylated mutant DNA and transformed into Escherichia coli (E. coli) cells, where the break is ligated by host repair enzymes (Liu & Naismith, 2008); - Targeted mutagenesis using mutagens. This method relies on the use of chemical mutagens (ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS)) or physical agents such as intense exposure to ultraviolet (UV) light. Genetic modifications occur, resulting in phenotypic changes. EMS is chosen to target GC-rich regions, while UV is chosen to target AT-rich regions.

[0068] After genetic modification using the above mutation procedure, interesting mutants were obtained. The method used for mutant strain screening involved diluting 1 unit volume of the mutant library stock with 1 unit volume of physiological water. The cell suspension was spread on a nutrient agar plate (provided by Condalab) to obtain single colonies. Several single colonies were then selected using a Qpix460 (Molecular Devices) and inoculated into nutrient culture medium. After 24 hours of incubation at 30°C, the cells were harvested, and the supernatant was used for mycosubtilin analysis by reverse-phase high-performance liquid chromatography as disclosed in Example 2. In different embodiments, other media such as LB medium, Landy medium, or Cooper medium can be used.

[0069] Using this methodology, the Bacillus subtilis strain I-5565 was selected after several rounds of point mutation procedures. * Genomic sequencing using 150 bp (libraries prepared using Nextera XT) revealed changes in the sequences of these seven genes, as disclosed below.

[0070] 1.2 Genetic characterization of B. subtilis strain I-5565 The genome of B. subtilis strain I-5565 was cloned using Illumina Paired end 2 * Sequencing was performed using a 150 bp (library prepared using Nextera XT). Genomic sequencing results confirm changes in the nucleotide sequences of seven genes: resE, rpoC, addB, araA, cotY03427, and 03457.

[0071] The effects of these nucleotide changes were then analyzed and the primary structures of the corresponding proteins were confirmed, as disclosed below: A change in the amino acid sequence from serine to asparagine caused by a mutation in the RseE_1 gene at position -1118; A change in the amino acid sequence due to the substitution of aspartic acid by asparagine in the RpoC gene at position −3466; a change in the amino acid sequence from alanine to valine due to a mutation in the AddB gene at position −886; A change in the amino acid sequence due to the substitution of glutamic acid by lysine caused by a mutation in the AraA gene at position -121; a change in amino acid sequence due to a substitution of proline by serine caused by a mutation in the CotY gene at position -154; a change in the amino acid sequence due to a substitution of serine by phenylalanine resulting from a mutation in gene 03427 at position -32; A change in the amino acid sequence due to a substitution of serine by phenylalanine caused by a mutation in gene 03457 at position -92.

[0072] Compared to B. subtilis LBS0, B. subtilis strain I-5565 produces more mycosubtilin and a variety of novel isoforms as shown in Example 2.

[0073] 1.3 pLIP1 Plasmid Construction Protocol First, εpbp-P was used to replace the native mycosubtilin operon promoter in B. subtilis strain I-5565. repUA hybrid plasmid containing -neo-εfenF and rep(R6K) was constructed as follows: The bLIP1 plasmid was digested with the restriction enzymes Xmil (Thermo Scientific; Reference No. FD1484) and Pael (Thermo Scientific; Reference No. FD0604), and a 4.5-kilobase pair (Kb) fragment was extracted and purified according to the protocol of the GeneJET Gel Extraction Kit (Thermo Scientific; Reference No. K0692). A pbp gene fragment (950 base pairs) was amplified from Bacillus subtilis I-5565 using primers containing similar restriction sites. The amplified fragment was extracted and purified. Ligation of the 4.5-kb fragment and the 950-bp PCR fragment was performed according to the protocol of the Rapid DNA Ligation Kit (Thermo Scientific; Reference No. K1422). The ligation product was transformed into ready-to-use competent E. coli JM109 cells (Promega, Reference No. L2005). Clones are selected on Luria-Bertani medium containing agar and 50 μg / mL neomycin / kanamycin. The final plasmid from positive clones is extracted according to the protocol of the GeneJET Plasmid Miniprep Kit (Thermo Scientific; reference number K0503). The final plasmid obtained is called pLIP1 (5.5 kb).

[0074] 1.4 Protocol for obtaining Bacillus subtilis I-5679 The B. subtilis strain I-5565 is then transformed with the pLIP1 plasmid using a native transformation protocol. Clones are selected on Luria-Bertani medium containing agar and 20 μg / mL neomycin / kanamycin. Positive clones are confirmed by colony PCR. The final construct is known as B. subtilis I-5679. Compared to B. subtilis I-5565, the B. subtilis I-5679 strain produces higher concentrations of mycosubtilin and greater amounts of various novel isoforms, as presented in Example 2.

[0075] Example 2: Production and purification of mycosubtilin from a newly engineered Bacillus subtilis strain 2.1 Composition of modified Landy medium 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 、 0.25g / L;K2HPO4, 1g / L;KCl, 0.5g / L;CuSO4·5H2O, 1.6mg / L;FeSO4.7H2O, 1.2mg / L;MnSO4·H2O, 0.4mg / L.

[0076] 2.2 Stock solutions To ensure reproducibility of the medium composition, sterile concentrated solutions were prepared. 10x glucose solution (400 g / L) was sterilized by autoclaving at 121°C for 10 minutes. 10x ammonium sulfate solution (80 g / L); 10x yeast extract solution (40 g / L); 10x inorganic salts solution No. 1 (KHPO 10 g / L; MgSO 2.5 g / L; KCl 5 g / L); and 1000x inorganic salts solution No. 2 (CuSO 5H O 1.6 g / L; FeSO 7H O 1.2 g / L; MnSO H O 0.4 g / L) were acidified with concentrated sulfuric acid to completely dissolve the salts. All of these stock solutions were sterilized by autoclaving at 121°C for 20 minutes.

[0077] 2.3 Producing 1 liter of MOPS-modified Landy medium 150 mL of glucose solution was taken under sterile conditions and poured into a 1 L reagent bottle. 100 mL of yeast extract solution, 100 mL of ammonium sulfate solution, 100 mL of inorganic solution No. 1, and finally 1 mL of inorganic solution No. 2 were added successively to each solution under sterile conditions. 20x MOPS (2 M) buffer was prepared by dissolving 3-N-morpholinopropanesulfonic acid (MOPS) (Sigma, reference number M3183) in water and adjusting the pH to 7.0 with NaOH. The solution was then sterilized with a 0.2 μm porosity filter. To prepare 1 liter of modified Landy's medium buffered to 100 mM with MOPS, 50 mL of 20x MOPS was added to the mixture.

[0078] Adjust the final pH to 7.0 using sterile 6M NaOH solution. Bring the volume to 1 liter with sterile water.

[0079] 2.4 Preparation of inoculum An inoculum was prepared from a strain stock stored at -80°C in 25% glycerol. The glycerol stock was inoculated into Luria-Bertani (LB) broth for 7-8 hours. The culture was then spread onto Luria-Bertani (LB) agar plates containing 20 μg / mL kanamycin (kanamycin sulfate, Sigma, reference number 60615) to obtain single colonies. The plates were incubated overnight at 37°C. A single colony from the overnight plate was then used to generate a P1 preculture, which was then inoculated into a 100 mL Erlenmeyer flask containing 20 μg / mL kanamycin in a final volume of 10 mL of Luria-Bertani (LB) broth. The culture was incubated at 30°C for 10-14 hours with agitation at 200 rpm. After the preculture P1 incubation period, the culture was centrifuged at 2000 g for 10 minutes at 25°C. The cells were washed with physiological water and finally suspended in sterile physiological water. The suspension is now ready for inoculation. In a 1 L Erlenmeyer flask in a final volume of 100 mL of modified Landy's medium, the inoculation OD 600 Generate a P2 preculture at 0.2°C. Incubate the culture at 30°C with agitation at 200 rpm for 7-8 hours.

[0080] 2.5 Cultivation in Erlenmeyer flasks Before inoculation, the P2 pre-culture cells are harvested, washed with physiological water, and then suspended in sterile physiological water. This suspension is used for inoculation. Inoculation OD 600 The pH is 0.1-0.2. The volume of the Erlenmeyer flask is 1 L containing 100 mL of modified Landy's medium. Similarly, ten 1 L Erlenmeyer flasks containing 100 mL of modified Landy's medium are prepared for the high-concentration mycosubtilin culture. The culture is incubated at 30°C with agitation at 200 rpm for up to 72 hours. After the incubation period, the culture supernatant is used for lipopeptide analysis.

[0081] 2.6 Concentration and purification of mycosubtilin The culture is mixed with acetonitrile (VWR Chemical, order number 83639.320) to a final concentration of 50% acetonitrile. The mixture is centrifuged at 8500 g for 10 minutes at 25°C. The cells are separated and the supernatant is used to concentrate mycosubtilin using a laboratory rotary evaporator (Buchi, Rotavapor R300). The sample is concentrated to a final concentration of 3 g / L.

[0082] 2.7 Quantitative analysis of mycosubtilin by UPLC Samples were analyzed using a complete Waters UPLC system (Aqcuity H class plus) (Waters SAS, Guyancourt, France) using a C18 column (2.1 × 150 mm, CORTECS.UPLC.C18.1.6 μm).

[0083] For mycosubtilin analysis, 3 μL of purified sample was injected and compared with a 500 mg / L iturin A standard (11774, Sigma-Aldrich, St. Louis, MO, USA) at a flow rate of 0.4 mL / min. Elution was performed in isocratic mode using a water / acetonitrile / formic acid 60 / 40 / 0.1 (v / v / v) solvent. The retention time and second derivative of the spectrum from 200 to 400 nm for each peak were automatically analyzed using Empower3 software to identify the eluted molecules.

[0084] As shown in FIG. 6, triplicate cultures of each strain were analyzed to compare mycosubtilin production among the three strains (LBSO, I-5565, and I-5679).

[0085] As shown in Figure 6, compared to its parent strain LBS0, mycosbutyrin production increased 6-fold in strain I-5565, while mycosbutyrin production in strain I-5679 (in which the native mycosbutyrin promoter is replaced by the constitutive prepU promoter) was 28.5-fold and 2.5-fold higher than LBS0 and I-5565, respectively. Comparing these results with Bacillus subtilis strain BBG125 (WO 2013 / 050700), which was previously obtained by suppressing surfactin production and inserting the PrepU promoter in front of the mycosbutyrin gene, strain I-5565 produced 5-fold more than strain BBG125, and strain I-5679 produced 12-fold more than strain BBG125. In conclusion, disruption of the resE, rpoC, addB, araA, cotY03427, and 03457 genes in strain I-5565 unexpectedly resulted in increased production far beyond previous results. More importantly, the combination of these seven gene disruptions with PrepU promoter insertion in strain I-5679 further increased mycosubtilin production by 2.5-fold compared to strain I-5565.

[0086] 2.8 Preparative HPLC analysis of mycosubtilin The isoforms were separated using a PuriFlash-interchim preparative HPLC column. Purification was performed on a US5C18HQ-250 / 300 interchim column (UPTISPHERE STRATEGY C18-HQ 5um 250x30mm HPLC column).

[0087] The parameters used for preparative HPLC were as follows: -Injection volume: 10 mL; - Mobile phase: water / acetonitrile with 0.1 TFA; -Flow rate: 35mL / min; -Fractional volume: 14 mL, threshold was 3 mAU; -Detector: 214nm.

[0088] The program used for isoform separation is shown in Table 1 below.

[0089] [Table 1]

[0090] The different peaks representing the different mycosubtyrin isoforms are shown in Figure 2. Each peak was collected in several 14 mL tubes. The number of tubes depended on the volume of the peak. A total of five experiments were performed (10 mL of sample per experiment). Each isoform sample was then analyzed by mass spectrometry (Q-tof analysis) as disclosed in Example 3. Antifungal analysis was performed on the different isoforms as disclosed in Example 4. Cytotoxicity analysis was also performed on the different isoforms as disclosed in Example 5.

[0091] Example 3: Qualitative analysis of mycosubtilin produced by the newly engineered Bacillus subtilis strain The parameters used for LC / MS-Qtof analysis were as follows: UPLC experiments were performed using an Agilent 1290 Infinity II 2D-LC system. A C18 Acquity UPLC BEH column (2.1 × 50 mm × 1.7 μm; Waters) was used at a flow rate of 0.3 mL / min and a temperature of 40 °C. The injection volume was 20 μL, and the diode array detector (DAD) scanned the wavelength spectrum from 190 to 600 nm. Agilent OpenLab CDS ChemStation software and Agilent 1290 Infinity 2D-LC acquisition software were used for LC analysis.

[0092] The parameters used for UPLC were as follows: -Injection volume: 10 μL; - Column temperature: 40°C; - Mobile phase: Water / acetonitrile with 0.1% formic acid.

[0093] The program used for isoform separation is shown in Table 2 below.

[0094] [Table 2]

[0095] LC / MS-Qtof analysis was performed using a 1290 Infinity II coupled to a Jet Stream ESI-Q-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; fragmentor voltage: 175 V; skimmer voltage: 65 V; ctopole RF: 750 V. Accurate mass spectra were recorded in the m / z range of 100–1200. Briefly, 10 μL of sample was injected and separated using a C18 Acquity UPLC BEH (2.1 × 50 mm × 1.7 μm; Waters) with 0.1% formic acid (FA) as solvent A and ACN + 0.1% FA as solvent B. The gradient started at 0% solvent B and increased to 30% within 5 min. Data were processed using MassHunter Qualitative Analysis software (Agilent Technologies). Table 3 shows the novel mycosubtilin C16 and C17 isoforms compared to the conventional C16 and C17 isoforms obtained from the LC / MS-Qtof analysis disclosed above.

[0096] [Table 3]

[0097] In addition to the conventional C16 isoform of mycosubtilin (C16-Myco), the C17 isoform of mycosubtilin (C17-Myco), and the C18 isoform of mycosubtilin (peaks 3, 5, 6, 7, and 8), three novel isoforms were detected. Peaks 1, 2, and 4, corresponding to C16-Gln1 and C17-Gln1, are novel mycosubtilin isoforms. Peak 1 has an asparagine substitution at the third position with glutamine, while peaks 2 and 4 have an asparagine substitution at the third position with glutamine.

[0098] [Table 4]

[0099] In addition to the eight major isoforms purified by preparative HPLC (shown in Table 3), several other minor isoforms were isolated by UPLC from the supernatants of strains I-5565 and I-5679. These minor isoforms were then identified and characterized using the sensitive MS-Qtof method previously disclosed herein. A total of 20 isoforms were identified in the supernatants of the novel B. subtilis strains, as shown in Table 4. Among them, 13 novel minor isoforms were discovered and are listed here: Gln7-C16; Gln1, Gln3-C16; Gln3, Gln7-C16; Gln1-C16; Gln1, Gln3, Gln7-C16; Gln7-C17; Gln1, Gln3-C17; Gln3, Gln7-C17; Gln1-C17; Gln3-C18; Gln7-C18; Gln1-C18, and A-C19. In addition to all of the novel isoforms discovered in this study, it was also found that the adenylation domain of mycosubtilin synthase can activate both Gln and Asn at positions 1, 3, and 7 of the peptide ring in overproducing mutants. The novel isoforms will be used in activity and cytotoxicity experiments to determine their effectiveness. The isoforms are dissolved in DMSO and used for activity studies, the results of which are disclosed in Examples 4 and 5.

[0100] Example 4: Antifungal activity of different mycosubtyrin isoforms Three different fungal strains were tested for their antifungal activity: B. cinerea, Aspergillus sp., and Z. tritici. In addition to two conventional isoforms, three novel isoforms were tested for their antifungal properties. A range of concentrations (μg ai / mL or ppm) of 0; 0.0037; 0.0146; 0.0586; 0.2344; 0.9375; 3.75, and 15 were used to determine the activity coefficient. Based on this concentration range, the EC 50 (concentration that reduces the growth of fungal strains by 50%) and EC 95 (the concentration that reduces the growth of the pathogen by 95%) was calculated and presented as disclosed in Table 5.

[0101] 4.1 Antifungal activity against B. cinerea A-C16 EC 50 The EC value for A-C16 was 0.933 ppm, while that for Gln1-C16 was 1.048 ppm, indicating that a 10% lower concentration of the former was required to have the same activity as the latter. 95 The activity of the novel isoform (Gln1-C16) was 1.457 ppm, compared with 1.282 ppm for Gln1-C16, indicating that a 17% higher concentration of the former was required to have the same activity as the latter. This novel isoform (Gln1-C16) is effective against this fungal strain.

[0102] The other two isoforms tested, Gln3-C16 and Gln1-C17, proved effective against this fungal strain, but at higher concentrations.

[0103] 4.2 Antifungal activity against Aspergillus spp.

[0104] A-C17 EC 50The EC value for A-C17 was 1.191 ppm, while that for Gln1-C17 was 1.019 ppm, indicating that a 14% higher concentration of the former was required to have the same activity as the latter. 95 The activity of Gln1-C17 was 1.905 ppm, compared with 1.290 ppm for Gln1-C17, indicating that a 32% higher concentration of the former was required to have the same activity as the latter. This novel isoform proved to be effective against this fungal strain.

[0105] The other two isoforms tested, Gln1-C16 and Gln3-C16, proved effective against this fungal strain, but at higher concentrations.

[0106] 4.3 Antifungal activity against Z. tritici A-C16 EC 50 The EC value for A-C16 was 4.258 ppm, compared to 1.906 ppm for Gln1-C16, indicating that a 55% higher concentration of the former was required to have the same activity as the latter. 95 The activity of Gln3-C16 was 26.106 ppm, compared to 17.874 ppm for Gln3-C16, indicating that the former required a 32% higher concentration to have the same activity as the latter. These isoforms proved effective against this fungal strain.

[0107] Other novel Gln1-C17 isoforms are effective against this fungal strain, but at higher concentrations.

[0108] [Table 5]

[0109] Example 5: Cytotoxicity test Cytotoxicity tests are performed using Vero cells and the CyQUANT™ XTT Cell Viability Assay Kit (supplied by Invitrogen™).

[0110] The sample was dissolved in 100% DMSO at a concentration of 20-50 g / L. The final concentration in DMEM medium was 40 μg / L. Cells were prepared in flasks containing DMEM cell culture medium containing 10% FBS as follows: A 175 mL flask containing 20-30 million cells was added with 25 mL of medium and 2 mL of trypsin. The medium was then discarded as the cells had adhered to the flask wall. The mixture was rinsed twice with PBS, and then trypsin was added to detach the cells from the flask wall. The mixture was incubated at 37°C for 5 minutes. To inactivate the trypsin, 6 mL of trypsin was added to the medium (twice the original volume of trypsin in the medium).

[0111] The medium was centrifuged at 300 g for 5 minutes in a 50 mL flask. The medium was centrifuged at 300 g for 5 minutes. The medium was discarded and the cells were resuspended. 100 μL of cells were added to 100 μL of dye, and then 12 μL was used for counting. Viability was compared to control cells without any treatment.

[0112] Tests are performed in 96-well plates. The following concentration ranges (mg / L) are used to determine the toxicity limit for each isoform: 0.14; 0.28; 0.56; 1.16; 2.25; 4.5.

[0113] The results shown in [[Fig. 7]] indicate that the percentage of viable Vero cells is significantly higher in the presence of the novel C16 and C17 isoforms compared to the normal A-C16 and A-C17 isoforms. From these data, the EC50 can be determined. The EC50 of Gln3-C16- exceeds 4.5 mg / L, the EC50 of Gln1-C16 is 4.5 mg / L, and the EC50 of A-C16 is 1.7 mg / L, which is at least 2.64-fold lower than that of the novel C16 isoform of mycosubtilin. The cytotoxicity profile of the C16 isoforms is Gln3-C16 < Gln1-C16 < A-C16. The C16 isoforms, namely Gln3-C16 and Gln1-C16, are significantly less toxic than the conventional A-C16 isoform. The same profile is observed for the mycosubtilin-based C17 isoforms. The EC50 of Gln3-C17 exceeds 4.5 mg / L, the EC50 of Gln1-C17 is approximately 3.4 mg / L, and the EC50 of A-C17 is approximately 1.7 mg / L, which is at least 2-fold lower than that of the novel C17 isoform of mycosubtilin. For the C17 isoforms, the cytotoxicity profile is Gln3-C17 < Gln1-C17 < A-C17.

[0114]

Table 6

Brief Description of the Drawings

[0115] [Figure 1] Classical formula of mycosubtilin A-C17 (n = 13). [Figure 2] Schematic diagram of preparative HPLC chromatogram. Peaks P3, P5, P6, P7, and P8 are classical mycosubtilin isoforms, while peaks P1, P2, and P4 are novel mycosubtilin isoforms. Modified amino acids in the peptide ring are labeled. [Figure 3] Formula of mycosubtilin isoform Gln3-C16. [Figure 4] Expression of the novel mycosubtilin isoform Gln1-C16. [Figure 5] Expression of the novel mycosubtilin isoform Gln1-C17. [Figure 6] Quantitative analysis of mycosubtilin produced by a novel engineered Bacillus subtilis strain. [Figure 7] Cell viability assay of Vero cells treated with different mycosubtyrin isoforms at different concentrations of each isoform.

Claims

1. A genetically modified Bacillus strain, characterized in that it is a Bacillus subtilis strain deposited on July 30, 2020 at the National Collection of Microbial Cultures (CNCM) of the Pasteur Institute (Paris, France) under the number CNCM I-5565.

2. 2. The strain according to claim 1, characterized in that it is a Bacillus subtilis strain modified by replacing the endogenous promoter with the constitutive PrepU promoter and deposited on May 5, 2021 at the Collection National des Cultures de Microorganisms (CNCM) of the Pasteur Institute (Paris, France) under the number CNCM I-5679.

3. A novel mycosubtilin isoform selected from the Gln1-C16 and Gln1-C17 isoforms.

4. A composition comprising at least one mycosubtilin selected from the Gln1-C16 and Gln1-C17 isoforms.

5. Use of at least one mycosubtilin selected from the Gln1-C16 and Gln1-C17 isoforms as an antifungal agent in the agricultural, food, chemical, detergent, petroleum and environmental industries.

6. Mycosubtilins selected from the Gln1-C16 and Gln1-C17 isoforms for use as antifungal agents in the cosmetic, medical and pharmaceutical fields.

7. 6. The use according to claim 5, wherein at least one mycosubtilin corresponding to the Gln1-C16 isoform is used as an antifungal agent against B. cinerea strains.

8. 6. The use according to claim 5, wherein at least one mycosubtilin corresponding to the Gln1-C17 isoform is used as an antifungal agent against Aspergillus strains.

9. 6. The use according to claim 5, wherein at least one mycosubtilin corresponding to the Gln1-C16 isoform is used as an antifungal agent against Z. tritici strains.

10. Use of at least one mycosubtilin selected from the Gln1-C16 and Gln1-C17 isoforms and the Gln3-C16 isoform as an antifungal agent against Z. tritici strains in the agricultural, food, chemical, detergent, petroleum, and environmental industries.

11. A method for producing at least one novel mycosubtyrin isoform selected from the Gln1-C16 and Gln1-C17 isoforms, comprising culturing the strain of any one of claims 1 or 2.

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