EXOPOLYSACCHARIDE-PRODUCING MICROORGANISM AND USE THEREOF

Abstract

The present invention relates to genes involved in exopolysaccharide production in Firmicutes. The invention provides mutations in those genes and corresponding mutant proteins to increase or stabilize exopolysaccharide production or prevent autolysis of the exopolysaccharide, particularly over the course of fermentation. The invention also provides suitable methods for fermenting the microorganisms and exopolysaccharides, and suitable uses of the mutant genes and mutant proteins of the microorganisms and exopolysaccharides.

Description

[Technical field]

[0001] The present invention relates to genes involved in exopolysaccharide production in Firmicutes. The invention provides mutations in those genes and corresponding mutant proteins to increase or stabilize exopolysaccharide production or prevent exopolysaccharide degradation, particularly over the course of fermentation. The invention also provides suitable methods for fermenting the microorganisms and exopolysaccharides, and suitable uses of the mutant genes and mutant proteins of the microorganisms and exopolysaccharides. [Background technology]

[0002] Microbial exopolysaccharides serve multiple protective functions for the producing cells, e.g., protection against desiccation, toxins and abiotic stresses, scavenging nutrients and extracellular enzymes, acting as an adhesive for the cells to surfaces, and maintaining structural purpose by creating a growth-promoting microenvironment, e.g., as found in biofilms.

[0003] Therefore, exopolysaccharides are a useful tool for improving the survival and colonization of beneficial microorganisms on surfaces, for example in the treatment of plants. The production of exopolysaccharides prevents or reduces the washing away of beneficial microorganisms from leaves, for example by rain or wind, and improves the colonization of leaves, shoots and roots. This is advantageous, for example, when applying Paenibacilli, which is beneficial to plants, and the colonization of the plant surface by Paenibacilli significantly protects the plant from fungal pathogens and improves nutrient intake by the roots. As an example, biofilm polysaccharides of P. polymyxa A26 can antagonize Fusarium graminearum (Timmusk S, Copolovici D, Copolovici L, Teder T, Nevo E, Behers L. Paenibacillus polymyxa biofilm polysaccharides antagonise Fusarium graminearum. Sci Rep. 2019 Jan 24;9(1):662. doi:10.1038 / s41598-018-37718-w). Biofilm formation of some Paenibacillus species can effectively help them colonize plant roots and help the host plants adapt and survive in harsh conditions.

[0004] In addition, exopolysaccharides and the microorganisms which produce them can increase soil fertility and improve yield consistency, for example by absorbing water and providing ammonia to the plant. In this regard, exopolysaccharides can help establish a microaerobic environment around the bacteria, thus allowing the activity of the oxygen-sensitive enzyme nitrogenase, which is necessary for the fixation of atmospheric nitrogen by the microorganism.

[0005] Other uses of exopolysaccharides rely on their ability to modify the rheological properties of liquids, in particular to increase the viscosity by being simultaneously biodegradable. This is useful, for example, in underground oil and gas extraction, in the preparation of cosmetics, food, feed and medicines. As an example, exopolysaccharides are applied as scaffolds or matrices in tissue engineering, drug delivery and wound dressings (Nwodo UU, Green E, Okoh AI. Bacterial exopolysaccharides: functionality and prospects. Int J Mol Sci. 2012; 13(11): 14002-14015. Published 2012 Oct 30. doi: 10.3390 / ijms131114002). Furthermore, exopolysaccharides from Paenibacillus sp. have been applied as antitumor, antioxidant or flocculant agents (He X, Li Q, Wang N, Chen S. Effects of an EPS Biosynthesis Gene Cluster of Paenibacillus polymyxa WLY78 on Biofilm Formation and Nitrogen Fixation under Aerobic Conditions. Microorganisms. 2021 Jan 30;9(2):289. doi:10.3390 / microorganisms9020289). This indicates that the biotechnological and industrial application fields of microbial exopolysaccharides are broad. Exopolysaccharides are built up from sugar units and can consist of either only one type of monomer (e.g., fructose in polysaccharide levan) or different monomers. Examples of polysaccharides include, but are not limited to, glucan, fructan, curdlan, gellan, xanthan, emulsan, dextran, cellulose, aliginate, colonic acid, curdlan, dextran, diutan, levan, succinoglycan, welan, and combinations thereof, among others.Similarly, production materials and methods for producing exopolysaccharides are described: WO 2015118516 and WO 2016044768 describe soil inoculation with exopolysaccharides, in particular for improving soil fertility. WO 2020163251 describes the application of exopolysaccharides to improve yield consistency. WO 2014176061 describes the application of exopolysaccharides for the treatment of subterranean formations, in particular in the extraction of oil and gas. WO 2014160350 also describes the treatment of wastewater with exopolysaccharides. Materials and methods for precipitating exopolysaccharides and trapping beneficial plant microorganisms therein are described in WO 2017151742. The genes and metabolism involved in exopolysaccharide production in Paenibacillus polymyxa have been studied in detail in Ruetering et al., Tailor-made exopolysaccharides - CRISPR-Cas9 mediated genome editing in Paenibacillus polymyxa, Synthetic Biology 2017, doi:10.1093 / synbio / ysx007, which is incorporated herein in its entirety, with particular reference to the composition of the exopolysaccharide sugar monomers and the genes involved in exopolysaccharide synthesis in Paenibacillus polymyxa.

[0006] Apart from their benefits, exopolysaccharides are a major cause of concern in industrial liquid phase fermentation. The increased viscosity of the fermentation medium caused by exopolysaccharides can reduce the oxygen and nutrient transfer rate, thereby reducing the fermentation yield and requiring higher energy to agitate the fermentation medium. Therefore, attempts are typically made to reduce or eliminate exopolysaccharide production in microorganisms.

[0007] Many microorganisms produce exopolysaccharides during fermentation, mainly during logarithmic growth. During the later stages of fermentation, especially at the end of logarithmic growth, the exopolysaccharides are degraded and consumed as nutrients, so that part of the metabolic energy consumed for exopolysaccharide production is recovered. Thus, the maximum exopolysaccharide content of the fermentation broth often precedes the maximum content of the target fermentation product. For example, in the fermentation of Paenibacillus, the maximum viscosity and therefore the maximum exopolysaccharide content is reached before the maximum content of Fusaricidin A, B or D is reached, so that the exopolysaccharide content must be exchanged for the Fusaricidin content. This is clearly undesirable, especially in plant protection applications. Furthermore, during storage of the fermentation broth after recovery, the exopolysaccharides were often degraded from the cells or residual enzymes within hours or days. Thus, downstream processing is often time-critical to obtain high levels of exopolysaccharides.

[0008] It was therefore an object of the present invention to alleviate or mitigate the aforementioned drawbacks of the prior art. In particular, it was an object of the present invention to provide modified nucleic acids comprising alleles which allow the corresponding microorganisms to produce a higher exopolysaccharide content in the fermentation broth compared to the corresponding wild type. Furthermore, it was an object of the present invention to provide such nucleic acids, alleles and microorganisms which allow the exopolysaccharide content of the fermenter content in the late fermentation phase to be increased compared to the corresponding wild type, preferably when a maximum content of at least one target fermentation product, preferably antibacterial substance, produced by the microorganism in addition to the exopolysaccharides is available. Summary of the Invention [Means for solving the problem]

[0009] The present invention provides a microorganism comprising a mutant degU gene and / or a mutant degS gene, and optionally further a mutant spo0A gene, which microorganism exhibits increased and / or stabilized exopolysaccharide production and / or reduced exopolysaccharide degradation.

[0010] The present invention also provides a method for increasing or stabilizing exopolysaccharide production in a microorganism, or for reducing or preventing exopolysaccharide degradation in a microorganism, comprising the steps of: a) a mutant degU gene, - the degU gene encodes a DegU protein that has reduced DNA-binding activity and / or lacks a functional DNA-binding domain, and / or - the degU gene encodes the DegU protein, the mutation of which, for each of the options aa) and ab), in descending order of priority, aa) Q218*, Q218K, Q218N, Q218D, Q218R, and / or ab) a mutated degU gene comprising or consisting of one or more of: D223*, D223*+M220N, D223*+M220N+E221G, D223*+M220N+V222G, D223*+M220N+E221G+V222G, D223*+M220D, D223*+M220E, D223*+M220H, D223*+M220F, D223*+M220W, D223*+M220S, D223*+M220A; b) a mutant degS gene, - the degS gene encodes a DegS protein that lacks a functional single binding domain, a functional phosphoacceptor domain and / or a functional ATPase domain, and / or - a mutant degS gene, wherein the degS gene encodes the DegS protein, the mutations of which comprise or consist of L99F, L99C, L99D, L99E, L99G, L99H, L99K, L99N, L99P, L99Q, L99R, L99S, L99W or L99Y; c) a mutant spo0A gene, ca) the mutation is located in the DNA binding domain or in the receiver domain and leads to reduced or abolished phosphorylation and / or reduced or abolished dimerization of the Spo0A protein; and / or cb) The mutation is - A257V, more preferably A257S, - I161R, more preferably I161L, - providing to the microorganism one or more of the following mutant spoOA genes consisting of or comprising, in order of decreasing priority: A257S+I161I, A257A+I161L, A257V+I161I, A257S+I161F or A257A+I161R.

[0011] The present invention also provides a) a mutant degU gene, - the degU gene encodes a DegU protein that has reduced DNA-binding activity and / or lacks a functional DNA-binding domain, and / or - the degU gene encodes the DegU protein, the mutation of which, for each of the options aa) and ab), in descending order of priority, aa) Q218*, Q218K, Q218N, Q218D, Q218R, and / or ab) a mutated degU gene comprising or consisting of one or more of: D223*, D223*+M220N, D223*+M220N+E221G, D223*+M220N+V222G, D223*+M220N+E221G+V222G, D223*+M220D, D223*+M220E, D223*+M220H, D223*+M220F, D223*+M220W, D223*+M220S, D223*+M220A; b) a mutant degS gene, - the degS gene encodes a DegS protein that lacks a functional single binding domain, a functional phosphoacceptor domain and / or a functional ATPase domain, and / or - a mutant degS gene, wherein the degS gene encodes the DegS protein, the mutations of which comprise or consist of L99F, L99C, L99D, L99E, L99G, L99H, L99K, L99N, L99P, L99Q, L99R, L99S, L99W or L99Y; c) a mutant spo0A gene, ca) the mutation is located in the DNA binding domain or in the receiver domain and leads to reduced or abolished phosphorylation and / or reduced or abolished dimerization of the Spo0A protein; and / or cb) The mutation is - A257V, more preferably A257S, - I161R, more preferably I161L, - providing an expression vector comprising an expression cassette for expressing one or more of the mutant spoOA genes consisting of or including, in order of decreasing priority, A257S+I161I, A257A+I161L, A257V+I161I, A257S+I161F or A257A+I161R;

[0012] Furthermore, the present invention provides a method for improving plant health, comprising application of a microorganism comprising a mutant degU gene and / or a mutant degS gene, and optionally further comprising a mutant spoOA gene, the microorganism comprising: a) plant material and / or b) increased and / or stabilized exopolysaccharide production and / or reduced exopolysaccharide degradation on a plant cultivation substrate.

[0013] The present invention also provides a method for producing a semiconductor device comprising the steps of: i) growing a microorganism comprising a mutant degU gene and / or a mutant degS gene, and optionally further a mutant spoOA gene, which microorganism exhibits increased and / or stabilized exopolysaccharide production and / or reduced exopolysaccharide degradation; and ii) optionally separating the microorganism from the exopolysaccharide.

[0014] The present invention also provides Use of a microorganism comprising a mutant degU gene and / or a mutant degS gene, and optionally further a mutant spoOA gene, which microorganism exhibits increased and / or stabilized exopolysaccharide production and / or reduced exopolysaccharide degradation. or a degU gene or protein, - the degU gene encodes a DegU protein that has reduced DNA-binding activity and / or lacks a functional DNA-binding domain, and / or - the degU gene encodes the DegU protein, the mutation of which, for each of the options aa) and ab), in descending order of priority, aa) Q218*, Q218K, Q218N, Q218D, Q218R, and / or ab) a degU gene or protein comprising or consisting of one or more of: D223*, D223*+M220N, D223*+M220N+E221G, D223*+M220N+V222G, D223*+M220N+E221G+V222G, D223*+M220D, D223*+M220E, D223*+M220H, D223*+M220F, D223*+M220W, D223*+M220S, D223*+M220A; and / or a degS gene or a DegS protein, - the degS gene encodes a DegS protein that lacks a functional single binding domain, a functional phosphoacceptor domain and / or a functional ATPase domain, and / or - a degS gene or a DegS protein, the degS gene encoding the DegS protein, the mutations of which include or consist of L99F, L99C, L99D, L99E, L99G, L99H, L99K, L99N, L99P, L99Q, L99R, L99S, L99W or L99Y; and / or a spo0A gene or a Spo0A protein, ca) the mutation is located in the DNA binding domain or in the receiver domain and leads to reduced or abolished phosphorylation and / or reduced or abolished dimerization of the Spo0A protein; and / or cb) The mutation is - A257V, more preferably A257S, - I161R, more preferably I161L, - use of a spo0A gene or Spo0A protein consisting of or comprising any of the following in decreasing order of preference: A257S+I161I, A257A+I161L, A257V+I161I, A257S+I161F or A257A+I161R, One of the following: - production of exopolysaccharide compositions, - treatment of plants, plant leaves, plant roots and / or plant seeds, - inoculation of soil, preferably to increase soil fertility; - Improved yield consistency, - Treatment of underground layers; - Wastewater treatment, - Preparation of pharmaceutical or cosmetic carriers, - preparation of pharmaceutical or cosmetic compositions, - preparation of skin hydrating compositions, - Preparation of food or feed additives, - preparation of antitumor agents; - Preparation of antioxidants, - Preparation of a flocculant, for use therein.

[0015] The present invention also provides a method for producing a semiconductor device comprising the steps of: a) a mutant degU gene, - the degU gene encodes a DegU protein that has reduced DNA-binding activity and / or lacks a functional DNA-binding domain, and / or - the degU gene encodes the DegU protein, the mutation of which, for each of the options aa) and ab), in descending order of priority, aa) Q218*, Q218K, Q218N, Q218D, Q218R, and / or ab) a mutated degU gene comprising or consisting of one or more of: D223*, D223*+M220N, D223*+M220N+E221G, D223*+M220N+V222G, D223*+M220N+E221G+V222G, D223*+M220D, D223*+M220E, D223*+M220H, D223*+M220F, D223*+M220W, D223*+M220S, D223*+M220A; b) a mutant degS gene, - the degS gene encodes a DegS protein that lacks a functional single binding domain, a functional phospoacceptor domain and / or a functional ATPase domain, and / or - a mutant degS gene, wherein the degS gene encodes the DegS protein, the mutations of which comprise or consist of L99F, L99C, L99D, L99E, L99G, L99H, L99K, L99N, L99P, L99Q, L99R, L99S, L99W or L99Y; c) a mutant spo0A gene, ca) the mutation is located in the DNA binding domain or in the receiver domain and leads to reduced or abolished phosphorylation and / or reduced or abolished dimerization of the Spo0A protein; and / or cb) The mutation is - A257V, more preferably A257S, - I161R, more preferably I161L, - the use of one or more of the following mutant spoOA genes, consisting of or comprising, in order of decreasing priority, A257S+I161I, A257A+I161L, A257V+I161I, A257S+I161F or A257A+I161R, The present invention provides a use for increasing or stabilizing exopolysaccharide production or preventing exopolysaccharide degradation of a microorganism selected from any of the taxonomic ranks of the phylum Firmicutes, the class Bacilli, the class Clostridia, or the class Negativicutes. [Brief description of the drawings]

[0016] [Figure 1]Figure 2 shows the evolution of the fermentation broth viscosity measured according to example 3. The fermentation broths were obtained from wild-type Paenibacillus polymyxa DSM 365 and its mutants during fermentation in a 21 l bioreactor as described in example 2. In the graphs, the viscosity profile of wild-type DSM 365 was always kept as a reference for comparison with the respective mutant strain. For the wild-type strain, the viscosity reaches a maximum of approximately 120 [mPa·s] at 100 / s after 13 hours of fermentation start, then decreases to approximately 50 [mPa·s] at 100 / s after 24 hours of fermentation start. After that, the viscosity does not change significantly. Fermentation broths of both DSM365 DegU mutants (strain DegU Q218* and strain DegU D223*+M220N+E221G+V222G) reach a viscosity of approximately 120 [mPa·s] at 100 / s 16 and 24 h after the start of fermentation, respectively, and the viscosity increases to approximately 160 [mPa·s] at 100 / s at 36 and 28 h, respectively. Thereafter, both broth viscosities remain above 120 [mPa·s] at 100 / s. The fermentation broth viscosity of the DegS L99F mutant of DSM365 reaches approximately 140 [mPa·s] at 100 / s 12 h after the start of fermentation, peaks at approximately 200 [mPa·s] at 100 / s, and remains above approximately 130 [mPa·s] at 100 / s. The DSM365 strain carrying both the degU Q218* and degS L99F mutations reached a fermentation broth viscosity of approximately 120 [mPa s] at 100 / s approximately 14 hours after the start of fermentation, and the fermentation broth viscosity continued to increase to approximately 180 [mPa s] at 100 / s 28 hours after the start of viscosity. The DSM365 strain carrying only the spo0A A257V mutation reached a fermentation broth viscosity of approximately 120 [mPa s] at 100 / s 22 hours after the start of fermentation, and increased the fermentation broth viscosity to a peak of 140 [mPa s] at 100 / s 32 hours after the start of fermentation. The triple mutant DSM365 degU Q218*+degS L99F+spo0A A257V reached a fermentation broth viscosity of approximately 120 [mPa·s] at 100 / s 16 h after the start of fermentation, with a peak fermentation broth viscosity of approximately 180 [mPa·s] at 100 / s, followed by a fermentation broth viscosity of at least approximately 150 [mPa·s] at 100 / s. [Diagram 2] FIG. 2 shows an exemplary comparison of the carbon transfer rate (CTR, measured by mass spectrometry) and broth viscosity (measured according to Example 3, same data as in FIG. 1) of the wild-type strain Paenibacillus DSM365. [Figure 3-1] 1 shows a sequence alignment of SEQ ID NO:2 with the sequence from Uniprot entry A0A074LBY4_PAEPO for the DegS protein. Numbers are indicated according to position in the Uniprot entry A0A074LBY4_PAEPO sequence. The number of asterisks above each amino acid in the A0A074LBY4_PAEPO sequence indicates the degree of conservation, with more stars indicating stronger conservation. The amino acids shown below each amino acid in SEQ ID NO:2 indicate possible substitutions that are allowed at each position, with "-" indicating a gap (a deletion relative to the A0A074LBY4_PAEPO sequence). The possible substitutions are listed in order of their priority, with more preferred substitutions shown closer to their respective positions in SEQ ID NO:2. [Figure 3-2] Continued from Figure 3. [Figure 3-3] Continued from Figure 3. [Diagram 3-4] Continued from Figure 3. [Figure 4-1] 1 shows a sequence alignment of SEQ ID NO:1 with the sequence from Uniprot entry E3EBP5_PAEPS for the DegU protein. Numbers are indicated according to position in the Uniprot entry E3EBP5_PAEPS sequence. The number of asterisks above each amino acid in the E3EBP5_PAEPS sequence indicates the degree of conservation, with more stars indicating stronger conservation. The amino acids shown below each amino acid in SEQ ID NO:1 indicate possible substitutions that are allowed at each position, with "-" indicating a gap (a deletion relative to the E3EBP5_PAEPS sequence). The possible substitutions are listed in order of their preference, with more preferred substitutions shown closer to their respective positions in SEQ ID NO:1. [Figure 4-2] Continued from Figure 4. [Figure 4-3] Continued from Figure 4. [Figure 5-1]1 shows a sequence alignment of SPO0A protein with SEQ ID NO:3 and the sequence from Uniprot entry A0A074LZY6_PAEPO. Numbers are shown according to the position in the Uniprot entry A0A074LZY6_PAEPO sequence. The number of asterisks above each amino acid in the A0A074LZY6_PAEPO sequence indicates the degree of conservation, with more stars indicating stronger conservation. The amino acids shown below each amino acid in SEQ ID NO:3 indicate possible substitutions that are allowed at each position, and "-" indicates a gap (a deletion relative to the A0A074LZY6_PAEPO sequence). The possible substitutions are listed in order of their priority, with more preferred substitutions shown closer to their respective positions in SEQ ID NO:3. [Figure 5-2] Continued from Figure 5. [Figure 5-3] Continued from Figure 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] [Table 1]

[0018] Detailed Description of the Invention The technical teachings of the present invention are expressed herein using linguistic means, in particular by using scientific and technical terms. However, those skilled in the art will understand that the linguistic means, however detailed and precise they may be, may only approximate the complete content of the technical teachings, if only because there are multiple ways of expressing the teachings, each of which is necessarily impossible to fully express all conceptual connections, since each expression must necessarily be complete. With this in mind, those skilled in the art will understand that the subject matter of the present invention is the sum of the individual technical concepts shown herein or necessarily expressed in a pulse-prototype manner by the inherent constraints of the present specification. In particular, those skilled in the art will understand that the expression of the individual technical concepts is made herein as a shorthand for detailing each possible combination of the concepts as far as technically practical, so that, for example, the disclosure of three concepts or embodiments A, B and C is a shorthand for the concepts A+B, A+C, B+C, A+B+C. In particular, alternatives regarding features are described herein with reference to a list that aggregates the alternatives or examples. Unless otherwise stated, the invention described herein includes any combination of such alternatives. Selection of more or less preferred elements from such lists is part of the invention and is subject to the preference of one skilled in the art to realize to the minimum extent the advantages conveyed by each feature. Such multiple combined embodiments represent appropriately preferred forms of the invention.

[0019] To the extent that reference is made herein to a database entry, e.g., a Uniprot entry, the entry was published on May 1, 2021 at 10:00 CET. This also applies to the sequences published under the corresponding database entry identifier.

[0020] Nucleic acids and amino acids are abbreviated using standard one- or three-letter abbreviations. Deletions are indicated with "-" and truncations with "*". Amino acid modifications are designated by the position of the modification in the respective parental sequences.

[0021] As used herein, singular terms and singular forms such as "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, use of the term "nucleic acid" actually includes, optionally, many copies of that nucleic acid molecule. Similarly, the term "probe" encompasses, optionally (and typically), many similar or identical probe molecules. It is also understood that, as used herein, the word "comprising" or variations such as "comprises" or "comprising" include the recited elements, integers, or steps or groups of elements, integers, or steps, but do not exclude any other elements, integers, or steps or groups of elements, integers, or steps.

[0022] As used herein, the term "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted as an alternative ("or"). The term "comprising" also encompasses the term "consisting of."

[0023] The term "about" when used in reference to a measurable value, such as the magnitude of mass, dose, time, temperature, sequence identity, and the like, refers to ±0.1%, 0.25%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or even 20% variation of the particular value and that particular value. Thus, when a given composition is described as comprising "about 50% X," it will be understood that in some embodiments the composition comprises 50% X, while in other embodiments the composition may comprise 40%-60% X (i.e., 50% ±10%).

[0024] As used herein, the term "gene" refers to a gene product, i.e. biochemical information that, when embodied in a nucleic acid, can be transcribed into a further nucleic acid, preferably RNA, and can also be translated, preferably, into a peptide or polypeptide. The term is therefore also used to indicate sections of nucleic acid that are analogous to said information, and the sequence of such a nucleic acid (also referred to herein as a "gene sequence").

[0025] Also, as used herein, the term "allele" refers to a variant of a gene characterized by one or more specific differences in the gene sequence compared to the wild-type gene sequence, regardless of the presence of other sequence differences. The alleles or nucleotide sequence variants of the present invention have at least, in order of increasing preference, 30%, 40%, 50%, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%-84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide "sequence identity" to the nucleotide sequence of the wild-type gene. Similarly, when "allele" refers to biochemical information for expressing a peptide or polypeptide, the nucleic acid sequence of each of the alleles has at least, in ascending order of preference, 30%, 40%, 50%, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%-84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid "sequence identity" to the respective wild-type peptide or polypeptide.

[0026] Mutations or modifications of amino acid or nucleic acid sequences can be either substitutions, deletions or insertions. The terms "mutation" or "modification" also encompass any combination of these. Below, all three specific methods of mutation are described in more detail with reference to amino acid sequence mutations. The corresponding teachings apply to nucleic acid sequences, whereby "amino acids" are replaced by "nucleotides". Mutations can be introduced into the nucleotide sequence of a gene by random or directed mutagenesis techniques. Random mutagenesis techniques include, for example, UV irradiation and exposure to chemicals, such as EMS. Directed mutagenesis techniques include primer extension, meganucleases, zinc finger nucleases and CRISPR-type template-directed mutagenesis.

[0027] "Substitutions" are described by the original amino acid followed by the number of the position in the amino acid sequence, followed by the substituted amino acid. For example, the substitution of histidine at position 120 with alanine is designated "His120Ala" or "H120A."

[0028] A "deletion" is described by adding the original amino acid followed by the number of the position in the amino acid sequence followed by a "-". Thus, a deletion of glycine at position 150 would be designated "Gly150-" or "G150-". Alternatively, the deletion may be indicated by, for example, "deletion of D183 and G184".

[0029] "Terminations" are described by adding the number of the position in the amino acid sequence after the original amino acid, followed by a "*". Thus, a termination of the amino acid chain at position 150, replacing the glycine at this position, would be named "Gly150*" for "G150*".

[0030] An "insertion" is described by giving the original amino acid followed by the number of the position in the amino acid sequence, followed by the original amino acid and the added amino acid. For example, the insertion of a lysine at position 180 next to a glycine would be named "Gly180GlyLys" or "G180GK". If multiple amino acid residues are inserted, for example Lys and Ala after Gly180, this can be indicated as Gly180GlyLysAla or G180GKA. If the substitution and insertion occur at the same position, this can be indicated as S99SD+S99A, or S99AD for short. It is clear that degeneracy in nomenclature occurs when an amino acid residue identical to an existing amino acid residue is inserted. For example, if a glycine is inserted after a glycine in the above example, this would be indicated by G180GG.

[0031] Variants containing multiple modifications are separated by "+", e.g., "Arg170Tyr+Gly195Glu" or "R170Y+Gly195E" represent the substitution of arginine and glycine with tyrosine and glutamic acid, respectively, at positions 170 and 195. Alternatively, multiple modifications may be separated by spaces or commas, e.g., R170Y G195E or R170Y,G195E, respectively.

[0032] When different modifications can be introduced at a position, the different modifications are separated by commas, for example, "Arg170Tyr,Glu" represents the substitution of arginine at position 170 with tyrosine or glutamic acid. Alternatively, the different modifications or optional substitutions can be indicated in square brackets, for example, Arg170[Tyr,Gly] or Arg170{Tyr,Gly} or abbreviated R170[Y,G] or R170{Y,G}.

[0033] A special aspect regarding amino acid substitutions are conservative mutations that are often expected to have minimal effect on protein folding, resulting in substantial maintenance of the peptide or polypeptide properties of the respective peptide or polypeptide variant compared to those of the parent peptide or polypeptide. Conservative mutations are those in which an amino acid is exchanged for a similar amino acid. For determining % similarity the following applies, which is also based on the BLOSUM62 matrix, one of the most used amino acid similarity matrices for database searching and sequence alignment: Amino acid A is similar to amino acid S Amino acid D is similar to amino acids E and N Amino acid E is similar to amino acids D, K, and Q Amino acid F is similar to amino acids W and Y Amino acid H is similar to amino acids N and Y Amino acid I is similar to amino acids L, M, and V The amino acid K is similar to the amino acids E, Q, and R. Amino acid L is similar to amino acids I, M, and V Amino acid M is similar to amino acids I, L, and V The amino acid N is similar to the amino acids D, H, and S. The amino acid Q is similar to the amino acids E, K, and R. Amino acid R is similar to amino acids K and Q The amino acid S is similar to the amino acids A, N, and T. The amino acid T is similar to the amino acid S Amino acid V is similar to amino acids I, L, and M Amino acid W is similar to amino acids F and Y The amino acid Y is similar to the amino acids F, H and W.

[0034] Conservative amino acid substitutions may occur throughout the entire length of a polypeptide sequence of a functional protein, such as a peptide or polypeptide. Preferably, such mutations do not involve functional domains of the peptide or polypeptide.

[0035] Variants of proteins or nucleic acids can be defined by their sequence identity when compared to a parent protein or parent nucleic acid. Sequence identity is usually indicated as "% sequence identity" or "% identity". In a first step, to determine the percent identity between two amino acid sequences, a pairwise sequence alignment is made between the two sequences, and the two sequences are aligned over their entire length (i.e., pairwise global alignment). The alignment is made using a program that implements the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, p. 443-453), preferably "NEEDLE" (European Molecular Biology Open Software Suite (EMBOSS)) with the program default parameters (gap open=10.0, gap extension=0.5 and matrix=EBLOSUM62). The preferred alignment for the purposes of the present invention is the alignment that allows the determination of maximum sequence identity.

[0036] The following example is intended to illustrate two nucleotide sequences, but the same calculations apply to protein sequences. Sequence A: AAGATACTG Length: 9 bases Sequence B: GATCTGA Length: 7 bases Therefore, the shorter sequence is sequence B.

[0037] Producing a pairwise global alignment showing both sequences over their full length gives: [ka]

[0038] The symbol "I" in the alignment indicates an identical residue (meaning a base for DNA or an amino acid for proteins). The number of identical residues is six.

[0039] The symbol "-" in the alignment indicates a gap. The number of gaps in sequence B introduced by the alignment is 1. The number of gaps at the boundary of sequence B introduced by the alignment is 2 and at the boundary of sequence A is 1.

[0040] The alignment length is 10, showing sequences aligned over the full length.

[0041] According to the present invention, when a pairwise alignment is generated showing a shorter sequence over its entire length, the result is: [ka]

[0042] According to the present invention, generating a pairwise alignment showing sequence A over its entire length results in: [ka]

[0043] According to the present invention, generating a pairwise alignment showing sequence B over its entire length results in: [ka]

[0044] The alignment length, which shows the shorter sequence over its entire length, is 8 (there is one gap included in the alignment length of the shorter sequence).

[0045] Thus, the alignment length showing sequence A over its entire length will be 9 (meaning that sequence A is a sequence of the invention) and the alignment length showing sequence B over its entire length will be 8 (meaning that sequence B is a sequence of the invention).

[0046] After aligning the two sequences, the second step is to determine an identity value from the alignment. Therefore, in accordance with the present description, the following percent identity calculation is applied: % identity = (identical residues / length of the alignment region showing each sequence of the invention over its full length) * 100. Thus, sequence identity for a comparison of two amino acid sequences according to the invention is calculated by dividing the number of identical residues by the length of the alignment region showing each sequence of the invention over its full length. Multiplying this value by 100 gives the "% identity". According to the example given above, the % identity is (6 / 9) x 100 = 66.7% if sequence A is a sequence of the invention; (6 / 8) * 100 = 75% if sequence B is a sequence of the invention.

[0047] The term "expression cassette" refers to a construct in which a nucleic acid sequence encoding an amino acid sequence to be expressed is operably linked to at least one genetic control element that allows or regulates its expression (i.e., transcription and / or translation). Expression can be, for example, stable or transient, constitutive or inducible. An expression cassette may contain coding regions for more than one polypeptide, resulting in the transcription of a polycistronic RNA.

[0048] The terms "express", "expressing", "expressed" and "expression" refer to the expression of a gene product (e.g., a biosynthetic enzyme of a gene of a pathway or reaction defined and described in this application) at the level of the enzymatic activity resulting from the encoded protein or at the level at which the pathway or reaction to which it refers allows metabolic flux through the pathway or reaction in the organism in which the gene / pathway is expressed. Expression may also be achieved by genetic modification of the microorganism used as the starting organism. In some embodiments, the microorganism may be genetically modified (e.g., engineered) to express a gene product at an increased level compared to that produced by the starting microorganism or that produced in an equivalent microorganism that has not been modified. Genetic modifications include, but are not limited to, altering or modifying regulatory sequences or sites associated with the expression of a particular gene (e.g., by adding a strong promoter, an inducible promoter, or multiple promoters, or by removing regulatory sequences so that expression is constitutive), changing the chromosomal location of a particular gene, altering nucleic acid sequences adjacent to a particular gene, such as a ribosome binding site or a transcription terminator, increasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators, etc.) involved in the transcription of a particular gene and / or the translation of a particular gene product, or any other conventional means of deregulating the expression of a particular gene using routine techniques in the art (e.g., including but not limited to the use of antisense nucleic acid molecules to block the expression of repressor proteins).

[0049] The terms "overexpress", "overexpressing", "overexpressed" and "overexpression" refer to the expression of a gene product, and in particular to increasing the expression of a gene product at a level higher than that present prior to genetic modification of the starting microorganism. In some embodiments, a microorganism can be genetically modified (e.g., genetically engineered) to express a gene product at an increased level compared to that produced by the starting microorganism. Genetic modifications include, but are not limited to, altering or modifying the regulatory sequences or regulatory sites associated with the expression of a particular gene (e.g., by adding a strong promoter, an inducible promoter, or multiple promoters, or by removing regulatory sequences so that expression is constitutive), changing the chromosomal location of a particular gene, modifying nucleic acid sequences adjacent to a particular gene, such as a ribosome binding site or a transcription terminator, increasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators, etc.) involved in the transcription of a particular gene and / or the translation of a particular gene product, or any other conventional means of deregulating the expression of a particular gene using routine techniques in the art (e.g., including but not limited to, the use of antisense nucleic acid molecules to block the expression of repressor proteins). Another way to overexpress a gene product is to increase the stability and thus the life span of the gene product. The terms "overexpress", "overexpressing", "overexpressed" and "overexpression" can also mean that a gene activity is introduced into a microorganism where the respective gene activity has not been observed before, for example, by introducing a recombinant gene, e.g., a heterologous gene, into the microorganism, preferably in one or more copies by genetic engineering.

[0050] The term "plant" is used herein in its broadest sense as it pertains to organic matter and is intended to encompass eukaryotic organisms that are members of the taxonomic kingdom Plantae, including, but not limited to, monocotyledonous and dicotyledonous plants, vascular plants, vegetables, cereals, flowers, trees, herbs, shrubs, grasses, vines, ferns, mosses, fungi, algae, etc., as well as clones, offshoots, and plant parts used for asexual reproduction (e.g., cuttings, tubers, shoots, rhizomes, rhizomes, clumps, crowns, bulbs, corms, tubers, rhizomes, plants / tissues produced in tissue culture, etc.). Unless otherwise stated, the term "plant" refers to the entire plant, any part thereof, or cell or tissue culture derived from a plant, including any of the whole plant, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, seeds, plant cells, and / or their progeny. A plant cell is a biological cell in a plant or plant part that has been taken from a plant or obtained through the culture of a cell taken from a plant.

[0051] Plants that are particularly useful for the purposes of the present invention include Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., celery (Apium graveolens), Arachis spp., Artocarpus spp., Asparagus spp., and the like. officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var.sativa, Avena hybrida), star fruit (Averrhoa carambola), Bambusa sp., Benincasa hispida, Brazil nut (Bertholletia excelsea), sugar beet (Beta vulgaris), Brassica spp. (e.g. Brassica napus, Brassica rapa subsp. ssp. [canola, rapeseed, turnip rape]), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp.), safflower (Carthamus tinctorius), chestnut species (Castanea spp.), kapok (Ceiba pentandra), endive (Cichorium endivia), cinnamon species (Cinnamomum spp.), watermelon (Citrullus lanatus), citrus species (Citrus spp.), coconut species (Cocos spp.), coffee species (Coffea spp.), taro (Colocasia esculenta), cola species (Cola spp.), coriander (Coriandrum sativum), hazel species (Corylus spp.), hawthorn species (Crataegus spp.), saffron (Crocus sativus), pumpkin species (Cucurbita spp.), Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g. Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Eragrostis tef, Erianthus spp., Eriobotrya japonica, Eucalyptus spp. sp.), Pitanga (Eugenia uniflora), Buckwheat (Fagopyrum spp.), Beech (Fagus spp.), Tall fescue (Festuca arundinacea), Fig (Ficus carica), Fortunella spp., Fragaria spp., Ginkgo (Ginkgo biloba), Glycine spp.) (e.g. Glycine max, Soja hispida or Soja max), cotton (Gossypium hirsutum), Helianthus spp. (e.g. Helianthus annuus, Hemerocallis fulva), Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), sweet potato (Ipomoea batatas), Juglans spp., lettuce (Lactuca sativa), Lathyrus spp., lentil (Lens culinaris), flax (Linum usitatissimum), litchi (Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp., Acerola (Malpighia emarginata), Mammea americana, Mangifera indica, Manihot spp. spp.), Sapodilla (Manilkara zapota), Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp.), Opuntia spp., Ornithopus spp., Oryza spp. (e.g., Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Parsley (Petroselinum crispum), Phalaris arundinacea, Phaseolus spp., Timothy grass (Phleum pratense, date palms (Phoenix spp.), common reeds (Phragmites australis), nightshades (Physalis spp.), pines (Pinus spp.), pistachios (Pistacia vera), peas (Pisum spp.), Poa spp., populus spp., Prosopis spp., cherry blossoms (Prunus spp.), Psidium spp., pomegranates (Punica granatum), pears (Pyrus communis), oaks (Quercus spp.), radishes (Raphanus sativus), and rhubarb (Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp.) (e.g. potato (Solanum tuberosum), Solanum integrifolium or tomato (Solanum lycopersicum)), sorghum (Sorghum bicolor), spinach (Spinacia spp.), myrtaceae (Syzygium spp.), Tagetes spp., tamarind (Tamarindus indica), cacao (Theobroma cacao), Trifolium spp., gamagrass (Tripsacum dactyloides), Triticosecale rimpaui, wheat (Triticum spp.) (e.g. wheat (Triticum aestivum), durum wheat (Triticum durum), riveted wheat (Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare, Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp.), amaranth, artichoke, asparagus, broccoli, brussels sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, rapeseed, okra, onion, potato, rice, soybean, strawberry, sugar beet, sugarcane, sunflower, tomato, pumpkin, tea and algae, among others. According to a preferred embodiment of the invention, the plant is a crop. Examples of crops include, among others, soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato, corn or tobacco.

[0052] According to the invention, plants are cultivated to obtain plant material. The cultivation conditions are selected taking into account the plant and may include, for example, greenhouse growth, field growth, hydroculture growth and hydroponic growth. The plants and plant parts, such as seeds and cells, may be genetically modified. In particular, the plants and plant parts, preferably seeds and cells, may be recombinant, preferably transgenic or cisgenic.

[0053] The term "plant material" refers to any tissue, organ or material produced by a plant, including but not limited to plant cells, stems, roots, flowers, plant propagation material, ovules, stamens, seeds, leaves, embryos, meristematic areas, callus tissue, anther cultures, gametophytes, sporophytes, pollen, microspores, protoplasts, hairy root cultures, straw, husks, fruits and nut shells. As used herein, "plant cells" include but are not limited to protoplasts, gametogenic cells, and cells that regenerate into whole plants. The term "plant propagation material" should be understood to mean all reproductive parts of a plant, such as seeds and vegetative plant material, such as cuttings and tubers (e.g., potatoes), that can be used for plant propagation. This includes seeds, roots, fruits, tubers, bulbs, rhizomes, shoots, shoots, and other parts of a plant, including seedlings and seedlings that will be transplanted after germination or emergence from the soil. These seedlings may also be protected prior to transplantation by total or partial treatment with a dipping in or injection of the plant health promoting composition of the present invention.

[0054] The present invention provides a microorganism comprising a mutant degU gene. When the mutant degU gene is expressed in a microorganism, a mutant DegU protein is produced. This is because the degU gene encodes the DegU protein. According to the present invention, the wild-type DegU protein is a member of the CheY-like superfamily (InterPro ID IPR011006), and using the InterPro designation, comprises a signal transduction response regulator (receiver domain) (IPR001789) and a transcription regulator LuxR domain (C-terminus) (IPR000792). According to the Pfam nomenclature, the wild-type DegU protein comprises a response regulator receiver domain (PF00072, Pao et al., J Mol Evol 1995, 136-154 Response regulators of bacterial signal transduction systems: selective domain shuffling during evolution) and a LuxR-type DNA binding HTH domain (PF00196). Preferably, the wild-type degU gene encodes a DegU protein whose amino acid sequence has at least 40%, more preferably at least 43%, more preferably at least 45%, more preferably at least 53%, more preferably at least 57%, more preferably at least 70%, more preferably at least 77%, more preferably at least 85%, more preferably at least 88% sequence identity to SEQ ID NO: 1, and preferably the sequence identity to SEQ ID NO: 1 is at most 95%, more preferably at most 92%. Particularly preferred wild-type DegU proteins have a sequence identity of 50-95%, more preferably 77-91% to SEQ ID NO: 1. It should be understood that SEQ ID NO: 1 is an artificial amino acid sequence specially constructed as a template for screening and annealing of amino acid sequences. This sequence can therefore be used for the identification of degU genes, regardless of the fact that the DegU activity of the polypeptide of SEQ ID NO: 1 is not shown herein. Particularly preferred as wild-type DegU genes in the method or plant according to the invention are:Any of the amino acid sequences defined by the following Uniprot identifiers in descending order of priority: E3EBP5_PAEPS, A0A4R6MUX9_9BACL, A0A268SA79_9BACL, A0A069DEZ2_9BACL, A0A0B0HVN5_9BACL, W4EI28_9BACL, A0A1X7GB62_9BACL, A0A089MEU3_9BACL, A0A0E4HEC8_9BACL, A0A4P8XUS1_9BACL, A0A0M2VKR6_9BACL, A0A089M364_9BACL, V9GIW8_ 9BACL, W7YTM0_9BACL, A0A098MFT1_9BACL, D3EMG0_GEOS4, A0A1B8VU54_9BACI, A0A2Z2KSF3_9BACL, A0A269W3P3_9BACL, A0A1R1EEL5_9BACL, X5A6E5_ 9BACL, A0A089IT67_9BACL, A0A1I0JV80_9BACL, A0A168QEL2_9BACL, A0A0D3VFM7_9BACL, A0A172ZLN3_9BACL, A0A167D848_9BACL, A0A1E3L0K1_9BACL , A0A2W1LCB1_9BACL, A0A0U2N3N5_9BACL, L0EHW2_THECK, A0A1T2X729_9BACL, A0A1B8UUC0_9BACL, H3S9W0_9BACL, A0A3D9SC72_9BACL, A0A401I4R6_ 9BACL, A0A1I6WHZ6_9BACL, A0A015NM30_9BACL, A0A0F5R725_9BACL, A0A2N5NDN0_9BACL, M9LLL0_PAEPP, A0A0D5NRR7_9BACL, A0A2S0UEL3_9BACL, A0A 4Q2M1I7_9BACL, A0A1H1WMP7_9BACL, A0A3A1US45_9BACL, A0A3G9JII4_9BACL, C6D5A1_PAESJ, A0A433XGQ7_9BACL, A0A1I3PZK5_9BACL, A0A1R1DAD8_9 BACL, A0A4P6F1N4_9BACL, A0A0Q4R517_9BACL, A0A172TIH8_9BACL, A0A2V4X724_9BACL, A0A1Y5KD60_9BACL, A0A368VSS2_9BACL, A0A1B8VZY5_9BACI,<h2 style=";text-align:left;direction:ltr">A0A371P0X4_9BACL, A0A231RB89_9BACL, A0A369BC27_9BACL, E0IEE4_9BACL, A0A2V2YZQ8_9BACL, A0A1G7PNR5_9BACL, A0A3S1BJF8_9BACL, A0A1A5YDL9_9BACL, A0A0U2WGN9_9BACL, A0A494X986_9BACL, A0A3D9KD00_9BACL, C6J2I4_9BACL, A0A3Q9IF25_9BACL, A0A3G3K2Z7_9BACL, A0A090XUD0_PAEMA, A0 A3D9I787_9BACL, A0A398CFS8_9BACL, A0A1B1N3Y9_9BACL, A0A081P3I6_9BACL, A0A3T1DDG1_9BACL, A0A1K1QXK1_9BACL, A0A3Q8SA76_9BACL, A0A1X7KWC6_9BACL, A0A229USY4_9BACL, A0A4Q9DKY4_9BACL, A0A4R5KE70_9BACL, A0A329L4V8_9BACL, A0A2W1N4T3_9BACL, A0A1I1BB61_9BACL, H6NT64_9BACL, A0A1I4LD00_9BACL、A0A329MBB6_9BACL、A0A3S1AKF3_9BACL、F5LST8_9BAC L、A0A1V4HGJ1_9BACL、A0A1H0L0T9_9BACL、A0A0Q7JPS4_9BACL、A0A1H4RQ8 6_9BACL、A0A3S0BTA6_9BACL、A0A1C0ZYC4_9BACL、A0A0C2RFX7_9BACL、V9W 4A0_9BACL、A0A2V5KBB6_9BACL、A0A3B0C3G8_9BACL、A0A4R4EFH3_9BACL、A0 A1U9KAL0_9BACL, A0A4R3KIF8_9BACI, A0A292YJB9_9BACL, A0A075RHH4_BRELA, A0A0D1XDF4_ANEMI, A0A1A5XJS0_9BACL, V6M9Z2_9BACL, A0A120HRZ5_9BACL, A0A419V950_9BACL, A0A3R9QNM1_9BACI, A0A1I4L117_9BACI, A0A1H0J2F9_9BACI, A0A3M8DYQ5_9BACL, A0A1I2EIB9_9BACI, A0A428N9S8_9BACIA0A2P6MHC1_9BACI, A0A1I4CG56_9BACL, C0Z730_BREBN, M8DFP6_9BACL, A0A345BZD8_9BACI, A0A419SF78_9BACL, A0A3M8BE38_9BACL, A0A1H9W953_9BACI , A0A4Q1ST01_9BACL, F5L9B2_CALTT, A0A1G8AEE7_9BACI, D6Y0E8_BACIE, A0A4Q0VW28_9BACI, A0A2T4U7P0_9BACI, A0A061NX68_9BACL, A0A061P3R3_9BAC L, A0A098EIU7_9BACL, A0A3M8P3C8_9BACL, A0A1H2UAM2_9BACI, A0A3A9KCQ9_9BACI, A0A1Y0IJ22_9BACL, A0A1G8E1Q8_9BACI, A0A1S2M8P6_9BACI, Q9K6U7 _BACHD, A0A4R3N1F6_9BACI, A0A437KCI7_9BACI, A0A2P8GCB9_9BACL, A0A1X9MFG9_9BACI, A0A1H9TTG1_9BACI, A0A327YHU2_9BACI and A0A368Y3Q5_9BACI. According to the invention, wild-type DegU protein sequences and corresponding degU genes encoding same are particularly preferred, which have a sequence identity of at least 45%, more preferably at least 51%, more preferably at least 54%, even more preferably 73-100% to the amino acid sequence shown by the Uniprot identifier E3EBP5_PAEPS. Without taking into account specific mutations to the DegU protein sequence described according to the invention, the mutant DegU protein preferably differs from the amino acid sequence shown by the Uniprot identifier E3EBP5_PAEPS by 0-20 amino acids, more preferably by 0-15 amino acids, even more preferably by 0-10 amino acids, even more preferably by 1-5 amino acids, said differences preferably meeting the constraints according to FIG. 4. If the mutant DegU sequence is longer than said sequence when aligned with the sequence according to the Uniprot identifier E3EBP5_PAEPS, the respective C-terminal or N-terminal extension is preferably not more than 10 amino acids, more preferably by 0-5 amino acids.

[0055] The present invention also provides a microorganism comprising a mutant degS gene. When the mutant degS gene is expressed in a microorganism, a mutant DegS protein is produced because the degS gene encodes the DegS protein. According to the present invention, the wild-type DegS protein is a member of the DegS-type signal transduction histidine kinase family (InterPro ID IPR016381), and using the InterPro designation, comprises a sensor DegS domain (IPR008595) and a histidine kinase domain (IPR005467). According to the Pfam nomenclature, the wild-type DegS protein comprises a sensor protein DegS domain (PF05384), a HisKA_3 histidine kinase domain (PF07730) and a HATPase_c GHKL domain (PF02518). Preferably, the wild-type degS gene encodes a DegS protein whose amino acid sequence has at least 40%, more preferably at least 43%, more preferably at least 46%, more preferably at least 50%, more preferably at least 58%, more preferably at least 64%, more preferably at least 79%, more preferably at least 84% sequence identity to SEQ ID NO:2, and preferably the sequence identity to SEQ ID NO:2 is at most 95%, more preferably at most 91%. Particularly preferred wild-type DegS proteins have a sequence identity of 50-95%, more preferably 58-89% to SEQ ID NO:2. It should be understood that SEQ ID NO:2 is an artificial amino acid sequence specially constructed as a template for screening and annealing of amino acid sequences. This sequence can therefore be used for the identification of degS genes, regardless of the fact that the DegS activity of the polypeptide of SEQ ID NO:2 is not shown herein. Particularly preferred as wild-type degS genes in the methods or plants according to the invention are any of the amino acid sequences defined by the following Uniprot identifiers in descending order of priority: A0A074LBY4_PAEPO, E3EBP6_PAEPS, A0A4R6MVR0_9BACL, A0A069DLG2_9BACL, A0A268SAI9_9BACL, A0A1X7GB86_9BACL, A0A0M2VLZ1_9BACL,<h2 style=";text-align:left;direction:ltr">A0A1R1EED0_9BACL、A0A0B0HR83_9BACL、A0A4P8XRM7_9BACL、W7YPT3_9BAC L、A0A433XGY7_9BACL、D3EMG1_GEOS4、V9GK22_9BACL、A0A269W177_9BACL、 A0A3Q8SA22_9BACL、A0A1E3L2X6_9BACL、A0A369BCF2_9BACL、A0A2S0UEJ3_ 9BACL、A0A090XSK7_PAEMA、A0A3S1DMQ5_9BACL、A0A1B1N3X4_9BACL、C6J2I3 _9BACL、A0A172ZLS7_9BACL、A0A1G7PLD1_9BACL、A0A2Z2KM36_9BACL、A0A3 Q9IDP6_9BACL、A0A0D3VFE7_9BACL、A0A168QEJ2_9BACL、A0A1B8VU57_9BAC I、W4EHN2_9BACL、A0A0E4CZI9_9BACL、A0A089L4Y0_9BACL、A0A098MEC1_9B ACL、A0A089IPW0_9BACL、A0A167D837_9BACL、A0A089NAN3_9BACL、X4ZSE0_9 BACL、A0A2W1M2J1_9BACL、A0A1I0JTZ3_9BACL、A0A4Q2LW12_9BACL、A0A1I3 PZ40_9BACL、A0A401I4T4_9BACL、A0A1B8UU84_9BACL、A0A1T2X709_9BACL、 A0A2N5NDJ2_9BACL、A0A0F5RAF5_9BACL、A0A3G9IYB3_9BACL、A0A1H1WM07_ 9BACL、H3S9W1_9BACL、A0A1I6WIJ5_9BACL、A0A0D5NQK3_9BACL、A0A015KKW1 _9BACL, A0A3D9SD21_9BACL, A0A1B8VZZ2_9BACI, M9LFD8_PAEPP, A0A2V4WBE9_9BACL, A0A0U2WI25_9BACL, A0A1R1DAI7_9BACL, A0A368VS81_9BACL, E0IEE5_9BACL, A0A371P0S2_9BACL, A0A4P6EY40_9BACL, L0EJK6_THECK, A0A3D9I772_9BACL, A0A1X7KY93_9BACL, A0A231R9F5_9BACL, A0A3A1UXN0_9BACL,<h2 style=";text-align:left;direction:ltr">A0A494XFI9_9BACL, A0A1Y5KD15_9BACL, A0A398CI63_9BACL, A0A3T1DDD1_9BACL, A0A0Q4RDM1_9BACL, C6D5A2_PAESJ, A0A2V2Z262_9BACL, A0A3G3K194_9BACL, A0A3D9KBV6_9BACL, A0A1A5YD71_9BACL, A0A433R8L8_9BACL, A0A172TIT6_9BACL, A0A1I1BCS1_9BACL, A0A081P3I5_9BACL, A0A4R5KGY1_9B ACL、A0A229USJ3_9BACL、A0A2V5JWK3_9BACL、A0A329MCI0_9BACL、A0A0U2 INE8_9BACL、A0A1K1QX93_9BACL、A0A2W1NWZ0_9BACL、A0A1I4LCQ8_9BACL、 A0A329L6X4_9BACL、A0A0C2V9A2_9BACL、A0A4Q9DIC7_9BACL、H6NT63_9BAC L、A0A1H4RQL7_9BACL、A0A3B0C2F8_9BACL、V9VZ78_9BACL、A0A1H0L1L7_9B ACL、A0A430JA16_9BACL、F5LST9_9BACL、A0A4R4EAF4_9BACL、A0A0Q7JRA6 _9BACL、A0A1V4HGJ0_9BACL、A0A1C0ZYJ1_9BACL、A0A4R3KJJ5_9BACI、M8DF K7_9BACL、A0A1U9KAR4_9BACL、A0A3M8DWV1_9BACL、A0A1A5XKA3_9BACL、A0 A1E5LA89_9BACL、A0A074LTT3_9BACL、A0A1I4CE81_9BACL、C0Z731_BREBN、 V6MBX1_9BACL、A0A1Y0IJ16_9BACL、A0A3M8BE71_9BACL、A0A4Q1STZ5_9BA CL、A0A1E5G3N9_9BACL、A0A075RB77_BRELA、A0A419SF93_9BACL、A0A1Z5HT H4_9THEO、F5L9B1_CALTT、A0A3S9T1P5_9FIRM、A0A2N5M9N1_9BACI、A0A235 FGA1_9BACI、A0A0M2U6G0_9FIRM、A0A4R6TU87_9BACI、A0A1E5LDM8_9BACI、A0A498RIM9_9FIRM, A0A120HRZ3_9BACL, A0A4Q0VV23_9BACI, A0A1I2EJ29_9BACI, A0A1U7MGK1_9FIRM , E6TSA5_BACCJ, A0A3E2JMS2_9BACI, A0A1I4L1V6_9BACI, A0A2P8HQR2_9BACI, Q9K6U6_BACHD, A0A402B According to the invention, wild-type DegS protein sequences and corresponding degS genes encoding same, which have at least 40%, more preferably at least 46%, more preferably at least 58%, even more preferably 80-100% sequence identity to the amino acid sequence represented by the Uniprot identifier A0A074LBY4_PAEPO, are particularly preferred. Without taking into consideration specific mutations to the DegS protein sequence described according to the present invention, the mutant DegS protein preferably differs from the amino acid sequence shown by the Uniprot identifier A0A074LBY4_PAEPO by 0-40 amino acids, more preferably by 0-20 amino acids, even more preferably by 0-10 amino acids, even more preferably by 1-5 amino acids, said differences preferably meeting the constraints according to Figure 3. If the mutant DegS sequence is longer than said sequence when aligned with the sequence according to the Uniprot identifier A0A074LBY4_PAEPO, each C-terminal or N-terminal extension is preferably not more than 10 amino acids, more preferably by 0-5 amino acids.

[0056] A microorganism according to the present invention may comprise either a mutant degU gene or a mutant degS gene, or the microorganism comprises both a mutant degU gene and a mutant degS gene. Similarly, a microorganism according to the present invention may comprise either a mutant DegU protein or a mutant DegS protein, or the microorganism comprises both a mutant DegU protein and a mutant DegS protein.

[0057] According to the present invention, the microorganism can contain a mutant spo0A gene. When the mutant spo0A gene is expressed in the microorganism, a mutant Spo0A protein is generated. This is because the spo0A gene encodes the Spo0A protein. According to the present invention, the wild-type Spo0A protein is a member of the sporulation transcription factor Spo0A (IPR012052), and using InterPro notation, contains a signal transduction response regulator receiver domain (IPR001789) and a sporulation initiation factor Spo0A C-terminal domain (IPR014879), which is part of the winged-helix-like DNA-binding domain superfamily (IPR036388). According to the Pfam nomenclature, the wild-type Spo0A protein contains a response regulator receiver domain (PF00072) and a sporulation initiation factor Spo0A C-terminal domain (PF08769). Preferably, the wild-type spo0A gene encodes a Spo0A protein whose amino acid sequence has at least 45%, more preferably at least 56%, more preferably at least 69%, more preferably at least 70%, more preferably at least 67%, more preferably at least 70%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75% sequence identity to SEQ ID NO: 3, and preferably the sequence identity to SEQ ID NO: 3 is at most 85%, more preferably at most 11%. Particularly preferred wild-type Spo0A proteins have a sequence identity of 50-85%, more preferably 76-84% to SEQ ID NO: 3. It should be understood that SEQ ID NO: 3 is an artificial amino acid sequence specially constructed as a template for screening and annealing of amino acid sequences. This sequence can therefore be used for the identification of spo0A genes, regardless of the fact that the Spo0A activity of the polypeptide of SEQ ID NO: 3 is not shown herein. Particularly preferred as wild-type spoOA genes in the methods or plants according to the invention are any of the amino acid sequences defined by the following Uniprot identifiers in descending order of priority: A0A074LZY6_PAEPO, E0RDX7_PAEP6, H6CM41_9BACL, A0A0D7WZ78_9BACL, A0A167DI09_9BACL,W7YKB3_9BACL, A0A168BRF7_9BACL, A0A1G5JWJ2_9BACL, A0A168P4Q5_9BACL, A0A168M3D7_9BACL, A0A1R1EUX4_9BACL, A0A2W6PE29_9BACL, A0A2V4WTN3_PAEBA, A0A328WGM0_PAELA, D3E6N2_GEOS4, G4HF05_9BACL, A0A1R0XBX0_9BACL, A0A098M8U8_9BACL, A0A3Q8SBT8_9BACL, A0A0M1P3N3_9BACL, R9LQX4 _9BACL、A0A2Z2KRN4_9BACL、A0A1B8WQN2_9BACI、A0A089MEU2_9BACL、A0A089LZP7_9BACL、A0A0F7FA95_PAEDU、A0A0E4HDK7_9BACL、A0A1G7R7Q0_9BAC L、A0A1H8N6P6_9BACL、X4ZFA8_9BACL、A0A3G9IQE6_9BACL、A0A369BNP1_9BACL、A0A1B1N0I3_9BACL、A0A015KRJ2_9BACL、A0A2N5N5F6_9BACL、A0A1T2XN U8_9BACL、A0A090ZFJ2_PAEMA、A0A3D9QX06_9BACL、E0ICH6_9BACL、A0A1G9E5Z0_9BACL、A0A3S1DUM5_9BACL、A0A0D5NPL4_9BACL、A0A368WCL4_9BACL、 A0A4Q2LM98_9BACL、A0A328U1G0_9BACL、A0A172TM01_9BACL、A0A1I2EKA8_9BACL、A0A1A5YCA7_9BACL、A0A371PM84_9BACL、A0A3A6PB13_9BACL、A0A2V2 YXM7_9BACL、L0EEN3_THECK、A0A3A1UXY9_9BACL、A0A3B0CH88_9BACL、A0A1V4HR00_9BACL、A0A1V0UWJ6_9BACL、H3SFG5_9BACL、A0A1X7JKH5_9BACL、A0A1V4HR00_9BACL 1I2FDS6_9BACL、A0A3D9IJB3_9BACL、A0A398CE46_9BACL、M9LB51_PAEPP、A0A3D9KSR7_9BACL、A0A081NWT7_9BACL、H6NL94_9BACL、A0A1C0ZWF8_9BACL、A0A4Y8M823_9BACL、A0A1X7HJ70_9BACL、A0A329MFB7_9BACL、A0A1G4P4T7_9BACL、A0A229UXF4_9BACL、A0A0U2WBQ5_9BACL、A0A3S0BM74_9BACL、K4ZP7 6_PAEA2、A0A2W1NBK6_9BACL、A0A172ZK56_9BACL、A0A3M8CIR1_9BACL、M8EE17_9BACL、A0A0Q3T5E2_BRECH、A0A0K9YRB7_9BACL、A0A1I3U483_9BACL、V 6MCA1_9BACL、C0ZC17_BREBN、A0A4R3KM88_9BACI、A0A3M8B6E4_9BACL、A0A2N3LN87_9BACI、A0A419SMW9_9BACL、A0A3M8D088_9BACL、A0A075R4A3_BRE LA、U1X7N0_ANEAE、A0A1H2UFN8_9BACL、A0A0D1VW72_ANEMI、A0A0X8D3E6_9BACL、A0A0U5AZK5_9BACL、A0A4R3L002_9BACL、A0A0Q3WXA1_9BACI、A0A0B0 IAE5_9BACI, A0A223KSV6_9BACI, W4PXN5_9BACI, A0A235BCM6_9BACL, A0A235FAK4_9BACI, A0A2T4Z9J8_9BACL, A0A1S2MEZ1_9BACI, Q9K977_BACHD, A0A1S2LUZ3_9BACI, A0A1U9KC16_9BACL, A7Z6J0_BACVZ, Q65HJ7_BACLD, W4QWX1_BACA3, A0A1I6TUX2_9BACL, A0A1I2L3I1_9BACL, A0A0H3E179_BACA1, SP 0A_BACSU、A8FF06_BACP2、A0A0J6EVC7_9BACI、A0A417YV34_9BACI、D5DS62_BACMQ、A0A4Q0VQU7_9BACI、A0A1H9PKN5_9BACI、A0A1I3QAI8_9BACL、A0A1 G6Q9T8_9BACL、W1SHY1_9BACI、A0A364K8M0_9BACL、A0A150F6K4_9BACI、M5PEN8_9BACI、A0A1S2M754_9BACI、A0A0A8X8S8_9BACI、A0A1R1RU53_9BACI、A0A1S2LYV1_9BACI、A0A1B3XQX6_9BACI、A0A1H8EQX3_9BACI、A0A2N5GRE3_9BACI、A0A4R1B005_9BACI、A0A4R1QFH8_9BACI、A0A1B1Z5W5_9BACI、K6BXH 2_9BACI、A0A160F753_9BACI、U5LDF9_9BACI、A0A0M0KYT7_9BACI、A0A061NL57_9BACI、A0A3A1QZJ5_9BACI、A0A2N5H854_9BACI、A0A160ISE8_9BACI、A0A160ISE8_9BACI 0A2I7SRN1_LACSH, A0A1M4TLQ2_9BACL, A0A4R2QSJ5_9BACL, A0A3L7K5H6_9BACI, A0A2N5M452_9BACI, W4QKM5_9BACI, A0A4R2PAA5_9BACL, A0A0J1IMN1_BACCI, R9C857_9BACI, A0A0M4FX23_9BACI, A0A165XSR5_9BACI, A0A179SV99_9BACI, A0A1Y0IS88_9BACL, A0A248TLE9_9BACI, A0A1H0WI33_9BACI, A0 A0H4PIL5_9BACI、I8AMT2_9BACI、A0A0D6ZAA3_9BACI、A0A3T0I1Q1_9BACI、A0A1I0SQQ4_9BACI、I3EAA8_BACMM、A0A0M0GB29_SPOGL、A0A1L8ZLZ8_9BACI I、A0A370GBM9_9BACI、A0A433H928_9BACI、A0A4R6U795_9BACI、A0A060LXS4_9BACI、A0A074LME5_9BACI、A0A0K9GWU6_9BACI、A0A150KM63_9BACI、K6C V08_BACAZ、A0A323TXM3_9BACI、A0A2N0Z9Q2_9BACI、J8AK67_BACCE、A0A073KUP4_9BACI、A0A292YQZ8_9BACL、A0A226QLR6_9BACI、A0A160FBJ9_9BACI 、C3BPR4_9BACI、E6TXR1_BACCJ、A0A1L3MQ53_9BACI、A0A0C2YCQ6_BACBA、Q8EQ49_OCEIH、A0A316D8M3_9BACL、A0A0J6FU61_9BACI、A0A1H8C0C3_9BACI、<h2 style=";text-align:left;direction:ltr">A0A084J373_BACMY、A0A1I4JPZ3_9BACI、A0A0M2SG37_9BACI、A0A150MMS1_9BACI、A0A1J6WGW3_9BACI、A0A0P6W2Q8_9BACI、A0A1I 0SZE6_9BACI、A7GSJ0_BACCN、A0A2C9Z3P6_BACHU、A0A398BG15_9BACI、A0A0V8JFI7_9BACI、A0A1I5NPW8_9BACI、A0A4R2B866_9BAC I、A0A023DE04_9BACI、A0A023CLR0_9BACI、A0A327YN47_9BACI、A0A0Q9XV74_9BACI、A0A147K7R5_9BACI、A0A443J408_9BACI、A0A A0A073K3V0_9BACI、A0A1X7D063_9B ACI、Q5WF68_BACSK、A0A3S4RLT9_9BACI、A0A150JT68_BACCO、F5L3H6_CALTT、A0A0M0GPU2_9BACI、S5Z7C0_BACPJ、A0A1Z2V3H9_9B ACI、A0A3A9KGU4_9BACI、A0A285CLU9_9BACI、A0A366XYH1_9BACI、A0A0D8BRF6_GEOKU、A0A265NFG5_9BACI、A0A428N868_9BACI、A0 A2P8HAG1_9BACI, A0A1H0B3U0_9BACI, A0A150M7C5_9BACI, A0A1G8D1C3_9BACI, A0A1G8BRD8_9BACI, A0A4Q4IIH6_9BACL, A0A4Y9AEG4_9BACI, A0A1I0FQG9_9BACI, A0A0F5HWK7_9BACI, A0A1H1BJ69_9BACI, W9A8X3_9BACI, A0A1H9LW77_9BACI, A0A494Z0K1_9BACI<h2 style=";text-align:left;direction:ltr"> A0A1M5CXL0_9BACI、A0A1G8JJN9_9BACI、A0A1G6IGH8_9BACI、A0A4Y7S8L6_9FIRM、A0A1X9MFG7_9BACI、A0A0A2UZF1_9BACI、A0A1H9ZLP9_9BACI、A0A1M4 XLK4_9CLOT、A0A0A5GIF6_9BACI、A0A0C2VIM1_9BACL、A0A2A2IDA3_9BACI、A0A366EJ45_9BACI、A0A317KZA9_9BACI、A0A0A5GEQ9_9BACI、A0A1E5LK88_9 BACI、A0A2S5GEL8_9BACL、A0A1G9LM94_9BACI、A0A1N6PFX1_9BACI、A0A1E7DMX2_9BACI、N4WSS3_9BACI、A0A1I1T1Z9_9BACI、A0A0A1MZ98_9BACI、A0A4R 3N0Q4_9BACI、A0A4Y8KST9_9BACL、A0A2U1K6N5_9BACI、A0A075LLD7_9BACI、A0A1I0V6R2_9BACI、A0A0U1KL95_9BACI、A0A2P6MK99_9BACI、C8WXF8_ALIA D、A0A1M6KIQ3_9CLOT、A0A1L8CTW3_9THEO、A0A1V2A9Q9_9BACI、A0A2T4UAN8_9BACI、A0A4Z0GKV9_9BACL、A0A1M6I6U1_9FIRM、A0A1I2VPT9_9BACL、A0A1 M6S6D3_9BACL、A0A1N7KMH0_9BACL、A0A140L8E0_9CLOT、A0A090J299_9BACI、V6IWU0_9BACL、A0A024P5H3_9BACI、A0A285NM88_9BACI、A0A143MRA0_9BACI CI、A0A0A5GCA3_9BACI、A0A0U1QSI9_9BACL、A0A0B5AS70_9BACL、A0A1M6C4X1_9CLOT、A0A2I0QX97_9BACI、A0A0P9EJT7_9BACL、A0A1U7MLA0_9FIRM、A0A1M6C4X1_9CLOT 2T0BRS8_9CLOT、A0A1H2T3C9_9BACL、A0A1H9A7M5_9BACI、A0A084JIX0_9CLOT、D9SLV6_CLOC7、A0A4R2RWP5_9FIRM、U2CLF1_9FIRM、A0A1G8VG90_9BACI。According to the invention, wild-type Spo0A protein sequences and corresponding spo0A genes encoding same are particularly preferred, which have a sequence identity of at least 55%, more preferably at least 60%, more preferably at least 62%, more preferably at least 70%, even more preferably 80-100%, even more preferably 95-100% to the amino acid sequence shown by the Uniprot identifier A0A074LZY6_PAEPO. Without considering specific mutations to the Spo0A protein sequence described according to the invention, the mutant Spo0A proteins preferably differ from the amino acid sequence shown by the Uniprot identifier A0A074LZY6_PAEPO by 0-20 amino acids, more preferably 0-15 amino acids, even more preferably 0-10 amino acids, even more preferably 1-5 amino acids, which differences preferably meet the constraints according to FIG. If the mutant Spo0A sequence is longer than said sequence when aligned with the sequence according to Uniprot identifier A0A074LZY6_PAEPO, then each C-terminal or N-terminal extension is preferably 30 amino acids or less, more preferably 0 to 10 amino acids.

[0058] The microorganism according to the present invention may comprise a mutant Spo0A protein or a mutant spo0A gene in addition to the mutant degU gene and / or the mutant degS gene. The microorganism may also comprise a mutant Spo0A protein or a mutant spo0A gene in addition to the mutant DegU protein and / or the mutant DegS protein.

[0059] The microorganisms of the invention exhibit increased and / or stabilized exopolysaccharide production and / or reduced exopolysaccharide degradation compared to an exopolysaccharide-producing control strain ("parent") lacking the mutations of the invention. However, the invention is not limited to the modification of exopolysaccharide-producing microorganisms. The invention also advantageously makes it possible to create microorganisms that exhibit increased and / or stabilized exopolysaccharide production and / or reduced exopolysaccharide degradation after one or more genes for exopolysaccharide production that were previously lacking are introduced. In particular, the invention makes it possible to prepare Firmicutes strains for producing exopolysaccharides by introducing degU, degS and / or spoOA of the invention, such that after further introduction of a heterologous exopolysaccharide gene cluster or modification of the native genes, the strains are able to produce one or more desired exopolysaccharides while exhibiting increased and / or stabilized exopolysaccharide production and / or reduced exopolysaccharide degradation.

[0060] One advantage of the present invention is that the increase or stabilization of exopolysaccharide production and / or prevention or reduction of exopolysaccharide degradation is possible by mutation of easily accessible genes in a wide variety of Firmicutes microorganisms. Thus, although the present invention is illustrated by the following specific examples, the methods described in the examples can be transferred to other microbial species to increase and / or stabilize exopolysaccharide production and / or reduce exopolysaccharide degradation. Furthermore, the mutations described herein do not require the deletion or insertion of large nucleic acid fragments that may affect the transcription of downstream genes. Instead of the need to change the transcription regulator binding sites of the various genes involved in the formation of exopolysaccharides or delete those genes, the present invention makes it possible to achieve the advantages described herein by mutation of the degU, degS and / or spoOA genes. These genes are involved in a complex web of gene regulatory pathways, and the effects caused by the introduction of the mutations of the present invention were not predictable. Similarly, the production and degradation of exopolysaccharides are themselves subject to complex regulatory mechanisms, which are inherently unpredictable. Moreover, in light of WO 2019221988, it was particularly surprising that mutations in the degS, degU and / or spo0A genes lead to increased or stabilized exopolysaccharide production and / or prevented or reduced exopolysaccharide degradation. In this document, a strain with a Spo0A-deficient background and containing mutated degU and degS genes in addition to Spo0A deficiency is used with the clear intention of reducing the viscosity caused by exopolysaccharides during fermentation. However, as shown herein and contrary to the seemingly plausible teachings of this document, introduction of mutations only in the degU, degS and / or spo0A genes in wild-type microorganisms increases or stabilizes exopolysaccharide production and / or prevents or reduces exopolysaccharide degradation. Thus, the effects described in the publication are not causally related to mutations in the degU, degS and spo0A genes.

[0061] The present invention, as a particularly noteworthy advantage, allows the stabilization of exopolysaccharides during fermentation production and / or the reduction of exopolysaccharide degradation by the microorganisms, especially during fermentation production. This can be detected in comparative analyses of batch fermentations. In such analyses, a batch fermentation of the microorganism according to the invention and a control fermentation using a suitable control microorganism are carried out to determine the time of maximum carbon transfer rate. From that point on, the viscosity of each fermentation broth is measured at predetermined intervals over the next 48 hours. The sum of the viscosity readings of the fermentation of the microorganism of the invention is then found to be higher than the sum of the same number of viscosity measurements of the comparative fermentation. The exopolysaccharides can be used by the microorganisms as a reserve carbon or energy source in later fermentation stages, as shown in the examples. Thus, even exopolysaccharides produced in large quantities during the early fermentation stages can be degraded during later fermentation conditions or subsequent storage and downstream processing, which can reduce the exopolysaccharide yield and change the product composition, especially when the product contains exopolysaccharides together with further components, preferably one or more microorganisms, their spores, and target substances produced by the microorganisms, preferably one or more fusaricidins. However, the present invention makes it possible to maintain a high viscosity and thus a high exopolysaccharide content throughout the entire fermentation and even possibly during further downstream processing or storage. Thus, compared to wild-type strains, there is no need to harvest the fermenter early to balance the exopolysaccharide yield against the yield of said further components. In effect, the present invention can be used to produce two product categories in a single fermentation, i.e. one or more exopolysaccharides and said one or more further components.

[0062] Preferably, the target fermentation product is or comprises a lipopeptide and / or a siderophore, preferably having antibacterial activity, preferably antifungal activity. Preferably, the lipopeptide is a non-ribosomal lipopeptide (NRP). Even more preferably, the lipopeptide and / or siderophore is of any of the following types: aculeacin, amphicin, amphomycin, anticapsin, aspartocin, bacillaene, bacillibactin, bacillomycin, bacillorin, bacillysin, bacitracin, caspofungin, selexin, cichofactin, cormycin, crystallomycin, daptomycin, difficidin, ecomycin, entolysin, fengycin, friulimicin, fusaricidin, gatavalin, hodersin, iturin, jollipeptin, kurstakin, laspartomycin, lichenisin, locillomycin, locilomycin, loxin, macrolactin. , maribacin, marihicin, massetolide, octapeptin, orfamide, paenibacterin, paenilarvin, paeniproxilin, paeniserin, pelgipeptin, plantazolicin, plipastatin, pneumocandin, polyketide, polymyxin, polypeptin, pseudodesmin (p The antibacterial agents include one or more of the following: seudodesmin, pseudomycin, pseudophomin, putisolvin, saltavalin, surfactin, syringafactin, syringomycin, syringopeptin, tensin, traacin, tridecaptin, tsushimacin, viscocin, and viscocinamide.In particular, the production of antibacterial target fermentation products obtainable from Paenibacillus strains is described in WO2016154297. A particularly preferred target fermentation product is fusaricidin. Fusaricidin is a group of antibiotics isolated from Paenibacillus spp., from the class of cyclic lipodepsipeptides, which often share the following structural features: a macrocyclic ring of six amino acid residues (three of which are L-Thr, D-allo-Thr and D-Ala) and a 15-guanidino-3-hydroxypentadecanoic acid tail attached to the N-terminal L-Thr residue by an amide bond (ChemMedChem 7,871-882,2012; J.Microbiol.Meth.85,175-182,2011). These compounds are cyclized by a lactone bridge between the N-terminal L-Thr hydroxyl group and the C-terminal D-Ala carbonyl group. The positions of the amino acid residues in the depsipeptide ring are usually numbered starting from the aforementioned L-Thr, which itself carries the GHPD chain, and ending with the C-terminal D-Ala. Non-limiting examples of fusaricidins isolated from Paenibacillus include LI-F03, LI-F04, LI-F05, LI-F07 and LI-F08 (J. Antibiotics 40(11), 1506-1514, 1987; Heterocycles 53(7), 1533-1549, 2000; Peptides 32, 1917-1923, 2011), and fusaricidins A (also called Ll-F04a), B (also called Ll-F04b), C (also called Ll-F03a) and D (also called Ll-F03b) (J. Antibiotics 49(2), 129-135, 1996; J. Antibiotics 49(2), 129-135, 1996). 50(3), 220-228, 1997). The amino acid chain of fusaricidin is not produced by ribosomes, but by nonribosomal peptide synthetases.Among the isolated fusaricidins, fusaricidin A has shown the most promising antibacterial activity against various clinically relevant fungi and Gram-positive bacteria such as Staphylococcus aureus (MIC range: 0.78-3.12 g / ml) (ChemMedChem 7, 871-882, 2012). Fusaricidins A, B, C and D have also been reported to inhibit phytopathogenic fungi such as Fusarium oxysporum, Aspergillus niger, Aspergillus oryzae and Penicillium thomii (J.Antibiotics 49(2), 129-135, 1996; J.Antibiotics 50(3), 220-228, 1997). Fusaricidins such as Li-F05, LI-F07 and LI-F08 have been found to have certain antifungal activity against various phytopathogenic fungi such as Fusarium moniliforme, F. oxysporum, F. roseum, Giberella fujkuroi, Helminthosporium sesamum and Penicillium expansum (J.Antibiotics 40(11), 1506-1514, 1987). Fusaricidin also has antibacterial activity against gram-positive bacteria, including Staphylococcus aureus (J. Antibiotics 49, 129-135, 1996; J. Antibiotics 50, 220-228, 1997). In addition, fusaricidin has antifungal activity against Leptosphaeria maculans, which causes black root rot of canola (Can. J. Microbiol. 48, 159-169, 2002).Furthermore, Fusaricidins A and B and two related compounds produced by certain Paenibacillus strains were found to induce resistance responses in cultured parsley cells and inhibit the growth of Fusarium oxysporum (WO 2006 / 016558; EP 1788074A1). In WO 2016 / 020371, whole culture broths, culture media and cell-free extracts of bacterial strains Lu16774, Lu17007 and Lu17015 were found to exhibit inhibitory activity against Alternaria spp., Botrytis cinerea and Phytophthora infestans, among others.

[0063] The microorganism according to the invention preferably comprises a mutant degU gene, which encodes a DegU protein with reduced DNA-binding activity and / or lacking a functional DNA-binding domain. This is preferably achieved by providing a mutant degU gene encoding a mutant DegU protein, the mutation affecting the LuxR-type DNA-binding HTH domain (PF00196). As shown in the following examples, simply providing a mutant degU gene is already sufficient to improve exopolysaccharide production and / or reduce exopolysaccharide degradation during the fermentation process. This was particularly surprising in view of WO2019221988, which explicitly publishes that such mutations of the degU gene do not result in an increase in the viscosity of the fermentation broth and are therefore not suitable for increasing and / or stabilizing exopolysaccharide production and / or reducing exopolysaccharide degradation during fermentation.

[0064] It is an additional advantage that the DegS, DegU and DegS+DegU variants of the present invention do not abolish or significantly reduce the sporulation ability of a microorganism, which is particularly advantageous for sporulation plant health compositions or other applications that rely on sporulation.

[0065] The DegU protein preferably has reduced DNA-binding activity and / or lacks a functional DNA-binding domain. The presence of these traits can be easily identified in a microorganism of the invention, preferably a microorganism of the genus Paenibacillus, by observing an increase in viscosity or retention of viscosity during fermentation compared to the corresponding wild-type strain.

[0066] As mentioned above, the wild-type DegU protein contains a DNA-binding HTH domain. This domain extends from amino acid position 171 to the end of the sequence according to the numbering of the protein sequence with the Uniprot identifier E3EBP5. Further information on the DNA-binding domain is available from the corresponding Pfam and InterPro databases. For example, in the case of the most preferred wild-type DegU protein sequence E3EBP5, the DNA-binding domain is predicted to contain four alpha-helical domains extending to positions 180-191, 195-202, 206-221 and 225-235. It is preferred that the DNA-binding domain of the DegU protein is mutated in the third or fourth position, and most preferred if it is mutated in the third alpha-helical domain. Here, the mutations in the protein sequence generally do not affect the correct folding and function of the remainder of the DegU protein.

[0067] Preferably, the DegU protein mutation is selected from the group consisting of, for each option a) and b), in decreasing order of preference: a)Q218*, Q218K, Q218N, Q218D, Q218R, b) comprising or consisting of one or more of D223*, D223*+M220N, D223*+M220N+E221G, D223*+M220N+V222G, D223*+M220N+E221G+V222G, D223*+M220D, D223*+M220E, D223*+M220H, D223*+M220F, D223*+M220W, D223*+M220S, D223*+M220A.

[0068] For the purposes of the present invention, the above numbering refers to the wild-type DegU protein sequence with Uniprot identifier E3EBP5. It is noted that, as above, the mutant DegU protein has at least 45%, more preferably at least 51%, more preferably at least 54%, and even more preferably 73-100% sequence identity to the amino acid sequence represented by Uniprot identifier E3EBP5_PAEPS, ignoring the mutations specifically listed above.

[0069] Both mutations a) and b) are simultaneously included in the predicted third alpha helix of the DNA binding domain. As shown in the examples, both mutations of type a) and b) both result in an increase in fermentation broth viscosity over the course of fermentation. Furthermore, the viscosity of the fermentation broth, even if it changes in the later fermentation stages, is not less than 50% of the maximum viscosity of the fermentation broth of the wild-type strain, and preferably not less than 70% of the maximum viscosity of the fermentation broth of the wild-type strain, as shown in the examples, measured 48 hours after the time of maximum carbon transfer rate during fermentation. The conditions for measuring the viscosity are shown in the examples below.

[0070] Preferably, the mutation of option a) results in a faster increase in viscosity compared to a microorganism comprising only a mutation according to option b), such that the viscosity plateau is reached sooner compared to a microorganism comprising only a mutation according to option b), whereas a microorganism comprising a DegU mutation according to option b) preferably results in a higher peak viscosity compared to a microorganism according to option a) (see also FIG. 1).

[0071] The mutated amino acids according to options a) and b), respectively, are listed above in the order of increasing frequency of each in natural homologues of the DegU protein. Since the interest of the present invention is to provide microorganisms with modified properties of the DegU protein compared to the wild type, the least frequent modifications are the most preferred modifications, with the preference decreasing as the frequency of each amino acid at each position increases.

[0072] The above mentioned mutations in the DegU protein can also be combined, except for the mutation Q218*. Thus, the present invention also relates to a microorganism comprising a mutant degU gene encoding a mutant DegU protein, the mutations being Q218K+D223*, Q218K+M220N+D223*, Q218K+M220N+E221G+D223*, Q218K+M220N+V222G+D223*, Q218K+M220N+E221G+V222G+D223*, Q218K+M220D+D223*, Q218K+M220E+D223*, Q218K+M220 H+D223*, Q218K+M220F+D223*, Q218K+M220W+D223*, Q218K+M220S+D22 3*, Q218K+M220A+D223*, Q218N+D223*, Q218N+M220N+D223*, Q218N+M2 20N+E221G+D223*, Q218N+M220N+V222G+D223*, Q218N+M220N+E221G+V 222G+D223*, Q218N+M220D+D223*, Q218N+M220E+D223*, Q218N+M220H+D 223*, Q218N+M220F+D223*, Q218N+M220W+D223*, Q218N+M220S+D223*, Q218N+M220A+D223*, Q218D+D223*, Q218D+M220N+D223*, Q218D+M220N +E221G+D223*, Q218D+M220N+V222G+D223*, Q218D+M220N+E221G+V222 G+D223*, Q218D+M220D+D223*, Q218D+M220E+D223*, Q218D+M220H+D22 3*, Q218D+M220F+D223*, Q218D+M220W+D223*, Q218D+M220S+D223*, Q2 18D+M220A+D223*, Q218R+D223*, Q218R+M220N+D223*, Q218R+M220N+E 221G+D223*, Q218R+M220N+V222G+D223*, Q218R+M220N+E221G+V222G+ D223*, Q218R+M220D+D223*, Q218R+M220E+D223*, Q218R+M220H+D223*,It includes or consists of any one of Q218R+M220F+D223*, Q218R+M220W+D223*, Q218R+M220S+D223*, and Q218R+M220A+D223*.

[0073] The microorganism according to the invention preferably comprises a mutant degS gene, which encodes a DegS protein lacking a functional single binding domain, a functional phosphoacceptor domain and / or a functional ATPase domain. As shown in the following examples, simply providing a mutant degS gene is already sufficient to improve exopolysaccharide production and / or reduce exopolysaccharide degradation during the fermentation process. This was particularly surprising in view of WO2019221988, which explicitly publishes that such mutations of the degS gene do not result in an increase in the viscosity of the fermentation broth and are therefore not suitable for increasing and / or stabilizing exopolysaccharide production and / or reducing exopolysaccharide degradation during fermentation.

[0074] The DegS protein preferably lacks a functional single binding domain, a functional phosphoacceptor domain and / or a functional ATPase domain. The presence of these traits can be easily identified in the microorganism of the invention, preferably a microorganism of the genus Paenibacillus, by observing an increase in viscosity or retention of viscosity during fermentation compared to the corresponding wild-type strain, which can be easily achieved, for example, by introducing a mutation in the sensor DegS domain (IPR008595).

[0075] As mentioned above, the wild-type DegS protein contains a sensor DegS domain. This domain extends from amino acid position 10 to 165 according to the numbering of the protein sequence with the Uniprot identifier A0A074LBY4_PAEPO. Further information on the DNA-binding domain is available from the corresponding Pfam and InterPro databases. For example, in the case of the most preferred wild-type DegS protein sequence A0A074LBY4_PAEPO, the DNA-binding domain is predicted to contain two alpha-helical domains extending from positions 5 to 81 and 84 to 186, with the amino acids at positions 175 to 186 already overlapping with the histidine kinase domain. To prevent interference with the folding of the histidine kinase domain, it is preferred if the DNA-binding domain of the DegS protein is mutated in such a way that the entire alpha-helical structure is kept intact.

[0076] Preferably, the mutant DegS protein differs from the corresponding wild-type sequence by one or more mutations selected from, in order of decreasing priority, L99F, L99C, L99D, L99E, L99G, L99H, L99K, L99N, L99P, L99Q, L99R, L99S, L99W or L99Y.

[0077] For purposes of the present invention, the foregoing numbering refers to the wild-type DegS protein sequence of Uniprot identifier A0A074LBY4_PAEPO. It is noted that, as stated above, the mutant DegS protein has at least 40%, more preferably at least 46%, more preferably at least 58%, and even more preferably 80-100% sequence identity to the amino acid sequence represented by Uniprot identifier A0A074LBY4_PAEPO, ignoring the mutations specifically listed above.

[0078] The specific mutations mentioned above are contained in the predicted second alpha helix of the DegS sensor domain. As shown in the Examples, all such mutations result in an increase in the viscosity of the fermentation broth over the course of fermentation. Furthermore, even if the viscosity of the fermentation broth changes during the later stages of fermentation, it does not fall below 50% of the maximum viscosity of the fermentation broth of the wild-type strain, and preferably does not fall below 70% of the maximum viscosity of the fermentation broth of the wild-type strain, as shown in the Examples. The conditions for measuring the viscosity are shown in the Examples below.

[0079] Preferably, the mutation of the degS gene according to the present invention results in a higher peak viscosity compared to the wild-type strain, and more preferably, the increase in viscosity is not delayed compared to the wild-type strain, such that at the time of maximum viscosity of the wild-type fermentation broth, the viscosity of the corresponding DegS mutant fermentation broth is at least 90% of the viscosity of the wild-type fermentation broth, preferably 100% to 300% of the viscosity of the wild-type fermentation broth (see also FIG. 1).

[0080] The mutated amino acids of the DegS mutant proteins are listed above in order of increasing frequency of each in natural homologues of the DegS protein. Since the interest of the present invention is to provide microorganisms with modified properties of the DegS protein compared to the wild type, the least frequent modifications are the most preferred modifications, with the preference decreasing as the frequency of each amino acid at each position increases.

[0081] The microorganism according to the invention preferably comprises a mutant spo0A gene, the mutation being located in the DNA binding domain or in the receiver domain, leading to reduced or absent phosphorylation of the Spo0A protein. As shown in the examples below, simply providing a mutant spo0A gene is already sufficient to improve exopolysaccharide production and / or reduce exopolysaccharide degradation during the fermentation process. This was particularly surprising in view of WO2019221988 and WO2016154297. According to the latter publication, the parent strains of all DegU / DegS mutants of WO2019221988 already contained a spo0A mutation. In WO2016154297, this spo0A mutation was described as the reason for a stable colony morphology, while the parent strains showed various morphologies, such as a viscous slime-like phenotype. In contrast to our findings, where a wild-type strain was used for targeted insertion of point mutations, no increase or stabilization of the viscosity of the fermentation broth was observed in WO2019221988, especially given that in the latter publication an already randomly mutated strain was used as the parent strain for further genetic optimization.

[0082] The mutant Spo0A protein preferably lacks a functional DNA-binding domain or receiver domain, and the presence of these traits can be easily identified in the microorganism of the invention, preferably a microorganism of the genus Paenibacillus, by observing an increase in viscosity or retention of viscosity during fermentation compared to the corresponding wild-type strain, which can be easily achieved, for example, by introducing a mutation in the Spo0A C-terminal domain (IPR014879).

[0083] As mentioned above, the wild-type Spo0A protein contains the sporulation initiation factor Spo0A C-terminal domain, which extends from amino acid position 158 to 261 according to the numbering of the protein sequence with the Uniprot identifier A0A074LZY6_PAEPO. Further information on the Spo0A C-terminal domain is available from the corresponding Pfam and InterPro databases mentioned above.

[0084] Preferably, the mutation in the mutant Spo0A protein is A257V, more preferably A257S, or - I161R, more preferably I161L, or - consisting of or containing, in descending order of priority, any of: A257S+I161I, A257A+I161L, A257V+I161I, A257S+I161F or A257A+I161R.

[0085] For the purposes of the present invention, the aforementioned numbering refers to the wild-type Spo0A protein sequence of Uniprot identifier A0A074LZY6_PAEPO. It is noted that, as stated above, the mutant Spo0A protein has at least 55%, more preferably at least 60%, more preferably at least 62%, more preferably at least 70%, even more preferably 80-100%, even more preferably 95-100% sequence identity to the amino acid sequence shown by Uniprot identifier A0A074LZY6_PAEPO, ignoring the mutations specifically listed above.

[0086] Preferably, the mutant Spo0A protein comprises one of the two aforementioned mutations at position 257, i.e. A257V, or more preferably A257S. This position is in the last predicted alpha-helix of the Spo0A C-terminal domain. Also preferably, the mutant Spo0A protein comprises one of the two aforementioned mutations at position 161, i.e. I161R, or more preferably I161L. This position is in the first predicted alpha-helix of the Spo0A C-terminal domain. More preferably, the mutant Spo0A protein comprises any of the aforementioned respective mutations at each of the aforementioned positions, i.e., in descending order of preference, A257S+I161I, A257A+I161L, A257V+I161I, A257S+I161F, or A257A+I161R. The mutated amino acids of the double mutants are listed in ascending order of their respective frequency in natural homologs of the Spo0A protein. Since the interest of the present invention is to provide microorganisms having modified properties of the Spo0A protein compared to the wild type, the least frequent modifications are the most preferred modifications, with the preference decreasing as the frequency of each amino acid at each position increases.

[0087] Typically, the aforementioned mutations in the Spo0A protein cause a delay in the time until the fermentation broth viscosity reaches its maximum, compared to the wild type. Furthermore, even if the viscosity of the fermentation broth changes in the later fermentation stages, it does not fall below 50% of the maximum viscosity of the fermentation broth of the wild type strain, preferably not below 70% of the maximum viscosity of the fermentation broth of the wild type strain, as shown in the examples. The conditions for measuring the viscosity are shown in the examples below.

[0088] As shown in the examples, the advantages obtainable by the present invention can be further improved by providing a microorganism that contains mutations in two genes rather than just one, in particular resulting in increased and / or stabilised exopolysaccharide production and / or reduced exopolysaccharide degradation.

[0089] Furthermore, the present invention provides a microorganism comprising both a mutant degU gene and a mutant degS gene, a mutant degU gene and a mutant spo0A gene, a mutant degS gene and a mutant spo0A gene, or a mutant degU gene, a mutant degS gene and a mutant spo0A gene. As exemplified in the examples, by providing a microorganism comprising mutations in two genes rather than only one gene, the advantages obtainable by the present invention can be further improved, in particular increasing and / or stabilizing exopolysaccharide production and / or reducing exopolysaccharide degradation. For example, a microorganism comprising both a mutant DegU protein and a mutant DegS protein can achieve a more stable viscosity in the later fermentation stage. Also, as shown in the examples, a microorganism comprising a mutant DegU protein, a mutant DegS protein and a mutant Spo0A protein can reach a maximum viscosity earlier than a corresponding Spo0A single gene mutant microorganism. Also, as an example, a microorganism carrying a mutant DegS can reach a significantly higher viscosity compared to a strain carrying a wild-type degS gene.

[0090] According to the invention, it is particularly preferred to cause an increase in the viscosity of the fermentation medium when the microorganism is grown in a liquid fermentation medium, such that within 48 hours of the maximum carbon transfer rate in batch fermentation, the viscosity of the fermentation medium remains 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80% higher than the maximum fermentation medium viscosity obtained in the fermentation of the corresponding wild-type strain, thus advantageously increasing exopolysaccharide production and / or advantageously reducing exopolysaccharide degradation of the microorganism of the invention.

[0091] The microorganism according to the invention is preferably - the phylum Firmicutes, the class Bacilli, the class Clostridia or the class Negativicutes, more preferably from the orders Bacillales, Clostridiales, Thermoanaerobacterales, Thermosediminibacterales or Selenomonadales, more preferably from the family Bacillaceae, Paenibacillaceae, Pasteuriaceae, Clostridiaceae, Peptococcaceae, Heliobacteriaceae, Syntrophomonadaceae, Thermoanaerobacteraceae, Tepidanaerobacteraceae or Sporomusaceae, More preferably, the genus Alkalibacillus, Bacillus, Geobacillus, Halobacillus, Lysinibacillus, Piscibacillus, Terribacillus, Brevibacillus, Paenibacillus, Thermobacillus, Pasteuria, Clostridium, the genus Ostridium, the genus Desulfotomaculum, the genus Heliobacterium, the genus Pelospora, the genus Pelotomaculum, the genus Caldanaerobacter, the genus Moorella, the genus Thermoanaerobacter, the genus Tepidanaerobacter, the genus Propionispora or the genus Sporomusa, More preferably, it is selected from the taxonomic ranks of the genera Bacillus, Paenibacillus or Clostridium.

[0092] In particular, microorganisms of the families Bacillaceae, Paenibacillaceae and Clostridiaceae are known to produce exopolysaccharides and are important microorganisms in industrial fermentation processes. Furthermore, spore-forming strains are known among these genera.

[0093] In agriculture, bacterial spores have been used in plant pest control compositions to reduce or prevent plant pathogenic fungal or bacterial diseases. Spore biopreparations are also applied to improve plant resistance to biotic and abiotic stresses, accelerate plant growth, and increase yields at harvest of plants, fruits, or legumes. Spore products have been applied to leaves, shoots, fruits, roots, or plant propagation materials and substrates on which plants grow (Toyota K. Bacillus-related Spore Formers: Attractive Agents for Plant Growth Promotion. Microbes Environ. 2015;30(3):205-207. doi:10.1264 / jsme2.me3003rh).Bochow, H., et al. "Use of Bacillus Subtilis as a Biocontrol Agent. IV. Salt-Stress Tolerance Induction by Bacillus Subtilis FZB24 Seed Treatment in Tropical Vegetable Field Crops, and Its Mode of Action / The use of Bacillus Subtilis for biological control. IV. Induction of salt stress tolerance by application of Bacillus subtilis FZB24 in tropical field vegetables and its mechanism of action." Journal of Plant Diseases and Protection, vol. 108, no. 1, 2001, pp. 21-30. JSTOR, www.jstor.org / stable / 43215378. Accessed 14 Dec. 2020.) (Hashem, Abeer & Tabassum, B. & Abd_Allah, Elsayed. (2019). Bacillus subtilis: A plant-growth promoting rhizobacterium that also impacts biotic stress.Saudi Journal of Biological Sciences.26.10.1016 / j.sjbs.2019.05.004.)。

[0094] Furthermore, bacterial spores have been applied in the field of nanobiotechnology and building chemistry, for example for self-healing concrete (repairing cracks), stabilizing mortar and reducing water permeability [JYWang, H.Soens, W.Verstraete, N.De Belie,Self-healing concrete by use of microencapsulated bacterial spores,Cement and Concrete Research,Volume 56,2014,139-152,ISSN 0008-8846,https: / / doi.org / 10.1016 / j.cemconres.2013.11.009][Ricca E,Cutting SM.Emerging Applications of Bacterial Spores in Nanobiotechnology.J Nanobiotechnology.2003;1(1):6.Published 2003 Dec 15.doi:10.1186 / 1477-3155-1-6].

[0095] In addition, bacterial spores have been applied in the area of ​​cleaning products such as for cleaning laundry, hard surfaces, sanitary equipment and for odor control in clinical and domestic environments (Caselli E. Hygiene: microbial strategies to reduce pathogens and drug resistance in clinical settings. Microb Biotechnol. 2017 Sep;10(5):1079-1083. doi:10.1111 / 1751-7915.12755. Epub 2017 Jul 5). As an example, spores have been used in cosmetic compositions, such as skin cleaning products (US20070048244), dishwashing agents (WO2014 / 107111), pipe degreasers (DE19850012), laundry malodor control (WO2017 / 157778 and EP3430113) or allergen removal (US20020182184). Spores can also be embedded in matrices of non-biological origin to catalyze subsequent matrix degradation.

[0096] In addition, bacterial spores have been applied in the areas of human and animal nutrition and health. As an example, various bacterial strains have been applied to broilers as part of an antibiotic replacement strategy (Neveling, D.P., Dicks, L.M. Probiotics: an Antibiotic Replacement Strategy for Healthy Broilers and Productive Rearing. Probiotics & Antimicro. Prot. 13, 1-11 (2021). https: / / doi.org / 10.1007 / s12602-020-09640-z). Other examples include aquaculture, pigs, and many more (Nayak, SK (Nayak, SK (2021), Multifaceted applications of probiotic Bacillus species in aquaculture with special reference to Bacillus subtilis. Rev. Aquacult., 13:862-906. https: / / doi.org / 10.1111 / raq.12503). The application of bacterial spores to human health has also been extensively described (e.g., US Patent Publication No. 20180289752; Lee, NK., Kim, WS. & Paik, HD. Bacillus strains as human probiotics: characterization, safety, microbiome, and probiotic carrier. Food Sci Biotechnol 28, 1297-1305 (2019). https: / / doi.org / 10.1007 / s10068-019-00691-9).

[0097] It is therefore a particular advantage of the present invention that the mutations described herein allow for increased and / or stabilized exopolysaccharide production and / or reduced exopolysaccharide degradation in such industrially relevant organisms. With the aid of microbial exopolysaccharides, bacterial spores are further protected from biotic and abiotic stresses and aided in germination and reproduction.

[0098] Particularly preferred are microorganisms of one of the following species:

[0099] Paenibacillus species: P. abekawaensis, P. abyssi, P. aceris, P. aceti, P. aestuarii, P. agarexedens, P. agaridevorans, P. alba, P. albidus, P. albus, P. alginolyticus cus, P. algorifonticola, P. alkaliterrae, P. alvei, P. amylolyticus, P. anaericanus, P. antarcticus, P. antibioticophila, P. antri, P. apiaries, P. apiarius ), P. apis, P. aquistagni, P. arachidis, P. arcticus, P. assamensis, P. aurantiacus, P. azoreducens, P. azotifigens, P. baekrokdamisoli, P. barcinonensis, P. barrensis, P. barengoltzii, P. beijingensis, P. borealis, P. bouchesdurhonensis, P. bovis, P. brasilensis, P. brassicae, P. bryophyllum, P. caespitis, P. camelliae, P. camerounensis.camerounensis, P. campinasensis, P. castaneae, P. catalpae, P. cathormii, P. cavernae, P. cellulosilyticus, P. cellulositrophicus, P. chartarius, P. chibensis, P. chinensis inensis, P.chinjuensis, P.chitinolyticus, P.chondroitinus, P.chungangensis, P.cineris, P.cisolokensis, P.contaminans, P.cookii, P.crassostreae, P.cucumis s), P. curdlanolyticus, P. daejeonensis, P. dakarensis, P. darangshiensis, P. darwinianus, P. dauci, P. dendritiformis, P. dongdonensis, P. donghaensis, P. dosanensis. doosanensis, P. durus, P. edaphicus, P. ehimensis, P. elgii, P. elymi, P. endophyticus, P. enshidis, P. esterisolvens, P. etheri, P. eucommiae, P. faecis, P. favisporusfavisporus, P. ferrarius, P. filicis, P. flagellatus, P. fonticola, P. forsythiae, P. frigorieristens, P. fujiensis, P. fukuinensis, P. gansuensis, P. gelatinlyticus atinilyticus, P. ginsengagri, P. ginsengarvi, P. ginsengihumi, P. ginsengiterrae, P. glacialis, P. glebae, P. glucanolyticus, P. glycanilyticus, P. gorillae, P. graminis. graminis, P. granivorans, P. guangzhouensis, P. harenae, P. helianthi, P. hemerocallicola, P. herberti, P. hispanicus, P. hodogayensis, P. hordei, P. horti, P. fumicaceus, P. humicus, P. hunanensis, P. ihbetae, P. ihuae, P. ihumii, P. illinoisensis, P. insulae, P. intestini, P. jamilae, P. jilunlii, P. kobensis, P. koleovorans, P. konkukensis.konkukensis, P. konsidensis, P. koreensis, P. kribbensis, P. kyungheensis, P. lactis, P. lacus, P. larvae, P. lautus, P. lemnae, P. lentimorbus, P. lentus, P. liaoningensis P.liaoningensis, P.limicola, P.lupini, P.luteus, P.lutimineralis, P.macerans, P.macquariensis, P.marchantiophytorum, P.marinisediminis, P.marinum, P.massi liensis, P. maysiensis, P. medicaginis, P. mendelii, P. mesophilus, P. methanolicus, P. mobilis, P. montanisoli, P. montaniterrae, P. motobuensis, P. mucilaginosus, P. P. nanensis, P. naphthalenovorans, P. nasutitermitis, P. nebraskensis, P. nematophilus, P. nicotianae, P. nuruki, P. oceanisediminis, P. odorifer, P. oenotherae, P.P.oralis, P.oryzae, P.oryzisoli, P.ottowii, P.ourofinensis, P.pabuli, P.paeoniae, P.panacihumi, P.panacisoli, P.panaciterrae, P.paridis, P.pasadenensi s), P. pectinilyticus, P. peoriae, P. periandrae, P. phocaensis, P. phoenicis, P. phyllosphaerae, P. physcomitrellae, P. pini, P. pinihumi, P. pinisoli, P. pinistramentii, P. p inistramenti, P. pocheonensis, P. polymyxa, P. polysaccharolyticus, P. popilliae, P. populi, P. profundus, P. prosopidis, P. protaetiae, P. provencensis, P. psy chroresistens, P. pueri, P. puernese, P. puldeungensis, P. purispatii, P. qingshengii, P. qinlingensis, P. quercus, P. radicis, P. relictisesami, P. residui, P. rhizoplanae, P.rhizoplanae, P. rhizoryzae, P. rhizosphaerae, P. rigui, P. ripae, P. rubinfantis, P. ruminocola, . P. sabinae, P. sacheonensis, P. salinicaeni, P. sanguinis, P. sediminis, P. segetis, P. selenii, P. selenitireducens, P. senegalensis, P. senegalimassiliensis, P. seodonensis, P. septentrio P.septentrionalis, P.sepulcri, P.shenyangensis, P.shirakamiensis, P.shunpengii, P.siam ensis), P. silagei, P. silvae, P. sinopodophylli, P. solanacearum, P. solani, P. soli, P. sonchi group), P.sophorae, P.spiritus, P.sputi, P.stellifer, P.susongensis, P.swuensis, P.taichungensis, P.taihuensis, P.taiwanensis, P.taofu P. taohuashanense, P. tarimensis, P. telluris, P. tepidiphilus, P. terrae, P. terreus, P. terrigena, P. tezpurensis, P. thailandensis, P. thermoaerophilusthermoaerophilus, P. thermophilus, P. thiaminolyticus, P. tianmuensis, P. tibetensis, P. timonensis, P. translucens, P. tritici, P. triticisoli, P. tuaregi, P. tumbae mbae, P. tundrae, P. turicensis, P. tylopili, P. typhae, P. tyrfis, P. uliginis, P. urinalis, P. validus, P. velaei, P. vini, P. vortex, P. vorticalis, P. vulneris is), P. wenxiniae, P. whitsoniae, P. wooponensis, P. woosongensis, P. wulumuqiensis, P. wynnii, P. xanthanilyticus, P. xanthinilyticus, P. xerothermodurans, P. P. xinjiangensis, P. xylanexedens, P. xylaniclasticus, P. xylanilyticus, P. xylanisolvens, P. yanchengensis, P. yonginensis, P. yunnanensis, P. zanthoxyli, P.P. zeae, preferably P. agarexedens, P. agaridevorans, P. alginolyticus, P. alkaliterrae, P. alvei, P. amylolyticus, P. anaericanus, P. antarcticus, P. assamensis, P. azo P. azoreducens, P. barcinonensis, P. borealis, P. brassicae, P. campinasensis, P. chinjuensis, P. chitinolyticus, P. chondroitinus, P. cineris, P. curdlanolyticus icus, P. daejeonensis, P. dendritiformis, P. ehimensis, P. elgii, P. favisporus, P. glucanolyticus, P. glycanilyticus, P. graminis, P. granivorans, P. hodo gayensis, P. illinoisensis, P. jamilae, P. kobensis, P. koleovorans, P. koreensis, P. kribbensis, P. lactis, P. larvae, P. lautus, P. lentimorbus, P. macerans, P.P. macquariensis, P. massiliensis, P. mendelii, P. motobuensis, P. naphthalenovorans, P. nematophilus, P. odorifer, P. pabuli, P. peoriae, P. phoenicis, P. phyllosphaerae, P. polymyxa, P. popilliae, P. rhizosphaerae P. rhizosphaerae, P. sanguinis, P. stellifer, P. taichungensis, P. terrae, P. thiaminolyticus, P. timonensis, P. tylopili, P. turicensis, P. validus, P. vortex, P. vulneris, P. wynnii, P. xylanilyticus,. Particularly preferred are Paenibacillus koreensis, Paenibacillus rhizosphaerae, Paenibacillus polymyxa, Paenibacillus amylolyticus, Paenibacillus terrae, Paenibacillus polymyxa polymyxa, Paenibacillus polymyxa plantarum, Paenibacillus nov.spec epiphyticus, Paenibacillus terrae. terrae), Paenibacillus macerans, Paenibacillus alvei, More preferred are Paenibacillus polymyxa, Paenibacillus polymyxa polymyxa, Paenibacillus polymyxa plantarum, Paenibacillus nov.spec epiphyticus, Paenibacillus terrae, Paenibacillus macerans, Paenibacillus alvei, Even more preferred are Paenibacillus polymyxa, Paenibacillus polymyxa polymyxa, Paenibacillus polymyxa plantarum and Paenibacillus terrae.

[0100] Bacillus species: B. abyssalis, B. acanthi, B. acidiceler, B. acidicola, B. acidiproducens, B. aciditolerans, B. acidopullulyticus, B. acidovorans, B. aeolius, B. aequo B. aequororis, B. aeris, B. aerius, B. aerolacticus, B. aestuarii, B. aidingensis, B. akibai, B. alcaliinulinus, B. alcalophilus, B. algicola, B. alkalicola , B.alkalilacus, B.alkalinitrilicus, B.alkalisediminis, B.alkalitelluris, B.alkalitolerans, B.alkalogaya, B.altitudinis, B.alveayuensis, B.amiliensis sis, B. andreesenii, B. andreraoultii, B. aporrhoeus, B. aquimaris, B. arbutinivorans, B. aryabhattai, B. asahii, P. aurantiacus, B. australimaris, B. azotoformans, B.azotoformans, B. bacterium, B. badius, B. baekryungensis, B. bataviensis, B. benzoevorans, B. beringensis, B. berkeleyi, B. beveridgei, B. bingmayongensis, B. bogoriensis, B. .bogoriensis, B.borbori, B.boroniphilus, B.butanolivorans, B.cabrialesii, B.caccae, B.camelliae, B.campisalis, B.canaveralius, B.capparidis, B.carboniphilus , B. casamancensis, B. caseinilyticus, B. catenulatus, B. cavernae, B. cecembensis, B. cellulosilyticus, B. chagannorensis, B. chandigarhensis, B. cheonanensis ), B.chungangensis, B.ciccensis, B.cihuensis, B.circulans, B.clausii, B.coagulans, B.coahuilensis, B.cohnii, B.composti, B.coniferum, B.coreaensis, B.B. crassostreae, B. crescens, B. cucumis, B. dakarensis, B. daliensis, B. danangensis, B. daqingensis, B. decisifrondis, B. decolorationis, B. depressus, B. delamifica B.deramificans, B.deserti, B.dielmoensis, B.djibelorensis, B.drentensis, B.ectoiniformans, B.eiseniae, B.enclensis, B.endolithicus, B.endophyticus, B. B.endoradicis, B.endozanthoxylicus, B.farraginis, B.fastidiosus, B.fengqiuensis, B.fermenti, B.ferrariarum, B.filamentosus, B.firmis, B.firmus, B.flavocaldarius, B.flexus, B.foraminis, B.fordii, B.formosensis, B.fortis, B.freudenreichii, B.fucosivorans, B.fumarioli, B.funiculus, B.galactosidialicus, B.galactosidilyticus, B. galliciensis, B. gibsonii, B. ginsenggisoli, B. ginsengihumi, B. ginsengisoli, B. glennei, B. glycinifermentans, B. gobiensis, B. gossypii, B. gotteiri B. gottheilii, B. graminis, B. granadensis, B. hackensackii, B. haikouensis, B. halmapalus, B. halodurans, B. halosaccharovorans, B. haynesii, B. hemicellulosilyticus us), B. hemicentroti, B. herbersteinensis, B. hisashii, B. horikoshii, B. horneckiae, B. horti, B. huizhouensis, B. humi, B. hunanensis, B. hwajinpoensis, B. idriensis (B.idriensis), B.indicus, B.infantis, B.infernus, B.intermedius, B.intestinalis, B.iocasae, B.isabeliae, B.israeli, B.jeddahensis, B.jeotgali, B.keksaeae, B.kexueae, B.kiskunsagensis, B.kochii, B.kokeshiiformis, B.koreensis, B.korlensis, B.kribbensis, B.krulwichiae, B.kwashiorkori, B.kyonggiensis, B .B. lacisalsi, B. lacus, B. lehensis, B. lentus, B. ligniniphilus, B. lindianensis, B. litoralis, B. loiseleuriae, B. lonarensis, B. longiquaesitum,. B. longisporus, B. luciferensis, B. luteolus, B. luteus, B. lycopersici, B. magaterium, B. malikii, B. mangrovensis, B. mangrovi, B. mannanilyticus, B. manus ensis, B. marasmi, B. marcorestinctum, B. marinisedimentorum, B. marisflavi, B. maritimus, B. marmarensis, B. massiliglaciei, B. massilioanorexius, B. massiliogabonensis siliogabonensis, B. massiliogorillae, B. massilionigeriensis, B. massiliosenegalensis, B. mediterraneensis, B. megaterium, B. mesonae, B. mesophyllum, B. mesophilus ... B.methanolicus, B.miscanthi, B.muralis, B.murimartini, B.nakamurai, B.nanhaiisediminis, B.natronophilus, B.ndiopicus, B.nealsonii, B.nematocida, B.niavensis, B.niabensis, B. niacini, B. niameyensis, B. nitritophilus, B. notoginsengisoli, B. novalis, B. obstructivus, B. oceani, B. oceanisediminis, B. ohbensis, B. okh ensis, B.okuhidensis, B.oleivorans, B.oleronius, B.olivae, B.onubensis, B.oryzae, B.oryzaecorticis, B.oryzisoli, B.oryziterrae, B.oshimensis, B.pakistanensis kistanensis, B. panacisoli, B. panaciterrae, B. paraflexus, B. patagoniensis, B. persicus, B. pervagus, B. phocaeensis, B. pichinotyi, B. piscicola, B. piscis, B. placorcidea B.plakortidis, B.pocheonensis, B.polygoni, B.polymachus, B.populi, B.praedii, B.pseudalcaliphilus, B.pseudofirmus, B.pseudoflexus, B.pseudomegaterium, B.B. psychrosaccharolyticus, B. pumilus, B. purgationiresistens, B. qingshengii, B. racemilacticus, B. rhizosphaerae, B. rigiliprofundi, B. rubiinfantis, B. ruricus ... uris, B.safensis, B.saganii, B.salacetis, B.salarius, B.salidurans, B.salis, B.salitolerans, B.salmalaya, B.salsus, B.sediminis, B.selenatarsenatis, B. B. senegalensis, B. seohaeanensis, B. shacheensis, B. shackletonii, B. shandongensis, B. shivajii, B. similis, B. simplex, B. sinesaloumensis, B. siralis, B. smithii (B.smithii), B.solani, B.soli, B.solimangrovi, B.solisilvae, B.songklensis, B.spongiae, B.sporothermodurans, B.stamsii, B.subterraneus, B.swezeyi, B.taeanensis, B.taeanensis, B. taiwanensis, B. tamaricis, B. taxi, B. terrae, B. testis, B. thaonhiensis, B. thermoalkalophilus, B. thermoamyloliquefaciens, B. thermoamylovorans, B. thermocopriae, B. thermolactis, B. thermophilus, B. thermoproteolyticus, B. thermoterrestris, B. thermozeamaize, B. thioparans, B. tianmuensis, B. tianshengyi enii, B. timonensis, B. tipchiralis, B. trypoxylicola, B. tuaregi, B. urumqiensis, B. vietnamensis, B. vini, B. vireti, B. viscosus, B. vitellinus, B. wakoensi s), B.weihaiensis, B.wudalianchiensis, B.wuyishanensis, B.xiamenensis, B.xiaoxiensis, B.zanthoxyli, B.zeae, B.zhangzhouensis, B.zhanjiangensis,. Preferably, Bacillus licheniformis, B. megaterium, B. subtilis, B. pumilus, B. firmus, B. thuringiensis, B. velezensis, B. linens, B. atrophaeus, B. amyloliquefaciens, B. aryabhattai, B. cereus, B. aquatilis, B. circulans, B. clausii, B. sphaericus, B. thiaminolyticus, B. mojavensis, B. vallismortis, B. coagulans, B. sonorensis, B. halodurans, B. pocheonensis, B. gibsonii, B. acidiceler, B. flexus, B. hunanensis, B. pseudomycoides, B. simplex, B. safensis, B. mycoides, Particularly preferred are B. amyloliquefaciens, B. licheniformis, B. thuringiensis, B. velezensis, B. subtilis and B. megatherium, Even more preferably, B. amyloliquefaciens, B. thuringiensis, B. velezensis and B. megatherium.

[0101] Clostridium species: C. autoethanogenum, C. beijerinckii, C. butyricum, C. carboxidivorans, C. disporicum, C. drakei, C. ljungdahlii, C. kluyveri, C. pasteurianum m), C. propionicum, C. saccharobutylicum, C. saccharoperbutylacetonicum, C. scatologenes, C. tyrobutyricum, preferably C. butyricum, C. pasteurianum and / or C. tyrobutyricum. m), C. aerotolerans, C. aminophilum, C. aminvalericum, C. celerecrescens, C. asparagforme, C. bolteae, C. clostridioforme, C. glycyrrhizinilyticum, C. (fungatella) hasewa C.(Hungatela)hathewayi, C.histolyticum, C.indolis, C.leptum, C.(Tyzzerella)nexile, C.perfringens, C.(Erysipelatoclostridium)ramosum, C.scindens, C.symbiosumsymbiosum, Clostridium saccharogumia, Clostridium sordellii, Clostridium clostridioforme, C. methylpentosum, C. islandicum and all members of Clostridium clusters IV, XIVa and XVIII, particularly preferred is C. butyricum.

[0102] Some suitable Bacillus and Paenibacillus strains are described and deposited in the following international patent applications: Spores of such microorganisms or any pesticidal variants thereof may be incorporated as spores in the compositions according to the invention: WO 2020200959: Bacillus subtilis or Bacillus amyloliquefaciens QST713 or fungicidal variants thereof, deposited under NRRL Accession No. B-21661. Bacillus subtilis QST713, its mutants, its supernatants and its lipopeptide metabolites and methods of their use to control plant pathogens and insects are fully described in U.S. Pat. Nos. 6,060,051, 6,103,228, 6,291,426, 6,417,163 and 6,638,910. In these patents, the strain is referred to as AQ713, which is synonymous with QST713; WO 2020102592: Bacillus thuringiensis strains NRRL B-67685, NRRL B-67687 and NRRL B-67688; WO 2019135972: Bacillus megatherium having deposit accession number NRRL B-67533 or NRRL B-67534; WO 2019035881: Paenibacillus sp. NRRL B-50972, Paenibacillus sp.) NRRL B-67129, Bacillus subtilis strain QST30002 and Bacillus subtilis strain NRRL B-50421 deposited under accession number; WO 2018081543: Bacillus psychrosaccharolyticus strains deposited under ATCC accession numbers PT A-123720 or PT A-124246; WO 2017151742: Bacillus subtilis assigned accession number NRRL B-21661; WO 2016106063: Bacillus pumilus NRRL B-30087; WO 2013152353: Bacillus sp. deposited as CNMC 1-1582; ​​WO 2013016361: Bacillus sp. strain SGI-015-F03 deposited as NRRL B-50760, Bacillus sp. strain SGI-015-H06 deposited as NRRL B-50761; WO 2020181053: Paenibacillus sp. NRRL B-67721, Paenibacillus sp. NRRL B-67723, Paenibacillus sp. NRRL B-67724, Paenibacillus sp. NRRL B-50374.

[0103] Most preferably, the microorganism is of the taxonomic genus Paenibacillus and is selected from the species Paenibacillus polymyxa, Paenibacillus polymyxa polymyxa, Paenibacillus polymyxa plantarum and Paenibacillus terrae. When the microorganism is a Paenibacillus microorganism, preferably the microorganism does not contain both the A257V Spo0A mutation and the Q218* DegU and / or L99F DegS mutations described according to the present invention. Even more preferably, the microorganism is not Paenibacillus sp. strain NRRL B-67304, Paenibacillus sp. strain NRRL B-67306 or Paenibacillus sp. NRRL B-67615. Strains NRRL B-67306 and NRRL B-67615 are described in WO2019221988. However, as mentioned above, this publication does not acknowledge the increased or stabilized exopolysaccharide production and / or reduced exopolysaccharide degradation as described in the present invention. All three strains are derived from Paenibacillus sp. strain NRRL B-67129, which contains the A257V mutation and further mutations in the spoOA gene according to Examples 22, 25 and Figure 16 of WO2016154297. More preferably, the microorganism is not derived from Paenibacillus strain NRRL B-67129 deposited with the NRRL on September 1, 2015.Thus, when the genome of a Paenibacillus microorganism of the invention (thus excluding any extrachromosomal nucleic acid, e.g., plasmids) is aligned to (a) the genome of Paenibacillus strain NRRL B-67129 and (b) the genome of a type strain of the species of that Paenibacillus microorganism, the sequence identity of the Paenibacillus microorganism is preferably higher to the type strain of each species than to Paenibacillus strain NRRL B-67129. The following type strains are preferred according to the invention:

[0104] [Table 2]

[0105] [Table 3]

[0106] [Table 4]

[0107] [Table 5]

[0108] [Table 6]

[0109] If the species cannot be determined with certainty for the microorganism, it is sufficient that the genome of the microorganism has greater sequence identity to the genome of any of the aforementioned preferred reference strains than to the genome of Paenibacillus strain NRRL B-67129.

[0110] The present invention also provides a method for increasing or stabilizing exopolysaccharide production in a microorganism, or for reducing or preventing exopolysaccharide degradation in a microorganism, comprising the steps of: a) a mutant DegU protein according to the present invention and as described herein, b) a mutant DegS protein according to the present invention as described herein, c) providing a microorganism with one or more of the mutant Spo0A proteins according to the present invention as described herein.

[0111] As described herein, the provision of such mutant proteins makes it possible to achieve the advantages conferred by the present invention, in particular the reduction of exopolysaccharide degradation in later fermentation stages and / or the stabilization of maximum fermentation broth viscosity. Thus, in case the microorganism further produces a substance of interest, such as one or more fusaricidins, the present invention advantageously allows for the harvesting of the fermentation broth when both the one maximum viscosity / exopolysaccharide content and the maximum concentration of the substance of interest have been reached, thereby eliminating the need to decide between either the maximum concentration of the substance of interest or the maximum exopolysaccharide content.

[0112] Similarly, the present invention also provides a method for producing microbial exopolysaccharides, comprising the steps of: i) fermenting a microorganism of the invention to produce a fermentation broth containing one or more exopolysaccharides, preferably one or more target fermentation products; and ii) concentrating one or more exopolysaccharides, and preferably also one or more target fermentation products, from said fermentation broth.

[0113] As described herein, a particular advantage of the microorganisms of the invention comprising alleles of the degU, degS and / or spoOA genes, as applicable, is that these microorganisms are capable of increasing or stabilizing exopolysaccharide production and / or reducing exopolysaccharide degradation. Thus, the invention advantageously allows for harvesting the fermentation at a point where both a high content of exopolysaccharide and a high content of the target fermentation product are recoverable. Using the teachings of the invention, harvest times can be extended until sufficient target fermentation product yields are reached without suffering losses in exopolysaccharide yields that would occur when using exopolysaccharide-degrading microorganisms.

[0114] Techniques for increasing exopolysaccharide production during fermentation and / or for concentrating exopolysaccharides are known to those skilled in the art. For example, Liang et al.,Recent Advances in Exopolysaccharides from Paenibacillus spp.:Production,Isolation,Structure,and Bioactivities, Mar.Drugs 2015,13,1847-1863,doi:10.3390 / md13041847;Sun et al.,Extraction of extracellular polymeric substances in activated sludge using sequential extraction. 2007, Texas either at the University of Alabama. Concentration typically involves precipitation or cation exchange resin extraction of the fermentation broth supernatant to obtain a crude exopolysaccharide fraction, which can be further purified, for example, by chromatography.

[0115] The present invention also provides a) a mutant DegU protein according to the present invention and as described herein, b) a mutant DegS protein according to the present invention as described herein, c) providing an expression vector comprising an expression cassette for expressing one or more of the mutant Spo0A proteins according to the present invention as described herein;

[0116] Such expression vectors make it possible to introduce the respective mutant genes into wild-type organisms, either in addition to or as a replacement for the respective wild-type genes. Thus, the expression vectors of the invention make it particularly easy to convert wild-type microorganisms into the microorganisms of the invention.

[0117] As mentioned above, the microorganisms according to the invention preferably have an agronomic relevance. Likewise, it is particularly advantageous that the invention provides a method for improving plant health, which method comprises: a) plant material and / or b) by application to a plant growing substrate.

[0118] The increased and / or stabilized exopolysaccharide production and / or reduced exopolysaccharide degradation of the microorganisms of the invention advantageously allows the residence time of the microorganisms at the place where they are initially applied to be increased. The probiotic microorganisms according to the invention can increase or stabilize exopolysaccharide production and / or reduce exopolysaccharide degradation. Thus, the microorganisms tend to be embedded in a larger exopolysaccharide coat compared to the corresponding wild-type microorganisms. The exopolysaccharides then prevent the movement of the microorganisms of the invention and protect them from, for example, being washed away by rainfall or blown away by wind.

[0119] Preferably, the microorganisms of the invention produce compounds beneficial to plants during fermentation or after application to plant material or plant growing substances, and therefore are preferably plant probiotic microorganisms.

[0120] The plant health promoting compositions of the present invention are preferably applied to plant material, preferably plant propagation material, by any of the following processes: dressing, spraying, coating, film coating, pelleting, dusting or immersion.

[0121] The plant probiotic microorganisms of the present invention are preferably applied to the surface of the plant, preferably to the leaves of the plant. Due to the improved exopolysaccharide production and / or reduced exopolysaccharide degradation, the probiotic microorganisms of the present invention will show increased immobility at the application site. This is particularly advantageous for providing an antipathogenic coating on the surface of the plant, most preferably on the leaf surface. Most preferably, the probiotic microorganisms of the present invention will colonize the surface of the plant, thereby further expanding their beneficial effect, preferably antipathogenic effect, on the plant. In this context, the microorganisms of the genus Paenibacillus of the present invention are particularly preferred, considering the protection they provide to the plant against fungal infection.

[0122] In addition to or as an alternative to the application of the probiotic microorganisms of the present invention to the plant surface, the probiotic microorganisms of the present invention are preferably applied to the plant cultivation substrate, most preferably to soil. Also, due to the improved exopolysaccharide production and / or reduced exopolysaccharide degradation of the microorganisms of the present invention, the microorganisms are retained at the application site for a longer period of time compared to the respective wild-type microorganisms. Therefore, most preferably, the probiotic microorganisms of the present invention colonize the plant cultivation substrate at the application site, thereby improving the quality of the plant growth substrate and making it more suitable for improving plant growth.

[0123] Thus, the present invention allows for improved soil fertility and / or improved yield consistency, thereby furthering the teachings of each of WO2015118516, WO2016044768 and WO2020163251, each of which is incorporated herein by reference.

[0124] Preferably, the plant beneficial compounds are the target fermentation products described herein. Such compounds and mixtures thereof have antibacterial, preferably antifungal properties. Thus, the microorganisms of the present invention, when applied to the plant cultivation substrate and / or plant material described herein, advantageously improve plant health and / or yield and / or yield consistency.

[0125] In particular, the microorganisms of the present invention are preferably used to prevent, delay and / or reduce infection of plant material, preferably whole plants, by phytopathogenic microorganisms, preferably fungi. Preferably, the microorganisms of the present invention are used to prevent, delay and / or reduce infection of plant material, preferably whole plants, by microorganisms selected from any of the following: - from the class Gammaproteobacteria, more preferably from the order Xanthomonadales, more preferably from the family Xanthomonadaceae, more preferably from the genus Xanthomonas; - from the class Sordariomycetes, more preferably from the order Hypocreales, more preferably from the family Nectriaceae, more preferably from the genus Fusarium; - from the class Sordariomycetes, more preferably from the order Glomerellales, more preferably from the family Glomerellaceae, more preferably from the genus Colletotrichum; - from the class Leotinomycetes, more preferably from the order Helotiales, more preferably from the family Sclerotiniaceae, more preferably from the genus Botrytis; - from the class Dothideomycetes, more preferably from the order Pleosporales, more preferably from the family Pleosporaceae, more preferably from the genus Alternaria; - from the class Dothideomycetes, more preferably from the order Pleosporales, more preferably from the family Phaeosphaeriaceae, more preferably from the genus Phaeosphaeria; - from the class Dothideomycetes, more preferably from the order Botryosphaeriales, more preferably from the family Botryosphaeriaceae, more preferably from the genus Macrophomina; - from the class Dothideomycetes, more preferably from the order Capnodiales, more preferably from the family Mycosphaerellaceae, more preferably from the genus Zymoseptoria; from the class Agraricomycetes, more preferably from the order Cantharellales, more preferably from the family Ceratobasidiaceae, more preferably from the genus Rhizoctonia or Thanatephorus; - from the class Pucciniomycetes, more preferably from the order Pucciniales, more preferably from the family Pucciniaceae, more preferably from the genus Uromyces or Puccinia; - from the class Ustilaginomycetes, more preferably from the order Ustilaginales, more preferably from the family Ustilaginaceae, more preferably from the genus Ustilago; - from the class Oomycota, more preferably from the order Pythiales, more preferably from the family Pythiaceae, more preferably from the genus Pythium; - from the class Oomycota, more preferably from the order Peronosporales, more preferably from the family Peronosporaceae, more preferably from the genera Phytophthora, Plasmopara or Pseudoperonospora; - from the class Eurotiomycetes, more preferably from the order Eurotiales or Onygenales, more preferably from the family Aspergillaceae, more preferably from the genera Aspergillus, Penicillium or Pseudopenicillium; Even more preferably, the genus Alternaria, the genus Botrytis, the genus Fusarium, the genus Sclerotinia or the genus Trichoderma.

[0126] The microorganisms of the present invention can be applied to the plant material and / or plant cultivation material in the form of active cells (i.e. non-sporulating cells that are metabolically active and capable of division). As described herein, the microorganisms can colonize the plant material and / or cultivation substrate and exert their beneficial properties. However, preferably, the microorganisms are applied in the form of spores of said microorganisms, optionally together with active cells of the microorganisms. The spores allow the microorganisms to withstand conditions that are unsuitable for the growth and survival of active cells. In particular, fermenter harvesting, downstream processing, storage and high pressure spraying are steps commonly used in the production of plant health products, each of which can cause a significant reduction in the content of live active cells. On the other hand, spores can easily survive these conditions and are therefore particularly suitable for the production of plant health products.

[0127] Accordingly, the present invention provides a plant health product comprising a microbial culture (preferably comprising spores and / or active cells) of one or more microorganisms of the present invention. The microorganisms may be included in the plant health product in the form of a mixed culture consisting of different species of microorganisms and / or different strains of a certain microorganism. Alternatively, the microbial culture is preferably a pure culture consisting of one microorganism of one species, and even more preferably consisting of one strain of one species of the microorganism of the present invention.

[0128] When at least one microorganism of the microbial culture produces spores, preferably, such spores are harvested.Recovery techniques such as centrifugal separation, filtration and device filtration are known to those skilled in the art.It is a particular advantage of the fermentation method of the present invention that high titer spores with high content of antifungal substances, especially fusaricidin, can be produced conveniently in a short time with low labor, and they have high antifungal activity.

[0129] It is also preferred to recover a cell-free suspension at the end of the fermentation process of the invention. Also, techniques for obtaining a cell-free suspension that are not known to the skilled person can be advantageously combined with the method for recovering the spores.

[0130] The present invention also provides plant health promoting compositions obtainable or obtained by the methods according to the present invention. As described herein, such compositions are surprisingly effective, and they are easy to produce, rapidly produced and cost-effective.

[0131] The plant health composition optionally further comprises a stabilizing agent (preferably one disclosed in WO2019222253A) and preferably one or more target fermentation products as described above.

[0132] Additionally, the plant health compositions of the present invention preferably include a) one or more microbial pesticides having fungicidal, bactericidal, virucidal and / or plant defense activator activity; b) one or more biochemical pesticides having fungicidal, bactericidal, virucidal and / or plant defense activator activity; c) one or more microbial pesticides having insecticidal, acaricidal, molluscicidal and / or nematicidal activity; d) one or more biochemical pesticides having insecticidal, acaricidal, molluscicidal, pheromone and / or nematicidal activity; e) further comprising one or more fungicides selected from respiratory inhibitors, sterol biosynthesis inhibitors, nucleic acid synthesis inhibitors, inhibitors of cell division and cytoskeleton formation or function, inhibitors of amino acid and protein synthesis, signal transduction inhibitors, lipid and membrane synthesis inhibitors, multi-site acting inhibitors, cell wall synthesis inhibitors, plant defense inducers and fungicides with unknown mechanism of action.

[0133] Further components a)-d) are described in WO2017137353, which is incorporated herein for purposes of listing the respective substances. Further component e) is described in WO2017137351, which is also incorporated herein for purposes of listing the respective fungicides.

[0134] The present invention also provides a method for producing exopolysaccharides, which comprises growing a microorganism according to the invention and optionally isolating the microorganism from the exopolysaccharides. Suitable methods for growing the microorganisms of the present invention, i.e. fermentation methods, are generally known to those skilled in the art. It is particularly advantageous that the present invention allows an improvement in the yield of exopolysaccharides to be achieved without fundamentally modifying the corresponding fermentation process.

[0135] As an extension of the advantages of the present invention, - production of exopolysaccharide compositions, - treatment of plants, plant leaves, plant roots and / or plant seeds, - inoculation of soil, preferably to increase soil fertility; - Improved yield consistency, - Treatment of underground layers; - Wastewater treatment, - Preparation of pharmaceutical or cosmetic carriers, - preparation of pharmaceutical or cosmetic compositions, - preparation of skin hydrating compositions, - Preparation of flocculants, - Preparation of food or feed additives, - preparation of antitumor agents; - Preparation of antioxidants Further provided is the use of a microorganism according to the invention for any of the following:

[0136] Particularly suitable methods of using exopolysaccharides and / or microorganisms to treat subterranean formations are described in WO2014176061. Particularly suitable methods of using exopolysaccharides and / or microorganisms to treat wastewater are described in WO2014160350, both of which are incorporated herein by reference.

[0137] The present invention also relates to a method for increasing or stabilizing exopolysaccharide production or preventing exopolysaccharide degradation of a microorganism selected from any of the taxonomic classes defined above, comprising: a) a mutant DegU protein according to the present invention, b) a mutant DegS protein according to the present invention; c) The mutant Spo0A protein according to the present invention The present invention provides for the use of one or more of the following:

[0138] Selected aspects of the present invention are further illustrated below by the following non-limiting examples. EXAMPLES

[0139] Example 1: Generation of mutants Strains and cultivation conditions A list of strains used for targeted integration of point mutations by CRISPR Cas9 in P. polymyxa is shown in Table 1. Targeted point mutations in the wild-type strain P. polymyxa DSM 365 were integrated according to the CRISPR Cas9 procedure described by Ruetering et. al (Ruetering et al., Tailor-made exopolysaccharides-CRISPR-Cas9 mediated genome editing in Paenibacillus polymyxa. Synth Biol (Oxf). 2017 Dec 21; 2(1): ysx007. doi: 10.1093 / synbio / ysx007). DSM 365 was obtained from the German Collection of Microorganisms and Cell Culture (DSMZ), Braunschweig, Germany. Cloning and propagation of plasmids was performed in either E. Coli DH5α or NEB (New England Biolabs, USA) Turbo. Transformation of P. polymyxa was performed by E. Coli S17-1 (DSMZ) mediated conjugation. Strains were grown in LB medium (10 g / L tryptone peptone, 5 g / L yeast extract, 5 g / L NaCl). 1.5% agar was used for plating. When necessary, media was supplemented with 50 μg / ml neomycin and / or 20 μg / mL polymyxin for counter selection of positive transformants and elimination of E. Coli after the conjugation procedure. Unless otherwise stated, P. polymyxa was grown at 30°C and 250 rpm, whereas E. Coli was grown at 37°C and 250 rpm. Strains were stored as frozen cultures containing 24% glycerol and kept at −80C for long-term storage.

[0140] [Table 7]

[0141] Joint Conjugation was performed between P. polymyxa (recipient strain) and E. Coli S17-1 (donor strain) carrying the desired plasmid according to the CRISPR Cas9 procedure described in Ruetering et al. 2017 (Ruetering M, Cress BF, Schilling M, Ruehmann B, Koffas MAG, Sieber V, Schmid J. Tailor-made exopolysaccharides-CRISPR-Cas9 mediated genome editing in Paenibacillus polymyxa. Synth Biol (Oxf). 2017 Dec 21; 2(1): ysx007. doi: 10.1093 / synbio / ysx007. PMID: 32995508; PMCID: PMC7445874). Verification of error-free conjugants was performed by colony PCR and sequencing of DNA fragments. Plasmid curing was performed by subculturing the positive mutants at 1:100 in LB liquid medium at 37°C.

[0142] Plasmid construction Targeted point mutations were achieved by the CRISPR-Cas9 mediated system. The selected gRNA sequences were chosen based on their location closest to the target position in the degU, degS or spo0A genes. Plasmids were assembled by isothermal Gibson assembly. The desired point mutations were introduced from the primers used for PCR of the homologous flanking. For degS and spo0A, some silent mutations were also introduced in the primers to improve the efficiency of the system. The homologous flanking was obtained by PCR of P. polymyxa genomic DNA approximately 1 kbp upstream and downstream of the target nucleotide. The Gibson assembly mixture was transformed into E. coli DH5α or Turbo and plated on LB plates containing 50 μg / ml neomycin. Screening of positive colonies was performed by colony PCR. Plasmids were isolated by miniprep and verified by sequencing for further confirmation. The error-free plasmid was used to transform E. coli S17-1, which would then mediate transformation into P. polymyxa.

[0143] Using the pCasPP vector system and homologous flanks, each carrying 1000 bp around the genomic sequence flanking the targeted point mutation region, the following mutations were generated (Table 2):

[0144] [Table 8]

[0145] Example 2: Fermentation conditions Characterization of the mutants was carried out in a 21 l bioreactor (Techfors, Infors) filled with 12 l of exopolysaccharide production medium adapted from Ruetering et.al 2017. The composition of the fermentation medium is listed in Table 3.

[0146] [Table 9]

[0147] Fermentation was carried out for 40 h at 30° C., pH was set to 6.8 and adjusted with H3PO4 (25%) and NaOH (1 M). As precultures, all variants were grown in baffled 1 L shake flasks containing 100 ml of modified TSB medium (30 g / L TSB from Becton Dickenson Art. No. 211825, 3 g / L yeast extract, 20.9 g / L MOPS buffer, 10 g / L glucose) at 33° C., 150 rpm / 2.5 cm throw for 24 h.

[0148] In the bioreactor, a target dissolved oxygen level was set at 30% or higher in the agitator-gas flow cascade. To prevent shearing of the exopolysaccharides produced, agitation was limited to 300–600 rpm while using an agitator mechanism consisting of two propellers and one Rushton, which was located near the agitator shaft. To maintain oxygen supply, aeration was performed at 5–30 l / min at a pressure of 0.5 bar. Struktol J673 (Schill+Seilacher “Struktol” GmbH, Germany) was used as an antifoam agent. Culture samples were taken every 4 h for rheological viscosity analysis and further offline analysis.

[0149] Example 3: Rheological analysis of culture broth viscosity Rheological analysis of broth viscosity was performed every 4 hours during fermentation using an Anton Paar MCR302 rheometer in double slid geometry (measuring cup: C-DG26.7 / SS / air, temperature: 30°C, sample volume: 5 ml of total culture broth). Samples were preconditioned in a preshear experiment for 100 seconds at a constant shear rate of 10 s-1. Ten data points were recorded every 10 seconds. After preconditioning, viscosity was measured as a function of shear rate. Thus, the shear rate was logarithmically increased from 1 s-1 to 100 s-1 while a total of 25 data points were recorded. Culture broth viscosity measured over the course of fermentation time is shown in Figure 1.

[0150] Example 4: Determination of carbon transfer rates Carbon transfer rate (CTR, mmol / l*h-1) was evaluated every 5 min in the headspace of the 21 L fermenter of Example 2 using a mass spectrometer according to the protocol of Anderlei et al. (Anderlei, Tibor & Zang, Werner & Papaspyrou, Manfred & Buechs, Jochen. (2004). Online respiration activity measurement (OTR, CTR, RQ) in shake flasks. Biochemical Engineering Journal. 17.187-194.10.1016 / S1369-703X(03)00181-5). Carbon transfer rate was used as an online indicator of the metabolic activity of the strain. Carbon transfer rate and viscosity profiles of Paenibacillus DSM365 are exemplarily shown in Figure 2. Maximum viscosity of the fermentation broth is reached after maximum CTR. This behavior is also found in the degU , degS , and spo0A mutants discussed in Fig. 1 (data not shown).

Claims

1. A microorganism comprising a mutant degU gene and / or a mutant degS gene, and optionally further comprising a mutant spoOA gene, A microorganism that exhibits increased and / or stabilized exopolysaccharide production and / or reduced exopolysaccharide degradation.

2. 2. The microorganism of claim 1, comprising a mutant degU gene, - the degU gene encodes a DegU protein that has reduced DNA-binding activity and / or lacks a functional DNA-binding domain, and / or - the degU gene encodes the DegU protein, mutations of which, for each of options a) and b), in decreasing order of priority, are: a) Q218*, Q218K, Q218N, Q218D, Q218R, and / or b) A microorganism comprising or consisting of one or more of D223*, D223*+M220N, D223*+M220N+E221G, D223*+M220N+V222G, D223*+M220N+E221G+V222G, D223*+M220D, D223*+M220E, D223*+M220H, D223*+M220F, D223*+M220W, D223*+M220S, D223*+M220A.

3. 2. The microorganism of claim 1, comprising a mutant degS gene, - the degS gene encodes a DegS protein that lacks a functional single binding domain, a functional phosphoacceptor domain and / or a functional ATPase domain, and / or a microorganism, the degS gene encoding the DegS protein, the mutations of which comprise or consist of L99F, L99C, L99D, L99E, L99G, L99H, L99K, L99N, L99P, L99Q, L99R, L99S, L99W or L99Y.

4. 2. The microorganism of claim 1, comprising a mutant spoOA gene, a) the mutation is located in the DNA binding domain or in the receiver domain and leads to reduced or abolished phosphorylation and / or reduced or abolished dimerization of the SpoOA protein; and / or b) the mutation is A257V, more preferably A257S, or I161R, more preferably I161L, or - A microorganism consisting of or comprising, in descending order of preference, A257S+I161I, A257A+I161L, A257V+I161I, A257S+I161F or A257A+I161R.

5. 2. The microorganism of claim 1, wherein the microorganism causes an increase in viscosity of the fermentation medium when grown in a liquid fermentation medium, such that the viscosity of the fermentation medium remains 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80% higher than the maximum fermentation medium viscosity obtained in fermentation of the corresponding wild-type strain, preferably for 48 hours after reaching a maximum carbon transfer rate (CTR) during batch fermentation.

6. The microorganism is - the phylum Firmicutes, the class Bacilli, the class Clostridia or the class Negativicutes, more preferably from the orders Bacillales, Clostridiales, Thermoanaerobacterales, Thermosediminibacterales or Selenomonadales, more preferably from the family Bacillaceae, Paenibacillaceae, Pasteuriaceae, Clostridiaceae, Peptococcusceae, Heliobacteriaceae, Syntrophomonadaceae, Thermoanaerobacteraceae, Tepidanaerobacteraceae or Sporomusaceae, More preferably, the genus Alkalibacillus, Bacillus, Geobacillus, Halobacillus, Lysinibacillus, Piscibacillus, Terribacillus, Brevibacillus, Paenibacillus, Thermobacillus, Pasteuria, Clostridium (Cl the genus Stridium, the genus Desulfotomaculum, the genus Heliobacterium, the genus Pelospora, the genus Pelotomaculum, the genus Cardanaerobacter, the genus Moorella, the genus Thermoanaerobacter, the genus Tepidanaerobacter, the genus Propionispora or the genus Sporomusa, more preferably of the genus Bacillus, Paenibacillus or Clostridium The microorganism according to claim 1, which is selected from the taxonomic ranks of

7. A method for increasing or stabilizing exopolysaccharide production in a microorganism, or a method for reducing or preventing exopolysaccharide degradation in a microorganism, comprising: a) the mutant DegU protein according to claim 2; b) the mutant DegS protein according to claim 3; c) The mutant Spo0A protein according to claim 4 to said microorganism.

8. a) the mutant DegU protein according to claim 2; b) the mutant DegS protein according to claim 3; c) The mutant Spo0A protein according to claim 4 An expression vector comprising an expression cassette for expressing one or more of the above.

9. The microorganism according to claim 1, a) plant material and / or b) Plant cultivation substrate 10. A method for improving plant health, comprising applying

10. A method for producing exopolysaccharides, comprising: i) growing the microorganism of claim 1, and ii) optionally separating the microorganism from the exopolysaccharide. A method comprising:

11. Use of the microorganism according to claim 1, or the degU gene or protein according to claim 2 and / or the degS gene or protein according to claim 3 and / or the spoOA gene or protein according to claim 4, - production of exopolysaccharide compositions, - treatment of plants, plant leaves, plant roots and / or plant seeds, - soil inoculation, preferably to increase soil fertility; - Improved yield consistency, - Treatment of underground layers, - wastewater treatment, - preparation of pharmaceutical or cosmetic carriers, - preparation of pharmaceutical or cosmetic compositions, - preparation of skin hydrating compositions, - preparation of the flocculant; - preparation of food or feed additives; - preparation of antitumor agents, - Preparation of antioxidants For either use.

12. A method for increasing or stabilizing the production of exopolysaccharides or preventing the decomposition of exopolysaccharides of a microorganism selected from any one of the taxonomic classes according to claim 6, a) the mutant DegU protein according to claim 2; b) the mutant DegS protein according to claim 3; c) The mutant Spo0A protein according to claim 4 Use of one or more of the following:

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