Improving mating competence in Firmicutes

Abstract

The present invention relates to microorganisms, genes, materials and methods for improving gene competence. In particular, the present invention provides individual genes / proteins and combinations thereof for improving mating competence in Firmicutes, as well as methods and uses comprising such genes, proteins and respective combinations.

Description

[Technical field]

[0001] The present invention relates to microorganisms, genes, materials and methods for improving gene competence. In particular, the present invention provides individual genes / proteins and combinations thereof for improving mating competence in Firmicutes, as well as methods and uses comprising such genes, proteins and respective combinations. [Background technology]

[0002] Firmicutes microorganisms are important microorganisms in industrial fermentation processes. It is therefore commonly necessary to manipulate the nucleic acids of such microorganisms, for example to enable them to produce a substance of interest or to prevent or reduce the production of undesirable substances during fermentation. However, the introduction of nucleic acids is notoriously difficult. Three main mechanisms of nucleic acid transfer have been applied so far: (1) transformation techniques that attempt to directly introduce naked DNA into the microorganism, for example by electroporation; (2) bacteriophage-mediated transduction; and (3) bacteria-mediated conjugation. Direct transformation techniques such as electroporation have not yielded consistently high efficiency in Firmicutes, and some genera, for example Paenibacillus, are even generally considered to be impossible to transform by conventional techniques. Bacteriophage-mediated transduction is relatively cumbersome and has limitations on the size of the nucleic acid transfer due to the limited capsule volume of the bacteriophage and the need for additional nucleic acid elements required for transfer by the bacteriophage transfer mechanism. Conjugation is therefore considered to be the transfer method of choice for microorganisms that have not been individually rendered competent by exhaustive mutagenesis. In conjugation, target nucleic acid is provided in a first microorganism ("donor microorganism") for transfer to a second microorganism ("target microorganism"), which is more suitable for genetic manipulation than the target microorganism. After mixing the donor and target microorganisms, a plasma bridge is formed between the donor and target microorganisms, which allows the transfer of linear DNA, plasmid DNA and / or chromosomal bacterial DNA. Although conjugation techniques overcome many of the sometimes insurmountable obstacles associated with transformation techniques, they generally have low transfer efficiencies. Thus, conjugation cannot efficiently transform a collection of different target microorganisms. However, this is required in high-throughput environments, for example for the manipulation and subsequent analysis of libraries of future production hosts. In particular, microorganisms of the genus Paenibacillus are known to have low conjugation efficiencies. Summary of the Invention [Problem to be solved by the invention]

[0003] It was therefore an object of the present invention to provide materials and methods, in particular genes, nucleic acids and proteins, for improving the transformability of Firmicutes microorganisms, preferably of the genus Paenibacillus. [Means for solving the problem]

[0004] The present invention relates to a) a mutant degS gene and optionally a mutant degU gene, or b) Mutant spo0A gene A microorganism comprising any one of Microorganisms are provided that exhibit increased mating competence compared to the corresponding wild-type strain.

[0005] The present invention also provides a method for increasing the mating competence of a microorganism, comprising the steps of: a) 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, and optionally a mutated 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 ab)D223*, D223*+M220N, D223*+M220N+E221G, D223*+M220N+V222G, D223*+M220N+E221G+V222G, D 223*+M220D, D223*+M220E, D223*+M220H, D223*+M220F, D223*+M220W, D223*+M220S, D223*+M220A A mutant degU gene comprising or consisting of one or more of: or b) a mutant spo0A gene, ba) the mutation is located in the DNA binding domain or in the receiver domain and leads to reduced or absent phosphorylation and / or reduced or absent dimerization of the Spo0A protein; and / or bb) The mutation is: - A257V, more preferably A257S, - I161R, more preferably I161L, - In descending order of priority, A257S+I161I, A257A+I161L, A257V+I161I, A257S+I161F or A257A+I161R The present invention also provides a method for the preparation of a mutated spo0A gene comprising any one of the following:

[0006] The present invention also provides a method for transferring genetic material between two microorganisms, comprising the steps of: 1) The first microorganism, a) a mutant DegS protein, - the DegS protein lacks a functional single binding domain, a functional phosphoacceptor domain and / or a functional ATPase domain, and / or - a mutant 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, and optionally a mutant DegU protein, - has reduced DNA-binding activity and / or lacks a functional DNA-binding domain, and / or - The mutation is, in descending order of priority for each of the alternatives aa) and ab), aa)Q218*, Q218K, Q218N, Q218D, Q218R ab) a mutant DegU 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; or b) a mutant Spo0A protein, ba) the mutation is located in the DNA binding domain or in the receiver domain and leads to reduced or absent phosphorylation and / or reduced or absent dimerization of the Spo0A protein; and / or bb) The mutation is: - A257V, more preferably A257S, - I161R, more preferably I161L, - providing a mutant 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, and 2) mating a first microorganism with a second microorganism that is mating competent, conjugating, wherein the first microorganism comprises the genetic material to be transferred prior to step 2.

[0007] Furthermore, the present invention relates to an expression cassette for expressing a counterselectable marker, a) 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, and optionally a mutated 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 ab) a mutant 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; or b) a mutant spo0A gene, ba) the mutation is located in the DNA binding domain or in the receiver domain and leads to reduced or absent phosphorylation and / or reduced or absent dimerization of the Spo0A protein; and / or bb) The mutation is: - A257V, more preferably A257S, - I161R, more preferably I161L, a mutant spo0A gene consisting of or comprising, in order of decreasing priority, any of A257S+I161I, A257A+I161L, A257V+I161I, A257S+I161F or A257A+I161R.

[0008] The present invention also provides a method for producing a semiconductor device comprising the steps of: a) 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, and optionally a mutated 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 ab) a mutant 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; or b) a mutant spo0A gene, ba) the mutation is located in the DNA binding domain or in the receiver domain and leads to reduced or absent phosphorylation and / or reduced or absent dimerization of the Spo0A protein; and / or bb) The mutation is: - A257V, more preferably A257S, - I161R, more preferably I161L, - the use of any of the mutant spoOA genes consisting of or comprising any of the following, in decreasing order of preference: A257S+I161I, A257A+I161L, A257V+I161I, A257S+I161F or A257A+I161R, The following classifications: - 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, - Use for increasing the mating competence of a microorganism, more preferably selected from the genera Bacillus, Paenibacillus or Clostridium. [Brief description of the drawings]

[0009] [Figure 1]Evaluation of gene competence of Paenibacillus polymyxa strains. Gene competence of the different variants was evaluated by conjugating the cured strains with E. Coli S17-1 carrying the pCasPP plasmid of Ruetering et.al 2017 (see example 1) as donor strain. The plasmid contains the SpCas9 gene expressed under the control of the constitutive sgsE promoter from Geobacillus stearothermophilus and does not contain a gRNA targeting the P. polymyxa genome. To obtain countable colonies, serial dilutions were prepared before plating the conjugated strains on selective LB plates containing antibiotics. The colony forming units of each strain are then normalized to the respective OD600 used for the conjugation. Finally, the fold competence is calculated relative to the wild type. [Figure 2-1] 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 2-2] Continued from Figure 2. [Figure 2-3] Continued from Figure 2. [Figure 2-4] Continued from Figure 2. [Figure 3-1]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 3-2] Continued from Figure 3. [Figure 3-3] Continued from Figure 3. [Figure 4-1] 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 4-2] Continued from Figure 4. [Figure 4-3] Continued from Figure 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] A brief description of the sequence

[0011] [Table 1]

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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."

[0017] 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%).

[0018] 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").

[0019] 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.

[0020] 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.

[0021] "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."

[0022] 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".

[0023] "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*".

[0024] 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 more than one amino acid residue is 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 in the above example a glycine is inserted after a glycine, this would be indicated by G180GG.

[0025] 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.

[0026] 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}.

[0027] 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. The following applies to the determination of % similarity. This is also based on the BLOSUM62 matrix, which is 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] Producing a pairwise global alignment showing both sequences over their full length gives: [ka] 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.

[0032] 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.

[0033] The alignment length is 10, showing sequences aligned over the full length.

[0034] According to the present invention, when a pairwise alignment is generated showing a shorter sequence over its entire length, the result is: [ka]

[0035] According to the present invention, generating a pairwise alignment showing sequence A over its entire length results in: [ka]

[0036] According to the present invention, generating a pairwise alignment showing sequence B over its entire length results in: [ka]

[0037] 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).

[0038] 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).

[0039] 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:

[0040] % 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.

[0041] 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.

[0042] 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).

[0043] 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.

[0044] The present invention provides microorganisms that exhibit increased mating competence compared to the corresponding wild-type strains. This is achieved by the microorganisms of the invention (a) comprising a mutant degS gene and preferably a mutant degU gene, but not a mutant spoOA gene, or (b) comprising a mutant spoOA gene without a mutant degS gene, preferably without a mutant degU gene. This was surprising in view of the publication by Hamoen et al., The pleiotropic response regulator DegU functions as a priming protein in competence development in Bacillus subtilis, PNAS 2000, 9246-9251, which describes that inactivation of the degS-degU operon reduced gene competence, while various inactivations of degS did not affect competence. Moreover, Verhamme et al., DegU coordinates multicellular behaviour exhibited by Bacillus subtilis, Molecular Microbiology, 2007, 554-568, describe that the development of gene competence in Bacillus subtilis is independent of DegS. Furthermore, Spo0A is known to downregulate AbrB gene expression, and AbrB gene is also a repressor of comK expression, and comK expression is also a key factor in competence development in Bacillus subtilis. Therefore, modification of the degS gene (and preferably also the degU gene) or modification of the spo0A gene would be expected to have at most no effect on competence or even to reduce it. Other microorganisms, particularly those in the genus Paenibacillus, do not even contain a homologue of the comK gene of Bacillus subtilis, so regulation of gene competence has been unpredictable.Finally, Example 3 of WO2019221988 describes the conjugation targeting of Paenibacillus strains containing DegS and / or DegU mutants without any apparent effect on conjugation efficiency. It was therefore surprising that mutating the genes encoding DegS, DegU and Spo0A could increase conjugation competence, and that this increase was only present when either the gene encoding Spo0A or the genes encoding DegS or DegU were mutated.

[0045] Therefore, the present invention provides a microorganism comprising a mutant degS gene. When the mutant degS gene is expressed in a microorganism, a mutant DegS protein is generated, and 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 InterPro notation, 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 to 40 amino acids, more preferably by 0 to 20 amino acids, even more preferably by 0 to 10 amino acids, even more preferably by 1 to 5 amino acids, said differences preferably meeting the constraints according to Figure 2. 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 to 5 amino acids.

[0046] The present invention further provides a microorganism comprising a mutant degU gene in addition to a mutant degS gene. When the mutant degU gene is expressed in a microorganism, a mutant DegU protein is generated, and 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 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 between 73 and 100% to the amino acid sequence indicated 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 indicated by the Uniprot identifier E3EBP5_PAEPS by 0 to 20 amino acids, more preferably by 0 to 15 amino acids, even more preferably by 0 to 10 amino acids, even more preferably by 1 to 5 amino acids, said differences preferably meeting the constraints according to FIG. 3. 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 to 5 amino acids.

[0047] 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, and 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, it 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, A0A2Z2K RN4_9BACL、A0A1B8WQN2_9BACI、A0A089MEU2_9BACL、A0A089LZP7_9BACL、A0A0F7FA95_PAEDU、A0A0E4HDK7_9BACL、A0A1G7R7Q0_9BACL、A0A1H8N6P6_9B ACL、X4ZFA8_9BACL、A0A3G9IQE6_9BACL、A0A369BNP1_9BACL、A0A1B1N0I3_9BACL、A0A015KRJ2_9BACL、A0A2N5N5F6_9BACL、A0A1T2XNU8_9BACL、A0A090 ZFJ2_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、A0A2V2YXM7_9BACL、L0EEN 3_THECK、A0A3A1UXY9_9BACL、A0A3B0CH88_9BACL、A0A1V4HR00_9BACL、A0A1V0UWJ6_9BACL、H3SFG5_9BACL、A0A1X7JKH5_9BACL、A0A1I2FDS6_9BACL、A0A1V0UWJ6_9BACL A3D9IJB3_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、K4ZP76_PAEA2、A0A2W1NBK 6_9BACL、A0A172ZK56_9BACL、A0A3M8CIR1_9BACL、M8EE17_9BACL、A0A0Q3T5E2_BRECH、A0A0K9YRB7_9BACL、A0A1I3U483_9BACL、V6MCA1_9BACL、C0ZC1 7_BREBN、A0A4R3KM88_9BACI、A0A3M8B6E4_9BACL、A0A2N3LN87_9BACI、A0A419SMW9_9BACL、A0A3M8D088_9BACL、A0A075R4A3_BRELA、U1X7N0_ANEAE、A 0A1H2UFN8_9BACL、A0A0D1VW72_ANEMI、A0A0X8D3E6_9BACL、A0A0U5AZK5_9BACL、A0A4R3L002_9BACL、A0A0Q3WXA1_9BACI、A0A0B0IAE5_9BACI、A0A223 KSV6_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, SP0A_BACSU, A8FF06_B ACP2、A0A0J6EVC7_9BACI、A0A417YV34_9BACI、D5DS62_BACMQ、A0A4Q0VQU7_9BACI、A0A1H9PKN5_9BACI、A0A1I3QAI8_9BACL、A0A1G6Q9T8_9BACL、W1SH Y1_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、K6BXH2_9BACI、A0A160F75 3_9BACI、U5LDF9_9BACI、A0A0M0KYT7_9BACI、A0A061NL57_9BACL、A0A3A1QZJ5_9BACI、A0A2N5H854_9BACI、A0A160ISE8_9BACI、A0A2I7SRN1_LACSH、A 0A1M4TLQ2_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, A0A0H4PIL5_9BACI, I8 AMT2_9BACI、A0A0D6ZAA3_9BACI、A0A3T0I1Q1_9BACI、A0A1I0SQQ4_9BACI、I3EAA8_BACMM、A0A0M0GB29_SPOGL、A0A1L8ZLZ8_9BACI、A0A370GBM9_9BAC I、A0A433H928_9BACI、A0A4R6U795_9BACI、A0A060LXS4_9BACI、A0A074LME5_9BACL、A0A0K9GWU6_9BACI、A0A150KM63_9BACI、K6CV08_BACAZ、A0A323T XM3_9BACI、A0A2N0Z9Q2_9BACI、J8AK67_BACCE、A0A073KUP4_9BACI、A0A292YQZ8_9BACL、A0A226QLR6_9BACI、A0A160FBJ9_9BACI、C3BPR4_9BACI、E6T XR1_BACCJ、A0A1L3MQ53_9BACI、A0A0C2YCQ6_BACBA、Q8EQ49_OCEIH、A0A316D8M3_9BACL、A0A0J6FU61_9BACI、A0A1H8C0C3_9BACI、A0A084J373_BACMY、<h2 style=";text-align:left;direction:ltr">A0A1I4JPZ3_9BACI、A0A0M2SG37_9BACI、A0A150MMS1_9BACI、A0A1J6WGW3_ 9BACI、A0A0P6W2Q8_9BACI、A0A1I0SZE6_9BACI、A7GSJ0_BACCN、A0A2C9Z3P 6_BACHU、A0A398BG15_9BACI、A0A0V8JFI7_9BACI、A0A1I5NPW8_9BACI、A0A 4R2B866_9BACI、A0A023DE04_9BACI、A0A023CLR0_9BACI、A0A327YN47_9BAC I、A0A0Q9XV74_9BACI、A0A147K7R5_9BACI、A0A443J408_9BACI、A0A498DDK 1_9BACI、A0A0K6GMP9_9BACI、A0A429XD58_9BACI、A0A1I1ZLD5_9BACI、A4I QR2_GEOTN、A0A073K3V0_9BACI、A0A1X7D063_9BACI、Q5WF68_BACSK、A0A3S 4RLT9_9BACI、A0A150JT68_BACCO、F5L3H6_CALTT、A0A0M0GPU2_9BACI、S5Z7 C0_BACPJ, A0A1Z2V3H9_9BACI, A0A3A9KGU4_9BACI, A0A285CLU9_9BACI, A0A366XYH1_9BACI, A0A0D8BRF6_GEOKU, A0A265NFG5_9BACI, A0A428N868_9BACI, A0A2P8HAG1_9BACI, A0A1H0B3U0_9BACI, A0A150M7C5_9BACI, A0A1G8D1C3_9BACI, A0A1G8BRD8_9BACI, A0A4Q4IIH6_9BACL, A0A4Y9AEG4_9BACI, A0 A1I0FQG9_9BACI, A0A0F5HWK7_9BACI, A0A1H1BJ69_9BACI, W9A8X3_9BACI, A0A1H9LW77_9BACI, A0A494Z0K1_9BACI, A0A1M5CXL0_9BACI, A0A1G8JJN9_9BACI, A0A1G6IGH8_9BACI, A0A4Y7S8L6_9FIRM, A0A1X9MFG7_9BACI, A0A0A2UZF1_9BACI, A0A1H9ZLP9_9BACI, A0A1M4XLK4_9CLOT, A0A0A5GIF6_9BACI,<h2 style=";text-align:left;direction:ltr">A0A0C2VIM1_9BACL, A0A2A2IDA3_9BACI, A0A366EJ45_9BACI, A0A317KZA9_9BACI, A0A0A5GEQ9_9BACI, A0A1E5LK88_9BACI, A0A2S5GEL8_9BACL, A0A1G9LM94_9BACI<h2 style=";text-align:left;direction:ltr"> A0A1N6PFX1_9BACI, A0A1E7DMX2_9BACI, N4WSS3_9BACI, A0A1I1T1Z9_9BACI, A0A0A1MZ98_9BACI, A0A4R3N0 Q4_9BACI, A0A4Y8KST9_9BACL, A0A2U1K6N5_9BACI, A0A075LLD7_9BACI, A0A1I0V6R2_9BACI, A0A0U1KL95_9 BACI, A0A2P6MK99_9BACI, C8WXF8_ALIAD, A0A1M6KIQ3_9CLOT, A0A1L8CTW3_9THEO, A0A1V2A9Q9_9BACI, A0A 2T4UAN8_9BACI, A0A4Z0GKV9_9BACL, A0A1M6I6U1_9FIRM, A0A1I2VPT9_9BACL, A0A1M6S6D3_9BACL, A0A1N7K MH0_9BACL, A0A140L8E0_9CLOT, A0A090J299_9BACI, V6IWU0_9BACL, A0A024P5H3_9BACI, A0A285NM88_9BAC I, A0A143MRA0_9BACI, A0A0A5GCA3_9BACI, A0A0U1QSI9_9BACL, A0A0B5AS70_9BACL, A0A1M6C4X1_9CLOT, A0 According to the invention, wild-type Spo0A protein sequences and corresponding spo0A genes encoding same, which have 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 represented by the Uniprot identifier A0A074LZY6_PAEPO, are particularly preferred.Without taking into consideration specific mutations to the Spo0A protein sequence described according to the present invention, the mutant Spo0A protein preferably differs from the amino acid sequence shown by the Uniprot identifier A0A074LZY6_PAEPO 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 Figure 4. If the mutant Spo0A sequence, when aligned with the sequence according to the Uniprot identifier A0A074LZY6_PAEPO, is longer than said sequence, each C-terminal or N-terminal extension is preferably not more than 30 amino acids, more preferably by 0-10 amino acids.

[0048] It is a particular advantage of the present invention that it allows to increase mating competence by mutation of one or two genes that are ubiquitously found in Firmicutes microorganisms. Thus, the teachings of the present invention are not only applicable to microorganisms of the genus Paenibacillus, as shown in the examples below, but can also be used to increase mating efficiency of other Firmicutes. Preferred microorganisms are described below.

[0049] 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.

[0050] 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 mating competence compared to the corresponding wild-type strain, which can be easily achieved, for example, by introducing a mutation in the sensor DegS domain (IPR008595).

[0051] 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.

[0052] 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.

[0053] 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.

[0054] The specific mutations discussed above are contained in the second predicted alpha helix of the DegS sensor domain, and as shown in the Examples, all such mutations result in increased mating competence.

[0055] 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.

[0056] The microorganism according to the present invention preferably comprises a mutant degU gene, which encodes a DegU protein having 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 the mutant degU gene is already sufficient to improve mating competence.

[0057] The DegU protein preferably has reduced DNA-binding activity and / or lacks a functional DNA-binding domain, the presence of which can be readily identified in a microorganism of the invention, preferably a microorganism of the genus Paenibacillus, by observing an increased mating competence compared to the corresponding wild-type strain.

[0058] As mentioned above, the wild-type DegU protein contains a DNA-binding HTH (helix-turn-helix) 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.

[0059] 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.

[0060] 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.

[0061] Both mutations of type a) and b) are involved together in the predicted third alpha helix of the DNA binding domain. As shown in the examples, both mutations of type a) and b) result in increased mating competence.

[0062] 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.

[0063] 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*,The microorganism according to the present invention preferably comprises or consists of any of Q218R+M220F+D223*, Q218R+M220W+D223*, Q218R+M220S+D223*, Q218R+M220A+D223*. The microorganism according to the present 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 mating competence.

[0064] 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 present invention, preferably a microorganism of the genus Paenibacillus, by observing an increase in mating competence compared to the corresponding wild-type strain, which can be easily achieved, for example, by introducing a mutation into the Spo0A C-terminal domain (IPR014879).

[0065] 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.

[0066] Preferably, the mutation in the mutant Spo0A protein is - A257V, more preferably A257S, - I161R, more preferably I161L, - consisting of or including, in descending order of priority, any of: A257S+I161I, A257A+I161L, A257V+I161I, A257S+I161F or A257A+I161R.

[0067] 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.

[0068] 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.

[0069] The present invention preferably comprises: - the expression of the wild-type (a) degU and degS genes, or the (b) spo0A gene, respectively, is lower than or equal to the expression of the mutant (a) degU and degS genes, or the (b) spo0A gene, respectively, or - providing a microorganism, in which expression of the wild-type (a) degU and degS genes, or respectively (b) spo0A gene, is inhibited or eliminated during expression of the mutant (a) degU and degS genes, or respectively (b) spo0A gene.

[0070] Such relative overexpression of each of the mutant degS, degU and spoOA genes relative to the corresponding wild-type counterparts can be achieved in a first and preferred option by a microorganism in which the respective genes encoding the wild-type DegS, DegU and SpoOA proteins, respectively, have been inactivated and in which the genes encoding the respective mutant proteins have been introduced. In such option, the microorganism is preferably one of the following: a) comprises a mutant degU and a mutant degS gene according to the invention, in which the wild-type degU and degS genes have been functionally inactivated, deleted or replaced by the mutant genes, or b) comprises a mutated spo0A gene according to the invention, in which the wild-type spo0A gene has been functionally inactivated, deleted or replaced by the mutated gene.

[0071] In the second option, the respective wild-type genes are still present in the microorganism of the invention. Such a microorganism is particularly advantageous since it is possible to switch between wild-type and mutant behavior when the wild-type or mutant genes are placed under the control of a regulatable promoter. In this way, competence can be selectively increased during the process of the desired nucleic acid transfer, while maintaining the low wild-type conjugation competence at all other stages, which advantageously limits horizontal gene transfer, for example during fermentation processes. Correspondingly, a microorganism according to the invention, - the mutant degU and degS genes or the mutant spoOA gene are operably linked to an inducible or repressible promoter, respectively; and / or - the wild-type degU and degS genes or the mutant spoOA gene are operably linked to a repressible or inducible promoter, Preferably, as a result, a microorganism is provided in which the expression of the mutant degU and degS genes or the mutant spoOA gene, respectively, can be freely increased or decreased compared to the expression of the corresponding wild-type genes.

[0072] Preferably, the mutant degU gene and the mutant degS gene or the mutant spoOA gene are each provided in a respective expression cassette located on an extrachromosomal nucleic acid, which further comprises a counterselection marker. As described below, such extrachromosomal nucleic acid makes it possible to confer increased competence to the microorganism for a selected period of time. In particular, such extrachromosomal nucleic acid can be advantageously removed from the microorganism after the conjugation event, for example before the creation of a cell bank sample of the product strain obtained by conjugation. Counterselection markers are known to those skilled in the art and are described, for example, in WO2021061694.

[0073] 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, they are selected from the taxonomic ranks of the genera Bacillus, Paenibacillus or Clostridium.

[0074] In particular, microorganisms of the families Bacillaceae and Paenibacillaceae are important microorganisms in industrial fermentation processes. Furthermore, among the microorganisms of these genera, spore-forming strains are known.

[0075] In agriculture, bacterial spores have been used in plant pest control compositions to reduce or prevent plant pathogenic fungal or bacterial diseases. Spore bioformulations 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 Biocontrol Agent. IV. Salt-Stress Tolerance Induction by Bacillus Subtilis FZB24 Seed Treatment in Tropical Vegetable Field Crops, and Its Mode of Action / Die Verwendung von Bacillus Subtilis zur biologischen Bekaempfung. IV. Induktion einer Salzstress-Toleranz durch Applikation von Bacillus subtilis FZB24 bei tropischem Feldgemuese und sein Wirkungsmechanismus.” Zeitschrift fuer Pflanzenkrankheiten und Pflanzenschutz / Journal of Plant Diseases and Protection, vol. 108, no. 1, 2001, pp. 21-30. JSTOR, www.jstor.org / stable / 43215378. Accessed December 14, 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.).

[0076] 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].

[0077] 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.

[0078] 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).

[0079] Particularly preferred are microorganisms of one of the following species: 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 ae), 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, Paenibacillus macerans, Paenibacillus alvei, more preferably Paenibacillus polymyxa, Paenibacillus polymyxa polymyxa, Paenibacillus polymyxa plantarum, Paenibacillus nov.spec epiphyticus, Paenibacillus terrae, Paenibacillus macerans, Paenibacillus alvei, more preferably Paenibacillus polymyxa, Paenibacillus polymyxa polymyxa, Paenibacillus polymyxa plantarum, Paenibacillus nov.spec epiphyticus, Paenibacillus terrae, Paenibacillus macerans, Paenibacillus alvei, and even more preferred are Paenibacillus polymyxa, Paenibacillus polymyxa polymyxa, Paenibacillus polymyxa plantarum and Paenibacillus terrae.

[0080] 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.lacisa B. lacisalsi, B. lacus, B. lehensis, B. lentus, B. ligniniphilus, B. lindianensis, B. litoralis, B. loiseleuriae, B. lonarensis, B. longiquaesitum, B. longispor B. longisporus, B. luciferensis, B. luteolus, B. luteus, B. lycopersici, B. magaterium, B. malikii, B. mangrovensis, B. mangrovi, B. mannanilyticus, B. manusensis, B. m anusensis, B. marasmi, B. marcorestinctum, B. marinisedimentorum, B. marisflavi, B. maritimus, B. marmarensis, B. massiliglaciei, B. massilioanorexius, B. B. massiliogabonensis, B. massiliogorillae, B. massilionigeriensis, B. massiliosenegarensis, B. mediterraneensis, B. megaterium, B. mesonae, B. mesophyllum, B. mesophyllum, B. mesophilus, B. methanolicus, B. miscanthi, B. muralis, B. murimartini, B. nakamurai, B. nanhaiisediminis, B. natronophilus, B. ndiopicus, B. nealsonii, B. nematocida ida), B. niabensis, B. niacini, B. niameyensis, B. nitritophilus, B. notoginsengisoli, B. novalis, B. obstructivus, B. oceani, B. oceanisediminis, B. ohbensis ), B.okhensis, B.okuhidensis, B.oleivorans, B.oleronius, B.olivae, B.onubensis, B.oryzae, B.oryzaecorticis, B.oryzisoli, B.oryziterrae, B.oshimensis, B.B. pakistanensis, B. panacisoli, B. panaciterrae, B. paraflexus, B. patagoniensis, B. persicus, B. pervagus, B. phocaeensis, B. pichinotyi, B. piscicola, B. p iscis, B. plakortidis, B. pocheonensis, B. polygoni, B. polymachus, B. populi, B. praedii, B. pseudocaliphilus, B. pseudofirmus, B. pseudoflexus, B. pseudomegatherium, B. pseudomegher ... udomegaterium, B.psychrosaccharolyticus, B.pumilus, B.purgationiresistens, B.qingshengii, B.racemilacticus, B.rhizosphaerae, B.rigiliprofundi, B.rubiinfantis infantis, B.ruris, B.safensis, B.saganii, B.salacetis, B.salarius, B.salidurans, B.salis, B.salitolerans, B.salmalaya, B.salsus, B.sediminis, B.selenatarsenatis, B.selenatarsenatis, B.senegalensis, B.seohaeanensis, B.shacheensis, B.shackletonii, B.shandongensis, B.shivajii, B.similis, B.simplex, B.sinesaloumensis, B.shirari B.siralis, B.smithii, B.solani, B.soli, B.solimangrovi, B.solisilvae, B.songklensis, B.spongiae, B.sporothermodurans, B.stamsii, B.subterraneus, B.swezei zeyi, B. taeanensis, B. taiwanensis, B. tamaricis, B. taxi, B. terrae, B. testis, B. thaonhiensis, B. thermoalkalophilus, B. thermoamyloliquefaciens, B. thermoamylovorans hermoamylovorans, B. thermocopriae, B. thermolactis, B. thermophilus, B. thermoproteolyticus, B. thermoterrestris, B. thermozeamaize, B. thioparans, B. tianmuensis, B.B.tianshenii, B.timonensis, B.tipchiralis, B.trypoxylicola, B.tuaregi, B.urumqiensis, B.vietnamensis, B.vini, B.vireti, B.viscosus, B.vitellinus, B.wakoensis, B. .weihaiensis, B. wudalianchiensis, B. wuyishanensis, B. xiamenensis, B. xiaoxiensis, B. zanthoxyli, B. zeae, B. zhangzhouensis, B. zhanjiangensis, preferably Bacillus licheniformis 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.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. spp. ... B. amyloliquefaciens, B. licheniformis, B. thuringiensis, B. velezensis, B. subtilis and B. megaterium, and even more preferably B. amyloliquefaciens, B. thuringiensis, B. velezensis and B. megaterium.

[0081] 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.

[0082] 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.

[0083] The present invention also provides a method for increasing the mating competence of a microorganism, comprising the steps of: a) a mutant DegS protein and preferably a mutant DegU protein according to the invention, or b) providing a microorganism with any of the mutant Spo0A proteins according to the invention.

[0084] As described herein, selective provision of such mutant proteins advantageously improves the mating competence of the microorganism.

[0085] Correspondingly, the present invention also provides a method for transferring genetic material between two microorganisms, comprising the steps of: 1) The first microorganism, a) a mutant DegS protein according to the invention and preferably a mutant DegU protein according to the invention, or b) providing a mutant Spo0A protein according to the invention; and 2) mating a first microorganism with a second microorganism that is mating competent, conjugating, wherein the first microorganism comprises the genetic material to be transferred prior to step 2.

[0086] As described herein, (a) providing a mutant DegS protein, preferably together with a mutant DegU protein (in either case without a mutant Spo0A protein), or (b) providing a mutant DegS protein and a mutant Spo0A protein, optionally without a DegU protein, increases the conjugation competence of a first microorganism (a "target microorganism"), which, when contacted with a conjugation-competent second microorganism (a "donor microorganism") carrying the nucleic acid to be transferred, is transferred by conjugation with high efficiency.

[0087] In the transfer method of the present invention, preferably, the mutant degU gene and the mutant degS gene or the mutant spoOA gene are each provided in a respective expression cassette located on an extrachromosomal nucleic acid of the target microorganism, the extrachromosomal nucleic acid further comprises a counterselection marker, and the transfer method further comprises a step of counterselecting against the counterselection marker. As described herein, the use of the counterselection marker can remove the extrachromosomal nucleic acid that confers conjugative competence, thereby increasing the stability of the microorganism after incorporation of the nucleic acid transferred by conjugation and preventing or limiting further horizontal gene transfer.

[0088] Correspondingly, the present invention also provides an expression cassette for expressing a counterselectable marker, a) a mutated degS gene according to the invention and preferably a mutated degU gene according to the invention, or b) providing an expression vector comprising the mutant spo0A gene according to the present invention.

[0089] Such expression vectors advantageously serve to provide the respective mutated genes for expression in the target microorganism intended as the recipient of the heterologous nucleic acid.

[0090] The present invention also provides a) a mutated degS gene according to the invention and preferably a mutated degU gene according to the invention, or b) Use of any of the mutant spoOA genes according to the invention, The present invention provides a use for increasing the mating competence of a microorganism selected from any of the taxonomic classes of preferred microorganisms listed herein.

[0091] By using the respective genes or by using the corresponding mutant proteins, the mating competence of microorganisms can be advantageously improved.

[0092] Aspects of the present invention are further illustrated below by way of non-limiting examples. EXAMPLES

[0093] 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.

[0094] [Table 2]

[0095] 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.

[0096] 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.

[0097] 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).

[0098] [Table 3]

[0099] Example 2: Assessment of gene competence The gene acceptance capacity of the various variants was evaluated by conjugating the cured strains with E. Coli S17-1 carrying the pCasPP plasmid as the donor strain, following the protocol described above. The plasmid contains the SpCas9 gene expressed under the control of the constitutive sgsE promoter from Geobacillus stearothermophilus and no gRNA targeting the P. polymyxa genome. To obtain countable colonies, serial dilutions were prepared before plating the conjugated strains on selective LB plates containing antibiotics. The colony forming units of each strain were then counted by the OD of each of the strains used for conjugation. 600 The increase in competence was calculated based on the number of CFUs after conjugation compared to the wild-type strain.

Claims

1. a) a mutant degS gene and optionally a mutant degU gene, or b) Mutant spo0A gene A microorganism comprising any one of A microorganism that exhibits increased mating competence compared to the corresponding wild-type strain.

2. Contains 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 The degS gene encodes a DegS protein, and the mutations include or consist of L99F, L99C, L99D, L99E, L99G, L99H, L99K, L99N, L99P, L99Q, L99R, L99S, L99W, or L99Y. The microorganism described in claim 1.

3. containing 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 a DegU protein, mutations of which, for each of options a) and b), in decreasing order of priority, are: 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; The microorganism described in claim 1.

4. 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, I161R, more preferably I161L, In descending order of priority, A257S+I161I, A257A+I161L, A257V+I161I, A257S+I161F or A257A+I161R The microorganism according to claim 1, consisting of or comprising any one of the following:

5. the expression of the wild-type (a) degU and degS genes, or each (b) spoOA gene, is lower than the expression of the mutant (a) degU and degS genes, or each (b) spoOA gene; or The microorganism described in claim 1, wherein expression of the wild-type (a) degU and degS genes, or each (b) spoOA gene, is inhibited or eliminated during expression of the mutant (a) degU and degS genes, or each (b) spoOA gene.

6. The microorganism is a) comprising a mutant degU and a mutant degS gene, preferably as defined in claims 2 and / or 3, wherein the wild-type degU and degS genes are functionally inactivated, deleted or replaced by the mutant genes, or b) A mutant spoOA gene, preferably as defined in claim 4, wherein the wild-type spoOA gene is functionally inactivated, deleted or replaced by the mutant gene. The microorganism according to claim 5, which is any one of the following:

7. The microorganism of claim 1, wherein the mutant degU gene and the mutant degS gene or the mutant spoOA gene are each provided within a respective expression cassette located on an extrachromosomal nucleic acid, and the extrachromosomal nucleic acid further comprises a counterselectable marker.

8. The microorganism is the phylum Firmicutes, the class Bacilli, the class Clostridia or the class Negativicutes, More preferably, the bacteria are from the order Bacillales, Clostridiales, Thermoanaerobacter, Thermosediminibacter or Selenomonadales; More preferably, the bacterium is selected from the group consisting of the family Bacillaceae, Paenibacillaceae, Pasteuriaceae, Clostridiaceae, Peptococcusceae, Heliobacteriaceae, Syntrophomonadaceae, Thermoanaerobacteraceae, Tepidanaerobacteraceae, and Sporomusaceae. More preferred are the genus Alkalibacillus, the genus Bacillus, the genus Geobacillus, the genus Halobacillus, the genus Lysinibacillus, the genus Piscibacillus, the genus Terribacillus, the genus Brevibacillus, the genus Paenibacillus, the genus Thermobacillus, the genus Pasteuria, the genus 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, the genus Bacillus, Paenibacillus, or Clostridium. The microorganism according to claim 1, which is selected from the taxonomic ranks of

9. 1. A method for increasing the mating competence of a microorganism, comprising: a) a mutant DegS protein and optionally a mutant DegU protein according to claim 2 and / or 3, or b) The mutant Spo0A protein according to claim 4 The method comprises providing the microorganism with either of the following:

10. 1. A method for transferring genetic material between two microorganisms, comprising: 1) a first microorganism; a) a mutant DegS protein according to claim 2 and optionally a mutant DegU protein according to claim 3, or b) providing a mutant SpoOA protein according to claim 4; and 2) mating the first microorganism with a second microorganism that is competent for mating, the first microorganism containing the genetic material to be transferred prior to step 2. A method comprising:

11. 11. The method of claim 10, wherein the first microorganism is the microorganism of claim 7, and the method of transfer further comprises the step of counterselecting against the counterselectable marker.

12. an expression cassette for expressing a counterselectable marker; a) a mutant degS gene according to claim 2 and optionally a mutant degU gene according to claim 3, or b) the mutant spoOA gene according to claim 4; and An expression vector comprising:

13. a) a mutant degS gene according to claim 2 and optionally a mutant degU gene according to claim 3, or b) The mutant spoOA gene according to claim 4 The use of any of the following:

9. Use for increasing the mating competence of a microorganism selected from any of the taxonomic classes of claim 8.

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