Regulation and integration of plasmid copy number

Abstract

The present invention relates to the field of plasmid copy number regulation, preferably in prokaryotic hosts, and provides materials and methods for user-controlled regulation of plasmid copy number. The present invention further provides host culture methods that utilize the means for copy number regulation provided by the present invention. In particular, the present invention allows for the integration of a target nucleic acid segment into another nucleic acid with high integration efficiency.

Description

Technical Field

[0001] The present invention relates to the field of plasmid copy number regulation, preferably in prokaryotic host organisms, and provides materials and methods for user-controlled regulation of plasmid copy number. The present invention further provides host culture methods that utilize the copy number regulation means provided by the present invention. In particular, the present invention enables a target nucleic acid segment to be integrated into another nucleic acid with high integration efficiency.

Background Art

[0002] Plasmids are extrachromosomal genomes that replicate in an autonomous and regulated manner. Plasmids are generally circular nucleic acids that have their own origin of replication that allows them to replicate independently of the replication of the host microorganism's chromosome. Plasmids are therefore often used as shuttle vectors that carry nucleic acid segments into a host and then travel back and forth. Plasmids are generally characterized by their inherent plasmid copy number. The replication mechanism on which plasmids depend ensures that the copy number of the plasmid in each host cell does not exceed or fall below the copy number specific to the plasmid. For example, the well-known plasmid pBR322 has a copy number of about 20. This copy number is generally maintained throughout the entire life cycle of the host cell.

[0003] When a plasmid contains a target gene expressed in a host, the plasmid copy number is intricately linked to the gene quantity of the target gene. Generally, it is desirable to achieve strong expression of one or more target genes during fermentation. One way to achieve such strong expression is to place the target gene(s) under the control of a strong promoter. However, it has been found that the available promoters are not strong enough for some host microorganisms. Another way to increase target gene expression is to provide additional copies of the promoter-gene expression cassette, thereby increasing the gene quantity. The target gene is therefore transcribed simultaneously from several locations, increasing its expression. However, increasing the copy number of the expression cassette in a plasmid is cumbersome and can lead to homologous recombination, which in turn can result in the uncontrolled loss of the target gene expression cassette.

[0004] Therefore, attempts have been made to manipulate the copy number of plasmids themselves. Homologous recombination between identical plasmids is unlikely to result in the loss of the target gene, especially when it contains only one copy of the target gene expression cassette. Several strategies have been proposed to increase plasmid copy number. For example, WO2009076196 and WO2002029067 provide specific high-copy-number plasmids. WO2007035323 provides a plasmid in which the origin of replication can be swapped, thereby allowing the insertion of an origin of replication known to result in a desired plasmid copy number into a selected plasmid vector.

[0005] High plasmid copy numbers come at a great cost. Generally, the size of the target gene expression cassette is small compared to the overall plasmid size. Furthermore, plasmids typically require the presence of selection markers, usually antibiotic resistance genes, to ensure plasmid maintenance in the host during culture. Therefore, an increase in plasmid copy number imposes a high metabolic burden on the host, which must divert resources to plasmid backbone replication. This metabolic burden can be a decisive factor limiting host viability under harsh conditions. For example, if target gene expression is already generating a metabolic burden, maintaining a high plasmid copy number can damage host viability during fermentation, leading to premature fermentation cessation or insufficient yield of the target gene product. Moreover, when a host is placed in a new medium, for example, inoculated into a fermenter to start batch fermentation (or after thawing cryopreserved cultures), the host experiences strong metabolic stress to adapt to the new environment. In such situations, the need to maintain a high plasmid copy number required by the plasmid's autonomous replication mechanism is excessive for host cells, leading to significant growth delays and thus potentially reducing the fermentation's time-space yield.

[0006] Therefore, attempts have been made to restrict plasmid replication of high-copy-number plasmids for a predetermined period using temperature-sensitive replication origins. One example of such a temporary restriction of plasmid replication is presented in Olson et al., Journal of Biological Engineering 2012, 6:5 and WO9318164. The latter literature provides a temperature-sensitive plasmid variant of plasmid pWV01 for Gram-positive bacteria, particularly lactic acid bacteria. This plasmid cannot replicate above approximately 37°C. However, in such systems, plasmid replication is either fully enabled or completely blocked by the general temperature. Therefore, great care must be taken to prevent accidental plasmid loss during growth stages where maintaining a low plasmid copy number (instead of zero plasmid per host cell) is desired.

[0007] Further uses of plasmids involve the manipulation of the host's non-plasmid genetic material, also known as the host chromosome. Plasmids can be incorporated into host chromosomes by homologous recombination. Plasmids can also be excised from their integration site, for example, by homologous recombination as described in Figures 6, 7A and 7B of WO9318164 and its accompanying instructions. Through such integration and optional partial excision, plasmids can be used, for example, to inactivate desired nucleic acid sequences on host chromosomes. Inactivation results from the presence of the entire inserted plasmid or its remainder after partial plasmid excision. Furthermore, by incorporating the entire plasmid and / or leaving a segment of the plasmid nucleic acid after partial plasmid excision, it becomes possible to introduce desired nucleic acid stretches, preferably containing a target gene expression cassette or introducing any other desired nucleic acid element at a specific location on the host chromosome.

[0008] Plasmid integration, as the first step in such a procedure, relies on the nature of the plasmid to recombine with the host chromosome instead of continuing autonomous replication. Therefore, it is first necessary to establish the presence of the plasmid in the host cell. This then depends on the plasmid's ability to replicate autonomously. However, when attempting to force plasmid integration into the host chromosome, the plasmid's ability to replicate autonomously significantly reduces the likelihood of integration. Therefore, attempts have been made to use plasmids that lose replication activity in a temperature-dependent manner in such integration assays. However, while it is possible to hinder the plasmid's ability to regenerate by significantly lowering or raising the culture temperature compared to the host's optimal temperature, such temperature changes place the host cell under severe metabolic stress. For example, at low temperatures, the host cell may be metabolically largely active, while at high temperatures, host cell proteins may denature. In both cases, the likelihood of plasmid integration decreases. Therefore, it has been found that plasmid integration assays are unreliable, and that a great deal of effort is required to check whether a putative clone obtained by such an assay actually contains the plasmid, even if a single plasmid has been accurately integrated at the exact desired location. [Overview of the project] [Problems that the invention aims to solve]

[0009] Therefore, the object of the present invention is to address the aforementioned shortcomings of the prior art. In particular, the present invention provides an easy and reliable method for plasmid copy number control. The present invention also provides a reliable method for plasmid incorporation. The present invention provides plasmids and hosts useful in such a method. [Means for solving the problem]

[0010] Therefore, the present invention is a method for controlling plasmid copy number, i) Providing a prokaryotic host containing a plasmid having an origin of replication that can be activated by a plasmid replication initiation protein (Rep), and ii) A step of adjusting the plasmid copy number to a desired value by regulating the expression of the Rep protein. This provides a method that includes [something].

[0011] The present invention also, - Origins of replication that can be activated by plasmid replication initiation proteins (Reps), - rep gene encoding Rep protein A controlled replication plasmid containing, a) The rep gene is operablely linked to a heterologous promoter, and / or b) The plasmid provides a plasmid containing a repressor gene encoding a Rep expression repressor, which is operably ligated to a heterologous promoter.

[0012] And the present invention is, - Origins of replication that can be activated by plasmid replication initiation proteins (Reps), - In some cases, a promoter operably linked to a repressor gene encoding a repressor for Rep expression. This provides a replication help-dependent plasmid that contains the functional rep gene encoding the Rep protein.

[0013] The present invention also provides plasmid replication control hosts comprising controlled replication plasmids described herein. Furthermore, i) Replication-assisted plasmids described herein, and ii) Expression cassette for the expression of rep genes located on the host chromosome and / or helper plasmids in the host. A plasmid replication support host containing [the specified substance] is provided.

[0014] Also, i) A step of providing a host starter culture containing the plasmid according to the present invention, ii) The step of culturing the host to achieve an increase in the number of host cells, and iii) A step to adjust the plasmid copy number in cultured host cells during or after step ii). A culture method including this is also provided.

[0015] moreover, i) A step of providing a plasmid according to the present invention containing a selection marker in a prokaryotic plasmid recipient host. ii) A step to inhibit plasmid replication while maintaining the selection of the selection marker. An implementation method including this is provided.

[0016] especially, i) A plasmid donor host comprises a controlled replication plasmid or replication support-dependent plasmid according to the present invention, which contains a selectable marker. - The plasmid recipient host does not contain the rep gene. A step of providing a plasmid donor host and a plasmid recipient host, ii) The step of introducing a plasmid from a plasmid donor host to a plasmid recipient host, and iii) A step of inhibiting plasmid replication in the plasmid recipient host, either simultaneously with step ii) or after step ii), while applying to the plasmid recipient host a selection pressure that enforces the presence of a selectable marker in the plasmid recipient host An integration method comprising the same is provided. [BRIEF DESCRIPTION OF THE DRAWINGS]

[0017] [Figure 1] It is a schematic diagram of the pE194 replication origin including regulatory genes and constructs. Non-coding sequences are depicted as light gray squares, and ORFs are represented as black arrows. Promoters are indicated by bent arrows. The produced protein in an oligomeric state is depicted in dark gray. SSO: single-strand origin; DSO: double-strand origin; cop gene: encodes a "copy control protein". Cop forms a dimer and binds the repressor to its own promoter. Transcription from the Pcop promoter results in mRNA containing the cop and repF genes. repF gene: encodes the RepF protein, which binds to the DSO and a nick within the DSO region as an initiation factor for plasmid replication. ctRNA: countertranscript RNA transcribed from the minus strand overlapping the 5'UTR of the repF ORF, which inhibits RepF translation initiation. [Figure 2]A) Schematic diagram of Bacillus subtilis 168 strain carrying the indicated plasmid pKS100 (pE194 replication origin, cop-repF), and plasmid pKS101 (pE194(ts) replication origin; marked as cop-repF*), or plasmid pKS102 (without pE194 replication origin) integrated at the amyE locus (Bs#073). LuxABCDE: Luciferase gene; amy: Amylase amyE homology region. B) Bacillus subtilis strains were cultured at the indicated temperature with agitation in LB medium supplemented with erythromycin (50 μg / ml). Samples were taken after 8 hours of growth, and the average plasmid copy number per cell was determined by quantitative real-time PCR. Relative PCN: Relative plasmid copy number (per cell) of the indicated plasmid compared to the chromosome terminus. Bs#073 is an integrated control strain with one plasmid backbone per chromosome terminus (dotted line). [Figure 3] A) Schematic diagram of Bacillus subtilis 168 strain carrying the indicated plasmid pKS100 (pE194 replication origin, cop-repF), as well as Bacillus subtilis strain GXC1 in which the PxylA-cop expression cassette and plasmid KS100 are integrated into the genome. LuxABCDE: Luciferase gene; amy: Amylase amyE homology region. B) Cultures of the indicated strains were incubated at 37°C in LB medium containing erythromycin (50 μg / ml) with no xylose (0%), 0.125% xylose or 0.5% xylose. Samples were taken after 8 hours, and quantitative real-time PCR was performed to determine the average plasmid copy number per cell. The dotted line indicates the ratio of the copy number of one plasmid per chromosome terminus to the integrated plasmid DNA. Relative PCN: Relative plasmid copy number (per cell) of the indicated plasmid compared to the chromosome terminus. PxylA: Promoter of the xylA gene from Bacillus megaterium. [Figure 4]A) A schematic diagram of Bacillus subtilis GCX1 strain with a PxylA-cop expression cassette integrated into its genome, containing plasmid pKS100 (pE194 origin, cop-repF) and plasmid pKS101 (pE194(ts) origin; marked as cop-repF*). LuxABCDE: luciferase gene; amy: amylase amyE homology region. B) The luminescence signal and cell density (OD 600nm) of Bacillus subtilis GCX1 cultures containing plasmids pKS10 and pKS101 were measured after 8 hours of incubation at 30°C in LB medium supplemented with erythromycin (50 μg / ml). Relative luminescence signal (RLU) is plotted against various concentrations of the indicated inducing molecule xylose. The dotted line shows the RLU of reference control strain Bs#073 with the lux operon integrated into its genome. [Figure 5] A) Schematic diagrams of Bacillus subtilis PCX1 strain containing plasmid pKS111, which has plasmid pKS100 (pE194 origin of replication, cop-repF) and plasmid pKS101 (pE194(ts) origin of replication; marked as cop-repF*), and Bacillus subtilis strain 168 containing plasmid pKS100. LuxABCDE: Luciferase gene; amy: Amylase amyE homology region. B) Luminescence signal and cell density (OD 600nm) of Bacillus subtilis PCX1 cultures containing plasmid pKS111, which also contains plasmid pKS100 or pKS101, were measured after 8 hours of incubation at 30°C in LB medium supplemented with erythromycin (50 μg / ml) and kanamycin (20 μg / ml). Relative luminescence signal (RLU) is plotted against various concentrations of the indicated inducing molecule xylose. 168 reference strains of Bacillus subtilis containing plasmid pKS100 served as controls and were cultured without kanamycin. [Figure 6]A) A schematic diagram of Bacillus subtilis strain 168 containing plasmid pBAio-lumiBs, which has the following genetic elements: pE194 origin of replication (shown as cop-repF), an additional copy of the cop gene under the control of the PxylA promoter, the LuxABCDE operon (luciferase gene under the constitutive promoter Pveg), and the amylase amyE homology region (amy). B) The luminescence signal and cell density (OD 600 nm) of cultures of Bacillus subtilis 168 containing plasmid pBAio-lumiBs were measured after 8 hours of incubation at 30°C in LB medium supplemented with erythromycin (50 μg / ml). The relative luminescence signal (RLU) is plotted against various concentrations of the inducing molecule xylose shown. The RLU signal decreases in a concentration-dependent manner with the inducing molecule xylose. [Figure 7] A) A schematic diagram of the Bacillus licheniformis P308 strain containing the plasmid pBAio-lumiBl, which has the following genetic elements: pE194 origin of replication (shown as cop-repF), an additional copy of the cop gene under the control of the PxylA promoter, the LuxABCDE operon (luciferase gene under the constitutive promoter Pveg), and the amylase amyB homology region (amy). Bacillus licheniformis P308 containing pBAio lumiBl is cultured in LB medium containing kanamycin (20 μg / ml) and the indicated xylose concentration for 8 hours at 37°C. B) The relative luminescence output (RLU) of plasmid pBAio-lumiBl is plotted against the indicated xylose concentration. The RLU of plasmid pBAio-lumiBl decreases when cop expression is induced. C) Average plasmid copy number of pBAio lumiBl in Bacillus licheniformis P308 during cop expression at the indicated xylose concentrations. Samples were removed after 8 hours of culture, and the average plasmid copy number per cell was determined by quantitative real-time PCR. Relative PCN: Relative plasmid copy number (per cell) of the plasmid compared to the chromosome end. The dotted line indicates the level of single-copy chromosome integration. [Figure 8]This figure shows the 100% Campbell recombination efficiency achieved by a combination of cop overexpression and temperature shift. A) Schematic diagram of two states of luminescent Bacillus licheniformis LBL cells containing the pKS137 plasmid with a luxA homology region. The plasmid replicates autonomously, the genome-coding luxABCDE sequence is intact, and all cells exhibit luminescence. When plasmid replication is stopped, integration of pKS137 into the genome occurs via homologous recombination by the homologous region (luxA') to the luxA sequence, disrupting this gene which encodes a luciferase subunit. Cells with this genotype can no longer produce functional luciferase and do not exhibit luminescence. B) Luminescence-based evaluation of plasmid integration rates in Bacillus licheniformis. Both Bacillus licheniformis strains LBL containing plasmid pKS137 are cultured in liquid culture the day after seeding on LB agar plates supplemented with 20 μg / ml kanamycin. Supplementation with 0.5% inducing molecule xylose is indicated by "+", and the culture temperature is shown as either 37°C or 45°C. Results for the following conditions are shown: no xylose on the culture day and no xylose on the agar plate (white bars), no xylose on the culture day and 0.5% xylose on the agar plate (right diagonal lines), 0.5% xylose on the culture day and no xylose on the agar plate (black), or 0.5% xylose on the culture day and 0.5% xylose on the agar plate (network). [Modes for carrying out the invention]

[0018] The technical teachings of the present invention are presented herein using linguistic means, particularly in scientific and technical terminology. However, as those skilled in the art will understand, linguistic means, while detailed and precise, have inherent limitations, and therefore can only approximate the complete content of the technical teachings, even if only because there are multiple ways of expressing the teachings (each inevitably unable to fully express all conceptual relationships). With this in mind, those skilled in the art will understand that, due to the inherent limitations of written description, the present invention is the sum of individual technical concepts presented or expressed herein in a manner in which the parts do not necessarily represent the whole. In particular, those skilled in the art will understand that, for example, the disclosure of three concepts or embodiments A, B, and C would be expressed as the simple notation concepts A+B, A+C, B+C, and A+B+C, and that individual technical concepts are presented herein as an abbreviation of a detailed description of each possible combination of concepts, to the extent that they are technically perceptible. In particular, feature alternatives are described herein in terms of a list of alternatives or instantiations. Unless otherwise indicated, the present invention as described herein includes any combination of such alternatives. The selection of generally preferred components from such a list is part of the present invention and depends on the preference of those skilled in the art for the minimal realization of the advantages(s) conveyed by each feature. Multiple such combined instantiations constitute a well-preferred form of the present invention.

[0019] As used herein, singular and singular terms such as “a,” “an,” and “the” refer to multiple objects unless the context specifically indicates otherwise. For example, the use of the term “a nucleic acid” may, in practice, include many copies of that nucleic acid molecule. Similarly, the term “probe” may (and typically) include many similar or identical probe molecules. Furthermore, as used herein, the word “comprising” or variations such as “comprises” or “comprising” are understood to mean the inclusion of the stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps.

[0020] As used herein, the terms "and / or" refer to and encompass any possible combination of one or more of the related enumerated items, and, if interpreted as an alternative ("or"), the absence of any combination. The term "comprising" also encompasses the term "consisting of."

[0021] When used in relation to measurable values, such as mass, dose, time, temperature, sequence identity, etc., the term "about" refers to variations of a particular value, including ±0.1%, 0.25%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or even 20% of that value. Therefore, if a given composition is described as containing "about 50%X", it should be understood that in some embodiments the composition contains 50%X, while in other embodiments the composition may contain a range of 40% to 60%X (i.e., 50% ± 10%).

[0022] As used herein, the term “gene” means, when embodied in nucleic acids, a gene product, i.e., biochemical information that can be transcribed into further nucleic acids, preferably RNA, and also translated into peptides or polypeptides. The term is therefore also used to indicate a portion of nucleic acid similar to the information and a sequence of such nucleic acid (also referred to herein as “gene sequence”).

[0023] Furthermore, as used herein, the term “allele” refers to a gene variant 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 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” with respect to the nucleotide sequence of the wild-type gene, in order of increasing priority. Correspondingly, if "allele" refers to biochemical information for expressing a peptide or polypeptide, then each nucleic acid sequence of the allele has at least 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" with respect to each wild-type peptide or polypeptide, in order of increasing priority.

[0024] "Target gene" refers to a gene or allele having the desired characteristics. In the present invention, the target gene preferably encodes an enzyme, and more preferably the enzyme is selected from the group consisting of amylase, catalase, cellulase, chitinase, cutinase, galactosidase, beta-galactosidase, glucoamylase, glucosidase, hemicellulase, invertase, laccase, lipase, mannanase, mannosidase, nuclease, oxidase, pectinase, phosphatase, phytase, protease, ribonuclease, transferase, and xylanase, more preferably protease, amylase, or lipase, and most preferably protease.

[0025] Mutations or alterations in amino acid sequences or nucleic acid sequences may be substitutions, deletions, or insertions, and the terms “mutation” or “alteration” also include any combination thereof.

[0026] Protein or nucleic acid variants can be defined by their sequence identity compared to the parent protein or nucleic acid. Sequence identity is usually provided as "% sequence identity" or "% identity". In the first step, to determine the percentage of identity between two amino acid sequences, a pairwise sequence alignment is created between the two sequences, where the two sequences are aligned to their full length (i.e., a pairwise full-range alignment). The alignment is generated using a program that implements the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, p. 443-453), preferably using the program "NEEDLE" (European Molecular Biology Open Software Suite (EMBOSS)) with the program's default parameters (gapopen=10.0, gapextend=0.5, and matrix=EBLOSUM62). For the purposes of the present invention, a preferred alignment is one that can determine the highest possible sequence identity.

[0027] The following example is intended to illustrate two types of nucleotide sequences, but the same calculations apply to protein sequences: Seq A: AAGATACTG Length: 9 bases Seq B:GATCTGA Length: 7 bases. Therefore, the shorter sequence is sequence B.

[0028] The generation of pairwise full-range alignments showing both sequences across their full lengths results in the following: In the alignment of TIFF0007933689000001.tif22153, the "|" symbol indicates identical residues (meaning bases in the case of DNA or amino acids in the case of proteins). There are 6 identical residues.

[0029] The "-" symbol in the alignment indicates a gap. The number of gaps introduced by alignment within sequence B is 1. The number of gaps introduced by alignment at the edge of sequence B is 2, and at the edge of sequence A it is 1. The alignment length, which shows the aligned sequence over its full length, is 10.

[0030] The generation of pairwise alignments exhibiting shorter sequences over their full length according to the present invention results in the following: TIFF0007933689000002.tif22153

[0031] The generation of a pairwise alignment showing sequence A over its full length according to the present invention results in the following: TIFF0007933689000003.tif22153

[0032] The generation of a pairwise alignment showing sequence B over its full length according to the present invention results in the following: TIFF0007933689000004.tif22153

[0033] The alignment length showing the shorter sequence over its full length is 8 (there is one gap, which is included in the alignment length of the shorter sequence). Therefore, the alignment length showing sequence A over its entire length is 9 (meaning that sequence A is the sequence of the present invention). Therefore, the alignment length showing sequence B over its full length is 8 (meaning that sequence B is the sequence of the present invention).

[0034] After aligning the two sequences, the identity value is determined from the alignment in the second step. Therefore, in accordance with this disclosure, the following calculation of identity percentage is applied: % Identity = (Identical residue / Length of alignment region showing each sequence of the present invention over its full length) * 100 In other words, sequence identity relating to the comparison of two amino acid sequences according to the present invention is calculated by dividing the number of identical residues by the length of the alignment region that shows each sequence of the present invention over its full length. Multiplying this value by 100 gives the "% identity". Following the example provided above, the % identity is: for sequence A, which is the sequence of the present invention, (6 / 9)*100 = 66.7%; for sequence B, which is the sequence of the present invention, (6 / 8)*100 = 75%.

[0035] The term "hybridization," as defined herein, is the process by which substantially complementary nucleotide sequences anneal to one another. Hybridization can occur entirely in solution, i.e., both complementary nucleic acids are in solution. Hybridization can also occur with one complementary nucleic acid immobilized on a matrix such as magnetic beads, Sepharose beads, or any other resin. Furthermore, hybridization can occur with one complementary nucleic acid immobilized on a solid support such as a nitrocellulose or nylon membrane, or immobilized on a siliceous glass support, for example by photolithography (the latter known as a nucleic acid array, microarray, or nucleic acid chip). To undergo hybridization, nucleic acid molecules are generally denatured thermally or chemically to melt the double strands into two single strands and / or to remove hairpins or other secondary structures from the single-stranded nucleic acids.

[0036] The term "stringency" refers to the conditions under which hybridization occurs. Hybridization stringency is influenced by conditions such as temperature, salt concentration, ionic strength, and hybridization buffer composition. Generally, low-stringency conditions are selected to be approximately 30°C lower than the thermal melting point (Tm) of the specific sequence at a given ionic strength and pH. Moderate-stringency conditions are when the temperature is 20°C lower than Tm, and high-stringency conditions are when the temperature is 10°C lower than Tm. High-stringency hybridization conditions are typically used to isolate hybridizable sequences that have high sequence similarity to the target nucleic acid sequence. However, due to degeneracy of genetic coding, nucleic acids may encode substantially identical polypeptides with sequence deviations. Therefore, moderate-stringency hybridization conditions may be necessary in some cases to identify such nucleic acid molecules.

[0037] "Tm" is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe at a specified ionic strength and pH. Tm depends on the solution conditions as well as the base composition and length of the probe. For example, longer sequences hybridize specifically at higher temperatures. The maximum hybridization rate is obtained at temperatures approximately 16°C to a maximum of 32°C lower than Tm. The presence of monovalent cations in the hybridization solution reduces the electrostatic repulsion between the two nucleic acid strands, thereby promoting hybridization; this effect is observed at sodium concentrations up to 0.4 M (at higher concentrations, this effect is negligible). Formamide lowers the melting temperature of DNA-DNA and DNA-RNA double helix by 0.6–0.7°C per 1% of formamide, and the addition of 50% formamide allows hybridization to occur at 30–45°C, although the hybridization rate will be reduced. Base pair mismatches reduce the hybridization rate and the thermal stability of the double helix. On average, for large probes, Tm decreases by approximately 1°C for every 1% base mismatch. Tm can be calculated using the following equation, depending on the type of hybrid: • DNA-DNA hybrid (Meinkoth and Wahl, Anal. Biochem., 138: 267-284, 1984): Tm = 81.5℃ + 16.6 × log[Na+]{a} + 0.41 × %[G / C{b}] - 500 × [L{c}] - 1 - 0.61 × %formamide • DNA-RNA or RNA-RNA hybrid: Tm=79.8+18.5(log10[Na+]{a})+0.58(%G / C{b})+11.8(%G / C{b})2-820 / L{c} • OligoDNA or oligoRNA hybrid: For nucleotides less than 20: Tm = 2({ln}) Regarding nucleotides 20-35: Tm = 22 + 1.46({ln}) With respect to {a} or other monovalent cations, it is accurate only in the range of 0.01 to 0.4 M. {b} is accurate with respect to %GC only in the range of 30% to 75%. {c} L = length of base pairs in a double hemisphere. {d} Oligo: Oligonucleotide; {ln}: Effective length of primer = 2 × (number of G / C) + (number of A / T).

[0038] Nonspecific binding can be controlled using one of many known techniques, such as blocking the membrane with a protein-containing solution, adding heterologous RNA, DNA, and SDS to the hybridization buffer, and treatment with RNases. With respect to unrelated probes, a series of hybridizations can be performed by changing one of the following: (i) gradually decreasing the annealing temperature (e.g., from 68°C to 42°C) or (ii) gradually decreasing the formamide concentration (e.g., from 50% to 0%). Those skilled in the art know of various parameters that can be changed during hybridization and that will maintain or change the stringency conditions.

[0039] In addition to hybridization conditions, the specificity of hybridization typically depends on the function of post-hybridization washing. To remove background resulting from non-specific hybridization, samples are washed with a diluted salt solution. Important factors of such washing include the ionic strength and temperature of the final washing solution: lower salt concentrations and higher washing temperatures result in higher stringency of washing. Washing conditions are typically performed at or below hybridization stringency. Positive hybridization produces a signal at least twice as strong as the background signal. Generally, preferred stringency conditions for nucleic acid hybridization assays or gene amplification detection procedures are as described above. Higher or lower stringency conditions can also be selected. Those skilled in the art are aware of the various parameters that can be changed during washing and that will maintain or alter the stringency conditions.

[0040] For example, typical high-stringency hybridization conditions for DNA hybrids longer than 50 nucleotides include hybridization in 1×SSC at 65°C or in 1×SSC and 50% formamide at 42°C, followed by washing in 0.3×SSC at 65°C. Examples of moderate-stringency hybridization conditions for DNA hybrids longer than 50 nucleotides include hybridization in 4×SSC at 50°C or in 6×SSC and 50% formamide at 40°C, followed by washing in 2×SSC at 50°C. The length of the hybrid is the length expected for the hybridizable nucleic acid. When hybridizing nucleic acids of known sequences, the length of the hybrid can be determined by aligning the sequences and identifying the conserved regions described herein. 1×SSC is 0.15M NaCl and 15mM sodium citrate; hybridization and washing solutions may further contain 5×Denhardt's reagent, 0.5-1.0% SDS, 100 μg / mL denatured fragmented salmon sperm DNA, and 0.5% sodium pyrophosphate. Another example of high stringency conditions is hybridization in 0.1×SSC containing 0.1% SDS and optionally 5×Denhardt's reagent, 100 μg / mL denatured fragmented salmon sperm DNA, and 0.5% sodium pyrophosphate at 65°C, followed by washing in 0.3×SSC at 65°C.

[0041] For the purpose of defining the level of stringency, refer to Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York or Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989 and annual revisions).

[0042] As used herein, the term “nucleic acid construct” refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, modified to include a nucleic acid segment in a manner not found in nature, or synthesized.

[0043] The term "nucleic acid construct" is synonymous with "expression cassette" when the nucleic acid construct contains the regulatory sequences necessary for the expression of polynucleotides.

[0044] The term “regulatory sequence” is defined herein to include all sequences that affect the expression of a polynucleotide (including, but not limited to, the expression of a polynucleotide encoding a polypeptide). Each regulatory sequence may be innate or exogenous to its polynucleotide, or innate or exogenous to each other. Such regulatory sequences include, but are not limited to, promoter sequences, 5'-UTRs (also called leader sequences), ribosome-binding sites (RBSs, Shine-Dalgano sequences), 3'-UTRs, and transcription start and stop sites.

[0045] The term “functionally linked” or “operably linked” in relation to regulatory elements should be understood as meaning a sequential arrangement of a regulatory element (including, but not limited to, a promoter), a nucleic acid sequence to be expressed, and, where appropriate, further regulatory elements (including, but not limited to, terminators), such that each regulatory element can perform its intended function and enable, modify, promote, or otherwise influence the expression of the nucleic acid sequence. For example, a control sequence is positioned appropriately relative to the coding sequence of a polynucleotide sequence so that the control sequence directs the expression of the coding sequence of a polypeptide.

[0046] A "promoter" or "promoter sequence" is a nucleotide sequence located upstream of a gene, on the same chain as the gene that enables its transcription. The promoter is followed by the gene's transcription start site. The promoter is recognized by RNA polymerase (along with any necessary transcription factors) to initiate transcription. A functional fragment or functional variant of a promoter is a nucleotide sequence that is recognizable by RNA polymerase and capable of initiating transcription.

[0047] "Active promoter fragment," "active promoter variant," "functional promoter fragment," or "functional promoter variant" describes a fragment or variant of a promoter nucleotide sequence that still retains promoter activity.

[0048] In this specification, an "inducer-dependent promoter" is understood as a promoter whose activity is increased upon the addition of an "inducer molecule" to a fermentation medium, thereby enabling the transcription of a gene to which the promoter is operably linked. Therefore, for an inducer-dependent promoter, the presence of an inducer molecule triggers, via signal transduction, an increase in the expression of the gene operably linked to the promoter. Pre-activation gene expression due to the presence of the inducer molecule does not need to be lost, but may be present at a low level of basal gene expression that is increased after the addition of the inducer molecule. An "inducer molecule" is a molecule whose presence in the fermentation medium can influence the increase in gene expression by increasing the activity of an inducer-dependent promoter operably linked to a gene. Preferably, the inducer molecule is a carbohydrate or its analogue. In one embodiment, the inducer molecule is a secondary carbon source for Bacillus cells. In the presence of a carbohydrate mixture, cells selectively absorb the carbon source that provides the greatest energy and proliferative advantages to the cell (the primary carbon source). At the same time, cells suppress various functions involved in the catabolism and uptake of less desirable carbon sources (the secondary carbon source). Typically, glucose is the primary carbon source for Bacillus, with various other sugars and sugar derivatives used by Bacillus as secondary carbon sources. Examples of secondary carbon sources include, but are not limited to, mannose or lactose. In contrast, the activity of promoters independent of the presence of inducer molecules (referred herein to as “inducer-independent promoters”) can be constitutively active or increased independently of the presence of inducer molecules added to the fermentation medium.

[0049] The term “expression” or “gene expression” means the transcription of a specific gene or specific nucleic acid construct. In particular, the term “expression” or “gene expression” means the transcription of a gene or gene construct into structural RNA (e.g., rRNA, tRNA) or mRNA (with or without subsequent translation of the latter into a protein). The process includes the transcription of DNA and the processing of the resulting mRNA product.

[0050] The term "vector" is defined herein as a linear or circular DNA molecule containing one or more polynucleotides operably linked to a control sequence, resulting in the expression of a polynucleotide.

[0051] As used herein, the term “isolated DNA molecule” means a DNA molecule that is at least partially isolated from other molecules that would normally bind to it in its natural or spontaneous state. The term “isolated” preferably means a DNA molecule that is at least partially isolated from a portion of nucleic acid that would normally adjoin it in its natural or spontaneous state. Thus, a DNA molecule that has been fused to a regulatory or coding sequence that would not normally bind, for example, as a result of a recombination method, is considered isolated herein. Such molecules are considered isolated in that they are not in their natural state if they are incorporated into a host cell's chromosome or present in a nucleic acid solution containing other DNA molecules.

[0052] Polynucleotides, or fragments thereof, can be isolated and manipulated as disclosed herein using any number of methods well known to those skilled in the art. For example, polymerase chain reaction (PCR) techniques may be used to amplify a particular starting polynucleotide molecule and / or produce variants of the original molecule. Polynucleotide molecules, or fragments thereof, can also be obtained by other means, for example, by chemical means, as commonly done using an automated oligonucleotide synthesizer. Polynucleotides may be single-stranded (ss) or double-stranded (ds). "Double-stranded" generally refers to base pairings that occur between sufficiently complementary antiparallel nucleic acid strands to form a double-stranded nucleic acid structure under physiologically appropriate conditions. Embodiments of the method include a polynucleotide which is at least one selected from the group consisting of sense single-stranded DNA (ssDNA), sense single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), double-stranded DNA (dsDNA), double-stranded DNA / RNA hybrid, antisense ssDNA, or antisense ssRNA, and a mixture of any of these types of polynucleotides may be used.

[0053] The term “heterogeneous” (or exogenous, foreign, recombinant, or unnatural) polypeptide is defined herein as a polypeptide that is not natural to a host cell, a polypeptide that is natural to a host cell and has been structurally modified by recombinant DNA methods to alter a natural polypeptide, e.g., by deletion, substitution, and / or insertion, or a polypeptide that is natural to a host cell and whose expression is quantitatively altered or directed from a different genomic location than that of a natural host cell as a result of recombinant DNA methods, e.g., manipulation of the host cell's DNA by a stronger promoter. Similarly, the term “heterogeneous” (or exogenous, foreign, recombinant, or unnatural) polynucleotide refers to a polynucleotide that is not natural to a host cell, a polynucleotide that is natural to a host cell and has been structurally modified by recombinant DNA methods to alter a natural polynucleotide, e.g., by deletion, substitution, and / or insertion, or a polynucleotide that is natural to a host cell and whose expression is quantitatively altered as a result of recombinant DNA methods, e.g., manipulation of the regulatory elements of the polynucleotide by a stronger promoter, or a polynucleotide that is natural to a host cell but is not incorporated into the natural genetic environment as a result of genetic manipulation by recombinant DNA methods. With respect to two or more polynucleotide sequences or two or more amino acid sequences, the term “heterogeneous” is used to characterize the fact that two or more polynucleotide sequences or two or more amino acid sequences do not naturally occur with each other in a particular combination. In particular, when referring to promoter-gene combinations, the term “heterogeneous” means that a particular combination of promoter and gene is not found naturally. Specifically, a promoter is heterogeneous to a gene if (a) a promoter operably ligated to gene A in wild-type cells is then operably ligated to another gene B instead, or (b) a promoter not found naturally is operably ligated to a gene, or (c) a promoter is operably ligated to a gene with a sequence not found naturally.

[0054] As used herein, the term "host cell" includes any cell type that is susceptible to transformation, transfection, transduction, conjugation, etc., by nucleic acid constructs or expression vectors. Therefore, the term "host cell" includes cells that have the ability to act as a host or expression medium for a newly introduced DNA sequence, and in particular for the expression of target genes contained in that newly introduced DNA sequence. The host cells according to the present invention should be understood to be prokaryotes, preferably belonging to Gram-positive or Gram-negative genera of microorganisms, more preferably Acetobacter, Acidithiobacillus, Aeromonas, Agrobacterium, Alcaligenes, Arthrobacter, Azotobacter, Bacillus, Campylobacter, Chromobacterium, Citrobacter, Clostridium, Comamonas, Corynebacterium, Enterococcus, Escherichia Genus Escherichia, Flavobacterium, Fusobacterium, Geobacillus, Geobacter, Glucobacter, Helicobacter, Ilyobacter, Lactobacillus, Lactococcus Genus Lactococcus, Microlunatus, Mycobacterium, Neisseria, Oceanobacillus, Paenibacillus, Pantoea, Pseudomonas, Ralstonia, Rhizobium,The genera selected are Rhodococcus, Saccharopolyspora, Salmonella, Serratia, Sinorhizobium, Staphylococcus, Stenotrophomonas, Streptococcus, Streptomyces, Synechocystis, Thermus, Ureaplasma, Xanthomonas, and Zymomonas. Among Gram-negative hosts, proteobacteria are preferred, more preferably gammaproteobacteria, and of these, the genera Campylobacter, Escherichia, Flavobacterium, Fusobacterium, Helicobacter, Iliobacter, Neisseria, Pseudomonas, Salmonella, and Xanthomonas are preferred, with Pseudomonas or Escherichia coli being the most preferred hosts. However, more preferred are Gram-positive hosts, particularly those of the phylum Firmicutes, more preferably any of the genera Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces, even more preferably those of the order Lactobacillales or Bacillales, even more preferably those of the family Bacillaceae, Paenibacillaceae, or Lactobacillaceae, even more preferably those of the genera Lactobacillus, Paenibacillus, Oceanobacillus, Virgibacillus, or Bacillus, and the species Bacillus amyloricephasiens. amyloliquefaciens),The most preferred host is any of the following species: Bacillus clausii, Bacillus halodurans, Bacillus lentus, Bacillus licheniformis, Bacillus paralicheniformis, Bacillus pumilus, Bacillus subtilis, or Bacillus velezensis, most preferably belonging to the Bacillus licheniformis species. An advantage of the present invention is that the host may be virtually any prokaryote in which the plasmid of the present invention can be regenerated, provided that a functional rep gene is present. Therefore, the plasmid and host of the present invention are advantageously useful for use in a variety of fermentation processes.

[0055] As used herein, “recombinant” when referring to nucleic acids or polypeptides means that such substances have been altered as a result of the application of a recombinant method to humans, such as by polynucleotide restriction and ligation, polynucleotide overlap extension, or genomic insertion or transformation. A gene sequence open reading frame is recombinant if (a) its nucleotide sequence exists in a non-natural context, for example, by (i) cloning into any type of artificial nucleic acid vector, or (ii) by movement or copying to another location in the original genome, or (b) the nucleotide sequence is mutagenic so as to be different from the wild-type sequence. The term recombinant can also refer to an organism that contains recombinant material, for example, a plant containing recombinant nucleic acid is a recombinant plant.

[0056] The term “transgenic” refers to an organism, preferably a plant or part thereof, or nucleic acid, that contains a heterogeneous polynucleotide. Preferably, the heterogeneous polynucleotide is stably incorporated into the genome so that the polynucleotide is passed down through generations. The heterogeneous polynucleotide may be incorporated into the genome alone or as part of a recombinant expression cassette. “Transgenic” is used herein to refer to any cell or cell lineage whose genotype is altered by the presence of a heterogeneous nucleic acid (including those transgenic organisms or cells that were first altered in this way, as well as those produced by hybridization or asexual reproduction from the first transgenic organism or cell). A “recombinant” organism is preferably a “transgenic” organism.

[0057] As used herein, “mutagenized” refers to an organism or its nucleic acid having a change in the biomolecular sequence of its natural genetic material compared to the sequence of the genetic material of a corresponding wild-type organism or nucleic acid, where the change in the genetic material is induced and / or selected by human action. The method of inducing a mutation may involve inducing a mutation at a random location in the genetic material, or it may involve inducing a mutation at a specific location in the genetic material, for example, using a genoplasty method (i.e., a specific mutagenesis method). In addition to nonspecific mutations, in the present invention, nucleic acids may also be mutagenesized using mutagenesis means that are selective or even specific to a particular site, thereby producing artificially induced heritable alleles according to the present invention. Such methods, including site-specific nucleases such as clustered regularly interspaced short palindromic repeats / CRISPR-associated nucleases (CRISPR / Cas) using zinc finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENS) (Malzahn et al., Cell Biosci, 2017, 7:21), and manipulated crRNA / tracr RNA (e.g., as single guide RNAs or as modified crRNA and tracrRNA molecules forming dual molecular guides), as well as methods for targeting known genomic locations using these nucleases, are well known in the art (see the review by Bortesi and Fischer, 2015, Biotechnology Advances 33: pp. 41-52; and Chen and Gao, 2014, Plant Cell Rep 33: pp. 575-583, and their references).

[0058] As used herein, “genetically modified organism” (GMO) is an organism whose genetic characteristics include changes brought about by human effort to cause transfection resulting in the transformation of a target organism using genetic material from another organism or “source” organism, or synthetic genetic material or modified natural genetic material, or an organism that is a descendant of such organism that carries inserted genetic material. The source organism may be of a different type of organism (for example, a GMO plant may contain bacterial genetic material) or may be of the same type of organism (for example, a GMO plant may contain genetic material from another plant).

[0059] The terms “native” (or wild-type or endogenous) cell or organism, and “native” (or wild-type or endogenous) polynucleotide or polypeptide, respectively, refer to cells or organisms found in nature, and to such polynucleotides or polypeptides found in cells in their natural form and genetic environment (i.e., without any human intervention).

[0060] As used herein, “wild-type” or “corresponding wild-type plant” means the typical form of the organism or its genetic material as it normally occurs, for example, when distinguished from mutagenic and / or recombinant forms. Similarly, “control cell” or “wild-type host cell” refers to a cell lacking the specific polynucleotides of the present invention disclosed herein. The use of the term “wild-type” does not therefore imply that the host cell lacks recombinant DNA in its genome.

[0061] As described herein, the present invention provides a method for controlling plasmid copy number. The term “control” means that the average number of plasmid copies per host cell can be arbitrarily adjusted by the user. Thus, the present invention makes it possible to culture a host strain while altering the plasmid copy number during culture. This is not possible in cases where the plasmid copy number is self-regulated only by the plasmid’s wild-type replication mechanism.

[0062] The present invention provides a prokaryotic host containing a plasmid. The plasmid has an origin of replication that can be activated by a plasmid replication initiation protein (Rep). Such Rep proteins are generally known to those skilled in the art. Functionally, Rep proteins and their corresponding wild-type mechanisms of plasmid copy number control can be classified into two groups. In the first and preferred group, the Rep protein results in plasmid replication by binding typically to the origin of replication at any physiologically acceptable concentration of the Rep protein. Such plasmids, origins of replication, Rep proteins and copy number control products (likewise Cop and / or antisense RNA, as further described herein) are described in detail in Khan, Review of Microbiology and Molecular Biology, 1997, pp. 442–455, the contents of which are incorporated herein by reference in their entirety. Well-known plasmids include those belonging to the families pBR322, pUC19, pACYC177, and pACYC184, which enable replication in E. coli, and pUB110, pE194, pTA1060, and rhoAMbetaI, which enable replication in Bacillus species. Typical plasmids classified into the first group described by Khan belong to the pLS1 or pUB110 family. In the second group, Rep proteins, when expressed at high concentrations, act as their own repressors. Such Rep proteins and their mechanisms of plasmid copy number autoregulation are described by Ishiai et al., Proc. Natl. Acad. Sci USA, 1994, pp. 3839-3843, and Giraldo et al., Nature Structural Biology 2003, pp. 565-571. Unless specifically shown herein or to the extent technically reasonable, plasmid copy number control according to the present invention is achieved by either the first group or the second group of copy number regulation mechanisms; however, the first group is preferred.

[0063] In this invention, the expression of the Rep protein is regulated to adjust the plasmid copy number to a desired value. Therefore, the average plasmid copy number can be arbitrarily increased or decreased by interfering with the host cell. In particular, by directly and specifically interfering with the expression of the Rep protein, this invention therefore allows for modification of the plasmid copy number without exposing the host to physiologically harsh conditions such as extreme temperature shifts. A particular advantage of this invention is that the plasmid copy number can be adjusted by the user at substantially any time and is not ultimately determined by the incorporation of a selected origin of replication (described in WO2007035323), a special promoter for the expression of the rep gene, etc.

[0064] Rep protein expression is a) Increased expression of the rep gene encoding the Rep protein, b) Decreased expression of the rep gene encoding the Rep protein, c) Decreased expression of the repressor gene encoding the repressor of Rep expression. d) Increased expression of the repressor gene encoding the repressor that expresses Rep. Preferably adjusted by one or more of the following.

[0065] Rep protein expression can be regulated by altering the transcription rate of the gene encoding the Rep protein (rep) and / or the translation rate of the mRNA encoding the Rep protein. Therefore, Rep protein expression can be increased by increasing the transcription and / or translation rates of the rep gene and its respective mRNAs. Conversely, a decrease in Rep protein expression can be achieved by decreasing the transcription and / or translation rates of the rep gene and its respective mRNAs.

[0066] The increase in gene expression by increasing transcription efficiency according to the present invention is preferably achieved by placing each gene under the control of a heterologous inductive promoter. Such promoters for any host microorganism, particularly those explicitly stated herein, are known to those skilled in the art. Typically, the transcription rate of such promoters is regulated by one or more transcription factors that respond to external stimuli, preferably the addition of a substance to the host culture medium. It is an advantage of the present invention that the method of plasmid copy number control can be carried out using such well-tested and reliable promoters and inductors. Suitable inductors and corresponding promoters known to those skilled in the art include IPTG, lactose, xylose, tetracycline and their derivatives, such as doxocycline.

[0067] An inductive promoter, also called an inducer-dependent promoter, is understood herein as a promoter whose activity is increased when an "inducer substance" is added to the culture or fermentation medium, enabling the transcription of the gene to which the promoter is operably linked. Therefore, for an inducer-dependent promoter, the presence of an inducer molecule triggers an increase in the expression of the gene operably linked to the promoter via signaling. While the gene expression before activation by the inducer molecule does not need to disappear, a low level of basal gene expression may also be present, which is increased after the addition of the inducer molecule. An "inducer molecule" is a molecule whose presence in the fermentation medium can influence the increase in gene expression by increasing the activity of an inducer-dependent promoter operably linked to the gene. Preferably, the inducer molecule is a carbohydrate or its analogue. In one embodiment, the inducer molecule is a secondary carbon source for Bacillus cells. In the presence of a carbohydrate mixture, cells selectively absorb the carbon source that provides the greatest energy and proliferative advantages to the cell (the primary carbon source). At the same time, cells suppress various functions involved in the catabolism and uptake of less desirable carbon sources (the secondary carbon source). Typically, glucose is the primary carbon source for Bacillus, and various other sugars and sugar derivatives are used by Bacillus as secondary carbon sources. Examples of secondary carbon sources include, but are not limited to, mannose or lactose.

[0068] A well-known example of an inducible promoter is the E. coli-derived P promoter, which is regulated by araC, a dimer that changes conformation upon the addition of arabinose and binds to operator sites I1 and I2. BADThe Bacillus-derived mannose-inducible promoter system PmanP is regulated by the promoter and the activator manR. Inducible promoters, such as the lacUV5 promoter, the T7 phage promoter for expression in E. coli, and the Pspac-I and Ppac-I promoters in Bacillus, are negatively regulated by a lac repressor (encoded by the lacI gene) that, in the absence of inducing molecules, binds to its specific lac operator site within the promoter sequence, e.g., at the -35 to -10 sigA recognition site, or in the vicinity of the promoter sequence, i.e., at either 3' or 5', thereby blocking transcription. Another example is the Bacillus megatherium-derived PxylA-inducible promoter system, widely used in Bacillus expression systems. The PxylA promoter is negatively regulated by an xylR repressor protein containing an xylR operator site at 3' of the transcription start site. A synthetic inducible expression system containing theophylline riboswitches, applicable to a wide range of bacterial species, has been developed (Topp S, Reynoso CM, Seeliger JC et al., Synthetic riboswitches that induce gene expression in diverse bacterial species; Appl Environ Microbiol. 2010;76(23):7881~7884).

[0069] Examples of inductor-dependent promoters are shown in the table below, referring to their respective operons.

[0070] TIFF0007933689000005.tif201169

[0071] Accordingly, the reduction in gene expression by lowering transcription efficiency according to the present invention is preferably achieved by placing each gene under the control of a silencable promoter. Such promoters for any host microorganism, particularly those explicitly stated herein, are known to those skilled in the art. Typically, the transcription rate of such promoters is regulated by one or more transcription factors that respond to external stimuli, preferably the addition of a substance to the host culture medium. It is an advantage of the present invention that methods of plasmid copy number control can be carried out using such well-tested and reliable promoters and silencer substances. Suitable silencer substances and corresponding promoters known to those skilled in the art include tet-off promoter systems applying tetracycline and its derivatives, e.g., doxocycline; riboswitch promoter systems not limited to coenzyme B12 riboswitches; lysine riboswitches; and TPP riboswitches (Winkler WC, Breaker RR. Regulation of bacterial gene expression by riboswitches. Annu Rev Microbiol. 2005;59:487-517).

[0072] Therefore, in the present invention, the rep gene can be operably linked to a heterologous inducible promoter in the expression cassette. The rep gene under the control of the inducible promoter may be present in the plasmid and / or host chromosome. In both cases, the transcription rate of the rep gene and the corresponding expression of the Rep protein are maximized when the corresponding inducer is provided to the host strain, and in the absence of such an inducer, the transcription rate and therefore the expression of the Rep protein are reduced. Therefore, the expression of the Rep protein can be temporarily increased for a certain period, for example by adding an inducer during the fermentation process. This is a particular advantage of the present invention. For example, this makes it possible to regenerate host strain cultures from cryopreservation in the absence of an inducer, and to grow starter cultures containing only a low plasmid copy number. As shown above, microorganisms become metabolically difficult when they experience changes in the culture medium or other environmental conditions. By not forcing the host to maintain a high plasmid copy number under such conditions, the present invention allows the host to adapt to changes in growth conditions more quickly, thereby enabling a faster initiation of exponential growth. A further advantage of the present invention is that selective pressure is still maintained even under metabolic stress conditions, thereby preventing plasmid loss from host culture.

[0073] If the Rep protein belongs to the first and preferred group of Rep proteins, an increase in Rep protein expression results in an increase in plasmid copy number. In the second group, an increase in Rep protein expression results only in an increase in plasmid copy number within the Rep protein-specific concentration range. For such plasmid copy number regulatory systems, a lack of Rep protein results in plasmid loss from the host due to a lack of plasmid replication initiation factors. High concentrations of Rep protein typically lead to Rep self-inactivation by Rep protein dimerization. Therefore, with respect to the second group, the present invention still allows for a transient increase in plasmid copy number by increasing Rep expression from zero to the optimal Rep concentration, but also allows for increasing the Rep concentration beyond the optimal Rep concentration, thereby causing Rep to self-repress and temporarily reducing the plasmid copy number.

[0074] The rep gene may also be operably linked to a heterologous repressive promoter in the expression cassette. In this case as well, the rep gene under the control of the repressive promoter may reside in the plasmid and / or host chromosome. In both cases, the addition of the silencer substance reduces the transcription rate of the rep gene. Correspondingly, the expression of the Rep protein is maximized in the absence of the silencer substance and reduced in the presence of the silencer substance. With respect to the first group of Rep proteins, such a configuration is advantageous when a high plasmid copy number is desired for most stages of host culture, and a low copy number is desired only at specific stages, e.g., during medium changes, during regeneration after cryopreservation, or at the earliest stages of fermenter inoculation. By adding the silencer substance only at such exceptional stages with a low desired plasmid copy number, the present invention allows all other handling of the host to be performed at a higher plasmid copy number without the need to add the silencer substance. With respect to the second group of Rep proteins, such a configuration is advantageous for temporarily lowering the Rep concentration from a self-repressive level to a plasmid replication-permissible level.

[0075] Rep protein expression can also be modified by the expression of a repressor of rep gene expression under the control of a heterologous promoter, in addition to or instead of binding to an inductive or repressive promoter. Such a repressor may be a nucleic acid or a protein. In pLS1 family plasmids, for example, both types of repressors, namely copA antisense RNA that hybridizes to the Rep-coding mRNA and the CopB repressor protein of rep gene expression, are naturally used to regulate plasmid copy number. The use of a native repressor of rep gene expression has the additional advantage that at least one copy of the rep gene may still be under the control of the native promoter of Rep expression.

[0076] Preferably, at least one rep gene is operably ligated to a native rep gene promoter, and the repressor is a repressor of the native rep gene promoter. Therefore, it is possible, for example, to provide an unmodified high-copy-number plasmid in a host containing an expression cassette with a copy of the repressor gene in a chromosome or helper plasmid under the control of a heterologous-inducible or repressive promoter. An advantage of such a plasmid-host system is that once the repressor expression cassette is introduced, only the host strain needs to be genetically modified. The resulting host can then be used to control the copy number of any plasmid of a suitable rep gene promoter by expressing the repressor at a desired concentration at each time point to obtain a desired level of Rep expression and then a desired plasmid copy number.

[0077] The present invention also provides the use of heterogeneous constitutive promoters in addition to or instead of inductive or repressive promoters. This results in a quantity of repressor that gives the cell a certain copy number. The expression rate of the constitutive promoter can be adjusted according to desired expression levels, for example, Guiziou et al., (2016): Nucleic Acids Res. 44(15), pp. 7495-7508, or according to anti-sigma factors, as described in WO2013148321.

[0078] Repressor genes may be under the control of a xenoinducible promoter. In such cases, the addition of an inducer to the host leads to an increase in repressor gene expression, resulting in a decrease in Rep protein expression as described above. When repressor genes are under the control of a xenorepressive promoter, the addition of a silencer substance to the host leads to a decrease in repressor gene expression, resulting in an increase in Rep protein expression as described above.

[0079] Preferably, rep gene expression is under the control of both a repressor nucleic acid and a repressor protein (also referred to herein as the "Cop protein"). In such a system, one first gene encoding the first repressor is preferably located on a plasmid, and the expression cassette of the other gene encoding the second repressor is preferably located anywhere and preferably integrated into the host chromosome. Thus, the first repressor functions as a self-regulator of plasmid copy number. As the plasmid copy number increases, the gene amount of the first repressor also increases accordingly. This, in turn, leads to an increase in the concentration of the first repressor and therefore to the suppression of rep gene expression, thereby limiting the maximum plasmid copy number without requiring user intervention. As the plasmid copy number decreases, the gene amount of the first repressor also decreases accordingly. This, in turn, leads to a decrease in the concentration of the first repressor and therefore to an increase in rep gene expression, thereby preventing plasmid loss or a decrease in plasmid copy number below a minimum threshold, again without requiring user action. When rep gene expression is under the control of both a repressor nucleic acid and a repressor protein, the first repressor is an expression cassette comprising a repressor nucleic acid, most preferably an antisense RNA, and a promoter, to which the first repressor gene operably ligated is preferably located on a copy-number-controlled plasmid. This configuration is particularly advantageous because self-regulation does not depend on the metabolically compensatory production of the repressor protein, and instead the transcript of the first repressor gene is sufficient to repress Rep expression to the desired strength. A further advantage is that nucleic acid repressors are produced faster than protein repressors, generally have shorter half-lives, and can therefore be used to control Rep expression with only a very short delay. Furthermore, nucleic acid repressors in the form of antisense nucleic acids are independent of the sequence of the promoter driving rep gene expression. Thus, nucleic acid repressors can repress Rep expression by blocking or reducing the translation of the rep mRNA transcript, even when the rep gene is under the control of a heterologous promoter.

[0080] In response, the present invention also provides plasmids adapted for plasmid copy number control. In particular, the present invention provides a controlled replication plasmid comprising a replication origin activatable by a plasmid replication initiation protein (Rep) and a rep gene encoding the Rep protein, wherein the rep gene is operably ligated to a heterologous promoter, the promoter preferably being inductive or repressive. As described above, such plasmids can increase or decrease Rep protein expression by adding an inducer or silencer substance. If the rep gene is repressible by at least one repressor, this repressor is preferably provided not by a plasmid but by a repressor expression cassette integrated into the host, preferably the host chromosome, and / or located on a helper plasmid. As shown above, the present invention also provides plasmids comprising a rep gene under the control of a heterologous, preferably inductive or repressive promoter, and also comprising a gene encoding a nucleic acid repressor of Rep expression. Such an antisense nucleic acid repressor is independent of the promoter driving the expression of the rep gene.

[0081] The present invention also provides a controlled replication plasmid comprising a plasmid replication initiation protein (Rep) activatable origin of replication and a rep gene encoding the Rep protein, wherein the plasmid comprises a repressor gene encoding a repressor of Rep expression, operably ligated to a heterologous promoter. Again, the heterologous promoter is preferably inductive or repressive. If the repressor is a repressor protein, the rep gene is under the control of a promoter repressible by the repressor protein. In such cases, the rep gene is preferably operably ligated to a native rep gene promoter or a promoter derived from a native rep gene promoter that is still capable of ligating a repressor protein.

[0082] A particular advantage of plasmids containing a plasmid replication initiation protein (Rep)-activated origin of replication, a rep gene encoding the Rep protein, and a repressor gene encoding a repressor of Rep expression under the control of a heterologous inducible or repressive promoter is that the copy number of such plasmids can be controlled by the application of their respective inducers or silencers without altering the host genetic material. Therefore, such all-in-one plasmids are particularly versatile. Particularly preferred uses of such plasmids are described herein, in particular, in the figures and examples.

[0083] Similarly, the present invention provides a controlled replication plasmid comprising a plasmid replication initiation protein (Rep) activatable origin of replication and a rep gene encoding the Rep protein, wherein the rep gene is operably ligated to a heterologous promoter, and further comprises a repressor gene encoding a repressor of Rep expression, which is operably ligated to a heterologous promoter. Preferably, at least the heterologous promoter operably ligated to the repressor gene is inducible or repressive. One such plasmid, i.e., a plasmid comprising a nucleic acid repressor that represses a rep gene under the control of a heterologous promoter, is described above.

[0084] Furthermore, the present invention, - Origins of replication that can be activated by plasmid replication initiation proteins (Reps), and - In some cases, a promoter operably linked to a repressor gene encoding a repressor for Rep expression. This provides a replication support-dependent plasmid that contains the functional rep gene encoding the Rep protein.

[0085] As described herein, replication support-dependent plasmids still require the activity of the Rep protein. However, the Rep protein is not provided cis by a corresponding expression cassette located on the plasmid. Instead, the Rep protein is provided trans by a corresponding expression cassette located on the host chromosome and / or a helper plasmid. Such helper plasmids preferably do not depend on the activity of the Rep protein for replication and therefore replicate independently of the replication support-dependent plasmid. Providing the rep gene trans has the advantage that it is sufficient once the corresponding rep gene expression cassette is established on the host, preferably on its chromosome, and therefore it becomes available to any replication support-dependent plasmid or any other controlled replication plasmid introduced into the host. Replication support-dependent plasmids have specific advantages. Firstly, replication support-dependent plasmids replicate only in competent hosts. Therefore, replication support-dependent plasmids are particularly safe vectors that essentially prevent accidental transformation of hosts that do not express the Rep protein. Furthermore, replication support-dependent plasmids particularly facilitate the plasmid integration method described herein.

[0086] Replication-assisted plasmids do not contain a functional rep gene. The absence of a functional rep gene can be achieved by excising the rep gene from a wild-type plasmid containing the rep gene. Furthermore, the rep gene may be inactivated, for example, by introducing one or more immature stop codons or by other amino acid changes encoding a non-functional protein. Furthermore, genes encoding non-functional proteins may preferably lack a functional promoter. Most preferably, replication-assisted plasmids do not contain nucleic acid sequences having at least 95%, more preferably at least 90%, and even more preferably at least 80% sequence identity with a rep gene encoding any of the following amino acids identified by Uniprot identifiers according to Table 1.

[0087] The replication-assisted plasmid, when expressed in a host containing an expression cassette for Rep protein expression, preferably further comprises a gene encoding a repressor that suppresses Rep protein expression. As described above, such a host provides the rep gene in trans. By controlling the expression of the rep gene provided by the host via the repressor, the present invention enables control of plasmid copy number. In such cases, having the rep gene on a replication-assisted plasmid encoding a nucleic acid repressor for Rep protein expression is particularly advantageous. As described above, nucleic acid repressors are particularly useful, for example, for establishing a feedback mechanism to limit the maximum plasmid copy number without requiring user interference.

[0088] Particularly preferred functions applicable to controlled replication plasmids and replication support-dependent plasmids are described below.

[0089] As described above, heterologous promoters operably linked to repressor and / or rep genes respond to changes in the concentration of external regulators or metabolites. Heterologous promoters that respond to the concentration of external regulators are referred to herein as inducible or repressive promoters. Heterologous promoters that respond to changes in metabolite concentrations allow for the automatic induction or repression of genes operably linked to the promoter under given metabolic conditions. It is particularly advantageous to provide repressor genes under the control of a promoter to the plasmid, host chromosomes, and / or helper plasmids of the present invention so that the repressor genes are repressed in the later stages of fermentation, when host cells stop growing exponentially. At such stages near the end of batch fermentation, host viability is no longer a concern. Instead, it may be more desirable to maximize the “last-minute” expression of the target gene located on the plasmid. Induction of such “last-minute” expression can be achieved by removing the repression of plasmid copy number control, thereby increasing the plasmid copy number and therefore increasing the amount of the target gene. A suitable promoter that responds to such an end to the fermentation process is, for example, a promoter that is suppressed under strict limitations, or a promoter that is suppressed when spore formation occurs. An example of such a promoter is abrB in Bacillus subtilis.

[0090] The plasmid according to the present invention is preferably a rolling-circle replication plasmid as described above. Since the Rep protein does not behave as an autorepressor, such plasmids allow for particularly easy and reliable replication control. Therefore, changes in the concentration of the Rep protein in the host cell do not need to be as finely tuned as plasmids of the second Rep group.

[0091] Furthermore, the plasmid is preferably, - Selectable markers, - Target gene, - An expression cassette containing a promoter operably linked to a target gene, - Recombination site Includes one or more of the following.

[0092] The selectable marker ensures the presence of the plasmid in the host cell. The selectable marker, as described above, also ensures highly efficient integration of the plasmid of the present invention into the aforementioned nucleic acid, preferably the host chromosome, by homologous recombination.

[0093] The target gene preferably encodes an enzyme, more preferably selected from the group consisting of amylase, catalase, cellulase, chitinase, cutinase, galactosidase, beta-galactosidase, glucoamylase, glucosidase, hemicellulase, invertase, laccase, lipase, mannanase, mannosidase, nuclease, oxidase, pectinase, phosphatase, phytase, protease, ribonuclease, transferase, and xylanase, more preferably protease, amylase, or lipase, and most preferably protease. A particular advantage of the present invention is that the plasmids of the present invention are useful for the expression of various such target genes common in industrial fermentation processes.

[0094] The present invention also provides plasmid replication control hosts comprising controlled replication plasmids as described herein. Such hosts are particularly adapted to changes in plasmid copy number due to user-defined needs. In particular, such hosts allow for a temporary reduction in plasmid copy number when preparing cryopreserved cultures or regenerating cryopreserved culture samples.

[0095] The plasmid replication control host preferably provides in trans a repressor gene encoding a Rep expression repressor operably linked to a heterologous inducible or repressive promoter. This makes it possible to induce the expression of the host-provided rep gene to temporarily reduce the plasmid copy number, or to reduce the expression of the host-provided rep gene to temporarily increase the plasmid copy number. Preferably, the plasmid replication control host provides in trans a gene encoding a Rep expression repressor protein as described herein.

[0096] The present invention also, i) Replication-assisted dependent plasmid according to the present invention, and ii) Expression cassette for the expression of rep genes located on the host chromosome and / or helper plasmids in the host. It also provides plasmid replication support hosts that include this host.

[0097] As described above, such plasmid replication support hosts provide the Rep protein in trans and are therefore particularly well-suited for maintaining replication support-dependent plasmids. Thus, plasmid replication support hosts become part of a safety framework that prevents the accidental transfer of replication support-dependent plasmids to other strains that do not provide the Rep protein in trans.

[0098] Considering the above advantages, the present invention also, i) A step of providing a host starter culture containing the plasmid according to the present invention, ii) The step of culturing the host to achieve an increase in the number of host cells, and iii) A step to adjust the plasmid copy number in cultured host cells during or after step ii). We also provide a culture method that includes this.

[0099] As described herein, the plasmid may be a controlled replication plasmid or a replication support-dependent plasmid. In the latter case, the host should be a plasmid replication support host.

[0100] The culture method according to the present invention provides a starter culture. Such a starter culture is preferably a culture maintained under significantly different metabolic conditions than those experienced in step ii) of the culture method. Preferably, the starter culture is a culture sample regenerated from cryopreservation. Also preferably, the starter culture is a culture sample for inoculation into a fermenter, and therefore undergoes a change in culture volume from a maximum starter culture volume of 100 ml to a fermentation volume of at least 1 cubic meter.

[0101] In step ii), the starter culture is cultured to increase the number of host cells. Typically, this results in an increase in the optical density of the culture medium. The present invention is not limited to a specific medium or method for achieving an increase in the number of host cells. Instead, the culture method of the present invention is advantageously broadly applicable to industrial fermentation processes.

[0102] During or, less preferably, after step ii), the plasmid copy number in the cultured host cells is adjusted. The adjustment is preferably a transient reduction in plasmid copy number, an increase in plasmid copy number, or a reduction followed by an increase in copy number.

[0103] As described above, the culture method provided herein allows the host to grow by maintaining a low copy number during metabolically harsh conditions, such as when changing the culture medium or during regeneration after cryopreservation, and then increasing the plasmid copy number later when it is expected that the host can sustain a high plasmid copy number without impairing growth.

[0104] Plasmid copy number adjustment may be postponed after the start of step ii). Typically, the starter culture is placed in or on a new medium in which host cell proliferation is initiated. Plasmid copy number reduction is preferably performed at the start of step ii). In this way, the host cell culture is relieved of the metabolic burden of maintaining a high plasmid copy number. Plasmid copy number increase preferably begins when the host cell number has increased by at least 25%, more preferably at least 33%, even more preferably at least 45%, even more preferably at least 50%, even more preferably at least 75%, and even more preferably at least 90%. Under these conditions, the occupancy rate of host cells that have adapted well to the new culture conditions is high enough to bear the additional metabolic burden of increased plasmid replication.

[0105] The present invention also provides a method for preparing a starter culture in which the plasmid copy number in the host culture is adjusted, preferably reduced, while the host cells of the culture continue to grow. In this way, the present invention provides a particularly advantageous method that complements the above-described method for culturing starter cultures. By reducing the plasmid copy number in the host cell culture before cryopreservation, medium change, inoculation with a large amount of new medium, or other events that are significantly metabolically stressful, the host cells are relieved of some of the stress of plasmid proliferation. Using this method for preparing starter cultures, the present invention advantageously provides starter cultures in which the plasmid copy number has already been reduced. Such starter cultures thus have reduced initial metabolic burdens, such as maintaining a high plasmid copy number immediately after the start of culturing these starter cultures, for example immediately after the regeneration of such cryopreserved starter cultures, without any user interaction. The method for preparing starter cultures thus advantageously provides starter cultures in which an additional reduction of the plasmid copy number is not required in step ii) of the above-described method.

[0106] Furthermore, the present invention provides a starter culture comprising a plasmid replication control host or plasmid replication support host according to the present invention in a starter medium, wherein the plasmid copy number of the host increases after introducing the host into LB medium, i.e., a medium of 10 g tryptone, 10 g NaCl, and 5 g yeast extract per 950 g of water. Thus, such a starter culture of the present invention advantageously achieves a spontaneous increase in plasmid copy number without requiring further user intervention. It should be understood that the starter culture can be added to any suitable medium selected considering the metabolic requirements of the host and is not limited to growth in LB medium.

[0107] In the present invention, a low plasmid copy number is preferably at most half, more preferably at most 35%, more preferably at most 30%, more preferably at most 25%, more preferably at most 20%, and more preferably at most 10% of the maximum plasmid copy number, and the maximum plasmid copy number is a) The equilibrium plasmid copy number of the corresponding wild-type plasmid in the corresponding host, and b) Where applicable, the maximum plasmid copy number of the plasmid of the present invention in the corresponding host, achieved by increasing Rep expression through the application of an inducer or silencer substance. It is determined as the maximum.

[0108] Preferably, the low plasmid copy number is 1 to 15, and the high plasmid copy number is 20 to 100. More preferably, the low plasmid copy number is 1 to 10, and the high plasmid copy number is 30 to 60. For example, with respect to the pE194 replicon plasmid (pLS1 family), the preferred high plasmid copy number is 30 to 40 plasmids per host cell, and the preferred low plasmid copy number is 1 to 10 plasmids per host cell.

[0109] Furthermore, methods for integration are also provided herein. As described above, integration according to the present invention may be integration into the host chromosome, or it may be integration into a further plasmid. However, it is preferable that the host used in the integration method according to the present invention does not contain plasmids other than the plasmid of the present invention. Thus, the integration method does not tend to cause replication incompatibility between two different plasmids present in the host.

[0110] Step i) provides a prokaryotic plasmid recipient host containing the plasmid of the present invention. The plasmid is preferably a controlled replication plasmid and therefore includes an expression cassette for rep gene expression. The plasmid may also include a replication support-dependent plasmid. The plasmid used in the integration method according to the present invention includes a selection marker. The selection marker allows for confirmation of the presence of at least a selection marker nucleic acid segment in the recipient host.

[0111] In step ii), plasmid replication is inhibited while maintaining the selection of the selection marker. The sustained selective pressure enhances the retention of at least the selection marker nucleic acid segment in the recipient host. However, since the plasmid cannot replicate during step ii), the plasmid (in the absence of selective pressure) is lost from the recipient host. Therefore, the continued presence of the plasmid is crucial for the survival of the recipient host. Since the recipient host can only withstand the sustained selective pressure after plasmid integration, the host is forced to integrate the plasmid into its host chromosome under such circumstances.

[0112] The inhibition of plasmid replication according to the present invention can be achieved by one or more of the following methods: - If the rep gene is operablely linked to an inducible xenopromoter, remove the required inducible proto-substance. - If the rep gene is operably linked to a repressive xenopromoter, add a silencer substance. - If the repressor gene is operably linked to an inducible promoter, the production of the repressor of Rep expression by adding the inducible substance, - If the repressor gene is operably linked to an inhibitory promoter, it produces a repressor of Rep expression by removing the silencer substance.

[0113] Preferably, the repressor is a protein repressor, and the rep gene is under the control of a promoter repressible by the protein repressor. A particular advantage of the integration method of the present invention is that it can be applied to both commonly used plasmids without modifying the plasmid's replication operon. In this case, the recipient host provides the repressor gene in trans under the control of an inductive or repressive promoter. However, the integration method can also be applied without the host providing the repressor gene in trans. In this case, the plasmid provides the repressor gene under the control of an inductive or repressive promoter. Of course, both the plasmid and the host can provide the repressor gene under the control of an inductive or repressive promoter, thereby advantageously increasing the repressor concentration in the host during step ii) of the integration method.

[0114] As shown in the examples, a particular advantage of the present invention is that it reliably achieves complete plasmid integration into the host chromosome.

[0115] In a specific integration method according to the present invention, a plasmid is introduced from a plasmid donor host to a plasmid recipient host. This method is advantageous because the plasmid is cultured in the plasmid donor host, for example, at a high plasmid copy number, and then the plasmid is introduced into a recipient host where the plasmid cannot replicate. Plasmid introduction can be achieved by any suitable means, e.g., electroporation or conjugation. Conjugation is a particularly preferred method of introduction because it is extremely gentle on the recipient host. This is especially important due to the selective pressure on the recipient host. Under such selective pressure, the recipient host is subjected to strong stress to immediately begin expressing the plasmid's selective markers. Under such conditions, the survival of the recipient host is significantly impaired if the host also has to recover from the effects of electroporation. The inventors have observed that recipient host cells die immediately after electroporation, thereby reducing the effectiveness of antibiotics added to the recipient host cell culture. This, in turn, leads to a decrease in selective pressure and therefore reduces the recipient host's need to integrate the plasmid nucleic acid. Furthermore, many hosts with interesting properties in industrial fermentation processes cannot be easily electroporated.

[0116] Therefore, the present invention i) A plasmid donor host comprises a controlled replication plasmid or replication support-dependent plasmid according to the present invention, which contains a selectable marker. - The plasmid recipient host does not contain the rep gene. A step of providing a plasmid donor host and a plasmid recipient host, ii) The step of introducing a plasmid from a plasmid donor host to a plasmid recipient host, and iii) A step in which plasmid replication in the plasmid recipient host is inhibited while applying selective pressure to the plasmid recipient host that compels the presence of a selectable marker in the plasmid recipient host, either simultaneously with or after step ii). We provide an implementation method that includes this.

[0117] As described above, plasmid introduction is preferably brought about by conjugation. The three-step integration method described above is particularly advantageous because the absence of any rep gene in the recipient host puts extremely high pressure on the host to integrate the plasmid into the chromosome. This is because it is the only way to maintain the presence of the selection marker nucleic acid sequence. Without integration, the selection marker nucleic acid sequence would be lost in the daughter cells, and the daughter cells would then be unable to proliferate.

[0118] The plasmid preferably contains a recombination site to facilitate recombination with a compatible site in the nucleic acid of the replication recipient host. By providing the recombination site to the plasmid, a single event of homologous recombination between the plasmid and the host nucleic acid, preferably the host chromosome, is sufficient to achieve complete plasmid integration. This is a particular advantage as it ensures that all or substantially all surviving recipient host cells have integrated the complete plasmid. Therefore, the integration method of the present invention is not only efficient but also reliable in the production of recombinant recipient hosts.

[0119] The present invention is further illustrated below by examples and figures. The examples and figures are provided as means of further illustrating selected embodiments in the context of the present invention and are not intended to limit the understanding of the present invention or the claims.

[0120] [Examples] Unless otherwise noted, the following experiments were performed using standard equipment, methods, chemicals, and biochemicals used in the genetic engineering and fermentation production of compounds by microbial culture. See also Sambrook et al. (Sambrook, J. and Russell, DW Molecular cloning. A laboratory manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2001) and Chmiel et al. (Bioprocesstechnik 1. Einfuhrung in die Bioverfahrenstechnik, Gustav Fischer Verlag, Stuttgart, 1991). Standard molecular biology methods, not limited to Escherichia coli culture, DNA electroporation, isolation of genomic and plasmid DNA, PCR reactions, and cloning techniques, were performed essentially as described by Sambrook and Russell (Sambrook, J. and Russell, DW Molecular cloning. A laboratory manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2001).

[0121] [Table 1]

[0122] Electrocompetent Bacillus licheniformis cells and electroperforation DNA transformation of Bacillus licheniformis strains DSM 641 and ATCC 53926 is performed by electroporation. Preparation of electrocompetent Bacillus licheniformis cells and DNA transformation are carried out with the following modifications, as essentially described by Brigidi et al. (Brigidi, P., Mateuzzi, D. (1991). Biotechnol. Techniques 5, 5). During DNA transformation, after seeding on a selective LB agar plate, the cells are harvested in 1 ml LBSPG buffer and incubated at 37°C for 60 minutes (Vehmaanpera J., 1989, FEMS Microbio. Lett., 61: pp. 165-170).

[0123] To overcome the Bacillus licheniformis-specific restriction modification system in Bacillus licheniformis strains DSM 641 and ATCC 53926, plasmid DNA is isolated from Ec#098 cells as described below. For introduction into Bacillus licheniformis restrictase knockout strains, plasmid DNA is isolated from E. coli INV110 cells (Life Technologies).

[0124] Electrocompetent Bacillus pumilus cells and electroporation DNA transformation of Bacillus pumilus DSM14395 cells is performed by electroporation. Preparation and DNA transformation of electrocompetent Bacillus pumilus DSM14395 cells are performed as described for Bacillus licheniformis cells.

[0125] To overcome the Bacillus pumilus-specific restriction modification system, plasmid DNA was isolated from E. coli DH10B cells, and the plasmid DNA was methylated in vitro using a whole cell extract from Bacillus pumilus DSM14395 according to the method described for Bacillus licheniformis in patent DE4005025.

[0126] Plasmid isolation Plasmid DNA was isolated from Bacillus cells and Escherichia coli cells using standard molecular biology methods described in (Sambrook, J. and Russell, DW Molecular cloning. A laboratory manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2001), or by alkaline lysis (Birnboim, HC, Doly, J. (1979). Nucleic Acids Res 7(6): pp. 1513-1523). Compared to Escherichia coli, Bacillus cells were treated with 10 mg / ml lysozyme at 37°C for 30 minutes before cell lysis.

[0127] Oligonucleotide annealing for oligonucleotide double helix formation Oligonucleotides were adjusted to a concentration of 100 μM in water. 5 μl of forward oligonucleotide and 5 μl of the corresponding reverse oligonucleotide were added to 90 μl of 30 mM Hepes buffer (pH 7.8). The mixture was annealed by gradually lowering the temperature from 95°C to 4°C at a rate of 0.1°C / second, and then the reaction mixture was heated at 95°C for 5 minutes (Cobb, RE, Wang, Y., and Zhao, H. (2015). High-Efficiency Multiplex Genome Editing of Streptomyces Species Using an Engineered CRISPR / Cas System. ACS Synthetic Biology, 4(6), pp. 723-728).

[0128] KK E. coli strain Ec#098 E. coli strain Ec#098 is E. coli strain INV110 (Life Technologies) that possesses a DNA methyltransferase encoding the expression plasmid pMDS003 (WO2019016051).

[0129] Generation of Bacillus licheniformis gene ko strains For gene deletions in Bacillus licheniformis strains DSM641 and ATCC 53926 (US5,352,604) and their derivatives, selection was performed on LB agar plates containing 100 μg / ml ampicillin and 30 μg / ml chloramphenicol at 37°C. The deletion plasmids were then transformed into competent Escherichia coli strain Ec#098 according to Chung's method (Chung, CT, Niemela, SL, and Miller, RH (1989). One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. US A 86, pp. 2172-2175). Plasmid DNA was isolated from individual clones and used for subsequent introduction into Bacillus licheniformis strains. The isolated plasmid DNA possesses the respective DNA methylation patterns of Bacillus licheniformis strains DSM641 and ATCC53926, and is protected from degradation upon introduction into Bacillus licheniformis.

[0130] Bacillus licheniformis P304: Restricted Endonuclease Deficiency Electrocompetent Bacillus licheniformis DSM641 cells (US5,352,604) were prepared as described above, seeded on LB agar plates containing 5 μg / ml erythromycin at 30°C, and then transformed with 1 μg of pDel006 restrictase gene deletion plasmid isolated from Escherichia coli Ec#098.

[0131] The gene deletion procedure was performed as described below.

[0132] Plasmids containing Bacillus licheniformis cells were grown at 45°C on LB agar plates containing 5 μg / ml erythromycin to drive the incorporation of the deletion plasmid into the chromosome via Campbell recombination by one of the homology regions of pDel006 homologous to the 5' or 3' sequence of the restrictase gene. Clones were harvested and seeded at 30°C on LB agar plates containing 5 μg / ml erythromycin, then cultured in LB medium at 45°C for 6 hours without selective pressure. Individual clones were harvested and screened for successful genomic deletion of the restrictase gene by colony PCR analysis using oligonucleotide sequence numbers 14 and 15. Individual putative deletion-positive clones were harvested and the plasmids were cured by incubation in antibiotic-free LB medium at 45°C for two consecutive nights, and then seeded on LB agar plates for an overnight incubation at 37°C. Single clones were analyzed by colony PCR for successful genomic deletion of the restrictase gene. A single erythromycin-sensitive clone containing the correct deletion restrictase gene was isolated and named Bacillus licheniformis P304.

[0133] Bacillus licheniformis P308: Polygamma-glutamate synthesis gene deletion Electrocompetent Bacillus licheniformis P304 cells were prepared as described above, seeded on LB agar plates containing 5 μg / ml erythromycin at 30°C, and then transformed with 1 μg of pDel007 pga gene deletion plasmid isolated from E. coli INV110 cells (Life Technologies).

[0134] The gene deletion procedure was performed as described for the deletion of the restrictase gene.

[0135] The deletion of the pga gene was analyzed by PCR using oligonucleotide sequence numbers 17 and 18. The resulting Bacillus licheniformis strain lacking the pga synthesis gene was named Bacillus licheniformis P308.

[0136] Bacillus licheniformis strain LBL: Integration of the lux gene into the amyB locus. Electrocompetent Bacillus licheniformis P308 was prepared as described above, transformed with 1 μg of plasmid pKS068 isolated from E. coli INV100, and then seeded on LB agar plates containing 50 μg / ml erythromycin at 30°C.

[0137] The gene deletion procedure was performed as described for the deletion of the restrictase gene.

[0138] The deletion of the amyB gene and the incorporation of the lux expression cassette were analyzed by PCR using oligonucleotides SEQ ID NOs. 9 and 10. After iodine staining with Lugol's solution, the clearing zone was analyzed on an LB agar plate containing 1% starch. The resulting Bacillus licheniformis strain was named Bacillus licheniformis LBL.

[0139] Bacillus licheniformis Bli#002: aprE gene deletion Electrocompetent Bacillus licheniformis ATCC53926 cells were prepared as described above, seeded on LB agar plates containing 5 μg / ml erythromycin at 30°C, and then transformed with 1 μg of pDel003 aprE gene deletion plasmid isolated from E. coli Ec#098.

[0140] The gene deletion procedure was performed as described for the deletion of the restrictase gene. The deletion of the aprE gene was analyzed by PCR using oligonucleotide sequence numbers 20 and 21. The resulting Bacillus licheniformis strain lacking the aprE gene was named Bli#002.

[0141] Bacillus licheniformis Bli#005: Polygamma-glutamate synthesis gene deletion The polygamma-glutamate synthesis gene was deleted in Bacillus licheniformis Bli#002, similar to the deletion of the pga gene in Bacillus licheniformis P304. The difference was that the pDel007 plasmid was isolated from E. coli Ec#098 cells. The resulting strain was named Bli#005.

[0142] Bacillus subtilis strain GCX1 Wild-type Bacillus subtilis 168 was made competent according to Spizizen's method (Anagnostopoulos, C. and Spizizen, J. (1961). J. Bacteriol. 81, pp. 741-746) and transformed with plasmid pKS099, which was made linear using restriction endonuclease ApaI to incorporate the xylR-PxylA-cop cassette into the sacA locus along with the phleomycin resistance gene. Cells were spread on LB agar plates containing 5 μg / ml phleomycin and incubated overnight at 37°C. Proliferated colonies were collected, and the correct incorporation of the expression construct into the sacA locus was verified by PCR. The resulting Bacillus subtilis strain has the genotype Bacillus subtilis 168 sacA::xylR-PxylA-cop-phleor and was named GCX1.

[0143] Bacillus subtilis strain PCX1 Bacillus subtilis 168 wild-type cells were made competent according to the method of Spizizen (Anagnostopoulos, C. and Spizizen, J. (1961). J. Bacteriol. 81, pp. 741-746) and transformed with plasmid pKS111. The cells were spread on LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37°C. The resulting Bacillus subtilis strain was named PCX1 and possesses the replicated pKS111 plasmid.

[0144] Bacillus subtilis strain KDS Bacillus subtilis 168 cells containing pLS20cat (Itaya M. et al., 2006, Bioscience, Biotechnology, and Biochemistry, 70(3), pp. 740-742) were made competent according to Spizizen's method (Anagnostopoulos, C. and Spizizen, J. 1961. J. Bacteriol. 81, pp. 741-746) and transformed with the linear (ScaI-cleaved) plasmid pBS4S (Addgene # 55170 or BGSCID = ECE259), thereby replacing the thrC locus with a spectinomycin-resistant cassette. Cells were spread on LB agar plates containing 100 μg / ml spectinomycin and 5 μg / ml chloramphenicol and incubated overnight at 37°C. Growing colonies were collected and tested for threonine nutritional requirements. The obtained Bacillus subtilis strain is threonine-dependent and possesses the replicated pLS20cat plasmid. This strain was named Bacillus subtilis KDS.

[0145] Bacillus subtilis strain Bs#056 The prototrophic Bacillus subtilis strain KO-7S (BGSCID:1S145; Zeigler DR) was made competent according to the method of Spizizen (Anagnostopoulos, C. and Spizizen, J. (1961). J. Bacteriol. 81, pp. 741-746) and transformed with the linear DNA methyltransferase expression plasmid pMIS012 for the incorporation of DNA methyltransferase into the amyE gene, as described in WO2019016051 for the generation of Bacillus subtilis Bs#053. The cells were spread on LB agar plates containing 10 μg / ml chloramphenicol and incubated overnight at 37°C. The grown colonies were collected and lightly smeared onto both an LB agar plate containing 10 μg / ml chloramphenicol and an LB agar plate containing 10 μg / ml chloramphenicol and 0.5% soluble starch (Sigma), and then incubated overnight at 37°C. The starch plates were covered with iodine containing Lugol's solution, and positively integrated clones were identified by negative amylase activity. The genomic DNA of the positive clones was treated with lysozyme (10 mg / ml) at 37°C for 30 minutes, then isolated by a standard phenol / chloroform extraction method, and the correct integration of the MTase expression cassette was analyzed by PCR. The resulting Bacillus subtilis strain was named Bs#056.

[0146] Bacillus subtilis strain Bs#073 Bacillus subtilis 168 wild-type cells were made competent according to Spizizen's method (Anagnostopoulos, C. and Spizizen, J. (1961). J. Bacteriol. 81, pp. 741-746) and transformed with plasmid pKS102. This plasmid lacks a functional origin of replication within the Bacillus genus and, when integrated into the Bacillus subtilis 168 genome, can only be stably inherited via a homologous donated region to the amyE locus. Cells were spread on LB agar plates containing 50 μg / ml erythromycin and incubated overnight at 37°C. Growing colonies were collected and validated by PCR. The resulting Bacillus subtilis strain incorporated the complete plasmid pKS102 into the amyE locus and was named Bs#073.

[0147] plasmid pEC194RS - Bacillus temperature-sensitive plasmid Plasmid pE194 was PCR amplified using oligonucleotides SEQ ID NOs. 1 and 2, which contain adjacent PvuII sites, digested with restriction endonuclease PvuII, and ligated to vector pCE1 digested with restriction enzyme SmaI. pCE1 is a pUC18 derivative in which the BsaI site in the ampicillin resistance gene is silently removed. The ligation mixture was transformed into E. coli DH10B cells (Life Technologies). Transformants were spread on LB agar plates containing 100 μg / ml ampicillin and incubated overnight at 37°C. Plasmid DNA was isolated from individual clones and analyzed for precision by restriction digestion. The resulting plasmid was named pEC194S.

[0148] Type II assembled mRFP cassettes were PCR amplified from plasmid pBSd141R (accession number: KY995200) (Radeck, J., Meyer, D., Lautenschlager, N., and Mascher, T. 2017. Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis. Sci. Rep. 7: 14134) using oligonucleotides SEQ ID NOs: 3 and 4, which contain additional nucleotides at the restriction site BamHI. PCR fragments and pEC194S were restricted using the restriction enzyme BamHI, followed by ligation, and transformed into E. coli DH10B cells (Life Technologies). Transformants were spread on LB agar plates containing 100 μg / ml ampicillin and incubated overnight at 37°C. Plasmid DNA was isolated from individual clones and analyzed for precision by restriction digestion. The resulting plasmid pEC194RS has an mRFP cassette containing an open reading frame that is the opposite of the reading frame of the erythromycin resistance gene.

[0149] pEC194RS(ts) - Bacillus strictly temperature-sensitive plasmid Two single-nucleotide exchanges were introduced into the pE194 origin of replication of pEC194RS using the QuikChange® kit (Agilent) and oligonucleotides SEQ ID NOs. 64 and 65. These mutations correspond to the temperature-sensitive mutations described by Villafane et al. (Villafane, R. et al., 1987. Replication control genes of plasmid pE194. Journal of bacteriology, 169(10), pp. 4822-4829). Plasmid DNA was isolated from individual clones and its accuracy was analyzed by sequencing. The resulting sequence-validated plasmid was named pEC194RS(ts).

[0150] pEC194RSdelta(cop-repF) - non-replicating pEC194RS derivative pEC194RSdelta(cop-repF) was used as an embedded control. pEC194RSdelta(cop-repF) is non-replicating in Bacillus species due to the deletion of the cop-repF operon. The pEC194RS skeleton was amplified using the NcoI restriction site and adjacent oligonucleotides SEQ ID NOs. 28 and 29. The skeleton fragments were cleaved with NcoI and re-circularized by self-ligation. The reaction mixture was then used to transform E. coli DH10β cells (Life Technologies). The transformants were spread on LB agar plates containing 100 μg / ml ampicillin and incubated overnight at 37°C. Plasmid DNA was isolated from individual clones and sequenced to analyze its accuracy. The resulting sequence-validated plasmid was named pEC194RSdelta(cop-repF).

[0151] pDel003 - aprE gene deletion plasmid A gene deletion plasmid for the aprE gene of Bacillus licheniformis was constructed using plasmid pEC194RS and gene synthesis construct SEQ ID NO: 19, which includes a BsaI site compatible with pEC194RS and the genomic regions 5' and 3' of the aprE gene adjacent to it. Type II assembly using restriction endonuclease BsaI was performed as described (Radeck, J., Meyer, D., Lautenschlager, N., and Masker, T. 2017. Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis. Sci. Rep. 7: 14134). The reaction mixture was then used to transform E. coli DH10β cells (Life Technologies). Transformants were spread on LB agar plates containing 100 μg / ml ampicillin and incubated overnight at 37C. Plasmid DNA was isolated from individual clones and analyzed for precision by restriction digestion. The obtained aprE deletion plasmid is named pDel003.

[0152] pDel006 - Restrictase gene deletion plasmid A gene deletion plasmid for the restrictor restrictor gene (SEQ ID NO: 12) of Bacillus licheniformis DSM641 (SEQ ID NO: 11) was constructed using plasmid pEC194RS and gene synthesis construct SEQ ID NO: 13, which contains a BsaI site compatible with pEC194RS and the genomic regions 5' and 3' of the restrictor gene adjacent to it. Type II assembly using restriction endonuclease BsaI was performed as described above, and the reaction mixture was subsequently transformed into E. coli DH10β cells (Life Technologies). The transformants were spread on LB agar plates containing 100 μg / ml ampicillin and incubated overnight at 37°C. Plasmid DNA was isolated from individual clones and analyzed for precision by restriction digestion. The obtained restrictor restrictor plasmid was named pDel006.

[0153] pDel007 - Polygamma-glutamate synthesis gene deletion plasmid Deletion plasmids for the deletion of the genes involved in polygamma-glutamic acid (pga) production, namely ywsC(pgsB), ywtA(pgsC), ywtB(pgsA), and ywtC(pgsE) of Bacillus licheniformis, were constructed as described for pDel006. However, gene synthesis construct SEQ ID NO: 16 was used, which included a BsaI site compatible with pEC194RS and genomic regions 5' and 3' adjacent to the ywsC, ywtA(pgsC), ywtB(pgsA), and ywtC(pgsE) genes. The resulting pga deletion plasmid was named pDel007.

[0154] Plasmid p#0074 - xylR-PxylA promoter fragment The xylA gene, with its Bacillus megatherium DSM319 promoter PxylA, and the xylose repressor gene xylR, located in the opposite direction at 5' of PxylA, were ordered as a gene synthesis construct adjacent to the type II restriction endonuclease BpiI site, optimized for low-content restriction endonuclease sites (SEQ ID NO: 22).

[0155] Plasmid pBW424 - Low-copy Bacillus T2A - E. coli shuttle vector Plasmid pBW424 is a low-copy Bacillus-Escherichia coli shuttle vector based on the pBS72 origin of replication (Titok, MA et al., 2003, Plasmid, 49(1), pp. 53-62), which contains the kanamycin resistance gene for pUB110, a ColE1 origin of replication in E. coli, and a type II assembly cassette for subsequent cloning of the expression construct. The plasmid was constructed as described below using a restriction endonuclease BsaI site and adjacent gene synthesis construct optimized for a small number of restriction endonuclease sites.

[0156] Sequence ID 68 contains a pBS72 origin (accession number: AY102630) optimized for fewer endonuclease restriction sites compared to the published sequence. Sequence ID 69 contains a modified mRFP cassette from plasmid pBSd141R (accession number: KY995200; Radeck et al., 2017; Sci. Rep. 7: p. 14134) containing an adjacent BpiI type II restriction enzyme site, and the terminator region of the aprE gene from Bacillus licheniformis. Sequence ID 70 contains a modified kanamycin resistance gene from pUB110 and a modified ColE1 origin. Plasmid pBW424 was assembled using BsaI by type II restriction cloning as described by Radeck et al., 2017. The ligation mixture was transformed into E. coli DH10B cells (Life Technologies). Transformants were spread on LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37°C. Plasmid DNA was isolated from individual clones, and its accuracy was analyzed by restriction digestion after sequencing. The resulting plasmid was named pBW424.

[0157] Plasmid p#692 - Low-copy Bacillus T2A - E. coli shuttle vector containing a potent terminator The t1t2t0 terminator (derived from pMUTIN) was introduced at 5' of the type II assembly cassette of pBW424 to block potential readthrough from the kanamycin selection marker.

[0158] The terminator sequence t1t2t0 was incorporated into pBW424 by Gibson assembly (NEBuilder® HiFi DNA Assembly Cloning Kit, New England Biolabs). For this purpose, the terminator fragment (0.44kb) was amplified by PCR using oligonucleotides SEQ ID NOs. 71 and 72, with pMutin2 (accession number AF072806) as the template. The corresponding vector backbone of pBW424 was amplified using oligonucleotides SEQ ID NOs. 73 and 74. The pBW424 amplicon was purified using a PCR product purification kit (Roche). After subsequent digestion of the pBW424 PCR product with DpnI (New England Biolabs), both PCR fragments were gel-purified using the Qiaquick Gel Extraction Kit (Qiagen, Hilden, Germany) and annealed at 50°C for 1 hour in a 1:2 ratio. E. coli strain DH10B was transformed with an assembly reaction product and then seeded onto LB agar plates containing 20 μg / ml kanamycin. Plasmid DNA was isolated from individual clones, and its quality was analyzed by restriction digestion and sequencing. The resulting plasmid was named p#692.

[0159] Plasmid p#0268 - Cargo vector for Type II assembly Plasmid p#0268 is a derivative of the cargo vector derivative of pSEVA243 (Radeck et al., 2017; Sci. Rep. 7(1): 14134), containing a gene synthesis construct (SEQ ID NO: 23), the restriction endonuclease BpiI site, the adjacent lacZ gene, and terminator regions 5' and 3' that protect the clonal expression cassette from potential transcriptional activity. This modified lacZ cassette also adheres to the type II restriction sites BsaI, BsmBI, and AarI RE sites according to the type II assembly concept of Radeck et al., 2017.

[0160] Plasmid p#0268ter - Cargo vector for Type II assembly including upstream terminator Plasmid p#0268ter was used as a cargo vector. Plasmid p#0268ter contains a terminator upstream of the insert. Plasmid p#0268 was cleaved with BpiI, and the terminator-mRFP fragment was PCR amplified from p#0692 using oligonucleotides SEQ ID NOs. 77 and 78, and then cleaved with restriction endonuclease BsaI. The two portions were ligated, and the ligation mixture was transformed into E. coli DH10B cells (Life Technologies). The transformants were spread on LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37°C. Plasmid DNA was isolated from individual red clones, and its precision was analyzed by sequencing. The resulting plasmid was named p#268ter.

[0161] Plasmid p#0268cat - Cargo vector containing the 3' chloramphenicol resistance gene Plasmid p#0268cat was used as a cargo vector. Plasmid p#0268cat contains a chloramphenicol-resistant cassette downstream of the insert. p#0268 was cleaved at its unique SmaI site with the restriction endonuclease SmaI and ligated with PCR fragments containing a chloramphenicol-resistant cassette, PCR-amplified from pBS3Clux (Radeck et al., J Biol Eng. 2013 Dec 2;7(1):29), using oligonucleotides SEQ ID NO: 51 and SEQ ID NO: 52. The ligation mixture was transformed into E. coli DH10B cells (Life Technologies). Transformants were spread on LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37°C. Plasmid DNA was isolated from individual clones and its precision was analyzed by sequencing. The resulting plasmid was named p#268cat.

[0162] Plasmid p#0268ter-cat- is a cargo vector containing a 5' terminator along with a 3' chloramphenicol resistance gene. Plasmid p#0268ter-cat was used as a cargo vector. Plasmid p#0268ter-cat contains a terminator upstream of the insert and a chloramphenicol-resistant cassette downstream. Plasmid p#0268cat was cleaved with BpiI, and the terminator-mRFP fragment was PCR amplified from p#692 using oligonucleotides SEQ ID NOs. 77 and 78, and then cleaved with restriction endonuclease BsaI. The two portions were ligated, and the ligation mixture was transformed into E. coli DH10B cells (Life Technologies). The transformants were spread on LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37°C. Plasmid DNA was isolated from individual red clones, and its precision was analyzed by sequencing. The resulting plasmid was named p#268ter-cat.

[0163] Plasmid p#0268phleo-3' cargo vector containing the phleomycin resistance gene Plasmid p#0268phleo was used as a cargo vector. Plasmid p#0268phleo contains a phleomycin-resistant cassette downstream of the insert. p#0268 was cleaved at its unique SmaI site and ligated with a phleomycin-resistant cassette PCR-amplified from pBSc243B (Radeck et al., 2017; Sci. Rep. 7(1): 14134) using oligonucleotides SEQ ID NO: 49 and SEQ ID NO: 50. The ligation mixture was transformed into E. coli DH10B cells (Life Technologies). Transformants were spread on LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37°C. Plasmid DNA was isolated from individual clones, and its precision was analyzed by restriction digestion after sequencing. The resulting plasmid was named p#268phleo.

[0164] Plasmid p#0268ter-phleo- Cargo vector containing 5' terminator and 3' phleomycin resistance gene Plasmid p#0268ter-phleo was used as a cargo vector. Plasmid p#0268ter-phleo contains a terminator upstream of the insert and a phleomycin-resistant cassette downstream. Plasmid p#0268phleo was cleaved with restriction endonuclease BpiI, and the terminator-mRFP fragment, which was PCR amplified from plasmid p#0692 using oligonucleotides SEQ ID NOs. 77 and 78, was cleaved with restriction endonuclease BsaI. The two portions were ligated together, and the ligation mixture was transformed into E. coli DH10B cells (Life Technologies). The transformants were spread on LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37°C. Plasmid DNA was isolated from individual red clones, and its precision was analyzed by sequencing. The resulting plasmid was named p#268ter-phleo.

[0165] Plasmid pKS099 - A plasmid for incorporating the xylR-PxylA-cop expression cassette into the sacA gene locus of Bacillus subtilis. The genome-integrated plasmid pKS099 was used for the insertion of xylR-PxylAcop into the sacA gene of Bacillus subtilis and constructed in a two-step process. First, the xylR-PxylA fragment (SEQ ID NO: 22) of plasmid p#0074 was introduced into cargo vector p#0268ter-phleo by a type II assembly reaction with BpiI restriction endonuclease. The cop-coding region of pE194RS was then assembled using the oligonucleotide SEQ ID NO: 59 and SEQ ID NO: 60, which were PCR-amplified. The reaction mixture transformed E. coli DH10B cells (Life Technologies). The transformants were spread on LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37°C. Plasmid DNA was isolated from individual clones and its precision was analyzed by sequencing. The obtained plasmid was assembled using the genomic regions 5' and 3' of the sacA gene (amplified from the Bacillus subtilis genome using oligonucleotides SEQ ID NOs. 53, 54, 55, and 56) and the pBSd191R (Radeck et al., 2017; Sci. Rep. 7: p. 14134; BGSC ID: ECE702) skeleton vector (all adjacent to the BsaI site). A second type II assembly with restriction endonuclease BsaI was performed as described, and the reaction mixture was then transformed into E. coli DH10B cells. The transformants were spread on LB agar plates containing 100 μg / ml ampicillin and incubated overnight at 37°C. Plasmid DNA was isolated from individual clones and its precision was analyzed by sequencing. The obtained plasmid was named pKS099.

[0166] Plasmid pKS111: High-copy E. coli-low-copy Bacillus vector for cop expression Plasmid pKS111 functions as a low-copy expression vector in Bacillus for expressing the cop gene of pE194 under the control of the xylose-inducible promoter PxylA derived from Bacillus megatherium. pKS111 was constructed by T2A assembly using restriction endonuclease BpiI, containing the following components: plasmid p#0692 as the destination vector, the xylR-PxylA fragment of plasmid p#0074 (SEQ ID NO: 22), and the cop fragment amplified from pEC194RS using oligonucleotides SEQ ID NOs. 59 and 60. The reaction mixture was transformed into E. coli DH10B cells. The transformants were spread on LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37°C. Plasmid DNA was isolated from individual clones and sequenced for accuracy. The resulting plasmid was named pKS111.

[0167] Plasmid pBS3KcatluxGG Plasmid pBS3KcatluxGG served as a template for PCR amplification of a type II assembly-compatible luxABCDE operon. All BsaI, BsmBI, and AarI restriction enzyme recognition sites inside the luxABCDE operon of the vector pBS3Kcatlux (Popp, PF et al., 2017. 7(1), pp. 1-13) were removed by silent mutation using the QuikChange® kit (Agilent) and oligonucleotides SEQ ID NOs. 46, 47, and 48. The resulting vector with functional luciferase activity was named pBS3KcatluxGG.

[0168] Plasmid p#0268ter-Pveglux-Pveg-luxABCDE Plasmid p#0268ter-Pveglux combines a constitutive Pveg promoter derived from Bacillus subtilis, adjacent to a T2A restriction endonuclease site, with a luciferase reporter gene. The Pveg fragment was PCR-amplified from TMB3090 (Popp, PF et al., 2017. The Bacillus BioBrick Box 2.0: expanding the genetic toolbox for the standardized work with Bacillus subtilis. Scientific reports, 7(1), pp. 1-13) using oligonucleotides SEQ ID NOs. 062 and 063, which include a BpiI site overhang. The reporter gene luxABCDE was PCR-amplified using oligonucleotides SEQ ID NOs. 57 and 58, derived from pBS3KcatluxGG. Plasmid p#0268ter functioned as a cargo vector. This intermediate promoter-reporter cargo plasmid was constructed by a type II assembly reaction with restriction endonuclease BpiI as described (Radeck et al., 2017; Sci. Rep. 7(1): 14134), and the reaction mixture was subsequently transformed into E. coli DH10B cells (Life Technologies). The transformants were spread on LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37°C. Plasmid DNA was isolated from individual clones and its accuracy was analyzed by sequencing. The resulting sequence-validated plasmid was named p#0268ter-Pveglux.

[0169] Plasmid pKS100 - A luminescent plasmid based on pEC194RS for use with Bacillus subtilis. Plasmid pKS100 is based on plasmid pEC194RS, which further contains a 600 bp fragment homologous to the Bacillus subtilis amylase amyE gene, and a luciferase gene regulated by the Pveg promoter derived from plasmid p#0268ter-Pveglux. The amylase gene fragment was PCR amplified using oligonucleotides SEQ ID NOs. 066 and 067. 5' phosphorylated oligonucleotides SEQ ID NOs. 79 and 080 were annealed to form oligonucleotide double helixes. Plasmid pKS100 was constructed by a type II assembly using the above-mentioned BsaI restriction endonuclease, containing the following components: pEC194RS, PCR fragment amyE, oligonucleotide double helixes, and p#0268ter-Pveglux. The reaction mixture transformed E. coli DH10B cells (Life Technologies), and the transformants were spread on LB agar plates containing 100 μg / ml ampicillin and incubated overnight at 37°C. Plasmid DNA was isolated from individual clones and its precision was analyzed by restriction digestion and sequencing. The obtained plasmid was named pKS100.

[0170] Plasmid pKS101 - A luminescent plasmid based on pEC194RS(ts) for use with Bacillus subtilis. pKS101 was constructed as described for plasmid pKS100, but using plasmid pEC194RS(ts).

[0171] Plasmid pKS102 - Bioluminescent embedded plasmid in Bacillus subtilis pKS102 was constructed as described for plasmid pKS100, but using plasmid pEC194RS?(cop-repF).

[0172] Plasmid pKS200 - A luminescent plasmid based on pEC194RS for use with Bacillus licheniformis. Plasmid pKS200 was constructed as described for plasmid pKS100, but instead of the Bacillus subtilis amyE fragment, a 600 bp fragment homologous to the Bacillus licheniformis amyB gene was PCR-amplified from the Bacillus licheniformis P308 genome using oligonucleotide sequence numbers 75 and 76.

[0173] Plasmid p#0268-PsrfAAlux - PsrfAAluxABCDE Cargo Plasmid p#0268-PsrfAAlux was constructed in the same manner as described for plasmid p#0268ter-Pveglux, with the following differences: Instead of the Pveg promoter, the promoter of the Bacillus subtilis-derived srfAA gene was PCR amplified from Bacillus subtilis genomic DNA using oligonucleotides SEQ ID NOs. 30 and 31, which contain a BpiI restriction site. The reporter gene operon luxABCDE was PCR amplified from pBS3KcatluxGG using oligonucleotides SEQ ID NOs. 57 and 61.

[0174] Plasmid pKS068 - luxABCDE embedded amyB plasmid Plasmid pKS068 was used to integrate the luxABCDE expression cassette into the amyB amylase locus of Bacillus licheniformis. The 5' and 3' regions of the amyB gene of Bacillus licheniformis were PCR amplified from Bacillus licheniformis genomic DNA using oligonucleotides SEQ ID NOs. 5, 6, 7, and 8, respectively, which contain adjacent BsaI endonuclease restriction sites. Plasmid pKS068 was constructed by the above-described type II assembly using plasmid pEC194RS, the 5' amyB homology region, plasmid PsrfAAluxABCDE, and the 3' amyB homology region. The reaction mixture was transformed into E. coli DH10β cells (Life Technologies), and the transformants were spread on LB agar plates containing 100 μmg / ml ampicillin and incubated overnight at 37°C. Plasmid DNA was isolated from individual clones and its precision was analyzed by restriction digestion and sequencing. The obtained plasmid was named pKS068.

[0175] Plasmid pBAio - Bacillus All-in-One Vector The pBAio plasmid is an E. coli-Bacillus shuttle vector containing a temperature-sensitive replication origin of plasmid pEC194RS, a kanamycin-resistant cassette that functions in E. coli and Bacillus, and an additional copy of the cop gene under the control of the Bacillus megatherium PxylA promoter. Plasmid pBAio consists of the following genetic elements: i) The kanamycin resistance gene and ColE1 replication origin were PCR amplified from p#0692 using oligonucleotides SEQ ID NO: 34 (NcoI) and SEQ ID NO: 35 (PstI). ii) The terminator fragment was amplified from pKS111 using SEQ ID NO: 38 (SpeI) and SEQ ID NO: 39 (BamHI), iii) The xylR-PxylA-cop fragment was PCR amplified from pKS111 using SEQ ID NO: 44 (NsiI) and SEQ ID NO: 45 (SpeI). iv) Type II assembly cassettes were PCR amplified from pEC194RS using SEQ ID NO: 36 (BamHI) and SEQ ID NO: 37 (NcoI). v) The oriT fragment was PCR amplified from pLS20 (Itaya M. et al., 2006, Bioscience, Biotechnology, and Biochemistry, 70(3), pp. 740-742) using SEQ ID NO: 42 (NcoI) and SEQ ID NO: 43 (XbaI). vi) pE194 replication origins were PCR-amplified from pEC194RS using SEQ ID NO: 40 (SpeI) and SEQ ID NO: 41 (NcoI).

[0176] The fragments KanR+ColE1(i), xylR PxylA-cop(ii), terminator(iii), and type II assembly cassette(iv) were cleaved at the specified restriction sites and ligated to form an intermediate construct (pBAioint). The reaction mixture was used to transform E. coli DH10β cells, which were then spread onto LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37°C. Plasmid DNA was isolated from individual clones and its quality was analyzed by restriction digestion and sequencing. This intermediate plasmid (pBAioint) was ligated with the fragments oriT(v) and pE194 origin(vi), cleaved at its unique NcoI site, treated with phosphatase (rSAP by NEB), and purified using the SLG DNA clean-up Kit (SLG). The reaction mixture was used to transform E. coli DH10β cells. The transformants were then spread onto LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37°C. Plasmid DNA was isolated from individual clones, and its quality was analyzed by restriction digestion and sequencing. The resulting plasmid was named pBAio.

[0177] pBAio-lumiBs - A pBAio-based luminescent plasmid for use with Bacillus subtilis. Plasmid pBAio-lumBs was constructed as described for pKS100, but plasmid pBAio was used instead of plasmid pEC194RS.

[0178] pBAio-lumiBl - A pBAio-based luminescent plasmid for use with Bacillus licheniformis I constructed plasmid pBAio-lumBl as described for pKS200, but used plasmid pBAio instead of plasmid pEC194RS.

[0179] Plasmid pKS137 - luxA embedded plasmid based on pBAio Plasmid pKS137 was used to integrate a Campbell-based recombinant / luxABCDE operon into the luxA gene for functional inactivation. Plasmid pKS137 was constructed using plasmid pBAio and a 500 bp region homologous to luxA (PCR amplified from pBS3KcatluxGG using SEQ ID NOs. 32 and 33) adjacent to a BsaI site compatible with pBAio. Type II assembly using restriction endonuclease BsaI was performed as described above, and the reaction mixture was then transformed into E. coli DH10B cells. Transformants were spread on LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37°C. Plasmid DNA was isolated from individual clones and sequenced for accuracy. The resulting luxA gene-integrated plasmid was named pKS137.

[0180] [Examples] [Example 1] Temperature-dependent plasmid copy number of pEC194RS and pEC194RS(ts) derivatives The temperature-dependent copy number of plasmids derived from pE194 and pE194(ts) is determined by real-time quantitative PCR.

[0181] 168 strains of Bacillus subtilis containing plasmids pKS100 (based on pEC194RS) and pKS101 (based on pEC194RS(ts)), and Bacillus subtilis reference strain Bs#073 containing the genome-integrated pKS102 sequence (lacking the cop-repF operon) were cultured for 16 hours at 30°C with stirring in 2 ml of LB-Lennox medium containing 50 μg / ml erythromycin. The following day, the cultures were diluted to OD600 0.1 in LB-Lennox medium supplemented with erythromycin (50 μg / ml), divided into three 10 ml shaker flasks, and subsequently incubated at 30°C, 37°C, and 42°C, respectively. After 8 hours of incubation, when the cells reached the transition phase, the samples were removed and the cells were recovered by centrifugation.

[0182] The entire DNA is isolated from each sample, and plasmid copy number is quantified in real-time PCR (using Luna® Universal qPCR Master Mix and NEB). 3 This was determined by Analytik Jena. Oligonucleotides SEQ ID NOs. 26 and 27 within the bla gene encoding the ampicillin resistance gene in pEC194RS and pEC194RS(ts) derivatives, as well as SEQ ID NOs. 24 and 25 within the Bacillus subtilis rtp locus (locus tag BSU_18490) near the chromosome terminal, were used for RT-qPCR amplification. Purified plasmid DNA and purified chromosomal DNA served as absolute quantification standards for calculating PCR efficiency, which were 99% and 98%, respectively.

[0183] The average amount of plasmid per cell was calculated by comparing it to the amount at the end of a chromosome. The latter corresponds to a single cell with one chromosome.

[0184] The results are shown in Figure 2. Bacillus subtilis strain Bs#073, which has an integrated non-replicating plasmid skeleton, serves as a single-copy control under all temperature conditions.

[0185] Plasmid pKS100 has an average copy number of 38.5 and 34.1 at 30°C and 37°C, respectively, and is severely reduced to an average copy number of 4.1 at the non-acceptable temperature of 42°C. Plasmid pKS101, which has a more strictly temperature-sensitive origin of replication, has an average copy number of 25 plasmids at 30°C and an average of 2.9 copies per cell at 37°C. At 42°C, the average plasmid copy number is calculated to be less than 1, indicating plasmid loss. Cells without the plasmid cannot survive under antibiotic selection pressure, but contribute to their genomic DNA, which can be detected by qPCR analysis.

[0186] [Example 2] Plasmid copy number reduction - Expression of additional chromosomal copies of the cop gene Bacillus subtilis strain GCX1 possesses a cop gene expression cassette integrated into the sacA locus. Transcription of the cop gene on the chromosome is regulated by the xylose-inducible promoter PxylA derived from Bacillus megatherium, and therefore cop expression is xylose-dependently regulated. Bacillus subtilis GCX1 was transformed with plasmid pKS100, incubated overnight at 37°C on LB agar plates containing erythromycin, and single clones were collected. Bacillus subtilis strain 168 (see Example 1) with pKS100 and single clones of Bacillus subtilis strain GCX1 with plasmid pKS100 were cultured at 37°C for 16 hours with agitation, and then inoculated with 2 ml of LB-Lennox medium containing 50 μg / ml erythromycin. The following day, the cultures were diluted to OD600 0.1 with LB-Lennox medium supplemented with 50 μg / ml erythromycin. A 10 ml culture of Bacillus subtilis strain 168 containing pKS100 was transferred to a new 100 ml shaking flask and incubated with stirring at 37°C. A 10 ml culture of Bacillus subtilis strain GCX1 containing plasmid pKS100 was divided into three 100 ml shaking flasks supplemented with 0%, 0.125%, or 0.5% xylose, and incubated with stirring at 37°C. After 8 hours, the samples were removed and the cells were recovered by centrifugation. Total DNA was isolated from each sample, and the average plasmid copy number per cell was determined as described in Example 1. The results are shown in Figure 3.

[0187] In Bacillus subtilis strain 168, plasmid pKS100 has an average plasmid copy number of 34. In contrast, plasmid pKS100 has a lower average copy number of 15.4 copies per cell even in the absence of the inducing molecule xylose in Bacillus subtilis strain GCX1, where an additional copy of the cop gene is present in the genome. The lower average copy number of pKS100 in Bacillus subtilis strain GCX1 indicates a low level of leaked expression of the cop gene from the PxylA promoter in the absence of the inducing molecule xylose. In the presence of 0.125% or 0.5% xylose in the culture medium, the elevated inducible cop expression levels sufficiently block plasmid replication, as seen by an average copy number of 1 or less, respectively.

[0188] [Example 3] Plasmid copy number reduction - Expression of additional chromosomal copies of the cop gene Plasmids pKS100 and pKS101 possess a 600 bp region homologous to the Bacillus subtilis amylase amyE gene and contain the luxABCDE gene, which serves as a reporter system enabling quantification by luminescence measurement. Since the expression of the luxABCDE gene is regulated by the constitutive Pveg promoter of Bacillus subtilis, a decrease or increase in the luminescence signal directly reflects a change in plasmid copy number.

[0189] Bacillus subtilis strain GCX1, containing plasmids pKS100 (pE194 origin) and pKS101 (pE194(ts) origin), and reference Bacillus subtilis strain Bs#073, containing a single copy of the integrated luxABCDE gene (via pKS102), were cultured for 16 hours at 30°C with agitation, and then inoculated with 2 ml of LB-Lennox medium containing 50 μg / ml erythromycin. The following day, the cultures were diluted to OD600 0.1 with LB-Lennox medium supplemented with erythromycin (50 μg / ml). Each 200 μl culture was transferred to a black 96-well microtiter plate (Sarstedt), and xylose was supplemented to final concentrations of 0%, 0.0625%, 0.125%, 0.25%, or 0.5% xylose. Cells were incubated at 30°C with agitation of the culture medium using a Synergy Neo2 HTS Multi-Mode Microplate Reader (BioTek). After 8 hours of incubation, OD600 and luminescence were measured. The relative luminescence units (RLU) per OD600 at 8 hours were determined and plotted against the indicated xylose concentration (Figure 4). The RLU of the single-copy control strain Bs#073, into which the lux gene was incorporated, is shown as a dotted line.

[0190] The addition of the inducing molecule xylose activates cop gene expression from the genomically integrated cop gene, and the lower levels of RLU signaling obtained for both plasmids pKS100 and pKS101 reflect lower plasmid copy numbers. Concentrations of 0.25% and 0.50% xylose-inducing molecule reduce the RLU signal to below the Bs#073 integrated control (dotted line in Figure 4), reflecting plasmid loss within the cell population. Lower cop expression from the genomically integrated cop gene (0.063%, 0.125% xylose-inducing molecule) indicates stable lower plasmid copy numbers for plasmids pKS100 and pKS101, referencing the absence of the inducing molecule xylose.

[0191] This indicates that the reduction in plasmid copy number can be controlled in a manner dependent on the concentration of xylose-inducible molecules.

[0192] [Example 4] Plasmid copy number reduction - Expression of the cop gene from the second plasmid Similar to Example 3, the additional cop gene was placed under the control of the inducible PxylA promoter, but the additional cop expression cassette is encoded in the co-existing low-copy plasmid pKS111.

[0193] Bacillus subtilis strain PCX1 (containing pKS111) was transformed with plasmids pKS100 and pKS101, respectively, and incubated at 30°C on LB-Lennox agar plates containing 50 μg / ml erythromycin and 20 μg / ml kanamycin. As a control, Bacillus subtilis strain 168 containing plasmid pKS100 was used. Single clones were collected and cultured in 2 ml of LB-Lennox medium for 16 hours with agitation at 30°C. Background 50 μg / ml erythromycin was added to the medium for Bacillus subtilis strain 168, and background 50 μg / ml erythromycin and 20 μg / ml kanamycin were added to the medium for Bacillus subtilis strain PCX1. The following day, the cultures were diluted to OD600 0.1 in LB-Lennox medium supplemented with the corresponding antibiotics. Each 200 μl culture was transferred to a black 96-well microtiter plate (Sarstedt), and xylose was added to the final concentrations of 0%, 0.0625%, 0.125%, 0.25%, or 0.5% xylose. The cells were incubated at 30°C with agitation of the medium using a Synergy Neo2 HTS Multi-Mode Microplate Reader (BioTek). After 8 hours of incubation, OD600 and luminescence were measured.

[0194] The relative luminescence units (RLU) as an emission signal per OD600 at 8 hours were determined and plotted against the indicated xylose concentration (Figure 5).

[0195] Reference strain Bacillus subtilis 168 containing plasmid pKS100 exhibits comparable RLU signals regardless of the presence or absence of the inducing factor molecule xylose at increasing concentrations. The RLU signal of Bacillus subtilis strain PCX1 containing plasmid pKS100 without the inducing factor molecule is lower compared to the signal from the reference strain. This indicates leaky expression of the cop gene, which already produces a lower RLU signal (approximately 20%) due to a lower plasmid copy number. Increased cop expression with the addition of the inducing factor molecule xylose reduces the RLU signal in a xylose concentration-dependent manner. The same dependence is observed in Bacillus subtilis strain PCX1 containing plasmid pKS101 despite a lower starting level. This is consistent with the lower plasmid copy number of pKS101 compared to pKS100 (Example 1, Figure 2).

[0196] Compared to Example 3, which used Bacillus subtilis strain GCX1 with the cop expression cassette integrated into its genome, the effect of additional plasmid-based copies of the cop expression cassette can be observed. Plasmid pKS111, containing the pBS72 origin of replication, has approximately 6 copies per cell (Titok, MA et al., 2003. Plasmid, 49(1), pp. 53-62). This plasmid copy effect results in approximately 6-fold higher cop expression and therefore lower RLU signaling at a given xylose concentration, and thus lower plasmid copy numbers for plasmids pKS100 and pKS101 can be observed.

[0197] [Example 5] Plasmid copy number reduction due to expression of additional cop genes from the same plasmid - pBAio in Bacillus subtilis Plasmid pBAio-lumiBs is functionally similar to pKS100 but possesses additional cop genes under the control of the PxylA promoter, as described in Examples 2, 3, and 4. 168 strains of Bacillus subtilis were transformed with pBAio-lumiBs and cultured at 30°C on LB agar plates containing 20 μg / ml kanamycin. Single clones were collected and cultured for 16 hours at 30°C with agitation in 2 ml LB-Lennox medium containing 20 μg / ml kanamycin. The following day, the cultures were diluted to OD600 0.1 in LB-Lennox medium supplemented with 20 μg / ml kanamycin. Each 200 μl culture was transferred to a black 96-well microtiter plate (Sarstedt) and xylose was supplemented to final concentrations of 0%, 0.0313%, 0.0625%, 0.125%, 0.25%, or 0.5% xylose. Cells were incubated at 30°C with agitation of the culture medium using a Synergy Neo2 HTS Multi-Mode Microplate Reader (BioTek). After 8 hours of incubation, OD600 and luminescence were measured. Relative luminescence units (RLU) as a luminescence signal per OD600 at 8 hours were determined and plotted against the indicated xylose concentration (Figure 6). Changes in plasmid copy number are reflected in changes in the luminescence output of Pveg-lux encoded by plasmid pBAio-lumiBs. Increased expression of additional cop genes in plasmid pBAio-lumiBs using the inducing molecule xylose at gradually increasing concentrations reduces the RLU signal and therefore reduces the plasmid copy number of plasmid pBAio-lumiBs. Addition of 0.0313% xylose results in an RLU signal of 1 / 7th of plasmid pBAio-lumiBs (15% of the uninducing control).

[0198] [Example 6] Plasmid copy number reduction due to expression of additional cop genes from the same plasmid - pBAio in Bacillus licheniformis Plasmid pBAio-lumiBl is functionally identical to pBAio-lumiBs, and the additional cop gene present in pBAio-lumiBl is a 600 bp fragment homologous to the amyB locus of Bacillus licheniformis amylase.

[0199] Bacillus subtilis KDS was transformed with pBAio-lumiBl and cultured at 37°C on LB agar plates containing 20 μg / ml kanamycin and 5 μg / ml chloramphenicol. Single clones were collected and cultured for 16 hours at 37°C with stirring in 2 ml LB-Lennox medium containing 20 μg / ml kanamycin and 5 μg / ml chloramphenicol. In parallel, Bacillus licheniformis P308 was cultured for 16 hours at 37°C with stirring in 2 ml LB-Lennox medium. The following day, pBAio lumiBl was conjugated to Bacillus licheniformis P308 as described (Itaya M, 2006, Biosci Biotechnol Biochem; 70(3):740-742), and the mixed cells were spread on minimal salt agar plates supplemented with 2% glucose, 0.2% potassium glutamate, and 20 μg / ml kanamycin, and incubated at 37°C for 3 days. The tryptophan and threonine-dependent Bacillus subtilis donor strain KDS could no longer grow on these agar plates. A single Bacillus licheniformis clone containing pBAio-lumiBl was isolated and cultured in 2 ml LB-Lennox medium containing 20 μg / ml kanamycin for 16 hours at 37°C with stirring. The following day, the culture was diluted to OD600 0.1 in LB-Lennox medium supplemented with 20 μg / ml kanamycin. Each 200 μl culture was transferred to a black 96-well microtiter plate (Sarstedt), and xylose was added to the final concentrations of 0%, 0.0313%, 0.0625%, 0.125%, 0.25%, or 0.5% xylose. Cells were incubated at 37°C with agitation of the medium using a Synergy Neo2 HTS Multi-Mode Microplate Reader (BioTek). After 8 hours of incubation, OD600 and luminescence were measured. Relative luminescence units (RLU) as a luminescence signal per OD600 at 8 hours were determined and plotted against the indicated xylose concentration (Figure 7B). Changes in plasmid copy number are reflected in changes in the luminescence output of Pveg-lux encoded by plasmid pAio-lumiBl.Induction of cop expression by adding a 0.031% xylose-inducible molecule results in a signal that is two-fifths (40%) of the RLU signal in the uninducible control. Stronger cop expression due to increased xylose concentration results in an even lower, constant RLU signal level, corresponding to the single-copy insertion plasmid pBAio-lumiBl into the amyB locus.

[0200] In parallel with the luminescence experiment, samples were taken from 0.0%, 0.0313%, and 0.5% xylose cultures at 8 hours and recovered by centrifugation. Total DNA was isolated from individual samples, and the average plasmid copy number per cell was determined as described in Example 1. Oligonucleotides SEQ ID NOs. 81 and 82 within the luxB gene of plasmid pBAio-lumiBli, and oligonucleotides SEQ ID NOs. 83 and 84 within the gene BLi02076 (locus tag DSM13) near the chromosome terminal were used for RT-qPCR amplification. The results are shown in Figure 7C. The average plasmid copy number of pBAio lumiBl in Bacillus licheniformis without xylose-inducible molecules was calculated to be 19.3 copies per cell. This lower average copy number compared to plasmid pKS100 (identical pE194 origin of replication with an average of 38.5 copies per cell; Example 1) reflects leaked cop expression from promoter PxylA and, therefore, suppression of plasmid replication. The addition of 0.313% xylose resulted in an even lower average plasmid copy number of 11.6 per cell, consistent with the luminescence data (Figure 7B) mentioned above. Strong cop expression due to the addition of 0.5% xylose resulted in an average plasmid copy number of 0.8, slightly less than 1.0, indicating plasmid loss. Cells without plasmids cannot survive under antibiotic selection pressure, but contribute to their genomic DNA, which is detected by qPCR analysis.

[0201] [Example 7] 100% Campbell recombination efficiency achieved by combining overexpression of COP and shift to an unacceptable temperature in Bacillus licheniformis. Homologous recombination-based gene deletion or integration approaches using plasmids with temperature-sensitive replication origins, such as pE194, force the genome to undergo the initial Campbell recombination of the plasmid by the homologous region present in the plasmid by shifting culture conditions to an unacceptable temperature under selective pressure (e.g., antibiotics). This procedure is imperfect and results in a large number of cells that likely contain non-replicating plasmids at extremely low copy numbers, ultimately leading to significant screening effort to identify the correct clones.

[0202] To demonstrate higher efficiency of pBAio-based plasmids in homologous recombination with the host genome, a "photoswitch" was constructed. Under the control of a strong PsrfAA promoter derived from Bacillus subtilis, luciferase encoding a gene (luxABCDE reporter operon) was incorporated into the amylase amyB locus of Bacillus licheniformis P308 using plasmid pKS068 to create the Bacillus licheniformis strain LBL described in the strain section. Each cell of Bacillus licheniformis LBL exhibits a luminescence signal. Plasmid pKS137, a pBAio plasmid derivative having a 500 bp homologous region of luxA, was transformed into Bacillus licheniformis LBL by conjugation as described in Example 6 (see above). One colony was taken and inoculated into 10 ml LB Lennox supplemented with 20 μg / ml kanamycin and 0.5% xylose, and incubated at 37°C with agitation for 3 hours (pre-culture). Dilution series were prepared, and 100 μl of each was seeded onto LB agar plates containing 20 μg / ml kanamycin and 1% xylose. The agar plates were incubated overnight at 45°C. As a control, cells were treated with pre-culture and / or agar plates without xylose and incubated at 37°C, or a combination of both. The following day, the presence or absence of luminescence was examined for colonies to analyze plasmid integration status. The agar plates were exposed to AlphaImager® (Alpha Innotech) at default settings for 3 minutes, and luminescence was analyzed. A second image was taken using reflected light at high resolution for 8 milliseconds. The two images were overlaid using Fiji (Schindelin et al., 2012, Nature methods 9(7): pp. 676-682). Plasmid integration rate was determined by dividing the number of non-luminescent colonies by the total number of colonies. An average of 1000 colonies were analyzed per condition, and the experiment was performed in triple replication.

[0203] Clones with the plasmid incorporated do not exhibit luminescence. This is because homologous recombination between plasmid pKS137 and the luxA gene leads to the integration of the plasmid into the genome and, consequently, the disruption of the luxABCDE operon. A schematic diagram is shown in Figure 8A, and the results are shown in Figure 8B.

[0204] At 37°C, a maximum plasmid integration rate of 87% could be achieved by seeding on LB agar plates containing 1% xylose, thus inducing cop expression. Induction of cop gene expression in liquid culture "pre-treated" with 0.5% xylose had a mild effect on the overall integration efficiency. At 45°C – an unacceptable temperature – the average integration rate was 85%–90%, indicating that the first step is actually inefficient. The combination of growth of the pE194 origin at an unacceptable temperature (45°C) and induction of cop expression shows that the integration rate can increase to 100% (no luminescent colonies in the more than 1000 colonies analyzed).

[0205] [Example 8] Suicide vectors and methods The gene deletion (incorporation of similar genes) procedure that applies a temperature-sensitive replication origin—as similarly described herein for the pBAio plasmid containing an additional cop gene under the control of the inducible promoter PxylA—is used for genome editing. In genome editing, the direct introduction of a linear DNA fragment or suicide plasmid (which cannot replicate in host cells) containing homologous DNA sequences and bilaterally flanked selectable markers into recipient cells fails if determined by a transformant that takes up the DNA and correctly integrates it into the genome. The former has been described in large numbers for Bacillus licheniformis, Bacillus pumilus and other species, while the latter has been described for Bacillus subtilis laboratory strains with so-called innate transformant receptivity, where linear DNA is actively taken up by the cell simultaneously with high-frequency recombination (e.g., Bacillus subtilis strain 168). Similarly, “inducible transformant” systems have been described by plasmid-based or genome integration-based overexpression of transformant genes comS and / or comK that enhance DNA uptake. Furthermore, further knockout of the mecA gene in Bacillus licheniformis is known to transform host cells into recipient hosts with improved linear DNA transformability. It is clear that the recipient host needs to be genetically modified before use. Moreover, adverse effects on the host's production capacity (e.g., chemicals, polymers, proteins, enzymes, etc.) may occur. Therefore, it is desirable to apply efficient DNA introduction and gene deletion / incorporation procedures without the need to modify recipient cells.

[0206] The DNA introduction and gene deletion procedures include the following: A Bacillus subtilis donor host with nutritional requirements (e.g., deletion of the alanine racemase gene - alr / dal gene) and a conjugation plasmid pLS20 (WO0200907). The conjugation plasmid pLS20 further possesses selectable markers (e.g., chloramphenicol resistance marker, Itaya et al., 2006) and counter-selection markers (e.g., codBA gene, Kostner et al., 2013). The Bacillus subtilis host is an induced strain of Bacillus subtilis host Bs#056 in which the chloramphenicol resistance gene is replaced by a kanamycin resistance cassette adjacent to the lox site. The lox site was subsequently removed by introducing cre recombinase into plasmid pDR244 (Bacillus Genetic Stock Center BGSC: ECE274). The replication cassette of plasmid pE194 (nt920~nt1910; containing cop and repF genes under the control of the Pcop promoter, as well as antisense ctRNA) is incorporated into the threonine (thrC) locus using the plasmid derivative pBS4S (Radeck et al., 2013), in which the spectinomycin-resistant cassette is replaced by a kanamycin-resistant cassette adjacent to a lox site that enables cre-mediated marker recycling. To enable the adjustment of low copy numbers of the pE194 derivative described for Bacillus subtilis GCX1, an additional copy of the cop gene is preferentially placed under an inducible PxylA gene promoter incorporated into the sacA locus.

[0207] The deletion plasmid consists of the following elements: A) Antibiotic resistance genes (e.g., kanamycin resistance gene of plasmid pUB110) and ColE1 replication; B) oriT derived from pLS20 (Itaya M. et al., 2006, Bioscience, Biotechnology, and Biochemistry, 70(3), pp. 740-742); C) pE194 sequence (nt630-nt920) including SSO and DSO; D) Anti-selection marker (e.g., codBA gene, Kostner et al., 2013); E) lox site and 5' and 3' 500bp regions homologous to the recipient host region, and adjacent spectinomycin resistance cassette.

[0208] A gene-integrated plasmid consists of the following elements: A) Antibiotic resistance genes (e.g., the kanamycin resistance gene in plasmid pUB110) and ColE1 replication; B) oriT derived from pLS20 (Itaya M. et al., 2006, Bioscience, Biotechnology, and Biochemistry, 70(3), pp. 740-742); C) pE194 sequence (nt630-nt920) containing SSO and DSO; D) Anti-selection marker (e.g., the codBA gene, Kostner et al., 2013); E) Heterogene expression cassette (promoter-gene-terminator) located at 5' of the spectinomycin resistance cassette adjacent to the lox site. The gene expression cassette and spectinomycin resistance cassette are also adjacent to 5' and 3' 500bp regions homologous to the recipient host region.

[0209] Plasmid replication within cells is either dependent on the repF protein trans-encoded on the chromosome or plasmid-based.

[0210] Bacillus licheniformis recipient hosts that exhibit prototrophicity to D-alanine racemase activity are insensitive to 5'-fluorocytosine but sensitive to kanamycin and spectinomycin antibiotics.

[0211] The gene deletion plasmid was assembled by type II assembly, selected on an LB agar plate supplemented with 20 μg / ml kanamycin, 5 μg / ml chloramphenicol, 10 μg / ml threonine, and 100 μg / ml D-alanine, and then transformed into a Bacillus subtilis donor strain. After plasmid isolation, restriction enzyme digestion, and sequencing, Bacillus subtilis clones with the correctly assembled gene deletion plasmid were identified using colony PCR. Since repF is expressed from the chromosome as described above, the plasmid is stable as a replication plasmid.

[0212] Next, the deletion plasmid is introduced by conjugation from a Bacillus subtilis donor strain to a Bacillus licheniformis recipient strain, as essentially described in the example of WO0200907 for replicated pE194-based gene deletion / gene integration plasmids. Conjugation is modified according to Itaya et al., 2006, in which conjugation is performed in liquid culture rather than on a solid agar plate.

[0213] Bacillus subtilis donor strains containing the deletion plasmid are grown for 15-17 hours in LB-Lennox medium supplemented with 20 μg / ml kanamycin, 5 μg / ml chloramphenicol, 10 μg / ml threonine, and 100 μg / ml D-alanine, while Bacillus licheniformis recipient strains are grown in LB-Lennox medium at 37°C. The following day, the cultures are diluted to an optical density OD(600 nm) of 0.1 in fresh preheated medium, and culture is continued in antibiotic-free medium until both donor and recipient cell cultures reach an OD(600) of 0.8-1.0. 0.5 ml of each culture is taken out, mixed, and incubated without shaking at 37°C for 15 minutes to introduce the conjugating plasmid. The cell mixture is washed once with preheated transformation medium, then seeded onto TM agar plates supplemented with 200 μg / ml spectinomycin, and incubated at 37°C for 24–48 hours.

[0214] The transformation medium TM consists of Spizizen minimal medium containing 1.0% glucose, 0.2% potassium glutamate, and 0.1% casamino acid (Anagnostopoulos, C. and Spizizen, J. (1961). J. Bacteriol. 81, pp. 741-746).

[0215] Only Bacillus licheniformis somatic cells that have received a linear single-strand deletion plasmid and successfully incorporated a spectinomycin-resistant cassette containing double homologous recombination via two homologous DNA regions can proliferate under these conditions. Since the repF gene is absent in Bacillus licheniformis, plasmid replication of the donor plasmid is no longer possible, resulting in a conjugated "suicide" plasmid.

[0216] Bacillus subtilis donor cells cannot grow in the absence of D-alanine. The conjugated Bacillus licheniformis cells are then seeded onto TM agar plates containing 200 μg / ml spectinomycin and 20 μg / ml fluorocytosine and incubated at 37°C for 24 hours. The second step is to counter-select the deletion plasmid if the self-transmitting pLS20CAT conjugation plasmid is present and integration via Campbell recombination has occurred.

[0217] The resulting Bacillus licheniformis strain lacked a target gene and was resistant to spectinomycin, insensitive to 5-fluorocytosine, and sensitive to kanamycin.

[0218] In the next step, the spectinomycin resistance marker may be removed by resolbase (cre resolbase) mediated marker recycling.

[0219] In contrast to other physical methods of DNA delivery such as electroporation or protoplast transformation, zygote DNA delivery allows recipient cells to immediately undergo DNA uptake and recombination by enabling specific gene deletion / incorporation through this suicide approach.

[0220] Furthermore, this method allows for the construction of gene expression cassettes under the control of a strong promoter, which is absolutely necessary for the stable maintenance of extremely low copy numbers in Bacillus subtilis donor strains. As outlined, plasmid copy number is trans-regulated by repF expression levels in cells. Upon conjugation induction, the suicide plasmid is produced in recipient cells, and the strong gene expression cassette is directly integrated into the genome without a multicopy plasmid intermediate. This will result in the selection of cells with mutations within the gene expression cassette. The present invention includes, for example, the following embodiments: [Embodiment 1] A method for controlling plasmid copy number, i) Providing a prokaryotic host containing a plasmid having an origin of replication that can be activated by a plasmid replication initiation protein (Rep), and ii) A step of adjusting the plasmid copy number to a desired value by regulating the expression of the Rep protein. Methods that include... [Embodiment 2] Rep protein expression, a) A step of increasing the expression of the rep gene that codes for the Rep protein, b) A step of reducing the expression of the rep gene that encodes the Rep protein. c) A step of reducing the expression of the repressor gene encoding the repressor of Rep expression, d) A step to increase the expression of the repressor gene encoding the repressor of Rep expression. The method according to Embodiment 1, which is adjusted by one or more of the following. [Embodiment 3] a) The rep gene is operably linked to a heterologous promoter, and / or b) The plasmid contains a repressor gene encoding a repressor for Rep expression, which is operablely ligated to a heterologous promoter. The method according to Embodiment 2. [Embodiment 4] - A replication origin that can be activated by plasmid replication initiation protein (Rep), - rep gene encoding Rep protein A controlled replication plasmid containing, a) The rep gene is operablely linked to a heterologous promoter, and / or b) A plasmid comprising a repressor gene encoding a repressor for Rep expression, which is operably ligated to a heterologous promoter. [Embodiment 5] - A promoter operably linked to the rep gene encoding the Rep protein, and - A repressor gene encoding a repressor of Rep expression, under the control of a heterologous promoter. A controlled replication plasmid as described in Embodiment 4, comprising: [Embodiment 6] - A replication origin that can be activated by plasmid replication initiation protein (Rep), - In some cases, a promoter operably linked to a repressor gene encoding a repressor for Rep expression. A replication-supported plasmid containing the functional rep gene encoding the Rep protein. [Embodiment 7] - A gene encoding a repressor that suppresses Rep protein expression when expressed in a host containing an expression cassette for Rep protein expression. The replication support-dependent plasmid according to Embodiment 6, further comprising: [Embodiment 8] - At least one, preferably each, heterologous promoter responds to changes in the concentration of an external regulator or metabolite, and / or - The plasmid is a rolling circle replication plasmid, and / or - Plasmid, - Selectable markers, - Target gene, - An expression cassette containing a promoter operably linked to a target gene, - Recombination site A plasmid according to any one of embodiments 4 to 7, comprising one or more of the following. [Embodiment 9] A plasmid replication control host comprising a controlled replication plasmid as described in any of Embodiments 4, 5, or 8. [Embodiment 10]i) A replication-supported dependent plasmid according to any one of Embodiments 6 to 8, and ii) Expression cassette for the expression of rep genes located on the host chromosome and / or helper plasmids in the host. A plasmid replication support host containing the plasmid. [Embodiment 11]i) Providing a host starter culture containing the plasmid described in any of Embodiments 4 to 8, ii) The step of culturing the host to achieve an increase in the number of host cells, and iii) A step to adjust the plasmid copy number in cultured host cells during or after step ii). A culture method including the following. [Embodiment 12] i) A step of providing a plasmid according to any one of Embodiments 4 to 8, comprising a selection marker, in a prokaryotic plasmid recipient host. ii) A step to inhibit plasmid replication while maintaining the selection of the selection marker. An implementation method that includes this. [Embodiment 13] Inhibition of plasmid replication in the plasmid recipient host, a) A step in which the rep gene is not provided on the plasmid. b) A step of suppressing the expression of the rep gene, and c) A step that does not provide the inducer necessary for the expression of the rep gene. The integration method according to Embodiment 12, which is performed using one or more of the following. [Embodiment 14]i)- The plasmid donor host comprises a controlled replication plasmid or replication support-dependent plasmid as described in any of Embodiments 4 to 8, which includes a selectable marker. - The plasmid recipient host does not contain the rep gene. A step of providing a plasmid donor host and a plasmid recipient host, ii) The step of introducing a plasmid from a plasmid donor host to a plasmid recipient host, and iii) A step in which plasmid replication in the plasmid recipient host is inhibited while applying selective pressure to the plasmid recipient host that compels the presence of a selectable marker in the plasmid recipient host, either simultaneously with or after step ii). An implementation method that includes this. [Embodiment 15] The integration method according to any one of Embodiments 12 to 14, wherein the plasmid includes a recombination site that facilitates recombination with a matching site in the nucleic acid of a replication recipient host. [Embodiment 16] The integration method according to any one of Embodiments 12 to 15, wherein the plasmid is integrated into the genome of a plasmid recipient host or into another plasmid whose replication is independent of the rep gene.

Claims

1. A plasmid copy number control host system comprising a prokaryotic host of the genus Bacillus, wherein the host comprises a first plasmid, and the first plasmid is - A replication origin that can be activated by plasmid replication initiation proteins (Rep), and the rep gene that encodes the Rep protein. - The first cop gene encoding the promoter repressor It has, The host contains a further cop gene in the first plasmid or helper plasmid. A plasmid copy number control host system in which the aforementioned additional cop genes are under the control of an inducible promoter, and the rep genes are operably ligated to a native rep gene promoter.

2. The plasmid copy number control host system according to claim 1, wherein the first cop gene is under the control of an inducible promoter.

3. The plasmid copy number control host system according to claim 1 or 2, wherein the first plasmid comprises an embedded sequence containing a sequence for homologous recombination.

4. A plasmid copy number control host system according to any one of claims 1 to 3, wherein the first plasmid comprises a selectable marker.

5. A plasmid copy number control host system according to any one of claims 1 to 4, wherein the first plasmid comprises an expression cassette containing a target gene and a promoter operably ligated to the target gene.

6. The plasmid copy number control host system according to any one of claims 1 to 5, wherein the first plasmid loses its replication activity in a temperature-dependent manner when the host is cultured at a temperature above an unacceptable temperature.

7. The plasmid copy number control host system according to any one of claims 1 to 6, wherein the Rep protein comprises a RepF protein.

8. A method for controlling plasmid copy number, i) Providing a plasmid copy number control host system according to any one of claims 1 to 7, and ii) A step of inducing further expression of the cop gene to reduce the plasmid copy number to a desired level. Methods that include...

9. i) A step of providing a plasmid copy number controlled host system according to claim 6, ii) The step of culturing the host system at a non-permissible temperature or higher in the presence of the further cop gene inducer and under the selective pressure of the selection marker. An implementation method that includes this.

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