Novel genetic tools

Abstract

The invention relates to a system for controlling protein expression, comprising a repressor, in particular a controllable repressor.

Description

[0001] INM-431 WO INMPT23048WO 15.11.2024 Applicant: INM - Leibniz Institute for New Materials non-profit GmbH Campus D22 66123 Saarbrücken Novel genetic tools Description Field of the invention The invention relates to novel genetic tools for Lactobacillus. This particularly concerns a system for controlling the expression of proteins, comprising a repressor, in particular a vector with such a repressor. The vector can also additionally comprise at least one toxin / antitoxin system. State of the art Lactobacilli (Lactobacillus) are gram-positive, rod-shaped lactic acid bacteria (LAB) that typically occur as commensals in humans and animals. Their stress-resistant phenotypic properties enable them to colonize a broad spectrum of host microenvironments, such as the intestine, skin, vagina, nasal and pharyngeal cavities. They often offer health benefits such asINM-431 WO INMPT23048WO 15.11.2024 anti-inflammatory, antipathogenic, and immunomodulatory activity. For this reason, they are one of the largest classes of probiotics, and several species are used clinically to treat a variety of diseases such as ulcerative colitis, mastitis, atopic dermatitis, bacterial vaginosis, and periodontitis. Aside from their health benefits, lactobacilli are also essential for numerous fermentation processes in the food industry, for example, in the production of yogurt, cheese, sourdough bread, beer, and wine. Due to this ubiquity in everyday life, there is considerable interest in genetically improving and expanding the capabilities of these bacteria for both medical and industrial purposes. In the medical field, lactobacilli are being developed as live biotherapeutic products (LBPs) that produce and deliver drugs directly to the site of diseases such as ulcerative colitis.To study the pharmacokinetics and colonization of these therapeutic bacteria in the body, these bacteria were engineered to express reporter proteins that can be imaged in situ. In industry, lactobacilli are being considered as alternative hosts for the recombinant expression of E. coli because they offer two key advantages: (i) they do not produce endotoxins, and many strains have "Generally Recognized as Safe" (GRAS) status, minimizing the risk of purified therapeutic proteins causing toxic effects in humans, and (ii) the infrastructure for their culture is already well-established and optimized in the food industry. INM-431 WO INMPT23048WO 15.11.2024 Despite this potential, the main limitations for Lactobacillus engineering are the lack of well-characterized genetic tools and the insufficient understanding of the biochemical pathways.More than two decades of careful investigations and screenings in phylogenetically related bacteria have yielded a handful of reliable tools for use in Lactobacillus, such as constitutive and inducible promoters, operators, repressors, replicons, retention modules, signal peptides, and so on. Most of these tools have been developed in a few species that have proven amenable to genetic modification, with Lactiplantibacillus plantarum (or Lactobacillus plantarum) being one of the most commonly used. While genomic integration of genes has been demonstrated in these bacteria, the greatest functional diversity has been achieved using plasmids. However, the available, well-characterized genetic tools are limited compared to the toolbox of E.coli are still tiny and need to be expanded to construct the type of circuits required for applications in the medical or food industries. The object of the invention is to provide a way to control the expression of proteins in Lactobacillus. Solution INM-431 WO INMPT23048 WO 15.11.2024 This object is achieved by the inventions having the features of the independent claims. Advantageous developments of the inventions are characterized in the subclaims. The wording of all claims is hereby incorporated by reference into the content of this description. The inventions also encompass all reasonable and, in particular, all mentioned combinations of independent and / or dependent claims.For clarity, certain terms used in the description are defined and described as follows: "Associated with / operatively linked" refers to two nucleic acids that are physically or functionally linked. For example, a promoter or regulatory DNA sequence is said to be "associated with" a DNA sequence encoding RNA or a protein if the two sequences are operatively linked or arranged in such a way that the regulatory DNA sequence influences the expression level of the coding or structural DNA sequence. This also applies, for example, to a repressor, which can bind to an operator sequence. A "coding sequence" is a nucleic acid sequence that is transcribed into RNA, such as mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA. Preferably, the RNA is then translated in an organism to produce a protein."Expression cassette" means a nucleic acid sequence capable of directing the expression of a particular nucleotide sequence in a suitable host cell, and INM-431 WO INMPT23048 WO 15.11.2024 which comprises a promoter operably linked to the nucleotide sequence of interest, which is operably linked to termination signals. It also typically comprises sequences necessary for proper translation of the nucleotide sequence. The expression cassette containing the nucleotide sequence of interest may have at least one of its components that is heterologous compared to at least one of its other components. The expression cassette may also be a naturally occurring cassette but maintained in a recombinant form suitable for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e.The specific nucleic acid sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell through a transformation event. Expression of the nucleotide sequence in the expression cassette can occur under the control of a constitutive promoter or an inducible promoter that triggers transcription only when the host cell is exposed to a specific external stimulus. Furthermore, the expression cassette preferably comprises at least one ribosome binding site. This is preferably located in the 5'-3' direction between the promoter and the protein to be expressed, or the start codon (ATG) of the protein to be expressed. An expression cassette containing a nucleotide sequence of interest can be chimeric, i.e., at least one of its components is heterologous with respect to at least one of its other components.An expression cassette may also contain a native promoter driving the native gene, but maintained in a recombinant form suitable for heterologous expression. Such use of an expression cassette renders it non-naturally present in the cell into which it was introduced. An expression cassette may also optionally contain a transcriptional and / or translational termination region (i.e., termination region) that is functional in Lactobacillus. A variety of transcription terminators are available for use in expression cassettes. These terminators are responsible for terminating transcription following the heterologous nucleotide sequence of interest and ensuring correct mRNA polyadenylation.The termination region may be native to the transcription initiation region, it may be native to the operably linked nucleotide sequence of interest, it may be native to Lactobacillus, or it may be derived from another source (i.e., foreign or heterologous to the promoter, the nucleotide sequence of interest, Lactobacillus, or any combination thereof). Furthermore, the native transcription terminator of a coding sequence may also be used. Any available terminator known to function in Lactobacillus may be used in the present invention. The term "expression," when used in reference to a polynucleotide, such as a gene, an ORF, or a portion thereof, or a transgene in Lactobacillus, refers to the process of converting the genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) by "transcription" of the gene (i.e.,Gene expression can be regulated at many stages of the process. In the case of antisense or dsRNA constructs, for example, expression may refer only to the transcription of the antisense RNA or the dsRNA. In some embodiments, the term "expression" refers to the transcription and stable accumulation of sense RNA (mRNA) or functional RNA. The term "expression" may also refer to the production of proteins. A "gene" is a defined region within a genome that comprises a coding nucleic acid sequence and typically also contains other, primarily regulatory, nucleic acids responsible for controlling the expression, i.e., transcription and translation, of the coding portion.A gene can also contain other 5' and 3' untranslated sequences and termination sequences. Other elements that may be present include introns. The regulatory nucleic acid sequence of the gene is not normally operably linked to the corresponding naturally occurring nucleic acid sequence and would thus be a chimeric gene. A "repressor" is a sequence that encodes at least one protein or protein complex that can bind to at least part of a specific promoter and thus prevents the expression of the gene sequence operably linked to this promoter. Expression of the repressor prevents the expression of the gene sequence controlled by the promoter. The repressor can contain further sequences that make the binding of the repressor dependent on external stimuli or binding agents.This makes it possible to make the function of the repressor dependent on external stimuli such as temperature. A "heterologous" nucleic acid sequence or molecule is a nucleic acid sequence or molecule that is not naturally associated with the host cell into which it is introduced. A heterologous nucleic acid sequence or molecule may comprise a chimeric sequence, such as a chimeric expression cassette, in which the promoter and coding region originate from multiple parent organisms. The promoter sequence can be a constitutive promoter sequence, a chemically inducible promoter sequence, a temperature-inducible promoter sequence, or a stress-inducible promoter sequence. Heterologous is also referred to as endogenous.A "homologous" nucleic acid sequence is one that is naturally associated with a host cell into which it is introduced. "Homologue recombination" is the mutual exchange of nucleic acid fragments between homologous nucleic acid molecules. INM-431 WO INMPT23048WO 15.11.2024 "Identity" or "percent identity" refers to the degree of similarity between two nucleic acid or protein sequences. In sequence comparison, one sequence typically serves as the reference sequence against which the test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, partial sequence coordinates are specified if necessary, and program parameters for the sequence algorithm are set. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence based on the specified program parameters.The term "substantially identical," in the context of two nucleic acids or two amino acid sequences, refers to two or more sequences or subsequences that exhibit at least about 50% nucleotide or amino acid residue identity when compared and aligned for maximum similarity, as measured by one of the following sequence comparison algorithms or by visual inspection. In certain embodiments, substantially identical sequences exhibit at least about 60%, or at least about 70%, or at least about 80%, or at least about 85%, or even at least about 90% or 95% nucleotide or amino acid residue identity. In certain embodiments, substantial identity exists over a region of the sequences that is at least about 50 residues long, or over a region of at least about 100 residues, or the sequences are substantially identical over at least about 150 residues.In further embodiments, the sequences are substantially identical if they are identical over the entire length of the coding regions. The above also applies to regulatory sequences, such as promoters, based on their nucleotide sequence. Optimal alignment of the sequences to be compared can be achieved, for example, with the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), or by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computer-assisted implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection.An example of an algorithm suitable for determining percentage sequence identity and sequence similarity is the BLAST algorithm, mentioned in Altschul et al., J. Mol. Biol. 215: 403–410 (1990). The software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm first determines high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match a word of the same length in a database sequence or satisfy a positively scored threshold T when aligned to that word. T is referred to as the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits serve as the starting point for searching for longer HSPs containing these words.The word hits are then extended in both directions along each sequence as far as the cumulative alignment score can be increased. The cumulative scores are calculated for nucleotide sequences using the parameters M (reward value for a pair of matching residues; always > 0) and N (penalty value for mismatched residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of word hits in each direction is stopped when the cumulative alignment score deviates by the amount X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negatively scored residue alignments, or the end of one of the two sequences is reached. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of the alignment.The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands as default values. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)). In addition to calculating the percentage sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90: 5873–5787 (1993)). A measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which gives an indication of the probability with which a match between two nucleotide or amino acid sequences would occur by chance INM-431 WO INMPT23048WO 15.11.2024.For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest cumulative probability when comparing the test nucleic acid sequence with the reference nucleic acid sequence is less than about 0.1, preferably less than about 0.01, and most preferably less than about 0.001. Another indication that two nucleic acids are essentially identical is the fact that the two molecules hybridize to each other under stringent conditions. The term "specifically hybridize to" refers to the binding, duplex formation, or hybridization of a molecule only with a specific nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) of DNA or RNA."Binding(s) substantially" refers to the complementary hybridization between a probe nucleic acid and a target nucleic acid and includes minor mismatches that can be compensated for by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence. "Strong hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence-dependent and differ in various environmental parameters. Longer sequences hybridize specifically at higher temperatures. A comprehensive guide to nucleic acid hybridization can be found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization INM-431 WO INMPT23048 WO 15.11.2024 with Nucleic Acid Probes Part I Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays" Elsevier, New York. Generally, very stringent hybridization and washing conditions are chosen, approximately 5°C below the thermal melting point (Tm) for the specific sequence at a given ionic strength and pH. Typically, under "stringent conditions," a probe hybridizes to its target subsequence but not to other sequences. The Tm value is the temperature (at a defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are chosen to match the Tm value for a specific probe.An example of stringent hybridization conditions for hybridizing complementary nucleic acids that have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide with 1 mg heparin at 42°C, with hybridization carried out overnight. An example of very stringent wash conditions is 0.15 M NaCl at 72°C for about 15 minutes. An example of stringent wash conditions is a 0.2× SSC wash at 65°C for 15 minutes (see J. Sambrook, et al., Molecular Cloning: A Laboratory Manual, 3d Ed., Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (2001), for a description of the SSC buffer). Often, a high-stringency wash is preceded by a low-stringency wash to remove background probe signal. An example of a medium-stringency wash for a duplex of, for example, B. more than 100 nucleotides is 1x SSC at 45°C for 15 minutes.An example of a low-stringency wash procedure for a duplex of, e.g., more than 100 nucleotides is 4-6x SSC at 40°C for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically include salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C. Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide. In general, a signal-to-noise ratio of 2x (or higher) than an unrelated probe in the respective hybridization assay indicates the detection of specific hybridization. Nucleic acids that do not hybridize with each other under strict conditions are nevertheless essentially identical if the proteins they encode are essentially identical.This is the case, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy allowed by the genetic code. The following are examples of hybridization / washing conditions that can be used to clone homologous nucleotide sequences that are substantially identical to the reference nucleotide sequences of the present invention: A reference nucleotide sequence preferably hybridizes with the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 1X SSC, 0.1% SDS at 50°C, especially in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C withINM-431 WO INMPT23048WO 15.11.2024Wash in 0.5X SSC, 0.1% SDS at 50°C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 50°C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 65°C. Another indication that two nucleic acids or proteins are essentially identical is that the protein encoded by the first nucleic acid is immunologically cross-reactive with or specifically binds to the protein encoded by the second nucleic acid. Thus, a protein is typically essentially identical to a second protein if the two proteins differ only by conservative substitutions. A nucleic acid sequence is "isocoding" with a reference nucleic acid sequence if the nucleic acid sequence encodes a polypeptide that has the same amino acid sequence as the polypeptide encoded by the reference nucleic acid sequence.An "isolated" nucleic acid molecule or an isolated toxin is a nucleic acid molecule or toxin that exists outside of its natural environment by human activity and is therefore not a product of nature. An isolated nucleic acid molecule or toxin may be in purified form or may exist in a non-natural environment, e.g., in a recombinant microbial cell. A "nucleic acid molecule" or a "nucleic acid sequence" is a segment of single- or double-stranded DNA or RNA that can be isolated from any source. In the context of the present invention, the nucleic acid molecule is typically a DNA segment. In some embodiments, the nucleic acid molecules of the invention are isolated nucleic acid molecules. The terms "protein," "peptide," and "polypeptide" are used interchangeably herein.The term "codon-optimized" sequence refers to the nucleotide sequence of a recombinant, transgenic, or synthetic polynucleotide in which the codons are chosen to reflect the particular codon bias that a host cell may have. This is done so that the amino acid sequence of the polypeptide encoded by the codon-optimized polynucleotide is preserved. In certain embodiments, the nucleotide sequence of the recombinant DNA construct contains a sequence that has been codon-optimized for the cell (e.g., an animal, plant, or fungal cell) in which the construct is to be expressed. A "promoter" is a non-translated DNA sequence upstream of the coding region that contains the binding site for RNA polymerase and initiates transcription of the DNA. The promoter region may also contain other elements that act as regulators of gene expression."Regulatory elements" are sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements include a promoter operably linked to the nucleotide sequence of interest, and optionally termination signals. They typically also include sequences required for proper translation of the nucleotide sequence. Other regulatory elements include, for example, repressors, which encode proteins that influence the promoter. "Transformation" is a process for introducing heterologous nucleic acid into a host cell or organism. In particular, "transformation" means the stable integration of a DNA molecule into the genome (nucleus or plastid) of an organism of interest."Transformed / transgenic / recombinant" refers to a host organism, such as a bacterium or a plant, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host organism or present as an extrachromosomal molecule. Such an extrachromosomal molecule can be self-replicating. Transformed cells, tissues, or plants include not only the end products of a transformation process but also their transgenic progeny. A "non-transformed," "non-transgenic," or "non-recombinant" host refers to a wild-type organism, e.g., a bacterium that does not contain the heterologous nucleic acid molecule. Nucleotides are indicated by their bases using the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G). The amino acids are also labeled with theINM-431 WO INMPT23048WO 15.11.2024 the following standard abbreviations are given: Alanine (Ala; A), Arginine (Arg; R), Asparagine (Asn; N), Aspartic acid (Asp; D), Cysteine ​​(Cys; C), Glutamine (Gln; Q), Glutamic acid (Glu; E), Glycine (Gly; G), Histidine (His; H), Isoleucine (Ile; I), Leucine (Leu; L), Lysine (Lys; K), Methionine (Met; M), Phenylalanine (Phe; F), Proline (Pro; P), Serine (Ser; S), Threonine (Thr; T), Tryptophan (Trp; W), Tyrosine (Tyr; Y) and Valine (Val; V). The object is achieved, inter alia, by a system for controlling the expression of proteins in Lactobacillus, comprising a promoter in operative linkage to a nucleic acid encoding at least one protein, and additionally a heterologous Repressor in operative linkage to the promoter. The repressor is preferably a protein. The repressor is preferably a genetically encoded repressor. The repressor is preferably a heterologous repressor derived from bacteriophages that infect Lactobacillus.Preferably, it is a repressor encoded in the genetic switches that control lytic and lysogenic cycles in bacteriophages. In a particular embodiment, the repressor is derived from bacteriophages selected from the group øg1e, mv4, or A2. Preferred is a repressor that, compared to a vector without a repressor, reduces the expression of a protein under the same conditions and promoter sequences by at least a factor of 2, preferably by at least a factor of 5. Preferably, the repressor comprises a protein with an amino acid sequence that is substantially identical to sequence ID No. 1. Preferably, the repressor has an identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID No. 1.Preferably, the repressor is a repressor encoded by a nucleotide sequence comprising a sequence which is substantially identical to SEQ ID NO: 3. Preferably, the sequence has an identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 3. Surprisingly, it was found that this repressor in Lactobacillus enables very high repression of the linked promoter and thus the production of protein operatively linked to this promoter. For the repressor to be active, it is necessary that the repressor be expressed as a protein in the cell. This can be achieved in different ways. For example, expression can be induced by corresponding mRNA. Preferably, the repressor is operatively linked to a promoter. DerINM-431 WO INMPT23048WO 15.11.2024The promoter linked to the repressor can be any promoter active in Lactobacillus. These can be homologous or heterologous promoters, but heterologous promoters are preferred. Preferably, the promoter is a constitutive promoter. These are promoters that are not activated by any external stimulus other than the repressor. It may be necessary to adapt the repressor to bind to the promoter. Examples of promoters are P. tlpA (Seq ID No. 26) , P 23 (Seq ID No. 30) , P 48 (Seq. Id No. 29), P spp(SEQ ID No. 28) or PTuf (SEQ ID No. 27), as well as Ptec (SEQ ID No. 21). Preferably, the gene sequence of the repressor is operatively linked to another promoter, preferably to a constitutive promoter. These are promoters that are not activated by an external stimulus. This means that the repressor is always active. In a preferred embodiment, the repressor can change its conformation or structure upon exposure to a stimulus such that it no longer binds to the operator sequence and thus no longer suppresses the promoter, thus resulting in protein expression. Responsiveness to a stimulus can be achieved, for example, by appropriate modification of the repressor, for example, by removing, adding, or replacing certain protein sequences. The added or replaced protein regions change their conformation or structure upon exposure to the stimulus, thus affecting the repressor's ability to bind to the promoter.In a preferred embodiment of the repressor, the DNA-binding form of the repressor is formed from at least two units INM-431 WO INMPT23048 WO 15.11.2024. The units can be covalently linked or bind to each other via non-covalent bonds. Preferably, the repressor comprises a dimer formed from two units comprising the repressor according to the invention. In a preferred embodiment, the repressor comprises at least two domains, comprising at least one DNA-binding domain for binding to the operator sequence and at least one dimerization domain. Via the dimerization domain, the two units of the repressor can form a dimer, which then forms the active form and binds to the operator sequence in the DNA. Adaptation of the repressor according to the invention to different stimuli, such as temperature, light, and certain substances, is possible by adapting or exchanging the dimerization domain.Due to dimerization, the repressor itself already has a built-in switch that can be used for adaptation. Preferably, the sequence of the DNA binding domain has an identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID No. 1. Preferably, the nucleotide sequence of the DNA binding domain has an identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least INM-431 WO INMPT23048 WO 15.11.202499 or 100% identity with the sequence of SEQ ID No. 3.In one embodiment of the invention, the sequence of the dimerization domain has an identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID No. 2. In one embodiment of the invention, the nucleic acid sequence of the dimerization domain has an identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID No. 4. In a further embodiment of the invention, the sequence of the repressor has an identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID No. 5.This sequence comprises a dimerization domain and a DNA-binding domain. After expression, the repressor forms a dimer for binding to the operator sequence. In a further embodiment of the invention, the nucleic acid sequence of the repressor has an identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, INM-431 WO INMPT23048 WO 15.11.2024 at least 97. at least 98 at least 99 or 100% identity with the sequence of SEQ ID No. 6. This sequence comprises a dimerization domain and a DNA-binding domain. After expression, the repressor forms a dimer for binding to the operator sequence. The repressor can preferably be present in at least two different forms. Preferably, in a first form, it is able to bind to the promoter in such a way that expression is inhibited, while in the other, particularly second, form, it does not bind sufficiently to the promoter to inhibit expression of the protein. These forms are preferably a dimer and the dissociated form of the dimer. The repressor is operatively linked to an operator sequence or operator. This is a nucleotide sequence to which the repressor protein can bind in order to suppress a promoter associated with the operator sequence and thus the expression of the associated protein. This operator sequence can be present once, twice, three times, or more.be located upstream, within, and / or downstream of the associated promoter, preferably upstream and / or downstream. Particularly preferably, the operator sequence is present once, twice, three times, or more within a region of 50 nucleotides before to 50 nucleotides after, preferably 40 nucleotides before to 40 nucleotides after, particularly preferably 35 nucleotides before to 35 nucleotides after, the promoter. The sequence can also be part of the promoter sequence, as well as replace part of the promoter sequence. In a particularly preferred embodiment, the operator sequence is simple,located twice or more in the range from 35 nucleotides before the promoter to the ribosome binding site after the promoter. In a preferred embodiment, the operator sequence is located in the range from -35 to -10 nucleotides before the promoter and / or after the promoter to the ribosome binding site. The operator sequence is preferably operatively linked to a specific repressor. In a preferred embodiment, the operator sequence is substantially identical to a nucleotide sequence with SEQ ID NO: 11. Preferably, the operator sequence has an identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% with the sequence of SEQ ID NO: 11. This operator is operatively linked to the preferred repressor according to the invention. The invention also relates to an expression cassette for expression in Lactobacillus,which the system according to the invention enables. In a preferred embodiment, the expression cassette comprises at least one promoter, a nucleotide sequence linked to the promoter, and at least the nucleotide sequence of the repressor according to the invention, as well as preferably at least one promoter linked to the repressor. The expression cassette preferably comprises at least one operator sequence operatively linked to the repressor. In a preferred embodiment, the expression cassette comprises at least one ribosome binding site operatively linked to the promoter, which is preferably located between the promoter and the encoded protein. In a preferred embodiment, the distance between the ribosome cleavage site and the start codon of the encoded protein is at least 5 base pairs, in particular at least 6 base pairs, preferably from 7 to 24 base pairs.in particular 7 to 15 base pairs. The invention also relates to a vector, in particular a plasmid, comprising the system according to the invention, preferably the expression cassette according to the invention. These can be any vectors suitable for Lactobacillus. The vectors can also comprise further regulatory elements. Within the meaning of the application, Lactobacillus is preferably understood to mean a bacterium of the order of lactic acid bacteria (Lactobacillales), preferably of the family Lactobacillaceae, particularly preferably of the genus Lactobacillus, Lacticaseibacillus, Lactiplantibacillus. Preferred species are Lactobacillus acetotolerans, Lactobacillus acidifarinae, Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus algidus, Lactobacillus alimentarius, Lactobacillus alvei, Lactobacillus alvi, Lactobacillus amylolyticus, Lactobacillus amylophilus, Lactobacillus amylotrophicus,Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus animata, Lactobacillus antri, Lactobacillus apinorum, Lactobacillus apis, Lactobacillus apodemi, Lactobacillus aquaticus, Lactobacillus aviarius, Lactobacillus backii, Lactobacillus bifermentans, Lactobacillus bombi, Lactobacillus bombicola, Lactobacillus brantae, Lactobacillus brevis, Lactobacillus brevisimilis, Lactobacillus buchneri, Lactobacillus cacaonum, Lactobacillus camelliae, Lactobacillus capillatus, Lactobacillus casei (Lacticaseibacillus casei), Lactobacillus paracasei (Lacticaseibacillus paracasei), Lactobacillus zeae, Lactobacillus catenefornis, Lactobacillus ceti, Lactobacillus coleohominis, Lactobacillus colini, Lactobacillus collinoides, Lactobacillus composti, Lactobacillus concavus, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus crustorum, Lactobacillus curieae, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus dextrinicus, Lactobacillus diolivorans, Lactobacillus equi,Lactobacillus equicursoris, Lactobacillus equigenerosi, Lactobacillus fabifermentans, Lactobacillus faecis, Lactobacillus faeni, Lactobacillus farciminis, Lactobacillus farraginis, Lactobacillus fermentum, Lactobacillus floricola, Lactobacillus forum, Lactobacillus formosensis, Lactobacillus fornicalis, Lactobacillus fructivorans, Lactobacillus frumenti, Lactobacillus fuchuensis, Lactobacillus furfuricola, Lactobacillus futsaii, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus gastricus, Lactobacillus ghanensis, Lactobacillus gigeriorum, Lactobacillus ginsenosidimutans, Lactobacillus gorillae,INM-431 WO INMPT23048WO 15.11.2024Lactobacillus graminis, Lactobacillus guizhouensis, Lactobacillus halophilus, Lactobacillus hammesii, Lactobacillus hamsteri, Lactobacillus harbinensis, Lactobacillus hayakitensis, Lactobacillus heilongjiangensis. Lactobacillus helsingborgensis, Lactobacillus helveticus, Lactobacillus herbarum, Lactobacillus heterohiochii, Lactobacillus hilgardii,Lactobacillus hokkaidonensis, Lactobacillus hominis, Lactobacillus homohiochii, Lactobacillus hordei, Lactobacillus iatae, Lactobacillus iners, Lactobacillus ingluviei,Lactobacillus insectis, Lactobacillus insicii, Lactobacillusintermedius, Lactobacillus intestinalis, Lactobacillus iwatensis, Lactobacillus ixorae, Lactobacillus japonicus, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus kalixensis, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus kimbladii, Lactobacillus kimchicus, Lactobacillus kimchiensis, Lactobacillus kisonensis, Lactobacillus kitasatonis, Lactobacillus koreensis, Lactobacillus kullabergensis, Lactobacillus kunkeei, Lactobacillus larvae, Lactobacillus leichmannii, Lactobacillus letivazi, Lactobacillus lindneri, Lactobacillus malefermentans, Lactobacillus mali, Lactobacillus manihotivorans, Lactobacillus mellifer, Lactobacillus mellis, Lactobacillus melliventris, Lactobacillus micheneri, Lactobacillus mindensis,Lactobacillus mixtipabuli, Lactobacillus mobilis, Lactobacillus modestisalitolerans, Lactobacillus mucosae, Lactobacillus mudanjiangensis, Lactobacillus murinus, Lactobacillus nagelii, Lactobacillus namurensis, Lactobacillus nantensis, Lactobacillus nasuensis, Lactobacillus nenjiangensis, Lactobacillus nodensis, Lactobacillus odoratitofui, Lactobacillus oeni, Lactobacillus oligofermentans, Lactobacillus ori, Lactobacillus oryzae, LactobacillusINM-431 WO INMPT23048WO 15.11.2024otakiensis, Lactobacillus ozensis, Lactobacillus panis, Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus parabuchneri, Lactobacillus paracollinoides, Lactobacillus parafarraginis, Lactobacillus parakefiri, Lactobacillus paraalimentarius, Lactobacillus paraplantarum, Lactobacillus pasteurii, Lactobacillus paucivorans, Lactobacillus pentosus, Lactobacillus perolens, Lactobacillus plajomi, Lactobacillus plantarum (Lactiplantibacillus plantarum), Lactobacillus pobuzihii, Lactobacillus pontis,Lactobacillus porcinae, Lactobacillus psittaci, Lactobacillus rapi, Lactobacillus rennanquilfy, Lactobacillus rennini, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus rodentium, Lactobacillus rogosae, Lactobacillus rossiae, Lactobacillus ruminis, Lactobacillus saerimneri, Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus sanfranciscensis, Lactobacillus saniviri, Lactobacillus satsumensis, Lactobacillus secaliphilus, Lactobacillus selangorensis, Lactobacillus senioris, Lactobacillus senmaizukei, Lactobacillus sharpeae, Lactobacillus shenzhenensis, Lactobacillus sicerae, Lactobacillus silagei, Lactobacillus siliginis, Lactobacillus similis, Lactobacillus songhuajiangensis, Lactobacillus spicheri, Lactobacillus sucicola, Lactobacillus suebicus, Lactobacillus sunkii, Lactobacillus taiwanensis, Lactobacillus thailandensis, Lactobacillus tucceti, Lactobacillus ultunensis, Lactobacillus uvarum, Lactobacillus vaccinostercus, Lactobacillus vaginalis,Lactobacillus vermiforme, Lactobacillus vespulae, Lactobacillus vini, Lactobacillus wasatchensis, Lactobacillus xiangfangensis, Lactobacillus yonginensis, Lactobacillus zymae and Lactococcus lactis, preferably Lactobacillus reuteri, Lactobacillusparacasei (Lacticaseibacillus paracasei), LactobacillusINM-431 WO INMPT23048WO 15.11.2024plantarum (Lactiplantibacillus plantarum), Lactobacillusjohnsonii, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus casei (Lacticaseibacillus casei), Lactococcus lactis, preferablyLactopactibacillus plantarum (Lactiplantibacillus plantarum), particularly preferablyLactopactibacillus plantarum WCFS1 (Lactiplantibacillus plantarum WCFS1). The protein encoded by the nucleic acid can be a protein homologous or heterologous to Lactobacillus,preferably a heterologous protein. The protein can be adapted according to the use of the Lactobacillus. The invention further relates to a Lactobacillus comprising the system according to the invention. The system according to the invention can be arranged on at least one vector. The sequence for the repressor with the associated promoter, as well as the promoter operatively linked to the repressor and the protein to be expressed, can be arranged on a different vector. Preferably, all sequences are arranged on one vector. The invention further relates to a Lactobacillus comprising at least one expression cassette according to the invention. The invention further relates to a Lactobacillus comprising at least one vector comprising the at least one expression cassette. INM-431 WO INMPT23048WO 15.11.2024 In one embodiment of the invention, the expression cassette is operatively linked to at least one toxin / antitoxin system. This means,that, in addition to the expression cassette, at least one toxin and at least one antitoxin can be expressed. Such systems are known from antibiotic-free genetic stabilization. When a protein is expressed, a toxin and a corresponding antitoxin are also expressed. The toxin usually inhibits the growth of the Lactobacillus. The antitoxin neutralizes the toxin, although the toxin is degraded more slowly than the antitoxin. If a cell without the antitoxin gene is created during cell division, it is killed by the toxin. These systems are therefore preferably used for gene maintenance, especially plasmid maintenance, in cells over several generations. Since they do not require antibiotics, they are also suitable for use in living systems, such as the gastrointestinal tract.to be used. The at least one toxin / antitoxin system can be homologous or heterologous. In a preferred embodiment, the at least one toxin / antitoxin system is arranged on the same vector as the expression cassette. In one embodiment of the invention, the expression cassette is operatively linked to at least two toxin / antitoxin systems, preferably at least two different toxin / antitoxin systems. In the case of a vector, these elements are arranged on the vector. INM-431 WO INMPT23048 WO 15.11.2024 In one embodiment of the invention, the toxin / antitoxin system is linked to a promoter, preferably a different promoter than the promoter of the expression cassette. It is only important that the promoter is at least also active when the promoter of the expression cassette is activated. It is preferably a constitutive promoter,which is not triggered by an external stimulus. Thus, this plasmid retention is always active when the plasmid is in the cell. In this case, "operably linked" can also mean that the expression cassette and the TA systems are located on the same vector, and plasmid retention by the toxin / antitoxin system represents the operative link. Many toxin / antitoxin systems are known. Different types of such systems are distinguished (Singh G. et al., Current Research in Microbial Sciences 2, 2021, 100047, Bacterial toxin-antitoxin modules: classification, functions, and association with persistence). In type I TA systems, the antitoxin is an antisense RNA that inhibits the translation of the toxin mRNA. An example of this is the hok-sok module (Gerdes, K., Bech, FW, Jørgensen, ST, Løbner-Olesen, A., Rasmussen, PB, Atlung, T., Boe, L., Karlstrom, O., Molin, S., von Meyenburg, K.,1986. Mechanism of postsegregational killing by the hok gene product of the parB system of plasmid R1 and its homology with the relF gene product of the E. coli relB operon. EMBO J 5 (8), 2023–2029. In type II TA systems, toxin and antitoxin are proteins that form a complex with each other. Many of the toxins are endoribonucleases or inhibitors of DNA gyrase. In a type III TA system, the antitoxin is an RNA that interacts directly with the toxin protein. In a type IV TA system, antitoxin and toxin are proteins that do not interact directly with each other but bind competitively to a protein. In a type V TA system, the antitoxin is an endoribonuclease against the toxin's mRNA. In a type VI TA system, the toxin inhibits replication, while the antitoxin causes the toxin's degradation. In a type VII TA system, the antitoxin modifies the toxin. The TA system can be a type I, type II,Type III, Type IV, Type V or Type VI. Examples of type I TA systems are Hok and Sok, Fst and RNAII, TisB and IstR, LdrB and RdlD, FlmA and FlmB, lbs and Sib, TxpA / BmT and RatA, SymE and SymR, and XXCV2162 and ptaRNAl. Examples of type II TA systems are CcdB and CcdA, ParE and ParD, MaxF and MazE, yafO and yafN, HicA and HicB, Kid and Kis, Zeta and Epsilon, DarT and DarG, Txe and Axe, YafQ and DinJ, HigB and HigA, HipA and HipB, PhD and Doc, RelB and RelE, VapB and VapC, RnlA and RnlB, MqsR and MqsA.INM-431 WO INMPT23048WO 15.11.2024 An example of a type III TA system is ToxN and Tox1. An example of a type V TA system is GoT and GoS. An example of a type VI TA system is SocA and SocB. In a preferred embodiment, the at least one toxin / antitoxin system is a type II TA system, preferably selected from the group comprising Txe (SEQ ID No. 31) / Axe (SEQ ID No. 32), YafQ (SEQ ID No. 33) / DinJ (SEQ ID No. 34), HicA (SEQ ID No. 35) / HicB (SEQ ID No. 36),HigB (SEQ ID No. 51) / HigA (SEQ ID No. 52) and MazF (SEQ ID No. 53) / MazE (SEQ ID No. 54). The sequences are preferably arranged adjacently, although they may also overlap. Such a toxin / antitoxin system can increase the retention of a plasmid over several generations of Lactobacillus. Retention is significantly improved by the presence of at least two toxin / antitoxin systems, in particular two toxin / antitoxin systems, and especially two different toxin / antitoxin systems. A retention of at least 15% after 100 generations is preferred, in particular at least 20%. In the presence of at least two toxin / antitoxin systems, a retention of at least 40%, preferably over 50% after 100 generations, is preferred.INM-431 WO INMPT23048WO 15.11.2024In a preferred embodiment, the at least one toxin / antitoxin system achieves a G, 50 -value of at least 50, especially at least 60. The G 50-value the number of generations until the proportion of cells with plasmid falls below 50%. In the case of at least two toxin / antitoxin systems, a G 50A value of over 100 is preferred. These retentions are lower than those under the influence of antibiotics. However, these systems do not require the external addition of antibiotics. Furthermore, the loss of plasmid leads to the creation of a natural organism without genetic modification. A Lactobacillus with such a vector can therefore also be referred to as a temporary genetically modified organism (GMO). Over the course of generations, this organism loses the genetic modification. This is particularly of interest if the Lactobacillus is to be used in a medical or human environment. The vector can also contain other regulatory elements, for example, antibiotic resistance, which simplify production. The invention also relates to the use of the repressor according to the invention in Lactobacillus.The invention further relates to the use of the vector according to the invention in Lactobacillus, in particular in Lactobacillus plantarum. The invention also relates to a promoter (P. tec) for use in Lactobacillus with a nucleotide sequence substantially identical to Sequence ID No. 22. Preferred is an identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% with the sequence of SEQ ID No. 22. The promoter is particularly advantageous because it is directly associated with the repressor according to the invention. Thus, the sequence SEQ ID No. 22 already comprises the corresponding operator sequence SEQ ID No. 11. The invention also relates to an expression cassette with this promoter. The invention further relates to the use of the promoter in the system according to the invention.The invention further relates to Lactobacillus comprising the promoter according to the invention, as well as the use of the promoter in Lactobacillus.It is precisely the improved control of protein expression by the system and / or promoter according to the invention that enables many new applications. Expression can be controlled very efficiently and can also be made dependent on external stimuli such as temperature. This allows the repressor to be used more effectively in analysis and diagnosis. Materials and Methods INM-431 WO INMPT23048 WO 15.11.2024 Further details and features emerge from the following description of preferred embodiments in conjunction with the subclaims. The respective features can be implemented individually or in combination with one another. The possibilities for achieving the object are not limited to the embodiments. The embodiments are shown schematically in the figures. Identical reference numerals in the individual figures designate identical or functionally equivalent features.elements that correspond to each other in terms of their functions. The details are shown in Fig. 1 a) Structure of the cloning process. In the first cloning round, the operators were inserted into the promoter P. tlpAinserted. In the second round, the repressors were cloned into the corresponding plasmid. All genetic fragments were based on synthetic eBlocks from IDT (fluorescence = fluorescence; count = number; operator = operator; operator effect = operator effect; repressor effect = repressor effect). B) FACS data showing the effects of cloning each operator (O) and each operator plus repressor (O + R) into the plasmid on mCherry expression (fluorescence intensity = fluorescence intensity; count = number); Fig. 2 A) Expression levels of mCherry in terms of RFU for all three operators (O cng, O rep, and O cI) and all three operators plus repressors (cng, rep, and cI). B) Percentage of repression mediated by each repressor (columns from left to right: cng, rep, cI). C) Growth curves of all three repressors and the wild-type bacteria over 16 hours (hours = hours). D) Bacterial biomass of wild-type, P tlpAmCherry and bacteria carrying each repressor after overnight growth in an incubator. Each sample is based on a 5 mL culture. Data with error bars represent the means and standard deviations (SD). ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001 (biomass). Fig. 3 A) Schematic of the genetic modifications introduced into the PtlpA promoter (new operator). B) Expression levels of mCherry in terms of RFU (relative fluorescence units) for all three operator modifications (with and without the repressor in the plasmid) (columns from left to right: O1, O2, O3, respectively). C) Percentage of repression mediated by each operator modification (O1, O2, and O3) (columns from left to right: O1, O2, O3). D) Scheme of substitution of P tlpA by Ptec. (endogenous promoter) E) Expression level of mCherry in the form of RFU for P tec _mCherry and P tec_rep_mCherry bacteria. F) Percentage suppression and fold changes for PtlpA_O_rep (OR_rep), PtlpA O3_rep (O3_rep), and Ptec_rep (Seq ID No. 21). Data with error bars represent the means and SD (columns from left to right: OR_rep, O3_rep, P tec _rep). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.INM-431 WO INMPT23048WO 15.11.2024Fig. 4 A) Left: 5 ml MRS cultures based on Ptec_rep and P tec Bacteria. The cells were pelleted, and the pellets are shown below. Right: 95 ml LB cultures (supplemented with 5 ml 1M glucose) based on P tec _rep and P tec Bacteria. The cells were pelleted, and the corresponding pellets are shown below (media = medium). B) Bacterial pellets from P tec _rep, P tec and P tlpA Bacteria after overnight growth. C) Fluorescence microscopy images of bacteria P tec _rep, P tec and P tlpA. D) FACS data show the comparison between P tec _rep, P tec and P tlpABacteria (fluorescence intensity = fluorescence intensity; count = number). E) Expression levels of mCherry in terms of RFU for Ptec in L. plantarum WCSF1, PtlpA in L. plantarum WCSF1, and PtlpA in E. coli Nissle. Fold changes (factor of increase) by Ptec in L. plantarum WCSF1 and PtlpA in E. coli Nissle normalized to expression by PtlpA in L. plantarum WCSF1. Data with error bars represent the means and SD. ** p < 0.01, *** p < 0.001; Fig. 5 FACS data showing the comparison between bacteria carrying the Rep repressor (mCherry suppressed) and wild-type bacteria (fluorescence intensity = fluorescence intensity; count = number); Fig. 6 A) Graphic representation of the arabinose and sucrose operons identified in the genome of L. plantarumWCFS1 (chromosome = chromosome; plasmid = plasmid; ATP-dependent = ATP-dependent; sucrose-specific enzyme IIBC protein of the phosphotransferase system = sucrose-specific enzyme IIBC protein of the phosphotransferase system).B) mCherry-INM-431 WO INMPT23048WO 15.11.2024Expression levels in the strain with the P. ara promoter in MRS and LB media, determined using a microplate reader with a gain setting of 136 (~10x higher than gain 100) to detect signal above noise. C) mCherry expression levels in the strain with the Psuc promoter in MRS and M17 media, determined using a microplate reader with a gain setting of 100 (left column -Suc, right column +Suc). D) mCherry expression levels in the strain with the P suc -promoter in M17 media without sugar and with sucrose, arabinose, fructose, and glucose, determined using a microplate reader with the gain set to 100; Fig. 7 Growth curves of PtlpA_O3_rep_mCherry,P tec_rep_mCherry and wild-type bacteria for 16 hours. Data with error bars represent the means and SD (hours = hours); Fig. 8 A) Schematic of site-directed mutagenesis to remove the operator from the plasmid. B) Expression levels of mCherry in terms of RFU (from left to right) for P tlpA (unmodified), P tlpA _rep (repressor only), P tlpA _op (rep operator only) and P tlpA_op_rep (Operator plus Rep). Data with error bars represent means and SD. ns = not significant, ***p < 0.001. C) AlphaFold 3D structural prediction of the rep in monomeric form. D) AlphaFold 3D structural prediction of the Rep in dimeric form; Fig. 9 A) Growth curves of Ptec_mCherry and wild-type bacteria after 16 hours of growth in the INM-431 WO INMPT23048WO 15.11.2024 microtiter plate reader (Hours = hours; wt is the upper measurement). B) OD600 values ​​for Ptec_mCherry (right) and wild-type bacteria (left) after 16 hours of growth in the microtiter plate reader. C) Bacterial biomass (Biomass) of P tec mCherry (right) and wild-type bacteria (left) after overnight growth in an incubator. Each sample is based on a 5 mL culture. Data with error bars represent the means and SD. ns = not significant, * p < 0.05. Fig. 10 Placement of the operators in the promoter region. Each operator sequence was placed downstream of P tlpAplaced (sequence: Seq ID No. 23); Fig. 11 Fold changes of PtlpA (unchanged promoter) compared to P tlpA associated with each operator (e.g., effect of placing each operator within P tlpA). Data with error bars represent the means and SD (standard deviation); Fig. 12 OD600 values ​​after 16 hours of growth in the microplate reader for wild-type bacteria and bacteria encoding each repressor. Data with error bars represent the means and SD. ns = not significant; Fig. 13 Scheme of PCR with site-directed mutagenesis to remove the operator sequence from the promoter region of the plasmid (sequences: Seq ID Nos. 24, 25) (forward primer; reverse primer; direct cloning); Fig. 14 Agarose gel showing that excision of rep from the plasmid by site-directed mutagenesis occurred as expected. The "+ rep" product (1524 bp) is 891 bp larger than the "- rep" product (633 bp) due to the excision of the P48_rep gene. The bacterial pellets were used as templates for PCR.The 1 kb DNA ladder from Generuler was used as a reference; Strains, Media, and Plasmids: L. plantarum WCFS1 was used as the starting strain for characterizing promoter strength and plasmid retention. The strain was maintained in De Man, Rogosa, and Sharpe (MRS) media (Carl Roth GmbH, Germany, Art. No. X924.1). Genetically modified L. plantarum WCFS1 strains were cultured in MRS medium supplemented with 10 μg / ml erythromycin (Carl Roth GmbH, Art. No. 4166.2) at 37 °C at 250 revolutions per minute (rpm) for 16 hours. NEB-5-alpha competent E. coli cells (New England Biolabs GmbH, Germany, Art. No. C2987) were used for cloning specific plasmids. This strain was maintained in Luria-Bertani medium (LB, Carl Roth GmbH, Art. No. X968.1). Genetically modified E. coli DH5α strains were grown in LB media supplemented with 200 μg / ml erythromycin at 37 °C and 250 rpm for approximately 16 hours.The plasmid used, pLp_3050sNuc, was a gift from Prof. Geir Mathiesen (Addgene Plasmid #122030). E. coli Nissle was kindly provided by Prof. Rolf Müller. The sequences described in the description and shown in the figures are given in Table 2. INM-431 WO INMPT23048WO 15.11.2024 Molecular Biology PCR was performed using Q5 High Fidelity 2X Master Mix (NEB) and primers from Integrated DNA Technologies (IDT) (Leuven, Belgium) or Eurofins Genomics GmbH (Cologne, Germany). The primers are given in Table 3. The synthetic genes were purchased as eBlocks from IDT (Coralville, USA). These were codon-optimized using the IDT Codon Optimization Tool (Coralville, USA). The NEBuilder® HiFi DNA Assembly Cloning Kit, Quick Blunting Kit, and the enzyme T4 DNA Ligase were purchased from New England BioLabs (NEB, Germany). Plasmid extraction was performed using a kit from Qiagen GmbH (Hilden, Germany).DNA purification was performed using a kit from Promega GmbH (Walldorf, Germany). A 1 kb DNA ladder (Thermo Fisher Scientific) was used as a reference for the agarose gels. L. plantarum WCFS1 preparation of competent cells and DNA transformation: L. plantarum WCFS1 was inoculated into 5 mL of MRS medium without antibiotics and cultured overnight at 37 °C with shaking (250 rpm). The next day, 1 mL of the bacterial culture was transferred to a secondary culture consisting of 20 mL of MRS medium and 5 mL of 1% (w / v) glycine. The secondary culture was incubated at 37 °C and 250 rpm until the optical density of a sample measured at a wavelength of 600 nm (OD600) reached approximately 1. The cells were then pelleted by centrifugation at 4000 rpm for 12 minutes at 4°C. The bacterial pellet was then washed several times, with the bacteria being centrifuged at 4000 rpm for 8 minutes each. The first two washes were performed at 4000 rpm for 15 minutes.2024 were performed with 5 mL of ice-cold 10 mM MgCl2. The next two washes were performed with 5 mL of ice-cold Sac / Gly solution [10% (v / v) glycerol and 1 M sucrose, mixed in a 1:1 (v / v) ratio]. After discarding the supernatant, the pellet was resuspended in 500 μL of Sac / Gly solution, and the competent cells were distributed in 60 μL aliquots for DNA transformation. For transformation, 1 μg of dsDNA was added to the competent cells and incubated on ice for 10 minutes. The mixture was transferred to an ice-cold electroporation cuvette with a 2 mm gap (Bio-Rad Laboratories GmbH, Germany), and the cells were electroporated with a single pulse at 1.8 kV, followed immediately by the addition of 1 ml of MRS medium. The mixture was then incubated at 37 °C and 250 rpm for a recovery time of 3 hours. After recovery, the cells were centrifuged at 4000 rpm for 5 minutes, and 800 μl of the supernatant was discarded.The remaining 200 μl was used to resuspend the pellet, and the entire 200 μl was spread on MRS agar supplemented with 10 μg / ml erythromycin. The plates were incubated at 37°C for 1-3 days to allow the bacterial colonies to grow. Direct cloning in L. plantarum WCFS1 Plasmid engineering of L. plantarum WCFS1 was performed using our previously developed direct cloning method (Blanch-Asensio, M., Dey, S. and Sankaran, S., 2023. In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1. PLOS one, 18(2), p.e0281625.), which involves PCR-based amplification and circularization of recombinant plasmids, which were then transformed into the bacteria by electroporation. In short, the complementary overhangs for the HiFi assembly were either synthesized as custom-made eBlocks or generated by PCR.The HiFi DNA assembly reaction was performed according to the manufacturer's protocol. Subsequently, 5 μl of the assembled HiFi product was used as DNA template in the PCR reaction (100 μl final volume). After purification of the PCR product, 1000 to 2000 ng of linear DNA was phosphorylated using the Quick Blunting Kit and according to the manufacturer's protocol. The phosphorylated products were then ligated using the T4 ligase enzyme. Two ligation reactions were performed per cloning, each containing 500 ng of phosphorylated DNA, 2.5 μl of 10X T4 ligase buffer, and 1.5 μl of T4 ligase enzyme (autoclaved Milli-Q water was made up to 25 μl). The ligations were incubated at 25 °C for 3 to 5 hours and then at 70 °C for 30 minutes for enzyme inactivation.After incubation, the ligations were mixed and purified, with three rounds of elution performed to concentrate the DNA (each based on 10 µl of autoclaved Milli-Q water). The entire eluted mixture (approximately 1000 ng) was transformed into electrocompetent L. plantarum WCFS1 cells. For sequence verification, the DNA sequences of interest (100 µl final volume) were amplified using a bacterial pellet as a template. The PCR product was purified and sent to Eurofins Genomics GmbH (Cologne, Germany) for Sanger sequencing by selecting the additional DNA purification step prior to sequencing.INM-431 WO INMPT23048WO 15.11.2024 Site-directed mutagenesis Specific DNA sequences were removed from the plasmid by PCR, using the bacterial pellet as the PCR template and primers covering the entire region except the target sequence.The linear PCR product was circularized as previously described. Preparation of competent E. coli Nissle cells. Wild-type E. coli Nissle bacteria were grown overnight in LB medium at 37 °C and 250 rpm. The next day, the bacteria were subcultured into 100 ml of fresh LB medium and incubated at 37 °C and 250 rpm until the OD600 reached 0.4. The bacteria were pelleted by centrifugation at 4000 rpm for 5 minutes. After discarding the supernatant, the pellet was washed twice with 10 mL of ice-cold CaCl2 (200 mM) and once with 10 mL of a 1:1 combination of CaCl2 (200 mM) and glycerol (10% w / v). After the final wash, the pellet was resuspended in 1 mL of the CaCl2+glycerol mixture and 100 μL aliquots were prepared and stored at -80°C unless used immediately.E. coli DH5α and E. coli Nissle DNA transformationThe E.E. coli DH5α DNA transformation was performed according to the manufacturer's protocol for the NEBuilder® HiFi DNA Assembly Cloning Kit. INM-431 WO INMPT23048WO 15.11.2024 For E. coli Nissle DNA transformation, 200 ng of plasmid DNA was mixed with the competent cells by gentle pipetting and incubated on ice for 30 minutes. After incubation, a 45-second heat shock was performed by placing the cells in a 42°C water bath. The cells were then incubated on ice for another 5 minutes. Afterward, 900 μl of SOC medium was added to the cell mixture, and incubated for 1 hour at 37°C. The mixture was then pelleted by centrifugation at 4000 rpm for 5 minutes. 600 μl were immediately discarded, and the remaining 300 μl were used to resuspend the mixture. Finally, 150 μl were added to an LB agar plate supplemented with 200 μg / ml erythromycin and incubated overnight at 37 °C.Flow cytometric analysis: The manipulated strains were grown in 5 mL of MRS medium supplemented with 10 µg / mL erythromycin at 37 °C with shaking (250 rpm). The next day, the bacteria were cultured to an OD. 600of 0.01 in 5 mL of MRS medium (supplemented with 10 µg / mL erythromycin) and cultured at 37 °C with shaking (250 rpm) for 16 hours. The following day, 1 mL of the bacterial suspensions was harvested by centrifugation at 10,000 rpm. After carefully discarding the supernatant, the pellet was resuspended in 1 mL of sterile Dulbecco's 1X PBS. The mixtures were then serially diluted with a dilution factor of 104, and 5,000 bacterial events were recorded for analysis using the Guava easyCyte BG flow cytometer (Luminex, INM-431 WO INMPT23048WO 15.11.2024 USA). A predefined gate based on forward side scatter and side scatter threshold was used to remove debris and duplicates during event acquisition.mCherry fluorescence intensity was measured by excitation with a green laser at 532 nm (100 mW), and the Orange-G detection channel with a 620 / 52 nm filter was used for signal analysis. The gain settings for forward scatter (FSC) were 11.8, side scatter (SSC) 4, and Orange-G fluorescence 1.68. The compensation control for fluorescence acquisition was set to 0.01, with an acquisition rate of 5 decades. Luminex GuavaSoft 4.0 software for EasyCyte was used for data analysis and presentation. A microplate reader was used for the quantification of reporter gene expression. The cultivation of L. plantarum WCSF1 strains was performed in the same manner as described for flow cytometry analysis. 200 uL of the 1000-uL resuspended mixture (PBS with genetically modified bacteria) was added to a UV STAR Flat Bottom 96-well microtiter plate (Greiner BioOne GmbH, Germany).The samples were then analyzed in the Infinite 200 Pro microplate reader (Tecan Deutschland GmbH, Germany), and both the absorbance (600 nm wavelength) and the mCherry fluorescence intensity (Exλ / Emλ = 587 nm / 625 nm) were measured. The Z position and gain were set to 19000 µm and 100, respectively. The fluorescence values ​​were normalized to the optical density of the bacterial cells to calculate the relative fluorescence units (RFU) (formula RFU = fluorescence / OD). 600). The same procedure was applied for the genetically modified E. coli Nissle strains, but these were grown in LB media supplemented with 200 μg / mL erythromycin at 37°C and with shaking (250 rpm). The experiments were carried out in triplicate on three different days. Measurement of growth rate and calculation of biomass To measure the growth curves of the genetically modified strains, they were cultivated overnight in antibiotic-supplemented MRS medium at 37°C and with shaking (250 rpm). The following day, the bacterial cultures were cultured in 3 mL secondary cultures at an initial OD 600 = 0.01 and incubated at 30°C with shaking (250 rpm) until an OD 600of 0.4-0.5 was reached. Then, 200 µL of the cultures were distributed in a UV STAR Flat Bottom 96-well microtiter plate. The plate was placed in the microplate reader with constant shaking at an incubation temperature of 37°C. The kinetic assay was set so that the absorbance (600 nm) of the bacterial cultures was recorded at 15-minute intervals over 16 hours. The experiment was performed twice on two independent days, with two technical duplicates kept per experiment. To determine the bacterial biomass of the manipulated strains, they were cultured overnight in MRS media supplemented with antibiotics at 37°C and with shaking (250 rpm). The following day, the bacterial cultures were cultured in 5 mL secondary cultures at an initial OD 600= 0.01 and incubated for 16 hours at 37°C with shaking (250 rpm). The following day, all 5 mL cultures were pelleted in several rounds of centrifugation (10,000 rpm), and the biomass of the bacterial pellets was measured using an analytical balance (Denver Instrument). The experiments were performed in triplicate on three different days. Fluorescence microscopic analysis: The culture of the manipulated strains was performed in the same manner as described for the flow cytometry analysis. 10 μL of the resuspended 1000 μL mixture (PBS with manipulated bacteria) was dispensed onto 1.5 mm thick glass slides (PaulMarienfeld GmbH, Germany) and 1.5H glass coverslips (Carl Roth GmbH, Germany) were placed on top. The samples were then observed under the Plan Apochromat 100× oil immersion objective (BZ-PA100, NA 1.45, WD 0.13 mm) of the BZ-X800 fluorescence microscope (Keyence Corporation, Illinois, USA).The mCherry signal was acquired using the BZ-X TRITC filter (model OP-87764) at an excitation wavelength of 545 / 25 nm and an emission wavelength of 605 / 70 nm with a dichroic mirror wavelength of 565 nm. The images were adjusted to identical brightness and contrast settings and analyzed using ImageJ2 software. Statistical and Bioinformatics Analyses INM-431 WO INMPT23048WO 15.11.2024 Statistical analysis was performed using GraphPad Prism 7.0 software. InterPro was used to identify the DBD of rep. AlphaFold was used to predict the 3D structures of the rep repressor. Identification of promoter / repressor systems in the genome of L. plantarum Some of the most successful inducible gene expression systems in bacteria are based on sugars such as lactose, arabinose, xylose, rhamnose, maltose, etc.The reason for this is that sugars are the main carbon and energy source for bacterial growth and metabolism. Therefore, their transport into the cell and utilization are efficiently controlled by genetic switches in the genome. In this regard, L. plantarum WCFS1 possesses a number of versatile metabolic pathways that allow it to utilize a wide variety of sugars such as glucose, fructose, mannose, galactose, arabinose, sucrose, etc. for its growth. Previous studies have developed induction systems based on xylose, galactose, and lactose, using promoters from the L. plantarum genome or from other organisms such as B. subtilis. In these studies, the reported fold changes upon induction are relatively small (<15x), even after mutagenic optimization of the promoter sequences.Genetic switches corresponding to other sugar metabolism pathways, including those of fructose, arabinose, and sucrose, were analyzed to identify potential sugar-inducible promoters. Based on the genome sequence and previous literature on these pathways, the operons encoding enzymes related to the metabolism of these sugars were determined (Figure 6A). These were then analyzed using the bioinformatics tool BProm to extract potential promoter sequences. This analysis reliably identified promoter sequences involved in gene switches related to sucrose and arabinose metabolism. These promoters were then encoded in plasmids upstream of a fluorescent mCherry reporter protein.When grown in MRS medium with and without the inducing sugar, almost no fluorescence signal was observed in the strain encoding the arabinose promoter (P. ara ) (Figure 6B). On the other hand, the strain encoding the sucrose promoter (P suc) exhibited a weak fluorescence signal in the absence of sucrose and an almost threefold higher signal in the presence of sucrose (Figure 6C). Previous reports have shown that in the presence of glucose, global catabolite regulatory proteins can affect promoters associated with the metabolism of other sugars. Since MRS medium contains 2% (w / v) glucose, mCherry expression with these strains was subsequently tested in LB and M17 medium. In this case, a slight increase in fluorescence was observed in the strain encoding Para, with expression increasing almost threefold in the presence of arabinose (Figure 6B). In the strain with Psuc, it was found that the permeable and induced expression levels were not very different in the absence and presence of sucrose (data not shown).However, in M17 medium, it was observed that in the presence of sucrose, a significant increase in mCherry expression occurred, while Leaky expression decreased to almost undetectable levels, resulting in a large 114-fold change (Figure 6C). It was therefore speculated that, in addition to glucose, other sugars and / or metabolites also affected P. suc promoter. To test this, mCherry expression was tested in M17 medium supplemented with the sugars arabinose, fructose, and glucose. In all cases, varying expression levels were observed (Figure 6D), suggesting that the promoter is strongly influenced by the presence of other sugars, possibly by certain global regulators. These results indicate that the P sucpromoter causes very strong induction in the presence of sucrose in M17 medium, but its performance can be affected by the presence of other sugars. Further promoter engineering could be performed to alter regions to which global regulators bind (e.g., in the spacer region between the -10 box and the RBS) to make the promoter specific only for sucrose. Furthermore, constitutive expression of the sucrose-responsive repressor ScrR by encoding it in the plasmid could ensure that its expression is not affected by other sugars. Identification of promoter / repressor systems in Lactobacillus phages. Another source of genetic parts that has been successfully explored in model bacteria such as E. coli is bacteriophages. They can provide promoters and repressors that are appropriate for the host and at the same time orthogonal to the host machinery.Accordingly, a strategy was developed to search for and optimize reliable promoter / repressor systems in L. plantarum WCFS1. The search for potential transcriptional repressors was limited to repressors encoded in the genetic switches that control lytic and lysogenic cycles in bacteriophages. Such genetic switches are naturally adapted to their hosts due to coevolution and the arms race between bacteriophages and bacteria over millions of years.The strategy involved 1) identifying bacteriophages infecting lactobacilli with known and characterized genetic switches, 2) selecting 6 repressors with a known operator sequence, 3) designing all genetic parts required to 4) build a genetic platform, 5) test the repression mediated by each repressor, and finally 6) enhance this repression by introducing specific modifications to the operator / promoter regions. The six different phage-derived repressors tested in this study are: cng and cpg repressors of the ϕg1e phage infecting L. plantarum (Kakikawa, M., Watanabe, N., Funawatashi, T., Oki, M., Yasukawa, H., Taketo, A. and Kodaira, KI, 1998. Promoter / repressor system of Lactobacillus plantarum phage ϕg1e: characterization of the promoters pR49–pR–pL and overproduction of the Cro-like protein Cng in Escherichia coli.INM-431 TO INMPT23048TO 15.11.2024Gene, 215(2), pp.371-379; [ PMC free article ] [ PubMed ] Kakikawa , M. , Ohkubo , S. , Syama , M. , Taketo , A. and Kodaira , KI , 2000 . Gene,242(1-2), pp.155-166.), tec- and rep-Repressor of the mv4 phage induced by Lactobacillus delbrueckii (Coddeville,M., Auvray, F., Mikkonen, M. and Ritzenthaler, P., 2007. Single independent operator sites are involved in the genetic switch of the Lactobacillus delbrueckii bacteriophage mv4 Virology,364(2), pp.256-268.), and cI- and cro-repressors of the A2 phage infected by Lactobacillus casei (Gracia, P., Ladero,V., Alonso, JC and Suárez, JE, 1999. Cooperative interaction of CI protein regulates lysogeny of Lactobacillus casei by bacteriophage A2, 1999. A2 cro, the lysogenic cycle repressor, specifically binds to the genetic switch region of Lactobacillus casei bacteriophage A2. Virology, 262(1), pp. 220-229.). The repressors are listed in Table 1. A simple standard genetic module was designed and built to test the repression mediated by each repressor. The module was based on the previously reported constitutive promoter P. tlpA(Dey, S., Blanch-Asensio, M., Balaji Kuttae, S. and Sankaran, S., 2023. Novel genetic modules encoding high-level antibiotic-free protein expression in probiotic lactobacilli. Microbial Biotechnology.), which is the strongest reported promoter in L. plantarum WCFS1 and allows reliable characterization of repressor activity. Previous studies attempting to identify efficient repressors in lactobacilli were limited to promoters driving moderate expression levels (Heiss, S.; Hörmann, A.; Tauer, C.; Sonnleitner, M.; Egger, E.; Grabherr, R.; Heinl, S., Evaluation of novel inducible promoter / repressor systems for recombinant protein expression in Lactobacillus plantarum. Microbial Cell Factories 2016, 15 (1), 50.). The module for testing repressors included (i) P tlpA, which drives the expression of the reporter gene mCherry, (ii) repressor-specific operators inserted between the -10 box and the ribosome binding site (RBS) (Figure 10), and (iii) repressors constitutively expressed by a moderately strong promoter (P 48The module was constructed through two rounds of cloning to encode both the operator and the repressor in the plasmid. In the first round, the operator was inserted into the promoter, and in the second round, each repressor was cloned into the plasmid containing the corresponding operator (Figure 1A). Suppression of mCherry production was first assessed by quantifying the decrease in fluorescence intensity using flow cytometry (Figure 1C). This analysis initially showed that the insertion of the operator sequences attenuated the potency of PtlpA between 1.3- and 4.3-fold in all cases (Figure 1I), although the fluorescence intensities remained high enough to assess repressor activity. When the repressors were encoded in the plasmids containing the operator sequences, a decrease in fluorescence was observed only for the repressors cng, rep, and cI (Figure 1C).In particular, rep proved to be the most potent of the three repressors, with a fluorescence intensity comparable to that of the INM-431 WO INMPT23048WO 15.11.2024 wild-type strain, which was not modified to produce mCherry (Figure 5). Next, the degree of repression and the effects on cell growth by cng, rep, and cI were quantified. The repressors were assessed as follows: The repressors should i) be able to repress at least up to 70% of mCherry expression and ii) not affect bacterial growth. Regarding repression, only rep showed repression above 70%, with rep-induced repression reaching up to 92% (Figures 2A, 2B). Regarding the effects on cell growth, the growth curves of all three repressors were measured and compared with those of wild-type bacteria (Figure 2C). Only the growth curve of the cI repressor was noticeably lower than that of the others.Nevertheless, differences in OD were evident. 600after 16 hours of growth in the microplate reader was found to be non-significant (Figure 12). Furthermore, bacterial biomass was calculated after overnight growth in an incubator with continuous shaking. In this scenario, bacteria constitutively expressing the cI repressor grew very poorly, whereas rep and cng clones grew like bacteria carrying the PtlpA_mCherry plasmid (Figure 2D). Overall, only the rep repressor fulfilled both requirements. It was confirmed that repression depends on the presence of the operator sequence. This sequence is generally required for the DNA-binding domain (DBD) of the repressors to bind and promote repression. For this purpose, the operator was removed from the plasmid by INM-431 WO INMPT23048WO 15.11.2024 site-directed mutagenesis-PCR using a specific primer set and the bacterial pellet as a template for amplification (Figure 8A). The PCR product was then purified by direct cloning into L.plantarum WCSFS1 (Figure 13). After operator removal, mCherry expression increased dramatically, as both rep could no longer bind to repress and the operator was removed from PtlpA (Figure 8B), which is known to negatively affect expression (Figure 11). Interpro was also used to confirm the presence of a DBD based on a helix-turn-helix domain at the N-terminus of the protein. Furthermore, AlphaFold predicted the 3D structure of rep, which exhibited the typical structure of a repressor, a DBD at the N-terminus, and a dimerization domain at the C-terminus (Figure 8C). AlphaFold also predicted the protein-protein interaction of rep monomers, which is typically required for repression (Figure 8D). Next, attempts were made to increase the degree of repression by making specific changes to the promoter region. The first approach was the P. tlpA-engineering, in which changes were made to the placement of the operator. Three new variants were cloned: i) removal of 14 bp between the operator and the ribosome binding site (RBS) (O1, Seq ID No. 18), ii) placement of the operator within P tlpA , between boxes -35 and -10 (O2, Seq ID No. 19), and iii) adding an additional operator upstream of P tlpA (O3, Seq ID No. 20) (Figure 3A). The removal of part of the spacer between the operator and the RBS and the placement of the operator within P tlpA significantly reduced mCherry expression, even in the absence of the RBS (Figure 3B). On the other hand, adding an additional operator sequence upstream of P tlpAmCherry expression only slightly, but significantly improved repression, from 92.1% (original clone, Figure 2C) to over 99.1% (Figure 3C). The second approach to attempt to improve repression was to use P tlpA by the native promoter (P tec ) associated with rep from phage mv4 (Figure 3D). In this cloning, the (rep)-associated PtlpA was first replaced by Ptec in the plasmid encoding rep, and repression was almost complete (over 99.7%). Surprisingly, mCherry expression was extremely high (Figures 3F and 3E) when the repressor was removed from the plasmid by site-directed mutagenesis (Figure 14). In addition, the effects of these new clones (P tlpA _O3_rep and P tec_rep) on cell growth. Growth was found to be only slightly reduced compared to wild-type bacteria (Figure 7), similar to that observed with rep with a single operator and the cng repressor, and better than that observed with the cI repressor (Figure 2C). These results demonstrate that the suppression can be achieved either by changing the operator placement in P tlpA or by introducing endogenous P tec promoter can be amplified. While the original clone showed a fold change of ~15 (PtlpA_O_rep), the optimized clones showed a fold change of ~117 (PtlpA_O3_rep_mCherry) and ~475 (Ptec_rep) (Figure 3F). INM-431 WO INMPT23048WO 15.11.2024 Due to the surprising expression level of Ptec, it was further characterized and labeled with P tlpAcompared. After overnight growth at 37°C, a change in the color of the MRS medium was observed due to the high mCherry production. This color change was even more pronounced when these bacteria grew in LB media supplemented with glucose to promote bacterial growth in this suboptimal medium. Such a visible color change in the medium due to the expression of a fluorescent protein is regularly observed when these proteins are expressed in E. coli, but is unknown in lactobacilli. Even when these bacteria were spun down, the pellet was bright red, similar to E. coli (Figure 4A). As expected, such a strong constitutive promoter had an effect on cell growth compared to wild-type bacteria. However, this effect was only significant when the bacteria were grown overnight in an incubator and not in a plate reader (Figure 9). Compared to P tlpA caused P tecsignificantly stronger gene expression, which was confirmed by eye (Figure 4B), microscopy (Figure 4C), and flow cytometry (Figure 4D). Fluorescence spectroscopy with a plate reader revealed that Ptec was ~9 times stronger than PtlpA (Figure 4E), confirming that it is the strongest constitutive promoter discovered for heterologous gene expression in L. plantarum WCFS1. Since the visible color changes of the liquid culture and bacterial pellets were comparable to the expression in E. coli, the P tec-driven mCherry expression in L. plantarum WCFS1 was compared with expression in probiotic E. coli Nissle 1917, which is driven by the strong PtlpA promoter. After quantifying mCherry expression levels with the microtiter plate reader, it was found that the fold changes between PtlpA in Nissle and WCFS1 were above 17, while the fold changes between P tlpA in Nissle and P tecin WCFS1 was less than 2 (Figure 4E). Bacteriophages have proven to be a rich and diverse source of genetic material to expand the toolbox of bacterial synthetic biology (Lemire S, Yehl KM, Lu TK. Phage-based applications in synthetic biology. Annual review of virology. 2018 Sep 29;5:453-76.), including transcription systems (Wang W, Li Y, Wang Y, Shi C, Li C, Li Q, Linhardt RJ. Bacteriophage T7 transcription system: an enabling tool in synthetic biology. Biotechnology advances. 2018 Dec 1;36(8):2129-37.), integrases (Fogg PC, Colloms S, Rosser S, Stark M, Smith MC. New applications for phage integrases. Journal of molecular biology. 2014 Jul 29;426(15):2703-16.), anti-CRISPR proteins (Marino ND, Pinilla-Redondo R, Csörgő B, Bondy-Denomy J. Anti-CRISPR protein applications: natural brakes for CRISPR-Cas technologies. Nature methods. 2020 May;17(5):471-9.), endolysins (Abdelrahman F, Easwaran M, Daramola OI, Ragab S, Lynch S, Oduselu TJ, Khan FM, Ayobami A, Adnan F, Torrents E, Sanmukh S. Phage-encoded endolysins. Antibiotics. 2021 Jan28;10(2):124.) and of course repressors (Hochschild A, LewisM. The bacteriophage λ CI protein finds an asymmetric solution. Current opinion in structural biology. 2009 Feb 1;19(1):79- 86.). These components have been naturally adapted to their bacterial hosts over millions of years due to coevolution and the arms race between bacteriophages and bacteria (Hampton HG, Watson BN, Fineran PC. The arms race between bacteria and their phage foes. Nature. 2020 Jan 16;577(7790):327-36.). However, bacteriophage components in probiotic lactobacilli have remained largely unexplored. In this study, a genetic platform for testing transcriptional repressors was established. The use of the strong constitutive P. tlpAproved to be a suitable strategy, as testing the natural phage promoter / repressor systems could be risky, as the promoter must be functional in the test host. Of the six different repressors encoded in genetic switches in lactobacillus prophages, only the rep repressor showed promising results in terms of efficient and reliable reporter gene repression. These results demonstrate that lactobacillus prophages can be a promising, albeit challenging, source of genetic components to expand the genetic toolbox. Furthermore, the endogenous promoter associated with the repressor showed unprecedented gene expression, significantly narrowing the gap between model and non-model bacteria developed for therapeutic applications.This efficient promoter / repressor system could now be used in combination with other genetic components to build a more complex genetic circuit. For example, using repressor engineering strategies, it would be possible to transform rep into a switchable repressor that responds to sugar (Dimas RP, Jiang XL, Alberto de la Paz J, Morcos F, Chan CT. Engineering repressors with coevolutionary cues facilitates toggle switches with a master reset. Nucleic acids INM-431 WO INMPT23048WO 15.11.2024 research. 2019 Jun 4;47(10):5449-63.) or physical stimuli such as light (Li, X., Zhang, C., Xu, X., Miao, J., Yao, J., Liu, R., Zhao, Y., Chen, X. and Yang, Y., 2020. A single-component light sensor system allows highly tunable and direct activation of gene expression in bacterial cells. Nucleic acids research,48(6). pp.e33-e33;) or heat (Xiong LL, Garrett MA, Buss MT,Kornfield JA, Shapiro MG. Tunable temperature-sensitive transcriptional activation based on lambda repressor.ACSSynthetic Biology. 2022 Jun 16;11(7):2518-22.) Such switchable repressors would enable inducible gene expression, which would be desirable to circumvent the considerable metabolic burden and stress experienced by P. tecdue to its transcriptional strength in the cells. Furthermore, the unmodified repressor can be combined with other inducible gene expression systems to reverse the induction system as a NOT logic gate (Ohlendorf R, Vidavski RR, Eldar A, Moffat K, Möglich A. From dusk till dawn: one-plasmid systems for light-regulated gene expression. Journal of molecular biology. 2012 Mar 2;416(4):534-42. Taton A, Ma AT, Ota M, Golden SS, Golden JW. NOT gate genetic circuits to control gene expression in cyanobacteria. ACS synthetic biology. 2017 Dec 15;6(12):2175-82.). In combination with the Ptec promoter, such induction or inversion functions can be achieved at a remarkably high level of performance. A novel, strong promoter / repressor system was identified that functions very efficiently in the probiotic bacterium L. plantarum WCFS1.After improving the system, repression levels of >99% and fold changes of >100 were achieved. Furthermore, a super-strong constitutive promoter, Ptec, was identified, driving unprecedented expression levels in this strain, ~9-fold higher than the previously reported strongest promoter, P. tlpA These novel genetic components will significantly contribute to expanding the possibilities for the technical regulation of gene expression in L. plantarum.

[0002] INM-431 WO INMPT23048WO 11 / 15 / 2024Table 1 INM-431 WO INMPT23048WO 11 / 15 / 2024 INM-431 WO INMPT23048WO 11 / 15 / 2024

[0003] INM-431 WO INMPT23048WO 15.11.2024 Table 2 Sequences used. For sequences 18, 19, 20, and 21, promoter sequences are shown in italics, the Rep operator is shown in bold, the -35 and -10 boxes are shown in underlined, the RBS sequence (aggaga) is shown in bold italics, and the mCherry start codon ATG at the end is shown in capital letters. INM-431 WO INMPT23048WO 11 / 15 / 2024 INM-431 WO INMPT23048WO 11 / 15 / 2024 INM-431 WO INMPT23048WO 11 / 15 / 2024 INM-431 WO INMPT23048WO 11 / 15 / 2024

[0004] INM-431 WO INMPT23048WO 15.11.2024 Table 3: Primers used INM-431 WO INMPT23048WO 11 / 15 / 2024 49 Sequencingcgttactaaaggggaatggag GOI fw 50 Sequencingagtggaacgaaaactcac GOI rev

[0005] INM-431 WO INMPT23048WO 15.11.2024Zitierte Literatur Blanch-Asensio, M., Dey, S. and Sankaran, S., 2023. In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1. PLOS one, 18(2), p.e0281625. Kakikawa, M., Watanabe, N., Funawatashi, T., Oki, M., Yasukawa, H., Taketo, A. and Kodaira, K.I., 1998. Promoter / repressor system of Lactobacillus plantarum phage øg1e: characterization of the promoters pR49–pR–pL and overproduction of the Cro-like protein Cng in Escherichia coli. Gene, 215(2), pp.371-379. Kakikawa, M., Ohkubo, S., Syama, M., Taketo, A. and Kodaira, K.I., 2000. The genetic switch for the regulatory pathway of Lactobacillus plantarum phage φg1e: characterization of the promoter PL, the repressor gene cpg, and the cpg-encoded protein Cpg in Escherichia coli. Gene, 242(1-2), pp.155-166. Coddeville, M., Auvray, F., Mikkonen, M. and Ritzenthaler, P., 2007.Single independent operator sites are involved in the genetic switch of the Lactobacillus delbrueckii bacteriophage mv4. Virology, 364(2), pp.256-268. Gracia, P., Ladero, V., Alonso, J.C. and Suárez, J.E., 1999. Cooperative interaction of CI protein regulates lysogeny of Lactobacillus casei by bacteriophage A2. Journal of virology, 73(5), pp.3920-3929. Ladero, V., García, P., Alonso, J.C. and Suárez, J.E., 1999. A2 cro, the lysogenic cycle repressor, specifically binds to theINM-431 WO INMPT23048WO 15.11.2024genetic switch region of Lactobacillus casei bacteriophage A2. Virology, 262(1), pp.220-229. Dey, S., Blanch-Asensio, M., Balaji Kuttae, S. and Sankaran, S., 2023. Novel genetic modules encoding high-level antibiotic-free protein expression in probiotic lactobacilli. Microbial Biotechnology. Heiss, S.; Hörmann, A.; Tauer, C.; Sonnleitner, M.; Egger, E.; Grabherr, R.; Heinl, S., Evaluation of novel inducible promoter / repressor systems for recombinant protein expression in Lactobacillus plantarum. Microbial Cell Factories 2016, 15 (1), 50. Li, X., Zhang, C., Xu, X., Miao, J., Yao, J., Liu, R., Zhao, Y., Chen, X. and Yang, Y., 2020. A single-component light sensor system allows highly tunable and direct activation of gene expression in bacterial cells. Nucleic acids research, 48(6), pp.e33-e33. Dimas, R.P., Jiang, X.L., Alberto de la Paz, J., Morcos, F. and Chan, C.T.Y., 2019. Engineering repressors with coevolutionary cues facilitates toggle switches with a master reset. Nucleic acids research, 47(10), pp.5449-5463. Ohlendorf, R., Vidavski, R.R., Eldar, A., Moffat, K. and Möglich, A., 2012. From dusk till dawn: one-plasmid systems for light-regulated gene expression. Journal of molecular biology, 416(4), pp.534-542.INM-431 WO INMPT23048WO 15.11.2024Taton, A., Ma, A.T., Ota, M., Golden, M.O. and Golden, J.W., 2017.NOT gate genetic circuits to control gene expression in cyanobacteria. ACS Synthetic Biology, 6 (12), 2175-2182. Lemire S, Yehl KM, Lu TK. Phage-based applications in synthetic biology. Annual review of virology. 2018 Sep 29;5:453-76. Wang W, Li Y, Wang Y, Shi C, Li C, Li Q, Linhardt RJ. Bacteriophage T7 transcription system: an enabling tool in synthetic biology. Biotechnology advances. 2018 Dec 1;36(8):2129-37. Fogg PC, Colloms S, Rosser S, Stark M, Smith MC. New applications for phage integrases. Journal of molecular biology. 2014 Jul 29;426(15):2703-16. Marino ND, Pinilla-Redondo R, Csörgő B, Bondy-Denomy J. Anti- CRISPR protein applications: natural brakes for CRISPR-Cas technologies. Nature methods. 2020 May;17(5):471-9. Abdelrahman F, Easwaran M, Daramola OI, Ragab S, Lynch S, Oduselu TJ, Khan FM, Ayobami A, Adnan F, Torrents E, Sanmukh S. Phage-encoded endolysins. Antibiotics. 2021 Jan 28;10(2):124. Hochschild A, Lewis M.The bacteriophage λ CI protein finds an asymmetric solution. Current opinion in structural biology. 2009 Feb 1;19(1):79-86. Hampton HG, Watson BN, Fineran PC. The arms race between bacteria and their phage foes. Nature. 2020 Jan 16;577(7790):327-36.INM-431 WO INMPT23048WO 15.11.2024Xiong LL, Garrett MA, Buss MT, Kornfield JA, Shapiro MG. Tunable temperature-sensitive transcriptional activation based on lambda repressor. ACS Synthetic Biology. 2022 Jun 16;11(7):2518-22.

Claims

INM-431 WO INMPT23048WO 15.11.2024 Patent claims 1. A system for controlling the expression of proteins in Lactobacillus, comprising a promoter operatively linked to a nucleic acid encoding at least one protein, and additionally a heterologous repressor operatively linked to the promoter.

2. The system according to claim 1, wherein the repressor is a genetically encoded repressor. 3 . System nach einem der Ansprüche 1 oder 2, wobei der Repressor derived from bacteriophages, which Lactobacillus infizieren.

4. The system according to any one of claims 1 to 3, wherein the repressor comprises a protein having an amino acid sequence substantially identical to Sequence ID No.

1.

5. The system according to any one of claims 1 to 4, wherein the gene sequence of the repressor is operatively linked to another promoter.

6. The system according to any one of claims 1 to 5, wherein the repressor can change its conformation or structure upon exposure to a stimulus such that it no longer suppresses expression of the protein.

7. The system according to any one of claims 1 to 6, wherein the repressor is formed from at least two units. INM-431 WO INMPT23048WO 15.11.2024 8. Expression cassette for expression in Lactobacillus, welche das System nach einem der Ansprüche 1 bis 7 ermöglicht.

9. Expression cassette according to claim 8, wherein the Expressionskassette mindestens eine Promotor, eine mit dem Promoter-linked nucleotide sequence and at least the Nukleotidsequenz des Repressors umfasst.

10. Expressionskassette nach einem der Ansprüche 8 oder 9, wherein the expression cassette is operatively linked to at least one toxin / antitoxin system. 1 1. Vektor umfassend die Expressionskassette nach einem der Ansprüche 8 bis 10.

12. Lactobacillus umfassend das System nach einem der Claims 1 to 7.

13. Lactobacillus umfassend mindestens einen Vektor nach Claim 11.

14. Use of the repressor according to any one of claims 1 to 7 in Lactobacillus.

15. A promoter for use in Lactobacillus having a nucleotide sequence substantially identical to Sequence ID No.

22.

16. An expression cassette comprising the promoter of claim . INM-431 WO INMPT23048WO 15.11.2024 17. Lactobacillus umfassend den Promotor nach Anspruch 15.

18. Verwendung des Promotors nach Anspruch 15 in Lactobacillus.

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