Nutritional requirements of Staphylococcus bacteria

Abstract

This invention provides recombinant Staphylococcus bacteria (e.g., Staphylococcus epidermidis) whose growth is dependent on D-alanine, and a method for producing recombinant Staphylococcus bacteria. [Solution] In one embodiment, this specification features recombinant Staphylococcus bacteria comprising two inactivated alanine racemase genes (Δalr1Δalr2); and an inactivated D-alanine aminotransferase (dat) gene.

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 614,096, filed on January 5, 2018, the entire content of which is hereby incorporated by reference in its entirety for all purposes.

Background Art

[0002] Background of the Invention Many bacteria utilize two amino acids, D-alanine and D-glutamic acid, in the biosynthesis of the peptidoglycan layer, which is necessary for the construction of a functional cell wall in such bacteria. Gram-positive bacteria, including species of the genus Staphylococcus, utilize D-alanine and D-glutamic acid in the synthesis of the peptidoglycan layer in the cell wall.

[0003] The genetic code assigns codons to the 20 amino acids that make up proteins, 19 of which are chiral and are the L-isomers. These are considered "natural" or "standard" amino acids. Amino acids with the opposite chirality, i.e., the D-isomers, are considered non-natural and generally do not exist in the environment. When an organism such as a bacterium requires D-amino acids, one or more enzymes for producing such non-natural amino acids must be present in the bacterium or must be intentionally provided to the bacterium, or else it cannot survive.

[0004] Alanine racemase is an enzyme that catalyzes the conversion of L-alanine to D-alanine and is an important building block in the biosynthesis of the peptidoglycan layer of the bacterial cell wall. Alanine racemase is not normally present in eukaryotes but is commonly present in prokaryotes.

[0005] D-alanine is essential for bacterial cell wall formation and therefore essential for bacterial survival; therefore, bacteria possess enzymes that can catalyze D-alanine production. Because D-alanine is extremely important for bacterial survival, bacteria may have an excess of multiple enzymes for D-alanine biosynthesis. For example, bacteria may have multiple alanine racemase genes. In species with two genes, one may be constitutively expressed and anabolic, while the other is inductive and catabolic (Strych, U. et al. 2007. BMC Microbiol. 7:40; Strych U. et al., Curr. Microbiol. 41:290-294; Strych U. et al., FEMS Microbiol. Lett. 196:93-98). These genes supply D-alanine, which is necessary for cell wall biosynthesis, and knockout studies on some of these bacteria have shown that in the absence of exogenous D-alanine, the alanine racemase enzyme is essential for growth (Franklin, FC, and WA Venables. 1976. Mol. Gen. Genet. 149:229-237; Hols, P., et al. J. Bacteriol. 179:3804-3807; Palumbo, E., et al. FEMS Microbiol. Lett. 233:131-138; Steen, A., et al. J. Bacteriol. 187:114-124; Wijsman, HJ 1972. Genet. Res. 20:269-277).

[0006] Removing the ability of a microbial organism to produce the amino acids necessary for its growth results in a microorganism known as a trophication-dependent strain. If the survival and growth of such a microorganism are required, the amino acids necessary for growth must be supplied from an external source. The creation of nutrient-dependent microorganisms is well known, particularly for Escherichia coli (E. coli) (published on the World Wide Web at cgsc2.biology.yale.edu / Auxotrophs.php; Methods Enzymol. 2015;565:45-66. doi: 10.1016 / bs.mie.2015.05.012. Epub 2015 Jun 10. “Escherichia coli auxotroph host strains for amino acid-selective isotope labeling of recombinant proteins.” Lin MT, Fukazawa R, Miyajima-Nakano Y, Matsushita S, Choi SK, Iwasaki T, Gennis RB; Nicola Casali, Methods in Molecular Biology, Vol 235. www.springer.com / gp / book / 9781588291516, the contents of each are incorporated herein by reference in their entirety).

[0007] D-alanine-requiring strains of Staphylococcus aureus were created for the purpose of producing a vaccine against methicillin-resistant strains of Staphylococcus aureus (Moscoso M, et al. 27th ECCMID 22-25 April 2017, The Congress of ESCMID (P0473); Moscoso et al., Virulence (2018) Vol. 9(1): 604-620, the contents of each are incorporated herein by reference in their entirety). In this case, it was found that not only two alanine racemases, alr1 and alr2, but also a third enzyme needed to be knocked out.

[0008] When introducing bacteria into a target environment, it is desirable to be able to control the introduced bacteria after their introduction into the target environment. For example, it is desirable to be able to control the growth of the introduced bacteria against the growth of bacterial populations already present in the target environment.

[0009] Such control can be imposed by using antibiotics that are selectively toxic to the introduced bacteria but not toxic to the bacterial populations already present in the target environment. However, finding antibiotics with such selectivity is often impossible. Furthermore, the use of antibiotics is often undesirable because they can disrupt the target environment in undesirable ways, such as by inducing antibiotic resistance in bacteria constituting the existing bacterial population, or by disrupting the target environment, leading to undesirable conditions such as dysbiosis or diarrhea.

[0010] Therefore, it is beneficial to use methods that selectively control the growth of bacteria introduced into a target environment without relying on antibiotics. Such desirable control can be achieved by introducing nutrient requirements into the bacteria introduced into the target environment. This is particularly advantageous for the purpose of introducing bacteria into a target environment to increase or alter the microbiome (internal microflora) of that environment, and is especially advantageous when the target environment is the human microbiome.

[0011] Staphylococcus epidermidis, a Gram-positive bacterium, is a well-known bacterium that makes up the human microbiome (Zhang et al, Molecular Microbiology (2003) 49(6), 1577-1593, “Genome-based analysis of virulence genes in a non-biofilm-forming Staphylococcus epidermidis strain (ATCC 12228)”, incorporated herein by reference). Staphylococcus epidermidis is a facultative anaerobic bacterium and is part of the normal human microbiota. Staphylococcus epidermidis is usually non-pathogenic, but immunocompromised patients are at risk of developing infections. These infections are generally hospital-acquired (Levinson, W. (2010). Review of Medical Microbiology and Immunology (11th ed.). pp. 94-99, incorporated herein by reference). For individuals with catheters or other surgical implants, Staphylococcus epidermidis is a particular concern because it is known to form biofilms that proliferate on the surfaces of these devices.

[0012] Therefore, this specification addresses the nutritional requirements of Staphylococcus bacteria (e.g., Staphylococcus epidermidis) that depend on externally supplied nutrients such as D-alanine or D-glutamic acid for survival and growth. [Overview of the project]

[0013] Summary of the Invention This specification generally relates to recombinant Staphylococcus species (e.g., Staphylococcus epidermidis) whose growth depends on D-alanine. A key finding of this specification is the ability to selectively control the growth of bacteria (e.g., recombinant Staphylococcus species (e.g., Staphylococcus epidermidis)) in a target environment without the use of antibiotics. According to some embodiments of this specification, the nutrient requirement characteristic is useful for maintaining the presence of plasmids without requiring the presence of antibiotic resistance genes. Therefore, in some embodiments, recombinant Staphylococcus species do not contain antibiotic resistance genes. In some embodiments, polynucleotides enabling the expression of enzymes or other components that restore the ability to metabolize foreign nutrients are incorporated into the plasmid to be maintained in the microorganism. In some embodiments, recombinant Staphylococcus species are transformed with a pUBTR114-based vector. In further embodiments, the pUBTR114-based vector is pUBTR119*-Sal-GFP.

[0014] In some embodiments, this specification features recombinant Staphylococcus bacteria comprising two inactivated alanine racemase genes (Δalr1Δalr2); and an inactivated D-alanine aminotransferase (dat) gene. In some embodiments, the Staphylococcus bacteria depend on D-alanine for growth. In other embodiments, the Staphylococcus bacteria are Staphylococcus epidermidis and its subspecies. In some embodiments, the Staphylococcus bacteria further comprises one or more additional mutations. In some embodiments, the additional mutations comprise an inactivated glutamate racemase gene MurI. In some embodiments, the Staphylococcus bacteria further comprises a polynucleotide encoding a therapeutically active protein (e.g., a soluble therapeutic protein). In some embodiments, the therapeutically active protein exhibits enzymatic activity or biological activity. In some embodiments, the protein is a growth factor. In some embodiments, the protein is a hormone.

[0015] In another embodiment, this specification provides a method for producing recombinant Staphylococcus bacteria, comprising: (i) transforming competent cells of a Staphylococcus strain by introducing a plasmid containing D-alanine aminotransferase (dat) knockout (SEΔalr1Δalr2); (ii) detecting the presence of the knockout plasmid in the transformed cells; (iii) culturing the transformed cells confirmed in step (ii); and (iv) purifying the isolated colonies. In some embodiments, the method further comprises testing the D-alanine requirement of the isolated colonies. In some embodiments, the presence of the knockout plasmid in the transformants is detected using polymerase chain reaction (PCR). In some embodiments, the recombinant Staphylococcus bacteria are Staphylococcus epidermidis and its subspecies. In some embodiments, the method further comprises transforming the recombinant Staphylococcus bacteria using a pUBTR114-based vector. In some embodiments, the pUBTR114-based vector is pUBTR119*-Sal-GFP. In some embodiments, recombinant Staphylococcus bacteria are produced by the method described above.

[0016] In another embodiment, this specification features a kit comprising a recombinant Staphylococcus bacterium of any one of the embodiments or models described herein. In some embodiments, the kit further comprises a pUBTR114-based vector. [Brief explanation of the drawing]

[0017] [Figure 1]Figure 1 shows the observation of D-alanine requirement in a Staphylococcus epidermidis strain (SEΔalr1Δalr2Δdat) with triple gene knockout. After transformation with the SE1423 knockout plasmid, plasmid integration, and removal of the plasmid backbone, cells were seeded to obtain colonies. 25 colonies were inoculated onto two different plates, and the plates were cultured overnight at 30°C. Left: TSA plate; Right: TSA + anhydrotetracycline (2 μg / mL) + D-alanine (40 μg / mL). Three clones (#7, #12, and #18, highlighted with red circles) were only able to grow on TSA supplemented with D-alanine. [Figure 2A] Figures 2A and 2B show the results of PCR testing of the triple knockout strain (SEΔalr1Δalr2Δdat). Cells obtained from patches on plates of TSA + anhydrotetracycline (2 μg / mL) + D-alanine (40 μg / mL) were used as templates for the PCR reaction: clone #7; KO clone #12; KO clone #18; wild-type SE; SE1423 KO plasmid DNA (vector, as control). In Figure 2A, PCR was performed using primers 1423-5F and 1423-3R to distinguish between the wild-type SE1423 locus (2.3 Kb PCR product) and the SE1423 knockout (1.5 Kb PCR product). In Figure 2B, PCR was performed using primers 1423-F and 1423-R to detect a 0.7 Kb PCR product specific to the wild-type SE1423 locus. As predicted, no PCR products were generated from the SE1423 knockout plasmid or the putative SE1423 knockout SE clone. The results confirmed the success of SE1423 deletion in clones #7, #12, and #18. [Figure 2B]Figures 2A and 2B show the results of PCR testing of the triple knockout strain (SEΔalr1Δalr2Δdat). Cells obtained from patches on plates of TSA + anhydrotetracycline (2 μg / mL) + D-alanine (40 μg / mL) were used as templates for the PCR reaction: clone #7; KO clone #12; KO clone #18; wild-type SE; SE1423 KO plasmid DNA (vector, as control). In Figure 2A, PCR was performed using primers 1423-5F and 1423-3R to distinguish between the wild-type SE1423 locus (2.3 Kb PCR product) and the SE1423 knockout (1.5 Kb PCR product). In Figure 2B, PCR was performed using primers 1423-F and 1423-R to detect a 0.7 Kb PCR product specific to the wild-type SE1423 locus. As predicted, no PCR products were generated from the SE1423 knockout plasmid or the putative SE1423 knockout SE clone. The results confirmed the success of SE1423 deletion in clones #7, #12, and #18. [Figure 3] Figure 3 shows the results of polymerase chain reaction (PCR) of clones of Staphylococcus epidermidis NRRL B-4268 transformed with pUBTR119*-Sal-GFP. Eleven clone cells (numbered 1 through 11) were used as templates for PCR reactions using primers Sar-sGFP-F and Sar-sGFP-R to detect 1.1-Kb PCR products. Cells of SE NRRL B-4268 and pUBTR119*-Sal-sGFP plasmid DNA isolated from SCK6 served as negative (-) and positive (+) controls. All transformed clones were identified. [Figure 4]Figure 4 shows the PCR results of Staphylococcus epidermidis triple gene knockout strain (SEΔalr1Δalr2Δdat) clones transformed with pUBTR119*-Sal-GFP by antibiotic selection or D-alanine requirement complementation. Cells were used as templates in PCR reactions using primers Sar-sGFP-F and Sar-sGFP-R to detect 1.1-Kb PCR products. Cells from SE NRRL B-4268 and pUBTR119*-Sal-sGFP plasmid DNA isolated from SCK6 served as negative (-) and positive (+) controls. Clones 1-3 were obtained by antibiotic selection, and clones 4-26 were obtained by D-alanine requirement complementation. All clones were verified. [Figure 5] Figure 5 shows a Western blot for detecting His-tagged fusion proteins. Lanes 1-6: Staphylococcus epidermidis culture broth samples shown in Table 2; Lanes 7-10: Samples containing His-tagged fusion TP protein (~52 kDa) loaded at dilutions of 1 / 20, 1 / 10, 1 / 5, and 1 / 1, respectively. No signal was detected for His-tagged fusion GFP protein (29 kDa). [Modes for carrying out the invention]

[0018] Detailed description of the invention 1. Definition Unless otherwise indicated, all scientific and technical terms used herein have meanings that will be commonly understood by those skilled in the art to which this invention pertains.

[0019] The following references provide many general definitions of terms used in the present invention to those skilled in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). The following terms used in this specification have the meanings as defined below unless otherwise specified.

[0020] As used herein, the articles "a" and "an" are used to refer to one or more (i.e., at least one) of the grammatical objects of the article. By way of example, "an element" means one or more elements.

[0021] The term "including" is used herein in the sense of the phrase "including but not limited", and is used interchangeably.

[0022] The term "or" is used herein in the sense of the phrase "and / or", and is used interchangeably unless the context clearly indicates otherwise.

[0023] The term "such as" is used herein in the sense of the phrase "such as but not limited to", and is used interchangeably.

[0024] As used herein, the terms "auxotrophic" or "auxotrophy" refer to an organism's inability to synthesize certain compounds necessary for its growth. A auxotrophic organism is an organism that exhibits this characteristic.

[0025] As used herein, the terms “alrA” and “alr” refer to the D-alanine racemase gene, including the normal allele of the alrA gene. In some embodiments, the alr gene derived from Staphylococcus epidermidis (UniProtKB - Q8CNK7 (ALR_STAES)) encodes the D-alanine racemase protein (EC 5.1.1.1). In some embodiments, the locus names SE1674 (alr1) and SE1079 (alr2) refer to individual Staphylococcus epidermidis D-alanine racemase genes.

[0026] As used herein, the term "dat" refers to the D-alanine aminotransferase gene and includes normal alleles of the dat gene. In some embodiments, the dat gene derived from Staphylococcus epidermidis (UniProtKB - Q8CS41 (DAAA_STAES)) encodes the D-alanine aminotransferase protein (EC:2.6.1.21). In some embodiments, the locus name SE1423 (dat) refers to a specific Staphylococcus epidermidis D-alanine aminotransferase gene. As used herein, the term "murI" refers to the glutamate racemase gene and includes normal alleles of the murI gene. In some embodiments, the murI gene derived from Staphylococcus epidermidis (UniProtKB - Q8CPL0 (MURI_STAES)) encodes the glutamate racemase protein (EC:5.1.1.3). In some embodiments, the locus name SE0843 (murI) refers to a specific Staphylococcus epidermidis glutamate racemase gene.

[0027] As used herein, the term “genetic factor” means a polynucleotide containing a region encoding a polypeptide, or a polynucleotide region that controls replication, transcription or translation, or other processes important to polypeptide expression in a host cell, or a polynucleotide containing both a polypeptide-encoding region and a functionally linked region that controls expression. Genetic factors may be contained as episomal elements; that is, within vectors that replicate as molecules physically unrelated to the host cell genome. Genetic factors may also be contained within plasmids. Genetic factors may also be contained within the host cell genome, not in their natural state, but particularly in the form of purified DNA or within vectors, after operations such as isolation, cloning, and introduction into host cells.

[0028] As used herein, the term “host cell” means a cell that has been transformed or transfected by an exogenous polynucleotide sequence, or a cell that can be transformed or transfected.

[0029] In this invention, the term "isolated" refers to biomaterials (cells, nucleic acids, or proteins) that have been removed from their original environment (the environment in which they naturally occur). For example, polynucleotides that exist naturally in plants or animals are not isolated, but the same polynucleotides separated from nearby naturally occurring nucleic acids are considered "isolated."

[0030] An "isolated nucleic acid molecule" (e.g., an isolated promoter) is a molecule that has been separated from other nucleic acid molecules present in the natural origin of that nucleic acid molecule. For example, with respect to genomic DNA, the term "isolated" includes nucleic acid molecules that have been separated from the chromosome to which that genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid molecule does not have a sequence that is naturally adjacent to it in the genomic DNA of the organism from which the nucleic acid molecule originates.

[0031] As used herein, the term "knockout" refers to the inactivation of the effective expression of a gene product (e.g., an enzyme) by complete or partial removal of a gene, partial or complete removal of a non-coding regulatory region necessary for the effective expression of the gene product, insertion of a creotide into a gene-coding polynucleotide, or any other method that prevents the effective expression of the gene product.

[0032] As used herein, the terms “polypeptide” or “protein” refer to biomolecules or macromolecules consisting of chain-like linked amino acid residues. As used herein, the definition of polypeptide includes proteins (generally higher molecular weight) consisting of one or more long chains of amino acid residues, and small peptides (generally lower molecular weight) consisting of fewer amino acids. In other embodiments, a single amino acid, while not strictly a polypeptide, is still considered to fall within the scope of the present invention.

[0033] As used herein, “promoter” refers to the DNA sequence that indicates the transcription of a structural gene. Typically, the promoter is located in the 5' region of the gene, adjacent to the transcription start site of the structural gene. If the promoter is an inducible promoter, the transcription rate increases in response to an inducer. For example, a promoter can be controlled in a tissue-specific manner so that it is active only when transcribing a relevant coding sequence in a particular tissue type.

[0034] As used herein, the term “polynucleotide” generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA. Therefore, for example, as used herein, polynucleotides refer specifically to DNA that is a mixture of single-stranded and double-stranded DNA, single-stranded and double-stranded regions, or single-stranded, double-stranded and triple-stranded regions, single-stranded and double-stranded RNA, and a hybrid molecule containing RNA, DNA, and RNA, which may be single-stranded, more typically double-stranded, or triple-stranded, or a mixture of single-stranded and double-stranded regions. In addition, as used herein, polynucleotides refer to a triple-stranded region containing RNA or DNA, or both RNA and DNA. The nucleotide chains of such a region may be derived from the same molecule, or from multiple different molecules. The region may contain all of one or more molecules, but more typically, it may contain only the regions of some of those molecules. One type of molecule having a triple-helix region is often an oligonucleotide. As used herein, the term polynucleotide includes the DNA or RNA described above, which contains one or more modified bases. Therefore, DNA or RNA having a modified backbone for stability or other reasons is a “polynucleotide” as defined herein. Furthermore, DNA or RNA containing unusual bases such as inosine, or modified bases such as tritylated bases, are polynucleotides as defined herein, to name just two examples. Naturally, many practical modifications known to those skilled in the art have been made to DNA or RNA. As used herein, the term polynucleotide includes polynucleotides in chemically, enzymatically, or metabolically modified forms, as well as chemical forms of DNA and RNA characteristic of viruses and cells, particularly simple and complex cells. The term polynucleotide also includes low-molecular-weight polynucleotides, often referred to as oligonucleotides.The terms "polynucleotide" and "nucleic acid" are often used interchangeably.

[0035] As used herein, the term “therapeutic protein” refers to a protein, peptide, glycoprotein, or glycopeptide administered to a subject to treat a disease or functional impairment of the subject or to improve the subject’s health. In some embodiments, the subject is a human. In some embodiments, the therapeutic protein is a human protein. Using the methods described herein, the therapeutic protein is produced in a Staphylococcus bacterium, such as Staphylococcus epidermidis, that has been genetically modified to have a double knockout of the alanine racemase gene (e.g., alfr1 and alfr2) and a knockout of the alanine aminotransferase gene (dat, SE1423).

[0036] II. Composition This specification describes triple knockout Staphylococcus bacteria that are D-alanine-requiring strains. This specification provides engineered Staphylococcus bacteria, such as Staphylococcus epidermidis, that have been genetically modified to have double knockout of alanine racemase genes (e.g., alr1 and alr2) and knockout of alanine aminotransferase gene (dat, SE1423). This specification provides a triple knockout Staphylococcus epidermidis strain (SEΔalr1Δalr2Δdat) that exhibits desirable D-alanine requirement.

[0037] D-alanine is an essential component for bacteria with a peptidoglycan layer structure. The fundamental importance (essentiality) of D-alanine stems from its crucial role in the cross-linking of peptidoglycan strands, specifically the dipeptide D-alanyl-D-alanine. As described herein, a double knockout strain of Staphylococcus epidermidis (SEΔalr1Δalr2) for the alanine racemase gene has already been developed. However, this double knockout strain did not exhibit D-alanine requirements, in contrast to Bacillus subtilis, Escherichia coli, and several other bacterial species. It was hypothesized that the presence of glutamate racemase (interconversion between L-glutamate and D-glutamate) and D-alanine aminotransferase (interconversion between D-alanine and D-glutamate) in Staphylococcus epidermidis could provide a bypass for alanine racemase. Therefore, this specification provides a knockout of the alanine aminotransferase gene (dat, SE1423) in a double knockout strain (SEΔalr1Δalr2) that exhibits D-alanine requirement.

[0038] This specification provides bacterial host cells or homologs thereof that have been genetically engineered to have a deletion in the dat gene, thereby reducing D-alanine aminotransferase activity and making the cells D-alanine-requiring. In some embodiments, bacterial cells are genetically engineered to have a deletion in another gene or operon, which affects the dat operon, reducing D-alanine aminotransferase activity and thereby making the cells D-alanine-requiring.

[0039] In some embodiments, the D-alanine-requiring bacteria described herein, such as genetically modified Staphylococcus bacteria, such as triple knockout Staphylococcus epidermidis strains (SEΔalr1Δalr2Δdat), further include requirements for other amino acids, vitamins, and / or nucleotides. For example, in some embodiments, the D-alanine-requiring bacteria described herein may further include nutritional requirements for one or more of the following amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, the D-alanine-requiring bacteria described herein may further include nutritional requirements for vitamins, such as vitamin A, vitamin B (e.g., B-1 to B-12), vitamin C, vitamin D, vitamin E, and vitamin K. In some embodiments, the D-alanine-requiring bacteria described herein may further include nutritional requirements for nucleotides.

[0040] bacterial strain This specification provides genetically modified microorganisms, such as bacteria. The methods described herein are thought to be achievable in any Staphylococcus bacterial cell by inactivating or knocking out the gene encoding the protein homolog of the cell's dat, or by inactivating the expression or activity of this protein. For a strain to belong to the Staphylococcus genus, it is necessary that it is a Gram-positive cocci that forms clusters, produces catalase, has a suitable cell wall structure (including the presence of peptidoglycan and teichoic acid), and has a DNA G+C content of 30-40 mol%.For example, the Staphylococcus aureus (S. aureus) group, such as S. argenteus, Staphylococcus aureus (S. aureus), S. schweitzeri, S. simiae, etc.; the S. auricularis group, such as S. auricularis, etc.; the S. carnosus group, such as S. carnosus, S. condimenti, S. massiliensis, S. piscifermentans, S. simulans, etc.; the Staphylococcus epidermidis (S. epidermidis) group, such as S. capitis, S. caprae, Staphylococcus epidermidis (S. epidermidis), S. saccharolyticus, etc.; the hemolytic staphylococcus (S. haemolyticus) group, such as S. devriesei, Staphylococcus haemolyticus (S. haemolyticus), S. hominis, etc.; the S. hyicus-intermedius group, such as S. agnetis, S. chromogenes, S. felis, S. delphini, S. hyicus, S. intermedius, S. lutrae, S. microti, S. muscae, S. pseudintermedius, S. rostri, S. schleiferi, etc.; S. lugdunensis group, such as S. lugdunensis; S. saprophyticus group, such as S. arlettae, S. cohnii, S. equorum, S. gallinarum, S. kloosii, S. leei, S. nepalensis, S. saprophyticus, S. succinus, S. xylosus, etc.; S. sciuri group, such as S. fleurettii, S. lentus, S. sciuri, S. stepanovicii, S. vitulinus, etc.; simulans group, such as S. simulans; S. The Staphylococcus group includes, but is not limited to, S. warneri, for example, S. pasteuri, S. warneri, etc. In one embodiment, the genus Staphylococcus is Staphylococcus epidermidis.

[0041] Genetic constructs This specification utilizes standard molecular biology techniques, e.g., the techniques described in (Sambrook et al. 2001). pJB38 (Boss et al., 2013) was used as the plasmid backbone for the knockout vector, which is based on the allele-exchange E. coli-Staphylococcus shuttle vector pJB38 and further incorporates additional design features on the plasmid to improve functionality (Bose, JL, et al. Applied and environmental microbiology. 2013;79(7):2218-2224). Specific primers for producing SE1423 knockout were designed (shown as Table 1 in Example 1 below).

[0042] In some embodiments, plasmids were constructed using standard molecular biology techniques, employing Top10 E. coli as the cloning host, by cloning overlapping PCR products to the EcoRI-SalI site of pJB38. Clones were then selected and screened by PCR using primers 1423-5F and 1423-3R (Table 1) for detecting PCR products. Clones of suitable SE1423 knockout plasmids (pJB-1423KO) were introduced into dam- / dcm- E. coli Gm2163 strains for transformation. Plasmid DNA was isolated from two Gm2163 transformant clones using the Qiagen Midi Prep Kit and checked by restriction enzyme digestion with EcoRI and SalI as described above.

[0043] Recombinant Staphylococcus bacteria used In some embodiments, the Staphylococcus bacteria described herein (e.g., Staphylococcus epidermidis, genetically modified to have a double knockout of the alanine racemase gene (e.g., alfr1 and alfr2) and a knockout of the alanine aminotransferase gene (dat, SE1423)) further comprises a polynucleotide encoding a protein having therapeutic properties. In some embodiments, the protein is a soluble therapeutic protein. A soluble therapeutic protein is a therapeutic protein that is soluble in aqueous solution. In some embodiments, all, most, or part of the expressed therapeutic protein may be soluble in the Staphylococcus bacteria described herein. In some embodiments, the soluble therapeutic protein is an active protein having, for example, enzymatic activity or biological activity, such as ligand or receptor binding activity, the ability to activate intracellular signaling pathways, or the ability to induce an immune response in mammals such as humans. In some embodiments, the therapeutic protein is glycosylated, or otherwise modified in vitro by one or more glycosyltransferases, or modified to enhance resistance to proteases.

[0044] In some embodiments, the Staphylococcus bacteria of the present invention may be used as is to treat a disease, or may be modified to express a therapeutic polypeptide. For example, the Staphylococcus bacteria of the present invention can be used to treat a skin disease or abnormality. In another embodiment, the Staphylococcus bacteria of the present invention can be modified to express a therapeutic polypeptide or a fragment thereof to treat a skin disease or abnormality.

[0045] formulation The formulation for use according to the present invention is capable of producing a therapeutically effective amount of desired polypeptides, and contains, for example, a pharmaceutically effective amount of any amount of recombinant Staphylococcus bacteria, by weight ratio of genetically modified microorganisms, e.g., bacteria, at least about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.5%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about It is possible to include 6.0%, approximately 7.0%, approximately 8.0%, approximately 9.0%, approximately 10.0%, approximately 11.0%, approximately 12.0%, approximately 13.0%, approximately 14.0%, approximately 15.0%, approximately 16.0%, approximately 17.0%, approximately 18.0%, approximately 19.0%, approximately 20.0%, approximately 25.0%, approximately 30.0%, approximately 35.0%, approximately 40.0%, approximately 45.0%, approximately 50.0%, or more, and it will be even clearer that the upper limit is approximately 90.0% by weight of genetically modified microorganisms, such as bacteria.

[0046] In other embodiments, formulations for use according to the present invention may contain, for example, at least about 0.01% to about 30%, about 0.01% to about 20%, about 0.01% to about 5%, about 0.1% to about 30%, about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.2% to about 5%, about 0.3% to about 5%, about 0.4% to about 5%, about 0.5% to about 5%, about 1% to about 5%, or more by weight of recombinant Staphylococcus bacteria.

[0047] III. Method This specification relates to a method for producing recombinant Staphylococcus bacteria, comprising: (i) transforming competent cells of a Staphylococcus strain (SEΔalr1Δalr2) by introducing a plasmid containing D-alanine aminotransferase (dat) knockout; (ii) detecting the presence of the knockout plasmid in the transformed cells; (iii) culturing the transformed cells identified in step (ii); and (iv) purifying the isolated colonies. In a preferred embodiment, the presence of the knockout plasmid in the transformants is detected by polymerase chain reaction (PCR). In a particular embodiment, the method further comprises testing the D-alanine requirement of the isolated colonies.

[0048] IV. Kit The present invention also provides kits. In one embodiment, a kit of the present invention includes (a) recombinant Staphylococcus bacteria of the present invention and (b) instructions for use thereof. The compositions of the present invention are described above. In some embodiments, the compositions of the present invention include recombinant Staphylococcus bacteria whose growth depends on D-alanine.

[0049] All publications and patent applications referenced herein are incorporated herein by reference in their entirety for any purpose, as if each individual publication or patent application were specifically directed to be incorporated by reference for any purpose. The publications referenced herein are provided solely for disclosure prior to the filing date of this application. Nothing herein should be construed as the inventors referenced herein having no prior right to such disclosure, whether for prior disclosure or for any other reason.

[0050] The present invention is further illustrated by the following embodiments, but this should not be construed as a further limitation. All drawings and all references, patents and published patent applications cited herein, as well as the drawings, are expressly incorporated herein by direct reference. [Examples]

[0051] Examples The following examples further illustrate and demonstrate embodiments that fall within the scope of the present invention. These examples are given for illustrative purposes only and should not be construed as limiting the invention, as many modifications are possible without departing from the spirit and scope of the invention.

[0052] This specification provides the construction of a Staphylococcus epidermidis (S. epidermidis) expression system that, in some embodiments, can maintain the expression plasmid without the use of antibiotics. This experiment documents an extensive effort to develop a D-alanine-requiring Staphylococcus epidermidis strain. Initially, a double knockout strain of the alanine racemase gene (SEΔalr1Δalr2) was constructed, but it did not exhibit D-alanine requirement. As reported in Staphylococcus aureus (S. aureus) and Listeria monocytogenes, the presence of glutamate racemase (interconversion between L-glutamate and D-glutamate) and D-alanine aminotransferase (interconversion between D-alanine and D-glutamate) in Staphylococcus epidermidis was considered to potentially provide a bypass for alanine racemase. Therefore, in order to develop a triple knockout Staphylococcus epidermidis strain exhibiting D-alanine requirement (SEΔalr1Δalr2Δdat), the present invention discloses a double knockout of the alanine racemase gene (SEΔalr1Δalr2) in the first strain, in addition to the knockout of the alanine aminotransferase gene (dat, SE1423).

[0053] Example 1: Vector for deletion of SE1423 (D-alanine aminotransferase) The method used to induce SE1423 knockout (KO) is briefly described below. First, an SE1423 KO plasmid was constructed using pJB38 (Boss et al., 2013).

[0054] Primer Based on the genome sequence of Staphylococcus epidermidis strain 12228, oligonucleotide primers were designed for PCR to develop an SE1423 knockout (KO) vector. The primer sequences, their specific applications, and the size of the PCR products are shown in Table 1 below.

[0055] [Table 1]

[0056] PCR products of 0.5 Kb and 1.0 Kb from the 5' and 3' facile regions were prepared. These were then used as templates for overlap PCR to produce a larger PCR product (1.5 Kb) containing both the 5' and 3' facile regions. Using Top10 E. coli as the cloning host, the overlap PCR product was cloned into the EcoRI-SalI site of pJB38. Clones were selected and screened by PCR using primers 1423-5F and 1423-3R, and a 1.5 Kb PCR product was detected. Plasmid DNA was also isolated and digested with EcoRI and SalI, and both the vector backbone (7.0 Kb) and insert (1.5 Kb) fragments were detected. A clone of a suitable SE1423 knockout plasmid (pJB-1423KO) was introduced into dam- / dcm-E. coli Gm2163 strain for transformation. Plasmid DNA was isolated from two Gm2163 transformant clones using the Qiagen Midi Prep Kit and checked by restriction enzyme digestion with EcoRI and SalI as described above.

[0057] Example 2: Preparation of a triple knockout strain (SEΔalr1Δalr2Δdat) Using plates with TAS + chloramphenicol (10 μg / mL), the pJB-1423 KO plasmid isolated from Gm2163 was introduced into competent cells of Staphylococcus epidermidis strain (SEΔalr1Δalr2) for transformation. The presence of the pJB-1423 KO plasmid in the transformants was confirmed by detecting a 1.5 Kb PCR product using primers 1423-5F (EcoRI) and 1423-3R (SalI). While a 1.5 Kb PCR product was observed in all 26 clones tested, a 2.3 Kb PCR product was observed in the reaction containing cell lysates derived from SE host cells. Cells from the two confirmed clones were streaked onto new plates with TSA + Cm (10 μg / mL) + D-alanine (40 μg / mL). The plates were incubated at 43°C for 24 hours for plasmid integration by homologous recombination. The isolated colonies were streaked again at 43°C for purification. To obtain a circular plasmid backbone through a second homologous recombination, four isolated colonies were inoculated into 50 mL of TSB + D-alanine (40 μg / mL) in a 250-mL baffled shaking flask. The culture was shaken at 30°C for 24 hours. A 0.5 mL portion of the culture was transferred to a flask containing 50 mL of fresh medium. Subculturing was repeated three times. The cells from the flask were seeded onto TSA + anhydrotetracycline (ATC 2 μg / mL) + D-alanine (DA, 40 μg / mL). After culturing at 30°C for 2 days, approximately 100-200 colonies were obtained. -5 Colonies were formed on plates seeded with 100 μl of diluted culture. Further analysis of the colonies is described below.

[0058] Example 3: Verification of D-alanine requirement in triple knockout strain (SEΔalr1Δalr2Δdat) A total of 25 isolated colonies obtained from TSA+ATC+DA plates were inoculated onto TAS plates and TSA+ATC+DA plates. The plates were incubated overnight at 30°C. All clones grew well on the D-alanine-supplemented plate (TSA+ATC+DA). As shown in Figure 1, three clones (#7, #12, and #18) could not grow on TSA without D-alanine supplementation, exhibiting D-alanine requirement. This trophic requirement phenotype was again observed when cells obtained from a patch on the TSA+ATC+DA plate were re-inoculated onto the TSA plate. It should be noted that, as a result of the second homologous recombination, the plasmid backbone may be removed without knocking out SE1423, suggesting that some clones obtained from the TSA+ATC+DA plate retain the wild-type SE1423 locus.

[0059] Further analysis was performed on clones that require D-alanine. When the above 1423KO SE clones were inoculated onto TSA+Cm (10 μg / mL), they did not proliferate, indicating the removal of the plasmid backbone containing the chloramphenicol selection marker during the second homologous recombination. Loss of antibiotic resistance markers was also confirmed by PCR using primers JB-Cm-F and JB-Cm-R (Table 1) (data not shown). PCR using primers 1423-5F and 1423-3R detected a 1.5 Kb PCR product in the above KO clones, while the PCR product from the SE host was 2.3 Kb, as expected (Figure 2A). Wild-type SE cells produced a 0.7 Kb PCR product using primers 1423-F and 1423-R (both specific to the SE1423 coding sequence); this PCR product was not detected from the KO plasmid DNA or the putative KO clones (Figure 2B).

[0060] Therefore, based on all experimental data, we can conclude that we successfully deleted SE1423 (dat, D-alanine aminotransferase) in a double knockout strain of the alanine racemase gene, and created a triple knockout Staphylococcus epidermidis strain (SEΔalr1Δalr2Δdat). Furthermore, the desired D-alanine requirement was observed in the triple knockout strain.

[0061] D-alanine is required for the synthesis of bacterial cell peptidoglycans. In Bacillus subtilis and Escherichia coli, deletion of the alanine racemase gene was sufficient to induce D-alanine requirements. However, in Staphylococcus epidermidis, two alanine racemase genes (alr1, alar2) and the D-alanine aminotransferase gene dat(SE1423) must be knocked out to express this phenotype. Indeed, combining glutamate racemase and D-alanine aminotransferase has been reported to provide a promising bypass for alanine racemase in Staphylococcus aureus (S. aureus) MRSA132 (Moscoso et al., 2017 and 2018) and Listeria monocytogenes (Thompson et al., 1998). The Staphylococcus epidermidis genome contains a third putative alanine racemase homolog (SE1769), but under the experimental conditions used in this study, it is not necessary to knock out this gene due to D-alanine requirement.

[0062] Having successfully developed a D-alanine-requiring Staphylococcus epidermidis strain, the next step is to transform this strain using an expression vector containing the alanine racemase gene as a selection marker. The transformants will be selected by complementation with a host D-alanine-requiring plasmid.

[0063] Example 4: Development of a Staphylococcus epidermidis expression vector with a non-antibiotic selection marker. This example describes the development of a Staphylococcus epidermidis expression system that can maintain expressed plasmids without the use of antibiotics.

[0064] Transformation of the cloning host, Bacillus subtilis SCK6, by pJB38 For protein production in Staphylococcus epidermidis, we use the E. coli / Staphylococcus aureus shuttle vector pJB38 (Bose et al., 2013). One possible approach to developing a non-ABR (antibiotic-resistant) protein expression system is modification of pJB38. To investigate this option, we tested whether pJB38 could be introduced into the cloning host Bacillus subtilis SCK6 and transformed. Using the reagents and protocol of the Qiagen HiSpeed ​​Plasmid Midi Kit, pJB38 DNA isolated from E. coli DH5α strain was introduced into SCK6ΔalrA using the BTR protocol for competent cell preparation and transformation (see below). Transformed cells were seeded on LB agar medium + D-alanine (DA, 40 μg / mL) + chloramphenicol (Cm, 10 μg / mL). After 2 days of incubation at 30°C, small colonies began to appear. Colonies were counted after 3 days of incubation at 30°C. When 250 μL of competent cells were transformed with 0.6 μg of pJB38 DNA (in 5 μL), 61 colonies of varying sizes were observed on a plate seeded with 50 μL of cells. Based on this, the transformation efficiency was 5.2 x 10⁻⁶. 2 This results in cfu / μg DNA.

[0065] To confirm the authenticity of the transformants, 40 colonies were picked and inoculated onto new plates of LB agar + DA + Cm, and incubated overnight at 30°C. All colonies grew well. Six clones were each inoculated into 3 mL of LB + DA + Cm liquid medium. Miniprep plasmid DNA was prepared using the cells. The plasmids were digested with EcoRI + HindIII and SalI + SnaBI. The DNA bands predicted to be obtained from EcoRI + HindIII digestion were 5 Kb and 2 Kb. The bands from SalI + SnaBI digestion were 4.7 Kb and 2.3 Kb. The predicted digestion patterns were observed for three large colony clones (#2, #3, and #4) and two small-sized colony clones (#6 and #7). Small-sized colony clone #5 showed a larger band on the agarose gel, but the intensity of the small band was very weak. From these data, it could be concluded that pJB38 successfully transformed Bacillus subtilis SCK6.

[0066] To introduce pJB38 into Staphylococcus epidermidis and transform it, it is necessary to isolate the plasmid from a dam- / dcm-E. coli host and minimize the effects of host limitations and modifications on transformation efficiency. This effort focused primarily on using a pUBTR114-based vector.

[0067] Transformation of Staphylococcus epidermidis by vector pUBTR14-TP Transformed competent cells of Staphylococcus epidermidis strain NRRL B-4268 were prepared and transformed. pUBTR114-TP (pUBTR114 possessing the test protein gene) was isolated from Bacillus subtilis SCK6 using the Qiagen Midi Prep Kit (see Appendix II). Transformed Staphylococcus epidermidis cells were seeded onto tryptone soy agar (TSA) plates containing 10 μg / mL kanamycin. The plates were incubated overnight at 37°C. Five colonies were observed from transformation using approximately 950 ng of plasmid DNA. All five colonies were inoculated onto new kanamycin plates and regrow at 37°C. Cells were picked using a toothpick and suspended in 100 μL of Tris buffer (100 mM, pH 8.0). Lysates (0.5 μL) were isolated and used as templates in a 25-μL PCR reaction using Taq polymerase and the primer pair spamyQ-Nde-F2 / Sbf-TP-R (Table 2). Cell lysates from untransformed Staphylococcus epidermidis and plasmid DNA isolated from SCK6 were used as negative and positive controls, respectively. Cell lysates obtained from all five clones produced 1.5 Kb PCR products as predicted. Thus, these experiments demonstrated that a pUBTR114-based vector can be introduced into Staphylococcus epidermidis, transform it, and maintain it through kanamycin selection.

[0068] [Table 2]

[0069] Building pUBTR119-GFP Detectable GFP expression and secretion have been demonstrated in Staphylococcus epidermidis transformed with pJB38-sGFP. Therefore, for evaluation, we decided to clone the expression cassette "SarAP1-SsaA-His-sGFP" into pUBTR119-TP. This plasmid is similar to pUBTR114-TP. The difference between the two plasmids is that in pUBTR119-TP, there is a second promoter sequence upstream of the TP coding sequence, as well as a more user-friendly cloning site.

[0070] The forward primer Sar-GFP-F and reverse primer Sar-GFP-R (Table 2) were designed to amplify the 1.1-Kb fragment of the sGFP expression cassette from the plasmid pJB38-sGFP by PCR. The forward and reverse primers contained restriction enzyme sites PaeR7I and SbfI, respectively. Standard PCR conditions were used with PfuUltra DNA polymerase obtained from Agilent. The PCR products were run on agarose gel, excised, and purified using the Qiagen QIAquick gel Extraction Kit. The fragments were then digested with PaeR71-SbfI and purified again on the gel. pUBTR119-TP was isolated from Bacillus subtilis SCK6 using the Qiagen Midi Prep Kit and digested with restriction enzymes PaeR7I and SbfI to remove the 1.5-Kb test protein (TP) coding sequence. The remaining 4.1-Kb vector backbone was purified by gel. Using NEB's Quick Ligation Kit, an sGFP expression cassette was ligated to the pUBT119 skeleton at the PaeR71-SbfI site and introduced into Bacillus subtilis SCK6ΔalrA competent cells for transformation. The transformation mixture was seeded on LB plates and on LB + 10 μg / mL kanamycin + 40 μg / mL D-alanine plates and cultured overnight at 37°C. Approximately 100 colonies were observed on both plates, suggesting effective selection by kanamycin resistance or D-alanine requirement complementation. 100 colonies obtained from the LB plates and 150 colonies from the LB + kanamycin (Kan) + DA plates were inoculated onto LB and LB + Kan + DA plates, respectively. All showed good growth. Fifteen pools of 10 colonies each, obtained from LB+Kan+DA plates (clones #1-150), and a pool of 10 colonies from LB plates (#151-250), were screened by PCR using primers Sar-GFP-F / Sar-GFP-R to confirm the presence of the 1.1-Kb insert. Plasmid DNA of pJB38-sGFP and SCK6ΔalrA cells served as positive and negative controls, respectively.All pools were PCR-positive. Individual clones from one pool (pool #5) were screened by PCR as described above, and all were positive. These clones obtained from LB+Kan+DA were grown overnight in liquid LB+Kan+DA at 37°C. The cells were used for plasmid minipreps. All 10 clones contained plasmids when checked on an agarose gel. Plasmid DNA obtained from clones #3 and #4 was analyzed by three sets of restriction enzyme digestion: PaeR71+SbfI, EcoRV, and KpnI. Both clones showed the digestion patterns predicted. Midiprep DNA was prepared from the two clones. Sequencing confirmed the success of the cloning, and no mutations were shown. The construct was introduced into SE NRRL B-4268 and transformed using kanamycin selection as described above. After a long culture of 3 days, 9 colonies were observed. Two clones were examined by PCR. However, PCR using primers Sar-GFP-F / Sar-GFP-R failed to detect the sGFP expression cassette.

[0071] It is unclear why pUBTR114-TP can transform Staphylococcus epidermidis and be confirmed by PCR, while putative transformants derived from pUBTR119-GFP cannot be confirmed. pUBTR114-TP colonies were observed after overnight culture at 37°C, while pUBTR119-GFP colonies were only observed after 2-3 days of culture at 37°C. One difference between the two plasmids is the presence of an XhoI restriction enzyme site in pUBTR119-GFP. Bacillus subtilis has an XhoI methylation system (Jentsch, 1983). The restriction enzyme XhoI cannot digest the XhoI site of pUBTR119-TP, and therefore its isoschisomer, PaeR71, was used for cloning the GFP expression cassette. It was suspected that the restriction / modification system of Staphylococcus epidermidis targets pUBTR119-GFP in some way due to the methylated XhoI site. Therefore, the vector was modified by replacing the XhoI site with a different restriction enzyme site, SalI.

[0072] Development of a novel GFP expression vector: pUBTR119*-SAL-GFP The Xho site of pUBTR119-GFP was replaced with a different restriction enzyme (SalI) using overlap PCR. This plasmid contains a MluI-XhoI fragment (840 bp) and an XhoI-KpnI fragment (251 bp). Novel primers were designed for PCR amplification of the two fragments and for overlap PCR, including a nucleotide change from 5'-CTCGAG-3' to 5'-GTCGAC-3'. The overlap PCR product (1.1 Kb) was digested with MluI and KpnI and ligated with pUBTR119-GFP pre-digested with MluI-KpnI. The ligated reaction was introduced into SCK6 competent cells by kanamycin selection as described above and transformed. Numerous colonies were formed. The colonies were inoculated onto new plates of LB+Kan (10 μg / mL). Twelve clones were analyzed by PCR using primers Mlu-F2 and Sal-R2 (Table 2). Primer Sal-R2 is specific to the SalI site. The expected 0.84 Kb band of the PCR product was weak, but clearly present in the reactant for eight clones. All twelve clones were grown in liquid medium (LB+Kan) for plasmid DNA miniprep. All these clones showed plasmid bands of the correct size, and they were linearized by SalI digestion. Clone #4 was grown for miniprep. DNA was analyzed by restriction enzyme digestion: MluI + KpnI; and SalI. As predicted, two bands (4.5 Kb and 1.1 Kb) were observed from MluI + KpnI digestion, and SalI linearized the plasmid. The novel plasmid was named pUBTR119*-Sal-GFP.

[0073] Transformation of Staphylococcus epidermidis strains by pUBTR119*-Sal-GFP Wild-type SE NRRL B-4268 competent cells were transformed with pUBTR119*-Sal-GFP plasmid DNA and seeded on TSA+Kan (10 μg / mL) plates. After overnight incubation at 37°C, 2 and 9 colonies were observed from transformations using approximately 440 ng and 880 ng of plasmid DNA, respectively. All 11 clones were inoculated onto new TSA+Kan plates, and the cells were examined by PCR using primers Sar-GFP-F and Sar-GFP-R. Staphylococcus epidermidis cells and pUBTR119*-Sal-GFP plasmid DNA were used as negative and positive controls, respectively. A 1.1 Kb PCR product was generated in all reactions except the negative control (Figure 3).

[0074] D-alanine-requiring triple knockout strain (SEΔalr1Δalr2Δdat) was grown, and transformed competent cells were prepared using the same protocol as NRRL B-4268, except that D-alanine (40 μg / mL) was added to TSB medium. pUBTR119*-Sal-GFP contains both the kanamycin resistance gene and the alanine racemase gene as selection markers. Based on kanamycin selection on TSA+Kan (μg / mL) and D-alanine requirement complementation on TSA, the plasmid was introduced into the triple gene knockout mutant strain for transformation. Plates were cultured at 37°C. Colonies were observed after overnight culture: 3 colonies from transformation with 880 ng of plasmid DNA on the kanamycin-selective plate, and 25 colonies from the same amount of plasmid DNA on the TSA plate. Transformation of Staphylococcus epidermidis using D-alanine requirement complementation is considered to function more efficiently than transformation using kanamycin selection. After inoculation onto a new plate, all 28 colonies were capable of growth. This was also confirmed by PCR using primers specific to the GFP gene (Figure 4).

[0075] Cell culture for GFP expression To evaluate protein expression in SE triple knockout strains transformed with the pUBTR119*Sal-GFP construct, a shaking flask experiment was planned. The strains and media used in this experiment are shown in Table 3. The shaking flask culture protocols for growth and protein expression in Bacillus subtilis and Staphylococcus epidermidis are shown below. The strains were inoculated into 5 mL of the medium from which glucose had been removed from the medium described, and grown overnight at 37°C and 225 rpm. Using the overnight culture (0.5 mL), the strains were inoculated into 50 mL of the medium described with 2% glucose added in a 250-mL baffled flask. The cultures were grown at 37°C and 225 rpm for 24 hours. All strains showed good growth. Culture broth was collected by centrifugation of 1.5 mL of culture for 3 minutes at 13,000 rpm (17,900 xg) in an Eppendorf Centrifuge 5417C. Figure 5 shows a Western blot for detecting His-tagged fusion proteins.

[0076] [Table 3]

[0077] Next, SDS-PAGE and Western blotting protocols (below) were performed to detect GFP with a C-terminal His tag. The secreted His-tagged fused GFP protein contains 252 amino acid residues and has a molecular weight of 29 kDa. 4-12% protein gels were run and stained, but no protein bands of the predicted size were observed (data not shown). Samples were run on 16% protein gels and transferred to membranes for detection using anti-His antibodies. It has been previously shown that Bacillus subtilis SCK6 transformed with pUBTR114-TP expresses and secretes a test protein (52 kDa) with an N-terminal His tag. Culture broth samples stored at -20°C were loaded at various dilutions as positive controls: 1 / 1 (as other samples), 1 / 5, 1 / 10, and 1 / 20 dilutions. The only visible bands observed were from the positive control. The signal was weak at the 1 / 20 dilution control. No GFP protein signal was detected on the blot. Therefore, GFP was not effectively expressed in this culture. The medium TSB + 2% glucose, which has been experimentally tested as suitable for protein expression in Bacillus subtilis, may not be optimal for protein expression regulated by the SarAP1 promoter and / or protein secretion driven by the signal peptide SsaA.

[0078] A pUBTR114-based vector was introduced into Staphylococcus epidermidis and successfully transformed. A GFP expression cassette was cloned to construct pUBTR119*-Sal-GFP. The vector contains both the kanamycin resistance gene and the alanine racemase gene as selection markers. The kanamycin gene can be readily removed when desired. pUBTR119*-Sal-GFP was introduced into SE NRRL B-4268 using kanamycin selection and successfully transformed. It was also introduced into a triple-gene knockout D-alanine-requiring mutant via kanamycin selection and D-alanine-requiring complementation and transformed. All clones were confirmed by PCR using GFP-specific primers.

[0079] This experiment discloses the development of a non-antibiotic expression system for protein production in Staphylococcus epidermidis. First, a D-alanine-requiring Staphylococcus epidermidis strain was developed by successively knocking out two alanine racemase genes (alr1 and alr2) and the D-alanine aminotransferase gene (dat). Next, the ability of a BTR Gram-positive bacterial expression vector to transform Staphylococcus epidermidis and to complement the host's D-alanine requirement was evaluated. The expression vector was found to contain replication origin and selection markers that function in both Bacillus subtilis and Staphylococcus epidermidis. The highly transformable Bacillus subtilis SCK6ΔalrA served as a cloning host to facilitate vector construction. Once the vector was constructed and confirmed in the Bacillus genus, it was introduced into the D-alanine-requiring Staphylococcus epidermidis strain for protein expression (SEΔalr1Δalr2Δdat) for transformation.

[0080] The above experiments were conducted using, but are not limited to, the following methods.

[0081] Plasmid preparation from Bacillus subtilis For miniprep, I used the Qiagen QIAprep Spin Miniprep Kit (catalog # 27106); for midiprep, I used the Qiagen HiSpeed ​​Plasmid Midi Kit (catalog # 12643). The key point is the addition of lysozyme to the P1 buffer.

[0082] Mini Prep: 1. Isolated colonies were inoculated into 5 mL LB of culture medium with the necessary antibiotics added, and grown overnight at 37°C and 225 rpm. 2. Three milliliters of each overnight culture were transferred to a 1.5-mL Eppendorf tube and centrifuged for 1 minute at 13,000 rpm in an Eppendorf Centrifuge 5417C. The supernatant was discarded. 3. The pellet was resuspended in 250 μL of P1 Buffer. Lysozyme was added to a final concentration of 200 μg / mL; 5 μL of freshly prepared 10 mg / mL lysozyme aqueous solution was added. The sample was vortexed and incubated at 37°C for 30 minutes. 4. Follow the remaining protocol as instructed in the manufacturer's handbook.

[0083] Preparation and transformation of competent cells of Bacillus subtilis strain SCK6 SCK6 Competent Cell Preparation 1. Streak SCK6 onto an LB plate for isolation from a vial stored in glycerol at -80°C. Incubate overnight at 37°C. 2. Inoculate the isolated colonies into 5 mL LB containers in 18 x 150 mm glass tubes. Shake overnight at 225 rpm at 37°C. 3. Prepare a 1:100 dilution of the overnight culture, and OD 600 Measure. 4. In 15 mL LB + 1% xylose in a 125 mL baffled flask, the cultures were subjected to initial OD (Oral Dissociation). 600 Dilute until the ratio is 1.0. Shake at 225 rpm at 37°C for 2 hours. 5. Completely freeze the culture in 10% glycerol at -80°C: Add 3.6 mL of 50% glycerol to the flask and completely freeze the 450 μL portion in a 1.5 mL Eppendorf tube at -80°C.

[0084] SCK6 has an erythromycin resistance marker on its chromosome. If necessary, 1.0 μg / mL of erythromycin can be added at any step. For SCK6ΔalrA, 40 μg / mL of D-alanine is added to the culture medium.

[0085] Transition protocol 1. Thaw competent cells at room temperature and use 200 μL for each transformation. 2. The DNA for transformation (plasmid or ligation reaction solution) was directly added to a 1.5-mL Eppendorf tube containing 200 μL of competent cells. 3. The Eppendorf tube was placed in an 18x150 mm glass tube and left at 225 rpm for 90 minutes at 37°C. 4. The samples were seeded onto 1-4 LB plates containing the necessary antibiotics. The plates were incubated overnight at 37°C.

[0086] Growth and preparation of Bacillus subtilis and Staphylococcus epidermidis for protein expression. • Growth medium: For Staphylococcus epidermidis and Bacillus subtilis SCK6 wild-type hosts, TSB + 20 g / L glucose; and for transformants of pUBTR114 or pUBTR119 constructs, TSB + 20 g / L glucose + 10 μg / mL kanamycin. Using a -80°C glycerol scrape, inoculate 5 mL of growth medium for each of the above strains into an 18 x 150 mm glass tube. Grow overnight at 37°C at 225 rpm. • Using 0.5 mL of overnight culture, inoculate each strain into a 250 mL baffled flask containing 50 mL of the same growth medium described above. Grow at 37°C at 225 rpm for 24 hours. Samples are taken from flasks by dispensing 2 x 1.5 mL from each 24-hour culture. Centrifuge in an Eppendorf Centrifuge 5417C at 13,000 rpm (17,900 xg) for 3 minutes. Transfer the supernatant to a new Eppendorf tube for use in SDS-PAGE analysis and anti-His Western blotting. The pellet is also reserved. All samples are stored at -20°C. • Measure the A600 of the culture for the remaining 24 hours.

[0087] SDS-PAGE and Western blot transcription protocols for His-tagged proteins. Components / reagents used: Expedeon Corporation. 20X Teo-Tricine-SDS Electrophoresis Buffer #B50500 RunBlue SDS Gel 4-12%, 12 wells, 10cm x 10cm #NXG41212 • 10X DTT Reducing Agent #A32001 • 4X LDS sample buffer #B31010 Invitrogen Novex Mini-Cell XCELL SureLock Electrophoresis Cell • Transfer buffer: 20X Tris-Glycine Blotting Buffer # B86500 • BioRad Plus Protein Western Standard # 161-0376 ·Genscript One-Hour Western Kit # L00204T • Genscript His-tagged antibody pAB, rabbit #A00174; Store 10µl at -20°C

[0088] Sample preparation: • Sample mixture: ○X μL sample ○ 5 μL 4X LDS Sample Buffer ○ 2 μL 10X DTT reducing agent ○Y μL Deionized water ○Total volume = 20 μL • Preparation steps: ○ Mix the samples by stirring them with a vortex mixer. ○ Boil for 3 minutes. ○Centrifuge briefly and then cool to room temperature. ○ Vortex stir again.

[0089] Gel setup and electrophoresis: Add 40 mL of 20X electrophoresis buffer to 760 mL of Milli Q H2O. Remove the gel from the pouch and rinse it with deionized water. Next, place the gel in the electrophoresis unit with the shorter side of the plate facing inward. Once the gel is in place, add the electrophoresis buffer to the inner container (approximately 200 mL). Check for leaks before proceeding. Add the remaining electrophoresis buffer to the outer container. Rinse the wells with electrophoresis buffer. Load an appropriate amount of prepared sample from above, along with 5 μL of BioRad Western Standard. • Perform electrophoresis on the gel at room temperature and 150 volts for approximately 1 hour (allow sufficient time for the leading edge of the dye to reach the bottom edge of the gel).

[0090] Transfer setup: Prepare 1,000 mL of transfer buffer: 50 mL 20X Tris-Glycine blotting buffer + 200 mL methanol + 770 mL MQH2O. Cool the buffer to keep the transfer cool. • Soak the sponge in the transfer buffer. Before constructing the sandwich, equilibrate the gel in transfer buffer for 7 minutes, and equilibrate the nitrocellulose (NC) membrane / blotting membrane in transfer buffer for 10 minutes. • Assemble the sandwich: Place a pre-soaked Whatman paper on top of a Parafilm piece. Place a pre-soaked gel on top of the Whatman paper. Place a pre-soaked NC membrane on top of the gel. Using a glass Pasteur pipette, gently roll it over the membrane to remove air bubbles. Place a pre-soaked Whatman paper on top of the NC membrane. Gently roll it over again to remove air bubbles. Lift the sandwich and place it on the two sponges (which have been squeezed to remove all the transfer buffer) inside the blot module. If electrophoresing only one gel, pack the remaining blot module with the squeezed sponge so that it remains about 0.5 cm above the unit. If electrophoresing two gels, place the squeezed sponge on top of the first sandwich. Assemble the second sandwich exactly as described above. Place this on top of the sponge. Pack the remaining blot module with the sponge as described above. • Cover the gel / membrane sandwich in the blot module with a sufficient amount of transfer buffer. Use approximately 550 mL of MQH20 in the outer buffer container. • Perform electrophoresis at room temperature under 30 volts for 90 minutes.

[0091] Western blot: • GenScript One-Hour Western Kit protocol; signal coloration with TMB substrate. • 10 μL Anti-His Ab + 100 μL WB-1; 50 μL / gel used

[0092] Equal parts Those skilled in the art will recognize many equivalents to the specific embodiments of the present invention described herein and can verify them by routine experimentation alone. Such equivalents shall be included in the following claims.

[0093] References Bose JL et al., 2013. Genetic tools to enhance the study of gene function and regulation in Staphylococcus aureus. Applied and Environmental Microbiology 79:2218-2224. Jentsch S. 1983. Restriction and modification in Bacillus subtilis: Sequence specificities of restriction / modification systems BsuM, BsuE, and BsuF. Journal of Bacteriology. 156:800-808. Kost C. et al., 2012. PLOS One. Vol. 7, Issue 7. E41349. Moscoso M et al., 2017. Protective efficacy of a D-alanine auxotroph Staphylococcus aureus as a vaccine candidate against staphylococcal disease. 27th ECCMID, April 22, 2017, Vienna, Austria. Pucci M.J. et al., 1992. J of Bacteriology. p.336-342. Thompson R et al., 1998. Pathogenicity and immunogenicity of a Listeria monocytogenes strain that requires D-alanine for growth. Infection and Immunity 66:3552-3561.

[0094] Sequence Listing SEQUENCE LISTING <110> AZITRA INC <120> AUXOTROPHIC STRAINS OF STAPHYLOCOCCUS BACTERIUM <130> PA26-250 <150> US62 / 614,096 <151> 2018-01-05 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 1 atgcgaattc atgagcgata cttatttgaa tc 32 <210> 2 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 2 ctatgcgatt gaatatactt ttccttagca tcctcttcat taac 44 <210> 3 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primary <400> 3 gttaatgaag agatgctaa ggaaaagtat attcaatcgc atag 44 <210> 4 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primary <400> 4 agctgtcgac agcagcatac caatgtcaat c 31 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primary <400> 5 catacgaaga tcgaggctac 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 6 gtaccaactt gtccgtcttg 20 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 7 ttgatttaga caattggaag ag 22 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 8 aagtacagtc ggcattatct c 21 <210> 9 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 9 tttacatatg attcagaaac gtaagcggac agtttcg 37 <210> 10 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 10 tttttcttgg aattgtgctg cctgcaggtt agtgatggtg 40 <210> 11 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 11 acgtctcgag ctgatatttt tgactaaacc aaatg 35 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 12 ctgacctgca ggagatgatc cgctactaac gac 33

Claims

1. Two inactivated alanine racemase genes (alr1 and arl2): and Inactivated D-alanine aminotransferase gene (dat) Recombinant Staphylococcus bacteria, including those mentioned above.

2. Recombinant Staphylococcus bacteria according to claim 1, wherein the Staphylococcus bacteria depend on D-alanine for their growth.

3. The recombinant Staphylococcus bacterium according to claim 1, wherein the Staphylococcus bacterium is Staphylococcus epidermidis (S. epidermidis) and its subspecies.

4. The recombinant Staphylococcus bacterium according to claim 1, further comprising one or more additional mutations.

5. The recombinant Staphylococcus bacterium according to claim 4, wherein the additional mutation comprises an inactivated glutamate racemase gene, MurI.

6. A recombinant Staphylococcus bacterium according to any one of claims 1 to 5, wherein the bacterium is transformed with a pUBTR114-based vector.

7. The recombinant Staphylococcus bacterium according to claim 6, wherein the pUBTR114-based vector is pUBTR119*-Sal-GFP.

8. A method for producing recombinant Staphylococcus bacteria: (i) Transform competent cells of a Staphylococcus strain by introducing a plasmid containing D-alanine aminotransferase (dat) knockout, wherein the Staphylococcus strain contains inactive alanine racemase genes arl1 and arl2 (SEΔalr1Δalr2); (ii) Detecting the presence of a knockout plasmid in transformed cells; (iii) Culturing the transformed cells identified in step (ii); and (iv) Purify the isolated colonies. The method, including the method described above.

9. The method according to claim 8, further comprising testing the D-alanine requirement of isolated colonies.

10. The method according to claim 8, wherein the presence of a knockout plasmid in a transformant is detected by polymerase chain reaction (PCR).

11. The method according to claim 8, wherein the recombinant Staphylococcus bacteria are Staphylococcus epidermidis and its subspecies.

12. The method according to claim 8, further comprising transforming recombinant Staphylococcus bacteria with a pUBTR114-based vector.

13. The method according to claim 12, wherein the pUBTR114-based vector is pUBTR119*-Sal-GFP.

14. Recombinant Staphylococcus bacteria prepared by the method described in claim 8.

15. A kit comprising recombinant Staphylococcus bacteria according to any one of claims 1 to 8 or 14.

16. The kit according to claim 15, further comprising a pUBTR114-based vector.

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