Methods and compositions for reducing malodor

Abstract

The present technology relates generally to the field of reducing, preventing, or ameliorating the generation of undesirable body odors emanating from the skin. In particular, the present technology discloses ApatB-Staphylococcus haemolyticus bacterial strains, which have been modified to reduce or prevent the production of 3-methyl-3- sulfanylhexan-l-ol (3M3SH), compositions and pharmaceutical comprising one or more of said strains, methods of genetically engineering said strains, methods for reducing body odor using said strains, and methods for altering a microbiome using said strains.

Description

Atty. Dkt. No. 135523-0119METHODS AND COMPOSITIONS FOR REDUCING MALODORCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 719,454, filed November 12, 2024, the disclosure of which is incorporated by reference herein in its entirety for any and all purposes.TECHNICAL FIELD

[0002] The present technology relates generally to the field of reducing, preventing, or ameliorating the generation of undesirable body odors emanating from the skin. In particular, the present technology provides ApatB-Staphylococcus haemolyticus bacterial strains, which have been modified to prevent or reduce the production of volatile thioalcohols, such as 3-methyl-3-sulfanylhexan-l-ol (3M3SH), from precursor molecules commonly found in sweat.BACKGROUND

[0003] The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art to the compositions and methods disclosed herein.

[0004] Body odors, particularly those originating from sweat, represent a significant irritant for many humans, as evidenced by the multi-billion-dollar deodorant, body -wash, and perfume industries. Sweat, released from glands throughout the body, is a complex solution of salts, minerals, metabolites, and, potentially, pharmaceuticals or other exogenous compounds. Once secreted, sweat provides a nutrient filled solution for the skin microbiota to use as a nutrient source. Unfortunately, this metabolism, and the incumbent high levels of metabolic activity, results in the production of a number of undesirable byproducts, including volatile thioalcohols, which give off a distinct and unpleasant smell commonly referred to as body odor.

[0005] Existing technologies in the anti-body odor space possess a number of limitations. Antiperspirants, so named for their sweat-blocking properties, prevent the body from engaging in natural cooling mechanisms and can be environmentally costly to produce. Deodorants, meanwhile, do not prevent the generation of body odor, but merely conceal itAtty. Dkt. No. 135523-0119with a more potent odor and, in some cases, provide antimicrobial ingredients to reduce bacterial activity and abundance. Both antiperspirants and deodorants are environmentally costly to manufacture, produce significant amounts of plastic waste, and typically require multiple applications over a period of days to retain efficacy. Accordingly, there is a need for more effective deodorizing technologies that do not pose the same negative environmental and health impacts as current anti-body odor products.SUMMARY

[0006] In one aspect, the disclosure of the present technology provides a genetically engineered Staphylococcus haemolyticus strain whose genome comprises a disruption of a patB gene. In some embodiments, the strain exhibits decreased expression of the patB gene as compared to a corresponding wild-type Staphylococcus haemolyticus strain. In some embodiments, the disruption prohibits transcription of a full-length wild-type mRNA from the disrupted patB gene. In some embodiments, the disruption prohibits expression of a functional PatB enzyme, inactivates the PatB protein, modifies the PatB protein, or has a negative effect on the expression of the patB gene. In some embodiments, the genetically engineered strain has decreased PatB enzyme activity as compared to a corresponding wildtype Staphylococcus haemolyticus strain. In some embodiments, the modified PatB protein is a truncated protein. In some embodiments, the truncated protein has decreased catalytic activity compared to a corresponding wild-type Staphylococcus haemolyticus strain. In some embodiments, the disrupted patB gene comprises a deletion of all or at least a portion of the gene, optionally wherein the deletion comprises deletion of the patB promoter. In some embodiments, the disrupted patB gene comprises an insertion of a gene cassette. In some embodiments, the disrupted patB gene comprises a deletion of at least a portion of the gene. In some embodiments, the disruption comprises a knockout of the patB gene. In some embodiments, the disruption comprises an allelic replacement knockout. In some embodiments, the disruption comprises a nucleotide substitution. In some embodiments, the nucleotide substitution is a cytosine (C) to a thymine (T). In some embodiments, the nucleotide substitution is at the W190 tryptophan amino acid site in the patB gene. In some embodiments, the nucleotide substitution results in a premature stop codon. In some embodiments, the Staphylococcus haemolyticus strain comprises a disrupted patB gene having about 80% to about 100% sequence identity to SEQ ID NO: 15. In some embodiments, the premature stop codon in the patB gene results in a truncated PatB proteinAtty. Dkt. No. 135523-0119lacking one or more enzymatic activities. In some embodiments, the premature stop codon in the expressed mRNA of the patB gene has the sequence UAG, UGA, or UAA. In some embodiments, the disruption is introduced by homologous recombination, site-directed mutagenesis, a Transcription activator-like effector nuclease (TALEN), a meganuclease, a zinc finger nuclease, a CRISPR / Cas9 system, a CRISPR base editing system, a CRISPR prime editing system, a CRISPR / Cpfl system, a CRISPR / Csml system, or any combination thereof. In some embodiments, the disruption is introduced by a CRISPR base editing system. In some embodiments, the CRISPR based editing system is a cytosine base editor (CBE) system. In some embodiments, the fitness of the strain comprising the disruption is not negatively affected. In some embodiments, the strain has a comparable growth rate as compared to a corresponding wild-type Staphylococcus haemolyticus strain. In some embodiments, the strain comprises Staphylococcus haemolyticus deposited with the International Depositary Authority of Canada (ID AC) under ID AC Accession Number 240925-04, or a variant strain thereof. In some embodiments, the strain has a reduced capacity to produce 3-methyl-3-sulfanylhexan-l-ol (3M3SH) when contacted with apocrine sweat or a 3M3SH precursor as compared to a corresponding wild-type strain. In some embodiments, the disruption is a deletion or nucleotide substitution in the DNA sequence of the patB gene encoding a PatB protein, wherein the deletion or nucleotide substitution inactivates the PatB protein, truncates the PatB protein, or has a negative effect on expression of the patB gene. In some embodiments, the genetically engineered Staphylococcus haemolyticus strain has a 16S rRNA gene sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the sequence set forth in SEQ ID NO: 16. In some embodiments, the strain has the 16S rRNA gene sequence as set forth in SEQ ID NO: 16. In some embodiments, the genetically engineered Staphylococcus haemolyticus strain is derived from the strain deposited with the International Depositary Authority of Canada (ID AC) under ID AC Accession Number 240925-04, or a variant strain derived therefrom, wherein the genome has been engineered to comprise a disruption of a patB gene.

[0007] In another aspect, the disclosure of the present technology provides a genetically engineered Staphylococcus haemolyticus strain deposited with the International Depositary Authority of Canada (ID AC) under ID AC Accession Number 240925-04, or a variant strain derived therefrom, wherein the variant strain is obtained using the deposited strain as starting material, and wherein a reduced capacity of the deposited strain to produce 3-Atty. Dkt. No. 135523-0119methyl-3-sulfanylhexan-l-ol (3M3SH) when contacted with apocrine sweat or a 3M3SH precursor as compared to a corresponding wild-type strain is retained or improved in the variant strain as compared to the deposited strain.

[0008] In another aspect, the disclosure of the present technology provides a Staphylococcus haemolyticus strain, wherein the strain comprises a mutation in at least one endogenous polynucleotide selected from the group consisting of: (a) a nucleotide sequence having about 80% to about 100% sequence identity to SEQ ID NO: 14; and (b) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having about 80% to about 100% sequence identity to SEQ ID NO: 18, such that the strain has a reduced capacity to produce 3-methyl-3-sulfanylhexan-l-ol (3M3SH) when contacted with apocrine sweat or a 3M3SH precursor as compared to a corresponding strain that does not comprise the mutation. In some embodiments, the nucleotide sequence of (a) has about 97% to about 100% sequence identity to SEQ ID NO: 14, and / or the nucleotide sequence of (b) encodes a polypeptide comprising an amino acid sequence having about 97% to about 100% sequence identity to SEQ ID NO: 18. In some embodiments, the strain grows at an equivalent or improved rate as compared to a Staphylococcus haemolyticus strain lacking the mutation. In some embodiments, the strain is incapable of converting Cys-Gly-3M3SH to 3M3SH.

[0009] In one aspect, the disclosure of the present technology provides compositions comprising an effective amount of one or more of the genetically engineered Staphylococcus haemolyticus strains. In some embodiments, the one or more Staphylococcus haemolyticus strains is lyophilized. In some embodiments, the composition further comprising lyoprotectant. In some embodiments, the lyoprotectant is selected from one or more of sucrose, micellar casein, trehalose, or inulin. In some embodiments, the one or more Staphylococcus haemolyticus strains is viable. In some embodiments, the one or more Staphylococcus haemolyticus strains is nonproliferative. In some embodiments, the composition further comprises Staphylococcus epidermidis. In some embodiments, the composition further comprises an excipient. In some embodiments, the excipient is selected from one or more of oleic acid, stearic acid, almond butter, avocado butter, babassu butter, beeswax, cocoa butter, coconut butter, coconut oil, cupuacu butter, hemp seed butter, kokum butter, macadamia butter, mango butter, mowrah butter, olive butter, sal seed butter, shea butter, propylene glycol, glycerin, stearyl alcohol, myristyl alcohol, capric triglyceride, polyethylene glycol (PEG), Isopropyl Myristate, or sorbitol. In some embodiments, theAtty. Dkt. No. 135523-0119Staphylococcus haemolyticus strain is present at a concentration of about 106to about 1013bacteria per gram. In some embodiments, the Staphylococcus haemolyticus strain is present at a concentration of about 107to about 1011bacteria per gram. In some embodiments, the composition comprises about 90 wt. % to about 99.99 wt. % of the excipient. In some embodiments, the lyophilized Staphylococcus haemolyticus bacteria is present at about 10 wt. % to about 0.01 wt. %. In some embodiments, the composition comprises about 95 wt. % to about 99.75 wt. % excipient. In some embodiments, the lyophilized Staphylococcus haemolyticus bacteria is present at about 5 wt. % to about 0.25 wt. %. In some embodiments, the composition further comprises a fragrance selected from one or more of elettaria cardamomum seed oil, eucalyptus globulus seed oil, citrus aurantium bergamia fruit oil, mentha piperita oil, salvia sclarea oil, juniperus virginiana oil, abies sibirica oil, citrus aurantium amara, citrus aurantium dulcis lower oil, citrus nobilis oil, rosmarinus officinalis oil, melaleuca alternifolia oil, citrus limon tree oil, zingiber officinale root oil, Mangifera indica oil, Lavandula angustifolia oil, and Santalum album. In some embodiments, the composition is formulated for topical administration. In some embodiments, the composition is formulated for use as a topical deodorant. In some embodiments, the composition further comprises an antiperspirant. In some embodiments, the antiperspirant comprises one or more of aluminum salt, charcoal, and witch hazel. In some embodiments, the topical deodorant is formulated as a deodorant stick, a roll-on, a cream, a lotion, a gel, a powder, a water activated cream, a water activated powder, an injectable, a patch, or a spray. In some embodiments, the composition is a pharmaceutical composition and the effective amount of one or more of the genetically engineered Staphylococcus haemolyticus strains is a therapeutically effective amount.

[0010] In another aspect, the disclosure of the present technology provides a pharmaceutical composition comprising a therapeutically effective amount of one or more genetically engineered Staphylococcus haemolyticus strains, whose genome comprises a disruption of a patB gene, and pharmaceutically acceptable carrier or excipient, and optionally, a lyoprotectant. In some embodiments, the disruption of the patB gene comprises a nucleotide substitution, optionally wherein the nucleotide substitution comprises a cytosine (C) to thymine (T) substitution as compared to the wild type Staphylococcus haemolyticus strains. In some embodiments, the substitution is at the W190 tryptophan amino acid site in the patB gene. In some embodiments, the substitution introduces a premature stop codon. In some embodiments, the one or more genetically engineered Staphylococcus haemolyticusAtty. Dkt. No. 135523-0119strains are lyophilized. In some embodiments, the pharmaceutically acceptable carrier or excipient is present at about 90 wt. % to about 99.99 wt. % and the one or more lyophilized genetically engineered Staphylococcus haemolyticus strains is present at about 10 wt. % to about 0.01 wt. %. In some embodiments, the pharmaceutically acceptable carrier or excipient is present at about 95 wt. % to about 99.75 wt. % and the one or more lyophilized genetically engineered Staphylococcus haemolyticus strains is present at about 5 wt. % to about 0.25 wt. %. In some embodiments, the one or more genetically engineered Staphylococcus haemolyticus strain has a concentration of about 106to about 1013bacteria per gram. In some embodiments, the one or more genetically engineered Staphylococcus haemolyticus strains is present at a concentration of about 107to about 1011bacteria per gram. In some embodiments, the one or more genetically engineered Staphylococcus haemolyticus strains is deposited with deposited with the International Depositary Authority of Canada (ID AC) under ID AC Accession Number 240925-04.

[0011] In another aspect, the disclosure of the present technology provides a method of reducing 3-methyl-3-sulfanylhexan-l-ol (3M3SH) production from a skin microbiota of a subject, the method comprising applying: (i) a composition or a pharmaceutical composition comprising the genetically engineered Staphylococcus haemolyticus strain of any of the preceding embodiments to the skin of the subject; (ii) the composition of any of the preceding embodiments; or (iii) the pharmaceutical composition of any of the preceding embodiments to the skin of the subject.

[0012] In another aspect, the disclosure of the present technology provides a method for treating, reducing, or preventing malodor on the skin of a subject, comprising treating the skin of the subject with a therapeutically effective amount of the composition or the pharmaceutical composition of any of the preceding embodiments. In some embodiments, the composition or pharmaceutical composition is applied topically to the skin of the subject. In some embodiments, the composition or pharmaceutical composition is applied to a sweat-gland-containing area of skin of the subject. In some embodiments, the composition or the pharmaceutical composition is administered to the subject about 1 to about 2 times per day for about 1 to about 7 days. In some embodiments, the treated skin of the subject comprises a reduction in the amount of malodorous bacteria as compared to a corresponding untreated portion of skin. In some embodiments, the malodorous bacteria comprises at least wild-type Staphylococcus haemolyticus.Atty. Dkt. No. 135523-0119

[0013] In another aspect, the disclosure of the present technology provides a method of producing a genetically engineered Staphylococcus haemolyticus strain, comprising targeting the patB gene for disruption. In some embodiments, the disruption is introduced into the patB gene by homologous recombination, site-directed mutagenesis, a Transcription activator-like effector nuclease (TALEN), a meganuclease, a zinc finger nuclease, a CRISPR / Cas9 system, a CRISPR / Cpfl system, or a CRISPR / Csml system, and any combination thereof. In some embodiments, the disruption is introduced into the patB gene by the CRISPR base editing system. In some embodiments, the CRISPR based editing system is a cytosine base editor (CBE) system. In some embodiments, the CRISPR based editing system comprises transforming a wild type Staphylococcus haemolyticus strain with a 6 part plasmid. In some embodiments, the 6 part plasmid comprises one or more sequences selected from SEQ ID NOs: 2-7. In some embodiments, the 6 part plasmid comprises the sequence of SEQ ID NO: 1. In some embodiments, the strain exhibits decreased expression of the patB gene as compared to a corresponding wild-type Staphylococcus haemolyticus strain. In some embodiments, the disruption prohibits transcription of a full-length wild-type mRNA from the disrupted patB gene. In some embodiments, the disruption prohibits expression of a functional PatB enzyme. In some embodiments, the genetically engineered strain has decreased PatB enzyme or catalytic activity and the PatB protein is truncated as compared to a corresponding wild-type Staphylococcus haemolyticus strain. In some embodiments, the disrupted patB gene comprises a nucleotide substitution, wherein the substitution is a cytosine (C) to a thymine (T). In some embodiments, the strain is selected from Staphylococcus haemolyticus deposited with the International Depositary Authority of Canada (ID AC) under ID AC Accession Number 240925-04. In some embodiments, the genetically engineered Staphylococcus haemolyticus strain has a reduced capacity to produce 3-methyl-3-sulfanylhexan-l-ol (3M3SH) when contacted with apocrine sweat or a 3M3SH precursor as compared to a corresponding wild-type strain.

[0014] In another aspect, the disclosure of the present technology provides an altered microbiota comprising a genetically engineered Staphylococcus haemolyticus strain, or a variant strain thereof, whose genome comprises a disruption of a patB gene. In some embodiments, the microbiota has a reduced capacity to produce 3-methyl-3-sulfanylhexan-l-ol (3M3SH) when contacted with apocrine sweat as compared to a corresponding wildtype microbiota.Atty. Dkt. No. 135523-0119

[0015] In an aspect, the disclosure of the present technology provides a deodorant composition comprising: (i) a therapeutically effective amount of a viable Staphylococcus haemolyticus strain whose genome comprises a disruption of a patB gene; and (ii) a therapeutically acceptable carrier or excipient; wherein the deodorant composition is formulated as a water activated formulation for topical administration. In some embodiments, the deodorant composition comprises about 90 wt. % to about 99.99 wt. % of the therapeutically acceptable carrier or excipient. In some embodiments, the Staphylococcus haemolyticus strain is lyophilized and the deodorant composition comprises about 10 wt. % to about 0.01 wt. % of the lyophilized Staphylococcus haemolyticus bacteria. In some embodiments, the deodorant composition comprises about 95 wt. % to about 99.75 wt. % of the therapeutically acceptable carrier or excipient. In some embodiments, the deodorant composition comprises about 5 wt. % to about 0.25 wt. % of the lyophilized Staphylococcus haemolyticus bacteria. In some embodiments, the Staphylococcus haemolyticus strain is present at a concentration of about 106to about 1016of bacteria per gram. In some embodiments, the Staphylococcus haemolyticus strain is present at a concentration of about 107to about 1011of bacteria per gram. In some embodiments, the deodorant composition further comprises (iii) a lyoprotectant. In some embodiments, the Staphylococcus haemolyticus strain comprises the strain deposited with the International Depositary Authority of Canada (ID AC) under ID AC Accession Number 240925-04. In some embodiments, the disclosure of the present technology provides a deodorant stick comprising the deodorant composition.

[0016] In another aspect, the disclosure of the present technology provides a pharmaceutical composition comprising a therapeutically effective amount of one or more of the genetically engineered Staphylococcus haemolyticus strains of any of the preceding embodiments and a therapeutically acceptable carrier or excipient, for use in the treatment of malodor on the skin of a subject. In some embodiments, the pharmaceutical composition further comprising a lyoprotectant.

[0017] In one aspect, the disclosure of the present technology provides for a use of the one or more genetically engineered Staphylococcus haemolyticus strains of any of the preceding embodiments in the manufacture of a medicament for the treatment of malodor on the skin of a subject.Atty. Dkt. No. 135523-0119

[0018] In some embodiments of any aspect of the present disclosure, the Staphylococcus haemolyticus strain is gene edited to comprise a disruption of a patB gene. Accordingly, the disclosure provides a gene edited Staphylococcus haemolyticus strain whose genome comprises a disruption of a patB gene. In some embodiments, the strain does not comprise any heterologous DNA.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 is a diagram showing the sequence confirmation results for a patB mutant. For the engineered strain, Sn00430, the Sanger-confirmed mutated sequence (bottom) is aligned with the allele of the wild type ancestral strain, Sn00412 (top). Mutated positions between the wild type and engineered sequences are highlighted, and the premature stope codon sequence (TAA) at codon 190 in the engineered strain is bolded.

[0020] FIG. 2 is a chart showing patB loss of function mutants have decreased capacity to produce 3M3SH. The ability of the wild-type Staphylococcus haemolyticus strain (Sn00412) and its corresponding patB mutant (Sn00430) to produce 3M3SH is shown. When cultures were incubated with precursor molecule Cys-Gly-3M3SH, the engineered strain exhibited decreased production of 3M3SH relative to the wild-type strain. Bar heights represent averages amongst replicates, and error bars represent 2 standard deviations.DETAILED DESCRIPTION

[0021] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present technology are described below in various levels of detail in order to provide a substantial understanding of the present technology. The definitions of certain terms as used in this specification are provided below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this present technology belongs.I. Definitions

[0022] The following terms are used herein, the definitions of which are provided for guidance.Atty. Dkt. No. 135523-0119

[0023] As used herein, the singular forms “a,” “an,” and “the” designate both the singular and the plural, unless expressly stated to designate the singular only.

[0024] The term “about” and the use of ranges in general, whether or not qualified by the term about, means that the number comprehended is not limited to the exact number set forth herein, and is intended to refer to ranges substantially within the quoted range while not departing from the scope of the present technology. As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term.

[0025] As used herein, “administration” of an agent, drug, bacterial strain(s) or composition of the present technology to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including topically. In some embodiments, the compositions of the present technology are formulated for topical administration. In some embodiments, the compositions are formulated as a stick, a roll-on, a cream, a lotion, a gel, a powder, a water activated formulation (e.g., a water activated cream or a water activated powder), an injectable, a patch, or a spray. In some embodiments, the compositions are formulated for use as a probiotic. As used herein, administration includes self-administration and administration by another.

[0026] As used herein, “wild type Staphylococcus haemolylicus" or “wild type S. haemolylicus" or “ Staphylococcus haemolylicus" or “5. haemolylicus" refers to a naturally occurring Staphylococcus haemolyticus strain. In some embodiments, “wild type Staphylococcus haemolylicus" or “wild type S. haemolylicus" or “ Staphylococcus haemolylicus" or “5. haemolyticus ” refers to a bacterial strain having been deposited under IDAC Accession No. 240925-01 (z.e., S. haemolyticus strain Sn00412) , or compositions comprising the strain.

[0027] As used herein, “Strain Sn00430” or “Sn00430” refers to a bacterial strain having been deposited under IDAC Accession No. 240925-04, or compositions comprising the strain. Strain Sn00430 is a ApatB-S. haemolyticus strain derived from the wild-type S.Atty. Dkt. No. 135523-0119haemolyticus strain Sn00412. Strain Sn00412 is deposited under IDAC Accession No. 240925-01.

[0028] As used herein, “ApatB-Staphylococcus haemolyticus" or “ApatB-S. haemolyticus" refers to a Staphylococcus haemolyticus whose genome comprises a disruption of a patB gene or compositions comprising ApatB-Staphylococcus haemolyticus. As used herein, “Strain Sn00430” or “Sn00430” or "ApalB of Sn00412” refers to a strain that comprises a disruption of the patB gene where the wild-type strain is Strain Sn00412.

[0029] In some embodiments, the ApatB-Staphylococcus haemolyticus comprises a variant of strain Sn00430. In some embodiments, the ApatB-Staphylococcus haemolyticus comprises a single base pair point mutation. In some embodiments, the ApatB-Staphylococcus haemolyticus comprises a cytosine (C) to a thymine (T) point mutation. In some embodiments, the point mutation is in the patB coding region. In some embodiments, the point mutation is in the W190 tryptophan amino acid site in patB. In some embodiments, the ApatB-Staphylococcus haemolyticus exhibits a reduced PatB activity and / or patB expression. In some embodiments, the ApatB-Staphylococcus haemolyticus has a reduced capacity to produce thiol and / or 3M3SH when contacted with an appropriate substrate and / or precursor. In some embodiments, ApatB-Staphylococcus haemolyticus comprises a Staphylococcus haemolyticus strain whose genome comprises a disruption of a patB gene, the strain having been deposited under IDAC Accession No. 240925-04 ( / .< ., S. haemolyticus strain Sn00430) or a variant strain thereof, or compositions comprising the strain and / or one or more variant strains thereof. In some embodiments, ApatB-S. haemolyticus comprises a S. haemolyticus strain that expresses a truncated PatB protein, i.e. not the full-length PatB protein. In some embodiments, ApatB-S. haemolyticus comprises a S. haemolyticus strain with phenotypic loss-of-function for the patB gene (e.g.,patB~, which can be used interchangeably herein with ApatB-S. haemolyticus). In some embodiments, the disruption is a non-synonymous (missense) substitution that yields a full-length PatB polypeptide having reduced or abolished P-lyase enzymatic activity sufficient to decrease 3M3SH production as compared to a corresponding wild-type strain. In certain embodiments, the non-synonymous substitution alters a catalytic, substrate-binding, cofactor-binding, folding, or stability determinant of PatB without truncating the protein. In some embodiments, ApatB-S. haemolyticus comprises a single amino acid substitution such that 3M3SH production is eliminated.Atty. Dkt. No. 135523-0119

[0030] As used herein, “variant” or “variant strain” refers to a strain derived from a strain of the present technology by any means, such as, but not limited to, genetic engineering, radiation and / or chemical treatment, and / or selection, adaptation, screening, etc. In some embodiments, the variant is naturally occurring. In some embodiments, the variant is selected for or engineered. In some embodiments, the variant is a functionally equivalent variant, e.g., a variant that has the same, or improved, properties with respect to its capacity to produce a thiol or 3M3SH when contacted with an appropriate substrate and / or precursor as the mother or wild type strain. Such variants are a part of the present technology. In some embodiments, the term “variant” refers to a strain obtained by subjecting a strain of the present technology to any conventionally used mutagenization treatment including treatment with a chemical mutagen such as ethane methane sulphonate (EMPS) or A-methyl-N’-nitro-N-nitroguanidine (NTG), UV light, or to a spontaneously occurring variant. In embodiments in which variants are subjected to mutagenization, one of skill in the art may refer to the variant as a mutant. A variant may have been subjected to several mutagenization treatments (a single treatment should be understood as one mutagenization step followed by a screening / selection step). In some embodiments, less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, or less than 0.0001% of nucleotides in the bacterial genome have been changed (such as by replacement, insertion, deletion, or a combination thereof) compared to the mother strain. Such variants, which may be identified by using appropriate screening techniques, are a part of the present technology.

[0031] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount which results in the prevention or treatment of a condition, and / or symptom(s) thereof. In the context of therapeutic or prophylactic applications, the amount of a composition or formulation administered to the subject will depend on the type and severity of the condition and on the characteristics of the subject, such as general health, age, sex, body weight, and lifestyle, for example. It will also depend on the degree, severity, and type of condition, for example. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. In some embodiments, a single dose is administered. In some embodiments, multiple doses are administered. Additionally, or alternatively, in some embodiments, multiple therapeutic compositions or compounds or formulations (e.g., pharmaceutical compositions comprising multiple bacterial strains alone or in combination with additional active agents, such as deodorizing or anti-perspiring agents). In the methods describedAtty. Dkt. No. 135523-0119herein, compositions comprising the bacterial strains of the present technology may be administered to a subject having, or at risk of having, body odor. In some embodiments, the compositions comprising the bacterial strains of the present technology may be administered to a subject unknown to have, or unknown to be at risk of having, body odor. In some embodiments, the compositions comprising the bacterial strains of the present technology may be administered to multiple locations on the body of a subject. For example, an “effective amount” of the compositions of the present technology, includes levels at which the presence, frequency, or severity of body odor is at a minimum reduced or ameliorated. In some embodiments, an effective amount reduces, prevents, or ameliorates the presence of body odor, and / or the likelihood of developing body odor. In some embodiments, an effective amount is achieved by multiple administrations. In some embodiments, an effective amount is achieved with a single administration.

[0032] As used herein, the terms “freeze-dried” or “freeze-drying” and “lyophilized” or “lyophilization” are used interchangeably and refer to a process that removes water from a product after it is frozen and placed under a vacuum and the products produced therefrom.

[0033] As used herein, the term “spray drying” refers to a process in which a liquid product is atomized into a hot drying chamber causing the liquid to evaporate rapidly resulting in a fine powder. In some embodiments, the process of spray drying does not require the use of lyoprotectants.

[0034] As used herein, “desiccated” or “desiccation” refers to a process in which liquid content of a product is significantly reduced through drying. Non-limiting examples of processes that yield a desiccated product include freeze-drying or spray drying. In some embodiments, desiccated cells can be the Staphylococcus haemolyticus bacterial cells as disclosed herein.

[0035] “Desiccated” refers to cells that have had their moisture content significantly reduced through drying, without specifying the exact drying method. This term effectively covers cells that have been dehydrated through various processes, including both freeze-drying and spray-drying, while still implying that the cells may retain some level of viability and can potentially be rehydrated to resume their metabolic functions.

[0036] As used herein, the term “lyoprotectant(s)” refers to an agent that prevents or ameliorates any damage caused to a product during the lyophilization process. In someAtty. Dkt. No. 135523-0119embodiments, the lyoprotectant is a protein, a carbohydrate, or a combination thereof. In some embodiments, the lyoprotectant is a milk protein, and in particular a micellar casein. In some embodiments, the carbohydrate is sucrose. In some embodiments, the lypoprotectant is trehalose, inulin, or a combination thereof. In some embodiments, the lyoprotectant prevents or ameliorates any damage caused to a bacterial strain of the present technology during the lyophilization process.

[0037] As used herein, “acceptable carrier and / or diluent” or “acceptable excipient” includes but is not limited to any combination of solvents, dispersion media, coatings, antifungal agents, isotonic and absorption delaying agents, and the like. In some embodiments, the acceptable carrier comprises oleic acid, stearic acid, almond butter, avocado butter, babassu butter, beeswax, cocoa butter, coconut butter, coconut oil, cupuacu butter, hemp seed butter, kokum butter, macadamia butter, mango butter, mowrah butter, olive butter, sal seed butter, shea butter, propylene glycol, glycerin, stearyl alcohol, myristyl alcohol, capric triglyceride, polyethylene glycol (PEG), Isopropyl Myristate, sorbitol, or any combination thereof. The use of such agents with biologically active substances is well known in the art. Further details of excipients are provided below. Supplementary active ingredients, such as antifungal agents, antiperspirants, fragrances, and deodorants, can also be incorporated into the compositions.

[0038] As used herein, “acceptable excipient” or “excipient” refers to substances and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or a human. As used herein, the term includes all inert, nontoxic, liquid or solid fillers, or diluents that do not react with the strains of the present technology in an inappropriate negative manner, including solvents, dispersion media, coatings, antifungal agents, isotonic and absorption delaying agents, preservatives and the like, for example liquid pharmaceutical carriers e.g., sterile water, saline, sugar solutions, Tris buffer, ethanol and / or certain oils. In some embodiments, the excipient is polyethylene glycol (PEG), Isopropyl Myristate, or sorbitol. In some embodiments, the excipient is polyethylene glycol (PEG). In some embodiments, the excipient is Isopropyl Myristate. In some embodiments, the excipient is sorbitol.

[0039] As used herein, “deodorant” refers to a substance which removes and / or conceals an unpleasant smell, such as body odor. In some embodiments, the strains of the present technology are formulated as deodorant compositions. In some embodiments, theAtty. Dkt. No. 135523-0119deodorant compositions further comprise a fragrance. In some embodiments, the fragrance comprises one or more of elettaria cardamomum seed oil, eucalyptus globulus seed oil, citrus aurantium bergamia fruit oil, mentha piperita oil, salvia sclarea oil, juniperus virginiana oil, abies sibirica oil, citrus aurantium amara, citrus aurantium dulcis lower oil, citrus nobilis oil, rosmarinus officinalis oil, melaleuca altemifolia oil, citrus limon tree oil, zingiber officinale root oil, Mangifera indica oil, Lavandula angustifolia oil, and Santalum album. In some embodiments, the strains and compositions of the present technology are administered with a fragrance, either simultaneously or sequentially and either as separate formulations or a single formulation.

[0040] As used herein “antiperspirant” refers to a substance that inhibits, reduces, or prevents the release of sweat from glands. In some embodiments, the strains and compositions of the present technology are administered with an antiperspirant. In some embodiments, the strains of the present technology are formulated as a deodorant that further comprises an antiperspirant. In some embodiments, the antiperspirant comprises one or more of aluminum salt, charcoal, and witch hazel.

[0041] The terms “disruption” and “mutation” are used interchangeably herein to refer to a detectable and heritable change in the genetic material. Disruptions or mutations may include insertions, deletions, substitutions (e.g., transitions, transversion, homologous recombination), transpositions, inversions, knockouts and combinations thereof. For example, in some embodiments, a disruption or mutation may include: (a) the introduction, substitution, or removal of one or more nucleotides in a Staphylococcus haemolyticus patB gene (or an open reading frame (ORF) thereof), or the introduction, substitution, or removal of one or more nucleotides in a regulatory element required for the transcription or translation of the patB gene or ORF thereof; (b) a patB gene conversion; (c) a patB gene deletion; (d) the down-regulation of the patB gene; (e) specific mutagenesis of the patB gene; and / or (f) random mutagenesis of the patB gene. Disruptions or mutations of the patB gene, as used herein, include any genetic modification that prevents or reduces the capability of the S. haemolyticus strains of the present technology from producing a functional patB gene product. Such disruptions or mutations may include complete or partial deletion of any portion of patB, including a polypeptide-coding sequence, a promoter, an enhancer, a ribosome-binding site (RBS), or other regulatory elements, or mutagenesis of the same, where mutagenesis encompasses substitutions, insertions,Atty. Dkt. No. 135523-0119deletions, inversions, and any combinations and variations thereof that inactivate patB and prevent or reduce the production of a patB functional gene product. Mutations or disruptions may involve only a single nucleotide (e.g., a point mutation, nucleotide substitution, or a single nucleotide polymorphism) or multiple nucleotides. In some embodiments, the mutation or disruption may be a cytosine (C) to thymine (T) substitution. In some embodiments, the conversion may occur by a cytosine-based editor (CBE) tool. In some embodiments, the CBE precisely converts a cytosine (C) to thymine (T) without causing a double-stand break. In some embodiments, the cytosine (C) to thymine (T) occurs at the W 190 tryptophan amino acid site in PatB. In some embodiments, the cytosine (C) to thymine (T) introduces a premature stop code in the expressed mRNA ( / .< ., UAG, UGA, or UAA). In some embodiments, the premature stop codon introduces an early termination signal in the mRNA sequence. In some embodiments, the early termination signal leads to production of a truncated protein. In some embodiments, the truncated protein has a loss of catalytic activity. In some embodiments, the mutation or disruption causes a phenotypic change, for example, the expression level of the encoded product is altered, or the encoded product itself is altered. In some embodiments, a disruption or mutation may result in a disrupted gene with decreased levels of expression of a gene product (e.g., protein or RNA) as compared to the wild-type strain. In other embodiments, a disruption or mutation may result in an expressed protein with activity that is lower as compared to the activity of the expressed protein from the wild-type strain. In some embodiments, a gene disruption or mutation results in the removal of the gene from the genome via a substitution, such as homologous recombination. In some embodiments, a functional patB gene product enables the metabolic conversion of Cys-Gly-3M3SH to 3M3SH. Accordingly, in some embodiments, a disruption or mutation of the patB gene, as used herein, includes any genetic modification that prevents or reduces the capability of the S. haemolyticus strains of the present technology to convert Cys-Gly-3M3SH to 3M3SH. This includes, but is not limited to, disruption of one or more PatB enzymatic activities or alteration of PatB binding with one or more metabolites or co-factors or the decreases to the relative enzymatic activity with respect to alternative substrates. Disruptions or mutations outside of the patB coding region that accomplish the effect of disrupted PatB activity are also encompassed within this disclosure, including disruptions or mutations in enhancer, repressor, promoter or other regulatory elements that result in decreased transcription or translation of the patB gene. In some embodiments, the nucleotides encoding a single amino acid are altered (e.g., replaced or deleted) which results in an elimination of 3M3SHAtty. Dkt. No. 135523-0119production in the strain. Other methods may also be used to disrupt PatB activity, including, but not limited to engineered transcription factors, including Transcription Activator-Like Effectors (TALEs) that repress patB expression, antisense RNAs or small regulatory RNAs that repress patB translation, CRISPR-dCasl3 constructs that target patB RNA and prevent translation, and protein-based or small molecule constructs that post translationally inactivate or repress PatB protein activity. For example, the present disclosure explicitly contemplates constructs (e.g., recombinant proteins) that post-translationally modify (e.g., phosphorylate) and / or alter the enzymatic properties of the PatB protein to reduce or inhibit enzymatic activity or metabolite binding by the PatB protein. Any construct, and method of use thereof, that downregulates, prevents, or inhibits in any way the transcription, translation, or protein activity of PatB is encompassed within the present disclosure. As used herein, “A,” for example as used in “ patB” is intended to encompass any disruption(s) or mutation(s) to the patB gene described herein.

[0042] The term “engineered” or “genetically engineered” is used herein to refer to an organism that has been manipulated to be genetically altered, modified, or changed, e.g., by disruption / editing of the genome. For example, an “engineered” or “genetically engineered” bacterial strain encompasses a bacterial strain that has been manipulated to be genetically altered, modified, or changed.

[0043] As used herein, the term “fitness” or “bacterial fitness” refers to the capacity of a bacterium, bacterial strain, or bacterial species to survive and reproduce in a given environment.

[0044] A “knocked out gene” or a “gene deletion” refers to a gene including a null mutation (e.g., the wild-type product encoded by the gene is not expressed, expressed at levels so low as to have no effect, or is non-functional). In some embodiments, the knocked out gene includes heterologous sequences or genetically engineered non-functional sequences of the gene itself, which renders the gene non-functional. In other embodiments, the knocked out gene is lacking a portion of the wild-type gene. For example, in some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40% or at least about 60% of the wild-type gene sequence is deleted. In other embodiments, the knocked out gene is lacking at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95% or at least about 100% of the wild-type gene sequence. In other embodiments, the knocked out gene may include up to 100% of the wild-type geneAtty. Dkt. No. 135523-0119sequence (e.g., some portion of the wild-type gene sequence may be deleted) but also include one or more heterologous and / or non-functional nucleic acid sequences inserted therein.

[0045] The term “gene cassette” is used herein to refer to a DNA sequence encoding and capable of expressing one or more genes of interest (e.g., a metabolic gene, a selectable marker, or a combination thereof) that can be inserted between one or more selected restriction sites of a DNA sequence. In some embodiments, insertion of a gene cassette results in a disrupted gene. In some embodiments, disruption of the gene involves replacement of at least a portion of the gene with a gene cassette, which includes a nucleotide sequence encoding a gene of interest (e.g., a metabolic gene, a selectable marker, or a combination thereof).

[0046] As used herein, “probiotic” refers to bacteria that supplements or replaces elements of endogenous flora of a subject, which when administered to the subject confers a beneficial prophylactic and / or therapeutic effect on the subject. In some embodiments, the beneficial effect is a reduction or prevention in malodor on the skin of a subject.

[0047] As used herein, “prevention,” “prevent,” or “preventing” of a condition refers to, in a statistical sample, reduction in the occurrence or recurrence of the condition in treated subjects / samples relative to an untreated controls, or refers to delays in the onset of one or more symptoms of the condition relative to the untreated controls. In some embodiments, the condition is malodor (body odor) on the skin of the subject.

[0048] As used herein, to “reduce” or “reducing” a condition refers to results that, in a sample or specific subject, make the occurrence of the condition better or more tolerable in a sample or subject administered a therapeutic agent (e.g., a ApatB-Staphylococcus haemolyticus strain or compositions comprising the strain) relative to a control sample or subject. In some embodiments, the condition is malodor (body odor) on the skin of the subject.

[0049] As used herein “subject” and “patient” are used interchangeably. In some embodiments, the subject is an animal subject. In some embodiments, the animal subject is a mammal. In some embodiments, the mammalian subject is a human.Atty. Dkt. No. 135523-0119

[0050] As used herein, the term “simultaneous” administration refers to the administration of at least two agents by the same route and at the same time or at substantially the same time.

[0051] As used herein, the term “separate” administration refers to an administration of at least two agents at the same time or at substantially the same time by different routes.

[0052] As used herein, the term “sequential” administration refers to administration of at least two agents at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one agent before administration of the other agent(s) commences. It is thus possible to administer one of the agents over several minutes, hours, or days before administering another.

[0053] A “synergistic effect” refers to a greater-than-additive effect which is produced by a combination of at least two agents, and which exceeds that which would otherwise result from the individual administration of the agents. For example, use of bacterial strain(s) of the present technology in conjunction with other agents for deodorizing or anti-perspiring may result in a greater than additive effect. In some embodiments, the synergistic effect may permit the use of lower doses of bacterial strain(s) of the present technology and / or other agents than would be required if each were used alone.

[0054] “Treating,” “treat,” “treated,” or “treatment” of a condition includes: (i) inhibiting the condition, z.e., arresting its development; (ii) relieving the condition, z.e., causing its regression; (iii) slowing progression of the condition; and / or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the condition. In some embodiments, the condition is malodor (body odor) on the skin of the subject.

[0055] As used herein, “3-methyl-3-sulfanylhexan-l-ol” and “3M3SH” are interchangeably used to refer to the alcohol compound 3-methyl-3-sulfanylhexan-l-ol having a formula of C7H16OS (CAS Number 307964-23-4). In some embodiments, a 3M3SH precursor is a compound comprising 3M3SH with additional chemical structural components. In some embodiments, Cys-Gly-3M3SH is a 3M3SH precursor.

[0056] It is to be appreciated that the various modes of treatment or prevention of medical diseases and conditions as described are intended to mean “substantial,” which includesAtty. Dkt. No. 135523-0119total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved.II. Sweat and Body Odor

[0057] The armpit offers two microbial habitats: the skin surface and the skin subsurface. The skin surface is the smaller of the two skin microbial habitats, with about 2 m2of surface area. It is also a harsher environment for bacteria as it is frequently disinfected through washing and deodorant use. The skin subsurface has a much larger surface area of about 24 m2and includes the epidermis, dermis, hair follicles, and sweat glands, all of which harbor skin bacteria (Gallo RL. Human Skin Is the Largest Epithelial Surface for Interaction with Microbes. J Invest Dermatol. 2017 Jun; 137(6): 1213-1214. doi: 10.1016 / j jid.2016.11.045. Epub 2017 Apr 8. PMID: 28395897; PMCID: PMC5814118; Nakatsuji T, Chiang HI, Jiang SB, Nagarajan H, Zengler K, Gallo RL. The microbiome extends to subepidermal compartments of normal skin. Nat Commun. 2013;4: 1431. doi: 10.1038 / ncomms2441. PMID: 23385576; PMCID: PMC3655727). The hair follicle provides a stable, moist, well-perfused, and UV light-protected environment. Sweat glands interface with both of these habitats and produce sweat, which bacteria feed on. Sweat glands can be categorized into apocrine glands and eccrine glands, although several additional types exist.

[0058] Eccrine glands open onto the skin surface and produce eccrine sweat, a saline solution that contains trace chemicals originating from interstitial fluid, such as potassium, bicarbonate, lactate, urea, amino acids, and calcium. This sweat causes perspiration (Groscurth P. Anatomy of sweat glands. Curr Probl Dermatol. 2002;30: 1-9. Doi:10.1159 / 000060678. PMID: 12471693). Apocrine glands open into the very upper hair follicle instead of the skin surface and produce apocrine sweat, a thick, sterile, lipid-rich, odorless fluid containing proteins, sugars, ammonia, steroids, and notably, the chemical precursors for body odor (Baker LB. Physiology of sweat gland function: The roles of sweating and sweat composition in human health. Temperature (Austin). 2019 Jul 17;6(3):211-259. doi: 10.1080 / 23328940.2019.1632145. PMID: 31608304; PMCID:PMC6773238; SanMiguel A, Grice EA. Interactions between host factors and the skin microbiome. Cell Mol Life Sci. 2015 Apr;72(8):1499-515. doi: 10.1007 / s00018-014-1812-z. Epub 2014 Dec 30. PMID: 25548803; PMCID: PMC4376244). Under emotional stress, hormones trigger apocrine glands to release this fluid, resulting in the formation of strong body odor (Barzantny H, Brune I, Tauch A. Molecular basis of human body odourAtty. Dkt. No. 135523-0119formation: insights deduced from corynebacterial genome sequences. Int J Cosmet Sci. 2012 Feb;34(l):2-ll. doi: 10.1111 / j.1468-2494.2011.00669.x. Epub 2011 Jul 25. PMID: 21790661).

[0059] Staphylococcus haemolyticus (S. haemolyticus) is a species of gram-positive bacteria known to reside on humans. It is typically considered a non-pathogenic commensal bacterium that produces thioalcohol compounds, thus contributing to malodor in humans. Staphylococcus haemolyticus is most frequently observed in areas of the skin with high numbers of apocrine glands, such as the axilla (underarm). Wild-type S. haemolyticus expresses the patB gene, thereby producing the PatB enzyme that allows the bacteria to metabolize Cys-Gly-3M3SH into 3M3SH. Apocrine sweat is typically rich in Cys-Gly-3M3SH, thereby providing a nutrient rich environment for patB expressing S. haemolyticus and allowing for microbiome colonization of specific areas of skin with high numbers of apocrine glands. The patB gene is required for the cleavage of an amino acid from the precursor Cys-Gly-3M3SH.III. ApatB-Staphylococcus haemolyticus

[0060] The technology of the present disclosure relates to the use of ApatB-Staphylococcus haemolyticus (whose genome comprises a disruption of a patB gene), or variant strains thereof, to treat or prevent body odor, and methods of making the same. Methods for disruption of the patB gene include targeted approaches for introducing a disruption by, e.g., homologous recombination, site-directed mutagenesis, a Transcription activator-like effector nuclease (TALEN), a meganuclease, a zinc finger nuclease, a CRISPR / Cas9 system, a CRISPR base editing system, a CRISPR prime editing system, a CRISPR / Cpfl system, or a CRISPR / Csml system. Additionally, or alternatively, such methods may also include random mutagenesis methods for introducing a disruption by, e.g., irradiation, chemical mutagens, passing cloned genes through mutator strains, “error-prone” PCR mutagenesis, rolling circle error-prone PCR, or saturation mutagenesis. For illustrative purposes, and for proof of concept, a patB disruption in Staphylococcus haemolyticus (e.g., ApatB-Staphylococcus haemolyticus) was created via CRISPR base editing system as described in the Examples section. In some embodiments, ApatB-Staphylococcus haemolyticus includes a bacterial strain, or variant thereof, having been deposited under IDAC Accession No. 240925-04 (z.e., Sn00430), or compositions comprising the strain. In some embodiments, the wild-type S. haemolyticus strain is Sn00412. In someAtty. Dkt. No. 135523-0119embodiments, the ApatB-Staphylococcus haemolyticus mutant comprises a TAA or a TGA or a TAG mutant at the W190* position, wherein the mutation encodes a premature stop codon.

[0061] In some embodiments, bacterial strains, such as the genetically engineered Staphylococcus haemolyticus strains (e.g., ApatB-Staphylococcus haemolyticus, such as Sn00430) of the present technology are used in methods, compositions, and pharmaceutical compositions for treating or preventing body odor (malodor). In some embodiments, the composition or pharmaceutical composition comprises one or more of the genetically engineered Staphylococcus haemolyticus strains and a probiotic for preventing or controlling body odor. In some embodiments, compositions or pharmaceutical compositions of the present technology comprise vegetative bacterial cells. In some embodiments, the compositions or pharmaceutical compositions of the present technology comprise desiccated bacterial cells. In some embodiments, the compositions or pharmaceutical compositions of the present technology comprise freeze-dried bacterial cells. In some embodiments, the compositions or pharmaceutical compositions of the present technology comprise spray-dried bacterial cells. In some embodiments, the ApatB-S. haemolyticus strains (e.g., ApatB-Staphylococcus haemolyticus having ID AC Accession No. 240925-04 (Sn00430)) of the present technology do not grow significantly slower than the corresponding wild-type S. haemolyticus strain(s) (e.g., Sn00412). In some embodiments, the disruption of patB does not significantly negatively affect the fitness of the S. haemolyticus strains whose genome comprises the disruption. In some embodiments, the genetically engineered Staphylococcus haemolyticus strains decrease the amount of thiol production by at least about 50% when contacted with a thiol substrate compared to corresponding wild-type strains. In some embodiments, the genetically engineered Staphylococcus haemolyticus strains decrease the amount of thiol production by at least about 55% when contacted with a thiol substrate compared to corresponding wild-type strains. In some embodiments, the genetically engineered Staphylococcus haemolyticus strains decrease the amount of thiol production by at least about 60% when contacted with a thiol substrate compared to corresponding wild-type strains. In some embodiments, the genetically engineered Staphylococcus haemolyticus strains decrease the amount of thiol production by at least about 65% when contacted with a thiol substrate compared to corresponding wild-type strains. In some embodiments, the genetically engineered Staphylococcus haemolyticus strains decrease the amount of thiol production by at leastAtty. Dkt. No. 135523-0119about 70% when contacted with a thiol substrate compared to corresponding wild-type strains. In some embodiments, the genetically engineered Staphylococcus haemolyticus strains decrease the amount of thiol production by at least about 75% when contacted with a thiol substrate compared to corresponding wild-type strains. In some embodiments, the genetically engineered Staphylococcus haemolyticus strains decrease the amount of thiol production by at least about 80% when contacted with a thiol substrate compared to corresponding wild-type strains. In some embodiments, the genetically engineered Staphylococcus haemolyticus strains decrease the amount of thiol production by at least about 85% when contacted with a thiol substrate compared to corresponding wild-type strains. In some embodiments, the genetically engineered Staphylococcus haemolyticus strains decrease the amount of thiol production by at least about 90% when contacted with a thiol substrate compared to corresponding wild-type strains. In some embodiments, the genetically engineered Staphylococcus haemolyticus strains decrease the amount of thiol production by at least about 95% when contacted with a thiol substrate compared to corresponding wild-type strains. In some embodiments, the genetically engineered Staphylococcus haemolyticus strains decrease the amount of thiol production by at least about 96%, 97%, 98%, or 99% when contacted with a thiol substrate compared to corresponding wild-type strains. In some embodiments, the genetically engineered Staphylococcus haemolyticus strains as disclosed herein have a reduced capacity to produce 3-methyl-3-sulfanylhexan-l-ol (3M3SH) when contacted with apocrine sweat or a 3M3SH precursor as compared to corresponding wild-type strains. In some embodiments, the genetically engineered Staphylococcus haemolyticus strains decrease the amount of 3M3SH production by at least about 50% when contacted with apocrine sweat or a 3M3SH precursor as compared to corresponding wild-type strains. In some embodiments, the genetically engineered Staphylococcus haemolyticus strains decrease the amount of 3M3SH production by at least about 60% when contacted with apocrine sweat or a 3M3SH precursor as compared to corresponding wild-type strains. In some embodiments, the genetically engineered Staphylococcus haemolyticus strains decrease the amount of 3M3SH production by at least about 70% when contacted with apocrine sweat or a 3M3SH precursor as compared to corresponding wild-type strains. In some embodiments, the genetically engineered Staphylococcus haemolyticus strains decrease the amount of 3M3SH production by at least about 80% when contacted with apocrine sweat or a 3M3SH precursor as compared to corresponding wild-type strains. In some embodiments, the genetically engineered Staphylococcus haemolyticus strains decrease the amount of 3M3SHAtty. Dkt. No. 135523-0119production by at least about 90% when contacted with apocrine sweat or a 3M3SH precursor as compared to corresponding wild-type strains. In some embodiments, the genetically engineered Staphylococcus haemolyticus strains decrease the amount of 3M3SH production by at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% when contacted with apocrine sweat or a 3M3SH precursor as compared to corresponding wild-type strains.

[0062] In some embodiments, bacterial strains, such as the genetically engineered Staphylococcus haemolyticus strains (e.g., ApatB-Staphylococcus haemolyticus) of the present technology are used in methods, compositions, and pharmaceutical compositions for altering a microbiota. In some embodiments, the bacterial strains comprise a probiotic that is capable of engraftment to an existing microbiota, such that the bacterial strains persist for a period of time in the microbiota community. In some embodiments, the microbiota is a skin microbiota. In some embodiments, an agent is applied to disrupt the native microbiota, and then the bacterial strains and compositions of the present technology are applied, such that the disruption of the native microbiota increases engraftment of the bacterial strains.

[0063] In some embodiments, the genetically engineered Staphylococcus haemolyticus strain whose genome comprises a disruption of a patB gene has a 16S rRNA gene sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO: 16. In some embodiments, the bacterial strain for use in the present technology has the 16S rRNA gene sequence represented by SEQ ID NO: 16.

[0064] In some embodiments, the strain for use in the present technology is derived from a strain deposited with the International Depositary Authority of Canada (ID AC) under ID AC Accession Number 240925-04, wherein the genome has been edited to comprise a disruption of a patB gene. In some embodiments, the strain for use in the present technology is derived from a strain that has a 16S rRNA gene sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO: 16, wherein the genome has been engineered to comprise a disruption of a patB gene. Within a single genome, there are often multiple copies of 16S that are not always identical. SEQ ID NO: 16 is a consensus sequences of all of the 16S sequences into a single sequence. Sequences below are provided using standard IUPAC nomenclature, wherein A indicates Adenine, C indicates Cytosine, G indicates Guanine, T (or U) indicates Thymine (or Uracil), R indicates A or G, Y indicates C or T, S indicates G or C, W indicates A or T, K indicates G or T, M indicates AAtty. Dkt. No. 135523-0119or C, B indicates C or G or T, D indicates A or G or T, H indicates A or C or T, V indicates A or C or G, N indicates any base, and . or - indicates a gap.Atty. Dkt. No. 135523-0119

[0065] In addition to strain Sn00430, other strains of Staphylococcus haemolyticus that have been genetically engineered to comprise a disruption of a patB gene are also expected to be effective for reducing 3-methyl-3-sulfanylhexan-l-ol (3M3SH) production from a skin microbiota and / or for treating, reducing, or preventing malodor on the skin of a subject. In particular, strains that are biotypes of strain Sn00430 are also expected to be effective for reducing 3-methyl-3-sulfanylhexan-l-ol (3M3SH) production from a skin microbiota and / or for treating, reducing, or preventing malodor on the skin of a subject. As used herein, a “biotype” refers to a strain that is a closely related strain that has the same or very similar physiological and biochemical characteristics.

[0066] Similarly, in addition to strain Sn00430, other strains of Staphylococcus haemolyticus are also expected to be suitable for genetically engineering disruption of a patB gene in order to generate a strain effective for reducing 3-methyl-3-sulfanylhexan-l-ol (3M3SH) production from a skin microbiota and / or treating, reducing, or preventing malodor on the skin of a subject. In particular, strains that are biotypes of strain Sn00430 are expected to be suitable for engineering to disrupt a patB gene.

[0067] Strains that are biotypes of strain Sn00430 and other Staphylococcus haemolyticus strains that are suitable for use in the present technology may be identified by sequencing other nucleotide sequences for strain Sn00430 and other Staphylococcus haemolyticus strains that are suitable for genetically engineering to disrupt a patB gene to provide a strain of the present technology may be identified by sequencing other nucleotide sequences for strains.

[0068] For example, substantially the whole genome may be sequenced and a strain for use in the present technology or for use in generating a strain for use in the present technology may have at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity across at least 80% of its whole genome (e.g., across at least 85%, 90%, 95% or 99%, or across its whole genome). Other suitable sequences for use in identifying suitable strains may include hsp60 or repetitive sequences such as BOX, ERIC, (GTG)s or REP (Masco et al. (2003) Systematic and Applied Microbiology, 26:557-563). Suitable strains, such as biotype strains, may have sequences with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of the strain Sn00430.Atty. Dkt. No. 135523-0119

[0069] Alternatively, strains that are biotypes of strain Sn00430 and other Staphylococcus haemolyticus strains that are suitable for use in the present technology, or strains that are biotypes of strain Sn00430 and other Staphylococcus haemolyticus strains that are suitable for genetically engineering to disrupt a patB gene to provide a strain of the present technology may be identified by using strain Sn00430 and restriction fragment analysis and / or PCR analysis, for example by using fluorescent amplified fragment length polymorphism (FAFLP) and repetitive DNA element (rep)-PCR fingerprinting, or protein profiling, or partial 16S or 23 S rDNA sequencing. In some embodiments, such techniques may be used to identify other Staphylococcus haemolyticus strains for use in the present technology, following genetic engineering to disrupt patB.

[0070] In certain embodiments, strains that are biotypes of strain Sn00430 and other Staphylococcus haemolyticus strains that are suitable for use in the present technology, or strains that are biotypes of strain Sn00430 and other Staphylococcus haemolyticus strains that are suitable for genetically engineering to disrupt a patB gene to provide a strain of the present technology, may be strains that provide the same pattern as strain Sn00430 when analyzed by amplified ribosomal DNA restriction analysis (ARDRA), for example when using Sau3 Al restriction enzyme (for exemplary methods and guidance see, for example, Sriitkova et al. (2011) J Microbiol Methods, 87(1): 10-6). Alternatively, biotype strains are identified as strains that have the same carbohydrate fermentation patterns as strain Sn00430.

[0071] Bacterial strains that have similar growth patterns, metabolic type and / or surface antigens to strain Sn00430 may be useful in the present technology. A useful strain will have comparable PatB catalytic activity and / or 3M3SH production activity to strain Sn00430. In particular, a biotype strain will elicit comparable effects on the models shown in the Examples, which may be identified by using the culturing and assay protocols described in the Examples.

[0072] In another aspect, the present disclosure provides a Staphylococcus haemolyticus strain, wherein the strain comprises a mutation in at least one endogenous polynucleotide having about 80% to about 100% sequence identity to SEQ ID NO: 14, such that the strain has a reduced capacity to produce 3-methyl-3-sulfanylhexan-l-ol (3M3SH) when contacted with apocrine sweat or a 3M3SH precursor as compared to a corresponding strain that does not comprise the mutation. In some embodiments, the strain comprises a mutation in atAtty. Dkt. No. 135523-0119least one endogenous polynucleotide having about 80% to about 100% sequence identity to SEQ ID NO: 14. In some embodiments, the strain comprises a mutation in at least one endogenous polynucleotide having about 85% to about 100% sequence identity to SEQ ID NO: 14. In some embodiments, the strain comprises a mutation in at least one endogenous polynucleotide having about 90% to about 95% sequence identity to SEQ ID NO: 14. In some embodiments, the strain comprises a mutation in at least one endogenous polynucleotide having about 95% to about 99% sequence identity to SEQ ID NO: 14. In some embodiments, the strain comprises the mutation in at least one endogenous polynucleotide having about 97% to about 100% sequence identity to SEQ ID NO: 14. In some embodiments, the strain comprises a mutation in at least one endogenous polynucleotide having about 80% sequence identity to SEQ ID NO: 14. In some embodiments, the strain comprises a mutation in at least one endogenous polynucleotide having about 85% sequence identity to SEQ ID NO: 14. In some embodiments, the strain comprises a mutation in at least one endogenous polynucleotide having about 86% sequence identity to SEQ ID NO: 14. In some embodiments, the strain comprises a mutation in at least one endogenous polynucleotide having about 87% sequence identity to SEQ ID NO: 14. In some embodiments, the strain comprises a mutation in at least one endogenous polynucleotide having about 88% sequence identity to SEQ ID NO: 14. In some embodiments, the strain comprises a mutation in at least one endogenous polynucleotide having about 89% sequence identity to SEQ ID NO: 14. In some embodiments, the strain comprises a mutation in at least one endogenous polynucleotide having about 90% sequence identity to SEQ ID NO: 14. In some embodiments, the strain comprises a mutation in at least one endogenous polynucleotide having about 91% sequence identity to SEQ ID NO: 14. In some embodiments, the strain comprises a mutation in at least one endogenous polynucleotide having about 92% sequence identity to SEQ ID NO: 14. In some embodiments, the strain comprises a mutation in at least one endogenous polynucleotide having about 93% sequence identity to SEQ ID NO: 14. In some embodiments, the strain comprises a mutation in at least one endogenous polynucleotide having about 94% sequence identity to SEQ ID NO: 14. In some embodiments, the strain comprises a mutation in at least one endogenous polynucleotide having about 95% sequence identity to SEQ ID NO: 14. In some embodiments, the strain comprises a mutation in at least one endogenous polynucleotide having about 96% sequence identity to SEQ ID NO: 14. In some embodiments, the strain comprises a mutation in at least one endogenous polynucleotide having about 97% sequence identity to SEQ ID NO: 14. In some embodiments, the strainAtty. Dkt. No. 135523-0119comprises a mutation in at least one endogenous polynucleotide having about 98% sequence identity to SEQ ID NO: 14. In some embodiments, the strain comprises a mutation in at least one endogenous polynucleotide having about 99% sequence identity to SEQ ID NO: 14. In some embodiments, the strain grows at an equivalent or improved rate as compared to a Staphylococcus haemolyticus strain lacking the mutation. In some embodiments, the strain is incapable of converting Cys-Gly-3M3SH to 3M3SH. In some embodiments, the mutation comprises a nucleotide substitution. In some embodiments, the nucleotide substitution results in a premature stop codon in the endogenous polynucleotide having about 80% to about 100% sequence identity to the nucleotide sequence as set forth in SEQ ID NO: 14. In some embodiments, the premature stop codon is introduced at the codon that, in the wild-type sequence, encodes for tryptophan at position 190 of the PatB amino acid sequence (W190), which is set forth in SEQ ID NO: 18, such that the mutation introducing the stop codon at W 190 results in the formation of a truncated PatB protein having, such as, for example, the amino acid sequence as set forth in SEQ ID NO: 19. It is to be understood that the nucleic acid sequences provided in SEQ ID NOs: 14 and 15 and the PatB amino acid sequences provided in SEQ ID NOs: 18 and 19 are illustrative, nonlimiting Staphylococcus haemolyticus patB gene and PatB polypeptide sequences, and one of skill in the art would understand that there is a certain degree of nucleotide and amino acid diversity across strains of the same species that is encompassed by the disclosure of the present technology.

[0073] The disclosure of the present technology encompasses genetic engineering of any Staphylococcus haemolyticus nucleotide sequence encoding a PatB polypeptide that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 18, to introduce a disrupt! on / mutati on to the patB gene such that the strain has a reduced capacity to produce 3-methyl-3-sulfanylhexan-l-ol (3M3SH) when contacted with apocrine sweat or a 3M3SH precursor as compared to a corresponding strain that does not comprise the disrupt! on / mutati on. Thus, in some embodiments, the technology of the present disclosure provides a Staphylococcus haemolyticus strain, wherein the strain comprises a mutation in at least one endogenous nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence having about 80% to about 100% sequence identity to SEQ ID NO: 14; and (b) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having about 85% to about 100% sequence identity to SEQ ID NO: 18, such that the strainAtty. Dkt. No. 135523-0119has a reduced capacity to produce 3-methyl-3-sulfanylhexan-l-ol (3M3SH) when contacted with apocrine sweat or a 3M3SH precursor as compared to a corresponding strain that does not comprise the mutation. The mutation may include any of those described herein, including, but not limited to one or more nucleotide substitutions that introduce a premature stop codon into the patB gene sequence. In some embodiments, the wildtype allele of the nucleotide sequence comprising the mutation encodes a PatB polypeptide that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 18.Genome Engineering and Other Methods Encompassed by the Present Disclosure

[0074] The present disclosure contemplates any and all methods for disrupting PatB metabolic enzyme activity and is not limited to any particular method. For example, the present disclosure contemplates any methods for achieving disruptions or mutations including insertions, deletions, substitutions (e.g., transitions, transversion, homologous recombination), transpositions, inversions, knockouts and combinations thereof in the patB gene, in any regulatory elements of the patB gene, or in any other gene or region of interest to achieve the desired result. For example, in some embodiments, a disruption or mutation may include: (a) the introduction, substitution, or removal of one or more nucleotides in a Staphylococcus haemolyticus patB gene (or an open reading frame (ORF) thereof), or the introduction, substitution, or removal of one or more nucleotides in a regulatory element required for the transcription or translation of the patB gene or ORF thereof; (b) a patB gene conversion; (c) a patB gene deletion; (d) the down-regulation of the patB gene; (e) specific mutagenesis of the patB gene; and / or (f) random mutagenesis of the patB gene. Disruptions or mutations of the patB gene, as used herein, include any genetic modification that prevents or reduces the capability of the S. haemolyticus strains of the present technology from producing a functional patB gene product. Such disruptions or mutations may include complete or partial deletion of any portion of patB, including a polypeptide-coding sequence, a promoter, an enhancer, a ribosome-binding site (RBS), or other regulatory elements, or mutagenesis of the same, where mutagenesis encompasses substitutions, insertions, deletions, inversions, and any combinations and variations thereof that inactivate patB and prevent or reduce the production of a patB functional gene product. Mutations or disruptions may involve only a single nucleotide (e.g., a point mutation, nucleotide substitution, or a single nucleotide polymorphism) or multiple nucleotides. In someAtty. Dkt. No. 135523-0119embodiments, the mutation or disruption may be a cytosine (C) to thymine (T) substitution. In some embodiments, the conversion may occur by a cytosine-based editor (CBE) tool. In some embodiments, the CBE precisely converts a cytosine (C) to thymine (T) without causing a double-stand break. In some embodiments, the cytosine (C) to thymine (T) occurs at the W 190 tryptophan amino acid site in patB. In some embodiments, the cytosine (C) to thymine (T) substitution introduces a premature stop codon in the expressed mRNA (z.e., UAG, UGA, or UAA). In some embodiments, the premature stop codon introduces an early termination signal in the mRNA sequence. In some embodiments, the early termination signal leads to production of a truncated protein. In some embodiments, the truncated protein has a loss of catalytic activity. In some embodiments, the mutation or disruption causes a phenotypic change, for example, the expression level of the encoded product is altered, or the encoded product itself is altered. In some embodiments, a disruption or mutation may result in a disrupted gene with decreased levels of expression of a gene product (e.g., protein or RNA) as compared to the wild-type strain. In other embodiments, a disruption or mutation may result in an expressed protein with activity that is lower as compared to the activity of the expressed protein from the wild-type strain. In some embodiments, a gene disruption or mutation results in the removal of the gene from the genome via a substitution, such as homologous recombination.

[0075] In some embodiments, a disruption or mutation of the patB gene, as used herein, includes any genetic modification that prevents or reduces the capability of the S. haemolyticus strains of the present technology from converting Cys-Gly-3M3SH to 3M3SH. This includes, but is not limited to, disruption of one or more PatB protein enzymatic activities or alteration of PatB protein binding with one or more metabolites or co-factors. The skilled artisan would understand that any appropriate method may be used to prevent or reduce the capability of the S. haemolyticus strains of the present technology from converting Cys-Gly-3M3SH to 3M3SH.

[0076] Disruptions or mutations outside of the patB coding region that accomplish the effect of disrupted PatB activity are also encompassed within this disclosure, including disruptions or mutations in enhancer, repressor, promoter, or other regulatory elements that result in decreased transcription or translation of the patB gene. Other methods may also be used to disrupt PatB activity, including, but not limited to engineered transcription factors, including Transcription Activator-Like Effectors (TALEs) that repress patB expression,Atty. Dkt. No. 135523-0119antisense RNAs or small regulatory RNAs that repress patB translation, CRISPR-dCasl3 constructs that target patB RNA and prevent translation, and protein-based or small molecule constructs that post translationally inactivate or repress PatB protein activity. For example, the present disclosure explicitly contemplates constructs (e.g., recombinant proteins) that post-translationally modify the PatB protein (e.g., phosphorylate) to reduce or inhibit enzymatic activity or metabolite binding by the PatB protein. Any construct, and method of use thereof, that downregulates, prevents, or inhibits in any way the transcription, translation, or protein activity of PatB is encompassed within the present disclosure.

[0077] Provided herein are methods and compositions for modifying a target genomic locus in a cell to modulate the expression of the patB gene, the translation of patB RNA, or the enzymatic activity of PatB protein. Targeted genome engineering techniques described herein include the CRISPR (clustered regularly interspaced short palindromic repeats) / Cas (CRISPR-associated) system, meganucleases, zinc finger nucleases (ZFNs), and TAL effector nucleases (TALENs). Such techniques may be employed to bind to and / or cleave a genomic region of interest. In some embodiments, the genome editing techniques described herein generate a specific sequence change or gene edit (e.g., insertion, deletion, or substitution) in the 5’-UTR of the patB gene or a gene involved in the expression of the patB gene, the translation of patB RNA, or the enzymatic activity of PatB protein, such as generating a single nucleotide gene edit to form an out-of-frame start codon upstream of the gene’s ORF, thereby suppressing expression of the gene. In some embodiments, the gene edit (e.g., deletion, insertion, or substitution) results in production of an upstream, out-of-frame start codon that may result in the elimination of protein production or a nonfunctional protein. In some embodiments, the genome editing techniques described herein generate a specific sequence change or gene edit (e.g., insertion, deletion, or substitution) in the coding region or a non-coding region of the patB gene or a gene involved in the expression of the patB gene, the translation of patB RNA, or the enzymatic activity of PatB protein, such as generating a large deletion to form (1) an out-of-frame start codon upstream of the gene’s ORF, thereby suppressing expression of the patB gene or a gene involved in the expression of the patB gene, the translation of patB RNA, or the enzymatic activity of PatB protein, or (2) a non-functional protein product resulting from a frame shift downstream of the gene edit. In some embodiments, the large deletion is greater than 50 bases, greater than 100 bases, greater than 200 bases, greater than 500 bases, greater than 1000 bases, greater than 2000 bases, greater than 5000 bases, or greater than 10000 bases. In some embodiments,Atty. Dkt. No. 135523-0119the large deletion is generated in the patB gene. In some embodiments, a large deletion is a complete deletion of a gene or genetic element. For example, in some embodiments, an ORF is deleted.CRISPR / Cas Systems

[0078] In some embodiments, the methods of the present technology relate to the use of a CRISPR / Cas system that binds to a target site in a region of interest in a genome, wherein the CRISPR / Cas system comprises a CRISPR / Cas nuclease and an engineered crRNA / tracrRNA (or single guide RNA (sgRNA) or guide RNA (gRNA)). In some embodiments, the CRISPR system generally comprises (i) a polynucleotide encoding a Cas protein, and (ii) at least one sgRNA for RNA-guided genome engineering in S. haemolyticus cells.

[0079] Non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Casl2a (also known as Cpfl), Csyl, Csy2, Cys3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Smrl, Cmr3, Cmr4, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. In some embodiments, the Cas protein is a Streptococcus pyogenes Cas9 protein. In some embodiments, the Cas protein is a Casl2a (Cpfl) protein. In some embodiments, the Cas protein is a Csml protein. These enzymes are known. For example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2. The amino acid sequence of Francisella tularensis subsp. Novicida Cpfl protein may be found in the UniProt database under accession number A0Q7Q2. The amino acid sequence of Thermococcus onnurineus Csml protein may be found in the UniProt database under accession number B6YWB8.

[0080] The sgRNA molecules comprise a crRNA-tacrRNA scaffold polynucleotide and a targeting sequence corresponding to a genomic target of interest. In some embodiments, the CRISPR / Cas system recognizes a target site in the patB gene or a gene involved in the expression of the patB gene, the translation of patB RNA, or the enzymatic activity of PatB protein. In some embodiments, the CRISPR / Cas system recognizes a target in the patB gene or a regulatory region thereof. The CRISPR / Cas system as described herein may bind to and / or cleave the region of interest in a region upstream of the coding region of the patBAtty. Dkt. No. 135523-0119gene or a gene involved in the expression of the patB gene, the translation of patB RNA, or the enzymatic activity of PatB protein. In some embodiments, the CRISPR / Cas system generates a specific sequence change in the 5’-UTR of the patB gene or a gene involved in the expression of the patB gene, the translation of patB RNA, or the enzymatic activity of PatB protein, such as generating a single nucleotide gene edit to form an out-of-frame start codon upstream of the gene’s ORF. In some embodiments, the gene edit (e.g., deletion, insertion, or substitution) results in production of an upstream, out-of-frame start codon that may result in the elimination of protein production or a nonfunctional protein. In some embodiments, the CRISPR / Cas system generates a specific sequence change or gene edit (e.g., insertion, deletion, or substitution) in the coding region or a non-coding region of the patB gene or a gene involved in the expression of the patB gene, the translation of patB RNA, or the enzymatic activity of PatB protein, such as generating a large deletion to form (1) an out-of-frame start codon upstream of the gene’s ORF, thereby suppressing expression of the gene, or (2) a non-functional protein product resulting from a frame shift downstream of the gene edit. In some embodiments, the large deletion is greater than 50 bases, greater than 100 bases, greater than 200 bases, greater than 500 bases, greater than 1000 bases, greater than 2000 bases, greater than 5000 bases, or greater than 10000 bases.

[0081] The CRISPR / Cas system can be based on the Cas9 nuclease and an engineered single guide RNA (sgRNA) that specifies the targeted nucleic acid sequence. Cas9 is a large monomeric DNA nuclease guided to a DNA target sequence adjacent to the PAM (protospacer adjacent motif) sequence motif by a complex of two non-coding RNAs:CRISPR RNA (crRNA) and trans-activating crRNA (tacrRNA). The Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand whereas the RuvC-like domain cleaves the non-complementary strand and, as a result, a blunt cut is introduced in the target DNA. Heterologous expression of Cas9 together with an sgRNA can induce site-specific double strand breaks (DSBs) into genomic DNA of live cells. See, e.g., Mussolino, Nat.Biothechnol., 31:208-209 (2013). In some embodiments, the Cas9 protein is expressed in a S. haemolyticus cell as a fusion protein. In some embodiments, the Cas9 protein is tagged (e.g, FLAG- or GFP-tagged). In some embodiments, promoters (e.g, PXyl tet, pCap, BBa_J23100-BBa_B0030, pHla, pBla, or pHelp) may be used to drive Cas9 expression in a S. haemolyticus cell. In some embodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes, or S. thermophiles Cas9, and may include mutated Cas9 derived from theseAtty. Dkt. No. 135523-0119organisms. The enzyme may be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme (e.g., Cas9 enzyme) is codon-optimized for expression in a S. haemolyticus cell.

[0082] The CRISPR / Cas system can be based on the Cpfl nuclease and an engineered single guide RNA (sgRNA) that specifies the targeted nucleic acid sequence.

[0083] Cpfl is distinguished from Cas9 by a its single RuvC endonuclease active site, its 5' protospacer adjacent motif preference, and for creating sticky rather than blunt ends at the cut site. The Cpfl protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9. Cpfl does not have a HNH endonuclease domain, and the N-terminal of Cpfl does not have an alpha-helical recognition lobe, unlike Cas9. In some embodiments, the Cpfl protein is tagged (e.g., FLAG- or GFP-tagged). In some embodiments, promoters (e.g., PXyl tet, pCap, BBa_J23100-BBa_B0030, pHla, pBla, or pHelp) may be used to drive Cpfl expression in a S. haemolyticus cell. In some embodiments, the Cpfl enzyme is Francisella tularensis subsp. Novicida Cpfl, and may include mutated Cpfl derived from these organisms. The enzyme may be a Cpfl homolog or ortholog. In some embodiments, the CRISPR enzyme (e.g., Cpfl enzyme) is codon-optimized for expression in a S. haemolyticus cell.

[0084] The CRISPR / Cas system can be based on the Csml nuclease and an engineered single guide RNA (sgRNA) that specifies the targeted nucleic acid sequence.

[0085] Csml belongs to the CaslO family of endonucleases. Csml is the largest subunit of the Csm interference complex in the type III- A CRISPR system. Csml exhibits ssDNA-specific endo- and exonuclease activity. In some embodiments, promoters (e.g, PXyl tet, pCap, BBa_J23100-BBa_B0030, pHla, pBla, or pHelp) may be used to drive Csml expression in a S. haemolyticus cell. In some embodiments, the Csml enzyme is Thermococcus onnurineus Csml, and may include mutated Csml derived from these organisms. The enzyme may be a Csml homolog or ortholog. In some embodiments, the CRISPR enzyme (e.g., Csml enzyme) is codon-optimized for expression in a S. haemolyticus cell.

[0086] The single guide RNA (sgRNA) is the second component of the CRISPR / Cas system that forms a complex with a Cas nuclease. The sgRNA is created by fusing crRNA with tacrRNA. The sgRNA guide sequence located at the 5’ end confers DNA targetAtty. Dkt. No. 135523-0119specificity. By modifying the guide sequence, sgRNAs with different target specificities can be designed to target any desired endogenous gene. In some embodiments, the target sequence is about 1,000, about 975, about 950, about 925, about 900, about 875, about 850, about 825, about 800, about 775, about 750, about 725, about 700, about 675, about 650, about 625, about 600, about 575, about 550, about 525, about 500, about 475, about 450, about 425, about 400, about 375, about 350, about 325, about 300, about 275, about 250, about 225, about 200, about 175, about 150, about 125, about 100, about 90, about 80, about 70, about 60, about 50, about 40, about 30, about 20, or about 15 base pairs upstream of the transcription start site, or the target sequence may be any number of base pairs in-between these values upstream of the transcription start site. In some embodiments, the target sequence is about 1 to about 10 base pairs upstream of the transcription start site (e.g., positions -10, -9, -8, -7, -6, -5, -4, -3, -2, or -1). In some embodiments, the target sequence is located within the open reading frame of the gene of interest. In some embodiments, the target sequence is located within a coding region of the gene of interest. In some embodiments, the CRISPR / Cas system comprises at least one sgRNA. In some embodiments, a target sequence of the at least one sgRNA is about 1,000, about 975, about 950, about 925, about 900, about 875, about 850, about 825, about 800, about 775, about 750, about 725, about 700, about 675, about 650, about 625, about 600, about 575, about 550, about 525, about 500, about 475, about 450, about 425, about 400, about 375, about 350, about 325, about 300, about 275, about 250, about 225, about 200, about 175, about 150, about 125, about 100, about 90, about 80, about 70, about 60, about 50, about 40, about 30, about 20, or about 15 base pairs upstream of the transcription start site, or the target sequence may be any number of base pairs in-between these values upstream of the transcription start site. In some embodiments, the target sequence of the at least one sgRNA is about 1 to about 10 base pairs upstream of the transcription start site (e.g., positions -10, -9, -8, -7, -6, -5, -4, -3, -2, or -1). In some embodiments, the target sequence of the at least one sgRNA is located within the open reading frame of the gene of interest. In some embodiments, the target sequence of the at least one sgRNA is located within a coding region of the gene of interest. In some embodiments, the target sequences of the at least one sgRNA is located within the open reading frame of the gene of interest. In some embodiments, the target sequences of the at least one sgRNA is located within a coding region of the gene of interest. In some embodiments, the CRISPR / Cas system comprises one sgRNA, wherein the one sgRNA targets two regions of a gene having the same sequence, such as two inverted terminal repeats (ITRs). In some embodiments, the targetAtty. Dkt. No. 135523-0119sequences of the sgRNA are separated by at least 50 bases, at least 100 bases, at least 200 bases, at least 500 bases, at least 1000 bases, at least 2000 bases, at least 5000 bases, or at least 10000 bases.

[0087] In some embodiments, the CRISPR / Cas system comprises at least two sgRNAs. In some embodiments, a target sequence of at least one of the at least two sgRNAs is about 1,000, about 975, about 950, about 925, about 900, about 875, about 850, about 825, about 800, about 775, about 750, about 725, about 700, about 675, about 650, about 625, about 600, about 575, about 550, about 525, about 500, about 475, about 450, about 425, about 400, about 375, about 350, about 325, about 300, about 275, about 250, about 225, about 200, about 175, about 150, about 125, about 100, about 90, about 80, about 70, about 60, about 50, about 40, about 30, about 20, or about 15 base pairs upstream of the transcription start site, or the target sequence may be any number of base pairs in-between these values upstream of the transcription start site. In some embodiments, the target sequence of at least one of the at least two sgRNAs is about 1 to about 10 base pairs upstream of the transcription start site (e.g., positions -10, -9, -8, -7, -6, -5, -4, -3, -2, or -1). In some embodiments, the target sequence of at least one of the at least two sgRNAs is located within the open reading frame of the gene of interest. In some embodiments, the target sequence of at least one of the at least two sgRNAs is located within a coding region of the gene of interest. In some embodiments, the target sequences of at least two of the at least two sgRNAs are located within the open reading frame of the gene of interest. In some embodiments, the target sequences of at least two of the at least two sgRNAs are located within a coding region of the gene of interest. In some embodiments, the CRISPR / Cas system comprises two sgRNAs, wherein the two sgRNAs have non-overlapping target sequences. In some embodiments, the target sequences of the two sgRNAs are separated by at least 50 bases, at least 100 bases, at least 200 bases, at least 500 bases, at least 1000 bases, at least 2000 bases, at least 5000 bases, or at least 10000 bases.

[0088] It is not intended that the present technology be limited to any particular distance restraint with regard to the location of the guide RNA target sequence from the gene transcription start site. In some embodiments, the target sequence lies “in proximity to” a gene of interest, where “in proximity to” refers to any distance from the gene of interest, wherein the Cas-regulatory domain fusion is able to exert an effect on gene expression. In some embodiments, the target sequence lies upstream of the ORF of the gene of interest.Atty. Dkt. No. 135523-0119

[0089] The canonical length of the guide sequence is about 20 bp and the DNA target sequence is about 20 bp followed by a PAM sequence having the consensus NGG sequence. In some embodiments, sgRNAs are expressed in a S. haemolyticus cell using S. haemolyticus promoters.

[0090] When the DSBs are repaired by either non-homologous end joining (NHEJ) or homology directed repair (HDR), the sequence at the repair site can be modified or new genetic information can be inserted (e.g., donor DNA comprising a desired gene edit can be inserted into the target gene at the break site). Although HDR typically occurs at lower and more variable frequencies than NHEJ, it can be leveraged to generate precise, defined modifications at a target locus in the presence of an exogenously introduced repair template. Accordingly, exogenous repair templates, designed by methods known in the art, can also be delivered into a cell, most often in the form of a synthetic, single-stranded DNA donor oligo or DNA donor plasmid, to generate a precise change in the genome. Single-stranded DNA donor oligos are delivered into a cell to insert or change short sequences (SNPs, amino acid substitutions, epitope tags, efc.) of DNA in the endogenous genomic target region. The benefits of using a synthetic DNA donor oligo are that no cloning is required to generate the donor template and DNA modifications can be added during synthesis for different applications, such as increased resistance to nucleases. Traditionally, the maximum insert length recommended for use with a DNA donor oligo is about 50 nucleotides.

[0091] In some embodiments, the present technology provides an engineered, programmable, non-naturally occurring CRISPR / Cas system comprising a Cas9 protein and one or more single guide RNAs (sgRNAs) that target the genomic loci of the patB gene or a gene involved in the expression of the patB gene, the translation of patB RNA, or the enzymatic activity of PatB protein, and the Cas9 protein cleaves the genomic loci of the patB gene or a gene involved in the expression of the patB gene, the translation of patB RNA, or the enzymatic activity of PatB protein, whereby expression of the one or more gene products is altered. In some embodiments, Cas9 introduces multiple DSBs in the same cell ( / .< ., multiplexes) via expression of one or more distinct guide RNAs.

[0092] In some embodiments, the present technology provides a method for targeted genomic modification of S. haemolyticus cells to alter the expression of the patB gene or a gene involved in the expression of the patB gene, the translation of patB RNA, or theAtty. Dkt. No. 135523-0119enzymatic activity of PatB protein, the method comprising introducing into a S. haemolyticus cell containing and expressing a DNA molecule having a target sequence and encoding the patB gene or a gene involved in the expression of the patB gene, the translation of patB RNA, or the enzymatic activity of PatB protein an engineered CRISPR / Cas system comprising (a) an expression construct comprising a first polynucleotide encoding a Cas9 protein, or a variant thereof or a fusion protein therewith, and a second polynucleotide encoding a guide RNA comprising: (i) a crRNA-tracrRNA scaffold polynucleotide, and (ii) a targeting sequence operably linked to the crRNA-tracrRNA scaffold polynucleotide, where the targeting sequence corresponds to a genomic locus of interest, and (b) delivering the expression construct into the S. haemolyticus cell, where the first and second polynucleotides are expressed (transcribed) within the S. haemolyticus cell. This method can optionally further include visualizing, identifying, or selecting for S. haemolyticus cells having a genomic modification at the genomic locus of interest that is induced by the delivering the expression construct into the S. haemolyticus cells.

[0093] In some embodiments of the methods of the present technology, the Cas9 polypeptide and one or more guide RNA are encoded on a single vector. In some embodiments, the single vector is a plasmid. In some embodiments of the methods of the present technology, the Cas9 polypeptide and the one or more guide RNA are encoded on two separate vectors. In these methods, the steps generally follow the sequence of introducing into a S. haemolyticus cell containing and expressing a DNA molecule having a target sequence and encoding the patB gene or a gene involved in the expression of the patB gene, the translation of patB RNA, or the enzymatic activity of PatB protein an engineered CRISPR / Cas system comprising (a) a Cas9 polynucleotide or a conservative variant thereof, and a guide RNA comprising (i) a crRNA-tracrRNA scaffold polynucleotide, and (ii) a targeting sequence operably linked to the crRNA-tracrRNA scaffold polynucleotide, with the targeting sequence corresponding to a genomic locus of interest, and (b) delivering the two polynucleotides into the S. haemolyticus cell. In variations of this method, a donor polynucleotide having homology to the genomic target of interest is included in a cotransfection. In some variations of these methods, the transfected material can be either plasmid DNA or RNA generated by in vitro transcription. In still other variations, the methods for targeted genomic modification are multiplexed, meaning that more than one genomic locus is targeted for modification. In still other variations of these methods, theAtty. Dkt. No. 135523-0119transformation of the S. haemolyticus cells can be followed by visualizing, identifying, or selecting for S. haemolyticus cells having a genomic modification at the genomic locus of interest.Meganucleases

[0094] In some embodiments, the compositions and methods described herein employ a meganuclease DNA binding domain for binding to a region of interest in the genome of a S. haemolyticus cell. Meganucleases are engineered versions of naturally occurring restriction enzymes that typically have extended DNA recognition sequences (e.g., about 14 to about 40 base pairs in length). Meganucleases (also known as homing endonucleases) are commonly grouped into five families based on sequence and structure motifs: the LAGLID ADG family (“LAGLID ADG” is disclosed as SEQ ID NO: 20), the GIY-YIG family, the His-Cyst box family, the PD-(DZE)XK family, and the HNH family. In some embodiments, the meganuclease comprises an engineered homing endonuclease. The recognition sequences of homing endonucleases and meganucleases such as I-Sce, I-Ceul, PI-PspI, PI-5ce, I-5ceIV, \-('sm\, \-P n\, I-5ceII, I-Ppol, I-5ceIII, I-Crel, I-TevI, I-TevII, and LTevIII are known.

[0095] In some embodiments, the meganuclease is tailored to recognize a target in one or more of the patB gene or a gene involved in the expression of the patB gene, the translation of patB RNA, or the enzymatic activity of PatB protein. The meganucleases as described herein may bind to and / or cleave the region of interest in a region upstream of the coding region of the patB gene or a gene involved in the expression of the patB gene, the translation of patB RNA, or the enzymatic activity of PatB protein. Gene insertion or correction can be achieved by the introduction of a DNA repair matrix containing sequences homologous to the endogenous sequence surrounding the DNA break. Gene edits can be created either at or distal to the break. In some embodiments, the meganuclease generates a specific sequence change in the 5’-UTR of the patB gene or a gene involved in the expression of the patB gene, the translation of patB RNA, or the enzymatic activity of PatB protein, such as generating a single nucleotide gene edit to form an out-of-frame start codon upstream of the gene’s ORF.Atty. Dkt. No. 135523-0119TALENs

[0096] In some embodiments, the compositions and methods described herein employ transcription activator-like effector nucleases (TALENs) to edit S. haemolyticus genomes by inducing double-strand breaks (DSBs). TALENs are restriction enzymes that can be engineered to cleave specific sequences of DNA. TALENs are constructed by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (e.g., a nuclease domain such as that derived from the FokI endonuclease). Transcription activator-like effectors (TALEs) can be engineered according to methods known in the art to bind to a desired DNA sequence, and when combined with a nuclease, provide a technique for cutting DNA at specific locations. For example, after a target sequence in the patB gene or a gene involved in the expression of the patB gene, the translation of patB RNA, or the enzymatic activity of PatB protein is identified, a corresponding TALEN sequence is engineered and inserted into a plasmid. The plasmid is inserted into a target cell where it is translated to produce a functional TALEN, which then binds to and cleaves its target sequence. Such an approach can be employed to introduce an exogenous DNA sequence into the target gene as the DSB is being repaired through either homology-directed repair or non-homologous endjoining. For example, in some embodiments, the use of TALEN technology generates a specific sequence change (e.g., insertion, deletion, or substitution) in the 5’-UTR of the patB gene or a gene involved in the expression of the patB gene, the translation of patB RNA, or the enzymatic activity of PatB protein, resulting in the production of an out-of-frame start codon upstream of the gene’s ORF.ZFNs

[0097] In some embodiments, the compositions and methods described herein employ zinc finger nucleases (ZFNs) to edit S. haemolyticus genomes by inducing double-strand breaks (DSBs). ZFNs are artificial restriction enzymes generated by fusing a zinc finder DNA-binding domain to a DNA cleavage domain (e.g., a nuclease domain such as that derived from the FokI endonuclease). ZFNs can be engineered to bind and cleave DNA at specific locations. ZFNs contain two protein domains. The first domain is the DNA-binding domain, which contains eukaryotic transcription factors and the zinc finger. The second domain is a nuclease domain that contains the FokI restriction enzyme responsible for cleaving DNA. ZFNs can be engineered according to methods known in the art to bind to a desired DNA sequence and cleave DNA at specific locations. For example, after a targetAtty. Dkt. No. 135523-0119sequence in the patB gene or a gene involved in the expression of the patB gene, the translation of patB RNA, or the enzymatic activity of PatB protein is identified, a corresponding ZFN sequence is engineered and inserted into a plasmid. The plasmid is inserted into a target cell where it is translated to produce a functional ZFN, which then binds to and cleaves its target sequence introducing a double strand break (DSB). Such an approach can be employed to introduce an exogenous DNA sequence into the target gene as the DSB is being repaired through either homology-directed repair or non-homologous endjoining. For example, in some embodiments, the use of ZFN technology generates a specific sequence change in the 5’-UTR of the patB gene or a gene involved in the expression of the patB gene, the translation of patB RNA, or the enzymatic activity of PatB protein, such as the insertion of an out-of-frame start codon upstream of the gene’s ORF.Additional Methodologies for Suppressing PatB activity

[0098] The expression and activity of PatB may be reduced by introducing mutations / disruptions as described herein for patB, or may be reduced by other well-known means of reducing expression of polynucleotides and activity of polypeptides including but not limited to the use of interfering RNAs developed from nucleic acids encoding PatB. As known in the art, “interfering RNA” is RNA capable of causing gene silencing. Interfering RNA (RNAi), as used herein, includes any type of RNA molecule capable of downregulating or silencing expression of a target patB nucleic acid, including but not limited to sense RNA, antisense RNA, short interfering RNA (siRNA), micro RNA (miRNA), doublestranded RNA (dsRNA), hairpin RNA, and the like. In some embodiments, methods and constructs are provided for suppressing PatB metabolic enzyme activity, such as, but not limited to, antisense, sense co-suppression, RNAi, and artificial microRNA approaches.

[0099] RNAi techniques involve stable transformation using RNAi plasmid constructs (Helliwell and Waterhouse, Methods Enzymol. 392:24-35 (2005)). Such plasmids are composed of a fragment of the target gene to be silenced in an inverted repeat structure. The inverted repeats are separated by a spacer, often an intron. The RNAi construct may be driven by a suitable promoter, for example, the PXyl tet, pCap, BBa_J23100-BBa_B0030, pHla, pBla, or pHelp promoter, and integrated into the S. haemolyticus genome and subsequent transcription of the transgene leads to an RNA molecule that folds back on itself to form a double-stranded hairpin RNA (hpRNA).Atty. Dkt. No. 135523-0119

[0100] Artificial microRNA (amiRNA) techniques exploit the microRNA (miRNA) pathway that functions to silence endogenous genes. In this method, 21 nucleotide long fragments of the gene to be silenced are introduced into a pre-miRNA gene to form a pre-amiRNA construct. The pre-miRNA construct is transferred into the genome using transformation methods apparent to one skilled in the art. After transcription of the pre-amiRNA, processing yields amiRNAs that target genes, which share nucleotide identity with the 21 nucleotide amiRNA sequence.

[0101] In RNAi silencing techniques, two factors can influence the choice of length of the fragment. The shorter the fragment the less frequently effective silencing will be achieved, but very long hairpins increase the chance of recombination in bacterial host strains. The effectiveness of silencing also appears to be gene dependent and could reflect accessibility of target mRNA or the relative abundances of the target mRNA and the hpRNA in cells in which the gene is active. A fragment length of between 100 and 800 bp, preferably between 300 and 600 bp, is generally suitable to maximize the efficiency of silencing obtained. The other consideration is the part of the gene to be targeted. 5' UTR, coding region, and 3' UTR fragments can be used with equally good results. As the mechanism of silencing depends on sequence homology there is potential for cross-silencing of related mRNA sequences. Where this is not desirable a region with low sequence similarity to other sequences, such as a 5' or 3' UTR, should be chosen. The rule for avoiding cross-homology silencing appears to be to use sequences that do not have blocks of sequence identity of over 20 bases between the construct and the non-target gene sequences. Many of these same principles apply to selection of target regions for designing amiRNAs.

[0102] Antisense techniques involve introducing an antisense oligonucleotide that will bind to the messenger RNA (mRNA) produced by the gene of interest. The “antisense” oligonucleotide has a base sequence complementary to the gene’s messenger RNA (mRNA), which is called the “sense” sequence. Activity of the sense segment of the mRNA is blocked by the anti-sense mRNA segment, thereby effectively inactivating gene expression.

[0103] Sense co-suppression techniques involve introducing a highly expressed sense transgene resulting in reduced expression of both the transgene and the endogenous gene (Depicker and van Montagu, Curr. Opin. Cell Biol. 9: 373-82 (1997)). The effect depends on sequence identity between transgene and endogenous gene.Atty. Dkt. No. 135523-0119IV. Deodorant Compositions: Methods and Modes of Administration and Application

[0104] The following discussion is presented by way of example only, and is not intended to be limiting.

[0105] One aspect of the present technology includes methods of treating or preventing body odor in a subject. In deodorizing applications, compositions comprising one or more ApatB -Staphylococcus haemolyticus strains of the present technology (e.g., Sn00430) or variants thereof, are administered to a subject suspected of, or already suffering from body odor, in an amount sufficient to reduce or eliminate the odor. Without wishing to be bound by theory, the one or more ApatB-Staphylococcus haemolyticus strains of the present technology may persist on the skin of a subject for a period of time, whereupon they do not engage in 3M3SH production or have reduced 3M3SH production and can reduce overall rates of 3M3SH production or the total amount thereof, possibly by reducing the amount of Gly-Cys-3M3SH available to other Staphylococcus bacteria present on the skin. In some embodiments, the genetically engineered Staphylococcus haemolyticus strain of the compositions persists on the human skin for a period of time (e.g., at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 120 hours, at least about 144 hours, at least about 1 week).

[0106] Compositions or pharmaceutical compositions of the present technology (e.g., deodorant compositions) can be formulated for topical administration, including for administration to a subject as a topical deodorant. In some embodiments, the deodorant compositions or pharmaceutical compositions are a topical deodorant. In some embodiments, the deodorant compositions or pharmaceutical compositions are formulated into a topical deodorant. In some embodiments, the subject is a mammal. In some embodiments, the mammalian subject is human. The deodorant compositions or pharmaceutical compositions comprising the bacterial strains or the genetically engineered Staphylococcus haemolyticus strains (e.g., ApatB-Staphylococcus haemolyticus, Sn00430, or variant strain thereof) of the present technology can be applied to any dermal location on a subject, including but not limited to the armpit, groin, nipples, and / or anywhere that glands, such as apocrine sweat glands, are located. Additionally, the deodorantAtty. Dkt. No. 135523-0119compositions or pharmaceutical compositions as disclosed herein can be formulated using any appropriate means, including as a deodorant stick, a roll-on, a cream, a lotion, a gel, a powder, a water activated cream, a water activated powder, an injectable, a patch, or a spray. In some embodiments, the compositions or pharmaceutical compositions of the present technology comprise one or more lyophilized or desiccated bacterial strains of the present technology (e.g., ApatB-Staphylococcus haemolyticus, Sn00430, or a variant thereof), optionally in a powdered form, which can be mixed with water or a watercontaining composition and subsequently applied to a subject as a topical deodorant, optionally to the armpits. In some embodiments, the water or a water-containing composition can be naturally occurring sweat from the subject. In some embodiments, the deodorant compositions or pharmaceutical compositions as disclosed herein comprise lyophilized or desiccated strains of the present technology. In some embodiments, the topical compositions or pharmaceutical compositions comprise lyophilized or desiccated strains of the present technology at an amount of about 0.0001% to about 35% by weight of the total composition (wt. %). In some embodiments, the deodorant compositions or pharmaceutical compositions comprise the lyophilized or desiccated strains at an amount of about 0.001 wt. % to about 15 wt. %. In some embodiments, the deodorant compositions or pharmaceutical compositions comprise the lyophilized or desiccated strains at an amount of about 0.01 wt. % to about 10 wt. %. In some embodiments, the deodorant compositions or pharmaceutical compositions comprise the lyophilized or desiccated strains at an amount of about 0.1 wt. % to about 10 wt. %. In some embodiments, the deodorant compositions or pharmaceutical compositions comprise the lyophilized or desiccated strains at an amount of about 0.15 wt. % to about 10 wt. %. In some embodiments, the deodorant compositions or pharmaceutical compositions comprise the lyophilized or desiccated strains at an amount of about 0.2 wt. % to about 10 wt. %. In some embodiments, the deodorant compositions or pharmaceutical compositions comprise the lyophilized or desiccated strains at an amount of about 0.25 wt. % to about 10 wt. %. In some embodiments, the deodorant compositions or pharmaceutical compositions comprise the lyophilized or desiccated strains at an amount of about 0.25 wt. % to about 9 wt. %. In some embodiments, the deodorant compositions or pharmaceutical compositions comprise the lyophilized or desiccated strains at an amount of about 0.25 wt. % to about 8 wt. %. In some embodiments, the deodorant compositions or pharmaceutical compositions comprise the lyophilized or desiccated strains at an amount of about 0.25 wt. % to about 7 wt. %. In some embodiments, the deodorant compositions or pharmaceutical compositions comprise the lyophilized or desiccated strains at an amount ofAtty. Dkt. No. 135523-0119about 0.25 wt. % to about 6 wt. %. In some embodiments, the deodorant compositions or pharmaceutical compositions comprise the lyophilized or desiccated strains at an amount of about 0.25 wt. % to about 5 wt. %. In some embodiments, the deodorant compositions or pharmaceutical compositions comprise the lyophilized or desiccated strains at an amount of about 0.25 wt. % to about 4 wt. %. In some embodiments, the deodorant compositions or pharmaceutical compositions comprise the lyophilized or desiccated strains at an amount of about 0.25 wt. % to about 3 wt. %. In some embodiments, the deodorant compositions or pharmaceutical compositions comprise the lyophilized or desiccated strains at an amount of about 0.25 wt. % to about 2 wt. %. In some embodiments, the deodorant compositions or pharmaceutical compositions comprise the lyophilized or desiccated strains at an amount of about 0.25 wt. % to about 1 wt. %. In some embodiments, the deodorant compositions or pharmaceutical compositions comprise the lyophilized strains or desiccated at an amount of about 0.25 wt. % to about 0.50 wt. %. In some embodiments, the amount of acceptable carrier or excipient in the deodorant compositions or pharmaceutical compositions ranges from about 65 wt. % to about 99.9999 wt. %. In some embodiments, the amount of acceptable carrier or excipient in the deodorant compositions or pharmaceutical compositions ranges from about 90 wt. % to about 99.9999 wt. %. In some embodiments, the topical deodorant compositions and or pharmaceutical compositions comprise about 0.25 wt. % to about 5 wt. % lyophilized strains of the present technology and about 95 wt. % to about 99.75 wt. % acceptable carrier or excipient. Herein, any of the upper limits of the amounts of the components of the deodorant compositions or pharmaceutical compositions disclosed can be combined with any of the lower limits of the amounts of the components of the deodorant compositions or pharmaceutical compositions. In some embodiments, the excipient is polyethylene glycol (PEG), Isopropyl Myristate, or sorbitol, or a combination of excipients. In some embodiments, the topical deodorant comprises about 105to about 1014viable bacteria per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, the topical deodorant comprises about 106to about 1013viable bacteria per gram of the deodorant compositions or pharmaceutical composition. In some embodiments, the topical deodorant comprises about 107to about 1011viable bacteria per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, the topical deodorant comprises about 105viable bacteria per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, the topical deodorant comprises about 106viable bacteria per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, the topicalAtty. Dkt. No. 135523-0119deodorant comprises about 107viable bacteria per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, the topical deodorant comprises about 108viable bacteria per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, the topical deodorant comprises about 109viable bacteria per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, the topical deodorant comprises about IO10viable bacteria per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, the topical deodorant comprises about 1011viable bacteria per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, the topical deodorant comprises about 1012viable bacteria per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, the topical deodorant comprises about 1013viable bacteria per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, the topical deodorant comprises about 1014viable bacteria per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, the amount of viable bacteria as disclosed herein is a colony-forming unit (CFU). For example, in some embodiments, the topical deodorant comprises about 105to about 1014viable CFUs per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, the topical deodorant comprises about 106to about 1013viable CFUs per gram of the deodorant compositions or pharmaceutical composition. In some embodiments, the topical deodorant comprises about 107to about 1011viable CFUs per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, the topical deodorant comprises about 105viable CFUs per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, the topical deodorant comprises about 106viable CFUs per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, the topical deodorant comprises about 107viable CFUs per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, the topical deodorant comprises about 108viable CFUs per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, the topical deodorant comprises about 109viable CFUs per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, the topical deodorant comprises about IO10CFUs bacteria per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, the topical deodorant comprises about 1011viable CFUs per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, the topical deodorant comprises about 1012viable CFUs per gram of the deodorant compositions orAtty. Dkt. No. 135523-0119pharmaceutical compositions. In some embodiments, the topical deodorant comprises about 1013viable CFUs per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, the topical deodorant comprises about 1014viable CFUs per gram of the deodorant compositions or pharmaceutical compositions. In some embodiments, topical deodorant application of the strains, deodorant compositions, and pharmaceutical compositions of the present technology deposits about 0.1 grams to about 2 grams of the product at the application location. In some embodiments, about 0.1 grams, about 0.2 grams, about 0.4 grams, about 0.6 grams, about 0.8 grams, about 1 gram, about 1.2 grams, about 1.4 grams, about 1.6 grams, about 1.8 grams, or about 2 grams or more of the present technology is deposited at the application location.

[0107] In some embodiments, the compositions of the present technology include an acceptable carrier. In some embodiments, the acceptable carrier comprises oleic acid, stearic acid, almond butter, avocado butter, babassu butter, beeswax, cocoa butter, coconut butter, coconut oil, cupuacu butter, hemp seed butter, kokum butter, macadamia butter, mango butter, mowrah butter, olive butter, sal seed butter, shea butter, propylene glycol, glycerin, stearyl alcohol, myristyl alcohol, sunflower extract, capric triglyceride, or any combination thereof. Additional components of the compositions of the present technology may include a preservative selected from the group consisting of sucrose, sodium ascorbate, and glutathione. In some embodiments the preservative is a cryoprotectant selected from the group consisting of a nucleotide, a disaccharide, a polyol, and a polysaccharide. In some embodiments, the cryoprotectant is selected from the group consisting of inosine-5’-monophosphate (IMP), guanosine-5 ’-monophosphate (GMP), adenosine-5’ -monophosphate (AMP), uranosine-5’ -monophosphate (UMP), cytidine-5’ -monophosphate (CMP), adenine, guanine, uracil, cytosine, guanosine, uridine, cytidine, hypoxanthine, xanthine, orotidine, thymidine, inosine, trehalose, maltose, lactose, sucrose, sorbitol, mannitol, dextrin, inulin, sodium ascorbate, glutathione, and skim milk.

[0108] In some embodiments, the composition comprises a lyoprotectant. In some embodiments, the lyoprotectant is a protein, a carbohydrate, or a combination thereof. In some embodiments, the lyoprotectant is a milk protein, and in particular a micellar casein. In some embodiments, the carbohydrate is sucrose. In some embodiments, the lyoprotectant is present in an amount of about 0.1% by weight of the composition to about 15% by weight of the composition.Atty. Dkt. No. 135523-0119

[0109] In some embodiments, the compositions of the present technology comprise one or more ApatB-Staphylococcus haemolyticus strains of the present technology and an additional probiotic agent (wild-type Staphylococcus epidermidis). that may improve engraftment efficacy for the the ApatB-Staphylococcus haemolyticus strains of the present technology.

[0110] In some embodiments, the disclosure of the present technology relates to a method of manufacturing the deodorant compositions, comprising homogeneously dispersing the ApatB-Staphylococcus haemolyticus strains (e.g., Sn00430) of the present technology in an acceptable carrier or excipient. In some embodiments, the excipient is polyethylene glycol (PEG), Isopropyl Myristate, or sorbitol. In some embodiments, the excipient is polyethylene glycol (PEG). In some embodiments, the excipient is Isopropyl Myristate. In some embodiments, the excipient is sorbitol.[OHl] In another aspect, the present disclosure provides an article of manufacture comprising a composition of any of the preceding embodiments. In some embodiments, the article of manufacture is a deodorant stick. In some embodiments, the article of manufacture comprises one or more chambers for housing one or more lyophilized strains of the present technology. In some embodiments, the article of manufacture comprises one or more lyophilized strains of the present technology and a carrier in separate chambers, wherein the article of manufacture can be used by inducing the mixture of the one or more lyophilized strains and the carrier prior to application of the mixture to the skin of a subject.

[0112] The strains, compositions, and pharmaceutical compositions of the present technology can be administered to a subject one or more times per day. In some embodiments, the strains and compositions are administered more than once per day, including, for example, twice per day or three times per day, etc. In some embodiments, the strains and compositions are administered once per day. In some embodiments, the strains, compositions and pharmaceutical compositions are administered once every 2 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 3 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 4 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 5 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 6 days. In some embodiments, the strains, compositions, andAtty. Dkt. No. 135523-0119pharmaceutical compositions are administered once every 7 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 8 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 9 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 10 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 11 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 12 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 13 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 14 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 15 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 16 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 17 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 18 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 19 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 20 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 21 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 22 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 23 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 24 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 25 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 26 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 27 days. In some embodiments, the strains, compositions, and pharmaceutical compositions are administered once every 28 days.

[0113] In some embodiments, administering the strains and compositions of the present technology results in a reduction of malodorous bacteria, including, for example, wild-type Staphylococcus haemolylicus. at the application location.Atty. Dkt. No. 135523-0119V. Combination Treatment with ApatB-Staphylococcus haemolyticus

[0114] In some embodiments, the ApatB-Staphylococcus haemolyticus strains e.g., Sn00430) of the present technology may be combined with one or more additional agents for the prevention or treatment of body odor. Additional agents include, but are not limited to, deodorants, antiperspirants, and / or fragrances.

[0115] In some embodiments, an additional agent is administered to a subject in combination with the ApatB-Staphylococcus haemolyticus strains (e.g., Sn00430) of the present technology such that a synergistic effect is produced. For example, administration of ApatB Staphylococcus haemolyticus with one or more additional agents for the prevention or treatment of body odor will have greater than additive effects in the prevention or treatment of the body odor.

[0116] In any case, the multiple agents may be administered in any order or even simultaneously. If simultaneously, the multiple agents may be provided in a single, unified form, or in multiple forms (by way of example only, either as a single deodorant stick or as two deodorant sticks). One of the agents may be given in multiple doses, or both may be given as multiple doses. In addition, the combination methods, compositions and formulations are not to be limited to the use of only two agents.EXAMPLES

[0117] The present technology is further illustrated by the following examples, which should not be construed as limiting in any way.Materials and Methods

[0118] Bacterial Strains. A strain of wild type Staphylococcus haemolyticus (Sn00412) was isolated from the human axilla. Puritan swabs (Puritan Medical, 25-1506 1PF 100 INNER) pre-wetted in PBS (Gibco™ 10010031) were used to swab the human axilla for 30 seconds. Swabs were then resuspended in PBS and vortexed for one minute. A 1 : 10,000 dilution was performed, and 100 uL of the resulting dilution was plated on a TSA plate and grown overnight at 37 °C.

[0119] Genotyping Bacterial Strains. A single colony of wild type Staphylococcus haemolyticus Sn00412 was picked, inoculated in 5 ml B2 low broth, and incubated at 250Atty. Dkt. No. 135523-0119rpm and 37°C overnight (B2 low: 25 g yeast extract, 10 g tryptone, 10 g NaCl, 5 g glucose, and 1 g K2HPO4 in 1 L. wt / vol, pH 7.5, adjusted with NaOH Filter sterilized). ImL of each 5 mL overnight cultures was centrifuged for 1 minute at 12,000 x g, and the supernatant was discarded. The resulting cell pellet was resuspended in 90 pL of cold PBS, followed by the addition of 10 pL of lysostaphin solution (10 mg / mL, Lysostaphin Recombinant ENZ2691MG, Supplier: Carbosynth LLC). The mixture were briefly vortexed and then incubated at 37°C for 40 minutes. Both samples were run through a Monarch® Genomic DNA Purification Kit #T3010S according to the manufacturer's protocol (New England Biolabs, 240 County Road, Ipswich, MA 01938), and purified gDNA was analyzed by Plasmidsaurus (Plasmidsaurus, 1850 Millrace Drive, Suite 200 Eugene, OR 97403) with Nanopore sequencing, according to the company’s internal procedures.

[0120] To identify and analyze the phylogenetic relationship between the strains and the broader Staphylococcus haemolyticus species, complete chromosomal reference genomes for the Staphylococcus malodor clade will be downloaded from NCBI / RefSeq. Pangenome alignments will be constructed using Bakta vl.9.3. Panaroo vl.5.0 and utilized to construct pangenome alignments. To identify mutations between S. haemolyticus strains, genomic positions where at least 90% of the genomes do not have an allele will be first removed. For the remaining positions, pairwise Jukes-Cantor nucleotide differences will be calculated. A phylogeny will then be constructed using the equal -variance neighbor joining algorithm.

[0121] 3-methyl-3-sulfanylhexan-l-ol (3M3SH) Assay. To measure the capability of the bacterial strains of the present technology to produce 3M3SH, the following assay was performed. A single inoculated colony (e.g., WT S. haemolyticus strain Sn00412 or ApatB-S. haemolyticus strain Sn00430) was grown in 5 mL of B2 low media in a 50 mL Falcon tube at 250 RPM overnight at 37°C. The cells were pelleted at 4000 xg for 10 minutes (Thermo Fisher Sorvall X4R Pro-MD), and resuspended in 2 mL of IX M9 buffer (MP Biomedicals™ 113037022). A 200 pL reaction assay mixture was prepared in a 96-well assay plate (Falcon, 96-well tissue culture plate, Ref: 353072) as follows: 150 pL resuspended cells normalized to an Optical Density (OD) 600 nm of 5.0, 50 pL of 10 mM Cys-Gly-3M3SH (Dalton Pharma services, DC-003684) dissolved in IX M9 buffer and allowed to grow for five hours at 250 RPM 37°C. After the five hour incubation, samplesAtty. Dkt. No. 135523-0119were pelleted at 4000 xg for 10 minutes. After centrifugation, 50 pL of supernatant was transferred to a clear bottom 96 well plate and mixed with 3.2 pL of 25 mM DTNB (CAS: 68-78-3, REF: 22582, Thermo Scientific) dissolved in lx M9 buffer. Each well was worked up to a total volume of 150 pL by adding IX M9 buffer with 50mM Tris-HCl, pH 8.0. For the reagent-free negative control, the same process was carried out with cell free media. The reaction assay mixture was incubated at room temperature for 5 minutes.Photometric reactions was measured using a Promega Glomax Discovery (REF: GM3000, SN: 9700100744) at A405 nm. This experiment was carried out with three biological replicates per sample. The average and standard deviation were calculated based on biological replicates. The average absorbance for the cell free control was subtracted from all samples to remove background signal. All plotting was performed using MATLAB.

[0122] Cytosine Base Editor. A cytosine base editor plasmid as described below was utilized to catalytically obliterate the function of PatB in the wild type Staphylococcus haemolyticus Sn00412. Specifically, the cytosine based editor (CBE) genome tool was designed to convert cytosine (C) to a thymine (T) in DNA without causing double strand brakes using single guide RNA (sgRNA) targeting the W 190 tryptophan amino acid site in patB. Conversion of at least one cytosine (C) on the complementary stand encoding the W190 site of PatB (5’-TGG-3’ is the W 190 nucleic acid sequence of the sense strand, while 3 ’-ACC-5’ is the nucleic acid sequence of the complementary antisense strand) introduces a premature stop codon in the expressed mRNA as UAA or UGA or UAG. The CBE system is based on a modified form of the CRISPR / Cas9 system, where the Cas9 protein is rendered “nickase” by mutation of the RuvC nuclease domain. The nickase form of Cas9 (Cas9n) can only cut one strand of DNA, reducing the likelihood of introduction doublestrand breaks. The CBE system comprises a cytidine deaminase enzyme (i.e., APOBEC1 or AID). The enzyme is fused to the Cas9n and is responsible for converting cytosine (C) to uracil (U) in a targeted manner. Uracil is then interpreted as thymine (T) during DNA replication or repair, resulting in a C»G to T»A base pair substitution.

[0123] Microbiome Profiling Protocol. The following microbiome profiling protocol was carried out to determine whether the compositions comprising the bacterial strains of the present technology are present on the skin of a subject to whom the compositions are applied post-application and whether such application alters self-reported odor levels.Atty. Dkt. No. 135523-0119

[0124] Product application'. A single ~1 g dose of a topical formulation containing 0.5% (w / v) lyophilized material containing Staphylococcus haemolyticus \N\99* patB mutant cells will be applied to both axillae of six adult volunteers using a silicone applicator.Participants will complete brief questionnaires on perceived body-odor reduction immediately before sampling on study days 3 and 7 after the single application.

[0125] Sample collection'. On days 3 and 7, axillary samples are collected by gently pressing foam-tipped swabs (Puritan Medical, 25-1506 1PF 100) pre-wetted in PBS with 0.005% Tween-20 against the epidermis for ~30 s in a zig-zag pattern (one swab per axilla). Swab heads are placed into cryotubes containing 750 pL PBS with 20% glycerol (v / v), vortexed ~1 min, and stored at -80 °C until processing.

[0126] DNA extraction'. From each primary sample, 200 pL are incubated with lysozyme (Millipore Sigma Cat # 6876, final concentration of 1 mg / mL) at 37 °C for ~24 h, then treated with Proteinase K (NEB Cat # 8107, final 10 U / mL) and SDS (final 0.1% w / v) at 55 °C for ~3 h. DNA is purified using PureLink Pro 96 Genomic DNA Purification Kits (Thermo Fisher Cat # 182104) following the manufacturer’s protocol, with an added 100% ethanol wash and a brief 37 °C incubation prior to elution to improve yield.

[0127] Engraftment'. Extracted genomic DNA will be analyzed for strain prevalence via sequencing analysis to determine the relative abundance of the ApatB-S. haemolyticus strain and wild-type S. haemolyticus strain in the sample. These results will demonstrate relative bacterial abundance at a species level and will allow for differentiation between WT bacteria and ApatB-S. haemolyticus bacteria at multiple sample collection time points.

[0128] Quantification of Viable Cells in Lyophilized Powder Suspended in PBS'. At each viability timepoint, lyophilized S. haemolyticus powder will be reconstituted in PBS (pH 7.4) and quantified by plate count. A sterile 15 mL polypropylene conical tube is preweighed to the nearest 0.001 g, the balance is tared, a small amount of lyophilized powder is added, the tube is re-weighed, and the powder mass is recorded by difference. PBS (1.0 mL) is added and the tube vortexed, followed by an additional 3.0 mL PBS and vortexing to homogeneity. Serial 10-fold dilutions are prepared in PBS, and 100 pL from the IO5- 107dilutions are spread-plated in duplicate onto TSA plates. Plates are incubated at 30 °C for 24-48 h until colonies are countable. CFU mL1of the stock suspension is calculated fromAtty. Dkt. No. 135523-0119plate counts and dilution factors, then normalized to the recorded powder mass to report viable CFU g1of lyophilized powder.Example 1 : Creation of a Six Part Cytosine Base Editor Plasmid pLOOl 14.

[0129] pLOOl 14: A plasmid containing six-part inserts was constructed using Golden Gate assembly which utilizes Type IIS restriction enzymes for multi-part DNA cloning (SEQ ID NO: 1). Each DNA fragment was synthesized and amplified with Bsal sites. The fragments were cloned into a universal entry vector via Bsal assembly creating “part” plasmids. The plasmid was transformed into E. coli cells and colonies were screened and verified through restriction mapping and sequencing to confirm correct assembly in line with Clutch Biotechnologies, Inc. (23595 Cabot Blvd, Suite 110, Hayward, CA 94545) procedures.

[0130] Each part plasmid was categorized by function (Types 1-6) with unique BsmBI -generated overhangs to ensure correct assembly. In a one-pot reaction, the six-part plasmids were combined and digested with Bsal and T4 DNA ligase. Part 1 is a 2710 bp region (SEQ ID NO: 2) that was synthesized by Clutch Biotechnologies, Inc. (23595 Cabot Blvd, Suite 110, Hayward, CA 94545) and encodes parts of a pIMAY vector (Addgene Plasmid #68939). Specifically, part 1 encodes the repBCAD temperature sensitive Staph origin, the constitutive E. colil p promoter, Phelp (a strong constitutive Staph promoter) and a chloramphenicol resistance marker (cat). Part 2 is a 547 bp region (SEQ ID NO: 3) that was synthesized by Clutch Biotechnologies, Inc. (23595 Cabot Blvd, Suite 110, Hayward, CA 94545) and encodes the low copy E. coli origin, pl5A ori. Part 3 is a 152 bp region (SEQ ID NO: 4) that was synthesized by Clutch Biotechnologies, Inc. (23595 Cabot Blvd, Suite 110, Hayward, CA 94545) and encodes a leaky inducible Staph, promoter. Part 4 is a 5167 bp region (SEQ ID NO: 5) that was synthesized by Clutch Biotechnologies, Inc. (23595 Cabot Blvd, Suite 110, Hayward, CA 94545) and encodes the open reading frame (ORF) for the cytosine base editor. Part 5 is a 128 bp region (SEQ ID NO: 6) that was synthesized by Clutch Biotechnologies, Inc. (23595 Cabot Blvd, Suite 110, Hayward, CA 94545) and encodes a low strength constitutive Staph promoter. Part 6 is a 121 bp region (SEQ ID NO: 7) that was synthesized by Clutch Biotechnologies, Inc. (23595 Cabot Blvd, Suite 110, Hayward, CA 94545) and encodes a sgRNA (sgW190) that introduces a premature stop codon at W 190 of patB.Atty. Dkt. No. 135523-0119Example 2: Transformation of pLOOl 14 into E. coli ER2796 strain to produce unmethylated DNA.

[0131] The pLOOl 14 plasmid was transformed into E. coli ER2796, which lacks all endogenous E. coli DNA methyltransferases. Briefly, transformation into E. coli ER2796 strain helps evade Type IV restriction systems in Staphylococcus species because the strain is deficient in all endogenous DNA methyltransferases and thus does not produce methylated DNA. Type IV restriction enzymes in Staphylococcus species recognize and cleave methylated DNA, and plasmids cloned into DNA methylation deficient strains such as ER2796 result in unmethylated plasmids, making it less recognizable to Type IV restriction systems in Staphylococcus. This increases the likelihood that the DNA will remain intact and functional after introduction into Staphylococcus cells.

[0132] Next, high concentration DNA (1000 ng / ul) was extracted and prepared using the ZymoPure II Plasmid Midiprep Kit (Zymo Research Corporation, 17062 Murphy Avenue, Irvine, CA 92614, USA) according to the manufacture’s protocol. High concentration DNA significantly enhances transformation efficiency in Staphylococcus strains for at least the following reasons. First, higher DNA concentration increases the likelihood that Staphylococcus cells will encounter and take up the DNA during transformation, crucial for species with naturally lower transformation efficiency. Additionally, high purity DNA reduces the addition of contaminants such as proteins, salts, and other impurities that can inhibit the downstream electroporation process by interfering with the electrical field.Example 3: Plasmid Electroporation into S. haemolyticus Strains.

[0133] The S. haemolyticus strain Sn00412 was streaked onto TSA plates and then incubated overnight at 37°C. A single colony per strain was inoculated into 50 mL of BHIS (BD Difco™ 237500) and incubated overnight at 37°C in a shaking incubator at 250 RPM. A 1 : 10 dilution of the overnight culture in 1 mL volume was performed and the OD measured using a spectrophotometer at OD 578 nm (Thermo Scientific Spectronic Genesys 20 Model 4001 / 4). The overnight culture was then subcultured to an OD of 0.2 in 50 mL BHIS and incubated at 37°C in a shaking incubator at 250 RPM until an OD of 0.7 was achieved. Next, the subculture was placed on ice for 10 minutes followed by centrifugation at 3500 x g for 10 minutes at 4°C using a pre chilled centrifuge. The supernatant was discarded and the pellet resuspended in an equal volume of 10% ice-cold glycerol. TheAtty. Dkt. No. 135523-0119centrifugation and resuspension process was repeated five times. At completion of the fifth cycle, the pellet was resuspended in 100 pL of 10% ice-cold glycerol per 50 mL of starting culture and transferred to a sterile microcentrifuge tube ready for electroporation. If electroporation was not be performed on the same day, the solution was flash frozen using liquid nitrogen or an ethanol dry ice bath. The frozen electrocompetent cells were later prepared for electroporation by thawing on ice for 5 minutes and then incubating at room temperature for 5 minutes. Electrocompetent cells were centrifuged at 6,000xg for 1 min, and the supernatant was discarded. Electrocompetent cells were resuspended in 10% Glycerol with 0.5M Sucrose.

[0134] Next, 50 pl of electrocompetent cells and 500 fmol of plasmid DNA from Example 2 (suspended in water) was transferred to an electroporation cuvette with a 0.1 cm gap, incubated for 5 minutes at room temperature, and electroporated using a BioRad Micropulser II for 2.5 milliseconds according to the following parameters: Voltage: 2.1 kV, Resistance: 100 , and Capacitance: 25 pF. The cell suspension was then transferred to 1 mL of room temperature BHIS and incubated while shaking at 250 RPM for 3 hours at 30°C. The cell suspension was then centrifuged at 3500 x g for 10 minutes, the cell pellet was resuspended in 100 pL BHIS, and plated on pre-warmed 30°C TSA Cm 10 plates (10 pg / ml chloramphenicol) and incubated at 30°C for 48 hours.Example 4: Validation of ApatB-S. haemolyticus Strains.

[0135] 16S sequencing was performed by Quintara Biosciences, Inc. according to their internal procedure (3563 Investment Blvd, Suite 2, Hayward, CA 94545) on a single transformant. The 16S was amplified out of the transformant using the 27F forward 16S primer (SEQ ID NO: 8) and the 1492R reverse 16S primers (SEQ ID NOs: 9-11). The amplicon was sequenced using Sanger sequencing and then aligned to the S. haemolyticus reference genome. The results confirmed that the transformant was from the S. haemolyticus species.

[0136] Next, using the patB004 forward patB primer (SEQ ID NO: 12) and patB002 reverse patB primer (SEQ ID NO: 13), PCR was performed by Quintara Biosciences, Inc. (3563 Investment Blvd, Suite 2, Hayward, CA 94545) to verify that the transformant had a premature stop codon introduced at the W190 tryptophan amino acid site in the patB gene.Atty. Dkt. No. 135523-0119

[0137] These results confirmed that the patB-S. haemolyticus strain Sn00430 has a premature stop codon of TAA at W190 compared to the wild-type strain (FIG. 1).Example 5: patB-S. haemolyticus Strains Exhibit Wild-Type Growth Rates.

[0138] Triplicate biological replicates of wild-type (WT) S. haemolyticus and lpatB S. haemolyticus, will be grown in a 96-well plate, in 200 pL TSB liquid media for 10 hours at 37°C and 220 RPM, in a BioTek Epoch 2 Microplate Spectrophotometer with A600nm recorded at 10 minute intervals.Example 6: UspatB-S. haemolyticus Strains Reduce Body Odor.

[0139] The ability of the lpatB-S. haemolyticus strain(s), of the present technology to treat, prevent, or reduce body odor (malodor) in a subject will be determined using one or more double blind odor studies.

[0140] A comparative group odor analysis will be performed. Treatment group study subjects will receive a probiotic deodorant comprising one or more of the lpatB-S. haemolyticus strains to the armpits. Control group study subjects will receive either a negative control probiotic deodorant without any bacterial strains, a standard commercial deodorant, a standard commercial antiperspirant, or some combination thereof. Multiple control groups will be tested with any of the described control treatments. Each treatment will be given for a specified number of days (e.g., study day -1, or study days -2 and -1). Then at a selection of study days taken from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 18, 20, 22, 24, and 28 days post-treatment, self-reported body odor levels from each subject will be recorded. Body odor will be compared between treatment groups, such as treatment versus control, but also within treatment groups, such as treatment group body odor at study day 0 versus study days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 18, 20, 22, 24, and / or 28.

[0141] It is anticipated that the results of these studies will demonstrate that the lpatB-S. haemolyticus strains of the present technology will successfully reduce, treat, or prevent body odor at levels that are comparable to, or greater than, the reductions observed from treatment with the control(s). Additionally, it is anticipated that the effect of the lpatB-S. haemolyticus strains will persist longer and at a greater level than the control deodorant effect. Accordingly, these results will demonstrate that a probiotic deodorant comprisingAtty. Dkt. No. 135523-0119the SpatB-S. haemolyticus strains of the present technology is effective for treating, reducing, or preventing body odor.Example 7: SpatB-S. haemolyticus Strains have Reduced 3M3SH Production.

[0142] The ability of the SpatB-Staphylococcus haemolyticus strains of the present disclosure (Sn00430) to produce 3M3SH was assayed as described above in the 3-methyl-3-sulfanylhexan-l-ol (3M3SH) Assay section, using a comparable wild-type Staphylococcus haemolyticus strain as a control (Sn00412).

[0143] The PatB enzyme in S. haemolyticus is known to cleave Gly-3M3SH during thiol production. As shown in FIG. 2, the wild-type S. haemolyticus strain (Sn00412) successfully metabolized Cys-Gly-3M3SH and produced 3M3SH. In contrast, ApatB-S. haemolyticus (Sn00430) was largely incapable of generating 3M3SH. These results indicate that the ApatB-S. haemolyticus strain lacks a functional patB gene and cannot produce 3M3SH. Accordingly, these results demonstrate that a probiotic deodorant comprising one or more ApatB-S. haemolyticus strains of the present technology is effective for reducing, preventing, or ameliorating body odor.Example 8: Lyophilized SpatB-S. haemolyticus Strains Retain Viability.

[0144] The viability of the lyophilized SpatB-S. haemolyticus strain of the present disclosure will be tested. Briefly, 50mL of both SpatB-S. haemolyticus strain will be grown up overnight in TSB at 37°C. The overnight culture will be centrifuged (4,000 g, 10 min) and resuspended (lOx dilution) in PBS with 10% (m / v) trehalose and 10% (m / v) inulin. ImL of this suspension was poured into 1.5mL microfuge tubes. The mixture will be thoroughly vortexed and stored at -80 °C before freeze-drying or spray drying.

[0145] The samples will be lyophilized in open microfuge tubes using a VirTis Advantage Pro per the manufacturer’s protocol. During lyophilization, samples will first be kept at -40 °C with a condenser temperature of -55 °C and chamber pressure of 10 Pa for 18 hours, and then the condenser was turned off, and the samples remained at room temperature and 10 Pa for 5 hours. After lyophilization, samples will be kept in sealed 1.5mL microfuge tubes at room temperature and kept under pressure. Samples of lyophilized bacteria will be taken directly after lyophilization and at 1 day, 1 week, 1 month, and 4 months post lyophilization, and the colony forming units per gram will be measured via plating.Atty. Dkt. No. 135523-0119

[0146] It is anticipated that t^palB-S. haemolyticus species will retain high levels of viability even as late as 4 months post-lyophilization. Accordingly, it is anticipated that these results will demonstrate that the ^patB-S. haemolyticus strains of the present technology retain viability in long-term storage, and therefore a probiotic deodorant or pharmaceutical composition comprising the UspatB-S. haemolyticus strains of the present technology will be effective for treating, reducing or preventing body odor.Example 9: UspatB-S. haemolyticus Strains Reduce Body Odor.

[0147] The ability of UspatB-S. haemolyticus to reduce body odor will be tested. Briefly, a mixture of the UspatB-S. haemolyticus strain(s) alone or in combination in an excipient will be applied to one armpit of a subject while excipient without the bacterial strains will be applied to the other armpit of the subject. One week and one month later the subject will engage in exercise and an odor test will be performed by smelling the subject’s armpits. It is anticipated that the armpit receiving the UspatB-S. haemolyticus strain(s) either alone or in combination in excipient will be identified as substantially less odorous as compared to the armpit receiving excipient alone. Accordingly, it is anticipated that these results will demonstrate that a probiotic deodorant or pharmaceutical composition comprising the lpatB-S. haemolyticus strain(s) of the present disclosure will be effective for treating, reducing, or preventing body odor.EQUIVALENTS

[0148] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present technology is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, ofAtty. Dkt. No. 135523-0119course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0149] Each and every publication and patent mentioned in the above specification is herein incorporated by reference in its entirety for all purposes. Various modifications and variations of the described methods and system of the present technology will be apparent to those skilled in the art without departing from the scope and spirit of the present technology. Although the present technology has been described in connection with specific embodiments, the present technology as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the present technology which are obvious to those skilled in the art and in fields related thereto are intended to be within the scope of the following claims.Atty. Dkt. No. 135523-0119BIOLOGICAL DEPOSITS

[0150] The Applicant requests that a sample of the deposited microorganisms should be made available only to an expert approved by the Applicant.

[0151] Staphylococcus haemolyticus strain identified as Sn00412 deposited with the International Depositary Authority of Canada (ID AC), National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington Street, Winnipeg, Manitoba Canada R3E 3R2, on September 24, 2025, under ID AC Accession Number 240925-01.

[0152] Staphylococcus haemolyticus strain identified as Sn00430 deposited with the International Depositary Authority of Canada (ID AC), National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington Street, Winnipeg, Manitoba Canada R3E 3R2, on September 24, 2025, under ID AC Accession Number 240925-04.

[0153] The deposit was made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.

[0154] Name of Depositor: Taxa Technologies, Inc. Address of Depositor: 135 Mississippi St., San Francisco, CA, 94107.

[0155] In respect of all designated states to which such action is possible and to the extent that it is legally permissible under the law of the designated state, it is requested that a sample of the deposited biological material be made available only by the issue thereof to an independent expert, in accordance with the relevant Patent legislation, for example EPC Rule 32, UK Patent Rules 2007 (as amended) Rule 13(1) and Schedule 1 paragraphs 6 and 7, and generally similar provisions mutatis mutandis for any other designated state.Atty. Dkt. No. 135523-0119SEQUENCE LISTINGStaphylococcus haemolyticus pLOOl 14 plasmid: Six-part cytosine base editor plasmid nucleotide sequence (SEQ ID NO: 1):tagtctcctttttcgcttctttattccaattgctttattgacgttgagcctcggaaccgtcgactttatttggatcccctcgagttcatgaaaa actaaaaaaaatattgaaactctatcattgatagagtataattaacaaggaggaataaaaaatgagctcagagactggcccagtggct gtggaccccacattgaggcggcggatcgagccccatgagtttgaggtattcttcgatccgagagagctccgcaaggagacttgcct gctttacgaaattaattgggggggccggcactccatttggcgacatacatcacagaacactaacaagcacgtcgaagtcaacttcat cgagaagttcacgacagaaagatatttctgtccgaacacaaggtgcagcattacctggtttctcagctggagcccatgcggcgaatg tagtagggccatcactgaattcctgtcaaggtatccccacgtcactctgtttatttacatcgcaaggctgtaccaccacgctgaccccc gcaatcgacaaggcctgcgggatttgatctcttcaggtgtgactatccaaattatgactgagcaggagtcaggatactgctggagaa actttgtgaattatagcccgagtaatgaagcccactggcctaggtatccccatctgtgggtacgactgtacgttcttgaactgtactgca tcatactgggcctgcctccttgtctcaacattctgagaaggaagcagccacagctgacattctttaccatcgctcttcagtcttgtcatta ccagcgactgcccccacacattctctgggccaccgggttgaaatctggtggttcttctggtggttctagcggcagcgagactcccgg gacctcagagtccgccacacccgaaagttctggtggttcttctggtggttctgataaaaagtattctattggtttagccatcggcactaa ttccgttggatgggctgtcataaccgatgaatacaaagtaccttcaaagaaatttaaggtgttggggaacacagaccgtcattcgatta aaaagaatcttatcggtgccctcctattcgatagtggcgaaacggcagaggcgactcgcctgaaacgaaccgctcggagaaggtat acacgtcgcaagaaccgaatatgttacttacaagaaatttttagcaatgagatggccaaagttgacgattctttctttcaccgtttggaa gagtccttccttgtcgaagaggacaagaaacatgaacggcaccccatctttggaaacatagtagatgaggtggcatatcatgaaaag tacccaacgatttatcacctcagaaaaaagctagttgactcaactgataaagcggacctgaggttaatctacttggctcttgcccatatg ataaagttccgtgggcactttctcattgagggtgatctaaatccggacaactcggatgtcgacaaactgttcatccagttagtacaaac ctataatcagttgtttgaagagaaccctataaatgcaagtggcgtggatgcgaaggctattcttagcgcccgcctctctaaatcccgac ggctagaaaacctgatcgcacaattacccggagagaagaaaaatgggttgttcggtaaccttatagcgctctcactaggcctgacac caaattttaagtcgaacttcgacttagctgaagatgccaaattgcagcttagtaaggacacgtacgatgacgatctcgacaatctactg gcacaaattggagatcagtatgcggacttatttttggctgccaaaaaccttagcgatgcaatcctcctatctgacatactgagagttaat actgagattaccaaggcgccgttatccgcttcaatgatcaaaaggtacgatgaacatcaccaagacttgacacttctcaaggccctag tccgtcagcaactgcctgagaaatataaggaaatattctttgatcagtcgaaaaacgggtacgcaggttatattgacggcggagcga gtcaagaggaattctacaagtttatcaaacccatattagagaagatggatgggacggaagagttgcttgtaaaactcaatcgcgaag atctactgcgaaagcagcggactttcgacaacggtagcattccacatcaaatccacttaggcgaattgcatgctatacttagaaggca ggaggatttttatccgttcctcaaagacaatcgtgaaaagattgagaaaatcctaacctttcgcataccttactatgtgggacccctggc ccgagggaactctcggttcgcatggatgacaagaaagtccgaagaaacgattactccatggaattttgaggaagttgtcgataaagg tgcgtcagctcaatcgttcatcgagaggatgaccaactttgacaagaatttaccgaacgaaaaagtattgcctaagcacagtttacttt acgagtatttcacagtgtacaatgaactcacgaaagttaagtatgtcactgagggcatgcgtaaacccgcctttctaagcggagaaca gaagaaagcaatagtagatctgttattcaagaccaaccgcaaagtgacagttaagcaattgaaagaggactactttaagaaaattga atgcttcgattctgtcgagatctccggggtagaagatcgatttaatgcgtcacttggtacgtatcatgacctcctaaagataattaaagat aaggacttcctggataacgaagagaatgaagatatcttagaagatatagtgttgactcttaccctctttgaagatcgggaaatgattga ggaaagactaaaaacatacgctcacctgttcgacgataaggttatgaaacagttaaagaggcgtcgctatacgggctggggacgat tgtcgcggaaacttatcaacgggataagagacaagcaaagtggtaaaactattctcgattttctaaagagcgacggcttcgccaata ggaactttatgcagctgatccatgatgactctttaaccttcaaagaggatatacaaaaggcacaggtttccggacaaggggactcatt gcacgaacatattgcgaatcttgctggttcgccagccatcaaaaagggcatactccagacagtcaaagtagtggatgagctagttaa ggtcatgggacgtcacaaaccggaaaacattgtaatcgagatggcacgcgaaaatcaaacgactcagaaggggcaaaaaaacag tcgagagcggatgaagagaatagaagagggtattaaagaactgggcagccagatcttaaaggagcatcctgtggaaaatacccaa ttgcagaacgagaaactttacctctattacctacaaaatggaagggacatgtatgttgatcaggaactggacataaaccgtttatctgat tacgacgtcgatcacattgtaccccaatcctttttgaaggacgattcaatcgacaataaagtgcttacacgctcggataagaaccgag ggaaaagtgacaatgttccaagcgaggaagtcgtaaagaaaatgaagaactattggcggcagctcctaaatgcgaaactgataac gcaaagaaagttcgataacttaactaaagctgagaggggtggcttgtctgaacttgacaaggccggatttattaaacgtcagctcgtg gaaacccgccaaatcacaaagcatgttgcacagatactagattcccgaatgaatacgaaatacgacgagaacgataagctgattcg ggaagtcaaagtaatcactttaaagtcaaaattggtgtcggacttcagaaaggattttcaattctataaagttagggagataaataacta ccaccatgcgcacgacgcttatcttaatgccgtcgtagggaccgcactcattaagaaatacccgaagctagaaagtgagtttgtgtatAtty. Dkt. No. 135523-0119ggtgattacaaagtttatgacgtccgtaagatgatcgcgaaaagcgaacaggagataggcaaggctacagccaaatacttcttttatt ctaacattatgaatttctttaagacggaaatcactctggcaaacggagagatacgcaaacgacctttaattgaaaccaatggggagac aggtgaaatcgtatgggataagggccgggacttcgcgacggtgagaaaagttttgtccatgccccaagtcaacatagtaaagaaaa ctgaggtgcagaccggagggttttcaaaggaatcgattcttccaaaaaggaatagtgataagctcatcgctcgtaaaaaggactggg acccgaaaaagtacggtggcttcgatagccctacagttgcctattctgtcctagtagtggcaaaagttgagaagggaaaatccaaga aactgaagtcagtcaaagaattattggggataacgattatggagcgctcgtcttttgaaaagaaccccatcgacttccttgaggcgaa aggttacaaggaagtaaaaaaggatctcataattaaactaccaaagtatagtctgtttgagttagaaaatggccgaaaacggatgttg gctagcgccggagagcttcaaaaggggaacgaactcgcactaccgtctaaatacgtgaatttcctgtatttagcgtcccattacgag aagttgaaaggttcacctgaagataacgaacagaagcaactttttgttgagcagcacaaacattatctcgacgaaatcatagagcaaa tttcggaattcagtaagagagtcatcctagctgatgccaatctggacaaagtattaagcgcatacaacaagcacagggataaaccca tacgtgagcaggcggaaaatattatccatttgtttactcttaccaacctcggcgctccagccgcattcaagtattttgacacaacgatag atcgcaaacgatacacttctaccaaggaggtgctagacgcgacactgattcaccaatccatcacgggattatatgaaactcggatag atttgtcacagcttgggggtgactctggtggttctggaggatctggtggttctactaatctgtcagatattattgaaaaggagactggta agcaactggttatccaggaatccatcctcatgctcccagaggaggtggaagaagtcattgggaacaagccggaaagcgatatactc gtgcacaccgcctacgacgagagcaccgacgagaatgtcatgcttctgactagcgacgcccctgaatacaagccttgggctctggt catacaggatagcaacggtgagaacaagattaagatgctctaatgggtccaggcatcaaataaaacgaaaggctcagtcgaaaga ctgggcctttcgttttatctgttgtttgtcggtgaacgctctcttccttgcaaaatatacaggggattatatataatggaaaacaagTCT GAC CAT AC AC GACC T GT gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtg gcaccgagtcggtgctttttttgcggccgcgtgatctaataaaccgtgatggtaacttcagcatgcgttttagcgtttatttcgtttagttat cggcataatcgttaaaacaggcgttatcgtagcgtaaaagcccttgagcgtagcgtggctttgcagcgaagatgttgtctgttagatta tgaaagccgatgactgaatgaaataataagcgcagcgcccttctatttcggttggaggaggctcaagggagtatgagggaatgaaa ttccctcatgggtttgattttaaaaattgcttgcaattttgccgagcggtagcgctggaaaatttttgaaaaaaatttggaatttggaaaaa aatggggggaaaggaagcgaattttgcttccgtactacgaccccccattaagtgccgagtgccaatttttgtgccaaaaacgctctat cccaactggctcaagggtttaaggggtttttcaatcgccaacgaatcgccaacgttttcgccaacgttttttataaatctatatttaagtag ctttattgttgtttttatgattacaaagtgatacactaactttataaaattatttgattggagttttttaaatggtgatttcagaatcgaaaaaaa gagttatgatttctctgacaaaagagcaagataaaaaattaacagatatggcgaaacaaaaaggtttttcaaaatctgcggttgcggc gttagctatagaagaatatgcaagaaaggaatcagaacaaaaaaaataagcgaaagctcgcgtttttagaaggatacgagttttcgct acttgtttttgataaggtaattatatcatggctattaaaaatactaaagctagaaattttggatttttattatatcctgactcaattcctaatgat tggaaagaaaaattagagagtttgggcgtatctatggctgtcagtcctttacacgatatggacgaaaaaaaagataaagatacatgga ataatagtaatattatacaaaatggaaagcactataaaaaaccacactatcacgttatatatattgcacgaaatcctgtaacaatagaaa gcgttaggaacaagattaagcgaaaattggggaatagttcagttgctcatgttgagatacttgattatatcaaaggttcatatgaatattt gactcatgaatcaaaggacgctattgctaagaataaacatatatacgacaaaaaagatattttgaacattaatgattttgatattgaccgc tatataacacttgatgaaagccaaaaaagagaattgaagaatttacttttagatatagtggatgactataatttggtaaatacaaaagattt aatggcttttattcgccttaggggagcggagtttggaattttaaatacgaatgatgtaaaagatattgtttcaacaaactctagcgccttta gattatggtttgagggcaattatcagtgtggatatagagcaagttatgcaaaggttcttgatgctgaaacgggggaaataaaatgaca aacaaagaaaaagagttatttgctgaaaatgaggaattaaaaaaagaaattaaggacttaaaagagcgtattgaaagatacagagaa atggaagttgaattaagtacaacaatagatttattgagaggagggattattgaataaataaaagccccctgacgaaagtcgaagggg gtttttattttggtttgatgttgcgattaatagcaatacattctataatagaaggtatggaggatgttatataatgagacagaattatgatgat catatgtcaactaacggggcaggttagtgacattagaaaaccgactgtaaaaagtacagtcggcattatctcatattataaaagccagt cattaggcctatctgacaattcctgaatagagttcataaacaatcctgcatgataaccatcacaaacagaatgatgtacctgtaaagata gcggtaaatatattgaattacctttattaatgaattttcctgctgtaataatgggtagaaggtaattactattattattgatatttaagttaaac ccagtaaatgaagtccatggaataatagaaagagaaaaagcattttcaggtataggtgttttgggaaacaatttccccgaaccattata tttctctacatcagaaaggtataaatcataaaactctttgaagtcattctttacaggagtccaaatgccagagaatgttttagatacaccat caaaaattgtataaagtggctctaacttatcccaataacctaactctccgtcgctattgtaaccagttctaaaagctgtatttgagtttatca cccttgtcactaagaaaataaatgcagggtaaaatttatatccttcttgttttatgtttcggtataaaacactaatatcaatttctgtggttata ctaaaagtcgtttgttggttcaaataatgattaaatatctcttttctcttccaattgtctaaatcaattttattaaagttcatgggtttcactctcc ttctacattttttaacctaataatgccaaataccgtttgccacccctctctttgataattataatattggcgaaattcgcttctaaagatgaaa cgcaatattatatgcttgctttatcggccgtatgtgattataccagccccctcactacatgtcaagaataaactgccaaagcataatggg ataattaaccctcttttccataggctccgcccccctgacaagcatcacgaaatctgacgctcaaatcagtggtggcgaaacccgaca ggactataaagataccaggcgtttccccctggcggctccctcgtgcgctctcctgttcctgcctttcggtttaccggtgtcattccgctgAtty. Dkt. No. 135523-0119ttatggccgcgtttgtctcattccacgcctgacactcagttccgggtaggcagttcgctccaagctggactgtatgcacgaacccccc gttcagtccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggaaagacatgcaaaagcaccactggcagcagc cactggtaattgatttagaggagttagtcttgaagtcatgcgccggttaaggctaaactgaaaggacaagttttggtgactgcgctcct ccaagccagttacctcggttcaaagagttggtagctcagagaaccttcgaaaaaccgccctgcaaggcggttttttcgttttcagagc aagagattacgcgcagaccaaaacgatctcaagPart 1 of Staphylococcus haemolyticus pLOOl 14 plasmid: (SEQ ID NO: 2):CGTGATGGTAACTTCAGCATGCGTTTTAGCGTTTATTTCGTTTAGTTATCGGCAT AATCGTTAAAACAGGCGTTATCGTAGCGTAAAAGCCCTTGAGCGTAGCGTGGC TTTGCAGCGAAGATGTTGTCTGTTAGATTATGAAAGCCGATGACTGAATGAAAT AATAAGCGCAGCGCCCTTCTATTTCGGTTGGAGGAGGCTCAAGGGAGTATGAG GGAATGAAATTCCCTCATGGGTTTGATTTTAAAAATTGCTTGCAATTTTGCCGA GCGGTAGCGCTGGAAAATTTTTGAAAAAAATTTGGAATTTGGAAAAAAATGGG GGGAAAGGAAGCGAATTTTGCTTCCGTACTACGACCCCCCATTAAGTGCCGAG TGCCAATTTTTGTGCCAAAAACGCTCTATCCCAACTGGCTCAAGGGTTTAAGGG GTTTTTCAATCGCCAACGAATCGCCAACGTTTTCGCCAACGTTTTTTATAAATCT ATATTTAAGTAGCTTTATTGTTGTTTTTATGATTACAAAGTGATACACTAACTTT ATAAAATTATTTGATTGGAGTTTTTTAAATGGTGATTTCAGAATCGAAAAAAAG AGTTATGATTTCTCTGACAAAAGAGCAAGATAAAAAATTAACAGATATGGCGA AACAAAAAGGTTTTTCAAAATCTGCGGTTGCGGCGTTAGCTATAGAAGAATAT GCAAGAAAGGAATCAGAACAAAAAAAATAAGCGAAAGCTCGCGTTTTTAGAA GGATACGAGTTTTCGCTACTTGTTTTTGATAAGGTAATTATATCATGGCTATTAA AAATACTAAAGCTAGAAATTTTGGATTTTTATTATATCCTGACTCAATTCCTAAT GATTGGAAAGAAAAATTAGAGAGTTTGGGCGTATCTATGGCTGTCAGTCCTTTA CACGATATGGACGAAAAAAAAGATAAAGATACATGGAATAATAGTAATATTAT ACAAAATGGAAAGCACTATAAAAAACCACACTATCACGTTATATATATTGCAC GAAATCCTGTAACAATAGAAAGCGTTAGGAACAAGATTAAGCGAAAATTGGGG AATAGTTCAGTTGCTCATGTTGAGATACTTGATTATATCAAAGGTTCATATGAA TATTTGACTCATGAATCAAAGGACGCTATTGCTAAGAATAAACATATATACGAC AAAAAAGATATTTTGAACATTAATGATTTTGATATTGACCGCTATATAACACTT GATGAAAGCCAAAAAAGAGAATTGAAGAATTTACTTTTAGATATAGTGGATGA CTATAATTTGGTAAATACAAAAGATTTAATGGCTTTTATTCGCCTTAGGGGAGC GGAGTTTGGAATTTTAAATACGAATGATGTAAAAGATATTGTTTCAACAAACTC TAGCGCCTTTAGATTATGGTTTGAGGGCAATTATCAGTGTGGATATAGAGCAAG TTATGCAAAGGTTCTTGATGCTGAAACGGGGGAAATAAAATGACAAACAAAGA AAAAGAGTTATTTGCTGAAAATGAGGAATTAAAAAAAGAAATTAAGGACTTAA AAGAGCGTATTGAAAGATACAGAGAAATGGAAGTTGAATTAAGTACAACAATA GATTTATTGAGAGGAGGGATTATTGAATAAATAAAAGCCCCCTGACGAAAGTC GAAGGGGGTTTTTATTTTGGTTTGATGTTGCGATTAATAGCAATACATTCTATA ATAGAAGGTATGGAGGATGTTATATAATGAGACAGAATTATGATGATCATATG TCAACTAACGGGGCAGGTTAGTGACATTAGAAAACCGACTGTAAAAAGTACAG TCGGCATTATCTCATATTATAAAAGCCAGTCATTAGGCCTATCTGACAATTCCT GAATAGAGTTCATAAACAATCCTGCATGATAACCATCACAAACAGAATGATGT ACCTGTAAAGATAGCGGTAAATATATTGAATTACCTTTATTAATGAATTTTCCT GCTGTAATAATGGGTAGAAGGTAATTACTATTATTATTGATATTTAAGTTAAAC CCAGTAAATGAAGTCCATGGAATAATAGAAAGAGAAAAAGCATTTTCAGGTAT AGGTGTTTTGGGAAACAATTTCCCCGAACCATTATATTTCTCTACATCAGAAAGAtty. Dkt. No. 135523-0119GTATAAATCATAAAACTCTTTGAAGTCATTCTTTACAGGAGTCCAAATGCCAGA GAATGTTTTAGATACACCATCAAAAATTGTATAAAGTGGCTCTAACTTATCCCA ATAACCTAACTCTCCGTCGCTATTGTAACCAGTTCTAAAAGCTGTATTTGAGTTT ATCACCCTTGTCACTAAGAAAATAAATGCAGGGTAAAATTTATATCCTTCTTGT TTTATGTTTCGGTATAAAACACTAATATCAATTTCTGTGGTTATACTAAAAGTC GTTTGTTGGTTCAAATAATGATTAAATATCTCTTTTCTCTTCCAATTGTCTAAAT CAATTTTATTAAAGTTCATGGGTTTCACTCTCCTTCTACATTTTTTAACCTAATA ATGCCAAATACCGTTTGCCACCCCTCTCTTTGATAATTATAATATTGGCGAAAT TCGCTTCTAAAGATGAAACGCAATATTATATGCTTGCTTTATCGGCCGTATGTG ATTATACCAGCCCCCTCACTACATGTCAAGAATAAACTGCCAAAGCATAATGG GATAATTAACCCTCPart 2 of Staphylococcus haemolyticus pLOOl 14 plasmid: (SEQ ID NO: 3):TTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATC AGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGC GGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGC TGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCA GTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACCGCTGC GCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGC ACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCAT GCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCA AGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACC GCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAA ACGATCTCAAPart 3 of Staphylococcus haemolyticus pLOOl 14 plasmid: (SEQ ID NO: 4):TCTCCTTTTTCGCTTCTTTATTCCAATTGCTTTATTGACGTTGAGCCTCGGAACC GTCGACTTTATTTGGATCCCCTCGAGTTCATGAAAAACTAAAAAAAATATTGAA ACTCTATCATTGATAGAGTATAATTAACAAGGAGGAATAAAAAPart 4 of Staphylococcus haemolyticus pLOOl 14 plasmid: (SEQ ID NO: 5):ATGAGCTCAGAGACTGGCCCAGTGGCTGTGGACCCCACATTGAGGCGGCGGAT CGAGCCCCATGAGTTTGAGGTATTCTTCGATCCGAGAGAGCTCCGCAAGGAGA CTTGCCTGCTTTACGAAATTAATTGGGGGGGCCGGCACTCCATTTGGCGACATA CATCACAGAACACTAACAAGCACGTCGAAGTCAACTTCATCGAGAAGTTCACG ACAGAAAGATATTTCTGTCCGAACACAAGGTGCAGCATTACCTGGTTTCTCAGC TGGAGCCCATGCGGCGAATGTAGTAGGGCCATCACTGAATTCCTGTCAAGGTA TCCCCACGTCACTCTGTTTATTTACATCGCAAGGCTGTACCACCACGCTGACCC CCGCAATCGACAAGGCCTGCGGGATTTGATCTCTTCAGGTGTGACTATCCAAAT TATGACTGAGCAGGAGTCAGGATACTGCTGGAGAAACTTTGTGAATTATAGCC CGAGTAATGAAGCCCACTGGCCTAGGTATCCCCATCTGTGGGTACGACTGTACG TTCTTGAACTGTACTGCATCATACTGGGCCTGCCTCCTTGTCTCAACATTCTGAG AAGGAAGCAGCCACAGCTGACATTCTTTACCATCGCTCTTCAGTCTTGTCATTA CCAGCGACTGCCCCCACACATTCTCTGGGCCACCGGGTTGAAATCTGGTGGTTC TTCTGGTGGTTCTAGCGGCAGCGAGACTCCCGGGACCTCAGAGTCCGCCACACCAtty. Dkt. No. 135523-0119CGAAAGTTCTGGTGGTTCTTCTGGTGGTTCTGATAAAAAGTATTCTATTGGTTTA GCCATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGT ACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAA AGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACT CGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATG TTACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTT TCACCGTTTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGC ACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCA ACGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCT GAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTTTCT CATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCA GTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTG GCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAG AAAACCTGATCGCACAATTACCCGGAGAGAAGAAAAATGGGTTGTTCGGTAAC CTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAGTCGAACTTCGACTTA GCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCGA CAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCAA AAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGAT TACCAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTACGATGAACATCACC AAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATA AGGAAATATTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGC GGAGCGAGTCAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGAT GGATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGAA AGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAA TTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAAGACAAT CGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACTATGTGGGACCC CTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAAC GATTACTCCATGGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATC GTTCATCGAGAGGATGACCAACTTTGACAAGAATTTACCGAACGAAAAAGTAT TGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGA AAGTTAAGTATGTCACTGAGGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAA CAGAAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACAGT TAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGA GATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCT CCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATA TCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTG AGGAAAGACTAAAAACATACGCTCACCTGTTCGACGATAAGGTTATGAAACAG TTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTATCAA CGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCG ACGGCTTCGCCAATAGGAACTTTATGCAGCTGATCCATGATGACTCTTTAACCT TCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTGCAC GAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCA GACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCATGGGACGTCACAAACCGG AAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGCA AAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGAACTG GGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTGCAGAACGA GAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGA ACTGGACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCCCAATC CTTTTTGAAGGACGATTCAATCGACAATAAAGTGCTTACACGCTCGGATAAGA ACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAAtty. Dkt. No. 135523-0119GAACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCG ATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGA TTTATTAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACA GATACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTC GGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAG GATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGAC GCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTA GAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGACGTCCGTAAGATGATC GCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTC TAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATAC GCAAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGAT AAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAA CATAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTC TTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGACCCG AAAAAGTACGGTGGCTTCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTG GCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATT GGGGATAACGATTATGGAGCGCTCGTCTTTTGAAAAGAACCCCATCGACTTCCT TGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAA AGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCC GGAGAGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTT CCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGA ACAGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAG AGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGAC AAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGC GGAAAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATT CAAGTATTTTGACACAACGATAGATCGCAAACGATACACTTCTACCAAGGAGG TGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGG ATAGATTTGTCACAGCTTGGGGGTGACTCTGGTGGTTCTGGAGGATCTGGTGGT TCTACTAATCTGTCAGATATTATTGAAAAGGAGACTGGTAAGCAACTGGTTATC CAGGAATCCATCCTCATGCTCCCAGAGGAGGTGGAAGAAGTCATTGGGAACAA GCCGGAAAGCGATATACTCGTGCACACCGCCTACGACGAGAGCACCGACGAGA ATGTCATGCTTCTGACTAGCGACGCCCCTGAATACAAGCCTTGGGCTCTGGTCA TACAGGATAGCAACGGTGAGAACAAGATTAAGATGCTCTAATPart 5 of Staphylococcus haemolyticus pLOOl 14 plasmid: (SEQ ID NO: 6):GGGTCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCG TTTTATCTGTTGTTTGTCGGTGAACGCTCTCTTCCTTGCAAAATATACAGGGGAT TATATATAATGGAAAACAAGPart 6 of Staphylococcus haemolyticus pLOOl 14 plasmid: (SEQ ID NO: 7):TCTGACCATACACGACCTGTgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaa agtggcaccgagtcggtgctttttttgcggccgcgtgatctaat27F (forward primer) for amplifying the 16S of the transformant (SEQ ID NO: 8):GAGTTTGATYMTGGCTCAGAtty. Dkt. No. 135523-01191492R (reverse primer) for amplifying the 16S of the transformant (SEQ ID NO: 9):GYTACCTTGTTACGACTT1492R (reverse primer) for amplifying the 16S of the transformant (SEQ ID NO: 10): GCTACCTTGTTACGACTT1492R (reverse primer) for amplifying the 16S of the transformant (SEQ ID NO: 11): GTTACCTTGTTACGACTTpatB008 (patB forward) primer to verify introduction of premature stop codon at W 190 tryptophan amino acid site of patB (SEQ ID NO: 12):CACCTTGTATTTTCTCATGGpatB012 (patB reverse) primer to verify introduction of premature stop codon at W 190 tryptophan amino acid site of patB (SEQ ID NO: 13):CGTCTAGATAATCATTCATAGCAGNucleic acid sequence of patB (SEQ ID NO: 14):GTGACGTACAATTTTGATGAGATCATAGATCGTCGTTCTACTAATGCGATGAAT GTAGAAGGTTATAAAGGCTATTTATTTGGAGACGCAGATACTTCAGATTTAGAA GAACATGATGAACTGATTCGCATGTGGGTAGCTGATATGGATTTCGCGACACCT GAAGTTGTGTTAGATGCGATAAGAGACAGACTCGATAAAAAGATTCTAGGTTA TACCAACATATTTGGCACAGATTATTATGAGGCTTTTATGTCATGGACAGAACG ACGATTTGGTTATACATTTCCGCAAGAACACCTTGTATTTTCTCATGGGATTGTT GCAGGGTTAATTGAACTTGTAAGCTATATTTGTGACGATGATGATAAAGCGTTG ATTTTTACACCAAGCTACGGTCCTTTTAAAATGGCTTGTGATAAAAATAATATT AAGACGGTATATTCTCCTATGATTAATCATAATGGATACTATGAAATCGACTTT GAAGATGTCCGTCAAAAAGTGGAAACAGAAAATATTAAATTATGTATTTTTGCT AATCCGCATAATCCAACAGGTCGTGTATGGTCAGAAGATGAATTAAAGCAATT GGGACAAATTATGGTCGATAATGACGTATGGATCATCTCAGATGAAATTCACT GCGACATTAAACGTGACGGTCAAACACATGTACCTTTTGCTAAAGCAGTGCCA GATTACGATAAGATTGTTACCGCAATGTCTCAAAGTAAGGCATTTAATATAGCA GGCTTAATGTTCTCTAACCTTATTATTCCGAATAGAAGATTATTAAAGACATGG AAGTTACATCACTTTAGTTCTGAGAATCCATTAAGTATCGTTGCGACACAGGCA GCTTATGAAAAGGGCGAAGACTGGTTAGCTGCTATGAATGATTATCTAGACGA TAACTTTAAATACCTAGCACAATTTTTAGAACAAGAACTGCCACATGCAGAATT TAAAATTCCAGAAGCAACCTACCTCGCATGGGTTGATCTAAGCTATTACATTAA AGCAAAACACATAGATGAACCAATCGCTAAATATTTCATCAAACACGCCGGCG TTATCATTGAAGGACAAGAACAATTCGTTCATAACGCAGAAGGACATATCAGA ATAAACATCGCTGTTCCACGTGAAATCATGATAAAAGGACTGCAAAAGATTAA AGACGCATTAGTTTAANucleic acid sequence of mutated patB with introduction of an exemplary stop codon atAtty. Dkt. No. 135523-0119the W 190 codon shown in bold and underlined text (SEQ ID NO: 15):GTGACGTACAATTTTGATGAGATCATAGATCGTCGTTCTACTAATGCGATGAAT GTAGAAGGTTATAAAGGCTATTTATTTGGAGACGCAGATACTTCAGATTTAGAA GAACATGATGAACTGATTCGCATGTGGGTAGCTGATATGGATTTCGCGACACCT GAAGTTGTGTTAGATGCGATAAGAGACAGACTCGATAAAAAGATTCTAGGTTA TACCAACATATTTGGCACAGATTATTATGAGGCTTTTATGTCATGGACAGAACG ACGATTTGGTTATACATTTCCGCAAGAACACCTTGTATTTTCTCATGGGATTGTT GCAGGGTTAATTGAACTTGTAAGCTATATTTGTGACGATGATGATAAAGCGTTG ATTTTTACACCAAGCTACGGTCCTTTTAAAATGGCTTGTGATAAAAATAATATT AAGACGGTATATTCTCCTATGATTAATCATAATGGATACTATGAAATCGACTTT GAAGATGTCCGTCAAAAAGTGGAAACAGAAAATATTAAATTATGTATTTTTGCT AATCCGCATAATCCAACAGGTCGTGTATAATCAGAAGATGAATTAAAGCAATT GGGACAAATTATGGTCGATAATGACGTATGGATCATCTCAGATGAAATTCACT GCGACATTAAACGTGACGGTCAAACACATGTACCTTTTGCTAAAGCAGTGCCA GATTACGATAAGATTGTTACCGCAATGTCTCAAAGTAAGGCATTTAATATAGCA GGCTTAATGTTCTCTAACCTTATTATTCCGAATAGAAGATTATTAAAGACATGG AAGTTACATCACTTTAGTTCTGAGAATCCATTAAGTATCGTTGCGACACAGGCA GCTTATGAAAAGGGCGAAGACTGGTTAGCTGCTATGAATGATTATCTAGACGA TAACTTTAAATACCTAGCACAATTTTTAGAACAAGAACTGCCACATGCAGAATT TAAAATTCCAGAAGCAACCTACCTCGCATGGGTTGATCTAAGCTATTACATTAA AGCAAAACACATAGATGAACCAATCGCTAAATATTTCATCAAACACGCCGGCG TTATCATTGAAGGACAAGAACAATTCGTTCATAACGCAGAAGGACATATCAGA ATAAACATCGCTGTTCCACGTGAAATCATGATAAAAGGACTGCAAAAGATTAA AGACGCATTAGTTTAAStaphylococcus haemo!ylicus\6 consensus sequence (SEQ ID NO: 16):TTTATGGAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAATAC ATGCAAGTCGAGCGAACAGACAAGGAGCTTGCTCCTTTGACGTTAGCGGCGGA CGGGTGAGTAACACGTGGGTAACCTACCTATAAGACTGGGATAACTTCGGGAA ACCGGAGCTAATACCGGATAATATTTCGAACCGCATGGTTCGATAGTGAAAGAT GGTTTTGCTATCACTTATAGATGGACCCGCGCCGTATTAGCTAGTTGGTAAGGT AACGGCTTACCAAGGCGACGATACGTAGCCGACCTGAGAGGGTGATCGGCCAC ACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCT TCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTCT TCGGATCGTAAAACTCTGTTATTAGGGAAGAACATACGTGTAAGTAACTATGCA CGTCTTGACGGTACCTAATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGC GGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCG TAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCA TTGGAAACTGGAAAACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAG CGGTGAAATGCGCAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTG GTCTGTAACTGACGCTGATGTGCGAAAGCGTGGGGATCAAACAGGATTAGATA CCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCC CCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAA GGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGG TTTAATTCGAAGCAACGCGAAGAACCTTACCAAATCTTGACATCCTTTGACAAC TCTAGAGATAGAGCCTTCCCCTTCGGGGGACAAAGTGACAGGTGGTGCATGGTT GTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCAtty. Dkt. No. 135523-0119TTAAGCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGTTGACTGCCGGTGACA AACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGATTTGGG CTACACACGTGCTACAATGGACAATACAAAGGGCAGCGAAACCGCGAGGTCAA GCAAATCCCATAAAGTTGTTCTCAGTTCGGATTGTAGTCTGCAACTCGACTACA TGAAGCTGGAATCGCTAGTAATCGTAGATCAGCATGCTACGGTGAATACGTTCC CGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGCC GGTGGAGTAACCATTTGGAGCTAGCCGTCGAAGGTGGGACAAATGATTGGGGT GAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTTgRNA used to engineer Sn00430 (SEQ ID NO: 17):TCTGACCATACACGACCTGTStaphylococcus haemolyticus PatB amino acid sequence (W190 in bold) (SEQ ID NO: 18):VTYNFDEIIDRRSTNAMNVEGYKGYLFGDADTSDLEEHDELIRMWVADMDFATPE VVLDAIRDRLDKKILGYTNIFGTDYYEAFMSWTERRFGYTFPQEHLVFSHGIVAGLI ELVSYICDDDDKALIFTPSYGPFKMACDKNNIKTVYSPMINHNGYYEIDFEDVRQK VETENIKLCIFANPHNPTGRVWSEDELKQLGQIMVDNDVWIISDEIHCDIKRDGQTH VPF AI< A VPD YDK I VT AM SQSK AFN I AGLMF SN LI I PN RRLLKTWK LHHF S SENPLSI VATQAAYEKGEDWLAAMNDYLDDNFKYLAQFLEQELPHAEFKIPEATYLAWVDL SYYIKAKHIDEPIAKYFIKHAGVIIEGQEQFVHNAEGHIRINIAVPREIMIKGLQKIKD ALV*Staphylococcus haemolyticus truncated PatB amino acid sequence resulting from the introduction of a stop codon at W190 (SEQ ID NO: 19):VTYNFDEIIDRRSTNAMNVEGYKGYLFGDADTSDLEEHDELIRMWVADMDFATPE VVLDAIRDRLDKKILGYTNIFGTDYYEAFMSWTERRFGYTFPQEHLVFSHGIVAGLI ELVSYICDDDDKALIFTPSYGPFKMACDKNNIKTVYSPMINHNGYYEIDFEDVRQK VETENIKLCIFANPHNPTGRV*

Claims

Atty. Dkt. No. 135523-0119CLAIMSWhat is claimed is:

1. A genetically engineered Staphylococcus haemolyticus strain whose genome comprises a disruption of a patB gene.

2. The genetically engineered Staphylococcus haemolyticus strain of claim 1, wherein the strain exhibits decreased expression of the patB gene as compared to a corresponding wild-type Staphylococcus haemolyticus strain.

3. The genetically engineered Staphylococcus haemolyticus strain of claim 1 or claim 2, wherein the disruption prohibits transcription of a full-length wild-type mRNA from the disrupted patB gene.

4. The genetically engineered Staphylococcus haemolyticus strain of any one of claims 1-3, wherein the disruption prohibits expression of a functional PatB enzyme, inactivates the PatB protein, modifies the PatB protein, or has a negative effect on the expression of the patB gene.

5. The genetically engineered Staphylococcus haemolyticus strain of any one of claims 1-4, wherein the genetically engineered strain has decreased PatB enzyme activity as compared to a corresponding wild-type Staphylococcus haemolyticus strain.

6. The genetically engineered Staphylococcus haemolyticus strain of claim 4, wherein the modified PatB protein is a truncated protein.

7. The genetically engineered Staphylococcus haemolyticus strain of claim 6, wherein the truncated protein has decreased catalytic activity compared to a corresponding wild-type Staphylococcus haemolyticus strain.

8. The genetically engineered Staphylococcus haemolyticus strain of any one of claims 1-7, wherein the disrupted patB gene comprises a deletion of all or at least a portion of the gene, optionally wherein the deletion comprises deletion of the patB promoter.

9. The genetically engineered Staphylococcus haemolyticus strain of any one of claims 1-7, wherein the disrupted patB gene comprises an insertion of a gene cassette.Atty. Dkt. No. 135523-011910. The genetically engineered Staphylococcus haemolyticus strain of any one of claims 1-7, wherein the disrupted patB gene comprises a deletion of at least a portion of the gene.

11. The genetically engineered Staphylococcus haemolyticus strain of any one of claims 1-7, wherein the disruption comprises a knockout of the patB gene.

12. The genetically engineered Staphylococcus haemolyticus strain of any one of claims 1-7, wherein the disruption comprises an allelic replacement knockout.

13. The genetically engineered Staphylococcus haemolyticus strain of any one of claims 1-7, wherein the disruption comprises a nucleotide substitution.

14. The genetically engineered Staphylococcus haemolyticus strain of claim 13, wherein the nucleotide substitution is a cytosine (C) to a thymine (T).

15. The genetically engineered Staphylococcus haemolyticus strain of claim 14, wherein the nucleotide substitution is at the W190 tryptophan amino acid site in the patB gene.

16. The genetically engineered Staphylococcus haemolyticus strain of claim 15, wherein the nucleotide substitution results in a premature stop codon.

17. The genetically engineered Staphylococcus haemolyticus strain of claim 16, wherein the Staphylococcus haemolyticus strain comprises a disrupted patB gene having about 80% to about 100% sequence identity to SEQ ID NO: 15.

18. The genetically engineered Staphylococcus haemolyticus strain of claim 16, wherein the premature stop codon in the patB gene results in a truncated PatB protein lacking one or more enzymatic activities.

19. The genetically engineered Staphylococcus haemolyticus strain of claim 16, wherein the premature stop codon in the expressed mRNA of the patB gene has the sequence UAG, UGA, or UAA.

20. The genetically engineered Staphylococcus haemolyticus strain of any one of claims 1-19, wherein the disruption is introduced by homologous recombination, site-directed mutagenesis, a Transcription activator-like effector nuclease (TALEN), a meganuclease, a zinc finger nuclease, a CRISPR / Cas9 system, a CRISPR base editing system, a CRISPRAtty. Dkt. No. 135523-0119prime editing system, a CRISPR / Cpfl system, a CRISPR / Csml system, or any combination thereof.

21. The genetically engineered Staphylococcus haemolyticus strain of claim 20, wherein the disruption is introduced by a CRISPR base editing system.

22. The genetically engineered Staphylococcus haemolyticus strain of claim 21, wherein the CRISPR based editing system is a cytosine base editor (CBE) system.

23. The genetically engineered Staphylococcus haemolyticus strain of any one of claims 1-22, wherein the fitness of the strain comprising the disruption is not negatively affected.

24. The genetically engineered Staphylococcus haemolyticus strain of any one of claims 1-23, wherein the strain has a comparable growth rate as compared to a corresponding wildtype Staphylococcus haemolyticus strain.

25. The genetically engineered Staphylococcus haemolyticus strain of any one of claims 1-24, wherein the strain comprises Staphylococcus haemolyticus deposited with the International Depositary Authority of Canada (ID AC) under ID AC Accession Number 240925-04, or a variant strain thereof.

26. The genetically engineered Staphylococcus haemolyticus strain of any one of claims 1-25, wherein the strain has a reduced capacity to produce 3-methyl-3-sulfanylhexan-l-ol (3M3SH) when contacted with apocrine sweat or a 3M3SH precursor as compared to a corresponding wild-type strain.

27. The genetically engineered Staphylococcus haemolyticus strain of any one of claims 1-26, wherein the disruption is a deletion or nucleotide substitution in the DNA sequence of the patB gene encoding a PatB protein, wherein the deletion or nucleotide substitution inactivates the PatB protein, truncates the PatB protein, or has a negative effect on expression of the patB gene.

28. The genetically engineered Staphylococcus haemolyticus strain of any one of claims 1-27, wherein the genetically engineered Staphylococcus haemolyticus strain has a 16S rRNAgene sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the sequence set forth in SEQ ID NO: 16.Atty. Dkt. No. 135523-011929. The genetically engineered Staphylococcus haemolyticus strain of claim 28, wherein the strain has the 16S rRNA gene sequence as set forth in SEQ ID NO: 16.

30. The genetically engineered Staphylococcus haemolyticus strain of any one of claims 1-29, wherein the genetically engineered Staphylococcus haemolyticus strain is derived from the strain deposited with the International Depositary Authority of Canada (ID AC) under ID AC Accession Number 240925-04, or a variant strain derived therefrom, wherein the genome has been engineered to comprise a disruption of a patB gene.

31. A genetically engineered Staphylococcus haemolyticus strain deposited with the International Depositary Authority of Canada (ID AC) under ID AC Accession Number 240925-04, or a variant strain derived therefrom, wherein the variant strain is obtained using the deposited strain as starting material, and wherein a reduced capacity of the deposited strain to produce 3-methyl-3-sulfanylhexan-l-ol (3M3SH) when contacted with apocrine sweat or a 3M3SH precursor as compared to a corresponding wild-type strain is retained or improved in the variant strain as compared to the deposited strain.

32. A Staphylococcus haemolyticus strain, wherein the strain comprises a mutation in at least one endogenous polynucleotide selected from the group consisting of:(a) a nucleotide sequence having about 80% to about 100% sequence identity to SEQ ID NO: 14; and(b) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having about 80% to about 100% sequence identity to SEQ ID NO: 18, such that the strain has a reduced capacity to produce 3-methyl-3-sulfanylhexan-l-ol (3M3SH) when contacted with apocrine sweat or a 3M3SH precursor as compared to a corresponding strain that does not comprise the mutation.

33. The strain of claim 32, wherein the nucleotide sequence of (a) has about 97% to about 100% sequence identity to SEQ ID NO: 14, and / or the nucleotide sequence of (b) encodes a polypeptide comprising an amino acid sequence having about 97% to about 100% sequence identity to SEQ ID NO: 18.

34. The strain of claim 32 or 33, wherein the strain grows at an equivalent or improved rate as compared to a Staphylococcus haemolyticus strain lacking the mutation.Atty. Dkt. No. 135523-011935. The strain of any one of claims 32-34, wherein the strain is incapable of converting Cys-Gly-3M3SH to 3M3SH.

36. A composition comprising an effective amount of one or more of the genetically engineered Staphylococcus haemolyticus strains of any one of claims 1-35.

37. The composition of claim 36, wherein the one or more Staphylococcus haemolyticus strains is lyophilized.

38. The composition of claim 36 or claim 37 further comprising a lyoprotectant.

39. The composition of claim 38, wherein the lyoprotectant is selected from one or more of sucrose, micellar casein, trehalose, or inulin.

40. The composition of any one of claims 36-39, wherein the one or more Staphylococcus haemolyticus strains are viable.

41. The composition of any one of claims 36-40, wherein the one or more Staphylococcus haemolyticus strains are nonproliferative.

42. The composition of any one of claims 36-41 further comprising Staphylococcus epidermidis.

43. The composition of any one of claims 36-42 further comprising an excipient.

44. The composition of claim 43, wherein the excipient is selected from one or more of oleic acid, stearic acid, almond butter, avocado butter, babassu butter, beeswax, cocoa butter, coconut butter, coconut oil, cupuacu butter, hemp seed butter, kokum butter, macadamia butter, mango butter, mowrah butter, olive butter, sal seed butter, shea butter, propylene glycol, glycerin, stearyl alcohol, myristyl alcohol, capric triglyceride, polyethylene glycol (PEG), Isopropyl Myristate, or sorbitol.

45. The composition of any one of claims 36-44, wherein the Staphylococcus haemolyticus strain is present at a concentration of about 106to about 1013bacteria per gram.

46. The composition of claim 45, wherein the Staphylococcus haemolyticus strain is present at a concentration of about 107to about 1011bacteria per gram.Atty. Dkt. No. 135523-011947. The composition of any one of claims 43-46, comprising about 90 wt. % to about 99.99 wt. % excipient.

48. The composition of claim 47, wherein the lyophilized Staphylococcus haemolyticus bacteria is present at about 10 wt. % to about 0.01 wt. %.

49. The composition of claim 48, comprising about 95 wt. % to about 99.75 wt. % excipient.

50. The composition of claim 49, wherein the lyophilized Staphylococcus haemolyticus bacteria is present at about 5 wt. % to about 0.25 wt. %.

51. The composition of any one of claims 36-50, further comprising a fragrance selected from one or more of elettaria cardamomum seed oil, eucalyptus globulus seed oil, citrus aurantium bergamia fruit oil, mentha piperita oil, salvia sclarea oil, juniperus virginiana oil, abies sibirica oil, citrus aurantium amara, citrus aurantium dulcis lower oil, citrus nobilis oil, rosmarinus officinalis oil, melaleuca alternifolia oil, citrus limon tree oil, zingiber officinale root oil, Mangifera indica oil, Lavandula angustifolia oil, and Santalum album.

52. The composition of any one of claims 36-51, wherein the composition is formulated for topical administration.

53. The composition of claim 52, wherein the composition is formulated for use as a topical deodorant.

54. The composition of any one of claims 36-53, further comprising an antiperspirant.

55. The composition of claim 54, wherein the antiperspirant comprises one or more of aluminum salt, charcoal, and witch hazel.

56. The composition of any one of claims 36-55, wherein the topical deodorant is formulated as a deodorant stick, a roll-on, a cream, a lotion, a gel, a powder, a water activated cream, a water activated powder, an injectable, a patch, or a spray.

57. The composition of any one of claims 36-56, wherein the composition is a pharmaceutical composition and the effective amount of one or more of the genetically engineered Staphylococcus haemolyticus strains is a therapeutically effective amount.Atty. Dkt. No. 135523-011958. A pharmaceutical composition comprising a therapeutically effective amount of one or more genetically engineered Staphylococcus haemolyticus strains, whose genome comprises a disruption of a patB gene, and pharmaceutically acceptable carrier or excipient, and optionally, a lyoprotectant.

59. The pharmaceutical composition of claim 58, wherein the disruption of the patB gene comprises a nucleotide substitution, optionally wherein the nucleotide substitution comprises a cytosine (C) to thymine (T) substitution as compared to the wild-type Staphylococcus haemolyticus strain.

60. The pharmaceutical composition of claim 59, wherein the substitution is at the W190 tryptophan amino acid site in the patB gene.

61. The pharmaceutical composition of any one of claims 58-60, wherein the substitution introduces a premature stop codon.

62. The pharmaceutical composition of any one of claims 58-61, wherein the one or more genetically modified Staphylococcus haemolyticus strains are lyophilized.

63. The pharmaceutical composition of any one of claims 58-62, wherein the pharmaceutically acceptable carrier or excipient is present at about 90 wt. % to about 99.99 wt. % and the one or more lyophilized genetically engineered Staphylococcus haemolyticus strains is present at about 10 wt. % to about 0.01 wt. %.

64. The pharmaceutical composition of any one of claims 58-62, wherein the pharmaceutically acceptable carrier or excipient is present at about 95 wt. % to about 99.75 wt. % and the one or more lyophilized genetically engineered Staphylococcus haemolyticus strains is present at about 5 wt. % to about 0.25 wt. %.

65. The pharmaceutical composition of any one of claims 58-64, wherein the one or more genetically engineered Staphylococcus haemolyticus strains are present at a concentration of about 106to about 1013bacteria per gram.

66. The pharmaceutical composition of claim 65, wherein one or more genetically engineered Staphylococcus haemolyticus strains are present at a concentration of about 107to about 1011bacteria per gram.Atty. Dkt. No. 135523-011967. The pharmaceutical composition of any one of claims 58-66, wherein the one or more genetically engineered Staphylococcus haemolyticus strains are selected from the strain deposited with the International Depositary Authority of Canada (ID AC) under ID AC Accession Number 240925-04.

68. The pharmaceutical composition of any one of claims 58-67, comprising the genetically engineered Staphylococcus haemolyticus strain deposited with the International Depositary Authority of Canada (IDAC) under ID AC Accession Number 240925-04.

69. A method of reducing 3-methyl-3-sulfanylhexan-l-ol (3M3SH) production from a skin microbiota of a subject, the method comprising applying: (i) a composition or a pharmaceutical composition comprising the genetically engineered Staphylococcus haemolyticus strain of any one of claims 1-35 to the skin of the subject; (ii) the composition of any one of claims 36-57, or (iii) the pharmaceutical composition of any one of claims 58-68 to the skin of the subject.

70. A method for treating, reducing, or preventing malodor on the skin of a subject, comprising treating the skin of the subject with a therapeutically effective amount of the composition of any one of claims 28-48 or the pharmaceutical composition of any one of claims 49-59.

71. The method of claim 70, wherein the composition or pharmaceutical composition is applied topically to the skin of the subject.

72. The method of claim 70 or 71, wherein the composition or pharmaceutical composition is applied to a sweat-gland-containing area of skin of the subject.

73. The method of any one of claims 70-72, wherein the composition or the pharmaceutical composition is administered to the subject about 1 to about 2 times per day for about 1 to about 7 days.

74. The method of any one of claims 70-73, wherein the treated skin of the subject comprises a reduction in the amount of malodorous bacteria as compared to a corresponding untreated portion of skin.

75. The method of claim 74, wherein the malodorous bacteria comprises at least wildtype Staphylococcus haemolyticus.Atty. Dkt. No. 135523-011976. A method of producing a genetically engineered Staphylococcus haemolyticus strain, the method comprising targeting the patB gene for disruption.

77. The method of 76, wherein the disruption is introduced into the patB gene by homologous recombination, site-directed mutagenesis, a Transcription activator-like effector nuclease (TALEN), a meganuclease, a zinc finger nuclease, a CRISPR / Cas9 system, a CRISPR / Cpfl system, or a CRISPR / Csml system, and any combination thereof.

78. The method of claim 77, wherein the disruption is introduced into the patB gene by the CRISPR base editing system.

79. The method of claim 78, wherein the CRISPR based editing system is a cytosine base editor (CBE) system.

80. The method of claim 78, wherein the CRISPR based editing system comprises transforming a wild type Staphylococcus haemolyticus strain with a 6-part plasmid.

81. The method of claim 80, wherein the 6-part plasmid comprises one or more sequences selected from SEQ ID NOs: 2-7.

82. The method of claim 81, wherein the 6-part plasmid comprises the sequence of SEQ ID NO: 1.

83. The method of any one of claims 76-82, wherein the strain exhibits decreased expression of the patB gene as compared to a corresponding wild-type Staphylococcus haemolyticus strain.

84. The method of any one of claims 76-83, wherein the disruption prohibits transcription of a full-length wild-type mRNA from the disrupted patB gene.

85. The method of any one of claims 76-84, wherein the disruption prohibits expression of a functional PatB enzyme.

86. The method of any one of claims 76-85, wherein the genetically engineered strain has decreased PatB enzyme or catalytic activity and the PatB protein is truncated as compared to a corresponding wild-type Staphylococcus haemolyticus strain.Atty. Dkt. No. 135523-011987. The method of any one of claims 76-86, wherein the disrupted patB gene comprises a nucleotide substitution, wherein the substitution is a cytosine (C) to a thymine (T).

88. The method of any one of claims 76-87, wherein the strain is selected from Staphylococcus haemolyticus deposited with the International Depositary Authority of Canada (ID AC) under ID AC Accession Number 240925-04.

89. The method of any one of claims 76-88, wherein the genetically engineered Staphylococcus haemolyticus strain has a reduced capacity to produce 3-methyl-3- sulfanylhexan-l-ol (3M3SH) when contacted with apocrine sweat or a 3M3SH precursor as compared to a corresponding wild-type strain.

90. An altered microbiota comprising a genetically engineered Staphylococcus haemolyticus strain, or a variant strain thereof, whose genome comprises a disruption of a patB gene.

91. The altered microbiota of claim 90, wherein the microbiota has a reduced capacity to produce 3-methyl-3-sulfanylhexan-l-ol (3M3SH) when contacted with apocrine sweat as compared to a corresponding wild-type microbiota.

92. A deodorant composition comprising:(i) a therapeutically effective amount of a viable Staphylococcus haemolyticus strain of any of claims 1-35; and(ii) a therapeutically acceptable carrier or excipient;wherein the deodorant composition is formulated as a water activated formulation for topical administration.

93. The deodorant composition of claim 92, comprising about 90 wt. % to about 99.99 wt. % therapeutically acceptable carrier or excipient.

94. The deodorant composition of claim 93, wherein the Staphylococcus haemolyticus strain is lyophilized and the deodorant composition comprises about 10 wt. % to about 0.01 wt. % lyophilized Staphylococcus haemolyticus bacteria.

95. The deodorant composition of claim 94, comprising about 95 wt. % to about 99.75 wt. % therapeutically acceptable carrier or excipient.Atty. Dkt. No. 135523-011996. The deodorant composition of claim 95, wherein the deodorant composition comprises about 5 wt. % to about 0.25 wt. % lyophilized Staphylococcus haemolyticus bacteria.

97. The deodorant composition of any one of claims 92-96, wherein the Staphylococcus haemolyticus strain is present at a concentration of about 106to about 1016bacteria per gram.

98. The deodorant composition of claim 97, wherein the Staphylococcus haemolyticus strain is present at a concentration of about 107to about 1011bacteria per gram.

99. The deodorant composition of any one of claims 92-98 further comprising: (iii) a lyoprotectant.

100. The deodorant composition of any one of claims 92-99, wherein the Staphylococcus haemolyticus strain comprises the strain deposited with the International Depositary Authority of Canada (ID AC) under ID AC Accession Number 240925-04.

101. A deodorant stick comprising the deodorant composition of any one of claims 92-100.

102. A pharmaceutical composition comprising a therapeutically effective amount of one or more of the genetically engineered Staphylococcus haemolyticus strains of any one of claims 1-35, and a therapeutically acceptable carrier or excipient, for use in the treatment of malodor on the skin of a subject.

103. The pharmaceutical composition of claim 102 further comprising a lyoprotectant.

104. Use of the one or more genetically engineered Staphylococcus haemolyticus strains of any one of claims 1-35 in the manufacture of a medicament for the treatment of malodor on the skin of a subject.

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