USOO9453232B2

(12) United States Patent (10) Patent No.: US 9.453,232 B2 Qvit-Raz et al. (45) Date of Patent: *Sep. 27, 2016

(54) TOPICAL COMPOSITION COMPRISING (56) References Cited TRANSFORMED BACTERIA EXPRESSING A COMPOUND OF INTEREST U.S. PATENT DOCUMENTS 4,259,444 A 3/1981 Chakrabarty (71) Applicant: TOPGENIX, INC., Menlo Park, CA 5,207,998 A * 5/1993 Robinson ...... A61K 8/29 424/47 (US) 5,620,682 A 4/1997 Fogel 6,221,648 B1 4/2001 Le Page et al. (72) Inventors: Noga Qvit-Raz, Menlo Park, CA (US); 6,605,286 B2 8, 2003 Steidler et al. 6,787,147 B1 * 9/2004 Huner ...... A61K 8.44 Tahel Altman, Menlo Park, CA (US) 424/400 7,081.442 B2 7/2006 Seiberg et al. (73) Assignee: TOPGENIX, INC., Menlo Park, CA 2009/O1300.73 A1 5/2009 Reindl et al. 2009, O232751 A1 9, 2009 Lott et al. (US) 2012fO263661 A1 10/2012 Grune 2012/03 01452 A1 1 1/2012 Gueniche et al. (*) Notice: Subject to any disclaimer, the term of this 2012fO3O8525 A1 12/2012 Greenberg et al. patent is extended or adjusted under 35 2014/0044653 A1 2/2014 Qvit-Raz et al. U.S.C. 154(b) by 0 days. 2014/004.4677 A1 2/2014 Qvit-Raz et al. This patent is Subject to a terminal dis FOREIGN PATENT DOCUMENTS claimer. EP 1473028 A1 11, 2004 EP O975227 B1 11/2005 (21) Appl. No.: 14/863,236 EP 1789.529 A2 2, 2006 EP 1 322 318 B1 * 12/2010 ...... A61K 35,74 EP 1322318 B1 12/2010 (22) Filed: Sep. 23, 2015 EP 2364712 B1 3, 2013 FR 28032O1 A1 T 2001 WO WO86,02350 A1 4f1986 (65) Prior Publication Data WO WO89/O1970 A2 3, 1989 US 2016/OOOO701 A1 Jan. 7, 2016 WO WO96, 11277 A1 4f1996 WO WOO2,39974 A1 5, 2002 WO WOO3,O2O236 A2 3, 2003 WO WO2006/013441 A2 2, 2006 Related U.S. Application Data WO WO2007/039086 A1 4/2007 WO WO2011/15O127 A2 12, 2011 (63) Continuation of application No. 13/961,845, filed on WO WO2011, 151426 A2 12/2011 Aug. 7, 2013, now Pat. No. 9.234,204. WO WO2012/150269 A1 11, 2012 WO WO2013/044059 A2 3, 2013 (60) Provisional application No. 61/680,620, filed on Aug. 7, 2012, provisional application No. 61/836,594, filed OTHER PUBLICATIONS on Jun. 18, 2013. Shick et al., “Mycosporine-Like Amino Acids and Related Gadusols: Biosynthesis, Accumulation, and UV-Protective Func tions in Aquatic Organisms' 64 Annual Review of Physiology (51) Int. Cl. 223-262 (2002).* A6 IK 8/18 (2006.01) Rastogi et al., “Photoprotective compounds from marine organ CI2N 15/74 (2006.01) isms' 37 Journal of Industrial Microbiology and Biotechnology A61O 1704 (2006.01) 537-558 (2010).* A61O 1700 (2006.01) Balskus et al., “The Genetic and Molecular Basis for Sunscreen A 6LX 8/99 (2006.01) Biosynthesis in Cyanobacteria' 329 Science 1653-1656 (incl Supplemental Material) (2010).* A61O 19/00 (2006.01) Perez-Arellano et al., "Construction of Compatible Wide-Host A61O 19/08 (2006.01) Range Shuttle Vectors for Lactic Acid Bacteria and Escherichia A6 IK 35/744 (2015.01) coli 46 Plasmid 106-116 (2001).* A6 IK 8/27 (2006.01) (Continued) A6 IK 8/40 (2006.01) CI2N I/2O (2006.01) CI2P 21/04 (2006.01) Primary Examiner — Nancy J Leith CI2P 21/06 (2006.01) (74) Attorney, Agent, or Firm — Shay Glenn LLP (52) U.S. Cl. (57) ABSTRACT CPC ...... CI2N 15/746 (2013.01); A6IK 8/27 Compositions comprised of a population of transformed (2013.01); A61K 8/40 (2013.01); A61K 8/99 bacteria formulated for topical application to a subject are (2013.01); A61K 35/744 (2013.01); A61O described. The population of transformed bacteria are cre 1700 (2013.01); A61O 1704 (2013.01); ated from a non-pathogenic bacteria and transformed to A61O 19/007 (2013.01); A61O 19/08 express a compound of interest for a therapeutic or a (2013.01); A6 IK 2800/592 (2013.01) cosmetic purpose. In one embodiment, the composition is (58) Field of Classification Search for protection of the skin from ultraviolet rays. None See application file for complete search history. 20 Claims, 3 Drawing Sheets US 9,453,232 B2 Page 2

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Sufficiently earlier than effective US filed Kiatpapan et al.; Genetic manipulation system in and any foreign priority date). propionibacterium; J. Biosci. Bioeng.: 93(1); pp. 1-8; 2002 (year of Shick et al.; Mycosporine-like amino acids and related gadusols: pub. Sufficiently earlier than effective US filing date and any foreign biosynthesis, accumulation, and uv-protective functions in aquatic priority date). organisms; Ann. Rev. Physiol.; 64; pp. 223-262; 2002 (year of pub. Kim et al.; A xylose-inducible bacillus subtilis integration vector sufficiently earlier than effective US filed and any foreign priority and its application; Gene; 181(1-2; pp. 71-76; Nov. 28, 1996. date). Kim et al., Improvement of a nisin-inducible expression vector for Sorensen; Towards universal systems for recombinant gene expres use in lactic acid bacteria; Plasmid; 58(3): pp. 275-283; Nov. 2007. sion; Microbial Cell Factories; 9:27; 4 pages; Apr. 2010. US 9,453,232 B2 Page 3

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U.S. Patent Sep. 27, 2016 Sheet 2 of 3 US 9,453,232 B2

U.S. Patent Sep. 27, 2016 Sheet 3 of 3 US 9,453,232 B2

US 9,453,232 B2 1. 2 TOPCAL COMPOSITION COMPRISING photoaging. By way of another example, topical treatment of TRANSFORMED BACTERIA EXPRESSINGA psoriasis and eczema, and other skin disorders, with an COMPOUND OF INTEREST effective, long-term therapy is needed. Healthy human epidermis is colonized by thousands of CROSS-REFERENCE TO RELATED bacterial species, including bacterial members from mainly APPLICATIONS five orders harboring about 60% of the total skin microbi ome in all people. A healthy human epidermis is colonized This application is a continuation of U.S. application Ser. with trillions of bacterial cells, creating, on average, No. 13/961,845, filed Aug. 7, 2013, now U.S. Pat. No. approximately 10 bacteria per square centimeter. The skin 9,234,204, which application claims the benefit of U.S. 10 microflora has evolved into commensal relationship with the Provisional Application No. 61/680,620, filed Aug. 7, 2012, host, as they exploit the unique attributes of the skin and and of U.S. Provisional Application No. 61/836,594, filed keep the skin ecosystem in a healthy balance (Grice A. E., Jun. 18, 2013, each of which is incorporated herein by Science, 324: 1190-1192 (2009). A therapy that uses skin reference in its entirety. bacteria for different dermatological needs would be able to 15 maintain the natural ecosystem of the skin, and also enhance REFERENCE TO SEQUENCE LISTING, TABLE those natural skin bacteria to address specific dermatological OR COMPUTER PROGRAM needs. The use of probiotic micro-organisms for improving the A Sequence Listing was submitted electronically via EFS skin's immune function under stress conditions, leading to in parent application Ser. No. 13/961,845 in the form of a immune Suppression, specifically for normalizing the skin's text file, created Dec. 23, 2013, and named “093242-0016 immune activity and reducing the tendency to develop seqlist ST25.txt (88.802 bytes), and is resubmitted here hyper-reactions under Such conditions is described in the art, with. for example in EP Patent No. 1322318. Cosmetic use of probiotic microorganisms as an active agent useful for TECHNICAL FIELD 25 treating and/or preventing impairing radiance of the skin complexion has also been described (US2012/0301452). The subject matter described herein relates to the field of Use of Solely probiotics, is one approach for topical skin dermatology, and more particularly, to compositions and treatments, yet there remains a need for a longer term and/or methods of treatment that comprise transformed bacteria more potent approach. that express a molecule or compound for a topical thera 30 Transformed bacteria are being used intensively in mod peutic, cosmetic or dermatological purpose. ern biotechnology for the production of recombinant pro teins and various molecules for food, pharmaceutical, and BACKGROUND biocatalysis applications. Bacteria able to produce and secrete proteins encoded by heterologous genes are used There are a spectrum of dermatological disorders and 35 extensively for the industrial production of pharmaceutical conditions that are commonly treated with a topically proteins such as human and animal growth hormones, insu applied agent. In some treatments, the agent offers a thera lin, interferons, cytokines etc. Organisms other than E. coli peutic purpose, for example for treating or ameliorating thus far used or proposed for industrial production include psoriasis, eczema or dermatitis. In other treatments, the cultured mammalian and insect cells, yeasts and fungi, and agent offers a cosmetic or protective effect, such as a skin 40 various bacteria species, including a number of Bacillus spp. lightening agent or depigmenting agent or a Sun protective Among the bacteria already widely used for industrial agent. Topical application of agents for cosmetics and medi purposes are the lactic acid bacteria, which are employed as cal purposes has certain limitations. For example, the starter cultures for fermented foodstuffs, and as flavor applied agent can be swept off easily from the skin or the enhancers, and preservatives. These properties depend on formulation in which the agent is applied can include 45 the ability of these organisms to produce certain enzymes, chemicals that may interfere with the balance of the natural lactic acid and harmless antimicrobial polypeptides. Such as skin microbiota. Creams and ointments can be messy, nisin (see, for example. U.S. Pat. No. 6,221,648). greasy, cumbersome, and patients can only treat a limited The foregoing examples of the related art and limitations number of lesions on a limited area, and only on certain related therewith are intended to be illustrative and not anatomic sites. As a result, nearly 35% of prescriptions for 50 exclusive. Other limitations of the related art will become topical preparations remained behind the pharmacy counter, apparent to those of skill in the art upon a reading of the the patient opting to not pick up the topical prescription. In specification and a study of the drawings. contrast, prescriptions for systemic agents fared much better, with reports that only 14% went unredeemed (Storm, A. et BRIEF DESCRIPTION OF THE DRAWINGS al., J. Am. Acad. Dermatol., 59:27-33 (2008)). 55 There are also disadvantages with topical treatments in FIGS. 1A-1C are maps of exemplary plasmids or vectors terms of the patient understanding of how often and how for transforming a bacterium for expression of a compound much to apply. In the case of Sunscreens, as just one of interest. Sequences corresponding to the restriction example, people typically apply Sunscreens less than half as enzyme recognition sites indicated on the map of FIG. 1A thickly as and less often than recommended, thus compro 60 are provided below in Table 1 with their respective SEQID mising their protection substantially (Stern, R. S., N Engl. NOS Med, 350:1526-1534 (2004)). There remains a need for more effective topical treatment BRIEF SUMMARY compositions, for medical, cosmetic and preventative pur poses. By way of example, the need for more effective UV 65 The following aspects and embodiments thereof protection is recognized around the world, as it is the main described and illustrated below are meant to be exemplary cause for the increasing incidence of skin cancers and and illustrative, not limiting in Scope. US 9,453,232 B2 3 4 In one aspect, a composition comprising a population of express the compound of interest. In another embodiment, transformed bacteria formulated for topical application to a the second population of non-pathogenic bacteria is a popu subject is provided, where the population of transformed lation of attenuated bacteria or killed (dead) bacteria, intact bacteria is created from a non-pathogenic bacteria trans or fragments thereof, where the bacteria prior to attenuation formed to express a compound of interest. or killing expressed the compound of interest. In one embodiment, the population of non-pathogenic In still another embodiment, the compound of interest is bacteria comprises a bacteria that occurs naturally on the one for treating psoriasis. Exemplary compounds include, human skin; that is, the bacteria in the population are from but are not limited to, a compound selected from the group the human skin microbiome. In another embodiment, the consisting of retinoid, Vitamin A, beta-carotene, Vitamin D, population of non-pathogenic bacteria is a population of live 10 anti-inflammatory cytokines. bacteria that express the compound of interest, and in one embodiment, that chronically express the compound of In one embodiment, the compound of interest is an interest. In another embodiment, the population of non anti-oxidant. pathogenic bacteria is a population of attenuated bacteria or In yet another embodiment, the compound is selected killed (dead) bacteria, intact or fragments thereof, where the 15 from the group consisting of resveratrol, Vitamin E. Vitamin bacteria prior to attenuation or killing expressed the com C, -epigallocatechin-3-gallate, and retinyl palmitate (ret pound of interest. inoids), lutein, tamarind, flavonoids, pycnogenol, lycopene. In one embodiment, the compound of interest is one that In another embodiment, the compound of interest pro can absorb or reflect ultraviolet light. In another embodi vides a cosmetic effect. For example, the compound may be ment, the compound can absorb or reflect ultraviolet A selected from the group consisting of coenzyme Q10, tyro (320-400 nm), ultraviolet B (315-280 nm), or both. sinases, collagen, laminin, ceramids, linoleic acid, tretinoin, In yet another embodiment, the compound of interest tazarotene and collagen. In still another embodiment, the expressed by the transformed bacteria is selected from the cosmetic effect is anti-aging. group consisting of mycosporine, gadusols, oxo-my In still another embodiment, the compound of interest is cosporines, imino-mycosporines and mycosporine-like 25 one that treats eczema. Exemplary compounds of interest amino acids (MAAS), Scytonemin, melanines, UV-screen include a compound is selected from the group consisting of ing/observing amino acids-like molecules, flavonoids, beta cortisone, tacrolimus and ciclosporin. lanines, UV-screening/observing pigments (e.g. carotenoids/ In other embodiments, the composition comprising the cartenoproteins, Xanthopylls and porphyrin-based/heme population of transformed bacterial is formulated for topical porphyrin based), UV-screening/observing co-factors (e.g. 30 application to the face. In another embodiment, the compo tetrahydrobiopterin), phenylpropanoids, polyphenol (e.g. sition is formulation for topical application to the skin, tannins), pycnogenol, tyrosinases (and its Substrates and excluding mucosal surfaces of the human body. products), alpha hydroxy acids (AHAS), polysaccharides In another embodiment, the population of transformed (e.g. glycosaminoglycans, (GAGs) or mucopolysaccha bacteria is created from a population of non-pathogenic rides), skin related cofactors, Vitamin E, polymers, and 35 bacteria resident on the skin in humans. That is, the popu additional skin related natural compounds, such as: collagen, lation of non-pathogenic bacteria comprise a bacteria typi keratin, elastin, linoleic acid, laminin, tretinoin, tazarotene, cally resident on skin in healthy, non-diseased human Sargaduinoic acid, Sargachromenol, fucoxanthin, retinoid, beings. anti-inflammatory cytokines (as Il-2), cortisone, tacrolimus, The population of transformed bacteria is created, in some ciclosporin, resveratrol, gallocatechol, gallocatechin, epigal 40 embodiments, from nonpathogenic bacterial members locatechin gallate, retinoid, vitamin A, vitamin A deriva selected from those in the group consisting of Actinomyc tives, beta-carotene, Vitamin D. Vitamin A derivatives, mois etales, Anaerococcus, Bacillales, Bifidobacterium, Enhydro ture compounds; cortisone, tacrolimus and ciclosporin, bacter; Finegoldia, Carnobacterium, Coryneobacterium, DNA repair enzymes; photolyase, endonuclease and glyco Lactobacillus, Lactococcus, Leunconostoc, Macrooccus, Sylase. 45 Micrococcineae, Oenococcus, Pediococcus, Peptoniphilus, In yet another embodiment, the population of transformed Propionibacterium, Salinicoccus, Sphingomonas, Strepoco bacteria is formulated into the composition to provide at ccus, Tetragenoccus, and Weissella. least about 10° bacteria per cm, or at least about 10, 10. In other embodiments, the transformed bacteria in the 10, or 10° bacteria per cm. population of transformed bacterial are not Propionibacte In another embodiment, the composition comprises a 50 rium acnes, a pathogenic strain of Coryneobacterium, S. second population of transformed bacteria formulated for aureus, or S. epidermidis. topical application to a Subject, wherein the second popu In still another embodiment, the population of trans lation of transformed bacteria is either or both (i) created formed bacteria is created from a bacteria selected from from a non-pathogenic bacteria that is different from the first those in the group consisting of Lactobacillus casei, Lacto population of transformed bacteria in the composition or (ii) 55 bacillus reuteri, Lactobacillus acidophilus, Lactobacillus transformed to express a compound of interest that is jensenii, Bifidobacterium lognum, Bifidobacterium reuteri, different from the first compound of interest expressed by Bifidobacterium lactis, Bifidobacterium breve, Bifidobacte the first population of transformed bacteria in the composi rium animalis, Propionibacterium acidipropionici, Propi tion. In other embodiments, the composition comprises at Onibacterium feudenreichii, Propionibacterium thoenii, least one population of transformed bacteria, at least two 60 and Propionibacterium jensenii. populations of transformed bacteria, or two or more popu In yet another embodiment, the population of transformed lations of transformed bacteria. bacteria is created from a bacteria selected from those in a In one embodiment, the second population of non-patho phylum selected from the group consisting of gamma genic bacteria is from the human skin microbiome. In proteobacteria, alpha-proteobacteria, and bacteriodetes. another embodiment, the second population of non-patho 65 The topical composition that comprises the population of genic bacteria is a population of live bacteria that express the transformed bacteria can be, in various embodiments, a compound of interest, and in one embodiment, chronically cream, lotion, emulsion, gel, ointment, liquid or spray. In US 9,453,232 B2 5 6 one embodiment, the topical composition is formulated to “Hydrophobic' as used herein refers to substances that provide at least about 10° bacteria per cm. lack an affinity for water; tending to repel and not absorb In another aspect, a method of treatment is provided, water as well as not dissolve in or mix with water. wherein a composition as described herein is topically “Lipid soluble' as used herein refers to substances that applied to the skin of a subject, preferably a human Subject, 5 have a solubility of greater than or equal to 5 g/100 mL in for disease preventative, or for a therapeutic or cosmetic a hydrophobic liquid, Such as castor oil. purpose. In embodiment, topically applying excludes topi “Lipophilic' refers to compounds having an affinity for cally applying to a mucosal Surface (nasal, vaginal, rectal, lipids. Examples of lipophilic substances include but are not oral Surfaces) of a human body. limited to naturally occurring and synthetic oils, fats, fatty In addition to the exemplary aspects and embodiments 10 acids, lecithins, triglycerides and combinations thereof. described above, further aspects and embodiments will An “oil is a composition containing at least 95% wt of a become apparent by reference to the drawings and by study lipophilic Substance. of the following descriptions. "Skin' intends to denote all of the epidermis of an Additional embodiments of the present methods and individual, in particular a human being, and in some compositions, and the like, will be apparent from the fol- 15 embodiments to intend, where specified, particular regions lowing description, drawings, examples, and claims. As can of the skin, Such as the face, neck, arms, legs, abdomen, be appreciated from the foregoing and following descrip hands, back, buttocks, or feet. tion, each and every feature described herein, and each and “Water soluble' as used herein refers to substances that every combination of two or more of such features, is have a solubility of greater than or equal to 5 g/100 mL included within the scope of the present disclosure provided 20 Water. that the features included in Such a combination are not II. Topical Composition mutually inconsistent. In addition, any feature or combina The composition described herein is comprised of a tion of features may be specifically excluded from any population of transformed bacteria formulated for topical embodiment of the present invention. Additional aspects and application to a subject. Described in section A below are advantages of the present invention are set forth in the 25 exemplary non-pathogenic bacteria Suitable for creating the following description and claims, particularly when consid population of transformed bacteria. In section B, compounds ered in conjunction with the accompanying examples and of interest to be expressed by the population of transformed drawings. bacteria are described, and in section C techniques for creating the transformed bacterial population are set forth. In DETAILED DESCRIPTION 30 section D, topical compositions comprising the population of transformed bacteria are disclosed. I. Definitions A. Exemplary Bacteria Various aspects now will be described more fully here The population of bacteria in the compositions described inafter. Such aspects may, however, be embodied in many herein and for use in the described methods is created from different forms and should not be construed as limited to the 35 a non-pathogenic bacterium that has been genetically modi embodiments set forth herein; rather, these embodiments are fied to express, produce and/or secrete a compound of provided so that this disclosure will be thorough and com interest. In this section, exemplary non-pathogenic bacteria plete, and will fully convey its scope to those skilled in the are described. In one embodiment, the bacteria in the art. population are non-pathogenic and non-invasive microor Where a range of values is provided, it is intended that 40 ganisms, and can be in certain embodiments a gram-positive each intervening value between the upper and lower limit of food grade bacterial strain. In another embodiment, the that range and any other stated or intervening value in that populations of transformed bacteria are prepared from a stated range is encompassed within the disclosure. For bacterium that occurs naturally in the skin microbiome. example, if a range of 1 um to 8 um is stated, it is intended Human skin is populated with microorganisms that reside that 2 um, 3 um, 4 um, 5 Lim, 6 Lim, and 7 um are also 45 on the skin, referred to as the skin microbiome. The bacterial explicitly disclosed, as well as the range of values greater microorganisms resident on the skin (in a healthy (non than or equal to 1 Lum and the range of values less than or diseased) human) are usually non-pathogenic and commen equal to 8 um. sal (not harmful to the host) and/or mutualistic (offer a As used in this specification, the singular forms 'a,” “an.” benefit). The bacteria commonly resident on the human skin and “the include plural referents unless the context clearly 50 are set forth in below, and are indicated by phylogenetic dictates otherwise. Thus, for example, reference to an levels, described with their phylogenetic lineage, down to “excipient' includes a single excipient as well as two or the genus level (Grice, E. A. et al., Science, 324(5931): more of the same or different excipients. 11904.192 (2009); Costello, E. K., et al. Science, 326(5960): “Amphiphilic' refers to a molecule combining hydro 1694-1697 (2009), Grice E. A. and J. A. Segre, Nature philic and lipophilic (hydrophobic) properties. 55 Reviews Microbiology, 9:244-253 (2011)). “Diluents’ may be included in the formulations to dis The bacteria forming the population of bacteria in the Solve, disperse or otherwise incorporate another component composition, and that are transformed to express one or in the formulation. Examples of diluents include, but are not more compounds of interest, can be a collection of the same limited to, water, buffered aqueous solutions, organic hydro bacteria or a mixture of different bacteria, at different philic diluents, such as monovalent alcohols, and low 60 phylogenetic levels. In one embodiment, the populations of molecular weight glycols and polyols (e.g. propylene glycol, bacteria for transformation are a group of individuals of one polypropylene glycol, glycerol, butylene glycol). bacterial species in an area that is separate from other groups A “gel' is a colloid in which the dispersed phase has of bacteria, apart from rare migration events. In practice, the combined with the continuous phase to produce a semisolid size and nature of the area (e.g., size and location of area on material. Such as jelly. 65 skin, such as chin, forehead) is defined, often arbitrarily, for “Hydrophilic” as used herein refers to substances that a desired purpose. In another embodiment, the bacteria for have strongly polar groups that readily interact with water. transformation to prepare the composition are a community US 9,453,232 B2 7 8 of bacteria, intending a collection of populations of different Corynebacterium ammoniagenes, Corynebacterium casei, bacteria species that occur together in space and time. In one Corynebacterium flavescens, and Corynebacterium varia embodiment, the community of bacteria includes all species bile. (that is, across all trophic levels and/or phylogenetic levels), In one embodiment, the bacteria is not C. diphtheria C. or, alternatively, includes all trophically similar species (for 5 amicolatum, C. striatum, C. jeikeium, C. urealyticum, and C. example, all the plants in a rainforest). In another embodi xerosis, C. pseudotuberculosis, C. tenuis, C. striatum, or C. ment, the bacteria for transformation to prepare the compo minutissimum, as these may be pathogenic. sition are a metapopulation, intending a group of popula In one embodiment, the bacteria are from the suborder tions that are perceived to exist as a series of local Micrococcineae, including but not limited to the GRAS populations that are linked by migration between them. In 10 bacteria species Arthrobacter arilaitensis, Arthrobacter another embodiment, the bacteria for transformation to bergerei, Arthrobacter globiformis, Arthrobacter nicoti prepare the composition are a metacommunity, intending an anae, Kocuria rhizophila, Kocuria varians, Micrococcus assemblage of trophically similar individuals and species, luteus, Micrococcus Iylae, Microbacterium gubbeenense, each of which is perceived to exist as a series of local Brevibacterium aurantiacum, Brevibacterium casei, Brevi communities, linked by the dispersal of potentially interact 15 bacterium linens, Brachybacterium alimentarium, and ing species (Green, J. L. et al., Science, 320(5879): 1039-43 Brachybacterium tyrofermentans. (2008)). In one embodiment, the bacteria are from the order Bacteria resident on the skin of healthy humans include Actinomycetales, including but not limited to the GRAS bacterial species typically resident on the face of humans, bacteria species Streptomyces griseus Subsp. Griseus. In one Such as Actinobacteria, including bacterial in the genus embodiment, the bacteria Streptomyces griseus will not corynebacterium and in the genus propionibacterium. In express tyrosinase. other embodiments, bacteria resident on the skin of healthy In another embodiment, the bacteria are from the genus human Subjects include bacterial species typically resident Staphylococcus, including but not limited to, Staphylococ on skin other than the face, including for example bacteria cus agnetis, S. arlettae, S. auricularis, S. Capitis, S. Caprae, in the genus bacteroidetes and proteobacteria. Other bacte 25 S. camosus, Staphylococcus caseolyticus, S. chromogenes, ria in the skin microbiome include those listed herein below. S. Cohnii, S. condiment, S. delphini, S. deVriesei, S. equorum, In one embodiment, the bacteria are from the genus S. felis, S. fleurettii, S. gallinarum, S. haemolyticus, S. Propionibacterium, including but not limited to, Propioni hominis, S. hyicus, S. intermedius, S. kloosii, S. leei, S. bacterium acidifaciens, Propionibacterium acidipropionici, lentus, S. lugdunensis, S. lutrae, S. massiliensis, S. microti, Propionibacterium acidipropionici strain 4900, Propioni 30 S. muscae, S. nepalensis, S. pasteuri, S. pettenkoferi, S. bacterium acnes, Propionibacterium australiense, Propioni piscifermentans, S. pseudintermedius, S. pseudolugdunen bacterium avidum, Propionibacterium cyclohexanicum, sis, S. pulvereri, S. rostra, S. Saccharolyticus, S. saprophyti Propionibacterium feudenreichii subsp. Freudenreichii, P cus, S. Schleiferi, S. Sciuri, S. Simiae, S. simulans, S. Ste freudenreichii ssp. feudenreichii strain 20271, Propionibac panovicii, S. Succinus, S. vitulinus, S. waneri, and S. terium feudenreichii subsp. Shermanii, P. feudenreichii 35 xylosus. ssp. Shermani strain 4902, Pfreudenreichii ssp. Shermanii In another embodiment, the bacteria are a population of strain 4902. Propionibacterium granulosum, Propionibac bacteria classified as “generally regarded as safe” (GRAS) in terium innocuum, jensenii, Pjensenii strain 20278, Propi the order of Staphylococcus, including but not limited to, Onibacterium lymphophilum, Propionibacterium microaero Staphylococcus cannosus Subsp. Camosus, Staphylococcus philum, Propionibacterium propionicum, 40 camosus Subsp. Utilis, Staphylococcus cohnii, Staphylococ Propionibacterium thoenii, and P thoenii strain 20277. cus condimenti, Staphylococcus equorum Subsp. Equorum, In another embodiment, the bacteria are a population of Staphylococcus equorum Subsp. Linens, Staphylococcus bacteria classified as “generally regarded as safe” (GRAS) in fleurettii, Staphylococcus piscifermentans, Staphylococcus the Propionibacterium genus, including but not limited to Saprophyticus, StaphylocoCrus Sduri Subsp. Sduri, Staphy Propionibacterium acidipropionici, Propionibacterium 45 lococcus succinus Subsp. succinus, Staphylococcus succinus freudenreichii subsp. Freudenreichii, Propionibacterium Subsp. Casei, Staphylococcus vitulinus, Staphylococcus freudenreichii subsp. Shermanii, Propionibacterium wameri, and StaphylocoCrus xylosus. jensenii, and Propionibacterium thoenii. In one embodi In one embodiment, the bacteria is not S. aureus or S. ment, the bacteria is not Propionibacterium acnes. epidermidis. In one embodiment, the bacteria are from the genus 50 In another embodiment, the bacteria are from the genus Corynebacterium, including but not limited to, C. accolens, Streptococcus, including but not limited to, Streptococcus C. afermentan, C. amycolatum, C. argentoratense, C. acidominimus, Streptococcus adjacens, Streptococcus aga aquaticum, C. auris, C. bovis, C. diphtheria, C. equi (now lactiae, Streptococcus alactolyticus, Streptococcus angino Rhodococcus equi), C. flavescens, C. glucuronolyticum, C. sus, Streptococcus australis, Streptococcus bovis, Strepto glutamicum, C. granulosum, C. haemolyticum, C. 55 coccus caballi, Streptococcus canis, Streptococcus Caprinus, halofitica, C. jeikeium (group JK), C. macginleyi, C. matriu Streptococcus Castoreus, Streptococcus Cecorum, Strepto chotii, C. minutissimum, C. parvum (Propionibacterium coccus constellatus, Streptococcus constellatus Subsp. Con acnes), C. propinquum, C. pseudodiphtheriticum (C. hof Stellatus, Streptococcus constellatus Subsp. Pharyngis, mannii), C. pseudotuberculosis, (C. ovis), C. pyogenes, C. Streptococcus Cremoris, Streptococcus criceti, Streptococ urealyticum (group D2), C. renale, C. Spec, C. striatum, C. 60 cus cristatus, Streptococcus danieliae, Streptococcus defec tenuis, C. ulcerans, C. urealyticum, and C. xerosis. Bacterial tives, Streptococcus dentapri, Streptococcus dentirousetti, with lipophilic and nonlipophilic groups are contemplated, Streptococcus didelphic, Streptococcus difficilis, Streptococ and the nonlipophilic bacteria may include fermentative cus durans, Streptococcus dysgalactiae, Streptococcus dys corynebacteria and nonfermentative corynebacteria. galactiae Subsp. Dysgalactiae, Streptococcus dysgalactiae In another embodiment, the bacteria are a population of 65 Subsp. Equisimilis, Streptococcus entericus, Streptococcus bacteria classified as “generally regarded as safe” (GRAS) in equi, Streptococcus equi Subsp. Equi, Streptococcus equi the Corynebacterium genus, including but not limited to Subsp. Ruminatorum, Streptococcus equi Subsp. Zooepi US 9,453,232 B2 10 demicus, Streptococcus equines, Streptococcus faecalis, lus coryniformis, Lactobacillus coryniformis Subsp. coryni Streptococcus faecium, Streptococcus ferus, Streptococcus formis, Lactobacillus coryniformis Subsp. torquens, gallinaceus, Streptococcus gallolyticus, Streptococcus Lactobacillus crispatus, Lactobacillus curvatus, Lactobacil gallolyticus Subsp. Gallolyticus, Streptococcus gallolyticus lus curvatus Subsp. curvatus, Lactobacillus curvatus Subsp. Subsp. Macedonicus, Streptococcus gallolyticus Subsp. Pas melibiosus, Lactobacillus delbrueckii, Lactobacillus del teurianus, Streptococcus garvieae, Streptococcus gordonii, brueckii Subsp. bulgaricus, Lactobacillus delbrueckii Subsp. Streptococcus halichoeri, Streptococcus hansenii, Strepto delbrueckii, Lactobacillus delbrueckii Subsp. lactis, Lacto coccus henryi, Streptococcus hyointestinalis, Streptococcus bacillus divergens, Lactobacillus farciminis, Lactobacillus hyovaginalis, Streptococcus ictaluri, Streptococcus infan fermentum, Lactobacillus formicalis, Lactobacillus fructiv tarius, Streptococcus infantarius Subsp. Coli, Streptococcus 10 orans, Lactobacillus fructosus, Lactobacillus gallinarum, infantarius Subsp. Infantarius, Streptococcus infantis, Strep Lactobacillus gasseri, Lactobacillus graminis, Lactobacil tococcus iniae, Streptococcus intermedius, Streptococcus lus halotolerans, Lactobacillus hamsteri, Lactobacillus hel intestinalis, Streptococcus lactarius, Streptococcus lactis, veticus, Lactobacillus heterohiochi, Lactobacillus hilgardii, Streptococcus lactis Subsp. Cremoris, Streptococcus lactis Lactobacillus homohiochii, Lactobacillus iners, Lactobacil Subsp. Diacetilactis, Streptococcus lactis Subsp. Lactis, 15 lus intestinalis, Lactobacillus jensenii, Lactobacillus john Streptococcus lutetiensis, Streptococcus macacae, Strepto sonii, Lactobacillus kandleri, Lactobacillus kefiri, Lactoba coccus macedonicus, Streptococcus marinammalium, cillus kefiranofaciens, Lactobacillus kefirgranum, Streptococcus massiliensis, Streptococcus merionis, Strep Lactobacillus kunkeei, Lactobacillus lactis, Lactobacillus tococcus minor, Streptococcus mitis, Streptococcus morbil leichmannii, Lactobacillus lindneri, Lactobacillus malefer lorum, Streptococcus mutans, Streptococcus Oligofermen mentans, Lactobacillus mali, Lactobacillus maltaronicus, tans, Streptococcus Oxalis, Streptococcus Orisratti, Lactobacillus manihotivorans, Lactobacillus minor, Lacto Streptococcus ovis, Streptococcus parasanguinis, Strepto bacillus minutus, Lactobacillus mucosae, Lactobacillus coccus parauberis, Streptococcus parvulus, Streptococcus murinus, Lactobacillus nagelii, Lactobacillus Oris, Lacto pasteurianus, Streptococcus peroris, Streptococcus phocae, bacillus panis, Lactobacillus parabuchneri, Lactobacillus Streptococcus plantarum, Streptococcus pleomorphus, 25 paracasei, Lactobacillus paracasei Subsp. paracasei, Lac Streptococcus pluranimalium, Streptococcus plurextorum, tobacillus paracasei Subsp. tolerans, Lactobacillus parake Streptococcus pneumonia, Streptococcus porci, Streptococ firi, Lactobacillus parallimentarius, Lactobacillus paraplan cus porcinus, Streptococcus porcorum, Streptococcus pseu tarum, Lactobacillus pentosus, Lactobacillus perolens, dopneumoniae, Streptococcus pseudoporcinus, Streptococ Lactobacillus piscicola, Lactobacillus plantarum, Lactoba cus pyogenes, Streptococcus raffinolactis, Streptococcus 30 cillus pontis, Lactobacillus reuteri, Lactobacillus rhamno ratti, Streptococcus rupicaprae, Streptococcus saccharolyti sus, Lactobacillus rhamnosus strain 5 AE5a, Lactobacillus cus, Streptococcus salivarius, Streptococcus salivarius rimae, Lactobacillus rogosae, Lactobacillus ruminis, Lac Subsp. Salivarius, Streptococcus salivarius Subsp. Thermo tobacillus sakei, Lactobacillus sakei Subsp. camosus, Lac philus, Streptococcus sanguinis, Streptococcus Shiloi, Strep tobacillus sakei Subsp. sakei, Lactobacillus salivarius, Lac tococcus sinensis, Streptococcus sobrinus, Streptococcus 35 tobacillus salivarius Subsp. Salicinius, Lactobacillus suis, Streptococcus thermophilus, Streptococcus thoralten salivarius Subsp. salivarius, Lactobacillus Sanfranciscensis, sis, Streptococcus tigurinus, Streptococcus troglodytae, Lactobacillus sharpeae, Lactobacillus Suebicus, Lactobacil Streptococcus troglodytidis, Streptococcus uberis, Strepto lus trichodes, Lactobacillus uli, Lactobacillus vaccinoster coccus urinalis, Streptococcus vestibularis, and Streptococ cus, Lactobacillus vaginalis, Lactobacillus viridescens, Lac CuS Waius. 40 tobacillus vitulinus, Lactobacillus xylosus, Lactobacillus In another embodiment, the bacteria are a population of yamanashiensis, Lactobacillus vananashiensis Subsp. mali, bacteria classified as “generally regarded as safe” (GRAS) in Lactobacillus yamanashiensis Subsp. Yamanashiensis and the genus Streptococcus, including but not limited to, Strep Lactobacillus zeae. tococcus thermophilus strain Th4, Streptococcus gallolyti In another embodiment, the bacteria are a population of cus Subsp. Macedonicus, Streptococcus salivarius Subsp. 45 bacteria classified as “generally regarded as safe” (GRAS) in Salivarius, and Streptococcus salivarius Subsp. Thermophi the genus Lactobacillus, including but not limited to, Lac lus. tobacillus acidophilus strain NP 28, Lactobacillus acidophi In another embodiment, the bacteria are from the genus lus strain NP51, Lactobacillus subsp. lactis strain NP7, Lactobacillus, including but not limited to, Lactococcus Lactobacillus reuteri strain NCIMB 30242, Lactobacillus garvieae, Lactococcus lactis, Lactococcus lactis Subsp. cre 50 casei strain Shirota, Lactobacillus reuteri strain DSM moris, Lactococcus lactis Subsp. hordiniae, Lactococcus 17938, Lactobacillus reuteri strain NCIMB 30242, Lacto lactis, Lactococcus lactis Subsp. Lactis, Lactococcus pis bacillus acidophilus NCFM, Lactobacillus rhamnosus strain cium, Lactococcus plantarum, Lactococcus raffinolactis, HNO01, Lactobacillus rhamnosus strain HNO01 produced in Lactobacillus acetotolerans, Lactobacillus acidophilus, a milk-based medium, Lactobacillus reuteri strain DSM Lactobacillus agilis, Lactobacillus algidus, Lactobacillus 55 17938, Lactobacillus casei subsp. rhamnosus strain GG, alimentarius, Lactobacillus amylolyticus, Lactobacillus Lactobacillus acidophilus, Lactobacillus lactis, Lactobacil amylophilus, Lactobacillus amylovorus, Lactobacillus ani lus acetotolerans, Lactobacillus acidifarinae, Lactobacillus malis, Lactobacillus aviarius, Lactobacillus aviarius Subsp. acidipisds, Lactobacillus acidophilus, Lactobacillus ali araffinosus, Lactobacillus aviarius Subsp. aviarius, Lacto mennrius, Lactobacillus amylolyticus, Lactobacillus amy bacillus bavaricus, Lactobacillus bifermentans, Lactobacil 60 lovorus, Lactobacillus brevis, Lactobacillus buchneri, Lac lus brevis, Lactobacillus buchneri, Lactobacillus bulgari tobacillus cacaonum, Lactobacillus casei Subsp. Casei, cus, Lactobacillus camis, Lactobacillus casei, Lactobacillus Lactobacillus collinoides, Lactobacillus composti, Lactoba casei Subsp. alactosus, Lactobacillus casei Subsp. casei, cillus coryniformis Subsp. Coryniformis, Lactobacillus Lactobacillus casei Subsp. pseudoplantarum, Lactobacillus crispatus, Lactobacillus crustorum, Lactobacillus curvatus casei Subsp. rhamnosus, Lactobacillus casei Subsp. tolerans, 65 Subps. Curvatus, Lactobacillus delbrueckii Subsp. Bulgari Lactobacillus catenaformis, Lactobacillus cellobiosus, Lac cus, Lactobacillus delbrueckii Subsp. Delbruecki, Lactoba tobacillus collinoides, Lactobacillus confusus, Lactobacil cillus delbrueckii Subsp. Lactis, Lactobacillus dextrinicus, US 9,453,232 B2 11 12 Lactobacillus diolivorans, Lactobacillus fabifermentans, In another embodiment, the bacteria are from the genus Lactobacillus farcininis, Lactobacillus fermentum, Lacto Salinicoccus, including but not limited to, Salinicoccus bacillus fructivorans, Lactobacillus frumenti, Lactobacillus Ventosa, Salinicoccus albus, Salinicoccus alkaliphilus, gasseri, Lactobacillus ghanensis, Lactobacillus hammesii, Salinicoccus carnicancri, Salinicoccus halodurans, Salini Lactobacillus harbinensis, Lactobacillus helveticus, Lacto 5 coccus hispanicus, Salinicoccus iranensis, Salinicoccus bacillus hilgardii, Lactobacillus homohiochii, Lactobacillus jeotgali, Salinicoccus kunmingensis, Salinicoccus luteus, hordei, Lactobacillus jensenii, Lactobacillus johnsonii, Lac Salinicoccus qingdaonensis, Salinicoccus roseus, Salinicoc tobacillus kefiri, Lactobacillus kefiranofadens Subsp. Kefira cus Salsiraiae, Salinicoccus Sesuvii, and Salinicoccus Sia nofaciens, Lactobacillus kefiranofadens Subsp. Kefirgra mensis. num, Lactobacillus kimchii, Lactobacillus kisonensis, 10 In another embodiment, the bacteria are from the genus of Lactobacillus mali, Lactobacillus manihotivorans, Lactoba Macrococcus, including but not limited to, Macrococcus cillus mindensis, Lactobacillus mucosae, Lactobacillus caseolyticus. nagelii, Lactobacillus namurensis, Lactobacillus mantensis, In another embodiment, the bacteria are from the order Lactobacillus nodensis, Lactobacillus Oeni, Lactobacillus Bacillales, including but not limited to, the GRAS bacteria Otakiensis, Lactobacillus panis, Lactobacillus parabrevis, 15 species Bacillus amyloliquefaciens, Bacillus coagulans, and Lactobacillus parabuchneri, Lactobacillus paracasei Subsp. Bacillus subbtilis. Paracasei, Lactobacillus parakefiri, Lactobacillus parali In another embodiment, the bacteria in the population are mentarius, Lactobacillus paraplantarum, Lactobacillus not Finegoldia magna. pentosus, Lactobacillus perolens, Lactobacillus plantarum In another embodiment, the bacteria are from the genus of Subsp. Plantarum, Lactobacillus pobuzihi, Lactobacillus Anaerococcus, including but not limited to, the species ponds, Lactobacillus rapi, Lactobacillus reuteri, Lactoba Anaerococcus hydrogenalis, Anaerococcus lactolyticus, cillus rhamnosus, Lactobacillus rossiae, Lactobacillus sakei Anaerococcus murdochii, Anaerococcus octavius, Anaero Subsp carnosus, Lactobacillus sakei Subsp. Sakei, Lactoba coccus previotii, Anaerococcus tetradius, and Anaerococcus cillus sali varius Subsp. Salivarius, Lactobacillus Sanfran vaginalis. ciscensis, Lactobacillus Satsumensis, Lactobacillus seca 25 In another embodiment, the bacteria are from the genus of liphilus, Lactobacillus Sennaizukei, Lactobacillus Siliginis, Peptoniphilus, including but not limited to, the species Lactobacillus spicheri, Lactobacillus Suebicus, Lactobacil Peptoniphilus asaccharolyticus, Peptoniphilus coxii, Pep lus sunki, Lactobacillus tucceti, Lactobacillus vacdnoster toniphilus duerdenii, Peptoniphilus gorbachii, Peptoniphi rus, Lactobacillus versmoldensis, and Lactobacillus yama lus harei, Peptoniphilus indolicus, Peptoniphilus ivorii, nashiensis. 30 Peptoniphilus koenoeneniae, Peptoniphilus lacrimalis, Pep In another embodiment, the bacteria are from the genus toniphilus methioninivorax, Peptoniphilus Olsenii, and Pep Lactococcus, including but not limited to, Lactococcus toniphilus tyrrelliae. Schleifer; Lactococcus chungangensis, Lactococcus fijien In another embodiment, the bacteria are from the genus of sis, LactoCOccus garvieae, Lactococcus lactis, Lactococcus Enhydrobacter, including but not limited to, the species lactis Subsp. Cremoris, Lactococcus lactis Subsp. Hordniae, 35 Enhydrobacter aerosaccus. Lactococcus lactis Subsp. Lactis, Lactococcus lactis Subsp. In another embodiment, the bacteria are from the genus of Tructae, Lactococcus piscium, Lactococcus plantarum, and Sphingomonas, including but not limited to, the species Lactococcus raffinolacti. Sphingomonas abaci, Sphingomonas adhaesiva, Sphin In another embodiment, the bacteria are a population of gomonas aerolata, Sphingomonas aestuarii, Sphingomonas bacteria classified as “generally regarded as safe” (GRAS) in 40 alaskensis, Sphingomonas alpine, Sphingomonas aquatilis, the genus Lactococcus, including but not limited to, Lacto Sphingomonas aromaticivorans, Sphingomonas asaccharo coccus lactis Subsp. Cremoris, Lactococcus lactis Subsp. lytica, Sphingomonas astaxanthiinifaciens, Sphingomonas lactis, and Lactococcus raffinolactis. aurantiaca, Sphingomonas azotifigens, Sphingomonas Cap In another embodiment, the bacteria are from the genus sulate, Sphingomonas changbaiensis, Sphingomonas chlo Enterococcus, including but not limited to, the GRAS bac 45 rophenolica, Sphingomonas chungbukensis, Sphingomonas teria species Enterococcus durans, Enterococcus faecalis, cloacae, Sphingomonas Cynarae, Sphingomonas desiccabi and Enterococcus faecium. lis, Sphingomonas dokdonensis, Sphingomonas echinoides, In another embodiment, the bacteria are from the genus Sphingomonas endophytica, Sphingomonas faeni, Sphin Tetragenococcus, including but not limited to, Tetrageno gomonas fennica, Sphingomonas formosensis, Sphingomo coccus halophilus and Tetragenococcus koreensis. 50 nas ginsengisoli, Sphingomonas ginsenosidimutans, Sphin In another embodiment, the bacteria are from the genus gomonas glacialis, Sphingomonas haloaromaticamans, Weissella, including but not limited to, the GRAS bacteria Sphingomonas hankookensis, Sphingomonas herbicido species Weissella koreensis, Weissella paramesenteroides, vorans, Sphingomonas histidimilytica, Sphingomonas Weissella thailandensis, Weissella confiusa, Weissella beni indica, Sphingomonas insulae, Sphingomonas japonica, mensis, Weissella cibaria, Weissella fabaria, Weissella 55 Sphingomonas jaspsi, Sphingomonas jejuensis, Sphingomo ghanensis, and Weissella hellenica. nas injuensis, Sphingomonas kaistensis, Sphingomonas In another embodiment, the bacteria are from the genus koreensis, Sphingomonas laterariae, Sphingomonas leidvi, Leuconostoc, including but not limited to, the GRAS bac Sphingomonas macrogolitabida, Sphingomonas mac teria species Leuconostoc carnosum, Leuconostoc citreum, rogoltabidus, Sphingomonas mali, Sphingomonas melonis, Leuconostoc fallax, Leuconostoc holzapfelii, Leuconostoc 60 Sphingomonas molluscorum, Sphingomonas mucosissima, inhae, Leuconostoc kimchi, Leuconostoc lactis, Leuconos Sphingomonas natatoria, Sphingomonas Oligophenolica, toc mesenteroides Subsp. Cremoris, Leuconostoc mes Sphingomonas Oryziterrae, Sphingomonas panni, Sphin enteroides Subsp. Dextranicum, Leuconostoc mesenteroides gomonas parapaucimobilis, Sphingomonas paucinobilis, Subsp. Mesenteroides, Leuconostoc palmae, and Leuconos Sphingomonas phyllosphaerae, Sphingomonas pituitosa, toc pseudomesenteroides. 65 Sphingomonas polyaromaticivorans, Sphingomonas pruni, In another embodiment, the bacteria are from the genus Sphingomonas pseudosanguinis, Sphingomonas rosa, Oenococcus, including but not limited to, Oenococcus oeni. Sphingomonas roseiflava, Sphingomonas rubra, Sphin US 9,453,232 B2 13 14 gomonas Sanguinis, Sphingomonas Sanxanigenens, Sphin 16992=JCM 1194, Bifidobacterium choerinum, Bifidobac gomonas sediminicola, Sphingomonas soli, Sphingomonas terium choerinum DSM 20434, Bifidobacterium coagulans, Starnbergensis, Sphingomonas Stygia, Sphingomonas sub Bifidobacterium indicum, Bifidobacterium kashiwanohense, arctica, Sphingomonas suberifaciens, Sphingomonas sub Bifidobacterium kashiwanohense JCM 15439, Bifidobacte terranean, Sphingomonas taejonensis, Sphingomonas ter rium longum, Bifidobacterium longum 3 137 DFAAB, rae, Sphingomonas trueperi, Sphingomonas ursincola, Bifidobacterium longum AGR2137, Bifidobacterium Sphingomonas wittichii, Sphingomonas xenophaga, Sphin longum BORI, Bifidobacterium longum D2957, Bifidobac gomonas xinjiangensis, Sphingomonas yabuuchiae, Sphin terium longum DJO10A, Bifidobacterium longum gomonas Vanoikuyae, and Sphingomonas yunnanensis. NCC2705, Bifidobacterium longum subsp. Infantis, Bifido In another embodiment, the bacteria are GRAS species in 10 bacterium longum subsp. infantis 157F, Bifidobacterium the gamma-proteobacteria phylum, Such as Halomonas longum subsp. infantis ATCC 15697=JCM 1222, Bifidobac elongata, Hafnia alvei, excluding Hafnia alvei. terium longum subsp. infantis CCUG 52486, Bifidobacte In another embodiment, the bacteria are from the genus of rium longum Subsp. Longum, Bifidobacterium longum Alpha-proteobacteria phylum, including but not limited to, Subsp. longum 1-6B, Bifidobacterium longum Subsp. longum the GRAS species Acetobacter aceti Subsp. Aceti, Aceto 15 2-2B, Bifidobacterium longum subsp. longum 35B, Bifido bacter fabarum, Acetobacter lovaniensis, Acetobacter malo bacterium longum Subsp. longum 4.4B, Bifidobacterium rum, Acetobacter Orientalis, Acetobacter pasteurianus longum subsp. longum ATCC 55813, Bifidobacterium Subsp. Pasteurianus, Acetobacter pomorum, Acetobacter longum subsp. longum BBMN68, Bifidobacterium longum Syzygii, Acetobacter tropicalis Gluconacetobacter azotocap subsp. longum CECT 7347, Bifidobacterium longum subsp. tans, Gluconacetobacter diazotrophicus, Gluconacetobacter longum CMCC P0001, Bifidobacterium longum subsp. entanii, Gluconacetobacter europaeus, Gluconacetobacter longum F8, Bifidobacterium longum subsp. longum JCM hansenii, Gluconacetobacter johannae, Gluconacetobacter 1217, Bifidobacterium longum subsp. longum JDM301, Bifi oboediens, Gluconobacter Oxydans, and Gluconacetobacter dobacterium longum subsp. longum KACC 91563, Bifido xylinus. bacterium longum Subsp. Suis, Bifidobacterium magnum, In another embodiment, the bacteria are Zymomonas 25 Bifidobacterium magnum DSM 20222, Bifidobacterium mobilis subsp. Mobilis. coryneforme, Bifidobacterium crudilactis, Bifidobacterium In another embodiment, the bacteria are from the Bacte cuniculi, Bifidobacterium dentium, Bifidobacterium dentium riodetes phylum, including but not limited to, Bacteroides ATCC 27678, Bifidobacterium dentium ATCC 27679, Bifi xylamisolvens strain DSM 23964. dobacterium dentium Bd1, Bifidobacterium dentium JCM In another embodiment, the bacteria are from the genus of 30 1195, Bifidobacterium dentium JCVIHMP022, Bifidobacte Bifidobacterium, including but not limited to, Bifidobacte rium gallicum, Bifidobacterium gallicum DSM 20093, Bifi rium adolescentis, Bifidobacterium adolescentis ATCC dobacterium gallinarum, Bifidobacterium simiae, Bifido 15703, Bifidobacterium adolescentis L2-32, Bifidobacte bacterium stellenboschense, Bifidobacterium stercoris, rium angulatum, Bifidobacterium, angulatum DSM Bifidobacterium subtile, Bifidobacterium subtile DSM 20098=JCM 7096, Bifidobacterium animalis, Bifidobacte 35 20096, Bifidobacterium merycicum, Bifidobacterium mini rium animalis Subsp. Animalis, Bifidobacterium animalis mum, Bifidobacterium minimum DSM 20102, Bifidobacte subsp. animalis ATCC 25527, Bifidobacterium animalis rium mongoliense, Bifidobacterium pseudocatenulatum, Subsp. Lactis, Bifidobacterium animalis Subsp. lactis Bifidobacterium pseudocatenulatum D2CA, Bifidobacte AD011, Bifidobacterium animalis subsp. lactis ATCC rium pseudocatenulatum DSM 20438=JCM 1200, Bifido 27673, Bifidobacterium animalis subsp. lactis B420, Bifi 40 bacterium pseudolongum, Bifidobacterium pseudolongum dobacterium animalis subsp. lactis BB-12, Bifidobacterium AGR2145, Bifidobacterium pseudolongum subsp. Globo animalis subsp. lactis Bi-07, Bifidobacterium animalis sum, Bifidobacterium pseudolongum Subsp. Pseudolongum, subsp. lactis B1-04, Bifidobacterium animalis subsp. lactis Bifidobacterium psychraerophilum, Bifidobacterium pullo BLC1, Bifidobacterium animalis subsp. lactis BS 01, Bifi rum, Bifidobacterium pullorum ATCC 49618, Bifidobacte dobacterium animalis subsp. lactis CNCM 1-2494, Bifido 45 rium reuteri, Bifidobacterium ruminantium, Bifidobacterium bacterium animalis subsp. lactis DSM 10140, Bifidobacte saeculare, Bifidobacterium Saguini, Bifidobacterium scar rium animalis subsp. lactis HNO19, Bifidobacterium dovii, Bifidobacterium scardovii JCM 12489, Bifidobacte animalis subsp. lactis V9, Bifidobacterium asteroids, Bifi rium thermacidophilum, Bifidobacterium thermacidophilum dobacterium asteroides PRL2011, Bifidobacterium biavatii, subsp. Porcinum, Bifidobacterium thermacidophilum subsp. Bifidobacterium bifidum, Bifidobacterium bifidum ATCC 50 Thermacidophilum, Bifidobacterium thermophilum, Bifido 29521=JCM 1255, Bifidobacterium bifidum BGN4, Bifido bacterium thermophilum RBL67, Bifidobacterium tsurumi bacterium bifidum CECT 7366, Bifidobacterium bifidum ense, Bifidobacterium tsurumiense DSM 17777, Bifidobac DSM 20215, Bifidobacterium bifidum IPLA 20015, Bifido terium sp. Bifidobacterium breve M-16V. Bifidobacterium bacterium bifidum JCM 1254, Bifidobacterium bifidum animalis subsp. lactis strains HNO19, Bi-07, B1-04 and LMG 13195, Bifidobacterium bifidum NCIMB 41171, Bifi 55 B420, Bifidobacterium animalis subsp. lactis strain Bf-6, dobacterium bifidum PRL2010, Bifidobacterium bifidum Bifidobacterium longum strain BB536, and Bifidobacterium S17, Bifidobacterium bombi, Bifidobacterium boum, Bifido lactis strain Bb12. bacterium breve, Bifidobacterium breve ACS-071-V-Sch8b. In another embodiment, the bacteria are from the genus of Bifidobacterium breve CECT 7263, Bifidobacterium breve Carnobacterium, including but not limited to, Carnobacte DPC 6330, Bifidobacterium breve DSM 20213–JCM 1192, 60 rium alterfunditum, Carnobacterium divergens, Carnobac Bifidobacterium breve EX336960VC18, Bifidobacterium terium funditum, Carnobacterium gallinarum, Carnobacte breve EX336960VC19, Bifidobacterium breve rium inhibens, Carnobacterium jeotgali, Carnobacterium EX336960VC21, Bifidobacterium breve EX533959VC21, maltaromaticum, Carnobacterium maltaromaticum 38b. Bifidobacterium breve HPH0326, Bifidobacterium breve Carnobacterium maltaromaticum ATCC 35586, Carnobac JCP7499, Bifidobacterium breve S27, Bifidobacterium breve 65 terium maltaromaticum LMA28, Carnobacterium mobile, UCC2003, Bifidobacterium callitrichos, Bifidobacterium Carnobacterium pleistocenium, Carnobacterium viridians, catenulatum, Bifidobacterium Catenulatum DSM Carnobacterium sp., Carnobacterium sp. "eilaticum US 9,453,232 B2 15 16 021211, Carnobacterium sp. 11-1, Carnobacterium sp. Pediococcus sp. epsi2-MSE-E3-2, Pediococcus sp. epsi31 12266/2009, Carnobacterium sp. 13-3, Carnobacterium sp. MSE-E3-2, Pediococcus sp. FUA 3137, Pediococcus sp. 17-4, Carnobacterium sp. 22-6, Carnobacterium sp. 2673, FUA 3140, Pediococcus sp. FUA 3226, Pediococcus sp. Carnobacterium sp. 27L, Carnobacterium sp. 35L, Carno GS4. Pediococcus sp. IBUN 186, Pediococcus sp. 1E3, bacterium sp. 37-3-1, Carnobacterium sp. 38ANAV. Car Pediococcus sp. IJ-K1, Pediococcus sp. J-11, Pediococcus nobacterium sp. 40L, Carnobacterium sp. 7196, Carnobac sp. KDLLL3-1, Pediococcus sp. L04, Pediococcus sp. terium sp. A. Carnobacterium sp. A2S1 OL14, LAB4012, Pediococcus sp. Lact10, Pediococcus sp. LQC Carnobacterium sp. A4, Carnobacterium sp. A726, Carno 1953, Pediococcus sp. LQC 1957, Pediococcus sp. LQC bacterium sp. aG53, Carnobacterium sp. ARCTIC-P2, Car 1963, Pediococcus sp. LQC 1966, Pediococcus sp. LQC nobacterium sp. ARCTIC-P26, Carnobacterium sp. ARC 10 1972, Pediococcus sp. MB2C, Pediococcus sp. MB2D, TIC-P35, Carnobacterium sp. AT 12, Carnobacterium sp. Pediococcus sp. MFC1, Pediococcus sp. MMZ60A, Pedio AT7, Carnobacterium sp. B. Carnobacterium sp. B5, Car coccus sp. MUU10, Pediococcus sp. MUU13, Pediococcus nobacterium sp. BA-81, Carnobacterium sp. BBDP54, Car sp. MUU2, Pediococcus sp. MUU3, Pediococcus sp. nobacterium sp. BBDP71, Carnobacterium sp. BM-8, Car MUU4, Pediococcus sp. NBRC 106004, Pediococcus sp. nobacterium sp. BM-81, Carnobacterium sp. C-13, 15 NBRC 106014, Pediococcus sp. NBRC 106015, Pediococ Carnobacterium sp. c58, Carnobacterium sp. c653, Carno cus sp. NBRC 106028, Pediococcus sp. NBRC 106032, bacterium sp. CM1, Carnobacterium sp. D35, Carnobacte Pediococcus sp. NBRC 107178, Pediococcus sp. NBRC rium sp. D4, Carnobacterium sp. D5, Carnobacterium sp. 107186, Pediococcus sp. NBRC 107193, Pediococcus sp. EK-153, Carnobacterium sp. ES-11, Carnobacterium sp. NBRC 107213, Pediococcus sp. NBRC 107218, Pediococ FBT1-19, Carnobacterium sp. FBT1-22, Carnobacterium cus sp. NBRC 107221, Pediococcus sp. NBRC 107222, sp. FBT3-14, Carnobacterium sp. FBT3-9, Carnobacterium Pediococcus sp. NBRC 107244, Pediococcus sp. NBRC sp. FBT4-1, Carnobacterium sp. FBT4-18, Carnobacterium 107250, Pediococcus sp. NBRC 107256, Pediococcus sp. sp. G1516J1L, Carnobacterium sp. G4a-1, Carnobacterium NBRC 107260, Pediococcus sp. NBRC 107264, Pediococ sp. G5a-1, Carnobacterium sp. GCM1, Carnobacterium sp. cus sp. NBRC 107299, Pediococcus sp. NBRC 107306, H126a, Carnobacterium sp. Hg4-03, Carnobacterium sp. 25 Pediococcus sp. NBRC 107309, Pediococcus sp. NBRC I-Bh20-14, Carnobacterium sp. I-Bh4-26, Carnobacterium 107310, Pediococcus sp. NBRC 107331, Pediococcus sp. sp. KA-2, Carnobacterium sp. KA-8, Carnobacterium sp. NBRC 107343, Pediococcus sp. NBRC 107346, Pediococ KH1, Carnobacterium sp. KOPRI80142, Carnobacterium cus sp. NBRC 107350, Pediococcus sp. NIR1, Pediococcus sp. KOPRI80153, Carnobacterium sp. KOPRI80155, Car sp. NIR3, Pediococcus sp. omega41-FH-E3-2, Pediococcus nobacterium sp. L02-6127, Carnobacterium sp. LIV10, 30 sp. P14, Pediococcus sp. Pom3, Pediococcus sp. Pom4. Carnobacterium sp. LMG 26642, Carnobacterium sp. Pediococcus sp. Pom7, Pediococcus sp. Povš, Pediococcus LV62: W1, Carnobacterium sp. LV66, Carnobacterium sp. sp. Pov7, Pediococcus sp. Pov8, Pediococcus sp. QCH-42, M7-C10, Carnobacterium sp. MARL15, Carnobacterium Pediococcus sp. QCH-66, Pediococcus sp. QCH-67, Pedio sp. MKJ37, Carnobacterium sp. NFU35-25, Carnobacte coccus sp. QMA-03G, Pediococcus sp. QMA-06CH, Pedio rium sp. NJ-46, Carnobacterium sp. R-36982, Carnobacte 35 coccus sp. QMA-07G, Pediococcus sp. QMA-11, Pediococ rium sp. RI234, Carnobacterium sp. S171, Carnobacterium cus sp. QMA-21 BC, Pediococcus sp. QMA-23 BC, sp. S181, Carnobacterium sp. Sd5t18, Carnobacterium sp. Pediococcus sp. QMA-24BC, Pediococcus sp. QMA-27BC, Sd5t5, Carnobacterium sp. Sdot1, Carnobacterium sp. Pediococcus sp. Rrt8, Pediococcus sp. Rrt9, Pediococcus Sdot15, Carnobacterium sp. Sdot17, Carnobacterium sp. sp. Rrv1, Pediococcus sp. Rrv3, Pediococcus sp. S17, Sdot18, Carnobacterium sp. SR2-31-1, Carnobacterium sp. 40 Pediococcus sp. S18. Pediococcus sp. SD2, Pediococcus sp. St2, Carnobacterium sp. T301, Carnobacterium sp. UI49, Shahsavar, Pediococcus sp. Sigal, Pediococcus sp. Carnobacterium sp. UPAA77, Carnobacterium sp. T1R1C23, Pediococcus sp. T1R4C24, Pediococcus sp. Teó, UST050418-652, Carnobacterium sp. WFPIS001, Carno Pediococcus sp. YCO-02, Pediococcus sp. YCO-04, Pedio bacterium sp. WN1359, Carnobacterium sp. WN1370, Car coccus sp. YCO-09, Pediococcus sp. YCO-10, Pediococcus nobacterium sp. WN1371, Carnobacterium sp. WN1372, 45 sp. YCO-11, Pediococcus sp. YCO-12, Pediococcus sp. Carnobacterium sp. WN1373, Carnobacterium sp. YCO-13, Pediococcus sp. YCO-16, Pediococcus sp. YCO WN1374, Carnobacterium sp. Y6, Carnobacterium diver 17, Pediococcus sp. YCO-18, Pediococcus sp. YCO-23, gens, Carnobacterium maltaromaticum, Carnobacterium Pediococcus sp. YCO-25, Pediococcus sp. YCO-26, Pedio piscicola, Carnobacterium maltaromaticum Strain CB1 (vi coccus sp. YCO-28, Pediococcus sp. YXC-17, Pediococcus able and heat-treated), and Carnobacterium maltaromati 50 sp. Z-17, Pediococcus acidilactici strain NP3, Pediococcus cum strain CB1. acidilactici, Pediococcus acidilactici, Pediococcus parvu In another embodiment, the bacteria are from the genus of lus, and Pediococcus pentosaceus. Pediococcus, including but not limited to, Pediococcus In one embodiment, any bacteria, listed herein or other acidilactici, Pediococcus acidilactici 7 4, Pediococcus aci wise known that is pathogenic and/or is an opportunistic dilactici D3, Pediococcus acidilactici DSM 20284, Pedio 55 pathogenic species is excluded. In another embodiment, the coccus acidilactici MA 18/5M, Pediococcus argentinicus, bacteria selected for transformation and to be included in the Pediococcus cellicola, Pediococcus claussenii, Pediococcus composition is any one of the bacterial genus listed herein or claussenii ATCC BAA-344, Pediococcus damnosus, Pedio any one of the specific bacterial species listed herein, or any coccus damnosus 9-6b, Pediococcus ethanolidurans, Pedio collection of first and second bacteria listed herein. coccus inopinatus, Pediococcus lolii, Pediococcus lolii 60 In one embodiment, the bacterial for use in the compo NGRI 0510O. Pediococcus parvulus, Pediococcus parvulus sition is any bacteria capable of existing on skin, in particu CIRM750, Pediococcuspentosaceus, Pediococcuspentosa lar human skin, and more particularly bacteria that reside on ceus ATCC 25745, Pediococcus pentosaceus IE-3, Pedio human skin and are GRAS bacteria, excluding pathogenic coccus Siamensis, Pediococcus stilesii, Pediococcus sp. and/or opportunistic bacteria. 14.8.17, Pediococcus sp. BGM59, Pediococcus sp. 65 In one embodiment, the composition comprises a popu BZ-2005, Pediococcus sp. CAT-100BC, Pediococcus sp. lation of transformed bacteria and a population of bacteria CR-6S, Pediococcus sp. CRA51, Pediococcus sp. EDB-LI4. not transformed to express a compound of interest, e.g., the US 9,453,232 B2 17 18 composition is comprised of a transformed bacteria popu According to one variation, the compositions may also lation and a naturally occurring or probiotic bacteria. Com contain a divalent inorganic cation. The compositions may positions comprising more than one population of bacteria, be in any of the galenical forms usually available for the wherein each population is a collection of individual trans method of administration selected. The active molecule formed bacteria for expression of different compounds of 5 interest, as each individual cell able to express more than synthesized by the bacteria (which in one embodiment are one compound of interest or each individual cell express one skin bacteria) could be could either stay in the bacteria or compound, and the collection of different individuals secreted outside to the skin. expressing different molecules of interest express different Limiting factors can control the bacterial growth. Such limiting factors can exist naturally on the skin and in one compounds of interest, or wherein one population is trans 10 formed and one population is not transformed, are also embodiment may be included in the composition that is to be contemplated. In one embodiment, the composition com applied topically to a subject to be treated. One or more prises first and second populations of transformed bacteria limiting factors may be included in the formulation. In formulated for topical application to a Subject. In one another embodiment the limiting factors are added to embodiment, the second population of transformed bacteria is complementary products such as Soaps, body wash, sham is either or both (i) created from a non-pathogenic bacteria poo, lotion to enrich and nourish the composition, and to that is different from the first population of transformed keep it active or alive. Examples for limiting factors include bacteria in the composition or (ii) transformed to express a amino acids, biotin, nicotinamide and thiamine, pantothen compound of interest that is different from the first com ate, riboflavin, folic acid, keratin, lipids, lactate, and mela pound of interest expressed by the first population of trans nins. A preferred limiting factor may be the amino acid formed bacteria in the composition. L-alanin. Bacterial growth can be controlled by the mecha The bacteria may be included in a composition in a live, nism of origin of replication to limit bacterial cycles. Bac attenuated, semi-active or inactivated, or dead form. Accord terial cycles can be limited to 50 cycles, or bacterial cycles ing to one particular embodiment, these bacteria are used in can be limited to 2-40 cycles. Limitation of bacterial growth a live form, and are capable of chronically expressing the can also be achieved by physical environmental factors as compound of interest upon topical application of the com 25 pH and temperature. position in which they are formulated. They may also be B. Exemplary Compounds of Interest included in the form of cell component fractions or in the As described above, the bacteria species selected for the form of metabolites. The bacterial species(s), molecule(s) of composition is transformed using known recombinant tech interest or fraction(s) may also be introduced in the form of 30 niques to express a compound of interest. Exemplary com a lyophilized powder, of a culture Supernatant, of harvested pounds of interest are listed in Table 1 below, along with an compound, and/or where appropriate, in a concentrated indication of the skin disorder or condition or purpose for form. which the compound is used: TABLE 1. Purpose of Compound Active compound UV protection Mycosporine, gadusols, oxo-mycosporines, imino-mycosporines and (Sunscreen; UVA, mycosporine-like amino acids (MAA; glycosylated or covalently bound to UVB) oligosaccharides, oligosaccharide-linked MAAS). Intracellular or extracellular. Examples include: gadusol, deoxygadusol, 4-Deoxygadusol (S2), shinorine, porphyra-334, palythine, palythene, asterina-330, palythinol, mycosporine glycine, mycosporine serinol, mycosporine-taurine, mycosporine-glycine valine, mycosporine-2-glycine, mycosporine-glycine-glutamic acid, mycosporine-glutamic acid-glycine, mycosporine-methylamine-Serine, mycosporine-methylamine-threonine, usujirene, dehydroxylusujirene, playthenic acid-337, playthenic acid-335, palythine-Serine, palythine threonine, palythine-threonine-Sulphate, playthine-Serine-Sulphate, euhalothece, mycosporine-alanine (2-(e)-2,3-dihydroxipro-1-enylimino mycosporine-alanine), Scytonemin Molecules with sequence similarity to MAAS, Such as dehydroquinate synthase homolog (DHQS homolog) and ATP-grasp Melamines, including eumelanin-(or dihydroxyphenylalanine (DOPA) melanins), pheomelanin allomelanins, pyomelanine, dopamelanin, neuromelanin UV-screening observing amino acids-like molecules, such as urocanic acid Flavonoids, Anthocyanines and anthoxantins, and Anthocyanidins Betalanines, such as betacyanin and betaxanthins UV-screening observing Pigments, such as Carotenoids cartenoproteins, caroteins, lycopene, Xanthopylls, lutins, zeaxanthin, porphyrin-based heme-porphyrin based, chlorophyll-II UV-screening observing co-factors, such as tetrahydrobiopterin and phenylpropanoids Polyphenol, Tannins, Phlorotannins, dieckol, eckol, Flavan-3-ols or flavanols, pycnogenol Sargaduinoic acid, Sargachromenol, Sphaerophorin (depside) pannarin (depSidone) DNA repair enzymes, that repair damage caused by exposure to UV, like photolyase, endonuclease and DNA glycosylases Psoriasis Retinoid, Vitamin A, beta-caroten, Vit D and it's derivatives, Anti-inflammatory cytokines such as Interleukin-2 (IL-2) US 9,453,232 B2 19 20 TABLE 1-continued Purpose of Compound Active compound Dry Skin Polymers, such as polyol and glycerol; skin related natural compounds, such as collagen, keratin, elastin, linoleic acid, laminin, tretinoin, tazaroteine, Sargaduinoic acid, sargachromenol, fucoxanthin, retinoid Relief of oxidative Tyrosinases (and its substrates and products) stress caused by UV: alpha hydroxy acids (AHAS), such as glycolic acid, lactic acid and citric acid Anti-Oxidants, Polysaccharides; Glycosaminoglycans, (GAGs) or mucopolysaccharides; Anti-reactive oxygen Hyaluronan (also called hyaluronic acid or hyaluronate or HA) species (Anti-ROS) Skin related cofactors, such as Vitamin A, Vitamin C or L-ascorbic acid, or Anti-Aging, simply ascorbate; Biopterin: Coenzyme A (CoA, CoASH, or HSCOA): moisturizing Coenzyme Q10, ubiquinone, ubidecarenone, coenzyme Q; CoQ10; and cosmetics Molybdopterin Vitamin E; alpha, beta, gamma, delta-tocopherols and alpha, beta, gamma, delta-tocotrienols Polymers, such as Polyol and Glycerol, Skin related natural compounds, such as collagen, keratin, elastin, linoleic acid, laminin, tretinoin, tazaroteine, Sargaduinoic acid, sargachromenol, fucoxanthin, retinoid, anti-inflammatory cytokines (such as IL-2), cortisone, tacrolimus, cyclosporine, resveratrol, gallocatechol, gallocatechin, epigallocatechin gallate Eczema cortisone, tacrolimus, cyclosporine Wound healing? anaerobic bacteria delivering oxygen Diabetic wounds. Ulcers Intertrigof talcum, starch diaper rash

In one embodiment, a composition for use in protection of Group 9—UV-screening/observing co-factors, such as skin from ultraviolet radiation is contemplated. That is, the tetrahydrobiopterin and biopterin; composition is for use as a Sunscreen, to absorb or reflect Group 10 Phenylpropanoids; ultraviolet A radiation, typically at a wavelength of between 30 Group 11—Tannins: Phlorotannins, dieckol, eckol; and 320-400 nm, ultraviolet B radiation, typically at a wave Group 12—Sargaduinoic acid, sargachromenol, Sphaero length of between 315-280 nm, or both UVA and UVB absorbing and/or reflecting. The transformed bacteria in the phorin (depside), pannarin (depsidone); composition express one or more of the following exem Group 13—DNA repair enzymes that repair damage plary compounds of interest in Group 1, Group 2. Group 3. caused by exposure to UV. Such as photolyase, endonu Group 4, Group 5, Group 6, Group 7. Group 8, Group 9. 35 clease, and DNA glycosylase. Group 10, Group 11 and Group 12: In one embodiment, the compound of interest is any one Group 1—mycosporine, gadusols, oxo-mycosporines, of the compounds listed in any one of Groups 1-13 alone. imino-mycosporines and mycosporine-like Amino Acids In another embodiment, a composition for use in provid (MAA; glycosylated or covalently bound to oligosaccha ing relief of oxidative stress is contemplated, for use as a rides, oligosaccharide-linked MAAS); and/or intracellular or 40 cosmetic or anti-aging composition. The composition may extracellular gadusol, deoxygadusol, 4-Deoxygadusol (S2), provide relief form UV exposure, as an anti-oxidant com shinorine, porphyra-334, palythine, palythene, asterina-330, position. The transformed bacteria in the composition palythinol, mycosporine-glycine, mycosporine serinol, express one or more of the following exemplary compounds mycosporine-taurine, mycosporine-glycine-Valine, of interest in Group 1, Group 2, Group 3, Group 4. Group 5, mycosporine-2-glycine, mycosporine-glycine-glutamic 45 Group 6 and Group 7: acid, mycosporine-glutamic acid-glycine, mycosporine Group 1—Tyrosinases (and its Substrates and products); methylamine-serine, mycosporine-methylamine-threonine, Group 2 Alpha hydroxy acids (AHAs): Glycolic acid, usujirene, dehydroxylusujirene, playthenic acid-337, play thenic acid-335, palythine-serine, palythine-threonine, lactic acid, and citric acid; palythine-threonine-Sulphate, playthine-serine-Sulphate, Group 3—Polysaccharides: glycosaminoglycans, euhalothece, mycosporine-alanine (2-(e)-2,3-dihydroxipro 50 (GAGs), mucopolysaccharides, hyaluronan (also called 1-enylimino-mycosporine-alanine); hyaluronic acid or hyaluronate or HA); Group 2-Scytonemin; Group 4 Skin related cofactors: Vitamin C or L-ascorbic Group 3 Melanines: eumelanin- (or dihydroxyphenyl acid, or simply ascorbate, Vitamin A, Biopterin, Coenzyme alanine (DOPA) melanins), pheomelanin allomelanins, pyo A (CoA, CoASH, or HSCoA), Coenzyme Q10 (ubiquinone, melanine, dopamelanin, neuromelanin; 55 ubidecarenone, coenzyme Q, CoQ10), Molybdopterin; Group 4 UV-screening/observing amino acids-like mol Group 5 Vitamin E: alpha, beta, gamma, delta-tocoph ecules: urocanic acid; erols, alpha, beta, gamma, delta-tocotrienols; Group 5-Flavonoids: Anthocyanines and anthoxantins, Group 6 Polymers: Polyol, Glycerol: Anthocyanidins; Group 7 Additional skin related natural compounds, Group 6 -Betalanines: betacyanin, betaxanthins; 60 Such as collagen, keratin, elastin, linoleic acid, laminin, Group 7 Molecules with sequence similarity to MAAS: tretinoin, tazarotene, Sargacquinoic acid, Sargachromenol, dehydroquinate synthase homolog (DHQS homolog), ATP fucoxanthin, retinoid, anti-inflammatory cytokines (such as grasp. IL-2), cortisone, tacrolimus, ciclosporin, resveratrol, gallo Group 8 UV-screening/observing pigments: Carote catechol, gallocatechin, and epigallocatechin gallate. noids/cartenoproteins, carotens, lycopene, Xanthopylls, 65 In other embodiments, a composition for use in treating lutins, Zeaxanthin, porphyrin-based/heme-porphyrin based, active dermatitis, acne, burns, insect bites, hives, dandruff chlorophyll-II; and body odor is contemplated. A person of skill in the art US 9,453,232 B2 21 22 can identify compounds of interest to be expressed in the letrisiloxane, mexoryl XL, enzacamene, 4-Methylben transformed bacteria for treatment of these conditions. Zylidene camphor, padimate-O, octyl dimethyl PABA, Compounds having sequence similarity to the sequences O-PABA, terephthalylidenedicamphor sulfonic acid, mexo of the compounds listed in the table above are also contem ryl SX, 3,3'-(1,4-phenylenedimethylidene) bis7,7-dim plated and may be regarded as identical compounds. Two 5 ethyl-2-oxobicylclo2.2.1]hept-1-yl methanesulfonic acid), sequences are said to be “substantially identical and identi cinoxate, 2-ethoxyethyl 3-(4-methoxyphenyl) propenoate, cal” if the sequence of nucleotides or amino acid residues, diethanolamine-methoxycinnamate, dioxybenzone, benzo respectively, in the two sequences has similarity of at least phenone-8, (2-hydroxy-4-methoxyphenyl)-(2-hydroxyphe 40%, when aligned for maximum correspondence as nyl) methanone, triethanolamine salicylate, and trolamine described below. Alternatively, percent identity can be any 10 salicylate. integer from 20% to 100%. More preferred embodiments Nucleic acid sequences coding for the compound of include at least: 20%, 25%, 30%, 35%, 40%, 45%, 50%, interest can be identified by those of skill in the art, and 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, several examples are set forth herein as: 93%, 94%, 95%, 96%, 97%, 98 or 99% compared to a SEQ ID NO:1 DNA sequence for shinorine operon; reference sequence (e.g., SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 15 SEQ ID NO:2 DNA sequence for shinorine nostoc: 9, 10, 11, 12, 13, 14, 18) using the programs described SEQ ID NO:3 AA sequence for Amino acid adenylatio herein, Such as BLAST using standard parameters, as in Ava 3855; described below. This definition also refers to the comple SEQ ID NO:4 AA sequence for ATP-grasp enzyme-like ment of a test sequence, when the test sequence has Sub protein Ava 3856; stantial identity to a reference sequence. 2O SEQ ID NO:5 AA sequence for O-methyltransferase, Compounds having a conserved protein domain with family 3 Ava 3857: sequence similarity to the sequences of the domains of the SEQ ID NO:6 AA sequence for 3-dehydroquinate syn proteins of the compounds listed in the table above, are also thase Ava 3858: contemplated and may be regarded as identical compounds. SEQ ID NO:7 DNA. Ava 3858 3 dehydroquinate syn For sequence comparison, typically one sequence acts as 25 thase; a reference sequence, to which test sequences are compared. SEQ ID NO:8 DNA spacer14804128 4803953; When using a sequence comparison algorithm, test and SEQ ID NO:9 DNA. Ava 3857 O-methyltransferase: reference sequences are entered into a computer, Subse SEQ ID NO:10 DNA spacer24803114 4803099: quence coordinates are designated, if necessary, and SEQ ID NO:11 DNA Ava 3856 ATP-grasp enzyme-like sequence algorithm program parameters are designated. 30 protein; Default program parameters can be used, or alternative SEQ ID NO: 12 DNA Spacer3 4804128 4803953; parameters can be designated. The sequence comparison SEQID NO:13 DNAAva 3855 Amino acid adenylation: algorithm then calculates the percent sequence identities for SEQ ID NO:16 DNA forward primer for shinorine op the test sequences relative to the reference sequence, based eron; on the program parameters. 35 SEQ ID NO:17 DNA reverse primer for shinorine op Methods of alignment of sequences for comparison are eron; and well-known in the art. As the use of the following programs SEQ ID NO:18 DNA sequence for tyrosynase. (provided by the National Center for Biotechnology Infor C. Recombinant Molecular Techniques mation) may be used to determine homologies/identities: Techniques for transformation of any of the bacterial BLAST, gapped BLAST, BLASTN and PSI-BLAST, which 40 species listed herein or otherwise known in the art are may be used with default parameters. Optimal alignment of understood by skilled artisans. Several techniques are briefly sequences for comparison can be conducted by different described herein, and factors to consider in Such techniques methods known in the art, such as, but not limited to, the are now discussed, including 1. Modular organization; 2. algorithms of Waterman, Needleman, Pearson, or by manual Vector types; 3. Expression and improving impression tech alignment and visual inspection. 45 niques; 4. Expression of insert and validation; and 5. Trans Because of the degeneracy of the genetic code, a large formation. number of functionally identical nucleic acids encode any 1. Modular Organization given protein. One of skill will recognize the individual The transformed bacterial strains can be regarded as cell codon usage to a nucleic acid, peptide, polypeptide, or factory, or vehicles, producing by any method known in the protein sequence that alters, adds or deletes a single amino 50 art recombinant proteins (U.S. Pat. No. 4,259,444, incorpo acid or a small percentage of amino acids in the encoded rated by reference herein), and would typically include sequence to allow the coding of the compound of interest. cloning of the isolated nucleic acid molecule that encodes It will be appreciated that the composition can be used in for the compound of interest into an appropriate vector. The combination with existing topical compositions intended for expression vector is built in a modular organization, allow treatment of the same or another disorder or condition. For 55 ing independent design of each component in separate example, the composition described herein for use as a conditions and an easy exchange of all essential elements. In Sunscreen may be used in combination with a known Sun Such a modular vector the essential elements typically screen product, including: titanium dioxide (TiO2), Zinc include: replicon, promoter (constitutive or inducible with oxide (ZnO), para-aminobenzoic acid, avobenzone, butyl regulation system), gene of interest, marker or reporter, methoxydibenzoylmethane, ensulizole, 2-phenylbenzimida- 60 resistance or limiting factor, Multiple cloning site (MCS), Zole-5-Sulfonic acid, homosalate, homomenthylsalicylat, shine-delgarno (ribosomal binding site), and terminators, as meradimate, menthyl 2-aminobenzoate, menthylanthra shown in several systems, as the NICE system (Mierau I and nilate, octinoxate, menthyl 2-aminobenzoate menthylanthra Kleerebezem M., Appl Microbiol Biotechnol. 68:705-17 nilate, octisalate, 2-ethylhexyl salicylate, octyl salicylate, (2005), or based on cryptic plasmids (Shareck J. et al., Crit octocrylene, 2-ethylhexyl-2-cyano-3.3 diphenylacrylate, 65 Rev Biotechnol. 24:155-208 (2004). oxybenzone, benzophenone-3.2-hydroxy-4-methoxybenzo Suitable vectors can be chosen or constructed, containing phenone, Sulisobenzone, benzophenone-4, drometrizo appropriate regulatory sequences, including promoter US 9,453,232 B2 23 24 sequences, terminator fragments, polyadenylation studies shown expression vectors for LAB strains, as by sequences, enhancer sequences, and other sequences as Wang T. T. and Lee B. H., Plasmids in Lactobacillus. Crit appropriate. Additional sequences may be added as Rev Biotechnol. 17:227-72 (1997), and specifically in the described above which sequences include a ribosome bind food industry by Nguyen T. T. et al., J Agric Food Chem. ing site and a translation start codon. 5 59:5617-24 (2011). Appropriate bacterial expression vectors are known to the Exemplary essential building blocks of a vector are listed person skilled in the art as described in Molecular Cloning: in Table 2 below, allowing modular configuration of a a Laboratory Manual: 2nd edition, Sambrook et al., 1989, backbone plasmid, with different combinations, for a suit Cold Spring Harbor Laboratory Press, and there are several able expression of the molecule of interest: TABLE 2

Promoter- Promoter- Marker or Replicon inducible (inducer) constitutive reporter Terminator Ori + repA, Bacteriocin, dna PermB, cml Lacz p15A, p353-1, (from usp45; High Pldh.L., P1 chloramphenicol, terminator, p353-2, Temp), FOS, (SPL), P10 (alr) alanin lollypop P8014-2, gadC-GdR (low pH), (SPL), P11 racemase gene, structure, pA1-derived, grac-lac (IPTG), (SPL), P13 Abr, amp (Ap)- pAI, p.AM- lacAflacClacR (SPL), P14 amplicillin, amyS, Tcat194, beta-1, (Lactose), lacA/T7 (SPL), P15 ccpA, cloxacillin, termé67, pBG10, (Lactose), lacF, (SPL), P16 Cmr, ermL, term908, pBMO2, lacG, lacS-GalR (SPL), P17 ery fern Tpep.A, pC194, (Lactose), lacZ, (SPL), P20 erythromycin TpepN, pCI305, NICE system, (SPL), P21, resistance marker, Tsai A pCI528, niSAFRKP P21 (SPL), esta, genes for pD125, (Nisin), orfx of P22 (SPL), TTFC, pFX1/3, Sakacin Pregulon, P23, P23 gentamycin, GusA pG+, pGK12, PA170 (low pH, low (SPL), P25 (beta pGT633, temp.), pgm, phi31 (SPL), P27 glucuronidas), pLA106, (and ori: phi (SPL), P29 Kanamycin, Lacz, pLAB1000, infection), Porf1, (SPL), P3 lux AB, mSmR, pLB10, Porf330, PorfX, (SPL), P30 neomycin, nisI, pLC2, PpfkA, prtP or ptrM (SPL), P31 nsr, penicillin, PLF1311, (absence o (SPL), P32 PepN pLJ1, pILP1, peptides), Psap.A, (weak), P33 (aminopeptidase pLP825, Psap A (sakacin A), (SPL), P34 N), pepO, ptsH, pLPE323, PsapiP, Pslp.A, (SPL), P35 streptomycin, PLUL631, PspplP (Sakacin P), (SPL), P38 tetracycline pND302, Pssp.A., PsspO, Ptuf (SPL), P4 pND324, (CDM), Pusp45, (SPL), P40 pOri+, repfopphirlt (SPL), P41 pPM4, (Mitomycin C), (SPL), P42 pPSC, represserioperator (SPL), P43 pPSC20/22, phirlt (Mytomycin (SPL), P44 pSH71, C), sodA (Aeration), (SPL), P44 pSK11L, tec-Rro12 (high (weak), P46 pVS40, temp), hyA, tre, trpE (SPL), P47 pWC1, (absence o (SPL), pWS97, tryptophan), xylA P48(SPL), pWVO1, (Xylose) P5 (SPL), pWVO2, P59, P6 rep256, (SPL), P8 repD + E (SPL), P9 (SPL), Pami, Ppgm, Pspac, Pveg, PrRNA1-a, PrRNA1-b, PrRNA2-b, PrRNA3-a, PrRNA3-b, PrRNA4-a, PrRNA4-b, PrRNA5-a, PrRNA5-b, Pslp

60 The modular organization is a construct that is capable of Vectors for transformation of the bacteria can be designed expression of the coding sequence by the bacterial host cell. by skilled artisans. Examples are set forth in FIGS. 1A-C, In particular Such a vector is either an expression vector or and as a sequence in SEQ ID NO:15 (with restriction a chromosomal integration vector, such as for example enzyme recognition sites (FIG. 1A)), and as in SEQ ID described in Steidler L. et al., Nature Biotechnology, 21 (7): 65 NO:14 (one embodiment of a shinorine operon). In one 785-789 (2003), or by Pérez-Arellano I. et al., Plasmid embodiment, the vector contains a compatible backbone 46:106-16 (2001). origin of replication to the bacteria Strain in use, a compat

US 9,453,232 B2 27 28 2. Vector Types expression vectors are known to the person skilled in the art The four major types of vectors are plasmids, viral as described in Nouaille S. et al., Genetics and Molecular vectors, cosmids, and artificial chromosomes. Common to Research, 2:102-111 (2003), and in Maniatis, Sambrook and all engineered vectors are an origin of replication, a multi Fritsch, 1982. Molecular Cloning: A Laboratory Manual, cloning site, and a selectable marker. Any of these are 3. Expression and Improving Expression Techniques suitable for use herein. The term heterologous expression means that a protein, or The sequence codes for the molecule of interest can be gene of interest, is experimentally put into a cell that does inserted into a clone, vector, shuttle, plasmid, BAC, or can not normally make (i.e., express) that protein e.g., to a cell, also be integrated into the bacterial genome. or nucleic acid, protein, or vector, indicates that the cell, The copy number of the plasmid can be between 5-500 10 nucleic acid, protein or vector, has been modified by the copy numbers per cell. introduction of a heterologous nucleic acid or protein or the Exemplary plasmids and expression vectors include but alteration of a native nucleic acid or protein, or that the cell are not limited to: is derived from a cell so modified. Thus, for example, p252, p256, p353-2 (Leer et al. 1992), p8014-2, p.A1, recombinant cells express genes that are not found within pACYC, p.AJ01, p.A 1-derived (Vujcic & Topisirovic 1993), 15 the native (nonrecombinant) form of the cell or express pall, p.AM-beta1,2,3,5,8 (simon and chopin 1988), native genes that are otherwise abnormally expressed, pAR1411, pBG10, pBK, pBM02, pBR322, pBR328, pBS under-expressed or not expressed at all. Also, the nucleic slpGFP. pC194 (McKenzie et al. 1986, 1987; Horinouchi & acid is typically recombinantly produced, can have two or Weisblum 1982b), PC194/PUB110, pC3011, pC3011 more sequences from unrelated genes arranged to make a (Skaugen 1989), pCD034-1, pCD034-2, pCD256, pCI2000, new functional nucleic acid, e.g., a promoter from one pCI305, pCI528, pCIS3, pCL2.1, pCT1138, pID125, pE194, Source and a coding region to a molecule of interest, from pE194/PLS1, pEGFP-C1, pEH, pF8801, pFG2, pFK-series, another source. pGK-series, pGK12, pGK13, pIA, pIAV 15.6.7.9, pIL.CatT. The nucleic acid, generated recombinantly or syntheti pIL252/3, pIL253, pIL7, pISA (low for e. coli), p.JW563, cally, with a series of specified nucleic acid elements that pKRV3, pILAB1000 (Josson et al. 1990), pI B4 (Bates & 25 permit transcription of a particular nucleic acid in a host cell Gilbert 1989, pIBS, pI E16, pIEB124, pIEB590, is called an "expression cassette'. The expression cassette pLEB591, pIEB600, pIEB604, pIEP24Mcop, pI J1 (Taki can be part of a plasmid, virus, or nucleic acid fragment. guchi et al. 1989), pI KS, pITK2, pWCFS101 and Typically, the expression vector includes a nucleic acid to be pMD5057 (Bates & Gilbert, 1989: Skaugen, 1989; Leer et transcribed operably linked to a promoter. al., 1992; Vujcic & Topisirovic, 1993: Eguchi et al., 2000; 30 Moreover, expression cassettes can include a variety of Kaneko et al., 2000: Danielsen, 2002; Daming et al., 2003: components to regulate expression and localization of the de las Rivas et al., 2004; van Kranenburg et al., 2005), compound of interest of the invention. For example, expres pLP1/18/30, pIP18, pILP317, pILP317cop, pIP3537, sion cassettes can include promoter elements, sequences pLP3537xy 1, pIP402, pIP825, pILP825 and pLPE323, encoding signal sequences, a coding sequence for the com pLP82H, pIPC37, pILPE23M, pIPE323, pIPE350, pILPI 35 pounds of interest, terminators and anchor sequences. (Bouia et al. 1989), pI S1, pI S1 and pl. 194 (Lacks et al. Promoters—Expression of the heterologous compound of 1986; Horinouchi & Weisblum 1982a), plu1631, pIUL631 interest can be constitutive or inducible. The promoter to be from L. reuteri carrying an erythromycin-resistance gene, used can be, for example, inducible lactobacillus lac pro pM3, pM4, pMD5057, pMG36e, pND324, pNZ-series, moter, LdhL, Slp, ernB, orfx, or artificially constitutive pPSC series, pSH71 (de Vos, 1987), pSIP-series, pSK11L, 40 (Rud I. et al., Microbiology, 152: 1011-92 (2006). pSL2, PSN2, pSN2 (Khan & Novick 1982), pT181 (Koepsel Examples of promoters are listed in the table of the et al. 1987), (Khan & Novick 1983), pT181, pC194 and modular construction, and for example can be, but not pE 194 are not functional in B. subtilis (Gruss et al. 1987), limited to: Pse (van der Vossen et al., Appl. Environ. Micro pT181, pE194/pLS1, pC194/pUB110 and pSN2 (Khan, biol. 58:3142-3149 (1992)), P., (Elliot et al., Cell 36:211 2005), pTL, pTRK family, pTRT family, pTUAT35, 45 219 (1984)) promoters), Lactobacillus casei L(+)-lactate pUB110 and pC194 (McKenzie et al. 1986, 1987; Horin dehydrogenase promoter (Pouwels et al., 1993, Genetics of ouchi & Weisblum 1982b), puCL22, puLP8/9, pVS40, lactobacilli: plasmids and gene expression, Antonie Van pWC1, pWCFS101, pWVO2, pWV04, pWV05, Rep.A, sys Leeuwenhoek 64:85-107), Promoter of Bacillus amylase tem. BetL. (Weickert et al., J. Bacteriol. 171:3656-66 (1989)) or xylose In one embodiment, the lactose phosphotransferase sys 50 (Kim et al. Gene 181:71-76 (1996)) promoters as well as the tem, optionally linked to the E. coli bacteriophage T7 Lactococcus nisin promoter (Eichenbaum et al., Appl. Envi promoter, the L. lactis nis A promoter system; vectors com ron. Microbiol. 64:2763-2769 (1998)) can be used to drive prising promoters regulated by environmental conditions, inducible expression. Additional promoters can be: p32 such as for example the P170 promoter that is only active at promoter which controls expression of Lactococcus lactis low pH. Another exemplary vector is a cosmid, a hybrid 55 fructose-1,6-diphosphate aldolase (Van de Guchte et al., plasmid (often used as a cloning vector) that contains a 1990, Appl. Environ. Microbiol. 56:2606-2611), T7 gene 10 Lambda phage cos sequence. (cos sites--plasmid cosmid). promoter (Wells et al., 1993, Mol. Microbiol. 8:1155-1162), DNA sequences are originally from the lambda phage, and alpha amylase promoter sequence of Lactobacillus amylo cosmids can be used to build genomic libraries. Another virus (Pouwels et al., 1993, Genetics of lactobacilli: plas example is a bacterial artificial chromosome (BAC), which 60 mids and gene expression, Antonie Van Leeuwenhoek is a DNA construct, based on a functional fertility plasmid 64:85-107), and promoters which control expression of: (or F-plasmid), used for transforming and cloning in bacte LdhL, Slp, ermB, orfx, p.6 (pLA6), pI T71, T7, p11, lacTp, ria, usually E. coli. Suitable vectors can be chosen or dltp, ccpAp, plp, and inducible lactobacillus as lac promoter, constructed, containing appropriate regulatory sequences, LdhL, Slp, ernB, orfx, as shown by Kim J H, and Mills D including promoter sequences, terminator fragments, poly 65 A. Plasmid 58:275-83 (2007). adenylation sequences, enhancer sequences, marker genes Several recombinant techniques to improve expression, or and other sequences as appropriate. Appropriate bacterial cloning and expression of elements are known, including US 9,453,232 B2 29 30 molecular biology methods, nucleic acid and clone construc Known sequences of compounds of interest can be iden tion, mutagenesis, sequencing, introduction of DNA into tified in commercially available databases, as described in cells, and analysis of proteins, are described in detail in more detail herein. Current Protocols in Molecular Biology, Ausubel et al. eds., Exemplary sequences of molecules of interest within the John Wiley & Sons, 1992, and in Molecular Cloning: a vector are those with genes coding for molecules screening Laboratory Manual: 2nd edition, Sambrook et al., 1989, UV. In particular, genes coding for molecules screening UV Cold Spring Harbor Laboratory Press Also, it is possible to in the range of 100-500 nm are contemplated. Sequences for configure the number of promoters and their length, for molecules of interest within the vector are those with genes better expression (Yagur-Kroll S. et al., Bioeng Bugs, 2010 coding for molecules reducing oxidative stress, such as 10 genes coding for molecules reducing oxidative stress caused 1:151-3 (2010). by UV; anti-Oxidants, anti-reactive oxygen species (Anti The mechanism of replication of the replicon can be of ROS). Sequences coding for the genes of interest may be RCR mechanism or by theta-replicating plasmids. The resis Scy A-F. from Cyanobacteria sp. Sun screen compounds, tance gene can be based on, but not limited to: antibiotics, Such as shinorine, can be obtained from corals (Stylophora bacterium marker, heat-shock, or Sugar utilization abilities, 15 pistallata), fish (Scarus Schlegeli and Chlorurus Sordidus), Such as: thymidylate synthase (thy A), lactose phosphotrans algea (Porphyra umbilicalis), microalgea and, bacteria, as ferase (lacF), phosph-beta-galactosidase (lac G), or alanine from cyanobacterium Nostoc spp., (like as Nostoc flagelli recemase (alr). Terminator can be added at different posi forme or Nostoc sp. PCC 7524) Lyngbya spp., Anabaena tions to provide more efficient expression. A variety of signal spp., and Nodularia spp. Nostoc punctiforme PCC 73102 and anchor sequences are known to direct expression of Anabaena sp., Anabaena variabilis, Anabaena cylindrica polypeptides to the membrane, extracellular space or the cell PCC 7122, Cyanothece sp. PCC 7424, Cyanothece sp. PCC wall (e.g., by covalent attachment to peptidoglycan). 8802, Rivularia sp. PCC 7116, Chroococcidiopsis thermalis In addition to comprising the desired gene, the microor PCC 7203, Cylindrospermum stagnate PCC 7417, Stanieria ganism may also be manipulated to encode other sequence cyanosphaera PCC 7437, Crinalium epipsammum PCC elements which facilitate production of the desired expres 25 9333, Crinalium epipsammum PCC 9333, Anabaena sp. 90 sion of the molecule of interest by the bacterium. Such chromosome chANA01, Gloeocapsa sp. PCC 7428, Chlo sequence elements include, but are not limited to, promoter/ rogloeopsis fritschii, Trichodesmium erythraeum IMS101, regulatory sequences which facilitate constitutive or induc Microcystis aeruginosa PCC 7806, Microcystis aeruginosa ible expression of the protein or which facilitate over strain UVO27, Planktothrix rubescens NIVA-CYA 98, expression of the protein in the bacterium. Additional 30 Microcystis sp. NIVA-CYA 172/5, Nostoc sp. GSV224, or sequence elements may also include those that facilitate Oscillatoria nigro-viridis PCC 7112. secretion of the protein from the bacterium, accumulation of In one embodiment, the sequence for expression of the the protein within the bacterium, and/or programmed lysis compound of interest incorporates into the genome of the of the bacterium in order to release the protein from the bacteria. same. Many of the sequence elements referred to above are 35 The copy number of the plasmid can be between 5-500 known to those skilled in the art (Maniatis, Sambrook and copy numbers per cell. The promoters can be constitutive or Fritsch. 1982. Molecular Cloning: A Laboratory Manual). inducible. 4. Expression of Insert and Validation In one embodiment, it is possible to add to the vector Expression of heterologous genes is widely used in bio DNA and amino acid elements like His-tag to allow purifi technology, especially in industrial food fermentation, con 40 cation of the molecule of interest. It is also possible to add tributing to flavor, texture and preservation. an element like usp45, which allows exerting the molecule Sequences can be inserted in the vector by de-novo of interest out of the membrane. sequencing or by PCR amplification. de-novo synthesized is In one embodiment, codon usage can be improved to done by the Capillary Electrophoresis method, or based on better express the molecule of interest. Also GC 96 of the Sanger sequencing techniques (Sanger et al. (1974)), when 45 expression vector can be changed and/or reduced. the DNA sequence is copied with high fidelity because at In one embodiment, the vector includes a limiting factor, each base on the DNA template, DNA polymerase incorpo or in another embodiment, a limiting factor is incorporated rates only the nucleotide that is complementary to that base. into the bacterial genome via homologous recombination. Thymine (T) is complementary to adenine (A) and guanine 5. Transformation (G) is complementary to cytosine (C) because they can form 50 With regard to transformation techniques, appropriate hydrogen bonds with each other. bacterial host strains are selected for, e.g. their transforma The sequence of the cloned genes and synthetic sequences tion ability, ability for heterologous protein expression. The can be verified after cloning using, e.g., the chain termina bacterial host will be rendered competent for transformation tion method for sequencing double-stranded templates of using standard techniques, such as the rubidium chloride Wallace et al., Gene 16:21-26 (1981). 55 method or electroporation (Maniatis, Sambrook and Fritsch. Appropriate primers and probes for identifying the genes 1982. Molecular Cloning: A Laboratory Manual). encoding for the compounds of interest of the invention can Particular methods for the transformation of LAB strains be derived from the sequences described in the art. For a are provided in the experimental part hereinafter, but are general overview of PCR, see, Innis et al., PCR Protocols: illustrative of techniques known in the art, and are not A Guide to Methods and Applications, Academic Press, San 60 intended to be limiting. Diego (1990). Transformation of Lactococcus lactis by electroporation The concentration of molecule of interest expressed in the can be performed by modifying standard methods as host bacteria can be varied from 0.1 mM to 100 mM. This described in, e.g., Luchansky et al. (J. Dairy Sci. 74: concentration can be controlled by various parameters. Such 3293-3302 (1991). Briefly, freshly inoculated Lactobacillus as: the concentration of bacteria, the copy number of the 65 spp. are cultured in MRS broth (e.g., to 0.4–0.8 at ODoo at plasmid, the activity of the promoter, and the kinetics of the 37° C. and 5% CO). The bacterial cells are harvested, molecule of interest. washed and re-suspended in a cold (e.g., 4°C.) Solution of US 9,453,232 B2 31 32 Sucrose and MgCl2. Competent cells are then mixed with Excipients in the formulation are selected based on the DNA and placed in a chilled gap cuvette and electroporated. type of formulation intended. Standard excipients include Afterward, cells are allowed to recover in pre-warmed broth gelatin, casein, lecithin, gum acacia, cholesterol, tragacanth, (e.g., for about two hours at 37° C.), prior to being plated on Stearic acid, benzalkonium chloride, calcium Stearate, glyc selective agar plate containing an antibiotic other selective 5 eryl monostearate, cetostearyl alcohol, cetomacrogol emul agent. Sifying wax, Sorbitan esters, polyoxyethylene alkyl ethers, Optimization of electroporation in lactobacillus: To Sup polyoxyethylene castor oil derivatives, polyoxyethylene Sor port cloning and heterologous protein expression in these bitan fatty acid esters, polyethylene glycols, polyoxyethyl vaginal lactobacillus strains, electroporation methods were ene Stearates, colloidol silicon dioxide, phosphates, sodium 10 dodecyl sulfate, carboxymethylcellulose calcium, car developed for application to skin bacteria. Various param boxymethylcellulose sodium, methylcellulose, hydroxyeth eters, including culture media, cell growth stages, DNA ylcellulose, hydroxypropylcellulose, hydroxypropylmethy concentration, wash or electroporation buffer composition, cellulose phthalate, noncrystalline cellulose, magnesium cuvette gap size, and Voltage were evaluated to determine aluminum silicate, triethanolamine, polyvinyl alcohol, poly conditions that improved transformation frequencies for the 15 vinylpyrrolidone, Sugars, and starches. WT lactobacillus strains in our collection. E. coli-derived An emulsion is a preparation of one liquid distributed in plasmids were transformed into Lactobacillus strains by small globules throughout the body of a second liquid. The electroporation according to Luchansky et al. (J. Dairy Sci. dispersed liquid is the discontinuous phase, and the disper 74:3293-302 (1991)) with modifications. Briefly, freshly sion medium is the continuous phase. When oil is the inoculated Lactobacillus strain were cultured in MRS broth dispersed liquid and an aqueous solution is the continuous to 0.6-0.7 at OD at 37° C. and 5% CO. The bacterial cells phase, it is known as an oil-in-water emulsion, whereas were harvested, washed and re-suspended in 952 mM when water or aqueous solution is the dispersed phase and sucrose and 3.5 mM MgCl, at 4°C. Using a pre-chilled 0.2 oil or oleaginous Substance is the continuous phase, it is cm gap cuvette, competent cells were added with 12 ug of known as a water-in-oil emulsion. The oil phase may consist plasmid DNA) and electroporated immediately at 2.5 kV/cm 25 at least in part of a propellant, such as an HFA propellant. and 200 ohms. Afterward, cells were allowed to recover in Either or both of the oil phase and the aqueous phase may pre-warmed MRS broth for two hours at 37° C., prior to contain one or more surfactants, emulsifiers, emulsion sta being plated on selective MRS agar plate containing anti bilizers, buffers, and other excipients. Preferred excipients biotic, as 20 g/ml erythromycin. include Surfactants, especially non-ionic Surfactants; emul 6. Harvesting 30 Sifying agents, especially emulsifying waxes; and liquid Genetic manipulations allows over production, in differ non-volatile non-aqueous materials, particularly glycols ent cell lines, of various expressed heterologous desired such as propylene glycol. The oil phase may contain other protein. Over expression, recovering the biological recom oily pharmaceutically approved excipients. For example, binant molecule from the skin bacteria, and harvesting a materials such as hydroxylated castor oil or sesame oil may desired molecule, is the essence of the biotechnology indus 35 be used in the oil phase as Surfactants or emulsifiers. try, and is known to the person skill in the art (Eugene Russo, “Emollients' are an externally applied agent that softens Nature, 421 456-457 (2003). or soothes skin and are generally known in the art and listed The extracted molecule from the bacteria will be used for in compendia, such as the “Handbook of Pharmaceutical dermatological benefits, as for UV protection. Excipients', 4' Ed., a Pharmaceutical Press, 2003. These The harvesting procedure may include mechanical, as 40 include, without limitation, almond oil, castor oil, ceratonia bead-beating the bacterial cells, or chemically breaking extract, cetostearoyl alcohol, cetyl alcohol, cetyl esters wax, them, by using lysozyme. cholesterol, cottonseed oil, cyclomethicone, ethylene glycol Isolation of the expressed molecule can be at various palmitostearate, glycerin, glycerin monostearate, glyceryl cleaning levels, as from 5%-90%, and can be used by monooleate, isopropyl myristate, isopropyl palmitate, lano molecular and chemical techniques, e.g. HPLC, HIS-tag. 45 lin, lecithin, light mineral oil, medium-chain triglycerides, and known to the person skill in the art. mineral oil and lanolin alcohols, petrolatum, petrolatum and The concentration of the harvested compound can be lanolin alcohols, soybean oil, starch, Stearyl alcohol, Sun 1-50% of the biomass before extraction. flower oil, xylitol and combinations thereof. In one embodi Various compounds including bacterial cells and/or par ment, the emollients are ethylhexylstearate and ethylhexyl ticles of the bacterial cells may be dissolved or suspended in 50 palmitate. the extracts. The final extract may include lipoproteins, "Surfactants' are surface-active agents that lower surface lipopeptides, peptidoglycans, lipooligosaccharides, lipote tension and thereby increase the emulsifying, foaming, ichoic acids, and teichoic acids. During the lysis process, dispersing, spreading and wetting properties of a product. molecules in the bacterial cells, may become chemically Suitable non-ionic Surfactants include emulsifying wax, modified. Variose parameters, as pH, starting volume and 55 glyceryl monooleate, polyoxyethylene alkyl ethers, poly temperature, may be range to increase yield of harvesting. oxyethylene castor oil derivatives, polysorbate, Sorbitan D. Exemplary Topical Compositions esters, benzyl alcohol, benzyl benzoate, cyclodextrins, glyc The transformed, non-pathogenic populations of bacteria erin monostearate, poloxamer, povidone and combinations are formulated for topical application to the skin of a subject. thereof. In one embodiment, the non-ionic Surfactant is Without intending to be limiting, but for purposes of exem 60 Stearyl alcohol. plary embodiments, it is contemplated that the formulation “Emulsifiers' are surface active substances which pro may be a gel, ointment, lotion, emulsion, cream, foam, mote the Suspension of one liquid in another and promote the mousse, liquid, spray, Suspension, dispersion or aerosol. The formation of a stable mixture, or emulsion, of oil and water. formulation includes one or more excipients to provide the Common emulsifiers are metallic Soaps, certain animal and desired form and a desired viscosity, flow or other physical 65 vegetable oils, and various polar compounds. Suitable emul or chemical characteristics for effective application, cover sifiers include acacia, anionic emulsifying wax, calcium age and adhesion to the skin. Stearate, carbomers, cetostearyl alcohol, cetyl alcohol, cho US 9,453,232 B2 33 34 lesterol, diethanolamine, ethylene glycol palmitostearate, homopolymers and copolymers; and combinations thereof. glycerin monostearate, glyceryl monooleate, hydroxpropyl Suitable solvents in the liquid vehicle include, but are not cellulose, hypromellose, lanolin, hydrous, lanolin alcohols, limited to, diglycol monoethyl ether, alklene glycols, such as lecithin, medium-chain triglycerides, methylcellulose, min propylene glycol; dimethyl isosorbide, alcohols, such as eral oil and lanolin alcohols, monobasic sodium phosphate, isopropyl alcohol and ethanol. The solvents are typically monoethanolamine, nonionic emulsifying wax, oleic acid, selected for their ability to dissolve the drug. Other addi poloxamer, poloxamers, polyoxyethylene alkyl ethers, poly tives, which improve the skin feel and/or emolliency of the oxyethylene castor oil derivatives, polyoxyethylene sorbitan formulation, may also be incorporated. Examples of Such fatty acid esters, polyoxyethylene Stearates, propylene gly additives include, but are not limited, isopropyl myristate, col alginate, self-emulsifying glyceryl monostearate, sodium 10 ethyl acetate, C12-C15 alkyl benzoates, mineral oil, citrate dehydrate, Sodium lauryl Sulfate, Sorbitan esters, squalane, cyclomethicone, capric/caprylic triglycerides, and Stearic acid, Sunflower oil, tragacanth, triethanolamine, Xan than gum and combinations thereof. In one embodiment, the combinations thereof. emulsifier is glycerol Stearate. Foams consist of an emulsion in combination with a A “lotion' is a low- to medium-viscosity liquid formula 15 gaseous propellant. The gaseous propellant consists primar tion. A lotion can contain finely powdered Substances that ily of hydrofluoroalkanes (HFAs). Suitable propellants are in soluble in the dispersion medium through the use of include HFAs such as 1,1,1,2-tetrafluoroethane (HFA 134a) Suspending agents and dispersing agents. Alternatively, and 1,1,1,2,3,3,3-heptafluoropropane (HFA 227), but mix lotions can have as the dispersed phase liquid Substances tures and admixtures of these and other HFAs that are that are immiscible with the vehicle and are usually dis currently approved or may become approved for medical use persed by means of emulsifying agents or other Suitable are suitable. The propellants preferably are not hydrocarbon stabilizers. In one embodiment, the lotion is in the form of propellant gases which can produce flammable or explosive an emulsion having a viscosity of between 100 and 1000 vapors during spraying. Furthermore, the compositions pref centistokes. The fluidity oflotions permits rapid and uniform erably contain no volatile alcohols, which can produce application over a wide Surface area. Lotions are typically 25 flammable or explosive vapors during use. intended to dry on the skin leaving a thin coat of their Buffers are used to control pH of a composition. Prefer medicinal components on the skin's Surface. ably, the buffers buffer the composition from a pH of about A “cream' is a viscous liquid or semi-solid emulsion of 4 to a pH of about 7.5, more preferably from a pH of about either the “oil-in-water or “water-in-oil type'. Creams may 4 to a pH of about 7, and most preferably from a pH of about contain emulsifying agents and/or other stabilizing agents. 30 5 to a pH of about 7. In a preferred embodiment, the buffer In one embodiment, the formulation is in the form of a cream is triethanolamine. having a viscosity of greater than 1000 centistokes, typically Preservatives can be used to prevent the growth of fungi in the range of 20,000-50,000 centistokes. Creams are often and microorganisms. Suitable antifungal and antimicrobial time preferred over ointments as they are generally easier to agents include, but are not limited to, benzoic acid, butyl spread and easier to remove. 35 paraben, ethyl paraben, methyl paraben, propylparaben, The basic difference between a cream and a lotion is the Sodium benzoate, sodium propionate, benzalkonium chlo Viscosity, which is dependent on the amount/use of various ride, benzethonium chloride, benzyl alcohol, cetylpyri oils and the percentage of water used to prepare the formu dinium chloride, chlorobutanol, phenol, phenylethyl alco lations. Creams are typically thicker than lotions, may have hol, and thimerosal. In one embodiment, a concentration of various uses and often one uses more varied oils/butters, 40 a preservative that is effective to prevent fungal growth is depending upon the desired effect upon the skin. In a cream selected, without affecting the effectiveness of the compo formulation, the water-base percentage is about 60-75% and sition for its intended purposed upon topical application. the oil-base is about 20-30% of the total, with the other Penetration enhancers are frequently used to promote percentages being the emulsifier agent, preservatives and transdermal delivery of drugs across the skin, in particular additives for a total of 100%. 45 across the stratum corneum. Some penetration enhancers An “ointment' is a semisolid preparation containing an cause dermal irritation, dermal toxicity and dermal allergies. ointment base and optionally one or more active agents. However, the more commonly used ones include urea, Examples of suitable ointment bases include hydrocarbon (carbonyldiamide), imidurea, N, N-diethylformamide, bases (e.g., petrolatum, white petrolatum, yellow ointment, N-methyl-2-pyrrolidine, 1-dodecal-azacyclopheptane-2- and mineral oil); absorption bases (hydrophilic petrolatum, 50 one, calcium thioglycate, 2-pyyrolidine, N,N-diethyl-m- anhydrous lanolin, lanolin, and cold cream); water-remov toluamide, oleic acid and its ester derivatives, such as able bases (e.g., hydrophilic ointment), and water-soluble methyl, ethyl, propyl, isopropyl, butyl, vinyl and glyceryl bases (e.g., polyethylene glycol ointments). Pastes typically monooleate, Sorbitan esters. Such as Sorbitan monolaurate differ from ointments in that they contain a larger percentage and Sorbitan monooleate, other fatty acid esters such as of solids. Pastes are typically more absorptive and less 55 isopropyl laurate, isopropyl myristate, isopropyl palmitate, greasy that ointments prepared with the same components. diisopropyl adipate, propylene glycol monolaurate, propyl A "gel’ is a semisolid system containing dispersions of ene glycol monooleatea and non-ionic detergents such as Small or large molecules in a liquid vehicle that is rendered BRIJR 76 (stearyl poly(10 oxyethylene ether), BRIJR 78 semisolid by the action of a thickening agent or polymeric (stearyl poly(20)oxyethylene ether), BRIJR 96 (oleyl poly material dissolved or suspended in the liquid vehicle. The 60 (10)oxyethylene ether), and BRIJR 721 (stearyl poly (21) liquid may include a lipophilic component, an aqueous oxyethylene ether) (ICI Americas Inc. Corp.). component or both. Some emulsions may be gels or other The microorganisms may be delivered in effective wise include a gel component. Some gels, however, are not amounts per unit dose, (or per cm), of at least 10° colony emulsions because they do not contain a homogenized blend forming units (cfu) to 10' cfu per cm, in particular between of immiscible components. Suitable gelling agents include, 65 10 cfu to 10" cfu per cm. In accordance with the method but are not limited to, modified celluloses, such as hydroxy as described in Balskus, et al. (Science 2010) UV elements propyl cellulose and hydroxyethyl cellulose; Carbopol are produced to at least 0.5 mM to 1 mM for 10° cfu. Based US 9,453,232 B2 35 36 thereon, the skilled person in the art can calculate the range 320 nm and UVA radiation made up of wavelengths from of produced element at any other dose of cfu. 320-400 nm is the two significant causes of damage in The biological compositions may further include one or organisms. UVB is particularly harmful to organisms more beneficial compounds in the formulation, for example, because its absorption by DNA creates cyclobutane pyrimi UV protection and chemically (or biologically produced) dine dimers, which do damage to other DNA, lipids and may include added vitamin A. proteins within the body. It is a common cause of skin The compositions can be prepared by any known or cancer. UVA is particularly harmful to organisms because it otherwise effective method for formulating or manufactur penetrates deeper into the skin layers. The composition ing the selected product form. In one embodiment, the contemplated herein in one embodiment comprises one or composition is formulated for application to a skin epider 10 more transformed bacteria to express one or more com mal Surface of a Subject, intending to exclude mucosal pounds of interest to protect from one or both of UVA and Surfaces, such as nasal, vaginal, rectal, oral Surfaces. In one UVB. The composition can be applied to the skin in com embodiment, topical application excludes the oral cavity, as bination with existing Sunscreens of either a chemical (e.g., well as other mucosal surfaces of the body. aminobenzoic acid (PABA), avobenzone, cinoxate, dioxy The composition can be formulated to comprise the 15 benZone, ensulizole, homosalate, menthylanthranilate, transformed bacterial at a particular concentration to yield a meradimate, octocrylene, octyl methoxycinnamate, Octi desired concentration of the compound of interest. For salate, octyl salicylate, oxybenzone, padimate-O, phenyl example, the composition can comprise an amount of trans benzimidazole Sulfonic acid, Sulisobenzone, and minerals: formed bacterial Such that the microorganisms may be titanium dioxide, trolamine Salicylate, Zinc oxide or physical delivered in effective amounts per unit dose, (or per cm), of nature. at least about 10° colony forming units (cfu) to about 10' The composition of transformed bacteria preferably cfu per cm, in particular between about 10 cfu to about expresses a compound that protects from UV absorption by 10' cfu per cm. The composition may be formulated with the skin, and can be shinorine which a natural mycosporine the transformed bacteria in a proportion of at least about like amino acid (MAA) small molecule, absorbing UV 0.0001% (expressed by dry weight), in particular in a 25 radiation, which being synthesized by various organisms as proportion of from about 0.0001% to about 99%, and more cyanobacteria, fungi and algae. In one embodiment, the particularly in a proportion of from about 0.001% to about composition comprising the population of transformed bac 90% by weight, in particular from about 0.01% to about 80% teria is a Soap or a body wash composition that is applied to by weight, and especially from about 0.1% to about 70% by the skin. weight, relative to the total weight of the composition. In 30 The microorganisms may be delivered in effective general, a composition intended to be administered topically, amounts per unit dose, (or per cm), of at least about 10° may comprise, for living microorganisms, from about 10 to colony forming units (cfu) to about 10' cfu per cm, in about 10" cfu/g, in particular from about 10 to about 10' particular between about 10 cfu to about 10' cfu per cm. cfu/g, and more particularly from about 10 to about 10' In embodiments where the compound of interest is for UV cfu/g of microorganisms per gram of carrier, or at equivalent 35 protection, UV elements are produced to at least about 0.1 doses calculated for inactive or dead microorganisms or for mM to about 100 mM for 10°-10' cfu. Based thereon, the bacterial fractions or for metabolites produced. In one skilled person in the art can calculate the range of produced embodiment, the compositions that have to be administered element at any other dose of cfu. In the particular case of the topically, the concentration of each bacterial strain and/or compositions that have to be administered topically, the corresponding fraction and/or metabolite can be adjusted so 40 concentration of each bacterial strain and/or corresponding as to correspond to doses (expressed as bacterial equivalent) fraction and/or metabolite can be adjusted so as to corre ranging from about 5x10 to about 10" cfu/d, and in spond to doses (expressed as bacterial equivalent) ranging particular from about 107 to about 10" cfu/d. A composition from about 5x10 to about 10" cfu/d, and in particular from for topical application may generally comprise from about about 107 to about 10'cfu/d. 10° to about 10" cfu/g, in particular from about 10 to about 45 A composition for topical application may generally com 10" cfu/g, and more particularly from about 10° to about prise from about 10° to about 10" cfu/g, in particular from 10' cfu/g of bacteria. When a composition comprises com about 10 to about 10' cfu/g, and more particularly from pounds of interest, the contents of compounds of interest in about 10° to about 10' cfu/g of bacteria. the compositions correspond Substantially to the contents In one embodiment—the transformed bacteria is applied capable of being produced by about 10 to about 10" cfu, in 50 to an animal’s skin, such as pets; including dogs and particular about 10 to about 10" cfu, and more particularly cats—to prevent UV damage, improve odor, and address about 10° to about 10° cfu of compounds of interest per veterinarian dermatological needs. gram of carrier. III. Methods of Treatment In one embodiment, a composition for topical application In another aspect, methods of treating or preventing to the skin for UV protection is contemplated. Photoaging is 55 disorders or conditions associated with the skin are contem the alteration in the structure, function and appearance of the plated. The compositions described above comprising one or skin as a result of prolonged or repeated exposure to more populations of transformed bacteria expressing one or ultraviolet radiation from the sun. It accounts for 90% of age more compounds of interest are applied to the skin in an associated cosmetic skin problems in both men and women, amount effective to provide a therapeutically effective and moderate to severe photoaging signs were observed in 60 amount of the compound(s) of interest. As used herein, a 72% of men and 47% of women under 30 years of age. therapeutically effective amount is an amount of the topical Ultraviolet radiation is light in the non-visible area of the composition that when administered to a patient or Subject, spectrum that is of shorter wavelength and higher energy; it ameliorates, eliminates and/or inhibits the skin disorder or ranges roughly from 150 nm to 400 nm. Most of the highest condition in the local region or vicinity of the application of energy UV radiation (UVC radiation at wavelengths less 65 the topical composition. than 280 nm) is absorbed by ozone and stratospheric oxy In one embodiment, a method of protecting the skin from gen. UVB radiation comprised of wavelengths from 280 damage due to Sun exposure is provided. Methods of treat US 9,453,232 B2 37 38 ment for relief of oxidative stress caused by UV, methods of lotions with certain pH. lotions or creams containing active providing an anti-oxidant, an anti-reactive oxygen species compounds, bacteria and limiting factors etc. In another (Anti-ROS)), method for providing skin moisturizing, embodiment, the complementary product is a limiting factor method for promoting anti-aging, and methods for treating that will enhance the growth, activity and/or expression of psoriasis, eczema, active dermatitis, acne, wound healing the compound of interest to provide a lasting or continuous (including diabetic wounds or ulcers), intertrigof diaper rush, expression of the compound. The complementary product burns, insects bites, hives, dandruff (Scales), and methods for may include any compound beneficial to the activity of the providing odor control or removal are contemplated. original product, and enhance its activity for lasting efficacy. IV. Packaging of the Composition Another contemplated packaging is one wherein the After formulation, the composition is packaged in a 10 population of transformed bacteria is maintained as a layer manner Suitable for delivery and use by an end user. on a bandage or film that is combined with a second layer of In one embodiment, the composition is placed into an bandage/film that will allow activation of the bacteria, and appropriate dispenser and shipped to the end user. Examples that optionally may also limit reproduction/growth factors. of final container may include a pump bottle, Squeeze bottle, In another embodiment, the final product could be stored jar, tube, capsule or vial. 15 refrigerated, with the bacteria being in their active state. In some embodiments, the packaging is mindful of the In another embodiment, the bacteria is stored in a small nature of the transformed bacteria in the composition. For bead of water soluble cellulose. The beads can be mixed in example, Lactococci grown via respiration Survive markedly any solution Such as Sunscreen/moisturizing/body wash or better after long time storage than fermenting cells (Gaudu Soap. et al., Antonie van Leeuwenhoek, 82:263-269 (2002)). This V. Examples long time survival is probably due to the induction of The following example is illustrative in nature and is in no cytochromes which may protect the cells from oxidative way intended to be limiting. stress. The presence of intracellular glutathione, which is also protecting against oxidative stress, can also result in an Example 1 improved viability of Lactococcus lactis upon storage (Liet 25 al., Appl. Environ. Microbiol, 69(10):5739 (2003)). Another Lactococcus lactis with the element shinorine using vec approach to improve the viability of Lactococci upon Stor tor pBTOP1-shinorine1 age lays in the adaptation of the spray-drying process, and A. Bacteria in the use of process aids, such as microcrystalline cellulose, Bacteria of the L. lactis Strain are used. A stock Solution carboxymethylcellulose, hydroxypropylmethylcellulose 30 of the strain is stored in -20° C. in 50% glycerol in acetate Succinate, or sodium alginate, which may be used to GM17/M17 broth with 0.5% sugar. Bacteria are cultured in coat the bacterial particles (EP 17895.29 A2). These GM17 medium/M17 broth with 0.5% sugar or in MRS examples for Lactococcus are intended to be illustrative of medium. After 16 hours of incubation, bacteria are harvested the types of packaging approaches that a skilled artisan can by centrifugation and 10-fold concentrated in BM9 medium identify for any of the bacteria described herein. 35 at 2x10 bacteria/100 ul. On plate or slant, the strain will In another embodiment, the bacteria in the composition survive 2-3 weeks. are lyophilized or freeze dried, for reconstitution before or A stock preparation of the bacteria is prepared by inocu after application to the skin. In one embodiment, lyophiliza lating 5 mL broth with cells from the slant. The cells are tion or freeze drying is conducted with one or more excipi grown overnight at 30°C. Then 3 mL fully grown culture is ents, such as glycerol or other Sugar alcohols, to improve the 40 added to 1 ml 60% glycerol and stored at -80° C. shelf life of the transformed bacteria. In one embodiment, B. Genomic Integration into the Vector the lyophilized composition does not include trehalose pBTOP shinorine contains the complete operon for shi (C-D-glucopyranosyl-1,1-C-D-glucopyranosyde). norine DNA sequence (SEQID NO: 1,2,3,4,5,6,7,8,9,10,11, The packaging for the composition may be in a kit form 12,13,14) and is used production of the compound of of one or more containers. For example, a single bottle, tube, 45 interest. Shinorine's operon was integrated in the vector container, or capsule may be divided to two equal or unequal using two exemplary procedures: molecular cloning proce parts wherein one part contains the bacteria, in their packing dures (Balskus et al., Science, 329:1653-1656 (2010)), and form (freeze dried/inactive, etc.), and the other part contains de-novo sequencing synthesizing of the plasmid sequences an activation material, which can be a liquid or a gel. The together with the shinorine operon. single bottle or container can be designed so that an end user 50 B1: Molecular Cloning can dispense with a single force applied to the container all The sequence of shinorine, can be obtained from several or a portion of the contents in the two container parts, to Sources. Such as amplification from genomic Anabaena dispense onto the skin or other surface the transformed variabilis ATCC 29413, de-novo sequencing according to bacteria and the activation material. The kit may also be of the complete genome of Anabaena variabilis or Nostoc spp. the form that comprises two or more containers, one con 55 or amplification from growth culture of Anabaena variabi tainer with the population(s) of transformed bacteria and the lis. In accordance with the method as described in Balskus, other with a formulation for admixture with the populations et al. (supra), the complete shinorine gene cluster is PCR of transformed bacteria. In another example, two or more amplified from genomic DNA of Anabaena variabilis, using containers, one container with the population of transformed the forward primer ava3858-start 1 (with Ndel restriction bacteria, the other container with natural non pathogenic 60 Site-5'-GAGATCCCATATGAGTATCG TCCAAGCAAAG skin bacteria that are not transformed, and a third container 3'; SEQID NO: 16) and reverse primer ava3855-stop 1 (with with a formulation for admixture with the populations of XhoI restriction site 5'-GTACCTCGAGTCATGAATT transformed bacteria. In another example, the two or more ATTTTCCAGACAATCTTG-3' SEQ ID NO: 17). Primers containers composing the single bottle had one pump con are designed for ligation into pETOP1 vector so as to encode nected to two separate tubes, each draining from a different 65 untagged gene products. PCR reactions contained 25 uL of chamber. The kit may also include one or more complemen master mix, 2ng of DNA template, and 17 pmoles of each tary products, such as Soaps, body washes or moisturizing primer in a total volume of 50 uL. Thermocycling is carried US 9,453,232 B2 39 40 out in a PCR machine using the following parameters: for 24 hours. The 50 mL culture is diluted by 10x and grow denaturation for 1 min at 95°C., followed by 50 cycles of until OD600 is 0.2-0.3 (ca. 3 h.), Spin down cells for 20 min 30 sec at 95°C., 1 min at 50° C., 6 min at 70° C., and a final 6000xg, at 4° C. Wash cells with 400 uL 0.5 M sucrose, extension time of 10 min at 70° C. 10% glycerol (4° C.) and spin down (6000xg), Resuspend the cells in 200 uL 0.5 M sucrose, 10% glycerol, 50 mM Amplified fragments are digested with the restriction 5 EDTA (4° C.), keep the suspension on ice for 15 min and enzymes Ndel and XhoI for 2.5 hours at 37° C. Digests spin down. Wash cells with 100 mL 0.5 M sucrose, 10% contain 2 LL water, 6 uL of NEB Buffer 4 (10x), 6 uL of glycerol (4° C.) and spin down (6000xg). Resuspend the BSA (10x), 40 uL of PCR product, 3 ul of Ndel (20 U/uL), cells in 4 ml 0.5 M sucrose, 10% glycerol (4°C.) Use 40 uL and 3 uI of XhoI (20 U/LL). Restriction digests are purified per sample (on ice), or keep the cells in Small portions in directly using agarose gel electrophoresis; gel fragments are -80° C., let them defreeze on ice before use. further purified using the Illustra GFX kit. The digests are A protocol to transform the cells via electroporation: place ligated into linearized pBTOP1 expression vector using T4 10-100 uL cells in a pre-chilled electroporation cuvette with DNA ligase. Ligations are incubated at room temperature for 1 uL DNA (reconstituted in TE buffer), and keep the cuvette 2 hand contained 3 ul. water, 1 uLT4 Ligase Buffer (10x), 15 on ice. Use a Biorad GENE PULSER(R) with following 1 uL digested vector, 3 LIL digested insert DNA, and 2 ul, T4 adjustments: 2000 V. 25F, 20092. Pulse (normal reading is DNA Ligase (400 U/LL). 5 uL of each ligation is used to 4.5-5 msec), add 1 ml growth medium+20 mg MgCl+2 mM transform a single tube of the chosen bacterial strain. The CaCl. Keep the cuvette for 5 min on ice and incubate 1-1.5 identity of the resulting pBTOP1 constructs (shinorine h at 30° C. Plate 10 uL. 100 ul, 900 uL on M17 agar with sequence) is confirmed by sequencing of purified plasmid 2O glucose or lactose and limiting element (depends on plas DNA mid). Incubate 1-2 days at 30° C.; and grow the bacteria in B2: Sequencing liquid for spectrophotometer analysis, and plasmid extrac The full sequence of shinorine's operon was searched tion. using known databases as the NCBI and identified, and set The cells can also be transformed via heat shock. Three samples (transformed bacteria (L. lactis trans whichforth herein can be as used SEQID as a templateNO:1,2,3,4,5,6,7,8,9,10,11,12,13,18 for de-novo sequencing, to formed with molecule of interest inserted into vector (e.g. be synthesized as an insert to the plasmid, or can be SEQID NO 15), only bacterial cells (not transformed); and synthesized within the plasmid. bacterial strain with a designed vector without the shinorine C. Transformation sequence inserted are checked in a regular spectrophotom A vector shinorine harboring shinorine sequence (e.g. eter using UV wavelengths of 270 nm, 310 nm, 330 nm, and SEQID NO 1), or any vector constructed for the purpose of 30 360 nm, every 10 min for an hour up to 10 days, or 200-400 blocking UV radiation to be applied on human skin is nm every 10 min for an hour up to 10 days. transformed into L. lactis strain, according to the following While a number of exemplary aspects and embodiments protocol, including the following steps: Preparation of the have been discussed above, those of skill in the art will bacterial cells, transformation, plasmid extraction, growth of recognize certain modifications, permutations, additions and bacteria, Spectrophotometer measurements, and storage of sub-combinations thereof. It is therefore intended that the transformed bacteria following appended claims and claims hereafter introduced Preparation of the Cells: briefly, 1-10 ml L. lactis strain are interpreted to include all Such modifications, permuta from a -80° C. stock grown at 30° C. for 24 hours is tions, additions and Sub-combinations as are within their inoculated. The culture is diluted by 10x, grown at 30° C. true spirit and scope.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 91

SEO ID NO 1 LENGTH: 6459 TYPE: DNA ORGANISM: Anabaena variabilis

<4 OOs SEQUENCE: 1 atgaattatt titccagacaa tottgtaaac gotgtgctaa aacctgtaca toggggttcta 60

gaacaaacga ataatggittt coaggga cat caataatctt aatttcttga go cqccatta 12O cagaaaataa ttctacccaa actaaggtog gg.tcaggagc cataatatgt ttitt coct gg 18O

citcgaaatac agtgacttitt cotggatatg gttgtctitat at aggaataa gttgcttitta 24 O

aagttcc cac caaaa catca agaatacggc gattattittg acgttctaca cc aggcggga 3 OO

at attctago gotcc.gtgct titat caatga tigtaattaat tttittcttct acagttaaat 360 tttct atttic titcaggtgtg act agattat cittgaccaaa cataccgc.ca aaaactctgg 42O

agagaac acc aactaaataa acgtcatcaa tdggitttttgttitatic cagc agaatcggta 48O cgtaagaatc taatattgct agtaaagata cittcttgtcc ttgtctatot aactgctgtg 54 O

ctact tcata agctacgact coaccaaatg accaccc.ccc gacacgataa ggcc cttggg 6 OO

US 9,453,232 B2 51 52 - Continued gcaactacgg cattttgac taatticcgcc at accottac gtacttggit c agttggtaga 576. O gtc.cgtaaaa aggagaaatc taagaaaact tacgagaag catgatatgc acccaaacgg 582O tttitt cagct tcttgtggitt aactg.cgacc ttgatggcaa cactagoat c aattaa.gc.ca 588 O attaatgtag taggaatacg gatataatta Ctgctg.cgac gg tatgcaga gcaa.gcaaaa 594 O ccgacaa.cat citgitaatcaa gocaccacca act actaata citggttctitt gcgaactaat 6 OOO ttgaaatctg caaaga catc taccacttitc. tcaaatgttt gaatggit citt gtttggctica 6 O6 O gtaatagitaa tigggaaaag agt caagt ca atgcc.gtgat actggaaata tttittgaatt 612 O tgttgtact.gt agaactgact cacattagca totacaa.cag ccaa.gcaacg. tccaaaattit 618O ttgtagatat citgctaattig gtgatttitta atctgaaaaa citc catctac ataaactaag 624 O tcqtacticaa ttttitt cqta gcc ttcaatg tdaaaagctg. citgcttgcgc titcaaactitt 63 OO gcttggacga tigct catatt ctittgacctt tagtgcagta aatgactgta totgttgact 636 O ggaatatttg act caa.gc.ca aaattgaata attctagt cc tagaattaaa ccgattgatt 642O ggcataccaa ataactictaa titt ct catca agaaatagag gtaatcgct g g cagaatttg 648 O aggtaatcaa aacttgattg taaaaag.cag aacgttgatt atatataacg tacagcttitt 654 O taalatatagt gaatgactitg tagt cttgg ttgaactgta agacitctgca aatacgagca 66OO aact acatta agtttctact gttittgcagt gatgg tacta aatgaagttcc at catgattg 666 O atctgattgt agacagatat agtatgccag aagttagtag aaatttgctic aaaaatc.tta 672 O agattittctt cacgattitta aagataac at tatttgcgaa at atttgtac ataattatga 678 O. gatttittcta agaaat ct catalactataag ctgcaactta gtttatataa gcatcataat 6840 ttittgatctgaactgcaaac caaagaaatt agaggagagt ttgat attaa tttittat cat 69 OO aagtat cago act actaaaa accatgaatt ttaat caa.ca cacagcaaat gtttic cc cta 696 O attctgaatc aagaac agga gtatggalagg aaaatttaca gcagattgtt gttaattaga 7 O2O aatacaaaaa tigagtgcta aagataaagc acatct actt titatgagcgc agcagaaatg 708 O t cattggcat tdgctaaaac tta aggctitc taccalatact totaacaaaa cittaactaat 714. O ttgct citcat ttittaagtta gtgacactaa tdaaagt cct aag caatago ggacttitttg f2OO

Cagttgggca 721 O

<210s, SEQ I D NO 3 &211s LENGT H: 888 212. TYPE : PRT &213s ORGAN ISM: Anabaena variabilis

<4 OOs, SEQUENCE: 3 Met Glin. Thir Ile Asp Phe Asn. Ile Arg Llys Lieu. Lieu Val Glu Trp Asn 1. 1O 15

Ala Thr His Arg Asp Tyr Asp Lieu. Ser Glin Ser Lieu. His Glu Lieu. Ile 25 3O

Wall Ala Glin Val Glu Arg Thr Pro Glu Ala Ile Ala Wall. Thir Phe Asp 35 4 O 45

Lys Glin Glin Lieu. Thir Tyr Glin Glu Lieu. Asn His Lys Ala Asn Gln Lieu. SO 55 6 O

Gly His Tyr Lieu. Glin Thr Lieu. Gly Wall Glin Pro Glu Thir Lieu. Val Gly 65 70 7s 8O

Val Cys Lieu. Glu Arg Ser Lieu. Glu Met Wall Ile Cys Lieu. Lieu. Gly Ile 85 90 95

Lieu Lys Ala Gly Gly Ala Tyr Val Pro Ile Asp Pro Glu Tyr Pro Glin US 9,453,232 B2 53 54 - Continued

105 11 O

Glu Arg Ile Ala Tyr Met Lell Glu Asp Ser Glin Wall Lys Wall Luell Luell 115 12 O 125

Thir Glin Glu Lell Lell Asn Glin Ile Pro His His Glin Ala Glin Thir 13 O 135 14 O

Ile Wall Asp Arg Glu Trp Glu Ile Ser Thir Glin Ala Asn Thir 145 150 155 160

Asn Pro Ser Asn Ile Thir Asp Asn Luell Ala Wall Ile 1.65 17O 17s

Thir Ser Gly Ser Thir Gly Pro Lys Gly Ala Met Asn Thir His 18O 185 19 O

Gly Ile Cys Asn Arg Lell Lell Trp Met Glin Glu Ala Tyr Glin Ile Asp 195

Ser Thir Asp Ser Ile Lell Glin Thir Pro Phe Ser Phe Asp Wall Ser 21 O 215 22O

Wall Trp Glu Phe Phe Trp Thir Luell Luell Thir Gly Ala Arg Luell Wall Ile 225 23 O 235 24 O

Ala Pro Gly Gly His Asp Ser Ala Lell Ile Asp Luell Ile 245 250 255

Thir Glin Glu Glin Ile Thir Thir Luell His Phe Wall Pro Ser Met Luell Glin 26 O 265 27 O

Wall Phe Luell Glin Asn Arg His Wall Ser Ser Ser Luell Arg 285

Wall Ile Ser Gly Glu Ala Luell Ser Ile Asp Lell Glin Asn Arg Phe 29 O 295 3 OO

Phe Glin His Luell Glin Cys Glu Luell His Asn Luell Gly Pro Thir Glu 3. OS 310 315

Ala Ala Ile Asp Wall Thir Phe Trp Glin Cys Arg Asp Ser Asn Luell 3.25 330 335

Ser Wall Pro Ile Gly Arg Pro Ile Ala ASn Thir Glin Ile Ile 34 O 345 35. O

Lell Asp Ala Asp Lell Glin Pro Wall Asn Ile Gly Wall Thir Gly Glu Ile 355 360 365

Ile Gly Gly Wall Gly Wall Ala Arg Gly Lell Asn Glu Glu 37 O 375 38O

Lell Thir Glu Lys Phe Ile Ile Asn Pro Phe Pro Asn Ser Glu Phe 385 390 395 4 OO

Arg Luell Lys Thir Gly Asp Luell Ala Arg Lell Pro Asp Gly 4 OS 415

Asn Ile Glu Tyr Lell Gly Arg Thir Asp Glin Wall Ile Arg Gly 42O 425 43 O

Arg Ile Glu Ile Gly Glu Ile Glu Asn Wall Lell Ser Ser His Pro 435 44 O 445

Glin Wall Arg Glu Ala Wall Wall Ile Ala Arg Asp Asp Asn Ala Glin Glu 450 45.5 460

Lys Glin Ile Ile Ala Tyr Ile Thir Asn Ser Ile Pro Glin Luell 465 470 47s

Asp Asn Luell Arg Asp Phe Lell Ala Arg Luell Pro Asp Phe Met Ile 485 490 495

Pro Ala Ala Phe Wall Met Lell Glu His Luell Pro Lell Thir Pro Ser Gly SOO 505

Wall Asp Arg Lys Ala Lell Pro Pro Asp Lell Phe Asn Ser 515 52O 525 US 9,453,232 B2 55 56 - Continued

Glu His Asn Ser Tyr Wall Ala Pro Arg Asn Glu Wall Glu Glu Lys Luell 53 O 535 54 O

Wall Glin Ile Trp Ser Asn Ile Luell His Luell Pro Wall Gly Wall Thir 5.45 550 555 560

Glu Asn Phe Phe Ala Ile Gly Gly Asn Ser Luell Ala Luell His Luell 565 st O sts

Ile Ser Glin Ile Glu Glu Lell Phe Ala Glu Ile Ser Luell Ala Thir 585 59 O

Lell Luell Thir Asn Pro Wall Ile Ala Asp Luell Ala Wall Ile Glin Ala 595 605

Asn Asn Glin Ile His Asn Ser Pro Luell Wall Pro Ile Glin Pro Glin Gly 610 615

Lys Glin Glin Pro Phe Phe Ile His Pro Ala Gly Gly His Wall Luell 625 630 635 64 O

Phe Lell Ala Glin Ile Gly Thir Asp Glin Pro Phe Tyr 645 650 655

Gly Luell Glin Ala Glin Gly Phe Gly Asp Glu Ala Pro Luell Thir Arg 660 665 67 O

Wall Glu Asp Met Ala Ser Lell Tyr Wall Thir Ile Arg Glu Phe Glin 675 68O 685

Pro Glin Gly Pro Tyr Arg Wall Gly Gly Trp Ser Phe Gly Gly Wall Wall 69 O. 695 7 OO

Ala Glu Wall Ala Glin Glin Luell His Arg Glin Gly Glin Glu Wall Ser 7 Os

Lell Luell Ala Ile Lell Asp Ser Wall Pro Ile Lell Lell Asp Lys Glin 72 73 O 73

Pro Ile Asp Asp Wall Luell Wall Gly Wall Lell Ser Arg Wall Phe 740 74. 7 O

Gly Gly Met Phe Gly Glin Asp Asn Luell Wall Thir Pro Glu Glu Ile Glu 760 765

Asn Luell Thir Wall Glu Glu Lys Ile Asn Ile Ile Asp Ala Arg 770 775

Ser Ala Arg Ile Phe Pro Pro Gly Wall Glu Arg Glin Asn Asn Arg Arg 79 O 79.

Ile Luell Asp Wall Lell Wall Gly Thir Luell Lys Ala Thir Ser Tyr Ile 805 810 815

Arg Glin Pro Tyr Pro Gly Wall Thir Wall Phe Arg Ala Arg Glu Lys 825 83 O

His Ile Met Ala Pro Asp Pro Thir Luell Wall Trp Wall Glu Luell Phe Ser 835 84 O 845

Wall Met Ala Ala Glin Glu Ile Ile Ile Asp Wall Pro Gly Asn His 850 855 860

Tyr Ser Phe Wall Lell Glu Pro His Wall Glin Wall Lell Ala Glin Arg Luell 865 87O 87s 88O

Glin Asp Luell Glu Asn Asn Ser 885

<210s, SEQ ID NO 4 &211s LENGTH: 458 212. TYPE : PRT <213> ORGANISM: Anabaena variabilis

<4 OOs, SEQUENCE: 4.

Met Ala Glin Ser Leu Pro Leu Ser Ser Ala Pro Ala Thr Pro Ser Lieu. US 9,453,232 B2 57 58 - Continued

1O 15

Pro Ser Glin Thir Lys Ile Ala Ala Ile Ile Glin Asn Ile Cys Thir Luell 25

Ala Luell Luell Luell Lell Ala Lell Pro Ile Asn Ala Thir Ile Wall Phe Ile 35 4 O 45

Ser Luell Luell Wall Phe Arg Pro Glin Wall Ala Ala Asn Pro Glin SO 55 6 O

Thir Ile Luell Ile Ser Gly Gly Met Thir Lys Ala Lell Glin Luell Ala 65 70

Arg Ser Phe His Ala Ala Gly His Arg Wall Wall Lell Wall Glu Thir His 85 90 95

Trp Luell Thir Gly His Arg Phe Ser Glin Ala Wall Asp Phe 105 11 O

Thir Wall Pro Ala Pro Glin Asp Asn Pro Glin Ala Tyr Ile Glin Ala 115 12 O 125

Lell Wall Asp Ile Wall Glin Glu Asn Ile Asp Wall Ile Pro Wall 13 O 135 14 O

Thir Ser Pro Wall Gly Ser Asp Ser Luell Ala Pro Luell 145 150 155 160

Ser His Glu Wall Phe His Phe Asp Ala Asp Ile Thir Met 1.65 17O

Lell Asp Asp Lys Phe Ala Lell Thir Glin Ala Arg Ser Luell Luell 18O 185 19 O

Ser Wall Pro Ser Phe Ile Thir Ser Pro Glu Glin Wall Asn 195

Phe Asp Phe Ser Gly Glu Thir Arg Ile Lell Ser Pro 21 O 215

Tyr Asp Ser Wall Arg Arg Lell Asp Luell Thir Lys Lell Pro Thir 225 23 O 235 24 O

Pro Glu Glu Thir Ala Ala Phe Wall Arg Ser Luell Pro Ile Thir Pro Glu 245 250 255

Pro Trp Ile Met Glin Glu Phe Ile Pro Gly Glu Phe Thir 26 O 265 27 O

His Ser Thir Wall Arg Asn Gly Glu Luell Arg Luell His Cys Glu 27s 28O 285

Ser Ser Ala Phe Glin Wall Asn Glu Asn Wall Asn Asn Pro Glin Ile 29 O 295 3 OO

Thir Glu Trp Wall Glin His Phe Wall Glu Luell Lell Thir Gly Glin 3. OS 310 315

Ile Ser Phe Asp Phe Ile Glin Ala Glu Asp Gly Thir Wall Ala Ile 3.25 330 335

Glu Asn Pro Arg Thir His Ser Ala Ile Thir Thir Phe Tyr Asp His 34 O 345 35. O

Pro Glin Wall Ala Glu Ala Luell Ser Glin Ala Pro Thir Thir Glu Thir 355 360 365

Ile Glin Pro Luell Thir Thir Ser Pro Thir Trp Thir His Glu 37 O 375

Wall Trp Arg Luell Thir Gly Ile Arg Ser Phe Thir Glin Lell Glin Arg Trp 385 390 395 4 OO

Lell Gly Asn Ile Trp Arg Gly Thir Asp Ala Ile Glin Pro Asp Asp 4 OS 415

Pro Luell Pro Phe Lell Met Wall His His Trp Glin Ile Pro Luell Luell Luell 42O 425 43 O US 9,453,232 B2 59 60 - Continued

Lieu. Asn. Asn Lieu. Arg Arg Lieu Lys Gly Trp Thr Arg Ile Asp Phe Asn 435 44 O 445 Ile Gly Lys Lieu Val Glu Lieu. Gly Gly Asp 450 45.5

<210s, SEQ ID NO 5 &211s LENGTH: 279 212. TYPE: PRT <213> ORGANISM: Anabaena variabilis

<4 OOs, SEQUENCE: 5

Met Thir Asn. Wall Ile Wall Glin Pro Thr Ala Arg Pro Wall Thir Pro Luell 1. 5 1O 15

Gly Ile Lieu. Thir Lys Glin Lieu. Glu Ala Ile Wall Glin Glu Wall Glin 2O 25

His Pro Asp Lieu Pro Gly Glu Lieu. Ile Ala ASn Ile His Glin Ala Trp 35 4 O 45

Arg Lieu Ala Ala Gly Ile Asp Pro Tyr Luell Glu Glu Thir Thir Pro SO 55 6 O

Glu Ser Pro Glu Lieu Ala Ala Lieu. Ala Thir Thir Ala Thir Glu Ala 65 70

Trp Gly Glu. His Phe His Gly Gly Thr Thir Wall Arg Pro Luell Glu Glin 85 90 95

Glu Met Leu Ser Gly His Ile Glu Gly Glin Thir Lell Met Phe Wall 1OO 105 11 O

His Met Thr Lys Ala Lys Llys Val Lieu Glu Ile Gly Met Phe Thr Gly 115 12 O 125

Tyr Ser Ala Lieu Ala Met Ala Glu Ala Luell Pro Glu Asp Gly Luell Luell 13 O 135 14 O

Val Ala Cys Glu Val Asp Pro Tyr Ala Ala Glu Ile Gly Glin Ala 145 150 155 160

Phe Glin Glin Ser Pro His Gly Gly Lys Ile Arg Wall Glu Luell Asp Ala 1.65 17O 17s

Ala Lieu Ala Thr Lieu. Asp Llys Lieu Ala Glu Ala Gly Glu Ser Phe Asp 18O 185 19 O

Lieu Val Phe Ile Asp Ala Asp Llys Llys Glu Tyr Wall Ala Phe His 195 2OO

Llys Lieu. Lieu. Gly Ser Ser Lieu. Lieu Ala Pro Asp Gly Phe Ile Wall 21 O 215 22O

Asp Asn. Thir Lieu. Lieu. Glin Gly Glu Val Luell Pro Ala Glu Glu Arg 225 23 O 235 24 O

Ser Val Asn Gly Glu Ala Ile Ala Glin Phe ASn His Thir Wall Ala Ile 245 250 255

Asp Pro Arg Val Glu Glin Val Lieu. Lieu. Pro Luell Arg Asp Gly Luell Thir 26 O 265 27 O

Ile Ile Arg Arg Ile Glin Pro 27s

<210s, SEQ ID NO 6 &211s LENGTH: 410 212. TYPE: PRT <213> ORGANISM: Anabaena variabilis

<4 OOs, SEQUENCE: 6 Met Ser Ile Val Glin Ala Lys Phe Glu Ala Lys Glu Thir Ser Phe His 1. 5 15 US 9,453,232 B2 61 62 - Continued

Wall Glu Gly Tyr Glu Ile Glu Tyr Asp Luell Wall Tyr Wall Asp Gly 25

Ile Phe Glu Ile Glin Asn Ser Ala Luell Ala Asp Wall Tyr Glin Gly Phe 35 4 O 45

Gly Arg Luell Ala Ile Wall Asp Ala Asn Wall Ser Arg Luell Tyr Gly SO 55 6 O

Asn Glin Ile Glin Ala Tyr Phe Glin Tyr Tyr Gly Ile Glu Luell Arg Luell 65 70

Phe Pro Ile Thir Ile Thir Glu Pro Asp Lys Thir Ile Glin Thir Phe Glu 85 90 95

Arg Wall Ile Asp Wall Phe Ala Asp Phe Luell Wall Arg Lys Glu Pro 105 11 O

Wall Luell Wall Wall Gly Gly Gly Luell Ile Thir Asp Wall Wall Gly Phe Ala 115 12 O 125

Ser Thir Arg Ser Ser Asn Tyr Ile Arg Ile Pro Thir Thir 13 O 135 14 O

Lell Ile Gly Luell Ile Asp Ala Ser Wall Ala Ile Wall Ala Wall Asn 145 150 155 160

His Arg Luell Lys Asn Arg Luell Gly Ala His Ala Ser Arg 1.65 17O

Wall Phe Luell Asp Phe Ser Lell Luell Arg Thir Luell Pro Thir Asp Glin Wall 18O 185 19 O

Arg Asn Gly Met Ala Glu Lell Wall Ile Ala Wall Wall Ala His Glin 195

Glu Wall Phe Glu Lell Lell Glu Gly Glu Glu Lell Luell Arg Thir 21 O 215

His Phe Gly Asn Ile Asp Ala Thir Pro Glu Ile Glu Ile Ala His 225 23 O 235 24 O

Arg Luell Thir Lys Ala Ile His Met Luell Glu Lell Glu Wall Pro 245 250 255

Asn Luell His Glu Lell Asp Lell Asp Arg Wall Ile Ala Gly His Thir 26 O 265 27 O

Trp Ser Pro Thir Lell Glu Lell Ala Pro Arg Luell Pro Met Phe His Gly 27s 285

His Ala Wall Asn Wall Asp Met Ala Phe Ser Ala Thir Ile Ala Ala Arg 29 O 295 3 OO

Arg Gly Ile Thir Ile Ala Glu Arg Asp Arg Ile Lell Gly Luell Met 3. OS 310 315

Ser Arg Wall Gly Lell Ser Lell Asp His Pro Met Lell Asp Ile Asp Ile 3.25 330 335

Lell Trp Arg Gly Thir Glu Ser Ile Thir Luell Thir Arg Asp Gly Luell Luell 34 O 345 35. O

Arg Ala Ala Met Pro Pro Ile Gly Asp Wall Phe Wall Asn Asp 355 360 365

Lell Thir Arg Glu Glu Lell Ala Ala Ala Luell Ala Asp His Glu Luell 37 O 375 38O

Cys Thir Ser Pro Arg Gly Gly Glu Gly Wall Asp Wall Pro Wall 385 390 395 4 OO

Glin Glu Lell Ile Gly Ser Wall Lys 4 OS 41O

<210s, SEQ ID NO 7 &211s LENGTH: 1233

US 9,453,232 B2 69 70 - Continued gaagaaaaat tag tacaaat citggit cqaat attctgcatt tacctaaagt aggtgtgaca 168O gaaaacttitt togctattgg togg taatticc citcaaagctic tacatttaat ttct caaatt 1740 gaagagittat ttgctaaaga gatat cotta gcaac actitt taacaaatcc agtaattgca 18OO gatttagcca aggttatt ca agcaaacaac caaatccata attcacc cct agttccaatt 1860 caac cacaag gtaagcagca gcc tittcttt totatacatc ctdctggtgg to atgttitta 1920 tgct attitta aact cqcaca atatatagga act gaccaac cattt tatgg cittacaa.gct 198O

Caaggattitt atggagatga agc acccttg acgcgagttg aagatatggc tagt ctotac 2O4. O gtcaaaacta ttagagaatt to aaccc.cala gggcc titatic gtgtcggggg gtggt cattt 21OO ggtggagtic tagctt atga agtag cacag cagttacata gacaaggaca agaagitatict 216 O ttac tagdaa tattagattic titacgtaccg attctgctgg ataaacaaaa acc cattgat 222 O gacgtttatt tagttggtgt t ct ct coaga gtttittggcg gtatgtttgg tdaagataat 228O c tagt cacac ctdaagaaat agaaaattta actgtagaag aaaaaattaa ttacat catt 234 O gataaagcac ggagcgctag aat attc.ccg cctggtgtag alacgtcaaaa taatcgc.cgt 24 OO attcttgatgttittggtggg aactittaaaa goaacttatt cctatataag acaac catat 246 O cCaggaaaag. tcactgtatt t cagcCagg gaaaaacata t tatggct cc tacccgacc 252O ttagtttggg tagaattatt ttctgtaatg gcggctcaag aaattaagat tattgatgtc 2580 cctggaaacc attatt cott togttctagaa ccc catgitac aggttittagc acagogttta 264 O caagattgtc. toggaaaataa tt cataa 26.67

<210s, SEQ ID NO 14 &211s LENGTH: 6469 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic sequence

<4 OOs, SEQUENCE: 14 tcqagatgaa ttatttitcca gacaatcttg taaacgctgt gctaaaacct gtacatgggg 6 O ttctagaa.ca aacgaataat gig titt coagg gacat caata at cittaattt cittgagcc.gc 12 O cattacagaa aataatticta cccaaactaa got cqggtca ggagc cataa tatgtttitt c 18O cctggct cqa aatacagtga cittitt cotgg atatggttgt cittatatagg aataagttgc 24 O ttittaaagtt cocaccaaaa catcaagaat acgg.cgatta ttittgacgtt ctacaccagg 3OO cgggaatatt ctagogct co gtgctittatc aatgatgtaa ttaattittitt cittctacagt 360 taaattitt ct atttcttcag gtgtgactag attat cittga ccaaacatac cqc caaaaac 42O tctggagaga acaccalacta aataaacgt.c atcaatgggit ttttgttitat coagcagaat 48O cggtacgitaa gaatctaata ttgctagtaa agatact tct tdtcc ttgtc. tatgtaactg 54 O ctgtgctact t cataagcta cqact coacc aaatgaccac ccc.ccgacac gataaggcc c 6OO ttggggttga aattct citaa tagttittgac gtagagacta gccatat citt caact cqcgt. 660 caagggtgct t catct coat aaaatccttg agcttgtaag ccataaaatg gttggtcagt 72 O t cctatatat tdtgcgagtt taaaatagca taaaacatga ccaccagcag gatgtataca 78O aaagaaaggc tigctgcttac Cttgttggttgaattggaact aggggtgaat tatggatttg 84 O gttgtttgct tdaataacct toggctaaatc togcaattact ggatttgtta aaagttgttgc 9 OO taaggatat c tictittagcaa ataacticttcaatttgagaa attaaatgta gagctittgag 96.O ggaattacca ccaatagoga aaaagtttitc tdt cacacct actittaggta aatgcagaat 1 O2O

US 9,453,232 B2 87 88 - Continued cggCaccg.cg gtgcgggaac tecCgacgc.g ggcggaggtg gagt cqgtgc ticggcatggc cacgtacgac acggcc.ccct ggalacagcgc Ctcggacggc titcc.gcaac C acctggaggg 660

Ctggcgcggc gtcaac Ctgc acaac.cgcgt. ccacgtctgg gtggg.cgggc agatggCCaC 72 O cgggatgtcg cccaacgacc C9gtgttctg gctgcacaac gcc tacgt.cg acaagctgtg ggcc.gagtgg cagcgc.cgcc acccgggatc cggctacctic cc.cgc.cgc.cg ggacgc.ccga 84 O cgtggtggac Ctgaacgaca ggatgaagcc Ctggaacgac acctic ccc.gg ccgaccttitt 9 OO ggaccacacc gcc cactaca cct tcgacac cgact gaccc 96.O

Cctic cc.gcag gtcaggggta Cc 982

<210s, SEQ ID NO 19 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (9) <223> OTHER INFORMATION: n is a C, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (13) . . (24) <223> OTHER INFORMATION: n is a C, g, or t <4 OOs, SEQUENCE: 19 gcannnnnnt gcnn.nnnnnn nnnn 24

<210s, SEQ ID NO 2 O &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) ... (9) <223> OTHER INFORMATION: n is a C, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (12) ... (23) <223> OTHER INFORMATION: n is a C, g, or t

<4 OOs, SEQUENCE: 2O ggaginnnnng timnnnnnnnn nnn 23

<210s, SEQ ID NO 21 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) . . (7) <223> OTHER INFORMATION: n is a C, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (12) ... (21) <223> OTHER INFORMATION: n is a C, g, or t

<4 OOs, SEQUENCE: 21 acnn.nnnct c Cnnnnn.nnnn in 21

<210s, SEQ ID NO 22 US 9,453,232 B2 89 90 - Continued

&211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence <4 OOs, SEQUENCE: 22

<210s, SEQ ID NO 23 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence <4 OOs, SEQUENCE: 23 gtatac

<210s, SEQ ID NO 24 &211s LENGTH: 17 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (17 <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 24 gaccgannnn nnnnnnn 17

<210s, SEQ ID NO 25 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OOs, SEQUENCE: 25 cycgrg

<210s, SEQ ID NO 26 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OOs, SEQUENCE: 26 cycgrg

<210s, SEQ ID NO 27 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OOs, SEQUENCE: 27 tcgcga

<210s, SEQ ID NO 28 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence US 9,453,232 B2 91 92 - Continued

22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (9) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (13) . . (24) <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 28 gcannnnnnt gcnn.nnnnnn nnnn 24

<210s, SEQ ID NO 29 &211s LENGTH: 11 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) . . (8) <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 29 gacnnnnngt c 11

<210s, SEQ ID NO 3 O &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence & 22 O FEATURE; <223> OTHER INFORMATION: synthesized restriction sequence

<4 OOs, SEQUENCE: 30 grogyC

<210s, SEQ ID NO 31 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OOs, SEQUENCE: 31

<210s, SEQ ID NO 32 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence <4 OOs, SEQUENCE: 32 agtact

<210s, SEQ ID NO 33 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OOs, SEQUENCE: 33 aggc ct US 9,453,232 B2 93 94 - Continued

<210s, SEQ ID NO 34 &211s LENGTH: 7 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 34 tccingga

<210s, SEQ ID NO 35 &211s LENGTH: 17 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (17 <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 35 gaccgannnn nnnnnnn 17

<210s, SEQ ID NO 36 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence <4 OOs, SEQUENCE: 36 gtcgac

<210s, SEQ ID NO 37 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) . . (6) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (12) ... (23) <223> OTHER INFORMATION: n is a, c, g, or t <4 OO > SEQUENCE: 37 acnnnngtay C 23

<210s, SEQ ID NO 38 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) . . (8) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (12) ... (24) <223> OTHER INFORMATION: n is a, c, g, or t US 9,453,232 B2 95 96 - Continued

<4 OOs, SEQUENCE: 38 aagnnnnnc t thinnnn.nnnn nnnn 24

<210s, SEQ ID NO 39 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (6) ... (9) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (12) ... (26) <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 39 grtacnnnng timnnnnnnnn nnnnnn 26

<210s, SEQ ID NO 4 O &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence <4 OOs, SEQUENCE: 4 O tctaga

<210s, SEQ ID NO 41 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (8) ... (15 <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 41 cacctg.cnnn 15

<210s, SEQ ID NO 42 &211s LENGTH: 11 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (11 <223> OTHER INFORMATION: n is a, c, g, or t

<4 OOs, SEQUENCE: 42 cgt.ct cnnnn n 11

<210s, SEQ ID NO 43 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OOs, SEQUENCE: 43 US 9,453,232 B2 97 98 - Continued gatatc

<210s, SEQ ID NO 44 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) ... (22) <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 44 ctgaaginnnn 22

<210s, SEQ ID NO 45 &211s LENGTH: 7 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OOs, SEQUENCE: 45 cct cagc

<210s, SEQ ID NO 46 &211s LENGTH: 16 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (16) <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 46 gaggaginnnn nnnnnn 16

<210s, SEQ ID NO 47 &211s LENGTH: 11 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (11 <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 47 cgt.ct cnnnn n 11

<210s, SEQ ID NO 48 &211s LENGTH: 7 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: n is a, c, g, or t

<4 OOs, SEQUENCE: 48 Cctnagg US 9,453,232 B2 99 100 - Continued

<210s, SEQ ID NO 49 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (9) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (13) . . (24) <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 49 tgannnnnnt cannnnnnnn nnnn 24

<210s, SEQ ID NO 50 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (6) ... (9) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (12) ... (26) <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 50 grtacnnnng timnnnnnnnn nnnnnn 26

<210s, SEQ ID NO 51 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) . . (6) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (12) ... (23) <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 51 acnnnngtay cnn.nnnnnnn nnn 23

<210s, SEQ ID NO 52 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (9) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (13) . . (24) <223> OTHER INFORMATION: n is a, c, g, or t

<4 OOs, SEQUENCE: 52 tgannnnnnt cannnnnnnn nnnn 24 US 9,453,232 B2 101 102 - Continued

<210s, SEQ ID NO 53 &211s LENGTH: 7 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OOs, SEQUENCE: 53 cct cagc

<210s, SEQ ID NO 54 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OOs, SEQUENCE: 54 gatatc

<210s, SEQ ID NO 55 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence <4 OO > SEQUENCE: 55 gtatac

<210s, SEQ ID NO 56 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) . . (12) <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 56 c cannnnnnn nintgg 15

<210s, SEQ ID NO 57 &211s LENGTH: 7 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: n is a, c, g, or t <4 OO > SEQUENCE: 57 rigginc.cy

<210s, SEQ ID NO 58 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) ... (22) <223> OTHER INFORMATION: n is a, c, g, or t US 9,453,232 B2 103 104 - Continued

<4 OOs, SEQUENCE: 58 ctgaaginnnn 22

<210s, SEQ ID NO 59 &211s LENGTH: 7 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: n is a, c, g, or t <4 OO > SEQUENCE: 59 tcc.ncga

<210s, SEQ ID NO 60 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence <4 OOs, SEQUENCE: 60 tctaga

<210s, SEQ ID NO 61 &211s LENGTH: 6 & 212 TYPE DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence <4 OOs, SEQUENCE: 61 tgtaca

<210s, SEQ ID NO 62 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OOs, SEQUENCE: 62 gagotc

<210s, SEQ ID NO 63 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OOs, SEQUENCE: 63 grgCyc

<210s, SEQ ID NO 64 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OOs, SEQUENCE: 64 US 9,453,232 B2 105 106 - Continued gagotc

<210s, SEQ ID NO 65 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (8) ... (15 <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 65 cacctg.cnnn 15

<210s, SEQ ID NO 66 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OOs, SEQUENCE: 66 gcatgc

<210s, SEQ ID NO 67 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthesized restriction sequence

<4 OO > SEQUENCE: 67 rcatgy

<210s, SEQ ID NO 68 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OOs, SEQUENCE: 68 tgcgca

<210s, SEQ ID NO 69 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OOs, SEQUENCE: 69 cycgrg

<210s, SEQ ID NO 70 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OO > SEQUENCE: 7 O Ctcgag US 9,453,232 B2 107 108 - Continued

<210s, SEQ ID NO 71 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OOs, SEQUENCE: 71 cycgrg

<210s, SEQ ID NO 72 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) . . (8) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (12) ... (24) <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 72 aagnnnnnc t thinnnn.nnnn nnnn 24

<210s, SEQ ID NO 73 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence & 22 O FEATURE; <223> OTHER INFORMATION: synthesized restriction sequence

<4 OO > SEQUENCE: 73

<210s, SEQ ID NO 74 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OOs, SEQUENCE: 74 atgcat

<210s, SEQ ID NO 75 &211s LENGTH: 10 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) . . (7) <223> OTHER INFORMATION: n is a, c, g, or t

<4 OO > SEQUENCE: 75 caynnnnrtg 10

<210s, SEQ ID NO 76 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence US 9,453,232 B2 109 110 - Continued

<4 OO > SEQUENCE: 76 grgCyc

<210s, SEQ ID NO 77 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OO > SEQUENCE: 77

<210s, SEQ ID NO 78 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OO > SEQUENCE: 78 aggc ct

<210s, SEQ ID NO 79 &211s LENGTH: 8 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence < 4 OO SEQUENCE: 79 ttaattaa.

<210s, SEQ ID NO 8O &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence <4 OOs, SEQUENCE: 80

<210s, SEQ ID NO 81 &211s LENGTH: 12 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) ... (9) <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 81 rgcannnnnt gc 12

<210s, SEQ ID NO 82 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OOs, SEQUENCE: 82 rcatgy US 9,453,232 B2 111 112 - Continued

<210s, SEQ ID NO 83 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence <4 OOs, SEQUENCE: 83 atgcat

<210s, SEQ ID NO 84 &211s LENGTH: 10 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) . . (7) <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 84 caynnnnrtg 10

<210s, SEQ ID NO 85 &211s LENGTH: 10 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence & 22 O FEATURE; <221 > NAMEAKEY: misc feature <222s. LOCATION: (6) ... (10 <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 85 ggat.cnnnnn 10

<210s, SEQ ID NO 86 &211s LENGTH: 7 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 86 ggtnacc

<210s, SEQ ID NO 87 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OO > SEQUENCE: 87 gttaac

<210s, SEQ ID NO 88 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 9,453,232 B2 113 114 - Continued <223> OTHER INFORMATION: synthesized restriction sequence <4 OOs, SEQUENCE: 88 c catgg

<210s, SEQ ID NO 89 &211s LENGTH: 11 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (8) ... (11 <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 89 gct citt.cnnn n 11

<210s, SEQ ID NO 90 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OOs, SEQUENCE: 90 yccggr

<210s, SEQ ID NO 91 & 211 LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized restriction sequence

<4 OOs, SEQUENCE: 91 c catgg

40 What is claimed is: avobenzone, butyl methoxydibenzoylmethane, ensulizole, 1. A composition comprising a population of transformed 2-phenylbenzimidazole-5-Sulfonic acid, homosalate, bacteria formulated for topical application to a subject, the homomethylsalicylat, meradimate, methyl 2-aminobenzo population of transformed bacteria comprising non-patho ate, methylanthranilate, octinoxate, methyl 2-aminobenzo genic, Gram-positive bacteria that have been genetically 45 ate methylanthranilate, octisalate, 2-ethylhexyl salicylate, modified to express a mycosporine-like amino acid at a level octyl salicylate, octocrylene, 2-ethylhexyl-2-cyano-3.3di of about 0.1 mM to about 100 mM of 10°-10 cfu of the phenylacrylate, oxybenzone, benzophenone-3,2-hydroxy-4- transformed bacteria. methoxybenzophenone, Sulisobenzone, benzophenone-4, 2. The composition of claim 1 wherein the population of so drometrizoletrisiloxane, mexoryl XL, enZacamene, 4-Meth transformed bacteria comprises bacteria selected from the ylbenzylidene camphor, padimate-O, octyl dimethyl PABA, group consisting of Lactobacillus cesei, Lactobacillus reu XPABA, terephthalylidenedicamphor sulfonic acid, mexo teri, Lactobacillus acidophilus, Lactobacillus jensenii, Bifi ryl SX, 3,3'-(1,4-phenylenedimethylidene)bis 7,7-dimethyl dobacterium lognum, Bifidobacterium reuteri, Bifidobacte 2-oxobicylclo2.2.1]hept-1-yl methanesulfonic acid), cino rium lactis, Bifidobacterium breve, Bifidobacterium 55 Xate, 2-ethoxyethyl 3-(4-methoxyphenyl) propenoate, animalis, Propionibacterium acidipropionici, Propionibac diethanolamine-methoxycinnamate, dioxybenzone, benzo terium feudenreichii, Propionibacterium thoenii, Propioni phenone-8, (2-hydroxy-4-methoxyphenyl)-(2-hydroxyphe bacterium jensenii, Lactobacillus lactis, Lactobacillus nyl) methanone, triethanolamine salicylate, or trolamine rhamnosus, Lactococcus lactis, and Lactococcus plantarum. salicylate. 3. The composition of claim 2 wherein the population of 60 6. The composition of claim 1 further comprising a transformed bacteria is selected from the group consisting of sunscreen active ingredient that absorbs UVB radiation. transformed Lactobacillus lactis and Lactococcus lactis. 7. The composition of claim 6 wherein the sunscreen 4. The composition of claim 1 wherein the transformed active ingredient that absorbs UVB radiation is avobenzone. bacteria is present in a topical composition at a concentra 8. The composition of claim 1 further comprising a tion of at least 0.1% by weight of the total composition. 65 SUSCC. 5. The composition of claim 1 further comprising a 9. The composition of claim 8 wherein the sunscreen titanium dioxide, Zinc oxide, para-aminobenzoic acid, comprises Zinc oxide. US 9,453,232 B2 115 116 10. The composition of claim 1 wherein the mycosporine mycosporine-taurine, mycosporine-glycine-valine, like amino acid is selected from the group consisting of mycosporine-2-glycine, mycosporine- glycine-glutamic gadusol, deoxygadusol, 4-deoxygaduSol, shinorine, por acid, mycosporine-glutamic acid-glycine, mycosporine phyra-334, palythine, palythene, asterina-330, palythinol, methylamine-serine, mycosporine-methylamine-threonine, mycosporine-glycine, mycosporine serinol, mycosporine- 5 usujirene, dehydroxylusujirene, palythenic acid-337. taurine, mycosporine-glycine-valine, mycosporine-2-gly palythenic acid-335, palythenic-serine, palythine-threonine, cine, mycosporine-glycine-glutamic acid, mycosporine-glu palythine-threonine-Sulphate, palythenic-serine-Sulphate, tamic acid-glycine, mycosporine-methylamine-serine, euhalothece, mycosporine-alanine, 2-(e)-2,3-dihydroxipro - mycosporine-methylamine-threonine, usujirene, dehydroxy 1-enylimino-mycosporine-alanine, and Scytonemin. lusujirene, palythenic acid-337, palythenic acid-335, 10 palythenic-serine, palythine-threonine, palythine-threonine 17. A composition comprising a Sunscreen active ingre Sulphate, palythenic-serine-Sulphate, euhalothece, dient that absorbs UVB radiation and a population of mycosporine-alanine, 2-(e)-2,3-dihydroxipro-1-enylimino transformed bacteria formulated for topical application to a mycosporine-alanine, and Scytonemin. Subject, the population of transformed bacteria comprising 11. The composition of claim 1 wherein the population of 15 non-pathogenic, Gram-positive bacteria that have been transformed bacteria is formulated in the composition for genetically modified to express a mycosporine-like amino topical application as a cream, lotion, emulsion, gel, oint acid at a level of about 0.1 mM to about 100 mM for ment or liquid. 10°-10' cfu of the transformed bacteria. 12. A composition comprising a population of trans 18. The composition of claim 17 wherein the transformed formed bacteria formulated for topical application to a bacteria is present in a topical composition at a concentra Subject, the population of transformed bacteria comprising tion of at least 0.1% by weight of the total composition. non-pathogenic, Gram-positive bacteria selected from the 19. The composition of claim 17 further comprising a group consisting of Lactobacillus lactis and Lactococcus SUSCC. lactis that have been genetically modified to express a 20. The composition of claim 17 wherein the mycosporine-like amino acid at a level of about 0.1 mM to 25 mycosporine-like amino acid is selected from the group about 100 mM for 10°-10 cfu of the transformed bacteria. consisting of gadusol, deoxygadusol, 4-deoxygaduSol, shi norine, porphyra-334, palythine, palythene, asterina-330, 13. The composition of claim 12 wherein the transformed palythinol, mycosporine-glycine, mycosporine serinol, bacteria is present in a topical composition at a concentra mycosporine-taurine, mycosporine-glycine-valine, tion of at least 0.1% by weight of the total composition. mycosporine-2-glycine, mycosporine-glycine-glutamic 14. The composition of claim 12 further comprising a 30 acid, mycosporine-glutamic acid-glycine, mycosporine SUSCC. methylamine-serine, mycosporine-methylamine-threonine, 15. The composition of claim 12 wherein the sunscreen usujirene, dehydroxylusujirene, palythenic acid-337. comprises avobenzone or Zinc oxide. palythenic acid-335, palythenic-serine, palythine-threonine, 16. The composition of claim 12 wherein the palythine-threonine-Sulphate, palythenic-serine-Sulphate, mycosporine-like amino acid is selected from the group 35 consisting of gadusol, deoxygadusol, 4-deoxygaduSol, shi euhalothece, mycosporine-alanine, 2-(e)-2,3-dihydroxipro norine, porphyra-334, palythine, palythene, asterina-330, 1-enylimino-mycosporine-alanine, and Scytonemin. palythinol, mycosporine-glycine, mycosporine serinol, k k k k k