An engineered skin commensal

Abstract

Mutant strains of Corynebacterium glutamicum bacteria are formulated as human skin commensals.

Description

[0001] An engineered skin commensal

[0002]

[0001] Introduction

[0003]

[0002] Genetically engineered probiotics that produce therapies on-site on the surface of the skin would be valuable for treating many diseases and also has cosmetic applications. We previously Corynebacterium glutamicum, a soil bacteria and Generally Recognized as Safe (GRAS) organism, as a probiotic to apply to topically to the skin (B21-024-2WO, PCT / US22 / 19051; WO2022 / 187724). We disclosed that C. glutamicum, when applied to the skin with a moisturizer that alters the microenvironment and provides nutrients, could colonize the skin for over 8 hours and produce glutamate on the surface of the skin. This was a novel and non-obvious idea as the soil microbe cannot natively grow on the skin surface.

[0004]

[0003] Summary of the Invention

[0005]

[0004] We disclose genetic engineering that enable C. glutamicum to adopt characteristics of an optimized skin commensal bacteria, namely the ability to grow in highly acidic and lipidic conditions. Application in a moisturizer that is also acidic prevents skin colonization by other harmful bacteria, and improved tolerance to lipids provides a more robust colonizer and expanded therapeutic options at the surface of the skin.

[0006]

[0005] Culturing C. glutamicum for hundreds of generations in highly acidic and lipidic conditions that are considered “skin-mimicking” (pH~5.5 and Tween80~l%), we developed evolved strains that can grow rapidly in those conditions while the wild-type strain cannot grow in those conditions. The strains were whole-genome sequenced, and we identified mutations and amplifications that contribute to increased acid and lipid tolerance.

[0007]

[0006] We show the growth rate of one of these strains to be 0.3 in “skin-mimicking” conditions, which is 60% of the growth rate in optimal rich media (0.5) and compares to the lack of exponential growth in the wild-type strain in “skin-mimicking” conditions.

[0008]

[0007] The invention provides mutant strains of C. glutamicum, natively a soil microbe, optimized for colonizing skin. The strains may be further genetically engineered, such as disclosed in WO2022 / 187724, to produce therapies or cosmetic substances, such as for use in commercial applications in both health care and cosmetics.

[0009]

[0008] In aspects and embodiments the invention provides:

[0010]

[0009] 1. A composition comprising a living population of engineered, mutant Corynebacterium glutamicum bacteria strain engineered to adopt characteristics of an optimized skin commensal bacteria, namely the ability to grow in acidic and lipidic conditions, such as pH 3.5-6.5, e.g. pH~5.5, and Tween80 of 0.2-5%, e.g. ~1%.

[0010] 2. The composition of claim 1, wherein the mutant bacteria strain provides increased growth under acidic and lipidic condition compared with a corresponding wild-type strain. [Oil] 3. The composition of claim 1, wherein the mutant bacteria strain is engineered to comprise a plurality of human skin commensal mutations or amplifications or Table 1, 2 or 3.

[0012] 4. The composition of claim 1, wherein the mutant bacteria strain is engineered to comprise 1, 2, 3, 4, 5, 6, 7, 8 or 9 of genes: Ipps, cgp_3312, pmt, cgp_0753, atpB, atpE, cglR3, cgp3052 and cgp_r5123, and / or amplifications of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all of genes: cgp_1683, tatC, tatX, cgp_1686, cgp_1687, pafA2, pup, pafAl, mpa, pimT, pepC, cgp_1694, cgp_1695.

[0011]

[0013] 5. The composition of claim 1, wherein the mutant bacteria strain is engineered to comprise mutations and amplifications of Cgm-IA-IG or Cgm-2A-2E.

[0012]

[0014] 6. The composition of claim 1, 2, 3, 4 or 5, configured as a topical, human skin probiotic moisturizer formulation further comprising a nutrient source for the bacteria, wherein the formulation is acidic to prevent skin colonization by other harmful bacteria.

[0013]

[0015] 7. The composition of claim 6, disposed on the skin of a person.

[0014]

[0016] 8. The composition of claim 6, wherein the nutrient source comprises one or more sugars.

[0015]

[0017] 9. The composition of claim 6, further comprising a humectant, such as glycerin, hyaluronic acid, and propylene glycol, or an emollient, such as shea butter, cocoa butter, and octyldodecanol, or an occlusive, such as petrolatum, cetyl alcohol (hexadecan- l-ol), and lanolin.

[0018] 10. The composition of claim 6, engineered to produce a skin-bioactive agent, wherein the bacteria are producing the agent.

[0016]

[0019] 11. The composition of claim 10, wherein the bioactive agent is selected from a cosmetic, anti-aging, anti-oxidant, sun-screen, anti-inflammatory, antimicrobial, analgesic, and therapeutic compound.

[0017]

[0020] 12. The composition of claim 10, wherein the bioactive agent is selected from signal peptides, carrier peptides, neurotransmitter inhibitor peptides, and enzyme inhibitor peptides.

[0021] 13. The composition of claim 10, wherein the bioactive agent is selected from lysine, arginine, cysteine, histidine, alanine, serine, theronine, isoleucine, aspartic acid, valine, citrulline, GHK-Cu, GSH-Cu, mangenese tripeptide, Peptamide-6, carnosine, N-acetyl carnosine, tripeptide- 10-citrulline, palmitoyl-tripeptides, palmitoyl-tetrapeptides, palmitoylpentapeptides, acetyl-tetrapeptide, hexapeptide- 11, tetrapeptide PKEK, hexapeptide- 14, silk protein, aquaporin, alpha interferon, Hsp70, transforming growth factors, rice peptides, and soybean peptides.

[0022] 14. The composition of claim 10, wherein the bioactive agent is a serine protease inhibitor, such as LEKTI-D6, LEKTI-D5, elafin and secretory leukocyte protease inhibitor for treating Netherton Syndrome, atopic dermatitis, psoriasis, and acne rosacea.

[0018]

[0023] 15. The composition of claim 10, wherein the bioactive agent is an anti-inflammatory compound, such as IL-10 to treat diseases such as Netherton Syndrome, atopic dermatitis, psoriasis, acne rosacea, and chronic wound infection.

[0019]

[0024] 16. The composition of claim 10, wherein the bioactive agent is an anti-microbial peptide, such as pediocin and nisin to treat diseases such as Netherton Syndrome, atopic dermatitis, psoriasis, acne rosacea, and chronic wound infection.

[0020]

[0025] 17. The composition of claim 10, wherein the bioactive agent is an antibody, antibody fragment or nanobody, such as single chain variable fragment anti-tumor necrosis factor alpha to treat diseases such as Netherton Syndrome, atopic dermatitis, psoriasis, acne rosacea, and chronic wound infection.

[0021]

[0026] 18. A method of using composition of claim 6, comprising applying the formulation to the skin of a person in need thereof under conditions wherein the bacteria produce an effective amount of the agent on the skin over a predetermined time range.

[0022]

[0027] 19. A method of claim 18, wherein the time range is 4, 8, 12, 24, and 48 hours.

[0023]

[0028] 20. A method of claim 18, further comprising adjusting the concentration of the nutrient source to adjust bioproduction and colonization time, wherein the concentration of glucose and glycerol is varied from 2 to 20% of the moisturizer for increasingly longer colonization times.

[0024]

[0029] The invention encompasses all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited.

[0025]

[0030] Brief Description of the Drawings

[0026]

[0031] Fig 1. Engineered C. glutamicum: Growth in Skin- Mimicking Media. The difference in growth between the original strain and one of the engineered strains. The original strain does not grow at all, while the engineered strain has a growth rate of 0.3. The growth rate in optimal rich media is 0.5, so the the engineered strain has 60% of the growth strain in the high acid, lipidic environments of the skin-mimicking media as it does of the optimal rich media.

[0027]

[0032] Fig. 2. Adaptive Laboratory Evolution of C. glutamicum to “skin-mimicking” (BHI, pH 5.5, and 1% Tween 80).

[0028]

[0033] Figs. 3A-D. Evaluate AL strains in different media conditions; . Growth Rate is calculated as the slope of the LN of OD600 values during the growth phase. In all the different conditions, AL3, AL4 and AL6 had the best growth rates. A. Overnight in BHI, Overnight in Skin-BHI, Assay (OD-. l) in tubes; B. Overnight in BHI, Exponential in BHI, Assay (OD-.l) in tubes; C. Overnight in BHI, Overnight in Skin-BHI, Assay (OD~. 1) on plate; D. Overnight in

[0029] BHI, Exponential in BHI, Assay (OD~. 1) on plate

[0030]

[0034] Description of Particular Embodiments of the Invention

[0031]

[0035] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and / or. The examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.

[0032]

[0036] Examples. Safety and efficacy of applying to skin a probiotic moisturizer comprising the soil bacterium C. glutamicum producing glutamate

[0033]

[0037] We used common moisturizer ingredients (paraffin oil, water, Span60, dimethicone, and stearic acid) with the needed components for C. glutamicum to grow (glucose, glycerol, yeast extract and biotin) so that C. glutamicum could produce nutrients on the skin surface. The moisturizer is combined in a 1 : 1 mixture with Corynebacterium glutamicum resuspended at a concentration of IO10CFU / mL. Human testing was performed to determine both skin colonization of the soil bacterium and production of glutamate, an amino acid secreted and produced C. glutamicum. We used a control of moisturizer alone to determine local microbiome conditions. After 0, 2, 4, 8 and 24 hours we used a skin swab and determine the colony forming units in a 16 cm portion on the skin. We determined that C. glutamicum stably colonizes the surface of the skin for 8 hours before dropping back to control conditions after 24 hours. We also found increasing and continuous production of C. glutamicum over 8 hours before dropping back to control conditions after 24 hours. Highlighting the power of C. glutamicum continuous production, a topical glutamate formula of 0.1% glutamate was added to the skin, and was undetectable after 15 minutes likely because of absorption and degradation on the skin surface, demonstrating the utility of bacterial produced compounds continuously on the surface. Our safety data from human trials also demonstrate that applying this organism on human skin is non-irritating and non-allergenic, confirming both safety and efficacy.

[0034]

[0038] The acidity and sebum of the skin environment provides a defense mechanism against colonization by microbes. Adult skin pH measurements indicate that the pH of skin for women (mean pH=5.54) is slightly lower than men (mean pH=5.80)8. Therefore, we sought to engineer our strain to grow in a “skin mimicking” medium that is highly acidic (pH —5.5) and contains a sebum analog (1% Tween 80).

[0039] We conducted a 45 -day Adaptive Laboratory Evolution study on seven clonal isolates of C. glutamicum, gradually decreasing the pH and increasing the sebum analog over thousands of generations. The genomes of resultant strains of C. glutamicum which exhibited robust growth under skin-mimicking conditions were then sequenced. Our results demonstrate that wild-type C. glutamicum was unable to grow under these conditions, while .S’. epidermidis, a commensal skin bacterium, exhibited a growth rate of 0.29. Remarkably, our evolved C. glutamicum strain

[0035] (RVB-002) showcased a growth rate of 0.47, surpassing .S', epidermidis by 62%. This successful evolution of C. glutamicum demonstrates its potential to temporarily colonize skin environments to produce targeted therapeutic delivery.

[0036]

[0040] We tested our evolved strains in a variety of conditions. We tested four conditions for all of our strains. In Fig. 3A, we grew our strains in skin-mimicking conditions, passaged them again in skin-mimicking conditions, then grew the strains to OD-.l before measuring the growth rate in skin-mimicking media in test tubes for 18 hours. We found that some of our evoled strains grew even better than the most common skin microbe, S. epidermidis. In Fig. 3C, we performed the same experiment, but this time on microtiter plates. In Fig. 3B, we grew our strains in skin-mimicking conditions, grew them to exponential phase in rich BHI, then diluted the strains to OD-.l before measuring the growth rate in skin-mimicking media in test tubes for 12 hours. In Fig. 3D, we performed the same experiment, but in microtiter plates. Across all experiments, AL3, AL4, and AL6 had the highest growth rates. These strains all include amplications of these genes: cgp_1683, tatC,tatX, cgp_1686, cgp_1687, pafA2, pup, pafAl, mpa, pimT, pepC, cgp_1694, cgp_1695, indicating that these genes contribute towards this phenotype.

[0037]

[0041] Table 1. Exemplary C. glutamicum mutant strains providing improved growth on human skin compared to wild-type strain.

[0038] Cgm-IA

[0039] SNP cgp_0001 P420R (CCA > CGA)

[0040] INS cgp_0790, pyc intergenic (+217 / - 180)

[0041] INS cgp_0790, pyc intergenic (+223 / - 174)

[0042] INS IppS coding (414 / 1212 nt)

[0043] INS IppS coding (410 / 1212 nt)

[0044] Genetic Amplications: Sequence Change / Genes 50,800 bp x 2 cgp_1663,cgp_1664,cgp_1665,cgp_1668,cgp_1669,cgp_I670,cgp_1671,ppmC,ppmN,cgp_1675

[0045] ,lip3,cgp_1677,cobL,cgp_4020,cgp_1679,cgp_1680,pepE,cgp_1682,cgp_1683,tatC,tatX,cgp_16 86, cgp_1687,pafA2, pup, pafAl, mpa, pimT,pepC,cgp_1694,cgp_1695,cgp_1696,cgp_1697,hisG, hisE,cgp_1700,metH,cgp_1702,cgp_1703,cgp_1704,arsC2,cgp_1706,cgp_1707,cgp_1708,mshC

[0046] ,bac A,cgp_1711 ,lppL,pyrD,cgp_1714

[0047] Cgm-IB

[0048] SNP mag G94S (GGT > AGT)

[0049] SNP cgp_1121, cgp_l 122 intergenic (+341 / + 109)

[0050] SNP cgp_1275 E97K (GAA > AAA)

[0051] SNP tatX W6S (TGG > TCG)

[0052] SNP clpC, guaBl intergenic (-8 / -281)

[0053] SNP cgp_0811 T219A (ACC > GCC)

[0054] SNP mgtEl A176T (GCC > ACC)

[0055] SNP IppS E230* (GAA > TAA)

[0056] SNP cgp_2756 T139R (ACA > AGA)

[0057] Cgm-l C

[0058] SNP cgp_0811 T219A (ACC > GCC)

[0059] SNP mgtEl A176T (GCC > ACC)

[0060] SNP IppS E230* (GAA > TAA)

[0061] SNP cgp_2756 T139R (ACA > AGA)

[0062] Genetic Amplications: Sequence Change / Genes

[0063] 83,700 bp x cgp_1663,cgp_1664,cgp_1665,cgp_1668,cgp_1669,cgp_1670,cgp_1671,ppmC,ppmN,cgp_1675

[0064] ,lip3,cgp_1677,cobL,cgp_4020,cgp_1679,cgp_1680,pepE,cgp_1682,cgp_1683,tatC,tatX,cgp_16

[0065] 86,cgp_1687,pafA2,pup,pafAl,mpa,pimT,pepC,cgp_1694,cgp_1695,cgp_1696,cgp_1697,hisG, hisE,cgp_1700,metH,cgp_1702,cgp_1703,cgp_1704,arsC2,cgp_1706,cgp_1707,cgp_1708,mshC

[0066] ,bacA,cgp_1711 ,lppL,pyrD,cgp_1714,cgp_1715 ,cgp_1716,cgp_1717,cgp_1718,tetB,tetA,cgp_t

[0067] 5033,cgp_t5034,cgp_1722,cgp_1724,mcmB,mcmA,cgp_1727,cgp_1728,cgp_1730,cgp_1731,cg p_1732,cgp_1733,hemH,cgp_1735,cgp_1736,acn,acnR,cgp_1739,cgp_1740,cgp_1741,cgp_174

[0068] 2,cgp_1743,pacL

[0069] Cgm-ID

[0070] SNP cgp_0569 T281P (ACC > CCC)

[0071] INS cgp_1159 coding (1757 / 1905 nt)

[0072] SNP atpA P291L (CCA > CT A)

[0073] SNP cgp_2535 A136A (GCC > GCT) DEL cgp_3312 coding (307 / 1461 nt)

[0074] Genetic Amplications: Sequence Change / Genes

[0075] 50,800 bp x

[0076] 3cgp_1663,cgp_1664,cgp_1665,cgp_1668,cgp_1669,cgp_1670,cgp_1671,ppmC,ppmN,cgp_167 5,lip3,cgp_1677,cobL,cgp_4020,cgp_1679,cgp_1680,pepE,cgp_1682,cgp_1683,tatC,tatX,cgp_l 686, cgp_1687,pafA2, pup, pafAl, mpa, pimT,pepC,cgp_1694,cgp_1695,cgp_l 696, cgp_1697,hisG ,hisE,cgp_1700,metH,cgp_1702,cgp_1703,cgp_1704,arsC2,cgp_1706,cgp_1707,cgp_1708,msh C,bac A,cgp_l 711 ,lppL,pyrD,cgp_l 714

[0077] Cgm-IE

[0078] DEL pmt coding (350 / 1563 nt)

[0079] SNP atpD D322Y (GAT > TAT)

[0080] SNP cgtR3 D187N (GAT > AAT)

[0081] Cgm-I F

[0082] SNP cgp_0001 D509H (GAT > CAT)

[0083] SNP cmt5 E188Q (GAA > CAA)

[0084] DEL IppS coding (1146 / 1212 nt)

[0085] SNP glpQl, gntP intergenic (-139 / -283)

[0086] SNP cgp_3348, nagL intergenic (-284 / +254)

[0087] Genetic Amplications: Sequence Change / Genes

[0088] 12,600 bp x 2cgp_1683,tatC, tatX,cgp_1686,cgp_1687, pafA2,pup, pafAl, mpa, pirnT, pepC, cgp_1694, cgp_1695

[0089] Cgm-IG

[0090] INS cgp_1159 coding (1764 / 1905 nt)

[0091] INS cgp_1159 coding (1757 / 1905 nt)

[0092] Genetic Amplications: Sequence Change / Genes

[0093] 14,100 bp x 3cgp_1680, pepE,cgp_1682,cgp_1683, tatC,tatX, cgp_1686, cgp_1687,pafA2, pup, pafAl, mpa, pimT

[0094] Cgm-2A

[0095] SNP atpB, atpE intergenic (+99 / -35)

[0096] SNP cgp_0666 T192A (ACT >GCT)

[0097] SNP cgp_0753 G159E (GGA^GAA) SUB cgp_6007, psp2 intergenic (+337 / -311)

[0098] DEL pmt coding (350 / 1563 nt)

[0099] SNP rpoC C441Y (TGT >TAT)

[0100] INS sufR, cgp_1766 intergenic (-88Z-234)

[0101] Cgm-2B

[0102] SNP atpG G314R (GGC^CGC)

[0103] INS cgp_0223, tnp2f intergenic (-658 / - 119)

[0104] DEL cgp_0753 coding (178-201 / 966 nt)

[0105] DEL cgp_1016a coding (334 / 360 nt)

[0106] SNP cgp_1103 M82I (ATG-’-ATC)

[0107] DEL cgp_1467, cgp_1468 intergenic (-65 / +19)

[0108] DEL cgp_2380 coding (270 / 390 nt)

[0109] DEL metY, cstA intergenic (-354Z-66)

[0110] INS pmt coding (350 / 1563 nt)

[0111] INS pmt coding (356 / 1563 nt)

[0112] Genetic Amplications: Sequence Change / Genes

[0113] 57,900 bp x

[0114] 4cgp_1680, pepE, cgp_1682, cgp_1683 ,tatC,tatX,cgp_1686,cgp_1687,pafA2, pup, pafAl,mpa, pirn

[0115] T,pepC,cgp_1694,cgp_1695,cgp_1696,cgp_1697,hisG,hisE,cgp_1700,metH,cgp_1702,cgp_170

[0116] 3,cgp_1704,arsC2,cgp_1706,cgp_1707,cgp_1708,mshC,bacA,cgp_1711,lppL,pyrD,cgp_1714,c gp_1715,cgp_1716,cgp_1717,cgp_1718,tetB,tetA,cgp_t5033,cgp_t5034,cgp_1722,cgp_1724,mc mB,mcmA,cgp_1727,cgp_1728,cgp_1730,cgp_1731,cgp_1732,cgp_1733,hemH

[0117] Cgm-2C

[0118] SNP cgp_0811 T219A (ACC^GCC)

[0119] SNP cgp_1288 W105* (TGG^TAG)

[0120] SNP cgp_2756 T139R (ACA^AGA)

[0121] SNP cgp_3084 E221E (GAG^GAA)

[0122] SNP dtxR P127L (CCT^CTT)

[0123] SNP IppS E230* (GAA^TAA)

[0124] Cgm-2D

[0125] SNP atpA P292R (CCG^CGG) SNP cgp_2810, cgp_2811 intergenic (- 139 / + 196)

[0126] DEL cgp_2854 Al, 639 bp

[0127] SUB cgp_6007, psp2 intergenic (+337 / -311)

[0128] SNP cgtR3 D187N (GAT^AAT)

[0129] INS mgtEl coding (742 / 1293 nt)

[0130] INS pmt coding (350 / 1563 nt)

[0131] Genetic Amplications: Sequence Change / Genes

[0132] 12,500 bp x 2cgp_1683, tatC,tatX, cgp_1686, cgp_1687, pafA2, pup, pafAl, mpa, pimT, pepC, cgp_1694, cgp_1695

[0133] Cgm-2E

[0134] INS cgp_0223, tnp2f intergenic (-658 / -119)

[0135] DEL cgp_1016a coding (334 / 360 nt)

[0136] DEL cgp_1467, cgp_1468 intergenic (-65 / + 19)

[0137] DEL cgp_2380 coding (270 / 390 nt)

[0138] SNP cgp_2737 P120Q (CCG^CAG)

[0139] DEL cgp_2854 Al, 639 bp

[0140] SUB cgp_6007, psp2 intergenic (+337 / -311)

[0141] SNP cgtR3 N188K (AAC^AAG)

[0142] DEL metY, cstA intergenic (-354 / -66)

[0143] INS pmt coding (350 / 1563 nt)

[0144] INS pmt coding (356 / 1563 nt)

[0145] SNP rpsA N219D ( A AC -* GAC)

[0146] SNP rpsH G97G (GGC^GGT)

[0147]

[0042] Table 2.

[0148] Table 2. MutationsRES167

[0149]

[0150]

[0151] Table 2 (cont). MutationsMB0015

[0152]

[0153]

[0154]

[0155]

[0044] Table 3 (cont.) AmplifcationMBOOl

[0156]

Claims

CLAIMS:

1. A composition comprising a living population of engineered, mutant Corynebacterium glutamicum bacteria strain engineered to adopt characteristics of an optimized skin commensal bacteria, namely the ability to grow in acidic and lipidic conditions, such as pH 3.5-6.5, e.g. pH~5.5, and Tween80 of 0.2-5%, e.g. ~1%.

2. The composition of claim 1, wherein the mutant bacteria strain provides increased growth under acidic and lipidic condition compared with a corresponding wild-type strain.

3. The composition of claim 1, wherein the mutant bacteria strain is engineered to comprise a plurality of human skin commensal mutations or amplifications or Table 1, 2 or 3.

4. The composition of claim 1, wherein the mutant bacteria strain is engineered to comprise 1, 2, 3, 4, 5, 6, 7, 8 or 9 of genes: Ipps, cgp_3312, pmt, cgp_0753, atpB, atpE, cglR3, cgp3052 and cgp_r5123, and / or amplifications of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 or all of genes: cgp_1683, tatC, tatX, cgp_1686, cgp_1687, pafA2, pup, pafAl, mpa, pimT, pepC, cgp_1694, cgp_1695.

5. The composition of claim 1, wherein the mutant bacteria strain is engineered to comprise mutations and amplifications of Cgm-IA-IG or Cgm-2A-2E.

6. The composition of claim 1, 2, 3, 4 or 5, configured as a topical, human skin probiotic moisturizer formulation further comprising a nutrient source for the bacteria, wherein the formulation is acidic to prevent skin colonization by other harmful bacteria.

7. The composition of claim 6, disposed on the skin of a person.

8. The composition of claim 6, wherein the nutrient source comprises one or more sugars.

9. The composition of claim 6, further comprising a humectant, such as glycerin, hyaluronic acid, and propylene glycol, or an emollient, such as shea butter, cocoa butter, and octyldodecanol, or an occlusive, such as petrolatum, cetyl alcohol (hexadecan- l-ol), and lanolin.

10. The composition of claim 6, engineered to produce a skin-bioactive agent, wherein the bacteria are producing the agent.

11. The composition of claim 10, wherein the bioactive agent is selected from a cosmetic, antiaging, anti-oxidant, sun-screen, anti-inflammatory, antimicrobial, analgesic, and therapeutic compound.

12. The composition of claim 10, wherein the bioactive agent is selected from signal peptides, carrier peptides, neurotransmitter inhibitor peptides, and enzyme inhibitor peptides.

13. The composition of claim 10, wherein the bioactive agent is selected from lysine, arginine, cysteine, histidine, alanine, serine, theronine, isoleucine, aspartic acid, valine, citrulline, GHK- Cu, GSH-Cu, mangenese tripeptide, Peptamide-6, carnosine, N-acetyl carnosine, tripeptide- 10- citrulline, palmitoyl-tripeptides, palmitoyl-tetrapeptides, palmitoyl-pentapeptides, acetyltetrapeptide, hexapeptide- 11 , tetrapeptide PKEK, hexapeptide- 14, silk protein, aquaporin, alpha interferon, Hsp70, transforming growth factors, rice peptides, and soybean peptides.

14. The composition of claim 10, wherein the bioactive agent is a serine protease inhibitor, such as LEKTI-D6, LEKTI-D5, elafin and secretory leukocyte protease inhibitor for treating Netherton Syndrome, atopic dermatitis, psoriasis, and acne rosacea.

15. The composition of claim 10, wherein the bioactive agent is an anti-inflammatory compound, such as IL-10 to treat diseases such as Netherton Syndrome, atopic dermatitis, psoriasis, acne rosacea, and chronic wound infection.

16. The composition of claim 10, wherein the bioactive agent is an anti-microbial peptide, such as pediocin and nisin to treat diseases such as Netherton Syndrome, atopic dermatitis, psoriasis, acne rosacea, and chronic wound infection.

17. The composition of claim 10, wherein the bioactive agent is an antibody, antibody fragment or nanobody, such as single chain variable fragment anti-tumor necrosis factor alpha to treat diseases such as Netherton Syndrome, atopic dermatitis, psoriasis, acne rosacea, and chronic wound infection.

18. A method of using composition of claim 6, comprising applying the formulation to the skin of a person in need thereof under conditions wherein the bacteria produce an effective amount of the agent on the skin over a predetermined time range.

19. A method of claim 18, wherein the time range is 4, 8, 12, 24, and 48 hours.

20. A method of claim 18, further comprising adjusting the concentration of the nutrient source to adjust bioproduction and colonization time, wherein the concentration of glucose and glycerol is varied from 2 to 20% of the moisturizer for increasingly longer colonization times.

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