An engineered cutaneous commensal

Abstract

Mutant strains of Corynebacterium glutamicum were formulated into commensal bacteria for human skin.

Description

[0001] introduction

[0002] Genetically engineered probiotics that produce therapeutics in situ on the skin surface are valuable for treating a variety of diseases and also have applications in cosmetics. Previously, we used Corynebacterium glutamicum—a soil bacterium and a recognized safe (GRAS) organism—as a probiotic for topical application to the skin (B21-024-2WO, PCT / US22 / 19051; WO2022 / 187724). We disclosed that when Corynebacterium glutamicum is applied to the skin together with a microenvironment-altering and nutrient-providing moisturizer, it can colonize the skin for more than 8 hours and produce glutamate on the skin surface. This is a novel and non-obvious concept because soil microorganisms cannot normally grow on the skin surface. Invention Overview

[0004] We have disclosed a genetic engineering technique that enables Corynebacterium glutamicum to acquire optimized skin symbiotic characteristics, namely the ability to grow under highly acidic and oily conditions. This is achieved by applying a similarly acidic moisturizer to prevent the colonization of other harmful bacteria on the skin, increasing oil tolerance to result in more stable colonies, and providing a wider range of treatment options on the skin surface.

[0005] We cultured hundreds of generations of Corynebacterium glutamicum under highly acidic and oily conditions (pH approximately 5.5 and Tween 80 approximately 1%), which we considered “skin-mimicking” conditions, and developed evolved strains capable of rapid growth under these conditions, while wild-type strains could not. Whole-genome sequencing of the strains allowed us to identify mutations and amplifications that contributed to improved acid and oil tolerance.

[0006] We found that one of these strains grew at a rate of 0.3 under “skin-mimicking” conditions, which was 60% of the growth rate in the optimal enrichment medium (0.5), while the wild-type strain did not exhibit exponential growth under “skin-mimicking” conditions.

[0007] This invention provides a mutant strain of Corynebacterium glutamicum, originally a soil microorganism, optimized for skin colonization. The strain can be further genetically engineered, as disclosed in WO2022 / 187724, to produce therapeutic or cosmetic substances, for both medical and cosmetic commercial applications.

[0008] In several aspects and in several embodiments, the present invention provides:

[0009] 1. A composition comprising a live colony of an engineered mutant Corynebacterium glutamicum strain, said strain being engineered to obtain optimized skin symbiotic characteristics, namely the ability to grow under acidic and oily conditions such as pH 3.5-6.5, for example, pH about 5.5, and 0.2-5% Tween 80, for example, about 1% Tween 80.

[0010] 2. The composition of claim 1, wherein the mutant bacterial strain achieves enhanced growth under acidic and oily conditions compared to the corresponding wild-type strain.

[0011] 3. The composition of claim 1, wherein the mutant bacterial strain is engineered to contain a plurality of human skin symbiotic mutations or amplifications or Tables 1, 2 or 3.

[0012] 4. The composition of claim 1, wherein the mutant bacterial strain is engineered to comprise one, two, three, four, five, six, seven, eight, or nine of the following genes: lpps, cgp_3312, pmt, cgp_0753, atpB, atpE, cglR3, cgp3052, and cgp_r5123, and / or amplifications of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or all of the following genes: cgp_1683, tatC, tatX, cgp_1686, cgp_1687, pafA2, pup, pafA1, mpa, pimT, pepC, cgp_1694, and cgp_1695.

[0013] 5. The composition of claim 1, wherein the mutant bacterial strain is engineered to include mutations and amplifications of Cgm-1A-1G or Cgm-2A-2E.

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

[0015] 7. The composition of claim 6, which is applied to human skin.

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

[0017] 9. The composition of claim 6, further comprising a humectant such as glycerin, hyaluronic acid and propylene glycol, or a skin moisturizer such as shea butter, cocoa butter and octyldodecanol, or an octyldodecanol such as petrolatum, cetyl alcohol (hexadecane-1-ol) and lanolin.

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

[0019] 11. The composition of claim 10, wherein the bioactive agent is selected from cosmetic ingredients, anti-aging agents, antioxidants, sunscreens, anti-inflammatory agents, antimicrobial agents, analgesics, and therapeutic compounds.

[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, threonine, isoleucine, aspartic acid, valine, citrulline, GHK-Cu, GSH-Cu, manganese tripeptide, Peptamide-6, carnosine, N-acetylcarnosine, tripeptide-10-citrulline, palmitoyl tripeptide, palmitoyl tetrapeptide, palmitoyl pentapeptide, acetyl tetrapeptide, hexapeptide-11, tetrapeptide PKEK, hexapeptide-14, silk protein, aquaporin, α-interferon, Hsp70, transforming growth factor, 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, skin-derived antileukocyte protease (Elafin), and secretory leukocyte protease inhibitor, for the treatment of Natherton syndrome, atopic dermatitis, psoriasis, and rosacea.

[0023] 15. The composition of claim 10, wherein the bioactive agent is an anti-inflammatory compound, such as IL-10, for treating conditions such as Natherton syndrome, atopic dermatitis, psoriasis, rosacea, and chronic wound infections.

[0024] 16. The composition of claim 10, wherein the bioactive agent is an antimicrobial peptide, such as peicosin and nisin, for treating diseases such as Natherton syndrome, atopic dermatitis, psoriasis, rosacea, and chronic wound infections.

[0025] 17. The composition of claim 10, wherein the bioactive agent is an antibody, antibody fragment, or nanobody, such as a single-chain variable fragment of anti-tumor necrosis factor α, for treating diseases such as Natherton syndrome, atopic dermatitis, psoriasis, rosacea, and chronic wound infections.

[0026] 18. A method of using the composition of claim 6, comprising applying the formulation to the skin of a person in need, provided that the bacteria produce an effective amount of the active agent on the skin within a predetermined time range.

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

[0028] 20. The method of claim 18, further comprising adjusting the concentration of the nutrient source to regulate biological production and colonization time, wherein the concentrations of glucose and glycerol are varied from 2% to 20% of the humectant to gradually extend the colonization time.

[0029] This invention covers all combinations of the specific embodiments described herein, as each combination has been described in detail. Brief description of the attached diagram

[0031] Figure 1 Engineered Corynebacterium glutamicum: Growth in a skin-mimicking medium. Growth difference between the original strain and one of the engineered strains. The original strain did not grow at all, while the engineered strain had a growth rate of 0.3. The growth rate in the optimal enrichment medium was 0.5; therefore, the growth rate of the engineered strain in the high-acid, high-oil environment of the skin-mimicking medium (the growth strain) was 60% of its growth rate in the optimal enrichment medium.

[0032] Figure 2 Laboratory evolution of Corynebacterium glutamicum's adaptation to "skin mimics" (BHI, pH 5.5 and 1% Tween 80).

[0033] Figure 3A -D: Evaluation of AL strains under different culture conditions; growth rate is calculated as the slope of LN of OD600 value during the growth phase. AL3, AL4, and AL6 exhibited the best growth rates under all different conditions. A. Overnight in BHI, overnight in skin-BHI, measured in test tubes (OD ~ .1); B. Overnight in BHI, exponential growth in BHI, measured in test tubes (OD ~ .1); C. Overnight in BHI, overnight in skin-BHI, measured in wells (OD ~ .1); D. Overnight in BHI, exponential growth in BHI, measured in wells (OD ~ .1).

[0034] Description of specific embodiments of the invention

[0035] Unless otherwise stated or prohibited, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, and the term “or” means and / or. The embodiments and implementations described herein are for illustrative purposes only and are intended to inform those skilled in the art of various modifications or variations thereof, which should be included within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein, including their citations, are hereby incorporated in their entirety for all purposes.

[0036] Example. Safety and efficacy of applying a probiotic moisturizer containing Corynebacterium glutamicum, a soil bacterium that produces glutamate, to the skin.

[0037] We used common moisturizing ingredients (paraffin oil, water, Span 60, dimethicone, and stearic acid) with components needed for the growth of Corynebacterium glutamicum (glucose, glycerin, yeast extract, and biotin), allowing Corynebacterium glutamicum to produce nutrients on the skin surface. The moisturizer was then mixed with 10... 10 Corynebacterium glutamicum resuspended at a concentration of CFU / mL in a 1:1 mixture. Human tests were performed to determine skin colonization of the soil bacteria and glutamate production, an amino acid secreted and produced by Corynebacterium glutamicum. We used a moisturizer-only control to determine local microbiome conditions. Skin swabs were used at 0, 2, 4, 8, and 24 hours, and measurements were taken on 16 cm of skin. 2 Colony-forming units were observed in the samples. We determined that *Corynebacterium glutamicum* stably colonized the skin surface for 8 hours, then reverted to control conditions after 24 hours. We also found that *Corynebacterium glutamicum* production continuously increased within 8 hours, then reverted to control conditions after 24 hours. To highlight the continuous production capacity of *Corynebacterium glutamicum*, a topical glutamate preparation of 0.1% glutamate was added to the skin; it was undetectable after 15 minutes, likely due to absorption and degradation on the skin surface, demonstrating the utility of compounds continuously produced by the bacteria on the surface. Our safety data from human trials also indicate that application of this organism to human skin is non-irritating and non-sensitizing, confirming its safety and effectiveness.

[0038] The acidity and sebum of the skin environment provide a defense mechanism against microbial colonization. Adult skin pH measurements show that women's skin pH (average pH = 5.54) is slightly lower than men's (average pH = 5.80). 8 Therefore, we attempted to engineer our strain to grow in a highly acidic (pH 5.5) “skin-mimicking” medium containing sebum analogs (1% Tween 80).

[0039] We conducted a 45-day adaptive laboratory evolution study on seven clonal isolates of *Corynebacterium glutamicum*, targeting thousands of generations with progressively decreasing pH and increasing sebum analogs. The genomes of the resulting *Corynebacterium glutamicum* strains that exhibited robust growth under simulated skin conditions were then sequenced. Our results show that wild-type *Corynebacterium glutamicum* cannot grow under these conditions, while *S. epidermidis*—a symbiotic skin bacterium—exhibited a growth rate of 0.29. Notably, our evolved *Corynebacterium glutamicum* strain (RVB-002) demonstrated a growth rate of 0.47, exceeding *S. epidermidis* by 62%. This successful evolution of *Corynebacterium glutamicum* demonstrates its potential for transiently colonizing the skin environment to generate targeted therapeutic delivery.

[0040] We tested our evolved strains under various conditions. For all of our strains, we tested them under four conditions. Figure 3A In our study, we grew our strains under skin-simulated conditions, then passaged them under these conditions, and finally grew them to an OD of 1.1. We then measured the growth rate in test tubes over 18 hours in a culture medium simulating skin. We found that some of our evolved strains even grew better than the most common skin microorganism, *Staphylococcus epidermidis*. Figure 3C We conducted the same experiment in [previous context], but this time on microplates. Figure 3B In this study, we grew our strains under skin-simulated conditions, allowing them to reach the exponential phase in enriched BHI, then diluted the strains to OD0.1, and measured their growth rate in skin-simulated medium over 12 hours in test tubes. Figure 3D In our study, we performed the same experiments, but in microplates. AL3, AL4, and AL6 exhibited the highest growth rates across all experiments. These strains all contained amplifications of the following genes: cgp_1683, tatC, tatX, cgp_1686, cgp_1687, pafA2, pup, pafA1, mpa, pimT, pepC, cgp_1694, and cgp_1695, indicating that these genes contribute to the formation of this phenotype.

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

[0042] Cgm-1A

[0043] SNPcgp_0001P420R (CCA > CGA)

[0044] INScgp_0790, pyc gene intergenicity (+217 / -180)

[0045] INScgp_0790, pyc gene intergenicity (+223 / -174)

[0046] INSlppS encoding (414 / 1212 nt)

[0047] INSlppS encoding (410 / 1212 nt)

[0048] Genetic amplification: sequence changes / genes

[0049] 50,800 bp x 2

[0050] cgp_1663,cgp_1664,cgp_1665,cgp_1668,cgp_1669,cgp_1670,cgp_1671,ppmC,ppmN,cgp_1675,lip3,cgp_ 1677,cobL,cgp_4020,cgp_1679,cgp_1680,pepE,cgp_1682,cgp_1683,tatC,tatX,cgp_1686,cgp_1687,pafA 2,pup,pafA1,mpa,pimT,pepC,cgp_1694,cgp_1695,cgp_1696,cgp_1697,hisG,hisE,cgp_1700,metH,cgp_1 702,cgp_1703,cgp_1704,arsC2,cgp_1706,cgp_1707,cgp_1708,mshC,bacA,cgp_1711,lppL,pyrD,cgp_1714

[0051] Cgm-1B

[0052] SNPmagG94S (GGT > AGT)

[0053] Between SNP cgp_1121 and cgp_1122 genes (+341 / +109)

[0054] SNPcgp_1275E97K (GAA > AAA)

[0055] SNPtatXW6S (TGG > TCG)

[0056] SNPclpC, guaB1 gene inter-(-8 / -281)

[0057] SNPcgp_0811T219A (ACC > GCC)

[0058] SNPmgtE1A176T (GCC > ACC)

[0059] SNPlppSE230* (GAA > TAA)

[0060] SNPcgp_2756T139R (ACA > AGA)

[0061] Cgm-1C

[0062] SNPcgp_0811T219A (ACC > GCC)

[0063] SNPmgtE1A176T (GCC > ACC)

[0064] SNPlppSE230* (GAA > TAA)

[0065] SNPcgp_2756T139R (ACA > AGA)

[0066] Genetic amplification: sequence changes / genes

[0067] 83,700 bp x

[0068] cgp_1663, cgp_1664, cgp_1665, cgp_1668, cgp_1669, cgp_1670, cgp_1671, ppmC, ppmN, cgp_1675, lip3, cgp_1677, cobL, cgp_4020, cgp_1679, cgp_1680, pepE, cgp_1682, cgp_1683, tatC, tatX, cgp_1686, cgp_1687, pafA2, pup, pafA1, 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, bacA, cgp_1711, lppL, pyrD, cgp_1714, cgp_1715, cgp_1716, cgp_1717, cgp_1718, tetB, tetA, cgp_t5033, cgp_t5034, cgp_1722, cgp_1724, mcmB, mcmA, cgp_1727, cgp_1728, cgp_1730, cgp_1731, cgp_1732, cgp_1733, hemH, cgp_1735, cgp_1736, acn, acnR, cgp_1739, cgp_1740, cgp_1741, cgp_1742, cgp_1743, pacL

[0069] Cgm-1D

[0070] SNP cgp_0569 T281P (ACC > CCC)

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

[0072] SNP atpA P291L (CCA > CTA)

[0073] SNP cgp_2535 A136A (GCC > GCT)

[0074] DEL cgp_3312 coding (307 / 1461 nt)

[0075] Genetic Amplications: Sequence Changes / Genes

[0076] 50,800 bp x

[0077] 3cgp_1663,cgp_1664,cgp_1665,cgp_1668,cgp_1669,cgp_1670,cgp_1671,ppmC,ppmN,cgp_1675,lip3,cgp _1677,cobL,cgp_4020,cgp_1679,cgp_1680,pepE,cgp_1682,cgp_1683,tatC,tatX,cgp_1686,cgp_1687,paf A2,pup,pafA1,mpa,pimT,pepC,cgp_1694,cgp_1695,cgp_1696,cgp_1697,hisG,hisE,cgp_1700,metH,cgp_1 702,cgp_1703,cgp_1704,arsC2,cgp_1706,cgp_1707,cgp_1708,mshC,bacA,cgp_1711,lppL,pyrD,cgp_1714

[0078] Cgm-1E

[0079] DELpmt encoding (350 / 1563 nt)

[0080] SNPatpDD322Y (GAT > TAT)

[0081] SNPcgtR3D187N (GAT > AAT)

[0082] Cgm-1F

[0083] SNPcgp_0001D509H (GAT > CAT)

[0084] SNPcmt5E188Q (GAA > CAA)

[0085] DELlppS encoding (1146 / 1212 nt)

[0086] The difference between SNPglpQ1 and gntP genes was (-139 / -283).

[0087] SNPcgp_3348, nagL gene inter-gene (-284 / +254)

[0088] Genetic amplification: sequence changes / genes

[0089] 12,600 bp

[0090] Cgm-1G

[0091] INScgp_1159 encoding (1764 / 1905 nt)

[0092] INScgp_1159 encoding (1757 / 1905 nt)

[0093] Genetic amplification: sequence changes / genes

[0094] 14,100 bp

[0095] Cgm-2A

[0096] Between SNPatpB and atpE genes (+99 / -35)

[0097] SNPcgp_0666T192A (ACT→GCT)

[0098] SNPcgp_0753G159E (GGA→GAA)

[0099] SUBcgp_6007, psp2 gene inter-gene (+337 / -311)

[0100] DELpmt encoding (350 / 1563 nt)

[0101] SNPrpoCC441Y (TGT→TAT)

[0102] INSsufR, cgp_1766 gene intergenicity (-88 / -234)

[0103] Cgm-2B

[0104] SNPatpGG314R (GGC→CGC)

[0105] INScgp_0223, tnp2f gene intergenicity (-658 / -119)

[0106] DELcgp_0753 encoding (178-201 / 966 nt)

[0107] DELcgp_1016a encoding (334 / 360 nt)

[0108] SNPcgp_1103M82I (ATG→ATC)

[0109] Between the DELcgp_1467 and cgp_1468 genes (-65 / +19)

[0110] DELcgp_2380 encoding (270 / 390 nt)

[0111] Between DELmetY and cstA genes (-354 / -66)

[0112] INSPMT code (350 / 1563 nt)

[0113] INSPMT encoding (356 / 1563 nt)

[0114] Genetic amplification: sequence changes / genes

[0115] 57,900 bp x

[0116] 4cgp_1680,pepE,cgp_1682,cgp_1683,tatC,tatX,cgp_1686,cgp_1687,pafA2,pup,pafA1,mpa,pimT,pepC,cgp_16 94,cgp_1695,cgp_1696,cgp_1697,hisG,hisE,cgp_1700,metH,cgp_1702,cgp_1703,cgp_1704,arsC2,cgp_1706,c gp_1707,cgp_1708,mshC,bacA,cgp_1711,lppL,pyrD,cgp_1714,cgp_1715,cgp_1716,cgp_1717,cgp_1718,tetB,t etA,cgp_t5033,cgp_t5034,cgp_1722,cgp_1724,mcmB,mcmA,cgp_1727,cgp_1728,cgp_1730,cgp_1731,cgp_1732, cgp_1733,hemH

[0117] Cgm-2C

[0118] SNPcgp_0811T219A (ACC→GCC)

[0119] SNPcgp_1288W105* (TGG→TAG)

[0120] SNPcgp_2756T139R (ACA→AGA)

[0121] SNPcgp_3084E221E (GAG→GAA)

[0122] SNPdtxRP127L (CCT→CTT)

[0123] SNPlppSE230* (GAA→TAA)

[0124] Cgm-2D

[0125] SNPatpAP292R (CCG→CGG)

[0126] Between SNP cgp_2810 and cgp_2811 genes (-139 / +196)

[0127] DELcgp_2854Δ1,639 bp

[0128] SUBcgp_6007, psp2 gene inter-gene (+337 / -311)

[0129] SNPcgtR3D187N (GAT→AAT)

[0130] INSmgtE1 encoding (742 / 1293 nt)

[0131] INSPMT code (350 / 1563 nt)

[0132] Genetic amplification: sequence changes / genes

[0133] 12,500 bp

[0134] Cgm-2E

[0135] INScgp_0223, tnp2f gene intergenicity (-658 / -119)

[0136] DELcgp_1016a encoding (334 / 360 nt)

[0137] Between the DELcgp_1467 and cgp_1468 genes (-65 / +19)

[0138] DELcgp_2380 encoding (270 / 390 nt)

[0139] SNPcgp_2737P120Q (CCG→CAG)

[0140] DELcgp_2854Δ1,639 bp

[0141] SUBcgp_6007, psp2 gene inter-gene (+337 / -311)

[0142] SNPcgtR3N188K (AAC→AAG)

[0143] Between DELmetY and cstA genes (-354 / -66)

[0144] INSPMT code (350 / 1563 nt)

[0145] INSPMT encoding (356 / 1563 nt)

[0146] SNPrpsAN219D (AAC→GAC)

[0147] SNPrpsHG97G (GGC→GGT)

[0148] Table 2

[0149] Table 2 Mutant RES167

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

Claims

1. A composition comprising a live colony of an engineered mutant Corynebacterium glutamicum strain, said strain being engineered to obtain optimized skin symbiotic characteristics, namely the ability to grow under acidic and oily conditions such as pH 3.5-6.5, for example, about pH 5.5, and 0.2-5% Tween 80, for example, about 1% Tween 80.

2. The composition of claim 1, wherein the mutant bacterial strain achieves enhanced growth under acidic and oily conditions compared to the corresponding wild-type strain.

3. The composition of claim 1, wherein the mutant bacterial strain is engineered to contain a plurality of human skin symbiotic mutations or amplifications or Tables 1, 2 or 3.

4. The composition of claim 1, wherein the mutant bacterial strain is engineered to comprise one, two, three, four, five, six, seven, eight, or nine of the following genes: lpps, cgp_3312, pmt, cgp_0753, atpB, atpE, cglR3, cgp3052, and cgp_r5123, and / or amplifications of one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve of the following genes: cgp_1683, tatC, tatX, cgp_1686, cgp_1687, pafA2, pup, pafA1, mpa, pimT, pepC, cgp_1694, and cgp_1695.

5. The composition of claim 1, wherein the mutant bacterial strain is engineered to include mutations and amplifications of Cgm-1A-1G or Cgm-2A-2E.

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

7. The composition of claim 6, which is applied to human skin.

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 a skin moisturizer such as shea butter, cocoa butter and octyldodecanol, or an octyldodecanol such as petrolatum, cetyl alcohol (hexadecane-1-ol) and lanolin.

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

11. The composition of claim 10, wherein the bioactive agent is selected from cosmetic ingredients, anti-aging agents, antioxidants, sunscreens, anti-inflammatory agents, antimicrobial agents, analgesics, and therapeutic compounds.

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, threonine, isoleucine, aspartic acid, valine, citrulline, GHK-Cu, GSH-Cu, manganese tripeptide, Peptamide-6, carnosine, N-acetylcarnosine, tripeptide-10-citrulline, palmitoyl tripeptide, palmitoyl tetrapeptide, palmitoyl pentapeptide, acetyl tetrapeptide, hexapeptide-11, tetrapeptide PKEK, hexapeptide-14, silk protein, aquaporin, α-interferon, Hsp70, transforming growth factor, 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, skin-derived antileukocyte protease (Elafin), and secretory leukocyte protease inhibitor, for the treatment of Natherton syndrome, atopic dermatitis, psoriasis, and rosacea.

15. The composition of claim 10, wherein the bioactive agent is an anti-inflammatory compound, such as IL-10, for treating conditions such as Natherton syndrome, atopic dermatitis, psoriasis, rosacea, and chronic wound infections.

16. The composition of claim 10, wherein the bioactive agent is an antimicrobial peptide, such as peicosin and nisin, for treating diseases such as Natherton syndrome, atopic dermatitis, psoriasis, rosacea, and chronic wound infections.

17. The composition of claim 10, wherein the bioactive agent is an antibody, antibody fragment, or nanobody, such as a single-chain variable fragment of anti-tumor necrosis factor α, for treating diseases such as Natherton syndrome, atopic dermatitis, psoriasis, rosacea, and chronic wound infections.

18. A method of using the composition of claim 6, comprising applying the formulation to the skin of a person in need, provided that the bacteria produce an effective amount of the active agent on the skin within a predetermined time range.

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

20. The method of claim 18, further comprising adjusting the concentration of the nutrient source to regulate biological production and colonization time, wherein the concentrations of glucose and glycerol are varied from 2% to 20% of the humectant for gradually extending the colonization time.

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