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ABSTRACT The human microbiome has emerged as a key player in maintaining skin health, and dysbiosis has been linked to various skin disorders. Amidst growing concerns regarding the side

effects of antibiotic treatments, the potential of live biotherapeutic products (LBPs) in restoring a healthy microbiome has garnered significant attention. This review aims to evaluate the

current state of the art of the genetically or metabolically engineered LBPs, termed single-cell engineered LBPs (eLBPs), for skin repair and disease treatment. While some studies

demonstrate promising outcomes, the translation of eLBPs into clinical applications remains a significant hurdle. Substantial concerns arise regarding the practical implementation and

scalability of eLBPs, despite the evident potential they hold in targeting specific cells and delivering therapeutic agents. This review underscores the need for further research, robust

clinical trials, and the exploration of current advances in eLBP-based bioengineered bacterial chassis and new outlooks to substantiate the viability and effectiveness of eLBPs as a

transformative approach in skin repair and disease intervention. SIMILAR CONTENT BEING VIEWED BY OTHERS CELLULAR HUMAN TISSUE-ENGINEERED SKIN SUBSTITUTES INVESTIGATED FOR DEEP AND DIFFICULT

TO HEAL INJURIES Article Open access 17 June 2021 UTILIZATION OF EX VIVO TISSUE MODEL TO STUDY SKIN REGENERATION FOLLOWING MICRONEEDLE STIMULI Article Open access 27 October 2022 COMPARATIVE

ASSESSMENT OF COMMERCIALLY AVAILABLE WOUND GELS IN EX VIVO HUMAN SKIN REVEALS MAJOR DIFFERENCES IN IMMUNE RESPONSE-MODULATORY EFFECTS Article Open access 19 October 2022 INTRODUCTION The

human microbiome is a highly complex system that plays a crucial role in maintaining human health such as facilitating digestion, regulating the immune system, and synthesizing essential

vitamins and nutrients1,2. Dysbiosis, an imbalance in the microbiome in many body parts, has been linked to various pathological conditions, including skin disorders such as acne vulgaris,

psoriasis, and atopic dermatitis (AD)3,4,5. Antibiotic treatments for skin conditions have resulted in detrimental side effects such as antibiotic resistance, quorum cheater development,

commensals depletion, and recurrent infections6,7,8. Live biotherapeutic products (LBPs) offer a potential solution by reestablishing the equilibrium of a healthy microbiome and enhancing

overall health, including the skin9,10,11,12,13,14. Single-cell engineered LBPs (eLBPs) are genetically and/or metabolically engineered microorganisms that provide targeted therapeutics

directly at the disease site9. The microbial engineering strategy primarily uses bacteria as synthetic biology and bioengineered chassis has shown promise in treating a range of skin

conditions, including wound healing, skin regeneration, and cancer treatment15,16. Despite increasing interest in microbiome engineering, previous reviews have mostly focused on microbial

living materials, and only a few have considered the use of whole intact cells2,9,17,18,19. A wide array of LBPs and probiotics for the skin have also been thoroughly reviewed, but the

majority barely discussed eLBPs concerning skin therapy. The present review focuses on the current strategies and advancements in the development of eLBP-based treatments for skin repair and

disease through evidence obtained from clinical and preclinical testing over the past 10 years (2014-2023), providing a comprehensive overview of the current state of the art of eLBP

research for skin therapy and its future outlook. SKIN MICROBIOME The human skin is a complex system that comprises a rich variety of microorganisms known as the skin microbiome. In general,

skin microenvironments which comprised moist, dry, and sebaceous sites harbor distinct microbial ecosystems, yet exhibit similarities in terms of the species present. Sebaceous sites are

primarily dominated by _Cutibacterium_ and _Staphylococci_, while moist sites are predominantly inhabited by _Corynebacterium_ and _Staphylococcus_ species20. Dry skin areas contain

comparatively lower numbers of bacteria, but exhibit a more diverse composition, including a wide array of Proteobacteria, in addition to skin commensal species21. The resilience of the

deeper layers of the core skin microbiome is influenced by genetics, diet, and personal hygiene routines. Despite exposure to cleaning and cosmetic products, the surface microbial community

remains relatively stable22,23. Applying lotions on dry skin improves hydration and skin components but does not significantly change the composition of commensal microorganisms such as _C.

acnes_, _S. epidermidis_, and _S. hominis_24. Dysbiosis of the skin microbiome is topographically specific and is commonly associated with pathological conditions or diseases as shown in

Supplementary Table 13,25,26,27. This review will highlight instances where natural LBPs have been utilized as skin disease intervention and subsequently introduce the concept of eLBPs and

discuss recent preclinical and clinical trials involving eLBPs. Considering the current advancement of synthetic biology and biological engineering especially in bacterial strain

modification, this review delves deeper into the recent development of non-commensal bioengineered bacteria towards expanding the repertoire of bacterial chassis tailored for desired

biological and biotherapeutic applications. SKIN DISEASE INTERVENTION USING LIVE BIOTHERAPEUTIC PRODUCTS One prevalent strategy to modulate the composition and/or function of the human

microbiome (Fig. 1) involves the use of probiotics28,29,30. There is a growing body of evidence to suggest that probiotics can effectively impact the composition and metabolic activities of

the human microbiome including the skin12,13,14,31. Conventional oral probiotic treatments have been proven to significantly reduce the severity of AD, particularly in adults, by promoting

immune response and gut impermeability32. Yet, evolution in current probiotics revealed that topically applied probiotics are equally effective in improving anti-oxidation and lowering

inflammation, apart from regulating other age-related conditions through the equilibration of commensal microbes and alteration of functional metabolisms33,34. Beneficial skin commensals

have a prospective biotherapeutic role, particularly for the repair and differentiation of the epidermal barrier35. For instance, certain strains of commensal _S. epidermidis_ can express

serine protease glutamyl endopeptidase and β-defensins, as well as activate Gamma delta T cells and upregulate Perforin-2, all of which inhibit the formation of pathogenic _S. aureus_

biofilms in AD36,37,38,39, and induce interleukin (IL)-8 and neutrophils to combat inflammation in acne40,41. Additionally, scientists discovered that _S. epidermidis_ (strain MO34 and

MO38), which produces 6-N-hydroxyaminopurine (6-HAP), a molecule that hinders DNA polymerase activity, holds promise for providing defense against neoplasia. This breakthrough has unveiled a

fresh perspective on the role of skin commensals in host protection against cancer42. In another case, the clinical trial reports of AD patients treated with the commensal _Roseomonas

mucosa_ showed better skin epithelial barrier function and decreased _S. aureus_ load due to glycerophospholipids synthesis, which activated tissue-repair pathways43,44. Skin commensals

offer the potential for a more robust skin microbiome engineering, such as skin microbiome transplantation (SMT) to treat dysbiosis. SMT involves transplanting healthy skin microbiomes to

the dysbiotic area45. A proof-of-concept study showed that unidirectional SMT, which transferred DNA markers partially from the forearm to the back of the same individual, was feasible46. As

a result, SMT is being proposed as a solution to address underarm odour by replacing odour-causing commensals with new commensals obtained from a non-odorous donour47. Notably, ongoing

research on SMT remains in its early phases, and there is yet to be an established, standardized procedure for its implementation. Contrary to SMT, fecal microbiome transplantation (FMT)

stands as a well-established procedure and has shown promise in treating skin diseases, particularly through its influence on the gut-skin axis48,49,50. A recent clinical efficacy report of

FMT on AD patients revealed a significant reduction in the average Scoring Atopic Dermatitis Score (SCORAD) from baseline, with a remarkable 75% decrease observed after just four

treatments50. Besides single-cell bacteriotherapy and microbiome transplantation, there is growing evidence that specific groups of microorganisms, when isolated and enriched, can manipulate

the physiological functions of the host. For instance, an 11-member commensal consortium extracted from the fecal matter of healthy donors has been shown to stimulate the production of

interferon-γ-producing CD8 T cells in the intestine which completely ablate and inhibit the metastasis of adenocarcinoma and melanoma cells51. Another _Lactobacilli_ consortium showed

promise in reducing inflammatory lesions by reducing the abundance of _Staphylococci_ and _C. acnes_ in a placebo-controlled study34. While microbial consortia may have greater impacts in

manipulating host physiological functions, single-cell LBPs are relatively simpler to monitor and exploit, therefore are more practical for treating cutaneous diseases. Examples of LBPs

formulated as oral and topical applications to treat skin conditions and diseases are presented in Supplementary Table 2. Single-cell eLBPs are a cutting-edge field of research that utilizes

genetically or metabolically engineered live microorganisms to perform specific functions, such as producing therapeutic compounds or targeting specific pathogen9. eLBPs have the potential

to revolutionize the way we approach skin repair and disease treatment, as they can be tailored to target specific sites, cells and pathways in the body17,18. There is limited information

available regarding the use of eLBPs for skin treatment, and most studies have only tested their efficacy using in vitro or in vivo models (Table 1). This review primarily focuses on two

leading areas of eLBP research: 1) cutaneous wound treatments, and 2) malignant melanoma therapeutics (Fig. 2). CUTANEOUS WOUND TREATMENTS In wound healing, the skin undergoes overlapping

phases of hemostasis, inflammation, proliferation, and remodeling. In the inflammation stage, immune cells gather at the site of injury due to distress signals, cytokines, and chemokines

produced by damaged cells15. Chemokines like CXCL12, have been shown to have beneficial effects in healing cutaneous wounds and bind to CXCR4 receptors on immune cells and keratinocytes. To

translate this theoretical framework, Ilya Pharma developed a first-in-class drug candidate, which is a recombinant CXCL12-expressing _Lactobacillus reuteri_ R2LC (ILP100)15,52. Experiments

on mice and minipigs in both healthy and hyperglycemic conditions showed faster wound healing due to increased CXCL12 availability and increased TGF-β expression in macrophages, therefore

hastening the formation of granulation tissue and thin epithelial layers. In the Phase 1 clinical trial, ILP100 shortened the time to initial healing among patients by an average of 6 days

and by 10 days at the highest dose53. It additionally elevated the density of CXCL12+ cells within the wounds and enhanced local blood perfusion at the wound site. The ILP100 has progressed

to its Phase 2 clinical trial (Identifier: NCT05608187) and is actively enrolling patients with diabetic foot ulcers. This trial spans 26 weeks and includes a long-term follow-up period of 5

years to assess both the safety and biological efficacy of ILP100 in promoting wound healing among subjects. On the contrary, Zhao et al.54 bolstered wound healing mechanisms by applying

CXCL12-expressing _Lactococcus lactis_ in tandem with yellow light-emitting diodes (LEDs). Prior research has demonstrated that LED light of varying wavelengths exerts distinct effects on

skin repair and regeneration. Specifically, yellow light within the range of 570–600 nm can stimulate collagen synthesis, resulting in skin tightening55. This synergy expedited wound

closure, facilitated tissue remodeling, spurred re-epithelialization and hair follicle regeneration, and mitigated over-inflammation. Additionally, it upregulated pivotal proteins within the

Wnt and Notch signaling pathways as well as curtailed inflammatory factors such as interleukin 1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α). Remarkably, this combined treatment

effectively reduced skin pathogens _Ralstonia_ and _Acinetobacter_, substantially diminishing the risk of infection. In another instance, Li et al.56 devised an inventive approach by

combining CXCL12-expressing _L. lactis_ with a photosynthetic bacteria, _Synechococcus elongatus_ PCC7942, enclosed within a hydrogel matrix. The engineered _L. lactis_ feeds on sucrose

produced by _S. elongatus_ through photosynthesis, creating a synergistic effect that substantially accelerates the wound-healing process. Impressively, this topical hydrogel-encapsulated

microbial consortium led to faster wound closure in mice, reducing the wound area ratio to a mere 13.2% by the 14th day, as compared to control treatments. In addition to inducing

chemokines, another strategy employed to expedite wound healing involves augmenting the presence of growth factors, specifically vascular endothelial growth factor (VEGF). This biomolecule

is essential in the process of angiogenesis57. However, applying VEGF directly has not yielded conclusive benefits in clinical trials58. Lu et al.31 found that thermosensitive hydrogel

containing probiotic _L. lactis_ NZ9000 was able to improve the microenvironment of diabetic wounds and promote wound healing by regulating lactic acid levels. They later developed an

engineered _L. lactis_ carrying a VEGF-encoding gene and embedded it in a heparin-poloxamer hydrogel59. This approach enhanced the stability of VEGF in the oxidative environment of chronic

wounds and enabled the living system to produce and protect VEGF. As a result, it promoted the growth and movement of endothelial cells, and shifted M1 and M2 macrophages toward an

anti-inflammatory phenotype, leading to successful angiogenesis in diabetic wounds59. A prolonged or impaired wound healing may be exacerbated by the biofilm-forming pathogens infection like

_S. aureus_, which can be challenging to treat due to their reduced susceptibility to both the immune system and topical antimicrobial agents60,61. To address this issue, previous research

has explored magnetic hyperthermia as a potential therapeutic modality. Magnetic hyperthermia employed magnetic nanoparticles that absorbed energy from an alternating magnetic field, leading

to highly localized heat transmission that inactivated _S. aureus_ within cutaneous abscesses in murine models62. Building upon this innovative approach, Chen et al.63 modified a bacterial

system comprising the magneto-ovoid strain MO-1 (closely related to _Magnetococcus_ species) which contained magnetosomes and was coated with a polyclonal antibody. In a murine model

experiment, this system significantly improved wound healing by promoting the formation of MO-1-_S. aureus_ aggregates and eradicates the pathogen by hyperthermia63. MALIGNANT MELANOMA

THERAPEUTICS Melanoma is a type of skin cancer that is known for its complexity, aggressive nature, high metastasis rate, and frequent relapses. A commonly used method to treat melanoma is

phototreatment, where near-infrared (NIR) light and a photosensitizer (PTS) interact to destroy tumor spheroids64. However, due to the restricted penetration of NIR light and the low

specificity of PTS, melanoma tumors located deep within the skin and phototreatment margins often lead to quick relapse and metastasis. Therefore, Peng et al.16 developed a system of

transgenic _Escherichia coli_ to deliver recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene. The nanosized outer membrane vesicles (OMVs) produced by the

engineered _E. coli_ which have been modified with αvβ3 integrin targeting ligand and indocyanine green (ICG) were able to penetrate the stratum corneum and specifically target melanoma

cells. When exposed to NIR irritation, these engineered OMVs exhibited photothermal and photodynamic responses against primary melanoma spheroids and activated TRAIL-induced apoptosis in

disseminated tumor cells. This resulted in the complete eradication of melanoma16. A different approach utilized to ablate melanoma involved the metabolic engineering of anaerobic oncolytic

bacteria, specifically _Clostridium butyricum_65. A study conducted nearly five decades ago involving _C. butyricum_ revealed its promising oncolytic activity against carcinomas,

particularly when used in conjunction with radio-frequency therapy to briefly raise tumor temperatures to the range of 42 to 44 °C66. Since then, numerous related studies have been carried

out, with recent investigations utilizing a metabolic labeling substrate to enhance its mechanistic action. This substrate was developed by coupling the metabolic substrate of _C.

butyricum_, D-alanine, with a photosensitizer known as TPApy, which exhibited aggregation-induced emission. This new metabolic substrate was incorporated into bacterial peptidoglycan to

create engineered _C. butyricum_. Once injected into melanoma, this bacterium colonized the hypoxia region and activated the intratumoral immune system thus eradicating the tumor masses.

Following this, the peripheral area, which has more oxygen content, caused the bacteria to die off while the photosensitizer on the bacteria exerted a photodynamic effect under light

irradiation, further removing any remaining melanoma65. Alternatively, chemotherapy drugs, such as doxorubicin (DOX), can induce immunogenic cell death in tumor cells but have severe

long-term side effects due to non-specific drug distribution67,68. Natural polysaccharide polymers, like glycogen (GLY), can be used as nanocarriers for drug delivery, providing targeted

delivery and controlled drug release, as well as reduced DOX toxicity69. On that account, a recent innovative system (GDOX@HSEc) has been developed using engineered _E. coli_ to secret

heparin sulfatase-1 (HSulf-1) within, and anchoring DOX-linked GLY nanoparticles (GDOX NPs) on the surface67. The GDOX@HSEc combination demonstrated a spatiotemporally intratumoral

distribution of therapeutic agents. In this context, HSulf-1 was upregulated to restrict angiogenesis and metastasis. Meanwhile, GDOX nanoparticles successfully infiltrated tumor cells,

inducing intracellular DNA damage70. Specific constituents of the skin commensals can trigger a robust T cell reaction when they colonize the skin, such as CD8+ T cells elicited by _S.

epidermidis_71. These cells are recognized for their role in fostering skin homeostasis and expediting wound closure72. In light of this, Chen et al73. engineered _S. epidermidis_ NIHLM087

to express various versions of ovalbumin (OVA)-derived MHC class I (OT1) or MHC class II-restricted (OT2) peptides. The most effective approach combined soluble OT1 peptide with cell

wall-anchored OT2 peptides, proving highly efficient in triggering immune responses against OVA-expressing B16F10 melanomas in mouse skin. Notably, the absence of either OT1 or OT2 from _S.

epidermidis_, the absence of OVA from B16F10 melanomas, or the depletion of CD8+ T lymphocytes thwarted the observed anticancer effects, underlining the therapeutic potential of a cellular

immune response targeting shared antigens between bacteria and cancer cells74. In previous applications, _Salmonella typhimurium_ served as the foundational bacterial chassis for targeting

tumors. To enhance safety and ameliorate potential toxicity concerns, a strategic attenuation approach was employed, involving the targeted deletion of specific genes, _purI_ and _msbB_,

resulting in the creation of a modified strain denoted as VNP2000975. While these genetic modifications effectively introduced adenine dependency and mitigated lipopolysaccharide-related

toxicity, the clinical utilization of VNP20009 revealed a dose-dependent elevation in proinflammatory cytokines. As a result, this immunological response precipitated adverse effects in

patients, encompassing thrombocytopenia, anemia, bacteremia, hyperbilirubinemia, diarrhea, vomiting, and nausea. Presently, efforts are directed towards reducing its toxicity and enhancing

tumor localization. In one example, an attenuated _S. typhimurium_ expressing recombinant interferon-gamma (IFN-γ) successfully invaded melanoma cells and induced cytotoxicity in

melanoma-bearing mice, while showing minimal toxicity to normal cells76. A modified strain expressing a radiation-sensitizing microRNA vector encoding the inhibin alpha gene (INHA) was also

able to exert cytotoxicity in combination with radiotherapy by enhancing ROS production77. Additionally, combining programmed death 1 (PD-1) knockdown with small interfering RNA (siRNA) and

pimozide drug therapy effectively suppressed melanoma through caspase 3-mediated apoptosis, as compared to bacteriotherapy alone78. STATUS QUO IN CLINICAL TRANSLATION OF SKIN ELBPS Despite

substantial progress in eLBP research, only a small fraction of eLBPs for skin therapy has progressed to clinical trials, and they are still in the early stages. Azitra Inc., a pioneering

skin eLBP company, is set to enter Phase 1b clinical trials in the first half of 2023 for their leading proprietary drug candidate, ATR-12. This innovative treatment incorporates engineered

_S. epidermidis_ to produce the serine protease inhibitor Lympho-epithelial Kazal-type related inhibitor (LEKTI) and is aimed at addressing Netherton syndrome (Identifier number pending). In

a similar vein, their product ATR-04 is expected to commence Phase 1b clinical trials in the first half of 2024, featuring lyophilized _S. epidermidis_ engineered to be auxotrophic to

D-alanine as the active ingredient. This product seeks to alleviate the severity of papulopustular rosacea associated with epidermal growth factor receptor inhibitor (EGFRI) therapy

(Identifier: NCT04731259). Additionally, Ilya Pharma is currently conducting a Phase 2 clinical trial using _L. reuteri_ as a chassis to secrete CXCL12, a short-lived human cytokine, to

improve wound healing. The progression of these eLBPs toward clinical trials is a crucial milestone in becoming a viable component of biomedical strategies for addressing human skin

diseases. Crown Aesthetics, a forefront aesthetic manufacturer, has made an impressive addition to its product lineup with the introduction of the BIOJUVE skin biome products, protected by a

patent under the Xycrobe® technology (WO2017147507A1). These innovative products primarily consist of engineered _C. acnes_ subsp. defendens strain XYCW42 to express human cytokines (such

as IL-10, IL-6, IL-7, and IL-8) through an inducible promoter located in front of the _ftsAZ_ operon, enabling precise control over bacterial cell division79. In their clinical study

involving 121 subjects, a specifically designed skincare regimen was rigorously followed including the XYCM42 Ferment Based Serum, Live XYCM42 Gel, and Prebiotic Activator. According to the

results, participants demonstrated enhanced skin health with sustained pH balance, optimal transepidermal water loss levels, increased skin moisture, reduced redness, improved skin

elasticity, and a noticeable reduction in surface sebum, with particularly pronounced effects in the forehead and nose areas79. This significant progress represents a noteworthy milestone

for eLBPs within the domain of skin health and beauty products, reinforcing their potential not only in skin repair and therapy but also in advancing towards clinical trials and subsequent

commercialization. CHALLENGES TOWARDS COMMERCIALIZATION OF ELBPS To date, the majority of the development of eLBPs has been rooted in probiotics sourced from the gut microbiome, encompassing

lactic acid bacteria and commensal strains80. This trend can be attributed to the promising potential of eLBPs as biotherapeutics, stemming from their superior safety profile when compared

to conventional chemical drugs, especially for extended periods of use80,81. eLBPs not only serve as effective therapeutic agents for chronic diseases by colonizing damaged cells but also

widen the possibilities for tailored tumor-targeted treatments. The use of eLBPs in treating skin diseases is still in its early stages and must address specific limitations, as detailed by

Charbonneau et al.18 and Pedrolli et al.82, concerning regulatory, application, manufacturing, safety, stability and efficacy. In both the United States and Europe, the development of eLBPs

necessitates the establishment of quality by ensuring safety, reliability, robustness, and batch consistency. Nevertheless, regulatory guidance under the purview of the US Food and Drug

Administration (FDA) for LBPs remains broad28, and there is currently no published directive specifying toxicology requirements tailored to LBPs18. Therefore, the development pathway for a

specific clinical candidate must involve discussions with the relevant regulatory authorities in the region or country where the product is intended for development and use. Biocontainment

stands as a significant hurdle in the progression of eLBP research. To address this challenge, emerging methods are being explored, with auxotrophies being one notable example. These

auxotrophies aim to curtail the proliferation of engineered strains outside their designated environments9. For instance, a biocontained _Saccharomyces boulardii_ strain, engineered with

_THI6_ and _BTS1_ gene knockouts, exhibited constrained growth in the absence of thiamine concentrations exceeding 1 ng/mL and experienced severe growth impairment at temperatures below 20 

°C83. Apart from auxotrophies, biocontainment strategies encompass ‘deadman’ and ‘passcode’ kill switches in which the eLBPs are programmed to respond exclusively to precise environmental

signals. These switches can suppress the transcription of essential genes in the absence of specific triggers or initiate self-destruction of engineered strains through toxin production,

effectively preventing unintended cell proliferation84. Furthermore, beyond traditional exo- and endonucleases, CRISPR-associated nucleases (Cas) have been deployed in _E. coli_ to design

effective kill switches85. These kill switch gene circuits leverage Cas386 or Cas987, successfully achieving biocontainment with minimal escape frequencies. A notable advantage of Cas-based

kill switches lies in their use of guide RNAs (gRNAs) to selectively target specific DNA sequences or microorganism strains, enabling the precise elimination of the target strain from the

microbiome87,88. Such innovative biocontainment strategies not only address critical safety concerns but also pave the way for responsible and controlled advancements in the field of eLBPs.

The significance of robust biocontainment in eLBPs research cannot be overstated. Beyond averting unintended consequences and ecological disruptions, it highlights the ethical and safety

imperatives essential for the responsible development and utilization of these innovative therapeutic agents. Consequently, the ongoing exploration and enhancement of biocontainment

techniques bear profound implications for the progression and acceptance of eLBPs in clinical applications89. Apart from clearance or biocontainment, a major future challenge will be

addressing issues such as the potential spread of genetically modified LBPs into other bacterial or mammalian cell genomes, how to establish stable colonization of targeted sites,

interactions with commensal flora, and tissue targeting. These questions highlight the importance of investigating genetic stability in eLBPs under normal physiological conditions. Although

challenging, advancements in biological technologies will enhance the depth and breadth of disease prevention and treatment strategies through the use of newly available bacterial tools and

upgraded therapeutic approaches for eLBPs, ultimately alleviating safety concerns. On a more intricate note, biopharmaceutical manufacturers grapple with a host of challenges when

translating LBP concepts (both engineered and non-engineered products) into mass production and commercialization. These challenges span formulation and development, regulatory approval,

production and packaging, shipping and storage, patient application, and efficacy. Comprehensive discussions by Vargason and Anselmo et al.14 have shed light on the complex landscape

confronting biopharmaceutical manufacturers in this pursuit. To make eLBPs viable products that cater to patient needs, it is vital to address these multifaceted challenges. A core necessity

lies in foundational research on delivering foreign microbiota to the skin, encompassing queries about formulation design, the impact of common topical formulations on eLBP viability during

storage, and the quest for ideal formulations that balance manufacturing practicalities with patient usability. Moreover, analyzing clinical data reveals variations in LBP colonization on

the skin, prompting questions about the specific LBP and formulation parameters dictating microbial adherence, competition, growth, and long-term persistence90. These complexities

necessitate the development of delivery devices and formulations capable of sustaining LBP growth and persistence, even in challenging skin environments, aiming to minimize variability and

enhance the overall potential of LBPs in addressing chronic, recurrent, and difficult-to-treat skin conditions, while paving the way for successful mass production and commercialization. The

utilization of eLBPs also brings forth potential risks concerning human safety that demand careful consideration. One critical aspect entails the evaluation of virulence factors within

bioengineered strains, necessitating a meticulous screening process to identify genes capable of inducing pathogenicity91. Equally vital is the scrutiny of genes responsible for producing

enzymes involved in synthesizing toxic or allergenic compounds, or their precursors92. The introduction of foreign DNA may lead to the production of novel substances, either as proteins or

metabolic byproducts, which can exhibit harmful or allergenic properties. Assessing these newly expressed proteins involves examining amino acid sequences for potential homology and

evaluating their stability under various conditions, including heat, processing, and degradation. Despite these measures, precise tests for predicting allergenic responses in humans remain

elusive91. Moreover, the safety of eLBPs hinges not only on their composition but also on factors such as the mode of application and the genetic profile of the consumers. Certain vulnerable

subpopulations, such as immunocompromised individuals, infants, and the elderly, can exhibit heightened sensitivity, necessitating vigilant post-market surveillance of novel biotherapeutics

to avert potential adverse effects. The need for prolonged monitoring after introducing such biotherapeutic products to the market is evident, albeit complicated by technical challenges

arising from inconsistent intake. Similarly, in the case of engineered bacteria, vigilant and ongoing monitoring post-release into the market is advisable to preempt delayed adverse effects

and guarantee safe consumption93. Overall, the safety considerations surrounding the use of eLBPs accentuate the importance of comprehensive risk assessment and vigilant surveillance to

safeguard human health. As we explore the potential benefits of these innovative approaches, it is imperative to remain cognizant of the intricate web of factors that can impact their safety

profiles and to prioritize ongoing monitoring to ensure the long-term well-being of consumers. PROSPECTS FOR NON-COMMENSAL BACTERIAL CHASSIS AS NEW ELBPS Given the progress made in

microbial systems biology and synthetic biology94, leveraging non-pathogenic skin commensals seems promising for initiating the development of new bacterial chassis dedicated to advancing

skin treatment in the field of eLBP. Skin commensal bacterium, _S. epidermidis_ is an emerging bioengineered chassis as eLBP in skin disease treatment given the innate anti-staphylococcal

activity36,37 as well as the successful development of genetic tools for delivery therapeutic proteins such as recombinant filaggrin and LEKTI proteins important for treating AD and

microbial dysbiosis81,95,96. Nevertheless, genetic manipulation of beneficial strains derived from human-associated microbes presents notable challenges, primarily stemming from the

necessity to navigate innate restriction-modification systems. Consequently, these strains are often regarded as genetically intractable when compared to well-established models or known

bacterial systems2,97. The discovery of antimicrobial activities of a subset of commensal and non-commensal _Corynebacterium_ spp. suggests the possibility of utilizing these bacteria as

alternative bacterial chassis for biotherapeutic or biodiagnostic purposes, particularly in targeting skin pathogens like _S. aureus_98,99,100,101,102. A recent _Staph_-targeted study has

showcased the use of an engineered non-commensal _C. glutamicum_ which has been modified to respond to the quorum sensing (QS) molecule known as autoinducing peptide (AIP) produced by _S.

aureus_100. The expression of accessory regulatory proteins agrAC in tandem with a recombinant red fluorescent protein (RFP) conferred AIP-stimulated protein production in engineered _C.

glutamicum_ pResponse strain100. Given the inherent ability of _C. glutamicum_ to hinder the growth of _S. aureus_100, this model bacterium can be subjected to additional manipulation to

mount a response and combat _Staph_ infections, following a strategy akin to that demonstrated by Guan et al.96. In this approach, _S. epidermidis_ was engineered to produce lysostaphin

biomolecules, which effectively inhibit the growth of _S. aureus_96. Compared to _S. epidermidis_ and other closely related commensal _Corynebacterium_ spp., _C. glutamicum_ has been more

readily used in the food, feed and biopharmaceutical industry with prior approval from the FDA103. Metabolic engineering of AIP-responsive _C. glutamicum_ to produce important skin

biomolecules such as cobamide104 and arginine105 will aid in stimulating the production of filaggrin-derived natural moisturizing factors as well as reducing pathogenic _Staph_ growth in the

skin microbiome. With the expanded availability of CRISPR-Cas genome editing tools106,107, unwanted genes that may interfere with the host system can be accurately modified and modulated

hence providing a promising means for development of _C. glutamicum_ strains as eLBPs. Importantly, the increased interest in the employment of bioengineering and synthetic biology

approaches in developing new strains as eLBPs should bring about timely technological development in the race against infectious pathogens following the rules and regulations in countries

all over the world. FUTURE PERSPECTIVES In summary, recent advancements in the development of eLBPs and their clinical trials for tackling skin diseases have ignited significant enthusiasm

for delving into the therapeutic potential of these agents. All of these endeavours have been conducted with the highest level of diligence, ensuring the implementation of stringent safety

assessment protocols. The substantial body of evidence supporting the efficacy of non-engineered LBPs in maintaining a healthy skin microbiome and their potential as chassis organisms

underscores the promising prospects for utilizing eLBPs in skin disease intervention. Nonetheless, it is imperative to acknowledge the multifaceted challenges associated with advancing

eLBPs, encompassing issues related to manufacturing scalability, ensuring stability throughout the production process, and the implementation of robust biocontainment strategies. These

formidable challenges emphasize the need for comprehensive research and development efforts to effectively address them, facilitating a seamless transition into the clinical phase. The

significant progress achieved through the bioengineering of skin commensals, particularly _S. epidermidis_, and non-commensals like _L. lactis_ has established a promising foundation for

expanding eLBP development. This pioneering work serves as a blueprint for harnessing other non-pathogenic and non-commensal bacteria, precisely tailored to combat skin pathogens and

alleviate inflammatory responses. By leveraging microbial engineering and synthetic biology approaches, this emerging platform holds tremendous potential for revolutionizing the field of

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was financially supported by Universiti Kebangsaan Malaysia through Dana Impak Perdana 2.0 grant (DIP-2022-004). The funder played no role in the study design, data collection, analysis and

interpretation of data, or the writing of this manuscript. Figures 1 and 2 were created with BioRender.com. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Institute of Systems Biology

(INBIOSIS), Universiti Kebangsaan Malaysia, 43600 UKM, Bangi, Selangor, Malaysia Muhamad Aidilfitri Mohamad Roslan, Mohd Norfikri Omar & Ahmad Bazli Ramzi * Department of Biological

Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM, Bangi, Selangor, Malaysia Nur Azlina Mohd Sharif & Nurul Hanun Ahmad Raston *

Department of Fundamental Dental & Medical Sciences, Kulliyyah of Dentistry, International Islamic University Malaysia, 25200, Kuantan, Pahang, Malaysia Mohd Hafiz Arzmi * Melbourne

Dental School, The University of Melbourne, 3053, Melbourne, Victoria, Australia Mohd Hafiz Arzmi * UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia, 56000,

Cheras, Kuala Lumpur, Malaysia Hui-Min Neoh Authors * Muhamad Aidilfitri Mohamad Roslan View author publications You can also search for this author inPubMed Google Scholar * Mohd Norfikri

Omar View author publications You can also search for this author inPubMed Google Scholar * Nur Azlina Mohd Sharif View author publications You can also search for this author inPubMed 

Google Scholar * Nurul Hanun Ahmad Raston View author publications You can also search for this author inPubMed Google Scholar * Mohd Hafiz Arzmi View author publications You can also search

for this author inPubMed Google Scholar * Hui-Min Neoh View author publications You can also search for this author inPubMed Google Scholar * Ahmad Bazli Ramzi View author publications You

can also search for this author inPubMed Google Scholar CONTRIBUTIONS M.A.M.R. and A.B.R. are responsible for the design, writing and organization of this review. M.N.O., N.A.M.S., N.H.A.R.,

M.H.A., and N.H.M. contributed to the content of this work. Figures were designed and created by M.A.M.R. CORRESPONDING AUTHOR Correspondence to Ahmad Bazli Ramzi. ETHICS DECLARATIONS

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engineered live biotherapeutic products research for skin repair and disease treatment. _npj Biofilms Microbiomes_ 9, 95 (2023). https://doi.org/10.1038/s41522-023-00463-8 Download citation

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