Our team aims to address skin aging in menopausal women due to a decline in estrogen. For menopausal women, estrogen is especially important in skin care, and common beauty and skin care products on the market do not solve this problem. Our team selected Escherichia coli Nissle 1917 as the chassis microorganism and constructed three systems, including genistein production system, bacterial cellulose production system, and light-controlled induced suicide system. Genistein can supplement estrogen, bacterial cellulose is used for hydration, and the light-controlled suicide induced suicide system ensures biosecurity. Through special production and use methods, the products provide a safe and efficient skin care experience for middle-aged and elderly women.

The mother of one of our team members used face masks and various skin care products as she entered menopause, and started to drink soy milk every morning because of some research reports that the soy isoflavones in soybeans are able to fight aging.

However, after reviewing the information, we found that the effect of soy isoflavones supplementation by consuming foods containing soy products was not particularly obvious. Genistein is one of the most important isoflavones in soybean. So our team thought about whether it is possible to synthesise genistein, through synthetic biology, and it can be used on the skin, so our genistein mask project was conceived.

As women reach the age of 30, noticeable changes in skin health begin to occur. These changes are primarily due to a natural slowdown in metabolism, leading to the loss of moisture and elasticity, which results in drier and rougher skin [1]. This decline is driven by factors such as reduced sebum production, the breakdown of collagen and elastic fibers, weakened skin repair mechanisms, and a gradual decrease in estrogen levels. By the time many women reach their 40s, estrogen levels often drop significantly, further contributing to skin challenges [2]. With growing economic prosperity and higher living standards, women in midlife are increasingly focused on maintaining their appearance. They seek advanced skincare solutions to delay visible signs of aging and retain a youthful look.

Although common skincare methods have their advantages, they each come with significant limitations：

Medical cosmetic procedures deliver noticeable results, but they are expensive and carry considerable risks and side effects, such as infections, scarring, and hyperpigmentation, with results that are often not long-lasting [3].

Anti-aging products on the market (such as the masks), while convenient to use, generally show minimal effectiveness, and some may contain irritating ingredients that can harm the skin with prolonged use [4].

Additionally, maintaining a healthy lifestyle is beneficial for the skin, but the effects are slow, and it is difficult to reverse existing skin problems or address the deeper damage caused by aging [5].

During menopause, the sharp decline in estrogen leads to visible skin aging, such as dryness, sagging, and wrinkles. Estrogen is crucial for maintaining skin moisture and collagen synthesis. As levels drop, the skin’s hydration and elasticity decrease, accelerating the aging process [6]. To address this issue, our team, JIASHU-SouthChina, has developed a skincare solution using synthetic biology to supplement genistein, a phytoestrogen. Genistein effectively enhances skin hydration and boosts collagen production, helping alleviate skin aging in menopausal women.

To design a safe and effective bio-mask specifically for middle-aged and older women that provides deep hydration and estrogen supplementation.

EcN is a key strain in synthetic biology, valued for its lack of endotoxin secretion and ability to efficiently express foreign genes. These traits make it ideal for metabolic engineering and complex biosynthesis. In our project, we use EcN as the core chassis, engineering it to express key enzymes for genistein biosynthesis. Its stability, safety, and efficiency make it highly suitable for industrial applications, offering both productivity and cost advantages [7].

In order to improve our project more smoothly, we divided EcN into three system, the first system is about producing the genistein, it is the most important component of biological anti-aging. Using the bacterial cellulose as the main ingredient for hydration is the second system. As for the third sysstem, light-controlled induced suicide system for the biosecurity.

Genistein is one of the main active factors in soy abnormal flavonoids and is the most effective functional component in soy isoflavone products [8]. Structurally genistein flavone is similar to mammalian estrogen-estradiol, so it has a variety of physiological activities such as estrogen-like activity [9]. Not only that, it has a variety of physiological functions. It combines with estrogen receptors on bone cells to reduce bone loss and prevent osteoporosis; it can inhibit the structure of undesirable lumps and prevent the proliferation of lumps and cancer cells; it can make the body maintain normal levels of estrogen and delay menopause to achieve the role of delaying aging [10]. In this project, genistein is used as a phytoestrogen to supplement hormone loss.

Currently, genistein is obtained through three main methods [11]: natural plant extraction, chemical synthesis, and microbial synthesis. Traditional extraction techniques, such as alcohol extraction and chloroform reflux defatting, are widely used but have several limitations, including significant resource consumption, complex processing steps, and potential environmental hazards [12]. Chemical synthesis involves constructing the genistein molecule through intricate reaction pathways, making the process highly demanding and challenging to scale up for industrial production [13]. Microbial synthesis, on the other hand, employs genetic engineering to modify microorganisms for efficient production of genistein or its precursors. Although microbial synthesis faces technical challenges and higher production costs, its potential for scalability and sustainability makes it a promising approach [14]. Therefore, this study focuses on using microbial synthesis for the production of genistein, with the aim of overcoming current technical barriers and achieving efficient, large-scale production.

Isoflavone synthase (IFS), IFS requires the involvement of NADPH and O2 for the reaction to occur. IFS first oxidizes C-3 to form free radicals and secondly the benzene ring is rearranged (migrated) from C-2 to C-3 [15].

Cytochorome P450 reductase (CPR), Carrying molecular oxygen for further oxidation and finally adding a hydroxy group at C-2. resulting in the formation of 2,4',5,7-tetrahydroxyisoflavanone [16].

2-hydroxyisoflavanone dehydratase （HID), A dehydration reaction occurs on the basis of 2,4',5,7 tetrahydroxyisoflavones. A molecule of water on C-2 is removed to form the final product, genisteins [17].

Microbial synthesis offers several distinct advantages over traditional methods. By leveraging genetic engineering, it allows the efficient production of genistein without being constrained by the seasonal and geographical limitations that affect plant-based extraction. Through optimizing bacterial strains and co-culture conditions, microbial synthesis can be performed year-round, in any environment, regardless of temperature or location. Additionally, this method is safer, as it reduces reliance on hazardous chemicals and heavy metal catalysts, thus minimizing risks of explosions or environmental contamination that are common in chemical synthesis. Lastly, microbial synthesis is environmentally friendly, producing minimal waste and harmful byproducts, and is aligned with sustainable development goals by reducing resource consumption.

Bacterial cellulose (BC) is a porous, reticulated, nanoscale biopolymer synthesized under different conditions by certain microorganisms in the genera Acetobacter, Agrobacterium, Rhizobium, and Sarcina, and is named bacterial cellulose because it is synthesized by bacteria [18]. A collective term for polymers, named bacterial cellulose because it is synthesised by bacteria. It consists of unique filamentous fibres with fibre diameters ranging from 0.01 to 0.10 μm. In terms of the molecular composition of cellulose, BC, like plant fibres, is made up of β-D-glucans bonded into a straight chain by β-1,4-glycosidic bonds, and is also known as β-1,4-glucan [19].

It has good biocompatibility and biodegradability, adjustable biosynthesis, strong hydrophilicity, viscosity and stability, high tensile strength and modulus of elasticity, and high water-holding and air permeability. It has corresponding applications in different fields. Bacterial cellulose can be used as medical dressings, food moulding agents, thickening agents, dispersing agents, anti-solubilising agents, to improve taste as the skeleton of sausage casings and certain foodstuffs, and in the paper industry to improve the strength and durability of paper and vibrating membranes of high-grade audio equipment [20].

In bacterial cellulose synthesis, acsAB , acsC , and acsD are key genes involved in producing cellulose:

1. acsAB : This gene encodes a cellulose synthase enzyme complex, where AcsA is the catalytic subunit responsible for polymerizing glucose into cellulose, and AcsB aids in the polymerization process. Together, they form the main enzyme for cellulose biosynthesis [21].

2. acsC : Encodes a protein involved in exporting the synthesized cellulose outside the cell membrane. It acts as a scaffold or channel through which the cellulose fibrils are secreted.

3. acsD : Works together with acsC and helps in the crystallization and organization of cellulose fibers as they are secreted from the bacterial cell. It ensures proper fiber formation and stability during the synthesis process [22].

Bacterial cellulose offers exceptional properties, making it ideal for facial masks. It has an outstanding water-holding capacity, absorbing 60-700 times its dry weight, which helps retain moisture and keep skin hydrated. Its excellent biocompatibility ensures it is safe for all skin types, without causing allergies or discomfort, while its nano-fiber structure supports skin repair and absorption. Additionally, bacterial cellulose is eco-friendly and biodegradable, breaking down into harmless substances in natural conditions. Its strength, elasticity, antibacterial properties, and air permeability further enhance its suitability, making it a superior choice for beauty products.

To ensure the biosafety of our product, we introduced a light-induced suicide system into the engineered bacteria. After analyzing existing systems (such as chemically-induced systems, heat-sensitive systems, and oxygen-induced suicide systems) and consulting with experts in the field, we ultimately chose the light-controlled system. This system is cost-effective, safe, and reliable, making it ideal for large-scale applications.

The light-induced suicide system can be paired with widely available photon skin rejuvenation devices. When exposed to light in the 500-600 nm wavelength range, the photosensitive promoter within the system is activated, triggering the self-destruction mechanism. This system activates the MazF gene, which leads to the death of the engineered bacteria, preventing gene leakage [23].

Low-Level Laser Therapy (LLLT) is an emerging technology in the field of medical aesthetics that utilizes photobiomodulation to stimulate cell activity, promote healing, and reduce pain and inflammation through red and near-infrared light wavelengths. LLLT is widely used to treat conditions that require accelerated healing, pain relief, inflammation reduction, and tissue function restoration. Despite the skin being one of the most exposed organs to light, it responds exceptionally well to red and near-infrared wavelengths.During LLLT, photons are absorbed by chromophores in the mitochondria of skin cells, enhancing electron transport, generating adenosine triphosphate (ATP), releasing nitric oxide, increasing blood flow, and producing reactive oxygen species. This, in turn, activates multiple cellular signaling pathways and promotes stem cell activation, enhancing tissue repair and healing capabilities.In dermatology, LLLT has been shown to be effective in treating wrinkles, acne scars, hypertrophic scars, and burn healing. Additionally, as both a therapeutic and preventative measure, LLLT can reduce UV damage. In pigment disorders like vitiligo, LLLT can increase pigmentation by stimulating melanocyte proliferation and suppressing autoimmunity. Inflammatory conditions such as psoriasis and acne also benefit from LLLT [24].

Laclq promotor: Control of YF1 gene expression

YF1: Activation of downstream genes under blue-green light irradiation

FixJ: Driving the pFixK2 promoter after activation by YF1

pFixK2 promoter: drives the downstream cI gene upon FixJ activation.

cI:epresses the λ promoter.

λ promoter: repressed by cI, controls MazF gene expression.

MazF: is a suicide gene that encodes a toxin that cleaves bacterial mRNA, leading to cell death [25].

Our system controls bacterial growth or death through light signaling. In the absence of blue-green light, the YF1 protein remains inactive, preventing the expression of the MazF toxin gene, allowing bacteria to survive. When exposed to blue-green light, YF1 activates FixJ, which in turn activates the pFixK2 promoter. This leads to the expression of the cI gene, repression of the λ promoter, and ultimately the activation of MazF, resulting in bacterial death [26]. The pDawn promoter, sensitive to blue light, controls the MazF gene expression. MazF, a toxin that blocks protein synthesis by cleaving mRNA, causes bacterial death. This light-controlled system ensures that genistein-producing bacteria are safely eliminated if accidentally released into the environment [27].

We use E. coli Nissle 1917 as the chassis microorganism, where three systems work together synergistically. First, the bacteria are placed in a mold petri dish, with naringenin as the substrate. Under the catalytic actions of the enzymes CPR, IFS, and HID, the bacteria produce flavonoid genistein. Simultaneously, through the action of the acsAB genes, the engineered bacteria synthesize bacterial cellulose. The acsCD genes facilitate the secretion of this cellulose, forming a membrane-like structure. At this point, a bacterial cellulose facial mask infused with genistein is formed. Finally, when a photon skin rejuvenation device is applied to the mask, the emitted blue-green light activates the light-controlled inducible suicide system. The promoter pdawn is triggered by the blue light, which in turn activates the MazF suicide system, killing the bacteria to prevent gene leakage and ensuring biosafety throughout the project. This process ultimately achieves anti-aging and beauty effects [28].

As people's pursuit of beauty and health, the market demand for anti-aging products has expanded. The target customer of our products is mainly the middle-aged and old-aged women aged between 30 and 59 years old who are suffering from skin problems such as waxing, dryness and wrinkles due to the decline of estrogen. Our products are also targeted at the pre-menopausal and menopausal women to solve and improve their skin problems.

We used E. coli Nissle 1917 to cultivate bacteria in a mask-film apparatus for the production of high-quality cellulose and genistein.

In order to ensure the stability and convenience of our products, we use freeze-drying technology to process the bacterial cellulose. During the process the cellulose can be dehydrated and preserved while the bacteria go dormant. This greatly extends the shelf life of the product and simplifies transportation and storage requirements.

Designed with a dual-compartment structure, one of the compartments stores freeze-dried bacterial cellulose. The other compartment stores the serum. Contains a range of highly effective ingredients such as Hyaluronic Acid, Astaxanthin, Epidermal Growth Factor (EGF) and Squalane.

Our product is a skin care mask, designed to improve skin problems caused by the decline of estrogen during menopause. The packaging uses advanced freeze-drying technology, and the mask's sheet and serum are packaged separately to ensure that the product's activity and effectiveness are maintained to the maximum extent possible.

Facial Cleaning: Before applying the mask, thoroughly clean your face with a facial cleanser to ensure that your face is free of residual oils and dirt.

Activate the strain: Fold the membrane package in half, squeeze the package firmly so that the essence completely enters the half of the membrane cloth Let the membrane cloth soak in the essence fully for at least 20 minutes to ensure that it absorbs the active ingredients.

Apply Mask: Carefully unwrap the mask, remove the mask and apply to face. Adjust the position of the mask with your hands and squeeze out the air bubbles to keep the mask close to the skin of your face.

Photorejuvenation: After applying the mask, put on the photorejuvenation device and select the blue-green light mode to boost the skin's absorption of the serum.

Remove the mask: Remove the mask and wash your face with water to enjoy the effect of refreshed skin.Through these steps, users can maximize the skincare effect of our masks to deeply nourish and repair the skin.

A mask containing only dye-derived flavonoids: This option caters to time-constrained customers, as it is easy to use but offers relatively mild effects, suitable for quick daily care.

A live bacterial mask with a light-induced suicide system: This option provides better results but requires longer application time, making it more suitable for professional settings like beauty salons to ensure safety.

Our project has several unique advantages. First, unlike most chemically synthesized masks on the market, our mask is entirely biologically synthesized. The EcN strain we use is equipped with a light-controlled suicide system, allowing it to self-lyse after use, preventing environmental release and ensuring biosafety. In contrast, traditional non-woven masks are difficult to biodegrade. Additionally, the genistein (a phytoestrogen) in our mask provides antioxidant and anti-inflammatory benefits, making it more appealing to consumers. Furthermore, with the excellent water retention capacity of bacterial cellulose, our product requires no additional moisturizers or gels, offering a more natural skincare experience.

In the future, we plan to develop a synthetic pathway for large-scale genistein production, significantly reducing costs and cultivation time. By adding abundant carbon and nitrogen sources to the culture medium, we can supply enough energy for E. coli to further enhance production efficiency. We also aim to address the challenge of continuous naringenin supplementation and the slow growth of bacterial cellulose in molds, enabling the product to be scaled for broader market availability.

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