In the face of increasing anxiety and depression in modern society, our team has used synthetic biology techniques to successfully produce two natural active ingredients with mood-regulating functions: kaempferol and GABA (gamma-aminobutyric acid). Kaempferol is a kind of plant flavonoid, which can effectively relieve the symptoms of depression. GABA is an inhibitory neurotransmitter that reduces anxiety and calms the nervous system. We incorporate these two ingredients into the "Joy-filled Drink" we have developed. This innovative drink provides consumers with a natural, safe and effective mood regulation solution to relieve anxiety and stress in daily life.

According to statistics, around 350 million people worldwide suffer from depression, accounting for 4.4% of the global disease burden [1]. In China, the lifetime prevalence of depression is 6.8% [2]. Depression is a serious mental illness characterized by persistent low mood, loss of interest in daily activities, and symptoms such as low self-esteem, hopelessness, significant changes in appetite and weight, fatigue, insomnia, and lack of energy. It affects daily life by straining relationships, causing confusion about the future, and fostering fear of social interactions [3]. According to the World Health Organization, over 700,000 people die by suicide annually, with depression being a leading cause [4].

With the increasing pace of life, anxiety has become a common emotional response [5]. The pressures of work, study, and daily life leave many people in a prolonged state of anxiety. If left unmanaged, anxiety can accumulate, eventually leading to serious mental health problems. Studies show a clear link between early anxiety disorders and later depression [6]. Long-term anxiety weakens psychological defenses, making emotional regulation difficult and increasing the risk of depression. As anxiety persists, mental resources are depleted, making it harder to cope with stress and negative emotions, which heightens vulnerability to depression [7]. Therefore, managing and alleviating anxiety is key to preventing its progression into depression.

Although the current cure rate for depression is as high as 70% to 80%, there are still significant challenges in early detection and treatment [8]. Early-stage depression often presents as anxiety and sadness, symptoms that are frequently overlooked, causing patients to miss the optimal treatment window and leading to worsened conditions. Additionally, the high cost of psychotherapy, typically ranging from 1,000 to 2,000 yuan per session with a course lasting 4 to 8 months, is prohibitive for many individuals with anxiety or mild depression [9]. This financial burden often forces many to forgo consistent treatment.

Antidepressants are widely used in treating depression but are not suitable for early or mild cases due to significant side effects [10]. Selective serotonin reuptake inhibitors(SSRIs) can initially worsen depression and anxiety, and long-term use may lead to weight gain and sexual dysfunction. Tricyclic antidepressants have more severe side effects, affecting heart rate and weight. Monoamine oxidase inhibitors(MAOIs) are typically used as a last resort, with complex usage requirements, including strict dietary restrictions and avoiding combination with other drugs [11].

In response to these challenges, our project uses synthetic biology to develop a non-pharmaceutical intervention beverage, “Joy-filled Drink”. This drink combines the natural mood-regulating effects of kaempferol and GABA (γ-aminobutyric acid) to help alleviate anxiety and depressive symptoms. It is particularly suited for individuals with early-stage or mild depression, providing a safe, gentle, and with minimal side effects for managing emotional well-being and preventing the progression of depression.

Compared to other strains, Escherichia coli Nissle 1917 (EcN) is a safer and more reliable chassis microorganism [12]. Since our product, kaempferol, is designed for use as a food additive, using common E. coli strains risks contamination with endotoxins, which could cause foodborne illnesses such as abdominal cramps and diarrhea. Therefore, selecting a strain that is both safe and efficient is critical.

After reviewing literature and past iGEM projects, we selected E. coli Nissle 1917. This non-pathogenic strain was first isolated in 1917 from a soldier who did not contract diarrhea during a shigellosis outbreak. As one of the few Gram-negative bacteria clinically used as a probiotic, EcN is widely employed to prevent infectious diarrhea and inflammatory bowel diseases. Importantly, EcN does not produce endotoxins, ensuring that our kaempferol production is free from food safety risks. By using E. coli Nissle 1917, we guarantee that our product is endotoxin-free, ensuring biological safety and enhancing the reliability and competitiveness of our product in the food additive industry.

Kaempferol, with the chemical formula C15H10O6, is a natural flavonol belonging to the flavonoid family, which is widely distributed in various fruits, vegetables, and herbs, such as grapes, tomatoes, broccoli, tea, and ginkgo biloba [13]. This bioactive compound exhibits a diverse array of pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial, antidiabetic, and anticancer properties [14]. Its multifaceted benefits make kaempferol a compelling subject of study in the field of nutraceuticals and pharmacology, highlighting its potential therapeutic applications in promoting health and preventing diseases.

Kaempferol, a natural flavonoid, exhibits significant anti-anxiety effects through mechanisms such as reducing oxidative stress, inhibiting pro-inflammatory cytokines, and regulating the AKT/β-catenin signaling pathway [15]. It inhibits the activation of NADPH oxidase (NOX), reducing the production of reactive oxygen species (ROS) and enhancing antioxidant enzyme activity, which lowers oxidative stress markers. This process protects neurons from oxidative damage, alleviating anxiety symptoms. Additionally, kaempferol decreases the concentration of pro-inflammatory cytokines in the prefrontal cortex and inhibits the mRNA expression of CD11b, which is associated with neuroinflammation in anxiety.

Kaempferol also regulates glucose uptake, mitochondrial biogenesis, and protein synthesis through the AKT/PI3K and MAPK signaling pathways, promoting neuronal survival and function while reducing physical fatigue. These effects make kaempferol a promising natural compound for mood regulation [16].

Currently, the production of kaempferol faces three main challenges: extraction from plants is feasible but limited by low yield and time-consuming processes [17], making large-scale production difficult; chemical synthesis is effective but costly, and it generates harmful by-products, posing environmental concerns; biotechnological methods, such as microbial fermentation and plant cell culture, hold promise but are technically complex and require significant initial investment. These factors present considerable challenges to the efficient and safe production of kaempferol [18].

We leveraged metabolic engineering to produce kaempferol in E. coli using naringenin as the substrate. The conversion of naringenin to kaempferol involves several enzymes, with two key catalytic enzymes being flavanone 3-hydroxylase (F3H) and flavonol synthase (FLS). F3H first introduces a hydroxyl group at the C3 position of naringenin, forming dihydrokaempferol. Subsequently, FLS introduces a double bond between the C2 and C3 positions, resulting in the formation of biologically active kaempferol [19].

We first employed the polycistronic strategy (BBa_B0034) to link F3H and FLS, allowing both genes to be expressed simultaneously on a single vector. To further enhance kaempferol production, we utilized the fusion enzyme technique, which involves fusing F3H and FLS together through a linker (GGGS), bringing the two enzymes into close proximity in space to improve their cooperative interaction and metabolic efficiency [20]. Additionally, we optimized production conditions (such as temperature, cell density, and substrate concentration), tested different linkers, and introduced multiple copies of key enzyme genes to further increase kaempferol yield [21].

GABA is a key inhibitory neurotransmitter widely found in vertebrates, plants, and microorganisms [22]. It plays a critical role in maintaining balance in the central nervous system by inhibiting excessive neuronal excitability. As a natural neurotransmitter, GABA has well-known calming, sleep-promoting, and anti-anxiety effects, without causing drug dependence or side effects, making it a valuable therapeutic agent for alleviating anxiety and improving sleep [23].

1. Improve sleep, because γ-aminobutyric acid has the effect of inhibiting the nerve centre, so it has the effect of sleeping and sedation, and at the same time, it is the neurotransmitter without drug dependence and side effects [24];

2. Anti-anxiety, GABA is an inhibitory neurotransmitter in the central nervous system. Its function is to reduce neuronal activity and prevent overexcitation of nerve cells. GABA can bind to anti-anxiety brain receptors, activating them and preventing anxiety-related signals from reaching the brain’s control center, helping to relieve anxiety symptoms [25].

GABA can be produced through chemical synthesis, plant enrichment, and microbial fermentation [26]. Chemical synthesis, while effective, involves multiple steps and is often costly. Plant enrichment, though a promising method, is time-consuming and limited by the availability of plant resources. In contrast, microbial fermentation is considered the safest and most stable method for producing GABA. This approach uses genetically engineered microorganisms, such as E. coli , to efficiently produce GABA, making it suitable for use as a natural food additive. Our team adopts this method due to its safety and efficiency.

In our design, glutamate decarboxylase (GAD) is the key enzyme responsible for converting glutamate into GABA. Under the catalysis of the GAD-PLP (pyridoxal phosphate) complex, the carboxyl group of the glutamate molecule accepts a proton and forms a covalent bond with the pyridoxal phosphate portion of vitamin B6. This covalent bonding triggers a decarboxylation reaction, generating the intermediate racemic α-ketoglutarate semialdehyde (L-glutamate α-decarboxylase semialdehyde) [27]. This intermediate then undergoes intramolecular racemization to finally form GABA.

To enable this conversion, we constructed a recombinant plasmid containing the GadB gene, which encodes glutamate decarboxylase, and transformed it into E. coli for expression. The engineered GABA-producing strain can use glutamate as a substrate to produce GABA. To optimize GABA yield, we cultured the production strain under different pH conditions to identify the most suitable production environment.

Our project primarily targets individuals who are under high levels of stress, as well as those with depressive symptoms. High-stress groups typically include working professionals, students, and adults facing life pressures and uncertainties. These individuals are prone to emotional fluctuations, anxiety, and mental fatigue due to prolonged mental strain. At the same time, we also focus on those who already exhibit symptoms of depression, who often need a safe and effective solution to help manage emotional fluctuations and alleviate anxiety and depression symptoms. By providing emotional regulation solutions, our project aims to help these individuals better cope with stress and prevent further emotional deterioration.

In the real-world production of the "Joy-filled Drink," the process begins by cultivating engineered bacteria under controlled conditions to ensure the efficient synthesis of the target compounds, kaempferol and GABA. Once the bacteria reach the desired growth phase, they are transferred to a biofermentation facility where they are processed in specialized fermenters. These fermenters are optimized to maximize fermentation efficiency and boost the production of kaempferol and GABA. Following fermentation, the bacterial cells are lysed to release kaempferol and GABA. The next step involves a series of purification processes to remove impurities, ensuring high-purity active ingredients. These active compounds are then blended into our custom fruit tea formula to create the final product—"Joy-filled Drink."

Our product, “Joy-filled Drink” is a functional beverage that combines the natural compounds kaempferol and GABA to support individuals experiencing depression or high stress. In response to user needs, especially considering that compounds like tea polyphenols may cause insomnia in some people with depression, we offer an option to replace the tea base with alternative herbal ingredients. This allows the product to better suit the needs of our target customers.

Additionally, we provide different formulations for daytime and nighttime use. During the day, we offer a refreshing version that includes kaempferol, designed to improve mood and alertness. In the evening, we offer a more calming version that includes GABA to promote relaxation and help with sleep. These drinks are available in offline stores, which also offer aroma therapy capsules to help further alleviate stress.

The most important point is that our product is not a prescription drug or even a medicine, it is just a drink containing functional ingredients, so our drink does not require people to use it on a regular basis, just need to drink it in moderation when you feel stressed out, depressed, or insomnia recently. As a medicinal drink, our milk tea contains roughly 0.04% kaempferol, so long-term consumption does not lead to dependence on the substance. Similarly, there is no fixed dose of this milk tea, so users can choose whether to drink a large amount over a long period of time or a small amount for a short period of time, depending on their state.

We compared chemical lysis and strain inactivation, ultimately selecting ultrasonic lysis as the strain inactivation method. Ultrasonic lysis is simpler to operate, avoids chemical residues, and better preserves the activity of kaempferol and GABA. To further ensure safety, we chose E. coli Nissle 1917, a non-endotoxin-producing strain, as our chassis microorganism, making the project more reliable and compliant with legal regulations.

Due to the influence of the traditional shame culture in East Asia, the sense of shame is an inextricable topic, how to close the distance with the target customers and let them out of the influence of this shame is very difficult, we have to try to communicate with them and do not arbitrarily label, and can not be sold under the name of the treatment of depression.

Given that we are going to incorporate genetically modified kaempferol into our product and sell it to the public, safety issues and legal restrictions will be an inevitable topic; in addition to safety and legal issues, the cost of production will also affect our sales; small batch laboratory production, although efficient and pure, is costly; large scale production in factories, although less costly, will most likely not be viewed favorably or no factory will be willing to take orders due to legal risks. Although the cost of mass production in factories is lowered, our niche and novel project may not be favoured, or there may be legal risks, which may lead to no factories being willing to take orders.

Efficient and Low-Cost Production: Using synthetic biology, we optimized metabolic pathways in E. coli to produce kaempferol efficiently, significantly reducing production costs and overcoming the limitations of plant extraction.

Acceptability: We developed the “Joy-filled Drink”, a non-medicinal beverage containing kaempferol and GABA. This drink helps alleviate anxiety and stress while remaining affordable and easily accepted by consumers.

Safety Assurance: Ultrasonic lysis technology ensures the safe inactivation of engineered strains, and the use of the safe E. coli Ni ssle1917 strain minimizes potential environmental and health risks.

Using synthetic biology, we developed an efficient and cost-effective method to produce kaempferol and created the functional beverage “Joy-filled Drink” to help alleviate anxiety and low mood, preventing anxiety from progressing into depression. Our product is safe, accessible, and serves as a natural alternative to traditional medication. This project offers a practical and effective way to support mental health for people worldwide.

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