Depression is a long-term mental health condition causing uncontrollable negative emotions, sleep disturbances, physical problems, and even thoughts of death. Severe depression is not only a personal problem but also has significant impacts on interpersonal relationships and society.
According to a global investigation (World health organization. 2023)[1] launched by the World Health Organization (WHO) in 2023, 2.8 million people are suffering from depression worldwide, and the number of people with depression grows year by year. Similarly, a survey (Gallup, inc. 2023)[2] conducted by the American Institute of Public Opinion indicated that more and more people are experiencing negative emotions. According to their report, the percentage of the global population experiencing negative emotions rose 9% from 2006 to 2022.
Studies (Arslanoglou E., et al. 2019)[3] on elderly depression patients indicated that the reduction of negative emotions could significantly ease the non-emotional symptoms of depression and, consequently, decrease the negative impacts of depression. Hence, this year, the CCU-Taiwan iGEM team chose to tackle the problem of long-term negative emotions, which in turn will relieve depression and its serious consequences.
5-Hydroxytryptophan (5-HTP) is a natural compound that serves as a precursor to serotonin, a neurotransmitter crucial for regulating mood, sleep, and appetite (Maffei, M. E, 2021)[4]. Supplements containing 5-HTP are often used to alleviate symptoms of depression, anxiety, and insomnia by increasing serotonin levels in the brain (Turner, E. H., et al. 2005)[5].
However, conventional 5-HTP supplements rely on harvesting seeds from Griffonia simplicifolia, mainly cultivated in West Africa. This reliance raises sustainability concerns due to over-harvesting and habitat loss, impacting local ecosystems (Cunningham, A. B., et al., 2021)[6]. Fluctuating availability of Griffonia simplicifolia seeds also leads to inconsistent supply, affecting the reliability of these supplements. Moreover, variability in the potency and quality of natural 5-HTP extracts can result in unpredictable therapeutic outcomes for users, highlighting issues of effectiveness and reliability.
Our project aims to revolutionize serotonin supplementation by genetically modifying the probiotic Nissle 1917 (EcN) to produce 5-HTP directly in the gut. The 5-HTP produced will travel from the intestines to the brain through the bloodstream and be converted into serotonin. By enhancing 5-HTP synthesis locally in the gut, our genetically modified EcN could provide a reliable and accessible option for improving mental well-being.
In the serotonin biosynthesis pathway, tryptophan hydroxylase (TPH) catalyzes the formation of 5-HTP from L-tryptophan(Wang et al, 2018)[7]. TPH incorporates one atom of oxygen from molecular oxygen into the substrate and reduces the other atom to water. The two electrons required for reduction are supplied by oxidation of tetrahydrobiopterin (BH4) into BH4-4a-carbinolamine. The regeneration of BH4 from BH4-4a-carbinolamine is mediated by pterin-4a-carbinol-amine dehydratase (PCD) and dihydropteridine reductase (DHPR)(Pribat et. al. 2010)[8].
Accordingly, we will express the human THP1 protein, PCD protein, and the DHPR protein through polycistronic expression vector pST39 in EcN.
To enhance 5-HTP production, we applied a DNA scaffold to bring TPH1, PCD, and DHPR enzymes into close physical proximity. Accordingly, we fused Zinc finger binding domains Zif268, PBSII, and ZFa with TPH1, PCD, and DHPR enzymes, respectively (Giuseppe Raschellà et al,2017)[9]. The corresponding binding motif for each Zinc finger binding domain will be located on the DNA scaffold (Matthew P. DeLisa et al, 2012)[10].
To generate sufficient DNA scaffolds in EcN for assembly, we applied the rolling circle replication (RCR) mechanism to produce circular single-stranded DNA (ssDNA) containing sequences complementary to the binding motifs. The resulting circular ssDNA will automatically form the DNA scaffold through sequence complementation. To regulate the DNA scaffold formation, we applied the protocatechuate (PCA) induced promoter pPCA to control the expression of RepA, the replication initiation protein in RCR. Together, this will build a PCA-promoted 5-HTP biosynthesis pathway in EcN.
It is not easy to develop a habit of regularly consuming probiotics in this busy modern society. Therefore, we decided to combine our probiotic powder with a daily beverage in Taiwan, bubble tea. Bubble tea is a custom-made beverage that mixes various ingredients, such as tapioca balls, milk, fruits, jellied additives, etc. After mixing, the paper cup will be automatically sealed with plastic film to make it easy for customers to mix the ingredients and carry without spilling. We decided to use an edible film as a carrier of probiotics, which will be attached to the bottom of the sealing film. The edible film will be made by mixing starch, maltodextrin (Hsu, C., & Chiang, P., 2014)[11], and probiotic powder. The application of the edible film to the sealing film will be carried out by automated machinery with rollers and nozzles. This novel delivery method integrates our product seamlessly into daily routines, making mental health support more convenient and discreet than ever before.
Although the synthetic biology approach provides a direct answer to ease negative emotions, we recognized through research (Scientific Research Publishing. 2019)[12] and expert interviews that awareness of negative emotions and healthy interpersonal relationships also play a major role in people's mental health.
Since the location of our university, Chiayi County, is known to have the highest ratio of senior citizens in Taiwan, we cooperated with the village leader to share methods of identifying and processing negative emotions through creative activities with the elderly. We also cooperated with a physical therapist to hold a workshop using games to help the elderly learn how to support each other in an emotional emergency.
Moreover, we developed a stress-recognizing App called “POKEFISH” to reach more people efficiently. Through this App, users can measure their stress level simply by touching their finger to their phone camera. In the App, we also provide positive feedback (i.e., awards collection) and social connections (i.e., group broadcast) to encourage users to share their emotional status with trustworthy people.
The core ambition of the CCU-Taiwan iGEM team is to tackle the growing problem of depression and persistent negative emotions by enhancing serotonin synthesis. We genetically modify probiotic EcN to produce 5-HTP in the gut, providing a sustainable and accessible alternative to current supplements. Our approach optimizes 5-HTP synthesis using a DNA-mediated biosynthesis pathway and integrates this solution into daily life with an innovative edible film for beverages.
In addition to the synthetic biology strategy, we developed an app to help people monitor daily stress levels. We promoted mental well-being and society connection through workshops and activities for the elderly. Our project not only targets long-term negative emotions through 5-HTP-producing probiotics but also offers a sustainable, accessible solution, significantly impacting global mental health.