Description

Inspiration

Anxiety is spreading among us China high school students, seeping into every corner of our lives. According to a study published in Frontiers, In Shandong Province, the prevalence of anxiety symptoms among high school students during the COVID-19 pandemic was found to be 31.4%, with moderate to severe symptoms in 4.6% of students. After being shocked by this set of data, we conducted a deeper investigation. We have noticed that this problem isn’t just confined to the high school students. According to WHO survey, an estimated 4% of the global population currently experiencing anxiety disorder, and in 2019, 301 million people of the world population were suffering from it.

The traditional treatment methods for anxiety disorders mainly include medication and psychotherapy. Medications, such as benzodiazepines and SSRIs, can quickly alleviate symptoms but often come with side effects. Some medications also have a strong potential for dependence, and patients may experience withdrawal symptoms after discontinuation. In terms of psychotherapy, cognitive behavioral therapy (CBT) helps patients adjust negative thinking, but its effectiveness varies among individuals and typically requires a longer time to show results. Some patients may feel disappointed if they do not achieve the expected outcomes from traditional treatments. Despite the effectiveness of traditional methods in alleviating anxiety, their side effects and limitations have prompted a search for safer alternatives.

In our quest for relief, we turned to theanine, a compound found in tea leaves.

Figure 1. L-theanine is an amino acid commonly found in tea leaves, especially in green tea leaves.

Introduction to Theanine

L-theanine, molecular formula C7H14N2O3, is a non-protein amino acid found in tea leaves, especially green tea (Camellia sinensis). It has a unique flavor of umami with applications in food, and has been applied as safe additives in medicine, healthcare, and other food industries.

L-theanine is also scientifically known as γ-ethylamino-L-glutamate. Its chemical structure is closely related to glutamate, the primary excitatory neurotransmitter in the central nervous system. L-theanine and glutamate share a similar backbone, consisting of an amino group, a carboxylic acid group, and a side chain. However, L-theanine differs in having an ethylamide group (-CH2CH3) attached to the alpha carbon, which distinguishes it from glutamate.

Figure2. The chemical structures of L-Theanine and L-Glutamate. L-theanine differs in having an ethylamide group (-CH2CH3) attached to the alpha carbon.

L-Theanine's Role in Mitigating Anxiety

This structural similarity allows L-theanine to interact with glutamate receptors in the brain, and this interaction is believed to contribute to L-theanine's anxiolytic effects by modulating the activity of the glutamatergic system, which is involved in the regulation of stress and anxiety.
L-theanine suppresses the glutamine transporters that are responsible for bringing glutamine into neurons. By inhibiting these transporters, L-theanine reduces the conversion of glutamine to glutamate, leading to lower levels of glutamate within neurons. With less glutamate available, there is a decreased release of this excitatory neurotransmitter in response to stimuli. This reduction in glutamate signaling can help calm overactive neurons and may contribute to the relaxation effects associated with L-theanine.
L-theanine also interacts with glutamate receptors, such as NMDA and AMPA receptors. By acting as an antagonist at these sites, it can modulate the effects of glutamate, potentially reducing neuronal excitability and preventing overstimulation. The overall effect of L-theanine is to help balance neurotransmitter levels, particularly by reducing excess glutamate activity, which is implicated in anxiety and stress responses. This balance promotes a sense of calm and may help to mitigate anxiety symptoms.

Figure3. The mechanism of anxiety relief by L-Theanine, referenced from Wang et al, 2022.

L-theanine has been shown to improve attention and working memory, as well as reduce mind-wandering, which can contribute to a sense of calm and focus (Sohail et al, 2021).

A randomized controlled trial examining the effects of L-theanine on stress-related symptoms and cognitive functions in healthy adults found significant improvements in self-reported depression, anxiety, and sleep quality, along with enhancements in verbal fluency and executive function (Hidese et al, 2019). These findings suggest that L-theanine may have therapeutic potential for alleviating anxiety and improving cognitive function.

Conventional Producing Methods

Conventional synthesis methods for producing theanine has several drawbacks. The plant extraction method suffers from low yield and purity, high material costs, and a risk of contamination, which can lead to significant loss of theanine. The chemical synthesis method requires costly separation processes and results in low product yield. Similarly, the enzymatic synthesis method is hindered by high substrate costs and low efficiency due to the complexity of the catalytic systems involved.

All of the problems above prompted us to adopt the synthetic biology method: we aim to construct a de novo synthetic pathway in E.coli for theanine production, which is sustainable, cost efficient, and high-yielding.

Our Solution

Inspired by innovative research, we designed a pathway - employed five enzymes, which are γ-glutamylmethylamide synthetase (GMAS) from Methyloversatilis universalis, polyphosphate kinase (PPK) from E. coli, alanine transaminase from Bacillus subtilis (BsAld), alanine decarboxylase from Camellia sinensis (CsAlaDC), glutamine permease from Saccharomyces cereviside (GNP1) - in E. coli BL21.

Since synthetic theanine requires glutamate, ethylamine, which are used as precursors, and ATP, we have devised the following plan.

We planned to convert pyruvate, produced in the glycolysis, into ethylamine, utilizing CsAlaDC and BsAld, then use GMAS to catalyze it with glutamate, generated in the TCA cycle, to produce theanine. Meanwhile, we have designed an ATP regeneration system by utilizing PPK to convert ADP into ATP, and a theanine transport system utilizing GNP1

Figure4. Pathway we designed for producing theanine, including the employing of five enzymes, illustrating the mechanism of ATP regeneration system and theanine transport system.

Product Design

Producing the substance is not yet the end of our journey. Delivery to the end-users are also important. Our team conceived and developed a series of end-user products utilizing theanine. These include a humidifier that releases theanine into the air and chewy candies with additive food-grade theanine obtained from safety and certified suppliers.

We conducted relevant Human Practices (HP) activities and gathered insights on public preferences and concerns regarding new anxiety treatment products through surveys. Among the preferences for products related to theanine, such as capsules, energy bars, candies, pre-packaged beverages, instant mixes, and assistive devices (like inhalers and humidifiers), candies received the highest votes, accounting for 73.74%.

Our first product is a humidifier that releases theanine into the air. It works by using an ultrasonic generator to turn the liquid inside the device into a mist containing dissolved theanine. We believe that this design allows anxiety patients to unknowingly intake theanine through their respiratory mucosa, helping to alleviate anxiety.

Additionally, we completed research on the formulation of theanine candies in the kitchens. After considering factors such as candy shape, theanine content, and flavoring of inactive ingredients, we developed various flavored candy formulations through multiple trials. To ensure the appropriate intake of theanine, we designed and created a timed candy dispenser using 3D printing technology, assisting anxiety patients in consuming the correct dosage at suitable times.

Refer to our Hardware page for more details of the design, build, test and learn of all the products (Hardware ).

Beyond these products, our team envisions a new application of theanine that could further aid patients in alleviating anxiety. We plan to construct a synthetic pathway for theanine in E. coli Nissle1917 and launch the product in capsule form. By having anxiety patients regularly take these engineered bacterial capsules, we aim to maintain a reasonable quantity of E. coli Nissle1917 capable of biosynthesizing theanine in the alimentary canal. This engineered strain will synthesize an appropriate amount of theanine when detecting changes in biomarkers, which indicate anxiety episodes. For safety reasons, the engineered bacteria should be designed with relevant safety features, including but not limited to: the engineered bacteria should not have the ability to reproduce, ensuring that effective amounts can only be obtained by taking capsules regularly.

References

[1] Cao, R., Hu, S., Lu, Y., Wang, W., Fu, Z., & Cheng, J. (2023). Fermentative Production of L-Theanine in Escherichia coli via the Construction of an Adenosine Triphosphate Regeneration System. Fermentation, 9(10), 875–875.
https://doi.org/10.3390/fermentation9100875

[2] Wang, L., Brennan, M., Li, S., Zhao, H., Lange, K. W., & Brennan, C. (2022). How does the tea L-theanine buffer stress and anxiety. Food Science and Human Wellness, 11(3), 467–475.
https://doi.org/10.1016/j.fshw.2021.12.004

[3] Hidese S, Ogawa S, Ota M, Ishida I, Yasukawa Z, Ozeki M, Kunugi H. Effects of L-Theanine Administration on Stress-Related Symptoms and Cognitive Functions in Healthy Adults: A Randomized Controlled Trial. Nutrients. 2019 Oct 3;11(10):2362.
https://www.mdpi.com/2072-6643/11/10/2362

[4] Anas Sohail A, Ortiz F, Varghese T, Fabara SP, Batth AS, Sandesara DP, Sabir A, Khurana M, Datta S, Patel UK. The Cognitive-Enhancing Outcomes of Caffeine and L-theanine: A Systematic Review. Cureus. 2021 Dec 30;13(12):e20828.
https://doi.org/10.7759/cureus.20828

[5] Kakuda, T., et al. (2002). "Inhibitory effect of theanine on the binding of [3H]AMPA, [3H]kainate, and [3H]MDL 105,519 to glutamate receptors in rat cortical neurons." Biosci Biotechnol Biochem. 66 (12): 2683-2686.
https://pubmed.ncbi.nlm.nih.gov/12596867/

[6] Nathan, P. J., et al. (2006). "The neuropharmacology of L-theanine(N-ethyl-L-glutamine)." J Herb Pharmacother. 6 (2): 21-30.
https://pubmed.ncbi.nlm.nih.gov/17182482/

[7] Kakuda, T., et al. (2002). "Neuropsychopharmacological effects of theanine." Nutrition. 18 (9): 843-846.
https://www.sciencedirect.com/science/article/pii/S0899900702900114

[8] Kimura, K., et al. (2007). "L-Theanine reduces psychological and physiological stress responses." Biol Psychol. 74 (1): 39-45.
https://pubmed.ncbi.nlm.nih.gov/16930802/

[9] Wang, L., Brennan, M., Li, S., Zhao, H., Lange, K. W., Brennan, C., & ... (2022). How does the tea L-theanine buffer stress and anxiety. Food Science and Human Wellness, 11(1), 467-475.
https://doi.org/10.1016/j.fshw.2021.12.004

[10] Zhang, Zeng, et al. (2020). “Prevalence of Depression and Anxiety Symptoms of High School Students in Shandong Province during the COVID-19 Epidemic.” Frontiers in Psychiatry, vol. 11, 21 Dec. 2020.
https://doi.org/10.3389/fpsyt.2020.570096.