Depression is a very common but serious mental disorder in modern society. The patients may suffer from persistent sad moods, anxiety, irritability, difficulty concentrating, loss of interest, and even develop aches or pains without clear physical cause. Based on the latest research, around 24.6% of teenagers and 18.5% of university students were diagnosed in 2019.
     Depression could be long-term or repeat-attacked, which severely influences the everyday life of patients. Nowadays, treatments for depression can be divided into three categories: medications, psychotherapies, and brain stimulation therapies such as electroconvulsive therapy. To have it under control, systematic and standardized treatment with continuous maintenance of drug dosage is highly recommended in clinical practice.
    However, prolonged medication intake has many drawbacks. For critical patient that requires life-long medication, long-term drug use will bring them a double burden both economically and psychologically and may cause drug abuse or resistance to antidepressant drugs, which is not conducive to long-term and sustainable treatment. Meanwhile, for mild and moderate patients, long-term use of antidepressants may also do more harm than good. Thus, innovations in antidepressants are of urgent requirement.

    Recently, more and more research has shown the importance “Brain-gut axis” plays in various kinds of disease mechanisms, including the regulation of the central nervous system. It’s also found that the gut microbiota is remarkably different in depression patients, which show severe intestinal dysbiosis. Several studies have shown that gut probiotics play an important role in maintaining mental health and fighting depression. What’s more, it has been discovered that SAMe, a molecule naturally synthesized in cells, can alleviate symptoms of depression. SAMe has been marketed in several European countries since the mid-1980s for the treatment of depression. SAMe has similar efficacy to prescription antidepressants in the treatment of depression. And experimentally, SAM-E biosynthesis is very simple.
    Inspired by this, we have designed one kind of engineered bacteria that can colonize the human intestine after oral uptake to achieve a prolonged delivery of SAMe, which avoids frequent dosing and can apply an effective control relatively longer over depression.
    This engineered bacterium has been designed for long-term drug administration based on the intestinal probiotics Bifidobacterium longum. In addition to efficiently exerting the multiple antidepressant functions of probiotics, our engineered bacteria can also produce SAMe in a long-term, regular, and quantitative manner for the treatment. We plan to prepare a special product form, such as probiotic chocolate, and after patients’ intaking, the engineered bacteria will colonize in the intestines, having safety switches to prevent drug leakage. What’s more, taking special molecules can also stop drug delivery according to the patient's wishes.

    Recently, more and more research has shown the importance “Brain-gut axis” plays in various kinds of diseOur project uses synthetic biology methods to develop a kind of probiotic that could colonize the human gut and periodically express and secrete drugs. The drug we utilized, named SAMe, is a natural intracellular product with the same effect as prescription antidepressants, while SAMe acts milder and does not cause nerve resistance or drug abuse. We limited the possible side effects of the drug by delivering the drug periodically and efficiently, and at the same time maintaining appropriate blood concentration to achieve treatment. In addition, our product, as an engineered probiotic for regular long-term drug supply, can serve as a platform that provides a more convenient way for drug delivery in the future. By replacing the genes of the drug and transporter, this engineered system can be an efficient drug delivery platform, the industrialization and commercialization prospects of which are very broad

    In recent years, with the research on the human microbiome in various domains, it has become a hot spot in the field of biomedicine. Since 2007, China has continuously strengthened funding in the field of microbiome and microecology through the National Natural Science Foundation of China, the "973" program, the "863" program, the Strategic pilot Science and Technology Special Project of the Chinese Academy of Sciences, etc. The research and development funds for intestinal microecology are nearly 400 million RMB every year, with a focus on intestinal microecology.
    In recent years, more and more research evidence suggested that intestinal microecology can affect the nervous system and play an important role in the mental and cognitive systems. Therefore, developing a probiotic antidepressant that can benefit gut microecology is a promising option.
    Our product uses bifidobacterium longum as a probiotic chassis, which is expected to regulate the intestinal ecology and relieve the symptoms of patients with depression. With the deepening understanding of intestinal ecology in the scientific community, probiotics have been recognized by most countries, whether as health products or drugs. GRAS（generally recognized as safe）of the FDA(U.S.
    Food and Drug Administration), the European Pharmacopoeia, and “the strain that can be used in food” issued by the Chinese Ministry of Health have all identified Bifidobacterium longum as an effective and safe probiotic and encourage its use. However, the current application of bifidobacterium longum is limited to regulating intestinal health in general. Its function of specifically affecting the brain-gut axis to ease depressive symptoms has not been fully developed. Currently, only one genetically modified bifidobacterium longum product developed for the treatment of cancer is in clinical trials. There are no genetically modified probiotics on the market. Our product will fill the gap with unique advantages. In “The Declaration and Review of Probiotic Health Food” issued by the Administration for Market Regulation of China in 2019, Articles 74 and 75 point out that genetic modification of probiotic strains is an important new direction for the probiotic industry. Banning the use of genetic engineering in the field of probiotics goes against China's strategy of invigorating the country through science and technology, especially in the field of biotechnology and synthetic biology. Therefore, this product innovates new products while conforming to the national strategy, and has a stable guarantee and broad prospect.
    SAMe has been available as an over-the-counter dietary supplement under the Dietary Health and Supplements Act of 1999. Through the clinical studies on it, the US FDA's Agency for Healthcare Research and Quality (AHRQ) concluded that SAMe has similar efficacy to prescription antidepressants in the treatment of depression. In” the Chinese Guidelines for the Prevention and Treatment of Depressive Disorders (Second edition)”, experts designated SAMe as class B recommended drug. It follows that SAMe has been largely approved as an antidepressant.

1.The market for probiotics is vast and developing rapidly
    The global Probiotic Market is expected to reach $57.4 billion by 2022, according to the Probiotic Market Report, with a compound annual growth rate of 7.7% from 2016 to 2022.
    As the chart above shows, the global probiotic market has been expanding in size from 2012 to 2023, with three major products: probiotic food and beverages, probiotic dietary supplements, and probiotic animal feed, respectively, gaining market share. Food and beverage apps now dominate global demand, worth more than $30 billion in 2015. In the same year, infant formula apps in China alone generated nearly $29 million in revenue and will continue to grow at a high rate until 2023. It can be speculated that the increase in the per capita expenditure on maintaining the health of the intestinal system should be conducive to the growth of the probiotic market. It can be seen that probiotic products have a fast development speed and a good and broad development prospect.

2. Probiotic drugs are favored by capital
    The market demand for probiotics is huge, which cannot be separated from the vast development market and huge capital flow input. According to incomplete statistics, more than 30 micro-ecological drug companies around the world have raised more than 1.9 billion dollars, which can promote the research and development of hundreds of pipelines. In 2019, nearly $1 billion went to
    Finch Therapeutics, Vedanta Biosciences, Azitra, Biomx, DayTwo, and others. The global microbiological therapeutics market was valued at US $1.13 billion in 2018 and will grow at a compound annual growth rate (CAGR) of 19.03% over five years to reach US $2.7 billion by 2023, a forecast well reflected by the Capital Markets, according to an analysis by Us-Based Research and Markets. These data reflect the fact that the capital market is quite optimistic about the development of probiotic products. The influx of capital makes the industry dynamic and full of possibilities, which is conducive to the accumulation of important production resources for companies to fully tap the potential and opportunities of their products with the support of the capital market. We are confident that we can expand our development with financial support.

3. There are many gaps to be filled in the development of probiotic drugs, and technological breakthroughs can bring huge profits
    Despite the huge market demand and abundant financial support, as an emerging industry, there are still many gaps to be built and filled in the development of probiotics. So far, there have been hundreds of pipelines under development for microecological drugs, but none of them have succeeded. So far, however, no drug has been successfully approved by the FDA， but the existence of challenges also means the existence of opportunities. We are now facing a promising blue ocean field. Once we take the lead in achieving technological breakthroughs, we have the perfect opportunity to become a pioneer or leaders in the industry, and as a leader, we can shine in the development of probiotic drugs and win huge profits.

4. There is no probiotic drug for depression in the development process, and the product can achieve a monopoly
     At present, many biopharmaceutical companies in the world are trying to develop the probiotic market. As shown in the chart above, their research and development types focus on (CD&BD) and cancer, but there are no probiotic drugs in development for depression. Our products focus on strengthening and amplifying the natural antidepressant effects of intestinal probiotics and are more effective and scientific by simulating patients' manual dosing with an improved periodic dosing process. However, there is no rival product of anti-depressant probiotics in the current market, which is an advantage for us. It means that once our products are commercialized smoothly, we can achieve a monopoly and hold the priority position in the market.

    The first use of probiotics being dated back to 2000BC when man discovered long-period milk preservation without actually realizing microorganisms’ existence, the history of probiotics could almost go parallel with the evolution of the human race. After centuries of development, as the positive effect of probiotics on the human body is gradually uncovered, applications and research have been extended from fermentation to human health maintenance, intestinal microbiome, nervous system regulation, and disease mechanism. The publishments and citations of probiotic-related research emerge after entering the 21st century, covering almost every aspect of human life.
     Along with the booming research progress of probiotics throughout the globe in recent years, probiotics products are gradually accepted and favored by consumers, constituting a constantly growing multi-billion-dollar industry. Not only does medication using probiotics be explored in clinical practice, but more and more foods such as yogurt, cheese, snacks, cereals, and formulas are claimed to have supplements of probiotics for better health effects, as are cosmetic products and daily necessities such as toothpaste. With the current global market size of about 40 billion euros, most probiotics products in developed countries have formed a more mature market. The product size is also estimated to be nearly 85 billion yuan in China, with demand increasing significantly and the industry ushering in rapid development constantly. This trend was even fueled up after the COVID-19 pandemic outbreak in 2020, which deeply reinforced the recognition and emphasis on immunity supplements, making the probiotic industry buck against the economic downturn. Combining the probiotics and others such as medication delivery and therapy would be a growing general trend.
    As one of the most common and researched psychological disorders, depression has formed a relatively complete treatment system. Among multiple methods such as cognitive behavioral therapy (CBT), and interpersonal therapy (IPT), regular medication is mostly applied and nearly basic, highly recommended by clinical practice. However, for patients who need to take antidepressants for life, long-term medication may bring them double burdens both economically and psychologically, and might cause drug abuse or resistance to taking antidepressants, which is not conducive to long-term sustainable treatment. And for mild to moderate patients, long-term use of antidepressants may do more harm than good. A survey delivered by Claire et. showed antidepressant treatment may bring several side effects, including insomnia, discontinuation effects, weight gain, sexual dysfunction, and forth. Around 30.7% of participants even reported moderate-to-severe depression while on antidepressants. These adverse effects reflect on several emotional effects such as “feeling not like me”, and “reduced positive feelings”. The same results are also shown in our investigation when interviewing some patients participating in our online meeting. There is a growing need for a new antidepressant with fewer side effects and a shorter frequency of use.
    Research has been conducted on the role probiotics played in both depression pathology and therapy. The gut microbiome profiles of depression patients were found to be significantly different from healthy individuals, suggesting severe intestinal dysbiosis in the former. Thus, therapy based on the regulation of the gut microbiome, especially probiotics is getting more and more popular. Several studies have shown that gut probiotics play an important role in maintaining mental health and against depression by up-regulating the level of some neurotransmitters or secreting some anti-inflammation substance. Through the brain-gut axis, a new treatment for major depressive disorder (MDD) combining probiotics and antidepressants would shed new light on depression therapy.

    Probiotics are living microorganisms that have been proven to be beneficial to the health of their host. In recent years, probiotics is seen as a promising strategy to treat different diseases. Some of the commonly used probiotics are Escherichia coli Nissle 1917, Saccharomyces boulardii, and species belonging to the Lactobacillus and Bifidobacterium genera. In recent years, these wild-type probiotics have been assessed in numerous clinical trials both individually and in combination. Meta-analysis of these clinical trials has shown that probiotics are effective in preventing or alleviating the symptoms of a variety of diseases. There are many probiotic forms including conventional methods such as tablets, capsules, and powders, and non-conventional methods such as cheeses, yogurts, and milk. However, up to now, freeze-dried probiotic has been the preferred product form, as their processing technique preserves viability to the greatest extent.
    Genetically modified to target specific diseases with the methods of synthetic biology, engineered probiotics can lead the next generation of live biotherapeutics. Engineered probiotics provide breakthroughs for the treatment and diagnosis of some difficult diseases such as cancer, infection, and inflammation.
    In addition, some probiotics such as certain strains of Bifidobacterium longum have been reported to have great potential in alleviating the symptoms of various mental disorders through the microbiota–gut–brain axis (MGBA). Currently, the normally chosen antidepressant is selective serotonin reuptake inhibitor (SSRI) because of its favorable risk‐benefit ratio. In the case of people with depression who have not responded to a first SSRI antidepressant after six to eight weeks of adequate treatment, switching to an alternative antidepressant may be considered. This may be a different SSRI or a better‐tolerated newer‐generation antidepressant. Subsequently, an antidepressant of a different pharmacological class may be chosen, although this may be less well tolerated, for example, venlafaxine, a tricyclic antidepressant (TCA), or an MAOI. Given the extent of the burden of depression, the high rates of chronicity and the high number of people who do not respond to conventional treatments, the exploration of the therapeutic role and potential of probiotics in depressive disorder is extremely noteworthy and there is an urgent need to examine alternative medications.
    Fortunately, some new antidepressants are on the way. S‐adenosyl methionine (SAMe) is naturally synthesized in the cell and there is evidence that it is effective as a prescription antidepressant. SAMe has been marketed in some European countries since the mid‐1980s for the treatment of depression and other medical conditions. It benefits a wide range of people and has corresponding social significance. Inspired by that, we design an engineered bacteria product based on the probiotic Bifidobacterium longum, which can colonize in guts and synthesize SAMe periodically after our modification, as a drug-delivery platform for regular long-term administration. In contrast to controlled-release capsules, our product can secrete drugs continuously. As the engineered bacteria continue to synthesize drugs, the total amount of drugs secreted in the blood is closely related to their density and metabolic level. If the amount remains low, the therapeutic effect will not be achieved, while it remains too high, the patients will suffer from side effects. With an oscillator, we can maintain appropriate blood concentration and limit the possible side effects to a short period by delivering the drug periodically and efficiently. With parallel kill switches, we can achieve voluntary termination of drug delivery and ensure biosafety. We intend to take yogurt, candy, or chocolate as the final forms of our product, in hope of bringing happiness to patients with depression and reducing their resistance to medication.
    Considering the feature that our product can secrete drugs periodically, our project will serve as a platform to provide a more convenient way for future drug delivery. By replacing the genes of drugs, it can be highly commercialized for the remission and treatment of various chronic and difficult diseases.

    Since our product requires oral administration into the body to secrete the drug in the intestine, it may be difficult to be accepted by society. And engineered bacteria may also have the potential risk of leaking into the environment.
    Both the FDA (U.S. Food and Drug Administration) and the Chinese Ministry of Health have identified Bifidobacterium longum as a safe probiotic that can be taken orally, and there are now Bifidobacterium longum in the market for commercialization. There are now many products containing Bifidobacterium longum registered as food on the market, and Bifidobacterium longum is one of the most used strains among the many probiotic strains. Through the clinical studies on it, the US FDA's Agency for Healthcare Research and Quality (AHRQ) concluded that SAMe has similar efficacy to prescription antidepressants in the treatment of depression with fewer adverse effects.
    Not only our chassis bacteria and the drug are safe, but our engineered bacteria also contain a safety switch module. If the cycle of drug production of the engineered bacteria is disrupted or the patient's condition has improved, the patient can kill the engineered bacteria by taking AHL orally, which is harmless to the human body, so the patient does not need to worry about the engineered bacteria in the intestine will not under control.
    Also, if the engineered bacteria are eliminated via the faces, a safety switch will be activated to kill the engineered bacteria, which can effectively prevent the engineered bacteria from leaking into the environment.

    The global market scale of the probiotics industry is developing rapidly, and the probiotics industry in North America, Europe， and other regions have complete systems and occupy a dominant position in the global market. Some international companies such as Lallemand, Bio Gaia， and Danisco already have well-proven production technology and low production cost.
    However, there are still few probiotic products for depressive disorders in the market, so we do not have many competitors and we can fill the gap in the market if our product is successfully commercialized. We believe that after a period of technological improvement we can also reduce production costs and lower the price of our product to attract more consumers.

    Our product is positioned as a health product with a wide audience and can be taken by patients with mild depression and major depression. Due to the low adverse effects of SAMe, patients with mild depression can take it to relieve their condition. Since our engineered bacteria can keep secreting the drug in the body for a long period, patients can take the drug at long intervals, and patients with major depression who need to take the drug frequently and repeatedly can also take our product.
    Traditional antidepressants generally require frequent long-term dosing, which can cause some psychological burden on patients. Because, like many mental illnesses, major depressive disorder is recurrent and about 2/3 of people will relapse, NICE recommends that patients should continue to take antidepressants for 6 months after recovering from a depressive episode, and extend to 2 years if the patient has experienced two or more depressive episodes. However, our product does not require frequent dosing and is expected to be given as a snack, thus it will reduce the mental burden of the patient.

    The probiotics industry is in a promising direction for growth. According to Statista, the market size of the global probiotics industry shows year-on-year growth from 2016 to 2020. the global market size of probiotics in 2016 is $42.8 billion, and the global market size of probiotics grows to $56.6 billion by 2020, up 7.6% from the previous year.
     Our combination of the antidepressant effect of probiotics and drugs is an innovation with unlimited future development possibilities. We expect to advertise the antidepressant effect of Bifidobacterium longum itself and the medicinal effect of SAMe. Bifidobacterium longum secretes substances such as short-chain fatty acids and regulates intestinal flora, while our engineered bacteria also secrete the antidepressant drug SAMe, which can alleviate depression through multiple pathways. At the same time, our product does not require frequent medication, which can attract more patients and is very promising.

    With economic development and the boom of microbial health science sweeping the world, there is growing consumer demand for probiotic products, but not many companies are putting insights into microbial therapy for depression, we might take over the market and take action to protect the proprietary technology.
    Our biosynthesis of the effector molecule SAMe is advancing and is hard to replace. It is easily synthesized within microorganisms. The synthesis of SAMe from L-methionine requires only one step reaction and the raw material is easily available from the cytoplasm of microorganisms. In addition, SAMe has fewer adverse effects compared to other antidepressants.