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Description

Description

Abstract

Depression is recognized worldwide and especially in Japan, it is one of the most influential diseases, causing annual losses of approximately 2 trillion yen [1]. However, the only established treatments for depression are expensive psychotherapy and drug therapy with heavy side effects, and these high-risk treatments pose a challenge. To overcome this disadvantage, we have developed an innovative method of self control by synthesizing antidepressants (crocetin, crocin, picrocrocin) derived from saffron (Crocus sativus) in E. coli and providing people with cookies containing E. coli that have accumulated antidepressant ingredients in the bacteria. The goal of the project was to provide people with an innovative means of self-control.

At the beginning of the project, our goal was to create a low-cost, stable platform for the production of antidepressants by having E. coli biosynthesize the target components under conditions that did not include special substrates and extracting them. However, based on the findings of Human Practices and laboratory experiments, we modified our aim to implement a low-cost, easy-to-consume self-control method by eliminating the growth potential of E.coli that had accumulated antidepressant components synthesized within the bacteria and including them directly in cookies. What we have achieved is as follows:

We have made a prototype of cookies that are very similar to the cookies we ultimately aimed to create, and we have received a lot of positive feedback from people with mild depression. Our cookies will provide depressed patients with a previously unestablished means of self-control for depression, thereby reducing the number of depressed patients and the number of people who become depressed, both in Japan and around the world. Just as taking vitamin C helps to tone the body, the time is near when mental health can be obtained in a simpler and more convenient way.

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Issue

Depression

Depression is a mental disorder characterized by deep sadness, and loss of motivation and interest in things, to the point that it affects patients’ daily lives. Psychotherapy, which corrects cognitive distortions and helps patients learn how to build relationships, is widely used.

Suicide is a major problem in Japanese society, as evident from the fact that Japan’s suicide rate is the highest among the G7 countries and suicide is the leading cause of death among the working population in their teens to 30s. Depression causes an economic loss of approximately 2 trillion yen in Japan, and data show that only 25% of those affected attend psychiatric care [2][3][4]. Governments and local authorities are also concerned about depression and have been conducting research and policies on suicide for some time.

According to a WHO survey, depression is a growing problem worldwide, with an estimated 322 million people worldwide suffering from depression in 2015 [5]. While this is more than 4% of the global population, many of these people have problems with correct diagnosis and appropriate treatment. In addition, previous studies have shown that 80% to 100% of people who commit suicides have a history of mental illness [5][6].　

Since mood disorders, mainly depression, are the most common mental disorder among suicide victims, it would be impossible to reduce the number of suicides without measures against depression.

Problems with Current Depression Treatment

Currently, there are two main types of treatment for depression: pharmacotherapy and psychotherapy.

Pharmacotherapy is the treatment of depression using drugs such as SSRIs (selective serotonin reuptake inhibitors), SNRIs (selective serotonin noradrenergic reuptake inhibitors), tricyclic antidepressants, and NaSSAs (noradrenergic and specific serotonergic antidepressants) to relieve depression.

One problem with drug therapy is the side effects specific to antidepressants, which are a major burden for depressed patients, although they vary depending on the type of drug and individuals.

Psychotherapy and counseling are mainly focused on preventing recurrence. Therefore, it is often conducted on the premise that it is compatible with pharmacotherapy. Specific treatment methods exist, such as behavioral cognitive therapy and interpersonal therapy, but both are expensive methods of treatment and place a heavy burden on patients.

Project

About the antidepressant component derived from saffron

Saffron is a plant produced mainly in Iran and other countries, and is also known as the most expensive spice in the world. We have focused on the substances crocetin, crocin, and picrocrocin, which are mainly produced from saffron to treat depression. These substances have been clinically confirmed to have similar or stronger antidepressant effects than existing antidepressants, but with lower side effects [7][8][9].

Japan, where Japan-United is based, has been producing saffron for 300 years and is known as one of the top high-quality and highly ethical producers in the world [10]. However, it is difficult to distribute saffron to the general public because its customers include Michelin Guide-recognized restaurants. This is mainly due to the nature of saffron itself.

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The issues listed above are also common in Iran, the main producer of saffron, where unethical production, such as child labor, has become an issue [10].

We therefore initiated this project with the idea that by using synthetic biology as a means to produce the target substance in a low-cost and stable manner, we could overcome the shortcomings of saffron itself, of which Japan boasts the highest quality, and provide a low-cost, stable, and ethical supply of saffron-derived ingredients with antidepressant effects.

What did we do?

Crocetin, crocin, and picrocrocin have been shown to have strong clinical effects as described above. Our ultimate goal is to market functional foods and provide a means of self-control for depression. However, since the mechanism of action of crocetin, crocin, and picrocrocin has not been clarified, we felt it necessary to clarify them, and predicted their mechanism of action using docking-simulation.

This achievement is a big step forward in the social implementation of our product.

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Biosynthesis of Antidepressant Components

In order to stably produce scientifically proven antidepressants at low cost, we aimed to biosynthesize antidepressant components derived from saffron, such as crocetin, crocin, and picrocrocin. In order to reduce the cost as much as possible, the biosynthesis was targeted using E. coli without any special substrates. This is a very challenging project because there have been four similar iGEM projects in the past, all of which have failed. In particular, no study has ever successfully demonstrated the in vivo biosynthesis of picrocrocin (regardless of substrate), making it a unique challenge.

In the end, we were the first iGEM team to successfully biosynthesize crocetin in E. coli without the addition of any special substrates. As for crocin, it failed to biosynthesize, although the DBTL cycle helped to solve the problems that arose. However, there is a great possibility of success by making improvements in the future.

DBTL cycle and Proof of Concept

At the beginning of our project, we aimed to create a low-cost and stable platform for the production of antidepressants using E.coli. However, when the results of our experiments with E.coli began to emerge, we realized that there was a big obstacle to overcome in terms of social implementation.

Among the components we were aiming to produce, carotenoids (crocetin, crocin) required four times the amount of methanol and chloroform as the culture medium when extracted. Even if we could produce a sufficient amount of antidepressants for social implementation, the cost of extraction would be too high, and the advantage over existing antidepressant therapies would be weakened.

While we were wondering what to do, we noticed the news that Synlogic, which aims to use gene-edited E.coli for human consumption as a treatment for phenylketonuria (PKU), advanced to Phase III clinical trials in June of this year. We then engaged in discussions with various stakeholders, including Akaito, Limited and Fermelanta, Inc. Finally, we thought that E.coli, which was designed to synthesize antidepressants and accumulated antidepressants in the body, could be offered to society as a low-cost, easy-to-consume, self-control measure by directly including it in cookies as a functional food, and we started a new project.

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When we researched projects for social implementation at Entrepreneurship, we realized that the biggest obstacle for us was the Cartagena Act. In order for people to consume genetically modified microorganisms, it is necessary to clear regulations through safety inspections, including inspections of production facilities [11]. We focused on the fact that the Cartagena Act regulates the “proliferation” of genetically modified organisms, and hypothesized that if we could eliminate the growth potential of E. coli that has accumulated antidepressant components, we could lower the regulatory hurdle for human consumption of genetically modified E. coli. We began working on additional projects based on the hypothesis that if we could eliminate the ability of E.coli to grow, which had accumulated antidepressants, we would be able to lower the regulatory hurdle for human consumption of genetically modified E.coli.

What do we do?

When we realized the high cost of extraction, we held discussions with various stakeholders. We discussed or surveyed companies that actually produce saffron, companies aiming to produce active ingredients with microorganisms, experts in the treatment of depression, companies producing allergen-free confections, companies that have successfully implemented genome-edited foods in society, the general public, medical professionals, etc. We learned about the side effects of antidepressant drugs on patients, the prejudice against depression, and considered foods that are easy for patients to consume. These were very important experiences for making our project more socially relevant.

Also, very few people are discussing our hypothesis, the concept that the regulatory hurdle could be lowered by eliminating the ability of genetically modified organisms to multiply. We believe it is important to have these discussions not only with those in congress who create regulations, but also with a diverse public. We have had discussions on the above concepts with local legislators and with multiple generations through our Education activities, and have received many comments that it is important to have understandable explanations for genetically modified organisms in a way that makes sense to them. Through these discussions, we thought that it is important to gain the trust of more people, not just experts, in future regulation making.

Substance production with E.coli Nissle 1917 strain

The BL21 strain traditionally used for substance production using microorganisms is not suitable for direct human consumption from a probiotic point of view. Therefore, in line with our goal, we aimed to synthesize an antidepressant component derived from saffron using the E.coli Nissle 1917 strain, which is approved for human consumption by the FDA.

If we succeeded, the extraction step would no longer be necessary, thus enabling us to achieve our ultimate goal: cookies as a low-cost, easily ingestible means of self-control.

Finally, we have succeeded in producing carotenoids with E.coli Nissle 1917.

what is E.coli Nissle 1917 strain?

It is an E. coli originally isolated from the feces of a soldier who did not develop diarrhea during a severe outbreak of dysentery during World War I. It has been used in Europe for 90 years to treat inflammatory and infectious bowel diseases, and has been commercialized by Ardeypharm GmbH as Mutaflor® and administered to adults and infants as a probiotic therapy to treat intestinal infections and various inflammatory diseases of the gut The product has been administered to adults and infants. Its safety has been proven in more than 4,500 patients, with an incidence of minor gastrointestinal side effects of only 2-3%.

Due to its genetic tractability, there is currently growing interest in developing engineered therapies using the E. coli Nissle 1917 strain as a chassis, led by Synlogic. Various applications have been reported, ranging from species-specific antimicrobial agents to treatments for metabolic disorders and biosensors.

We focused on E.coli Nissle 1917 strain as a Drug Delivery System because it is safe from a probiotic perspective.

Creation of Non-proliferative E. coli

We introduced a plasmid that expresses a selective restriction enzyme on E. coli chromosomes into E. coli that has a plasmid with an enzyme related to the production of antidepressant components. We expected that induction of that restriction enzyme would result in degradation of the chromosome into multiple fragments, and that subsequent degradation by endogenous nucleases would result in complete chromosome degradation.

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We confirmed that the E. coli strains we designed no longer proliferate in both the BL21(DE3) and E. coli Nissle 1917 strains.

Since our strategy is standardized and the loss of growth ability allows for taking them outside, the application possibilities are vastly expanded and could be an innovative tool for the social implementation of various iGEM projects.

References