Fluoxetine and paroxetine are common drugs at present.

  Fluoxetine: It can be used for various depression patients, as well as patients with psychiatric disorders such as obsessive-compulsive disorder, but fluoxetine can cause some negative effects of abnormal sleep, abnormal vision or difficulty breathing.

  Paroxetine: The effect is fast, but there may be a transition to manic episodes, and rapid discontinuation of paroxetine can lead to adverse reactions.

  Since scientists demonstrated the existence of a gut-brain axis, they have studied the neurological and mental effects of gut microbiota on the brain. Numerous studies have shown a significant relationship between depression and gut microbiota. 5-HTP and γ-aminobutyric acid (GABA) produced by gut microbes can effectively help patients with depression recover from depression. Based on the results of this study, we designed a synthetic biological strain for 5-HTP and γ-aminobutyric acid production based on Escherichia coli Nissle1917.

  Glutamic acid decarboxylase can promote bacterial production of GABA, human tryptophan hydroxylase can promote bacterial production of 5-HTP. We engineered E. coli 1917 to add genes that overexpress these two substances to produce large amounts of GABA and 5-HTP. By delivering these bacteria to the gut of depressed patients, we can help them alleviate or get rid of depression. There are other issues we need to address. In order to protect the environment and life from GM contamination, we consider designing a suicide system to prevent the escape of the strain.

  In addition, after consumers take our probiotics, the probiotics will produce GABA and 5-HTP. When needed, when we drink tea, the tea will activate the lysis system, and more GABA and 5-HTP will be released into the intestine after bacterial lysis.

5-HTP Background

  5-HTP is an amino acid substance that can be used as a precursor of 5-HT in the human body. Studies have shown that 5-HTP can increase the concentration of serotonin in the brain, promote the formation of melatonin, improve the patient's mood, improve sleep quality, and relieve pain.

Specific synthesis process

  5-HTP, also known as 5-hydroxytryptophan, is the chemical precursor of serotonin, a neurotransmitter that regulates a series of metabolic and psychological functions in the human body. The human body produces serotonin through the following pathways: tryptophan → 5-HTP → serotonin. 5-HTP is decarboxylated to serotonin (5-hydroxytryptamine or 5-HT) by the enzyme aromatic-L-amino-acid decarboxylase with the help of vitamin B6. This reaction occurs in nerve tissue and the liver. The human tryptophan hydroxylase 1 (TPH1) gene was overexpressed in Nissle 1917 (EcN) to encode tryptophan hydroxylase 1, which catalyzes the production of tryptophan in humans. Tryptophan hydroxylases catalyze the biopterin-dependent monooxygenation of tryptophan to 5-hydroxytryptophan (5-HTP), which is subsequently decarboxylated to form the neurotransmitter serotonin (5-hydroxytryptamine or 5-HT). It is the rate-limiting enzyme in the biosynthesis of serotonin.

Y-aminobutyric acid (GABA) Background

  GABA amino acid is an important central nervous system inhibitory neurotransmitter with good water solubility and thermal stability. GABA is safe to eat and can be used in the production of beverages and other foods. GABA can block the information exchange between nerve cells and the brain or spinal cord, and reduce fear, anxiety and stress by blocking certain nerve signals, thereby achieving physiological effects such as improving sleep quality and lowering blood pressure.

Specific synthesis process

  We plan to produce γ-aminobutyric acid (GABA) by overexpressing the GadB (amino acid decarboxylase B) gene in EcN by converting glutamate to GABA [3]. GABA is primarily synthesized from glutamate by glutamate decarboxylase (GAD) with pyridoxal phosphate (the active form of vitamin B6) as a cofactor. This process converts glutamate (the principal excitatory neurotransmitter) to GABA (the principal inhibitory neurotransmitter). After synthesizing the two substances, our team used the double terminator BBa_B0015, which is a double terminator composed of BBa_B0010 and BBa_B0012, with the best termination effect.

Facing Problems

  Both GABA and 5-HTP are in the cell. Although GABA and 5-HTP can be transported by themselves across the membrane, the secretion rate of GABA and 5-HTP by the strain itself is too slow and the amount is too small. The absorption of GABA and 5-HTP in human body requires the release of GABA and 5-HTP by thales.

Solution

  Tea polyphenols and phenolic acids in tea will be metabolized to protocatechuic acid (PCA). We used PCA-specific promoters to ligate lytic genes. We selected the SRRz lytic gene to create our lytic module. The SRRz gene was derived from bacteriophage. It is composed of S, R and Rz. After consumers take our probiotics, the probiotics will produce GABA and 5-HTP. When we take tea leaves, the protocatechuic acid in tea leaves will start the lysis system, and GABA and 5-HTP will be released into the intestine after bacterial lysis.

  Because our genetically modified E. coli bacteria, when excreted, cause genetic contamination that affects us and our environment, we designed our own escape gene suicide system. We designed E. coli to leave the mucosa of the small intestine and die by suicide. We decided to use a common operator. Under normal conditions, when arabinose is present, the arabinose promoter initiates the expression of the following three enzymes to decompose arabinose. The plan is to insert an antitoxin gene after the production of these three enzymes, and then express a toxin gene after that. Arabinose is present in the small intestine, and arabinose is not broken down by the small intestine. So when Escherichia coli is in the small intestine, it can receive arabinose in the small intestine, express the antitoxin gene in the back, and offset the toxin gene in the back, and the bacteria can live normally. When Escherichia coli leaves the small intestine, the arabinose operon without arabinose regulation does not express the following antitoxin gene, only the toxin gene is expressed. The bacteria eventually kill themselves.

  The toxin gene KID and antitoxin gene kis contained in the toxin-antitoxin system parD of plasmid R1 were selected as the main components of the suicide system. On the basis of the original arabinose manipulation, we inserted a promoter specifically expressing the KIS anti-virulence gene in the D region of the last poorly expressed isomerase, and then inserted the kis gene. The KID gene, which expresses the P10 antitoxin protein KIS (in the presence of arabinose), is followed by insertion of the promoter that initiates the KID and KIS genes and the KID gene for the toxin protein.

  The chemoresponsive switch initiates the death switch in response to the chemical inducer anhydrous tetracycline (aTc), and the termination switch initiates additional death due to a sudden drop in temperature caused by host excretion. Atc-inducible promoter Ptet was expressed, and TetR was expressed after gRNA, and Ptet was required to initiate the promoter. In the presence of aTc, TetR cannot bind to the target promoter, so the cell will activate the Pet promoter to express CAS9 and gRNA, and select the optimized 2-grNA for expression, which can effectively reduce the accidental killing situation and improve the stability.

  The suicide system is our idea and innovation. Due to the relationship between time and environment, it has not been tested and verified experimentally, which is one of the directions we will strive for in the future.

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[2]PubChem. TPH1 - tryptophan hydroxylase 1 (human). https://pubchem.ncbi.nlm.nih.gov/gene/TPH1/human.

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