1. Strategic Design

In the context of Alzheimer's disease (AD) precipitated by amyloid-beta (Aβ) protein deposition associated with the APOE4 allele, the identification of targeted intervention strategies remains an ongoing challenge. The inhibition of the LilrB3-APOE4 interaction represents both a significant obstacle and a potential breakthrough in the current therapeutic landscape for AD. Our proposed strategy involves modulating the gut microbiota through the use of microbiome-derived small molecules that specifically bind to the LilrB3 receptor. This approach aims to occupy the LilrB3 receptor sites, thereby competitively inhibiting the binding of APOE4 to these receptors. In the selection of which small molecules can effectively bind to LilrB3 receptors, we propose the use of molecular docking simulation techniques, bioinformatics tools such as AutoDock, and screening of gut microbiota metabolites to identify multiple small molecules with favorable interactions with LilrB3. We employed surface plasmon resonance (SPR) Biacore technology to evaluate the binding affinities between these small molecules and LilrB3 protein, considering the inherent properties and toxicity of the small molecules to select appropriate candidates.

Through the application of molecular docking simulations using software such as AutoDock, in conjunction with surface plasmon resonance (SPR) Biacore assays for molecular interaction analysis, it was conclusively demonstrated that tetrahydrofolate exhibits a strong binding affinity to the LilrB3 receptor. Given the safety profile and non-toxicity of this small molecule, along with its natural production by Lactobacillus plantarum, we propose that tetrahydrofolate represents a promising candidate for mitigating Alzheimer's disease (AD) symptoms through its biosynthesis by Lactobacillus plantarum.Tetrahydrofolate plays a crucial role as a coenzyme in one-carbon metabolism, essential for the proper functioning of the nervous system. It aids in the synthesis of neurotransmitters and DNA methylation, thereby improving cognitive function and mood regulation[1]. Tetrahydrofolate can lower homocysteine levels, reducing oxidative stress and neuroinflammation, offering therapeutic benefits for individuals with Alzheimer's Disease. Additionally, tetrahydrofolate 's role in mood regulation can help address depressive symptoms in AD patients[1,2].

Subsequently, we initiated the design of plasmids. In terms of engineered bacterial selection, we intend to utilize Lactobacillus plantarum L168. Lactobacillus plantarum L168 has been demonstrated to confer significant benefits in regulating gut microbiota andimprove defects of social behvior . Moreover, Lactobacillus plantarum is a harmless probiotic bacterium that does not colonize the human body for an extended period, which greatly aids in controlling the dosage of small molecule drugs within the body. Additionally, probiotic ingestion is more acceptable than drug intake, leading to better compliance among elderly patients.

We initially identified the key enzyme gene sequences responsible for the biosynthesis of tetrahydrofolate in Lactobacillus plantarum by conducting extensive searches on NCBI, UniProt, and various scholarly databases. Through this investigation, we pinpointed the critical enzymes: folK and folE, which are sometimes collectively referred to as folKE in the literature[3]. Subsequently, we successfully integrated these genes into our meticulously designed plasmid, which was equipped with a fail-safe suicide switch.

We then transformed the engineered plasmid into Lactobacillus plantarum L168 to express the tetrahydrofolate. Our aim was to engineer a probiotic strain of Lactobacillus plantarum capable of delivering a small molecule to the brain. Upon ingestion, this probiotic would colonize the intestines and release tetrahydrofolate. Designed to traverse the bloodstream and the gut-brain axis, the molecule tetrahydrofolate would then bind to LilrB3 receptors in the brain. By outcompeting APOE4 for receptor binding, it would help curb amyloid protein deposition, potentially leading to improved Alzheimer's disease symptoms.

2. System Design

In order to screen for small molecules capable of binding to the LilrB3 receptor and competitively inhibiting the binding of APOE4 to the LilrB3 receptor, a series of screenings and discussions were conducted on the candidate small molecules.

Firstly, considering the necessity for the substance to cross the blood-brain barrier and bind to the LilrB3 receptor in the brain, it was determined that the size of the substance must be sufficiently small to enable it to penetrate the blood-brain barrier. Secondly, it was believed that the substance should ideally be a metabolic product of the human gut microbiota, implying that it is inherently compatible with the human body and unlikely to cause harm. Therefore, the search and screening for the target small molecules were focused on the metabolic products of the human gut microbiota. Additionally, a comprehensive screening was conducted on the toxicity, side effects, and safety of the small molecules.

Subsequently, the modeling group used software such as AutoDock to analyze the binding capacity of small molecules from the human gut microbiota metabolite library to the LilrB3 receptor, taking into account the issues of toxicity, side effects, and safety. Ultimately, tetrahydrofolate was identified as the optimal choice, as its supplementation in appropriate amounts is generally beneficial to the human body.

Further research was conducted on databases such as NCBI and UniProt, and multiple literature sources were reviewed to confirm that the Lactobacillus plantarum can produce tetrahydrofolate, with the key genes being the folK and folE genes, which are often denoted as folKE in some scientific literature. Literature indicates that overexpression of folK and folE can effectively increase the production of tetrahydrofolate by Lactobacillus plantarum[4]. The folK and folE gene fragments were then identified and cloned into the pSIP403 vector.

For the promoter, the P9 promoter, which has been proven to function well in Lactobacillus plantarum, was utilized. Furthermore, the plasmid is equipped with a safety feature—a "suicide switch" mechanism. This system consists of a hypoxia-inducible promoter that initiates the genetic cascade under low-oxygen conditions, a ribosome binding site (RBS) that ensures efficient translation initiation, and the gene encoding the SrpR repressor. The SrpR repressor functions to suppress transcription driven by the SrpR-dependent promoter, effectively silencing the expression of downstream genes when activated. This regulatory sequence is capped with a terminator sequence to ensure controlled gene expression.

Due to the initial difficulty in transferring the plasmid into Lactobacillus plantarum, after the synthesis of the plasmid was completed, it was first transferred into E.coli DH5α. After successful verification of the transfer via PCR, the plasmid was then transferred from E.coli DH5α to Lactobacillus plantarum L168, followed by qPCR and RT-PCR to verify the transcription levels of folK and folE. Subsequently, the functionality of the engineered bacteria was validated through experiments such as LC-MS.

1. Strategic Design

Inosine, a purine nucleoside, has shown potential therapeutic effects in the treatment of Alzheimer's disease (AD) and depression. It acts as a neuroprotective agent by promoting axonal regeneration and enhancing neuronal survival. Studies have indicated that Inosine can improve cognitive functions and alleviate depressive symptoms by modulating neurotransmitter levels and reducing oxidative stress. Additionally, inosine has been found to enhance synaptic plasticity and neurogenesis, which are critical for cognitive and emotional health[1,2].

In our research, we have demonstrated through molecular docking simulations and molecular interaction experiments that inosine can bind to the neuronal nitric oxide synthase (nNOS) receptor. Current antidepressant drugs on the market suffer from insufficient targeting, significant side effects, and delayed onset of action. Research has found that in the dorsal raphe nucleus (DRN), serotonin transporter (SERT) is highly colocalized with nNOS, but this colocalization is largely absent in the postsynaptic region[2]. After chronic stress, SERT-nNOS coupling in the DRN increases, leading to reduced membrane localization of SERT, increased intercellular serotonin concentration, activation of serotonin autoreceptors, and enhanced negative feedback inhibition of serotonergic neuron firing, resulting in reduced serotonin concentration in the postsynaptic synaptic cleft and inducing depression. The rapid antidepressant mechanism is facilitated by a strategic intervention that decouples the serotonin transporter (SERT) from neuronal nitric oxide synthase (nNOS), while simultaneously augmenting the presence of SERT on the membrane of dorsal raphe nuclei (DRN). This dual action results in a diminished level of intercellular serotonin, effectively attenuating the negative feedback loop. Consequently, the firing rate of serotonergic neurons is enhanced, precipitating a swift surge in serotonin levels within the postsynaptic synaptic cleft. This intervention elicits a prompt antidepressant response that is distinct from the desensitization of serotonin autoreceptors, offering a novel and efficacious approach to treating depression.

Therefore, we aim to target the nNOS receptor by using gut microbiota-produced small molecules, which can reach the brain via the gut-brain axis, bind to depression-related nNOS receptors, uncouple SERT from nNOS, and alleviate depression symptoms[3].

2. System Design

As illustrated in the figure, the synthesis of Inosine involves three sequential steps and requires three critical enzymes. According to UniProt, the key enzymes for the first and second steps are present in both Escherichia coli and Lactobacillus plantarum, with several enzymes capable of catalyzing the same reactions and producing inosine precursors. Consequently, we determined that the specificity of the first two key enzymes for inosine production might not be very high. Therefore, we selected the gene for the third key enzyme, Gsk, which exhibits higher specificity. The product of this gene is found in E. coli but not in Lactobacillus plantarum according to UniProt. This suggests that Lactobacillus plantarum may lack this enzyme. Following the overexpression of gsk, the gene transcription significantly enhances the availability of inosine precursors, thereby leading to a substantial increase in Inosine production.

pSIP403-P9-gsk-PfdhF-SrpR-PsrpR-LysKB317

In our system, we employed the gsk gene to produce inosine and target the neuronal nitric oxide synthase (nNOS) for the treatment of depression. For safety considerations, we incorporated a suicide switch (PfdhF-SrpR repressor-SrpR repressible promoter-LysKB317). This composite part includes a hypoxia-inducible promoter, a ribosome binding site (RBS), a coding sequence for the SrpR repressor that inhibits transcription of the SrpR-dependent promoter, and a terminator. Under low oxygen conditions, this switch induces the production of downstream inhibitory proteins, thereby inhibiting endotoxins and promoting the survival of Lactobacillus plantarum under hypoxic conditions