Parkinson's disease is a chronic disorder that causes dysfunction of the central nervous system's extrapyramidal neurons, resulting in symptoms of motor impairment[1]. The associated neurodegenerative process may precede the onset of symptoms by decades. Potential risk factors include environmental toxins, drugs, microtrauma to the brain, focal cerebrovascular injury, and genetic defects. The neuropathological hallmark of Parkinson's disease is the selective loss of dopaminergic neurons in the substantia nigra pars compacta, accompanied by widespread effects on other central nervous system structures and peripheral tissues[2]. Etiological mechanisms involving genetic, epigenetic, and environmental factors lead to conformational changes and deposition of key proteins, driven by abnormalities in the ubiquitin-proteasome system, mitochondrial dysfunction, and oxidative stress.

Figure 1. Pathogenic factors of Alzheimer’s disease and Parkinson’s disease. These factors include oxidative stress/mitochondrial stress, neuroinflammation and neurodegeneration, and plaques of insoluble proteins including Aβ, tau, and α-syn[3].

Levodopa is the most common drug for the treatment of Parkinson's disease[4]. It crosses the blood-brain barrier and enters the central nervous system, exerting its pharmacological effects through the action of dopa decarboxylase[4]. Levodopa (3,4-dihydroxyphenylalanine, L-DOPA) is an amino acid derivative similar to L-tyrosine (Tyr) in the human body. It is a precursor to various substances, including dopamine, melanin, and alkaloids, and is widely used in the treatment of diseases such as Parkinson's disease[5].

Figure 2. From: Levodopa treatment: impacts and mechanisms throughout Parkinson’s disease progression[6].

Currently, methods for synthesizing L-DOPA mainly include chemical synthesis, plant extraction, and enzymatic methods. Chemical synthesis typically uses raw materials such as vanillin and hydantoin to synthesize L-DOPA through multiple reaction steps[7]. However, this method is complex and has low selectivity, which is inconsistent with the development of green production. Plant extraction methods mainly rely on raw materials such as cat beans, but the maximum L-DOPA content in cat beans is only 9%, and the limited raw material supply makes it difficult to meet market demand[8].

Figure 3. L-dopa Synthesis - Asymmetric Hydrogenation Of L Dopa[9]

Microbial synthesis, as an environmentally friendly, simple, and efficient new synthetic pathway, has attracted increasing attention[10]. However, the conversion efficiency of chorismate to L-tyrosine in this process is a key step that limits the further efficient production of levodopa. Therefore, identifying more efficient key enzyme genes, such as tyrA, and enhancing tyrA gene expression are particularly important. This research is necessary to further improve the industrial application of biosynthesis[11].

However, the most common adverse reactions after taking levodopa are gastrointestinal symptoms such as nausea, vomiting, anorexia, and constipation[12]. As research progresses, the use of prebiotics and probiotics in Parkinson's disease has been shown to improve symptoms in patients with Parkinson's disease, providing new ideas for improving various non-motor symptoms.

Figure 4. Side effects of levodopa[13]

This study primarily overexpressed key enzymes in the L-DOPA biosynthesis pathway and the genes that synthesize the main precursors. L-DOPA was produced using a dual-module system. The precursor synthesis module was used to divert carbon flux from central carbon metabolism, directing more carbon flux toward product production and increasing flux in the shikimate pathway. The L-DOPA synthesis module was used to produce the product. The two modules worked together to promote L-DOPA production. After constructing the expression pathway, we screened three exogenous tyrA genes for L-DOPA synthesis. To further test whether RBS sequence regulation of this gene affects enzyme expression, we tested three RBS sequences in the selected tyrA genes, hoping to identify effective elements that could enhance L-DOPA production.

Finally, we transferred these effective elements into probiotics, overexpressing the optimized L-DOPA production pathway. Combined with the probiotic properties of these bacteria, this approach will facilitate the development of new therapeutics targeting host-gut microbial drug metabolism and other novel health interventions.

The first step in constructing a complete L-DOPA production pathway was to screen for key genes in the L-DOPA production pathway in E. coli. The construction of the ECN L-DOPA production pathway was divided into two modules, each consisting of a corresponding plasmid vector containing three key genes. The first module focused on enhancing the precursor supply. The corresponding genes were tktA, aroF, and aroE. The plasmid was named pCDFDuet-tktA-aroF-aroE. This enhanced the rate-limiting enzymes in the PPP and glycolysis pathways and strengthened the metabolic flux of PEP and E4P to the shikimate pathway. The second module, corresponding to the genes tyrA, hpaB, and hpaC, was named pETDuet-TyrA-hpaB-hpaC. This enhanced the production of key precursors in the shikimate pathway that lead to L-DOPA production and the key enzymes for L-DOPA synthesis. Product production was achieved by enhancing pathway carbon flux and product synthesis, respectively. The second step compared the effectiveness of the three tyrA gene sources: PCR was performed on the plasmid from the second module, and the plasmid was ligated with the tyrA genes from the other two sources to construct new plasmids. The three constructed Module 2 plasmids were transformed with the Module 1 plasmid, yielding three strains. Each strain differed only in the source of the tyrA gene. Fermentation testing was performed to select the optimal tyrA gene source and use this plasmid for subsequent transformations.

The third step was to optimize the RBS sequence of the tyrA gene from the optimal source. Based on this plasmid, primer loop P was designed to replace the original RBS sequence of the gene, generating two new plasmids. These two plasmids were then transformed with the Module 1 plasmid to generate two new strains. Compared to the plasmid constructed in the previous round, the only difference between these three strains was the RBS sequence preceding the tyrA gene. Fermentation testing was performed to select the RBS sequence with the best strength. In the fourth step, after obtaining the optimal tyrA gene and RBS sequence, the effective element is transferred into probiotics, in which the optimized levodopa production pathway is overexpressed for production. Combined with its own probiotic properties, it will help develop new therapies targeting host-gut microbial drug metabolism and other new health interventions.

Figure 5. Technology Roadmap（E4P: D-erythrose 4-phosphate; PEP: phosphoenolpyruvate; DAHP: 3-deoxy-arabino-heptulonate 7-phosphate; DHS: 3-dehydroshikimate; CHA: chorismate; Tyr: L-tyrosine; tktA: transketolase Ⅰ gene; aroF: DAHP synthase gene; aroE: dehydroshikimate reductase gene; tyrA: CHA prephenate dehydrogenase gene; hpaBC: 4-hydroxyphenylacetate 3-hydroxylase gene）

This study primarily overexpressed key enzymes in the L-DOPA biosynthesis pathway and the genes that synthesize the main precursors. This dual-module approach enabled L-DOPA production. The precursor synthesis module was used to divert carbon flux from central carbon metabolism, directing more carbon flux toward product production and increasing shikimate pathway flux. The L-DOPA synthesis module was used to direct product production. The two modules worked together to promote L-DOPA production. After constructing the expression pathway, we screened three exogenous tyrA genes for L-DOPA synthesis. To further test whether RBS sequence regulation of these genes affects enzyme expression, we tested three RBS sequences in the selected tyrA genes, hoping to identify an effective element that could enhance L-DOPA production. This effective element could be transferred into probiotics to overexpress the optimized L-DOPA production pathway. Combined with the probiotic properties of these bacteria, this could potentially facilitate the development of novel therapeutics targeting host-gut microbial drug metabolism and other novel health interventions.

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9. L-dopa Synthesis - Asymmetric Hydrogenation Of L Dopa, HD Png Download - kindpng
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13. Management and treatment of Parkinson’s Disease