The mainstream industrial method for VB6 synthesis is the oxazole method, which requires the use of large amounts of organic and inorganic reagents. This not only causes environmental pollution but also poses a threat to the health of operators. The disposal of waste and by-products further increases costs. In terms of biosynthesis, the low catalytic efficiency of natural enzymes and the strict regulation of metabolic pathways have hindered the production of pyridoxine (PN) in microbial fermentation processes.
In this project, we proposed the construction of a gene expression system closely related to vitamin synthesis, enabling efficient extracellular expression, thereby greatly improving the production efficiency of related raw materials. Promoting research on engineered Escherichia coli strains will help in the production of microorganisms with low inherent metabolic flux, producing vitamins and other bioproducts.
Standard Part#
The synthase protein sequences were inserted into the pRSFDuet plasmid. It was designed to express two key synthase proteins, PDXA and PDXJ.
Composite Part: pRSFDuet-PDX-GFP#
We constructed an efficient expression system for VB6 biosynthesis enzymes and the corresponding linear expression vector. This cell-free expression system can determine the success and quantity of synthase expression through the indication of green fluorescent protein, as fluorescence intensity is correlated with enzyme synthesis quantity.
In the future, this linear expression vector construction plan can be used for environmentally friendly synthesis research of other substances, and by optimizing it, indicators such as product yield and purity can be improved. The pRSFDuet-1-PdxA-PdxJ expression plasmid was constructed, and the synthetic sequence was first digested with enzymes to obtain specific enzyme cutting and ligation sites. The ligated product was transformed into E. coli BL21(DE3) competent cells and screened with ampicillin to obtain a certain number of transformants. The plasmid was verified through enzyme digestion. As shown in the figure below, the size of the digested target gene fragment met the expected requirements.
In this project, we first synthesized the expression sequence based on the gene sequences of PDXA and PDXJ, optimized the codons, and after enzyme digestion, constructed the linear expression vector. The T7 promoter sequence and related expression elements were introduced through PCR for efficient expression in cell lysates. Conventional methods were used for detection and analysis. According to our design, when green fluorescent protein is expressed, the solution in the test tube will appear yellow-green after simple treatment, indicating that the two key genes PDXA and PDXJ can also be successfully expressed in the cell-free system. Preliminary yield estimation can be made based on the fluorescence. By combining previous research experiences, we constructed the biosynthesis pathway of VB6, as shown in the figure below.
We constructed recombinant expression vectors of the key genes in the VB6 synthase system and achieved corresponding expression in both E. coli and cell-free expression systems, providing a foundation for VB6 biosynthesis. Additionally, in subsequent experiments, we successfully obtained VB6, effectively verifying the success of the cell-free expression system and the feasibility of a new, efficient, environmentally friendly method for VB6 synthesis.
In this project, we combined previous experiences and focused on comparative research on synthesis time and yield, confirming the feasibility of in vitro VB6 biosynthesis and laying the foundation for subsequent environmentally friendly large-scale production. The above figure shows that the reaction reached its peak in vitro after 6 hours, achieving an expression level of 30 mg/L in a small-scale expression system. Compared to intracellular (E. coli expression system) production, the yield increased significantly (p = 0.0109, figure below), showing promise for the development of a faster and better environmentally friendly VB6 production system.
To further verify the feasibility of this synthesis system, we also validated the reaction product using high-performance liquid chromatography (HPLC).
In the figure, A shows the characteristic peak map of the standard sample, with peak 4 being the characteristic peak of VB6; B shows the characteristic peak of the target VB6 detected in the biosynthesis system (at the arrow).
The experimental results in this project show that we successfully constructed circular and linear expression vectors of PDXA and PDXJ, which were validated in both E. coli and cell-free expression systems. This project also successfully developed a set of cell-free expression systems capable of rapid expression of target genes. In the first phase, with high concentrations of PDXA and PDXJ, we further explored the new biosynthesis of VB6 using glutamine, glyceraldehyde 3-phosphate, and 5-phosphoribosyl pyrophosphate as raw materials. The experimental results confirmed the success of this project.
Reference
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