Overview#
In this study, we focused on constructing and implementing a cell-free expression system for the synthesis of VB6 synthase. On this basis, we further utilized two key synthases, with glutamine, glyceraldehyde 3-phosphate, and 5-phosphoribosyl pyrophosphate as raw materials, to attempt a simplified synthesis of VB6. The experimental results largely confirmed the feasibility of VB6 biosynthesis. This synthesis method does not use toxic or harmful raw materials, is environmentally friendly, and environmentally friendly with no pollution. On top of that, it simplifies the steps and improves efficiency, offering important reference points for the biosynthesis of vitamins and having a positive impact on human health and the development of environmentally friendly products.
Research Objectives and Design#
1. Vector#
After determining the gene sequences of the PdxA and PdxJ proteins, we searched through relevant literature and ultimately selected pRSFDuet-1 as the vector to construct the template DNA for the CFPS system.
2. Template DNA#
In the prokaryotic expression system, such as the E. coli bacterial expression system, the expression of exogenous genes usually requires an inducer to initiate express. The most commonly used inducer is IPTG (isopropyl-β-D-thiogalactoside), which is a highly potent and stable inducer. When IPTG is present, it binds to the Lac repressor (repressor protein), causing the repressor protein to change its conformation, leading to the dissociation of the repressor from the gene, allowing RNA polymerase to proceed unhindered, and initiating transcription.
In this project, the hydrophobicity, signal peptides, transmembrane domains, and basic structures of the pdxA and pdxJ protein sequences were theoretically evaluated. Based on the evaluation results, a plasmid construction strategy was designed, and the plasmid was synthesized using full gene synthesis and subcloned into the pRSFDuet-1 expression vector. The plasmid was then transformed into E. coli competent cells, cultured, induced to express, collected, and finally verified by SDS-PAGE to confirm the successful expression of the recombinant pdxA and pdxJ proteins.
DNA Template#
In the prokaryotic expression system, such as the E. coli bacterial expression system, the expression of exogenous genes usually requires an inducer to initiate express. The most commonly used inducer is IPTG (isopropyl-β-D-thiogalactoside), which is a highly potent and stable inducer. When IPTG is present, it binds to the Lac repressor (repressor protein), causing the repressor protein to change its conformation, leading to the dissociation of the repressor from the gene, allowing RNA polymerase to proceed unhindered, and initiating transcription.
In this project, the hydrophobicity, signal peptides, transmembrane domains, and basic structures of the pdxA and pdxJ protein sequences were theoretically evaluated. Based on the evaluation results, a plasmid construction strategy was designed, and the plasmid was synthesized using full gene synthesis and subcloned into the pRSFDuet-1 expression vector. The plasmid was then transformed into E. coli competent cells, cultured, induced to express, collected, and finally verified by SDS-PAGE to confirm the successful expression of the recombinant pdxA and pdxJ proteins.
Key Experiments#
1. Cell-Free Expression System#
Our team used Thermo Fisher’s MembraneMax™ Protein Expression Kit to prepare the cell-free expression system needed for subsequent experiments.
2. Synthesis of VB6 Synthase (PDXA and PDXJ) through the Cell-Free Expression System#
After confirming that the template DNA and cell-free expression system were both ready, we proceeded with the VB6 synthesis experiment. The yield of pyridoxal (VB6) was verified using methods such as spectrophotometry.
3. Synthesis of VB6 through Simplified Reaction Steps#
Based on our literature review, we used glutamine, glyceraldehyde 3-phosphate, and 5-phosphoribosyl pyrophosphate as raw materials, employing simplified steps to synthesize VB6.
Reference
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