Background#
With the advancement of life sciences research, the role of vitamins in the body is being constantly updated. Some studies have shown that the large-scale use of certain vitamins may produce unexpected effects in cellular pathways within the body. A recent study published in a subsidiary journal of The Lancet indicates that the pharmacological use of vitamin D may be beneficial for pre-diabetic patients to prevent sarcopenia.
“Vitamins are small molecular compounds that cannot be synthesized by the human body or are synthesized in insufficient amounts and must be obtained from external sources,” Zhang Dawei, a researcher at the Tianjin Institute of Industrial Biotechnology of the Chinese Academy of Sciences, told the Science and Technology Daily. He emphasized that vitamins are crucial for human growth and metabolism and are irreplaceable.
Vitamin B6 (VB6), also known as pyridoxine, includes pyridoxal, pyridoxamine, and exists in the body in the form of phosphate esters. It functions as a coenzyme in the synthesis or metabolism of proteins, carbohydrates, certain neurotransmitters, nucleic acids and DNA, vitamin B12, vitamin B2, and lipids. Plants and microorganisms have natural pathways to resynthesize vitamin B6, but humans and animals must obtain it from dietary supplements or feed additives. Currently, the synthetic processes for VB6 are relatively complex, with intermediate products generated during synthesis exhibiting significant toxicity and corrosiveness. Therefore, there is an urgent need for a pollution-free, rapid, and highly efficient synthesis method to meet the current demand for VB6 synthesis.
Our project has developed a cell-free expression system to achieve the expression of key genes involved in VB6 biosynthesis, thereby enabling efficient and green synthesis of VB6. Initially, the project successfully constructed a cell-free expression system that effectively expressed PDXA and PDXJ. Building on this foundation, the project then established a strategy for extracellular biosynthesis of VB6, achieving small-scale synthesis of VB6. This approach holds great potential for promotion and application.
Construction of Linear Expression Vectors and Expression#
Validation in Cell-Free Systems#
We designed and synthesized linear expression vectors containing the GFP reporter gene based on the gene sequences of PDXA and PDXJ. Gene expression was validated using cell extracts in an extracellular solution.
The validated linear expression vectors were added to the reaction system, and the expression of the target gene was assessed by detecting the green fluorescent protein. As shown in the figure below, after a period of reaction, part of the mixture was transferred to another clean PCR tube, and the supernatant appeared distinctly yellow-green after centrifugation, indicating the expression of the target protein.
Establishment and Validation of a Green Synthesis System for Vitamin B6#
Based on the initial establishment of a cell-free expression system, the project further constructed a synthesis system for VB6, using glutamine and other materials as substrates. By utilizing a critical enzyme system, the project largely achieved efficient synthesis of VB6. Time and yield studies were also conducted, and the efficiency of VB6 synthesis under the cell-free system was compared with that under current cellular factory conditions. The results showed that the expression efficiency of the cell-free system is higher, with a significant difference between the two methods.
To further validate the feasibility of this synthesis system, we also verified the reaction products using high-performance liquid chromatography (HPLC).
Summary#
In this study, we established a cell-free expression system capable of efficiently producing key genes for VB6 synthesis enzymes and enabling the extracellular biosynthesis of VB6. The results demonstrate that the project can produce microgram-levels of VB6, effectively verifying the feasibility of the new method for VB6 biosynthesis. This method is simple, rapid, highly efficient, and pollution-free, carrying significant importance and potential for widespread application.