Vitamins are essential nutrients with extensive applications in the pharmaceutical, food, animal feed, and cosmetics industries. For instance, Vitamin B6, whose synthetic precursor pyridoxine (PN), is currently produced through chemical synthesis using expensive and toxic chemicals. Additionally, the low catalytic efficiency of natural enzymes and the strict regulation of metabolic pathways hinder the microbial fermentation process for producing PN. In this project, we aim to construct a gene expression system closely related to vitamin synthesis, enabling its efficient expression outside the cell, which significantly improves the production efficiency of the relevant raw materials. Promoting research on engineered E. coli strains facilitates the production of microbial strains for vitamins and other bioproducts that have inherently low metabolic flux.
1. Background Information#
Depending on their different properties and functions, vitamins can be used as acidifiers, antioxidants, nutritional fortifiers, and dietary supplements in foods. In the food industry, vitamins can improve processing, enhance the quality and nutritional value of food, and meet the daily needs of the human body. The development of biomanufacturing technologies has driven iterative innovation in vitamin manufacturing. Under the guidance of biotechnology, the methods of vitamin production are steadily upgrading.
The Importance of VB6#
Vitamin B6 (VB6), also known as pyridoxine, includes pyridoxal, pyridoxamine, and exists in the body in the form of phosphate esters. It acts 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 for resynthesizing vitamin B6, but humans and animals must obtain it from dietary supplements or feed additives. Currently, the synthesis process of VB6 is relatively complex, and the intermediate products generated during synthesis exhibit significant toxicity and corrosiveness.
Problems Existing in VB6 Synthesis:#
Complex Synthesis Route: The synthesis of VB6 typically requires multiple steps involving intricate chemical reactions and precise operational control. Each reaction step demands specific conditions and catalysts, and any issue at any stage can affect the quality and yield of the final product.
Low Catalyst Efficiency: Catalysts play a crucial role in the synthesis of VB6 by accelerating reaction rates, enhancing reaction selectivity, and improving yields. However, commonly used catalysts often suffer from low efficiency and poor stability. For example, some catalysts tend to deactivate easily during the reaction process, necessitating frequent replacement and increasing production costs; others require stringent reaction conditions, limiting their applicability.
Difficult Separation and Purification: After synthesis, VB6 products generally need to undergo separation and purification to remove impurities and unreacted raw materials. However, due to the similar physicochemical properties between VB6 and impurities, the separation and purification processes are often challenging. For instance, incomplete crystallization and overlapping chromatographic peaks may occur during crystallization and chromatographic separation, leading to lower product purity.
Large Amounts of Waste Emissions: The synthesis of VB6 generates substantial amounts of wastewater, waste gas, and solid waste, causing severe environmental pollution. These wastes contain large quantities of organic compounds, heavy metals, and other harmful substances. If discharged without treatment, they can severely damage water bodies, air, and soil. For example, wastewater treatment consumes considerable financial and energy resources, yet the treated water quality may still fall short of discharge standards; volatile organic compounds and malodorous substances in waste gases can degrade the quality of life for nearby residents; and the disposal of solid waste requires significant land resources.
High Raw Material Costs: The synthesis of VB6 involves the use of various raw materials such as pyridine, formaldehyde, and malonic acid. Prices of these raw materials fluctuate widely, and their supply can be influenced by market demand and supply dynamics, potentially leading to shortages. For instance, pyridine, a key raw material in VB6 synthesis, is significantly affected by the petrochemical industry. An increase in the price of pyridine would directly lead to higher production costs for VB6.
Moreover, high equipment investment is another critical issue currently faced.
2. How to Face and Solve This Problem?#
Microbial Fermentation Synthesis is a Critical Direction for Vitamin Manufacturing Traditional vitamin production methods include chemical extraction and chemical synthesis. The former involves extracting vitamins from plants or other rich sources using chemical solvents; the latter synthesizes vitamins based on their chemical structure through chemical reactions.
Microbial fermentation synthesis represents a critical direction for vitamin manufacturing. The application of biomanufacturing technology can effectively achieve energy conservation, emission reduction, and resource saving, serving as a vital breakthrough for the green development of vitamin manufacturing. For instance, traditional vitamin B6 production primarily relies on chemical synthesis, which requires the use of highly corrosive phosphorus oxychloride and toxic solvents like benzene. The process also poses safety hazards and environmental pollution due to the difficulty in handling by-products. “In comparison, biological fermentation methods are relatively mild, with reaction temperatures mostly ranging from 30 to 37 degrees Celsius, eliminating the need for high temperatures and pressures,” said an expert. The raw materials used in biological fermentation methods generally do not include toxic chemical reagents, making them more aligned with green development requirements in terms of energy consumption, emissions, and environmental protection.
Improving Vitamin Production Efficiency by Modifying “Cell Factories” With the support of information technology and biotechnology, current vitamin production is continuously advancing towards higher efficiency. Biotechnologies, including gene editing, can purposefully enhance vitamin synthesis pathways within production strains, thereby strengthening the strains’ production capabilities. According to Yu Xihua, strain improvement makes “cell factories” more efficient. They only need inexpensive and readily available raw materials, such as corn starch and lignocellulose, to achieve efficient conversion of target products through fermentation. Synthetic biology technology is considered a disruptive technology that could transform future production methods, aligning with the goals of carbon peak and carbon neutrality as well as sustainable development concepts. Previously, the team led by Academician Deng Zixin of the Chinese Academy of Sciences successfully developed a method for producing vitamin E that is low-cost, highly efficient, highly profitable, and low-polluting using synthetic biology technology, making China a major producer of vitamin E.
The Cell-Free Expression System is an in vitro system that uses exogenous DNA or mRNA as a template, leveraging the cellular synthesis machinery, protein folding factors, and other associated enzyme systems present in cell extracts. By adding amino acids, T7 polymerase, and energy sources, it enables protein expression. This system effectively overcomes the limitations of cells, offering flexible and controllable reaction conditions, and supports high-throughput expression. It allows for the simultaneous parallel expression of multiple proteins under various conditions in multi-well plates, making it suitable for high-throughput proteomics studies and significantly enhancing experimental efficiency and research speed.
To address the aforementioned issues, under the guidance of a professor and doctoral supervisor at Southeast University, literature was reviewed to identify two key enzymes and their corresponding genes involved in the biosynthesis of VB6. Through the use of an in vitro cell-free expression system, there is potential to achieve a greener production method for VB6, reducing pollution and increasing efficiency. Therefore, we constructed a linear vector containing reporter genes and coding genes for specific target proteins. This vector can express specific VB6 synthetases for the production of VB6 via enzymatic engineering fermentation. Initially, we constructed the vector to simultaneously express the reporter gene and the target gene, where the fluorescent protein allows for intuitive detection, and the target protein can be used for VB6 synthesis, making green production possible.
Inspiration Source#
In Escherichia coli, the biosynthetic pathway for vitamin B6 involves the following steps: the initiation reaction, catalyzed by EPD and DXS enzymes, converts glucose into glyceraldehyde-3-phosphate and phosphoenolpyruvate; the intermediate reaction, through a series of enzymatic actions, transforms glyceraldehyde-3-phosphate and phosphoenolpyruvate into 4-hydroxy-L-threonine (4HTP); and the terminal reaction, catalyzed by PDXA and PDXJ enzymes, converts 4HTP into vitamin B6. This method requires further processing to obtain the product, and the yield is relatively low. Simplifying the operational steps and improving production efficiency would be more conducive to the green production of VB6. We utilized a cell-free expression system to first synthesize PDXA and PDXJ, then conducted the entire synthesis process in test tubes to complete the in vitro biosynthesis of VB6.
Reference#
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