Construction Of Multi-enzyme Pathway In Vitro

In vitro multi-enzyme catalysis is a very rapid development of synthetic biology research methods. It is process that uses purified enzymes and coenzyme,through a certain biochemical reaction, to converte the substrate into the desired product. Using metabolic catalytic methods in vitro, it is possible to improve the efficiency of the research without elucidating the complex cellular metabolism of the organism. In view of the advantages of the multi-enzyme catalytic method in vitro, the carbon fixation pathway of threonine cycle was validated and the process of synthesis of 5-aminolevulinic acid (5-ALA) synthesis was optimized in vitro.Acetyl-CoA is a central metabolite and an important precursor for the synthesis of many valuable products. However, the carbon yield of acetyl-CoA is relatively low due to the carbon loss at the pyruvate decarboxylation step. Therefore, it was of great significance to construct an effecient pathway for acetyl-CoA synthesis. In this study, carbon fixation pathway via threonine cycle was used to synthesize acetyl CoA in vitro. When pyruvate was used as the substrate, serine deaminase (TdcB) was the last step of the cycling pathway in the in vitro characterization. The results showed that when all enzymes except serine deaminase were added to the reaction system, the measured acetyl CoA concentration was about 1.5 mmol/L at an nearly equilibrium state. After serine deaminase was added, serine to pyruvate and re-entry into the circulation, acetyl CoA concentration was increased 0.2 mmol/L. This result indicated that carbon fixation was achieved via the threonine cycle in vitro.5-Aminolevulinic acid (ALA) is the precursor for the biosynthesis of tetrapyrroles and has broad agricultural and medical applications. Currently ALA is mainly produced by chemical synthesis and microbial fermentation. Cell free multi-enzyme catalysis is a promising method for producing high value chemicals. Succinyl CoA synthase from E. coli was only able to tolerate 2 mmol/L of succinic acid, affecting the production of 5-ALA in vitro. The succinyl CoA synthase from Advenella mimigardefordensis DPN7T was screened by literature to withstand 50 mmol/L succinic acid. In the external supply of ATP, 5-ALA can produce 130 mmol/L by adding succinic acid and ATP. In the case of ATP regeneration by sodium hexametaphosphate, 5-ALA can be produced 23 mmol/L by the addition of sodium hexametaphosphate, and the addition of succinic acid to sodium hexametaphosphate can produce 40 mmol/L 5-ALA.