Optimization of Hydrogenobyrinic Acid Synthesis in a Cell-Free Multienzyme Reaction by Novel S -Adenosyl-methionine Regeneration.

Hydrogenobyrinic acid, a modified tetrapyrrole composed of eight five-carbon compounds, is a key intermediate and central framework of vitamin B 12 . Synthesis of hydrogenobyrinic acid requires eight S -adenosyl-methionine working as the methyl group donor catalyzed by 12 enzymes including six methyltransferases, causing the great shortage of S -adenosyl-methionine and accumulation of S -adenosyl-homocysteine, which is uneconomic and unsustainable for the cascade reaction. Here, we report a cell-free synthetic system for producing hydrogenobyrinic acid by integrating 12 enzymes using 5-aminolevulininate as a substrate and develop a novel S -adenosyl-methionine regeneration system to steadily supply S -adenosyl-methionine and avoid the accumulated inhibition of S -adenosyl-homocysteine by consuming a cheaper substrate (l-methionine and polyphosphate). By combination of the reaction system optimization and S -adenosyl-methionine regeneration, the titer of hydrogenobyrinic acid was improved from 0.61 to 29.39 mg/L in a 12 h reaction period, representing an increase of 48.18-fold, raising an efficient and rapidly evolutional alternative method to produce high-value-added compounds and intermediate products.