Cell-free synthesis of 1,3-propanediol from glycerol with a high yield

Currently established fermentation processes converting glycerol to 1,3-propanediol reach a yield of only about 0.6 mol/mol with about 40% of the used glycerol being converted to unrequested side products and biomass. From economic and ecological points of view, the yield of 1,3-PDO is one of the most important parameters for bioconversion. The feasibility of engineering an enzymatic in vitro production process for 1,3-propanediol from glycerol was investigated. A very high production yield of about 0.95 mol/mol was reached. Current 1,3-propanediol production processes from glycerol established in industry and technology add up to fermentation processes exclusively. Those exhibit the disadvantage of reaching a production yield of about 0.6 mol/mol, which equals 60% of the theoretically possible maximum. In the present work, a cell-free in vitro system for nearly total glycerol conversion was developed. For this purpose, the enzymes glycerol dehydratase (from Lactobacillus reuteri), propanediol oxidoreductase isoenzyme (from Escherichia coli), and hydrogenase I (from Pyrococcus furiosus) were produced and partly purified. Through the combined activity of the mentioned enzymes, glycerol was successfully transformed into 1,3-propanediol at a yield close to 1 mol 1,3-propanediol/mol glycerol. The required cofactor NADPH for this reaction was continuously regenerated by hydrogenase I using hydrogen. The mechanism-based suicide-effect of glycerol dehydratase was partially overcome through continuous feeding of coenzyme B12, ATP, and magnesium to the reaction process. In this way, the durability of the glycerol dehydratase could be extended from few minutes to several hours. A novel in vitro approach for a highly efficient production process for 1,3-propanediol from glycerol was demonstrated.