Preparative synthesis of Poly[(R)-3-hydroxybutyrate] monomer for enzymatic cell-free polymerization

The synthesis of Poly[(R)-3-hydroxybutyrate] monomer (R)-3HB-CoA using crotonic anhydride and CoA as the starting materials in an aqueous solution was reported. By mixing crotonic anhydride and CoA at a molar ratio of 1:1, crotonyl-CoA was efficiently generated by an abiotic ester-exchange reaction. The crotonyl-CoA was then R-specifically hydrated using (R)-specific enoyl-CoA hydratase (PhaJ) to generate (R)-3HB-CoA. The hydratase activity of PhaJ was determined by monitoring the absorbance at 263 nm caused by the double bond of crotonyl-CoA. An activity assay of PhaC was performed by measuring the free CoA released from (R)-3HB-CoA during P(3HB) polymerization, using 5,50-dithiobis(2-nitrobenoic acid (DTNB). An high performance liquid chromatography analysis revealed that the free CoA released by PhaC successfully reacted with the crotonic anhydride, and was then converted to (R)-3HB-CoA by PhaJ. However, the progression of the polymerization was not observed because the crotonic anhydride seriously inhibited the PhaC activity.