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

INTRODUCTION Polyhydroxyalkanoates (PHAs) are produced as a carbon and energy storage material in various bacteria, and have received much attention as biodegradable and biocompatible plastics. PHAs can be produced from renewable carbon sources such as sugars, fatty acids and plant oils by fermentation.1 Poly[(R)-3-hydroxybutyrate], P(3HB), is the most widely studied and best characterized PHA. The physical properties of P(3HB) are similar to those of isotactic polypropylene. PHA synthase (PhaC) is an enzyme that catalyzes a PHA polymerization reaction. To form P(3HB), PhaC requires an R-enantiomer 3-hydroxybutyryl coenzyme A ((R)-3HB-CoA) as a substrate and polymerizes the acyl moiety of (R)-3HB-CoA. In 1995, Gerngross and Martin2 demonstrated the first enzymatic cell-free synthesis of P(3HB) by using purified PhaC and (R)-3HB-CoA. Subsequently, other groups have evolved the cell-free synthesis in terms of monomer supply and polymer yield.3–7 Cell-free synthesis is useful not only for polymer production but also for the functional analysis of PhaC. However, technical difficulties in the preparation of (R)-3HB-CoA monomer still limit the ability of cell-free synthesis. Conventional preparation methods require the purification of (R)-3HB-CoA because the synthesis reactions are performed in organic solvents and use large quantities of starting chemicals relative to CoA.8,9 These solvents and excess chemicals seriously inhibit enzyme activity when the as-prepared monomers are used for cell-free synthesis. Thus, it is desirable to develop a method for the preparation of (R)-3HB-CoA that does not use organic solvents and large quantities of starting chemicals. Apart from the cell-free synthesis, chemical recycling of waste plastics should be promoted from the viewpoint of sustainability. By thermal degradation, P(3HB) can be converted into crotonic acid with a high yield.10,11 Furthermore, the intermolecular dehydration of two crotonic acids generates crotonic anhydride. Recently, it has been shown that crotonic anhydride is generated as a minor product from P(3HB) by thermal degradation.12 It is, thus, worth utilizing crotonic acid and its anhydride as recovered feedstock. In this study, we focused on the synthesis of (R)-3HB-CoA using crotonic anhydride and CoA as the starting materials in an aqueous solution (Figure 1a). 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. This method was compared with (R,S)3HB-CoA synthesis, using b-butyrolactone and CoA as the starting materials (Figure 1b) in terms of the reaction kinetics. Here, we demonstrate that the as-prepared (R)-3HB-CoA by the method described here can be used for cell-free synthesis and achieves a very high P(3HB) molecular weight.