Analysis of the in vitro biocatalytic production of poly-(β)-hydroxybutyric acid

Poly-(β)-hydroxybutyric acid (PHB) is a biodegradable, thermoplastic biopolyester produced as a carbon-energy storage source by bacteria that are challenged by nutrient limitation. While production of PHB for industrial applications is generally accomplished via fermentation, in vitro enzymatic synthesis can be an attractive alternative. We now report a detailed analysis of the in vitro biosynthesis of PHB using the three-enzyme system from Wautersia eutropha. We have developed quantitative HPLC methodology which resolves CoA, acetyl-CoA, acetoacetyl-CoA, and β-hydroxybutyryl-CoA with baseline resolution and then utilized this methodology to elucidate the fluctuations in metabolite concentrations and the effect of each individual enzyme on the overall system. The experimental results were then used to design the first kinetic model for in vitro PHB biosynthesis. All previous modeling studies of PHB production have been designed to analyze in vivo biosyntheses, typically in bacteria but also in yeast and plants. From a biotechnological viewpoint, in vitro enzymatic synthesis has the advantage of obviating limitations related to cellular uptake and possible toxicity of substrates, thereby making it possible to utilize a much wider range of substrates for production of novel homopolymers or copolymers. Our results can therefore serve as a basis for further development of the potential of in vitro enzymatic syntheses of polyesters as a complement to the more widely used approach of in vivo production of these polymers.