In vitro synthesis of poly(3-hydroxybutyric acid) by using an enzymatic coenzyme A recycling system

Abstract

Purified recombinant poly(hydroxyalkanoic acid), PHA, synthase from Chromatium vinosum was used to examine in vitro poly(3-hydroxybutyric acid) (P(3HB)) formation. In combination with purified propionyl-coenzyme A transferase of Clostridium propionicum a two-enzyme in vitro P(3HB) biosynthesis system was established which allowed the synthesis of P(3HB) from free d-(−)-3-hydroxybutyric acid as substrate. The coenzyme A residue for the activation of this hydroxyacid was provided by acetyl-coenzyme A. By adding acetyl-coenzyme A synthetase to this system, a three-enzyme in vitro P(3HB) biosynthesis system was established. Coenzyme A that was released during the polymerization reaction was coupled to acetate which again served as the coenzyme A donor for the activation of 3-hydroxybutyric acid. The energy for the in vitro P(3HB) synthesis was provided by ATP hydrolyses resulting in acetyl-coenzyme A synthesis catalyzed by the acetyl coenzyme A synthetase. In this way the in vitro synthesis of P(3HB) became independent of the consumption of the expensive coenzyme A. By this procedure a handy system is available to produce in vitro PHA on a semipreparative scale.