Chain termination in polyhydroxyalkanoate synthesis: involvement of exogenous hydroxy-compounds as chain transfer agents

We have identified a range of compounds which, when present during poly(3-h ydroxybutyrate) [P(3HB)] accumulation by Ralstonia eutropha (reclassified f rom Alcaligenes eutrophus), can act as chain transfer agents in the chain t ermination step of polymerization. End-group analysis by P-31 NMR of polyme r derivatized with 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane reve aled that all these compounds were covalently linked to P(3HB) at the carbo xyl terminus. All chain transfer agents possessed one or more hydroxyl grou ps, and glycerol was selected for further investigation. The number-average molecular mass (M-n) of P(3HB) produced by R. eutropha from glycerol was s ubstantially lower than for polymer produced from glucose, and we identifie d two new end-group structures. These were attributed to a glycerol molecul e bound to the P(3HB) chain via the primary or secondary hydroxyl groups. W hen a primary hydroxyl group of glycerol is involved in chain transfer, the end-group structure is in both [R] and [S] configurations, implying that c hain transfer to glycerol is a random transesterification and that PHA synt hase does not catalyse chain transfer. 3-Hydroxybutyric acid is the most pr obable chain transfer agent in vivo, with propagation and termination react ions involving transfer of the P(3HB) chain to enzyme-bound and free 3-hydr oxybutyrate, respectively. Only carboxyl end-groups were detected in P(3HB) extracted from exponentially growing bacteria. It is proposed that a compo und other than 3-hydroxybutyryl-CoA acts as a primer in the initiation of p olymer synthesis. (C) 1999 Elsevier Science B.V. All rights reserved.