Abstract

Polyhydroxyalkanoates (PHAs) are produced as carbon and energy storage materials in various bacteria, with some members known to be biodegradable and biocompatible materials. (R)-3-hydroxybutyrate homopolymer [P(3HB)] is the most common bacterial PHA found in nature. PHA synthase (PhaC) catalyzes the polymerization reaction of (R)-3-hydroxybutyryl (3HB) moiety in (R)-3HB-coenzyme A (CoA) to PHAs with the release of CoA. At the end of the polymerization reaction, chain transfer (CT) reaction occurs wherein the polymer chain is transferred to a CT agent, resulting in covalent binding of the CT agent to the PHA chain at its carboxy end. CT reaction takes place as well as initiation and chain-propagation reactions in the biological polymerization system of (R)-3HB-CoA with PHA synthase as follows: Initiation: E-SH + 3HB-S-CoA  3HB-S-E + CoA-SH Chain propagation: (3HB)n-1-S-E + 3HB-S-CoA  (3HB)n-S-E + CoA-SH Chain transfer: (3HB)n-S-E + X-OH  (3HB)n-O-X + E-SH where E-SH, CoA-SH, 3HB-S-CoA, (3HB)n, and X-OH denote PHA synthase with thiol groups as the active site, free CoA, (R)-3-hydroxybutyryl-CoA as monomer, a polymer chain with n 3HB units, and a CT agent, respectively. Hydroxy compounds, such as 3HB, glycerol, and water, have been proposed as potential naturally occurring CT agents, whereas low molecular weight poly(ethylene glycols) (PEGs) are known as artificial CT agents. In CT reaction, the PHA polymer chain is transferred from PhaC to a CT agent. Accordingly, the frequency of CT reaction determines PHA molecular weight. We aimed to clarify the effect of endogenous ethanol as a CT agent for P(3HB) synthesis in recombinant Escherichia coli, by comparing with that of exogenous ethanol. Ethanol supplementation to the culture medium reduced P(3HB) molecular weights by up to 56% due to ethanol-induced CT reaction. NMR analysis of P(3HB) polymers purified from the culture supplemented with 13C-labeled ethanol showed the formation of a covalent bond between ethanol and P(3HB) chain at the carboxy end. Cultivation without ethanol supplementation resulted in the reduction of P(3HB) molecular weight with increasing host-produced ethanol depending on culture aeration. On the other hand, production in recombinant E. coli BW25113(ΔadhE), an alcohol dehydrogenase deletion strain, resulted in a 77% increase in molecular weight. Analysis of five E. coli strains revealed that the estimated number of CT reactions was correlated with ethanol production. These results demonstrate that host-produced ethanol acts as an equally effective CT agent as exogenous ethanol, and the control of ethanol production is important to regulate the PHA molecular weight.