18-Crown-6 ether increases molecular weight of poly(3-hydroxybutyrate) synthesized in Escherichia coli

Molecular weight of polyhydroxyalkanoate (PHA) is a critical factor governing their physical properties, particularly mechanical strength. It is empirically known that molecular weight of PHA varies depending on factors such as the microbial host and the type of PHA synthase (PhaC). However, methods for controlling the molecular weight of PHA are limited. In this study, we found that the addition of a cyclic polyether, 18-crown-6 ether (18c6), to the culture medium of recombinant Escherichia coli increased molecular weight of poly(3-hydroxybutyrate) [P(3HB)]. Cells expressing an engineered class I PHA synthase, PhaC_AR, and propionyl-coenzyme A transferase from Megasphaera elsdenii were cultivated with the supplementation of 3HB for P(3HB) production. The P(3HB) molecular weight increased by up to 20-fold in an 18c6 concentration-dependent manner. The addition of 18c6 reduced E. coli growth, but it had no significant effect on PhaC expression levels. Furthermore, in vitro assay indicated that 18c6 does not affect the enzymatic activity of PhaC. Therefore, these results suggest that the effect of 18c6 is not attributable to its direct interaction with PhaC. The addition of 18c6 also increased the molecular weight of poly(3-hydroxypropionate), but did not increase that of poly(3-hydroxyhexanoate), indicating that the effect of 18c6 varies depending on the monomer used for the polymer synthesis. The addition of 18c6 could potentially be utilized as a versatile method for producing high-molecular-weight PHAs.