The Effect of Co-Substrate Feeding on Polyhydroxyalkanoate (PHA) Homopolymer and Copolymer Production in Recombinant Escherichia coli LS5218

Polyhydroxyalkanoates (PHAs) are aliphatic polyesters produced by a wide variety of bacteria as carbon and energy storage sources. Copolymers of the short chain length (SCL) 3-hydroxybutyrate (3HB) repeating unit and medium chain length hydroxyalkanoate (MCL-HA) repeating units have better thermal and mechanical properties than poly-3HB homopolymers. In this study, a MCL monomer supplier gene phaJ4 from Pseudomonas putida KT2440, an engineered PHA synthase gene (phaC1) from Pseudomonas sp. 61-3, and the SCL monomer supplier genes, phaA and phaB from Ralstonia eutropha, were coexpressed under the lac promoter in recombinant E. coli LS5218 to produce SCL-MCL copolymers. The recombinant strains were co-fed lauric acid (a fatty acid) and glucose as a carbon source and the ratio of SCL to MCL monomers varied dependant on differences in the timing of the addition of each carbon source during the fermentation. The PHA copolymers produced exhibited a range from 0.4 mol% to 35 mol% MCL repeating units giving rise to wide range of physical properties. The molecular weights and thermal properties of different polymers were studied by gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). The results suggested the polymers produced by co-feeding with lauric acid and glucose were mixtures of 3HB homopolymers and 3HB-co-MCL copolymers. This conclusion was confirmed by acetone fractionation.