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The high production cost of bio-based plastic polyhydroxyalkanoates (PHAs) limits their use as commercial products. Thus, systems for PHAs production from waste substrates could reduce production costs. Crude glycerol is a by-product of biodiesel fuel production and thus represents an inexpensive, abundant and promising carbon source for production of valorized fermentation products. In this study, industrial crude glycerol by-product from palm oil biodiesel production was used as the carbon source for production of PHAs by recombinant Escherichia coli. Crude glycerol supplemented at 1-5 % (v/v) supported production of poly(3-hydroxybutyrate) (P(3HB)) in E. coli-ABC, which harbors the PHA synthetic genes for β-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB) of Ralstonia eutropha and Polyhydroxyalkanoate (PHA) synthase (PhaC) of Aeromonas hydrophila. The highest P(3HB) content and productivity of 14 wt% of cell dry weight and 0.6 g/L, respectively, were obtained at 1 % (v/v) glycerol concentration. Production of P(3HB- co-3-hydroxyhexanoate) (P(3HB- co-3HHx)) was achieved using E. coli-ABCJ, harboring genes for PhaA, PhaB, PhaC, and the ( R)-specific enoyl-CoA hydratase (PhaJ) of A. hydrophila. This led to the copolymer content of 3 wt% of cell dry weight with 1 mol% of 3HHx. Molecular weight and degradation temperature of the polymers were in the range of 110-130 kDa and 295-299 °C, respectively. These results indicated that crude glycerol could be an attractive carbon source for economical production of PHAs with properties for industrial application. [ABSTRACT FROM AUTHOR]

Ralstonia eutropha H16 is a well-studied bacterium with respect to biosynthesis of polyhydroxyalkanoates (PHAs), which has attracted attentions as biodegradable bio-based plastics. However, this strain shows quite poor growth on glycerol of which bulk supply has been increasing as a major by-product of biodiesel industries. This study examined enhancement of glycerol assimilation ability of R. eutropha H16 by introduction of the genes of aquaglyceroporin ( glpF) and glycerol kinase ( glpK) from Escherichia coli. Although introduction of glpFK into the strain H16 using a multi-copy vector was not successful, a recombinant strain possessing glpFK within the chromosome showed much faster growth on glycerol than H16. Further analyses clarified that weak expression of glpK alone allowed to establish efficient glycerol assimilation pathway, indicating that the poor growth of H16 on glycerol was caused by insufficient kination activity to glycerol, as well as this strain had a potential ability for uptake of extracellular glycerol. The engineered strains expressing glpFK or glpK produced large amounts of poly[( R)-3-hydroxybutyrate] [P(3HB)] from glycerol with much higher productivity than H16. Unlike other glycerol-utilizable wild strains of R. eutropha, the H16-derived engineered strains accumulated P(3HB) with no significant decrease in molecular weights on glycerol, and the polydispersity index of the glycerol-based P(3HB) synthesized by the strains expressing glpFK was lower than those by the parent strains. The present study demonstrated possibility of R. eutropha H16-based platform for production of useful compounds from inexpensive glycerol. [ABSTRACT FROM AUTHOR]