Production of polyhydroxyalkanoate (PHA) from renewable carbon sources in recombinant Ralstonia eutropha using mutants of original PHA synthase

Abstract

Ralstonia eutropha biopolyester synthase (PhaC Re) is a key enzyme for catalyzing formation of polyhydroxybutyrate (PHB) or polyhydroxyalkanoate (PHA) copolyesters from ( R)-3-hydroxybutyryl-CoA (3HB-CoA) and/or ( R)-3-hydroxyalkanoyl-CoA (3HA-CoA). Previously, in Escherichia coli, we found a good correlation between the accumulation of PHB and PhaC Re activity using seven PhaC Re mutants, all of which exhibited lower activities compared to the wild-type PhaC Re. In this study, these PhaC Re mutants were individually introduced into a PHB-negative mutant strain (PHB −4) of R. eutropha, as well as a new activity-revertant (termed E11S5) of a primary mutant E11. Recombinant R. eutropha PHB −4 strains harboring PhaC Re mutant or the wild-type expression plasmids were cultivated using renewable carbon sources. No parallel relationship in the accumulation levels of PHA (PHB and PHA copolyesters) between recombinant hosts, E. coli JM109 (for PHB) and R. eutropha PHB −4 (for PHB or PHA copolyesters) was observed. This suggests, in contrast with the case of E. coli, that the PHA accumulation in R. eutropha is not governed solely by PhaC Re activity. Enhanced PHA accumulation was achieved by some mutants (including E11 and E11S5) throughout all cultivation conditions. It is likely that monomer compositional changes of PHA copolyesters generated by recombinants may be attributable to the changed substrate specificities of mutants toward 3HB-CoA and 3HA-CoA monomer substrates channeled from the related metabolic pathways.