(R/S)-lactate/2-hydroxybutyrate dehydrogenases in and biosynthesis of block copolyesters by Ralstonia eutropha

Bacterial polyhydroxyalkanoates (PHAs) are promising bio-based biodegradable polyesters. It was recently reported that novel PHA block copolymers composed of ( R )-3-hydroxybutyrate (3HB) and ( R )-2-hydroxybutyrate (2HB) were synthesized by Escherichia coli expressing PhaC AR , a chimeric enzyme of PHA synthases derived from Aeromonas caviae and Ralstonia eutropha . In this study, the sequence-regulating PhaC AR was applied in the natural PHA-producing bacterium, R. eutropha . During the investigation, ( R / S )-2HB was found to exhibit strong growth inhibitory effects on the cells of R. eutropha . This was probably due to formation of excess 2-ketobutyrate (2KB) from ( R / S )-2HB and the consequent l -valine depletion caused by dominant l -isoleucine synthesis attributed to the excess 2KB. Deletion analyses for genes of lactate dehydrogenase homologs identified cytochrome-dependent d -lactate dehydrogenase (Dld) and [Fe-S] protein-dependent l -lactate dehydrogenase as the enzymes responsible for sensitivity to ( R )-2HB and ( S )-2HB, respectively. The engineered R. eutropha strain ( phaC AR + , ldhA Cd -hadA Cd + encoding clostridial ( R )-2-hydroxyisocaproate dehydrogenase and ( R )-2-hydoroxyisocaproate CoA transferase, ∆ dld ) synthesized PHA containing 10 mol% of 2HB when cultivated on glucose with addition of sodium ( RS )-2HB, and the 2HB composition in PHA increased up to 35 mol% by overexpression phaC AR . The solvent fractionation and NMR analyses showed that the resulting PHAs were most likely to be block polymers consisting of P(3HB- co -3HV) and P(2HB) segments, suggesting that PhaC AR functions as the sequence-regulating PHA synthase independently from genetic and metabolic backgrounds of the host cell. Key points (R/S)-2-hydroxubutyrates (2HB) caused l-valine deletion in Ralstonia eutropha (R)- and (S)-lactate/2HB dehydrogenases functional in R. eutropha were identified The engineered R. eutropha synthesized block copolymers of 2HB-containing polyhydroxyalkanoates on glucose and 2HB Graphical Abstract