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

ABSTRACT: 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: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00253-023-12797-6.