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In this study, a methylotrophic bacterium, Methylobacterium rhodesianum MB 126, was used for the production of the chiral compound (R)-3-hydroxybutyrate (R-3HB) from methanol. R-3HB is formed during intracellular degradation of the storage polymer (R)-3-polyhydroxybutyrate (PHB). Since the monomer R-3HB does not accumulate under natural conditions, M. rhodesianum was genetically modified. The gene (hbd) encoding the R-3HB-degrading enzyme, R-3HB dehydrogenase, was inactivated in M. rhodesianum. The resulting hbd mutant still exhibited low growth rates on R-3HB as the sole source of carbon and energy, indicating the presence of alternative pathways for R-3HB utilization. Therefore, transposon mutagenesis was carried out with the hbd mutant, and a double mutant unable to grow on R-3HB was obtained. This mutant was shown to be defective in lipoic acid synthase (LipA), resulting in an incomplete citric acid cycle. Using the hbd lipA mutant, we produced 3.2 to 3.5 mM R-3HB in batch and 27 mM (2,800 mg liter(-1)) in fed-batch cultures. This was achieved by sequences of cultivation conditions initially favoring growth, then PHB accumulation, and finally PHB degradation.

Production of the Chiral Compound ( R )-3-Hydroxybutyrate by a Genetically Engineered Methylotrophic Bacterium