Poly-β-hydroxybutyrate biosynthesis in the facultative methylotroph methylobacterium extorquens AM1: Identification and mutation of gap11, gap20, and phaR

Methylobacterium extorquens AM1, a serine cycle facultative methylotroph, accumulates poly-β-hydroxybutyrate (PHB) as a carbon and energy reserve material during growth on both multicarbon- and single-carbon substrates. Recently, the identification and mutation of the genes involved in the biosynthesis and degradation of PHB have been described for this bacterium, demonstrating that two of the genes of the PHB cycle (phaA and phaB) are also involved in C₁ and C₂ metabolism, as part of a novel pathway for glyoxylate regeneration in the serine cycle (N. Korotkova and M. E. Lidstrom, J. Bacteriol. 183:1038-1046, 2001; N. Korotkova, L. Chistoserdova, V. Kuksa, and M. E. Lidstrom, J. Bacteriol. 184:1750-1758, 2002). In this work, three new genes involved in PHB biosynthesis in this bacterium have been investigated via mutation and phenotypic analysis: gap11, gap20, and phaR. We demonstrate that gap11 and gap20 encode two major granule-associated proteins (phasins) and that mutants with mutations in these genes are defective in PHB production and also in growth on C₂ compounds, while they show wild-type growth characteristics on C₁ or multicarbon compounds. The phaR mutant shows defects in both PHB accumulation and growth characteristics when grown on C₁ compounds and has defects in PHB accumulation but grows normally on C₃ and C₄ compounds, while both PHB accumulation and growth rate are at wild-type levels during growth on C₂ compounds. Our results suggest that this phenotype is due to altered fluxes of acetyl coenzyme A (CoA), a major intermediate in C₁, C₂, and heterotrophic metabolism in M. extorquens AM1, as well as the entry metabolite for the PHB cycle. Therefore, it seems likely that PhaR acts to control acetyl-CoA flux to PHB in this methylotrophic bacterium.