Deletion of Glyoxylate Shunt Pathway Genes Results in a 3-Hydroxybutyrate Overproducing Strain of Ralstonia Eutropha

The glyoxylate shunt pathway was examined in the polyhydroxyalkanoate (PHA) producing bacterium, Ralstonia eutropha. The deletion of the aceB gene, encoding malate synthase, which catalyzes the second reaction of the glyoxylate shunt, resulted in severe growth defects in cultures containing acetate or fatty acids as the main carbon source. Further deletions of phaB genes, encoding acetoacetyl-CoA reductase enzymes, resulted in little or no growth of R. eutropha on acetate. Metabolomic studies revealed that the aceB deletion strain overproduced pyruvate and 3-hydroxybutyrate, both at 15X higher levels than the wild-type strain, when cultured in acetate as the main carbon source. To further enhance production of these compounds, the PHA biosynthetic pathway was disabled in the R. eutropha aceB mutant by an in-frame deletion of the phaC (PHA synthase) gene. The resulting strain produced even higher levels of 3-hydroxybutyrate at the onset of nitrogen limitation. This study has provided insight to the construction of a strain of R. eutropha that potentially produces value-added compounds when grown on carbon feedstocks containing acetate or fatty acids.