Establishment of an alternative phosphoketolase-dependent pathway for fructose catabolism in Ralstonia eutropha H16.

The β-proteobacterium Ralstonia eutropha H16 utilizes fructose and gluconate as carbon sources for heterotrophic growth exclusively via the Entner-Doudoroff pathway with its key enzyme 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase. By deletion of the responsible gene eda, we constructed a KDPG aldolase-negative strain, which is disabled to supply pyruvate for energy metabolism from fructose or gluconate as sole carbon sources. To restore growth on fructose, an alternative pathway, similar to the fructose-6-phosphate shunt of heterofermentative bifidobacteria, was established. For this, the xfp gene from Bifidobacterium animalis, coding for a bifunctional xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp; Meile et al. in J Bacteriol 183:2929-2936, 2001), was expressed in R. eutropha H16 PHB(-)4 Δeda. This Xfp catalyzes the phosphorolytic cleavage of fructose 6-phosphate to erythrose 4-phosphate and acetylphosphate as well as of xylulose 5-phosphate to glyceralaldehyde 3-phosphate and acetylphosphate. The recombinant strain showed phosphoketolase (PKT) activity on either substrate, and was able to use fructose as sole carbon source for growth, because PKT is the only enzyme that is missing in R. eutropha H16 to establish the artificial fructose-6-phosphate shunt. The Xfp-expressing strain R. eutropha H16 PHB(-)4 Δeda (pBBR1MCS-3::xfp) should be applicable for a novel variant of a plasmid addiction system to stably maintain episomally encoded genetic information during fermentative production processes. Plasmid addiction systems are often used to ensure plasmid stability in many biotechnology relevant microorganisms and processes without the need to apply external selection pressure, like the addition of antibiotics. By episomal expression of xfp in a R. eutropha H16 mutant lacking KDPG aldolase activity and cultivation in mineral salt medium with fructose as sole carbon source, the growth of this bacterium was addicted to the constructed xfp harboring plasmid. This novel selection principle extends the applicability of R. eutropha H16 as production platform in biotechnological processes.