Formation of polyester blends by a recombinant strain ofPseudomonas oleovorans: Different poly(3-hydroxyalkanoates) are stored in separate granules

WhenPseudomonas oleovorans (GPo1) is grown on sodium octanoate under ammonium limiting conditions, it is able to accumulate a copolyester consisting of medium chain length 3-hydroxyalkanoic acids (PHA_m). 3-Hydroxybutyrate is only incorporated in trace amounts. WhenP. oleovorans is equipped with the PHB biosynthetic genes ofAlcaligenes eutrophus (GPo1[pVK101::PP1]), it forms a polyester containing major amounts of 3-hydroxybutyrate. The resulting polymer however is a blend of PHA_m and PHB, rather than a copolymer of 3-hydroxybutyrate and medium chain length 3-hydroxyalkanoic acids [11]. To establish whether PHA_m and PHB molecules are stored in the same or separate granules by this recombinantP. oleovorans strain, we studied polymer forming cells by freeze-fracture electron microscopy. This approach is possible because previous freeze-fracture electron microscopy studies on PHA_m and PHB accumulating strains have shown that PHA_m and PHB granules can be distinguished from each other: PHA_m granules from mushroom-like structures, whereas PHB granules from needle structures during freeze-fracturing. In this paper we show that stationary phase cells of GPo1[pVK101::PP1] contained both mushroom and needle-like structures, indicating that PHA_m and PHB chains were stored in separate granules. To be able to determine whether the separation of PHA_m and PHB is complete, the respective granules were separated on sucrose gradients. A total cell extract of GPo1[pVK101::PP1] which was subjected to sucrose gradient centrifugation revealed two white bands of different densities: the upper band with a density of 1.05 g/mL consisted exclusively of PHA_m granules, while the lower band with a density of 1.19 g/mL consisted of PHB granules only. Thus, when bacteria synthesize both PHA_m and PHB, the resulting polymer chains are segregated completely and stored in separate granules.