Impact of various β-ketothiolase genes on PHBHHx production in Cupriavidus necator H16 derivatives

Poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] (PHBHHx) is a type of biopolyester of the polyhydroxyalkanoate group (PHA). Due to a wide range of properties resulting from the alteration of the (R)-3-hydroxyhexanoate (3HHx) composition, PHBHHx is getting a lot of attention as a substitute to conventional plastic materials for various applications. Cupriavidus necator H16 is the most promising PHA producer and has been genetically engineered to produce PHBHHx efficiently for many years. Nevertheless, the role of individual genes involved in PHBHHx biosynthesis is not well elaborated. C. necator H16 possesses six potential physiologically active β-ketothiolase genes identified by transcriptome analysis, i.e., phaA, bktB, bktC (h16_A0170), h16_A0462, h16_A1528, and h16_B0759. In this study, we focused on the functionality of these genes in vivo in relation to 3HHx monomer supply. Gene deletion experiments identified BktB and H16_A1528 as important β-ketothiolases for C6 metabolism in β-oxidation. Furthermore, in the bktB/h16_A1528 double-deletion strain, the proportion of 3HHx composition of PHBHHx produced from sugar was very low, whereas that from plant oil was significantly higher. In fact, the proportion reached 36.2 mol% with overexpression of (R)-specifc enoyl-CoA hydratase (PhaJ) and PHA synthase. Furthermore, we demonstrated high-density production (196 g/L) of PHBHHx with high 3HHx (32.5 mol%) by fed-batch fermentation with palm kernel oil. The PHBHHx was amorphous according to the differential scanning calorimetry analysis. KEY POINTS: • Role of six β-ketothiolases in PHBHHx biosynthesis was investigated in vivo. • Double-deletion of bktB/h16_A1528 results in high 3HHx composition with plant oil. • Amorphous PHBHHx with 32.5 mol% 3HHx was produced in high density by jar fermenter.