Controlling 3-hydroxyhexanoate mole fraction in poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) by altering enoyl-CoA hydratase (phaJ) ribosome-binding site in Cupriavidus necator H16

Polyhydroxyalkanoate (PHA) is a bioplastic attracting interest as an alternative to petroleum-based plastics. Particularly, poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-co-3HHx)), which shows notable polymeric properties, is usually produced using the engineered Cupriavidus necator. Currently, production of P(3HB-co-3HHx) is primarily possible by engineering phaC, however, relatively rare study of controlling the expression of enoyl-CoA hydratase (phaJ_Pa), which is directly involved in 3-hydroxyhexanoate (3HHx) monomers synthesis, was shown to control 3HHx mole fraction. As a result, we aimed to verify this by constructing vectors housing phaC_BP-M-CPF4 and phaJ_Pa with different ribosome-binding site (RBS) to control PhaJ translation. When different constructions were applied, the fluctuation in the 3HHx molar fraction was directly related to the phaJ_Pa RBS sequence and it was shown that varying the RBS sequence to AAAGGAGATATAG produces increased 3HHx mole fraction (3.6–6.2%). When fermentation was performed for 168 h to verify the capacity of the engineered strain (H16/pSJ-3) for mass production, it produced 194.9 g/L dry cell weight and 155.4 g/L of P(3HB-co-9.5 mol% 3HHx). Overall, this study presents a different approach of altering polymer properties for manipulating the 3HHx mole fraction of P(3HB-co-3HHx) by controlling PhaJ translation.