Biosynthesis of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from CO₂ by a Recombinant Cupriavidusnecator

The copolyester of 3-hydroxybutyrate (3HB) and 3-hydoxyhexanoate (3HHx), PHBHHx, is one of the most practical kind of bacterial polyhydroxyalkanoates due to its high flexibility and marine biodegradability. PHBHHx is usually produced from vegetable oils or fatty acids through β-oxidation, whereas biosynthesis from sugars has been achieved by recombinant strains of hydrogen-oxidizing bacterium Cupriavidus necator. This study investigated the biosynthesis of PHBHHx from CO₂ as the sole carbon source by engineered C. necator strains. The recombinant strains capable of synthesizing PHBHHx from fructose were cultivated in a flask using complete mineral medium and a substrate gas mixture (H₂/O₂/CO₂ = 8:1:1). The results of GC and ¹H NMR analyses indicated that the recombinants of C. necator synthesized PHBHHx from CO₂ with high cellular content. When 1.0 g/L (NH₄)₂SO₄ was used as a nitrogen source, the 3HHx composition of PHBHHx in the strain MF01∆B1/pBBP-ccr_MeJ4a-emd was 47.7 ± 6.2 mol%. Further investigation demonstrated that the PHA composition can be regulated by using (R)-enoyl-CoA hydratase (PhaJ) with different substrate specificity. The composition of 3HHx in PHBHHx was controlled to about 11 mol%, suitable for practical applications, and high cellular content was kept in the strains transformed with pBPP-ccr_MeJ_Ac-emd harboring short-chain-length-specific PhaJ.