Engineering of Pseudomonas putida to produce medium-chain-length polyhydroxyalkanoate from crude glycerol

The development of biodegradable polymers is crucial for addressing environmental issues and waste management challenges, and a medium-chain-length polyhydroxyalkanoate(MCL-PHA) exhibits significant application potential in diverse industrial and environmental contexts owing to its versatility and biodegradability. Here, Pseudomonas putida was metabolically engineered to produce MCL-PHA from crude glycerol. To increase the precursor pool, we first deleted the phaC1ZC2 operon and introduced a plasmid-based overexpression of phaC2 and phaG, and the MCL-PHA content derived from glycerol increased to 18.27 wt% at 60 h. Subsequently, by optimizing the acoA expression through promoter selection and UTR design, the MCL-PHA content further increased to 19.93 wt% at 72 h. Additionally, a notable increase in MCL-PHA production was achieved using PhaC2 designed to have no substrate-trapping effect (PhaC2^A477A478). This improvement was guided by filling structural data gaps using AlphaFold2 and docking simulations that revealed the substrate-trapping phenomenon. High-level production of MCL-PHA was achieved through fed-batch fermentation using the final engineered P. putida from refined glycerol, which yielded 34.9 g/L of MCL-PHA with 44.64 wt% at 180 h. Furthermore, using crude glycerol as the sole carbon source enabled the production of 49.5 g/L of MCL-PHA with 45.41 wt% at 180 h in fed-batch culture.