Engineering Cupriavidus cauae PHS1 as a non-model thermotolerant chassis for the direct conversion of palm oil and waste frying oil into polyhydroxybutyrate
Thermotolerant microbial hosts offer advantages for industrial bioprocesses, yet direct oil-based bioconversion requires efficient coupling of extracellular lipid hydrolysis with intracellular carbon assimilation. In the thermotolerant bacterium Cupriavidus cauae PHS1, direct utilization of triacylglycerol-rich substrates is constrained by the lack of extracellular lipolytic activity and limited glycerol metabolism. Here, the underexplored non-model bacterium C. cauae PHS1 was engineered as a thermotolerant chassis for direct conversion of palm oil (PO) and waste frying oil (WFO) into polyhydroxybutyrate (PHB) at 42 °C. Heterologous expression of a thermostable secretory lipase from C. necator H16 conferred extracellular oil-hydrolysis capability, and adaptive laboratory evolution was applied to improve glycerol metabolism. This combined strategy enabled direct utilization of triacylglycerol-derived carbon and enhanced PHB accumulation from both a model oil feedstock and a complex waste oil feedstock. Under non-optimized culture conditions, the engineered strain produced PHB from 10 g/L PO, reaching 5.11 g/L cell dry weight (CDW) with 64.18% PHB content, and from 10 g/L WFO, achieving 4.03 g/L CDW with 52.61% PHB content. These results support the potential of C. cauae PHS1 as a promising thermotolerant chassis for sustainable PHB biomanufacturing from complex waste oil feedstocks.