Enhancing oil feedstock utilization for high-yield low-carbon polyhydroxyalkanoates industrial bioproduction.

Polyhydroxyalkanoates (PHAs) are biodegradable and environmentally sustainable alternatives to conventional plastics, yet their adoption has been hindered by the high production costs and scalability challenges. This study employed unbiased genomics approaches to engineer Cupriavidus necator H16, an industrial strain with intrinsic capabilities for PHA biosynthesis, for enhanced utilization of oil-based feedstocks, including food-grade palm oil and crude waste cooking oil. The engineered strain demonstrated significant improvements in PHA production, achieving a 264 g/L yield (25.4 % increase) and a 100 g/g conversion rate of palm oil (12 % increase) in 60-h fed-batch fermentation at 150 m production scale, the highest yield and conversion rate using food-grade palm oil as carbon source reported to the best of our knowledge. Notably, the carbon footprint of PHA production was reduced by 29.7 % using the engineered strain, and could be further reduced by adopting waste cooking oil. Mechanistic studies revealed that the H16_A3043/H16_A3044 two-component system plays a central role in regulating stress response and biogenesis, the deletion of which unlocked the regulatory constraint and enhanced oil feedstock consumption. This mutation, supplemented with the necessary lipase engineering as revealed during the scale-up troubleshooting, confered higher PHA production in a robust fermentation process scalable through 0.5 L, 200 L, 15 m and 150 m. Additionally, the engineered strain demonstrated efficient utilization of waste cooking oil for PHA production. This study bridges laboratory-scale advancements and industrial feasibility, demonstrating a scalable, sustainable, and economically viable pathway for biopolymer production, contributing to the global shift toward a circular bioeconomy.