Enhanced oxalate decarboxylase biomanufacturing via multi-level optimization and integrated life cycle assessment

Oxalate decarboxylase (OXDC) holds substantial potential for applications in food safety, feed supplementation, and environmental remediation. Nevertheless, its industrial production is still restricted by antibiotic-dependent plasmid maintenance, refined substrate consumption, and a high environmental burden. Here, we developed a resource-efficient and sustainable biomanufacturing strategy. A multi-strategy computational framework identified key mutation sites, and a quadruple mutant (K51L-W55Y-D83R-W234Y) exhibited 24 % higher catalytic activity and 42 % improved thermal stability than the wild type. Molecular dynamics analysis attributed these improvements to enhanced dynamic coupling and structural compactness while preserving the Mn²⁺ catalytic center. To eliminate antibiotic use, a hok/sok stabilization system was introduced for stable plasmid maintenance. Meanwhile, intracellular ATP regeneration mediated by polyphosphate kinase led to a 35.32 % increase in ATP levels, which in turn resulted in a 21.62 % improvement in OXDC titer. We substituted refined nitrogen and carbon sources with agro-industrial residues, enabling cost reduction and improved resource utilization. In a 5 L fed-batch fermentation, the production of OXDC reached 1.132 g/L. Life cycle assessment showed that the optimized process reduced total environmental impacts by 86.9 % compared to the conventional antibiotic-dependent process, with notable reductions in climate change and metal depletion driven by material consumption. Overall, this work establishes a scalable and energy-efficient bioprocess by coupling protein engineering with cellular and process-level optimization, providing a generalizable framework for the sustainable production of energy-intensive recombinant enzymes.