Microbial upcycling of unrefined glycerol to beta-carotene by tailored metabolic engineering of extremophilic microorganism Deinococcus radiodurans R1

Unrefined glycerol, an abundant byproduct of biodiesel production, has emerged as a promising carbon source for microbial biorefineries. Despite its potential, large-scale application remains limited by cytotoxic impurities such as residual methanol and peroxides. Here, we report the engineering of extremophilic microorganism Deinococcus radiodurans R1 for efficient β-carotene production from unrefined glycerol. To establish a base strain, the crtY gene was overexpressed and the crtLm genes were deleted to complete the β-carotene biosynthetic pathway, enabling production of 0.46 ± 0.03 mg/L (0.10 ± 0.01 mg/g DCW). Further pathway optimization through deleting the crtO gene and fine-tuning of rate-limiting steps by gene overexpression and promoter replacement enhanced β-carotene production to 5.97 ± 0.35 mg/L (2.03 ± 1.03 mg/g DCW), representing a 33.2-fold increase. To enable glycerol utilization, the glpK, glpD, and tpiA genes were integrated into the genome, which resulted in the production of 11.56 ± 1.03 mg/L (2.27 ± 0.20 mg/g DCW) β-carotene from 10 g/L unrefined glycerol, approximately three-fold and 64-fold higher than the yields obtained using glucose and the base strain, respectively. In fed-batch fermentation, the engineered strain achieved 27.23 mg/L (2.60 mg/g DCW) from 25.45 g/L unrefined glycerol within 102 h. These findings demonstrate that the final engineered strain is promising and worthy of as a robust platform for cost-effective and sustainable biorefinery applications.