Optimization of Fermentation Conditions for Elevating Limonene Production with Engineered Rhodosporidium toruloides

Limonene is a valuable monoterpenoid with diverse applications in food, medicine, agriculture, etc. Currently, the commercial production of limonene from citrus pomace cannot meet the evergrowing market demand. With the development of synthetic biology, microbial biosynthesis is offering a sustainable alternative to plant-based extraction. However, the performance of engineered strains can be affected by many factors including the microbial host, the cultivation conditions, etc. Therefore, in the present study, potential factors that influence the limonene production of the engineered strains were investigated through single-factor and orthogonal experiments. The optimized medium was MM, with a working volume of 50 mL, inoculum OD600 = 0.6, 22?, pH 6, and a dodecane coverage (v/v) of 20%. The final limonene titer was improved from 52.5 to 358.1 mg/L, a 586% net increase in titer and 34.8-fold improvement in production efficiency (mg/OD600) on 250-mL flasks. The results here demonstrated that R. toruloides could be explored as an efficient limonene producer and could facilitate the study of the biosynthesis of other terpenes using R. toruloides.

Automated summary

Potential factors influencing the production of limonene in engineered strains were investigated through single-factor and orthogonal experiments. The optimized conditions included using MM as the medium, a working volume of 50 mL, inoculum OD600 = 0.6, 22°C incubation temperature, pH 6, and a dodecane coverage of 20%. The final limonene titer was improved from 52.5 to 358.1 mg/L, showing a 586% increase in titer and a 34.8-fold improvement in production efficiency (mg/OD600) in 250-mL flasks. These results suggest that R. toruloides could be a promising producer of limonene and facilitate the biosynthesis study of other terpenes.