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 °C, 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.

Limonene is a valuable monoterpenoid that with diverse applications. Microbial biosynthesis offers a sustainable alternative to plant-based extraction, which is prone to hinderance by climate change and acreage. In the present study, the effects on limonene production of carbon source, inoculum, working volume, temperature, pH, dodecane coverage, and media were investigated with engineered strains of Rhodosporidium toruloides. Through single-factor and orthogonal experiments, the limonene titer was improved from 52.2 to 358.1 mg/L on 250-mL flask, a 586% increase. The optimized medium was MM, with a working volume of 50 mL, inoculum OD600=0.6, 22 °C, pH 6, and dodecane coverage (v/v) of 20%. The results here demonstrated that the R. toruloides could be explored as an efficient limonene producer and facilitate other monoterpenoids biosynthesis with R. toruloides.