Mining and application of constitutive promoters from Rhodosporidium toruloides

Rhodosporidium toruloides is an oleaginous yeast under development with promising industrial applications. Since promoters of different strengths have been demonstrated as an efficient strategy to fine-tune gene expression in synthetic biology, a set of constitutive promoters with strengths varying over 2 orders of magnitude were identified in R. toruloides through transcriptome analysis under different growth conditions. Promoter candidates were first cloned and characterized using an enhanced green fluorescent protein (EGFP) as a reporter under eight conditions, and 31 promoters were identified with strength varied from 0.1 to 19.0 folds of the commonly used strong promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (P_GPD1). The resultant promoters were then used to optimize the linoleic acid biosynthetic pathway in R. toruloides in different media, including the use of lignocellulosic hydrolysate as the fermentation substrate, and improved the production of linoleic acid by up to 214.2% in minimal medium, with the highest production of 350.3 mg/L in Yeast Peptone Dextrose medium. This work has enriched the promoter library of R. toruloides, and helped develop R. toruloides as a platform organism for applications in biomanufacturing and synthetic biology.