Efficient co-expression of multiple enzymes from a single promoter mediated by virus 2A sequence in the oleaginous yeast Rhodosporidium toruloides

The red yeast Rhodosporidium toruloide is a versatile host for production of lipids and carotenoids. Genetic tools are underdeveloped for red yeasts due to their unique genetics and physiology. Currently expression of a heterogonous gene in red yeasts is largely based on integration of the designed cassette by Agrobacterium mediated transformation, yet this method is somewhat restricted when multiple genes are required to be expressed due to the lack of functional genetic elements. Here we demonstrate that virus 2A sequence is effective to mediate co-expression of multiple enzymes in R. toruloides. Two different 2A sequences, Porcine teschovirus-1 2A (P2A) and foot-and-mouth disease virus 2A (F2A), were evaluated. It was found that P2A sequence was more effective for co-expression of two antibiotic selection markers. Co-expression of three antibiotic resistance proteins was successful from a single promoter mediated by P2A sequence. When three heterogeneous enzymes responsible for beta-carotene biosynthesis were co-expressed, recombinant R. toruloides strains produced up to 4.5-fold more beta-carotene than that of the parental one. The use of 2A sequence can facilitate cassette construction to engineer advanced cell factories for production of lipids and related oleochemicals