Development of a multigene expression system using 2A peptides in <i>Rhodosporidium toruloides</i>

In eukaryotes, gene expression typically requires individual promoter and terminator for each gene, making the expression of multiple genes tedious and sometimes too difficult to handle. This is especially true for underdeveloped nonmodel organisms with few genetic engineering tools and genetic elements such as Rhodosporidium toruloides. In contrast, polycistronic expression offers advantages such as smaller size and ease of cloning. Here we report the development of a multigene expression system using 2A peptides in R. toruloides. First, twenty-two 2A peptides were evaluated for their cleavage efficiencies, which ranged from 33.65% to 93.32%. Subsequently, the 2A peptide of ERBV-1 with the highest efficiency was selected to enable simultaneous expression of four proteins. In addition, we demonstrated the optimization of the α-linolenic acid biosynthetic pathway using ERBV-1 peptide mediated polycistronic expression, which increased the α-linolenic acid production by 104.72%. These results suggest that using ERBV-1 peptide is an efficient strategy for multigene expression in R. toruloides.