Isolation of autonomously replicating sequence and gene knockout using an episomal plasmid in Rhodosporidium toruloides

Abstract:

【Objective】 Episomal expression vectors typically have higher copy number to achieve strong gene expression than chromosomal expression vectors. Moreover, they are more convenient and flexible for DNA manipulation. However, the episomal plasmids suitable for the application in Rhodosporidium toruloides remain to be determined, and the expression of heterologous genes or CRISPR/Cas9-based genome editing needs to be achieved by integration, which is a key reason for the slow progress in its genetic modification. Thus, this work aims to construct an episomal plasmid of R. toruloides, which facilitates the expression of heterologous genes and promotes the gene editing in a time-saving manner. 【Methods】 First, the possible autonomously replicating sequences (ARSs) in the phenylalanine ammonia-lyase gene (PAL) of R. toruloides were mined. Specifically, PAL and its upstream and downstream sequences were amplified in segments and constructed into a plasmid containing the β-isopropyl malate dehydrogenase gene (LEU2). The recombinant plasmids were then introduced into LEU2-deficient R. toruloides by the electroporation method. An ARS was then identified according to transformation efficiency. Then, the BTS1 gene encoding geranylgeranyl pyrophosphate synthase was selected as the knockout target, and its gRNA was constructed into the episomal plasmid based on the identified ARS. The color change of the transformant was observed to verify whether the episomal plasmid was successfully applied to the CRISPR/Cas9 system of R. toruloides. 【Results】 In this work, an ARS was identified, based on which an episomal plasmid was constructed and applied to CRISPR/Cas9 editing in R. toruloides. Finally, the episomal plasmid-based gene knockout of R. toruloides was successfully achieved. 【Conclusion】 This work enriched the existing tool library and provided a research basis and technical support for the application of R. toruloides in synthetic biology.