Precise excision of a selectable marker gene in transgenic Coccomyxa strains by the piggyBac transposase

Abstract

The piggyBac transposon isolated from the cabbage looper moth Trichoplusia ni, is integrated into the host genome, and then excised from it without leaving a footprint. The piggyBac transposon system has been used as a genomic engineering tool in a variety of organisms. In this study, we used two improved versions of the piggyBac transposase (PBase) to create marker-free transgenic strains of the unicellular green alga Coccomyxa sp. strain KJ as follows: Uracil-auxotrophic (Ura−) mutants of strain KJ defective in the gene for uridine monophosphate synthase (KJUMPS) were isolated on agar plates containing 5-fluoroorotic acid. Subsequently, cDNA of KJUMPS (cKJUMPS) was cloned between the promoter and terminator of the elongation factor 1 alpha gene to construct a cKJUMPS expression cassette. A DNA fragment carrying the cKJUMPS expression cassette flanked with piggyBac transposon terminal repeats was then constructed (TR_cKJUMPS) and introduced into an Ura− mutant, and Ura+ transformants were isolated. One of the Ura+ transformants was named strain TR2-7. Hyperactive PBase (hyPBase) is a mutant PBase with increased excision and integration frequencies. Herein, we synthesized a coding sequence for hyPBase (KJhyPBase), which has optimized codons for expression in strain KJ, and its expression cassette was introduced into strain TR2-7. Fourteen transformants stably carried the KJhyPBase expression cassette, and TR_cKJUMPS was excised from seven of these. We also introduced an expression cassette of KJhyPBase_Ex, which encodes the excision-competent/integration-defective R372A/K375A/D450N mutant of KJhyPBase (KJhyPBase_Ex), into strain TR2-7, and found that the excision frequency of TR_cKJUMPS in KJhyPBase_Ex transformants was significantly higher than that in KJhyPBase transformants. In further experiments, we purified His-tagged KJhyPBase_Ex, and transfected it into strain TR2-7 using electroporation. Under these conditions, TR_cKJUMPS was precisely excised at a frequency of 8.8×10−8cell−1. The present data extend applications of the present piggyBac transposase-catalyzed excision system in green algae.