Chloroplast Dual Divergent Promoter Plasmid for Heterologous Protein Expression in Tetraselmis suecica (Chlorophyceae, Chlorodendrales)

Carla L. Gutiérrez, Carla Muñoz, Margarita San Martín, Jean Paul Cadoret, VITALIA BEATRIZ HENRIQUEZ QUEZADA

Research output: Contribution to journalArticlepeer-review

Abstract

The eukaryotic green microalga Tetraselmis suecica is commonly used for aquaculture purposes because of its high stress tolerance and ease of culture in a wide spectrum of environments; they are therefore suitable candidates for biotechnology applications. To date, no data are available regarding chloroplast transformation vectors based on specific endogenous promoters and homologous targeting regions. We report on the identification of Tetraselmis suecica genes encoding the ribulose bisphosphate carboxylase/oxygenase large subunit protein, the photosystem II D1 protein and the ATP synthase CF1-beta subunit protein together with their untranslated regions (5′UTR, 3′UTR). The full-length ORFs of the putative genes with their regulatory sequences were obtained. We were also able to identify the downstream 3′ end of the large subunit ribosomal RNA gene (23S) along with the 5S RNA end-to-end with the psbA gene on the complementary strand. The intergenic region between these genes appears to be a good target site for the integration of target proteins. Moreover, we identified a back-to-back promoter region among the rbcL and atpB genes. To assess the bidirectionality activities of both promoters, a dual reporter vector was constructed for Tetraselmis suecica transformation containing the cat and TurboGFP genes driven by the 5′rbcL/5′atpB divergent promoter. The vector included the 23S-5S and psbA nucleotide sequences as flanking regions. These flanking regions provided suitable insertion sites within the chloroplast genome for cassette integration via homologous recombination. Simultaneous expression of the chloramphenicol-resistant conferring gene and the gene coding for TurboGFP driven by 5′rbcL/5′atpB showed a potent natural bidirectional promoter as a reliable genetic tool.

Original languageEnglish
Pages (from-to)1066-1076
Number of pages11
JournalJournal of Phycology
Volume56
Issue number4
DOIs
StatePublished - 1 Aug 2020
Externally publishedYes

Keywords

  • cat gene
  • chloroplast
  • divergent promoters
  • Tetraselmis suecica
  • tGFP

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