Abstract
Chloroplasts offer significant potential for multigene engineering in microalgae, but the lack of well-characterized regulatory elements and limited understanding of plastid transcriptional mechanisms have hindered progress. Here, through a comparative conservation analysis across fifteen species of microalgae and higher plants, we identified bidirectional promoter (BDP) intergenic regions (IRs) showing diverse evolutionary trajectories, from lineage-specific rearrangements of atpA/rbcL (BDP1) and chlL/petB (BDP2), to strict conservation of the psbH/psbN IR (BDP3), and complete loss of rpoB-1/psbF (BDP4). Based on promoter signature analysis, we selected three candidate regions (BDP1, BDP2, and BDP3) from the Chlamydomonas reinhardtii chloroplast genome for functional characterization. A semi-rational screen revealed that BDP1 supports expression of two transgenes, mVenus and tdTomato in opposing orientations; BDP2 drives balanced expression, but low protein accumulation; and BDP3 exhibits minimal activity, suggesting UTR-dependent post-transcriptional regulation. Strikingly, methyl-jasmonate selectively enhanced tdTomato expression from BDP1, offering a chemical method to regulate chloroplast transgene expression. Collectively, these results underscore the evolutionary diversity and functional potential of natural BDPs, particularly BDP1, as powerful tools for multigene engineering and chemical modulation in microalgae and higher plants. This study also provides fundamental insights into chloroplast transcription, establishing a basis for future investigations into its regulatory mechanisms.
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Data availability
All data supporting the findings of this study are included in the main text and Supplementary Information. Oligonucleotide sequences are listed in Supplementary Data 1. Next-generation sequencing (NGS) results for all plasmid constructs are provided in Supplementary Data 2. Flow cytometry, RT–qPCR, and confocal microscopy datasets are available in Supplementary Data 3. Raw NGS reads have been deposited in the NCBI Sequence Read Archive under BioProject accession number PRJNA1379576. The plasmid vectors pABDP1, pABDP2, pABDP3, pAMVe, and pATDt have been deposited in Addgene under accession numbers 249500, 249501, 249502, 249494, and 249499, respectively.
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Acknowledgements
The authors thank Dr. Karen Cristine Goncalves dos Santos for her valuable technical advice and input during this study, and Melodie B. Plourde for her generous support and patience in assisting with confocal microscopy acquisition and analysis. During the preparation of this work, the authors used ChatGPT -4.0, a free AI language model, to correct grammatical errors and enhance readability. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the final version of this publication. This research was funded by Canada Research Chair on plant specialized metabolism Award No. CRC-2023 00353 to IDP. The authors extent their gratitude to the Canadian taxpayers and to the Canadian government for supporting the Canada Research Chairs Program. Additional funding was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) through award No. RGPIN/3218-2021 to IDP. This work was also supported by NSERC award No. EQPEQ 472990-2015 (Research tools and instruments program) for the acquisition of the qPCR system.
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A.W.T.: Conceptualization, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing-Original draft, Writing Reviewing and Editing; N.M.: Conceptualization, Formal analysis, Methodology, Project administration Supervision, Writing-Original draft, Writing Reviewing and Editing; E.F.: Formal analysis, Investigation, Methodology, Writing Reviewing and Editing; A.B.: Investigation, Methodology, Writing Reviewing and Editing; F.A.: Investigation, Writing Reviewing and Editing; F.M.-M.: Conceptualization, Methodology, Project administration, Writing Reviewing and Editing; I.D.P.: Conceptualization, Funding, acquisition, Methodology, Project administration, Resources, Supervision, Validation, Writing- Reviewing and Editing.
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Tazon, A.W., Mérindol, N., Fantino, E. et al. Engineering bidirectional chloroplast promoters for tunable co-expression of multiple genes in microalgae (Chlamydomonas reinhardtii). Commun Biol (2026). https://doi.org/10.1038/s42003-025-09478-7
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DOI: https://doi.org/10.1038/s42003-025-09478-7
