Engineering bidirectional chloroplast promoters for tunable co-expression of multiple genes in microalgae (Chlamydomonas reinhardtii).

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.