PRK introns provide modular tools for enhanced transgene expression in Chlamydomonas reinhardtii

Abstract

Chlamydomonas reinhardtii is an attractive chassis for photosynthetic biotechnology, but reliable nuclear transgene expression remains a major limitation. Despite advances in genetic engineering, achieving robust nuclear transgene expression in this organism remains challenging. One effective strategy is the introduction of introns into transgene sequences to trigger Intron-Mediated Enhancement (IME). Genomic sequences retaining endogenous introns often show higher expression than their intronless counterparts, as we previously observed for the phosphoribulokinase (PRK) gene. Here, we investigated the IME activity of PRK introns, using the first intron of RBCS2 (RBCS2i1), the current reference IME element in Chlamydomonas, as a benchmark. The first two PRK introns emerged as the best candidates. We assessed their IME activity using two reporter systems, two genetic backgrounds, and multiple promoter-terminator combinations, and observed enhancements up to 35-fold relative to their intronless counterparts. Finally, we validated their performance using patchoulol synthase, a challenging transgene whose expression requires introns and enables the production of the high-value sesquiterpene patchoulol. PRKi1 enabled detectable patchoulol production under conditions in which a single RBCS2i1 intron was insufficient. Overall, our results establish PRKi1 and PRKi2 as versatile, MoClo-compatible IME tools and provide a modular, Biofoundry-compatible framework for optimizing transgene expression for synthetic biology and biotechnological applications in C. reinhardtii.