Expressing exogenous gene in Chlamydomonas reinhardtii chloroplast with viral replication elements

The advancement of Chlamydomonas reinhardtii chloroplasts as synthetic biology chassis remains constrained by low product yields, a limitation potentially rooted in chloroplast genomic heterogeneity. To address this replicative bottleneck, a geminivirus-inspired expression platform was developed in C. reinhardtii by leveraging replication machinery from the beet curly top virus, building upon its validated efficacy in Nicotiana tabacum. System validation demonstrated robust expression of a linearized mVenus cassette, achieving a higher expression level (2-4 folds) than conventional homologous recombination method. Furthermore, high-value carotenoids, such as zeaxanthin and canthaxanthin, were increased by 1.69 and 1.22 folds respectively, with the virus-derived system by expressing bacterial carotenogenic genes. Notably, transgenes can be stably expressed for a long period (12 months) without compromising growth or photosynthetic performance in transformants. This virus-based platform establishes a novel approach for stable high-titer compound synthesis in microalgae, positioning chloroplast engineering as a viable platform for bioproduction.