Engineering a complete mevalonate pathway in Chlamydomonas reinhardtii for enhanced isoprenoid production

Isoprenoids are a diverse class of natural compounds with significant industrial applications. While microbial production offers advantages, limitations arise from the absence of the mevalonate (MVA) pathway in many high-yield hosts. Here, we report the successful construction of a complete and functional MVA pathway in the eukaryotic microalga Chlamydomonas reinhardtii, a model organism with high photosynthetic efficiency and simple growth requirements. Employing a two-step transformation strategy and optimized gene expression cassettes, we achieved robust expression of both upstream and downstream MVA pathway enzymes. Notably, the introduction of the upstream module alone led to a dramatic seven-fold increase in isopentenyl pyrophosphate (IPP) levels, while co-expression of the complete pathway resulted in a 50 % increase in total carotenoid production. Transcriptomic analysis revealed significant upregulation of genes involved in carotenoid biosynthesis, confirming the redirection of metabolic flux towards terpenoid synthesis. Our findings demonstrate that a strategically engineered MVA pathway can overcome inherent limitations in C. reinhardtii metabolism, establishing this microalga as a powerful platform for the efficient, scalable production of diverse isoprenoids and other high-value compounds.