A Rapid and Reversible Molecular "Switch" Regulating Protein Expression in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii, a prominent chassis in synthetic biology, faces limitations in regulating the expression of exogenous genes. A destabilization domain (DD)/Shield‐1 system, originally derived from mammals, offers a ligand‐dependent control of stability, making it a valuable tool. This system utilises the destabilization domain to induce rapid degradation of target protein unless stabilised by Shield‐1, a synthetic ligand. Upon the addition of Shield‐1,the degradation is halted, leading to the accumulation and stabilisation of the target protein. This system has demonstrated successful regulation of foreign protein expression in mammals, parasites, and plants. In this study, the DD/Shield‐1 system was harnessed to regulate the expression of the paromomycin resistance gene and luciferase encoding gene in Chlamydomonas, revealing its capability for rapid, stable, and reversible protein expression regulation in microalgae, serving as a molecular switch. Furthermore, this regulation exhibits reagent dependency, enhancing its applicability in practical production. A strain with induced expression of the gene‐editing protein, LbCas12a, was successfully constructed and then tested for gene editing. The findings not only enrich the toolkit for Chlamydomonas molecular studies but offer a promising technique for regulating the expression and validating the functionality of exogenous proteins in microalgae. Summary statement: This study successfully implemented a DD/Shield‐1 system in Chlamydomonas reinhardtii to regulate the expression of the paromomycin resistance gene, luciferase encoding gene, and LbCas12a encoding gene, demonstrating its effectiveness in modulating exogenous gene expressions in a precise and tunable manner. With the established system, an engineering strain with inducible expression of CRISPR/Cas12a exhibits great potential in gene‐editing studies. The system provided post‐translational control over foreign protein expressions, in which protein levels showed a positive correlation with Shield‐1 dosage. This system enriches the molecular toolkit for Chlamydomonas by providing rapid, stable, and reversible regulation, which holds promise for both basic research and industrial applications in microalgae. [ABSTRACT FROM AUTHOR]