Use of plant chloroplast RNA-binding proteins as orthogonal activators of chloroplast transgenes in the green alga Chlamydomonas reinhardtii

The genomes of microalgal chloroplasts offer many advantages as a scaffold for the expression of foreign proteins for applications in biotechnology. To realize this potential, it will be necessary to develop robust systems to regulate, optimize, and tune the expression of microalgal chloroplast transgenes . The most promising systems to date employed native cis- elements and cognate trans -factors to drive chloroplast transgene expression. This type of approach, however, is complicated by crosstalk with endogenous regulatory networks. In that context, orthogonal regulatory systems – those that act independently of host regulators – are essential for maximizing the biofactory potential of microalgal chloroplasts. Toward that end, we tested the ability of nucleus-encoded chloroplast gene activators from land plants to activate plastid transgenes in microalgae . Our experiments employed two helical repeat RNA-binding proteins, maize PPR10 and Arabidopsis HCF107, that activate specific chloroplast genes at the post-transcriptional level. We show that expression of HCF107 and PPR10 from nuclear transgenes in Chlamydomonas reinhardtii increases expression of chloroplast reporter genes harboring the cognate 5′-untranslated region from plants. Both proteins stabilized RNA downstream from their binding sites and enhanced translational efficiency much as they do in their native plant context. However, fold-activation was less than that in plants, a limitation we suspect is due to suboptimal expression of the nuclear transgenes. This scheme provides a foundation from which to design diverse orthogonal systems to regulate gene expression from chloroplast transgenes in microalgae. • Two chloroplast gene activators from plants have similar effects when expressed in C. reinhardtii . • Both proteins stabilize RNA downstream from their binding sites and stimulate translation. • This approach represents a framework for developing diverse orthogonal regulatory systems in algal chloroplasts.