Establishing the use of TPR/PPR proteins for synthetic biology of microalgae

There is considerable interest in exploitation of microalgae as production platforms for industrial biotechnology, but this requires robust and controllable tools for metabolic engineering [2]. The aim of this project is to generate synthetic gene circuits to control expression of transgenes in the algal chloroplast, using endogenous regulatory mechanisms. Plants and algae encode multiple genes in the nucleus for PPR (pentatricopeptide repeat) and TPR (tetratricopeptide repeat) proteins, many of which have been shown to be RNA binding proteins required for normal expression of organelle transcripts. In Chlamydomonas reinhardtii, several mutants are known with defects in different TPR/PPR proteins, and their targets have been identified, such as NAC2 ([1]; see rotation project description). The PhD project has three objectives: i. Use the TPP-riboswitch-regulated NAC2 gene to regulate one or more genes for isoprenoid biosynthetic enzymes introduced into the C. reinhardtii chloroplast genome. Some of the compounds show cytotoxicity, so by being able to suppress the gene until the cells have grown this would avoid the problem. ii. Establish a similar regulatory circuit using one or more of the other TPR/PPR proteins, either using the same riboswitch, a modified version responsive to a different ligand, or an inducible promoter [3]. Combination of the two circuits could then result in an AND logic gate (where both must be functional to get expression of the chloroplast gene) or an OR gate (where only one must function). iii. Ideally, synthetic biology tools such as these should operate independently of context, such as in different organisms [2]. To test this, PPR/TPR proteins would be identified in other algal genomes by bioinformatics and co-expression analysis to establish if there are any in common, or whether they are all species-specific. The C. reinhardtii genetic circuits would then be tested in a species in which it is possible to transform the chloroplast genome. Currently this is not well established for other algae, but if necessary it could be done in the higher plant Nicotiana tabacum (tobacco).