New tools and strategies for metabolic engineering of the algal chloroplast

Microalgae are attractive as cell factories for production of bioactive metabolites, therapeutic proteins and high-value metabolites. Of the several microalgae that have been explored as potential biotechnological platforms, the unicellular chlorophyte Chlamydomonas reinhardtii, is the most genetically tractable given its long history as a model species for molecular- genetic studies of cell biology. In particular, the chloroplast of C. reinhardtii represents a novel sub-cellular compartment for synthesis and accumulation of recombinant products. It possesses a small, genetically tractable genome and lacks any gene-silencing mechanisms. This allows stable and high-level expression of multiple transgenes. However, the exploitation of this microalgal platform requires further advances in the molecular tools available for metabolic engineering, together with new strategies for large-scale cultivation such as simple methods for ensuring ‘crop protection’ to reduce invasion and growth of contaminating species. Due to the increasing scarcity of phosphate reserve, phosphite is an alternative P source at economical cost and can reduce demand on non-renewable phosphate fertiliser. A key goal of my project was therefore to develop a strain improvement strategy based on the expression in the chloroplast of the bacterial gene ptxD encoding an NAD(P)-dependent phosphite oxidoreductase to allow utilization of phosphite as a source of phosphorus (P). The approach is based on the fact that most organisms cannot use phosphite, therefore, growing the transgenic microalga in phosphite provides a selective advantage over competing species. The ptxD gene was successfully introduced into chloroplast and shown to produce a functional enzyme that allowed growth on phosphite media. This allows engineered strains to be grown in non-sterile medium without significant spoilage by bacteria or fungi, thereby avoiding costly medium sterilization and culture management. Furthermore, it was demonstrated that ptxD can serve as a new non-antibiotic selectable marker for chloroplast transformation, allowing direct selection of transformants for their phosphite-utilising activity. This increases the repertoire of available selectable markers and reduces the use of antibiotics. Having developed these tools, the metabolic engineering of the chloroplast was attempted by introducing a synthetic gene encoding limonene synthase (LS). Limonene is a high-value terpenoid that has applications as a pharmaceutical, a flavour and a fragrance. Whilst the LS protein was successfully produced in the chloroplast, detailed GC-MS analysis failed to detect limonene synthesis suggesting issues with either functionality of the enzyme or availability of substrate. However, the work adds important new tools to the molecular toolbox for advancing C. reinhardtii chloroplast as an expression platform.