Expanding the toolkit of the Chlamydomonas reinhardtii chloroplast for biotechnological applications

Microalgae have gained increasing interest over the last ten years for their exploitation for biotechnological applications. A commonly used model organism is Chlamydomonas reinhardtii and many recombinant proteins have been successfully expressed in this green microalga. The chloroplast is of special interest as it allows rather high expression levels and targeted gene integration. Tools for chloroplast transformation and expression systems have advanced remarkably over the last years and the field is developing increasingly faster.

In this thesis, existing tools for genetic modification of C. reinhardtii were used to expand further the toolkit, mainly for the synthesis of high-value diterpenes of plant origin, and to examine the potential of using these microalgae in an industrial setting. A strain expressing a large recombinant enzyme, a diterpene synthase, resulting in in vivo synthesis of the diterpene cis-abienol, was generated. The potential for light-driven product synthesis via a chloroplast re-located cytochrome P450 was successfully shown. It was also demonstrated that typically fragile, wall-deficient transgenic strains can be grown on a 100 L pilot scale. This is an important finding as the gap between lab- and large-scale studies needs to be closed to allow a transition to an industrial setting. It was also shown that there is still untapped potential in the algal chloroplast - by utilising a bacterial export signal peptide the chloroplast toolkit was expanded to the thylakoid lumen for recombinant protein production.

Three proof of concept studies presented herein and one study looking at pilot scale cultivation of previously generated transgenic strains show that the potential seen in microalgae for biotechnological applications is justified and further developments and improvement of the existing strains could make this a viable competitive platform for diverse applications.