There is considerable interest in exploitation of microalgae such as Chlamydomonas reinhardtii as production platforms for industrial biotechnology. Chlamydomonas has a single chloroplast, which is the site of photosynthesis and other important biosynthetic processes. Several high-value non-natural compounds have been successfully produced in this organelle by genetic engineering approaches. Application of synthetic biology design principles and utilisation of the design-build-test-learn cycle in metabolic engineering requires implementation of standardised and modular DNA building blocks. Here, we describe generation of the new synthetic biology toolkit for construction of transgene expression cassettes and their delivery into the chloroplast genome. The Golden-Gate cloning framework was used to generate a library of parts for controlling gene expression (promoters, 5’UTRs, intercistronic expression elements, 3’UTRs) and homology arms for targeting expression cassettes to neutral integration sites in the genome. The parts were successfully validated and characterised by using reporter genes. We also engineered the nuclear genome of the Chlamydomonas strain to allow regulation of the transgene expression in the chloroplast by using vitamin-dependent promoters and riboswitches. Finally, we demonstrated construction of novel metabolic pathways for production of plant diterpenoids that are used in the pharmaceutical, agricultural, food additive and fragrance industries.
