Rational Promoter Engineering Enables Robust Terpene Production in Microalgae.

Microalgal biotechnology promises sustainable light-driven production of valuable bioproducts and addresses urgent demands to attain a sustainable economy. However, to unfold its full potential as a platform for biotechnology, new and powerful tools for nuclear engineering need to be established. Chlamydomonas reinhardtii, the model for microalgal synthetic biology and genetic engineering has already been used to produce various bioproducts. Nevertheless, low transgene titers, the lack of potent expression elements, and sparse comparative evaluation prevents further development of C. reinhardtii as a biotechnological host. By systematically evaluating existing expression elements combined with rational promoter engineering, we established novel synthetic expression elements, improved the standardized application of synthetic biology tools, and unveiled an existing synergism between the PSAD 5' UTR and its corresponding chloroplast targeting peptide. Promoter engineering strategies, implemented in a newly designed synthetic algal promoter, increased the production of the sesquiterpene (E)-α-bisabolene by 18-fold compared to its native version and 4-fold to commonly used expression elements. Our results improve the application of synthetic biology in microalgae and display a significant step toward establishing C. reinhardtii as a sustainable green cell-factory.