Molecular Genetic Tools and Emerging Synthetic Biology Strategies to Increase Cellular Oil Content in Chlamydomonas reinhardtii.

Microalgae constitute a highly diverse group of eukaryotic and photosynthetic microorganisms that have developed extremely efficient systems for harvesting and transforming solar energy into energy-rich molecules such as lipids. Although microalgae are considered to be one of the most promising platforms for the sustainable production of liquid oil, the oil content of these organisms is naturally low, and algal oil production is currently not economically viable. Chlamydomonas reinhardtii (Chlamydomonas) is an established algal model due to its fast growth, high transformation efficiency, and well-understood physiology and to the availability of detailed genome information and versatile molecular tools for this organism. In this review, we summarize recent advances in the development of genetic manipulation tools for Chlamydomonas, from gene delivery methods to state-of-the-art genome-editing technologies and fluorescent dye-based high-throughput mutant screening approaches. Furthermore, we discuss practical strategies and toolkits that enhance transgene expression, such as choice of expression vector and background strain. We then provide examples of how advanced genetic tools have been used to increase oil content in Chlamydomonas. Collectively, the current literature indicates that microalgal oil content can be increased by overexpressing key enzymes that catalyze lipid biosynthesis, blocking lipid degradation, silencing metabolic pathways that compete with lipid biosynthesis and modulating redox state. The tools and knowledge generated through metabolic engineering studies should pave the way for developing a synthetic biological approach to enhance lipid productivity in microalgae.