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

Abstract

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 organismis 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-theart 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.

Site-Specific Gene Knock-Out and On-Site Heterologous Gene Overexpression in Chlamydomonas reinhardtii via a CRISPR-Cas9-Mediated Knock-in Method

“…C. reinhardtii has been extensively studied for fundamental research and industrial use based on its genome sequence data and well-developed molecular tool kit (Scaife et al, 2015;Crozet et al, 2018;Salomé and Merchant, 2019). Moreover, the genetic modification techniques are highly developed and the engineering strategies of metabolic pathways are well established (Plucinak et al, 2015;Baier et al, 2018b;Fu et al, 2019;Kong et al, 2019).…”

Good News for Nuclear Transgene Expression in Chlamydomonas

“…Chlamydomonas has emerged as a valuable model organism for basic research e.g., on photosynthesis and chloroplast biogenesis [2], the biology of cilia and basal bodies [3], the cell cycle [4], the plant heat stress response [5], or the circadian clock [6]. Moreover, Chlamydomonas has received much attention because of its ability to produce molecular hydrogen [7] and lipids [8], both with promise as biofuels. Important developments for major research fields in life sciences have their origin in basic Chlamydomonas research.…”

Heterologous Gene Expression System Using the Cold-Inducible CnAFP Promoter in Chlamydomonas reinhardtii

“…In the case of bacterial systems, cold-inducible promoters such as the cold-shock protein A (cspA) promoter are used to overexpress proteins at low temperature, thereby increasing protein solubility and stability [16]. However, cold-inducible promoters that can be used in C. reinhardtii have not yet been developed [17]. Recently, there have been attempts to produce useful recombinant proteins such as human growth factor, interferon β, or proinsulin using microalgae [18][19][20], so the development of cold-inducible expression systems may be necessary.…”