Genetic compensation of triacylglycerol biosynthesis in the green microalga Chlamydomonas reinhardtii

Genetic compensation through transcriptional adaptation has been proposed to explain phenotypic differences between gene knockouts and knockdowns. With the rapid development of reverse genetic tools such as CRISPR/Cas9 and RNAi in microalgae, it is increasingly important to assess whether genetic compensation affects the phenotype of engineered algal mutants. While exploring triacylglycerol (TAG) biosynthesis pathways in Chlamydomonas reinhardtii , it was discovered that knockout of certain genes catalyzing rate-limiting steps of TAG biosynthesis, type-2 diacylglycerol acyltransferase genes ( DGTTs ), triggered genetic compensation under abiotic stress conditions. Genetic compensation of a DGTT1 null mutation by a related PDAT gene was observed regardless of the strain background or mutagenesis approach, e.g., CRISPR/Cas 9 or insertional mutagenesis. However, no compensation was found in the PDAT knockout mutant. The effect of PDAT knockout was evaluated in a Δ vtc1 mutant, in which PDAT was up-regulated under stress. Knockout of PDAT in the Δ vtc1 background induced a 12.8-fold upregulation of DGTT1 and a 272.3% increase in TAG content in Δ vtc1 / pdat1 cells, while remaining viable. These data suggest that genetic compensation contributes to the genetic robustness of microalgal TAG biosynthetic pathways, maintaining lipid and redox homeostasis in the knockout mutants under abiotic stress with a mechanism distinct from that found in plants. This work demonstrates examples of genetic compensation in microalgae, implies the physiological relevance of genetic compensation in TAG biosynthesis, and provides guidance for future genetic engineering and mutant characterization efforts.