Regulation of fatty acid desaturation and lipid engineering

Overview & Synopsis

FAD3 gene encodes an omega-3 desaturase able of desaturating phosphatidylcholine-esterified linoleic fatty acid (C18:2-PC) to alpha linolenic fatty acid (C18:3-PC). This enzyme is particularly active during seed maturation and allows the accumulation of omega-3 fatty acids in the triacylglycerols (TAG) contained in the lipid bodies accumulated in the seed. It appears that the C18:3 (Omega-3) content is directly correlated to the expression of the FAD3 gene. The objective of this thesis is to better understand the transcriptional regulation of the FAD3 gene by generating combinatorial mutants of the FAD3 promoter and by identifying cis-regulatory elements correlated to lipid phenotypes. We initially decided to employ a combinatorial multiplexed CRISPR/Cas9 mutagenesis method targeting the FAD3 promoter of Arabidopsis thaliana, to understand the effect of promoter editing in a model organism. A similar mutagenesis method was used in Camelina sativa to evaluate the feasibility of this strategy in a hexaploid agronomic plant. Global analysis of the edited population by Illumina sequencing showed that although combinatorial mutagenesis was effective for some sites, others are not very sensitive to mutations, probably due to sgRNA efficiency or chromatin accessibility. In Arabidopsis, FAD3 editing led to the appearance of phenotypes with altered lipid contents. In contrast, the link between induced mutations at the FAD3 locus and phenotypes could not be established. In Camelina, we used PacBio sequencing technology to sequence the entire CsFAD3 promoters in individual plants, which allowed us to identify complete haplotypes with significant mutations validated in the progeny of mutants. Allelic combinations were linked to increased C18:3 accumulation phenotypes. The prospects of this work will be to identify the elements that led to the modification of lipid phenotypes in Arabidopsis and to validate the new CsFAD3 alleles generated in Camelina in a reporter gene system to verify whether they are indeed the cause of the increased C18:3 content phenotype.