Fig. 3
Metabolic engineering. a The URA5 gene, including its promoter, was deleted to generate the Δura5 strain (using single guide RNAs (sgRNAs 1 and 2). The 3’ end of the coding sequence (90 bp) and terminator were not deleted, as they contained the terminator elements of the downstream gene. The complete URA5 coding sequence, with its native promoter and terminator, was used as a selection marker. The D9FAD and D12FAD overexpression was accomplished by inserting a second copy fused to AKRp and AKRt, and the selection marker into the upstream region of URA5 locus in Δura5 strain (sgRNAs 3 and 4), thus generating the strains D9OE and D12OE, respectively. The D12FAD knockout was carried out by inserting the URA5 in the Δ12 desaturase locus (sgRNA9). The D9FAD promoter exchange was performed by separate insertion of AKRp or TEFp, and simultaneous deletion of the native promoter to modify their transcriptional regulation (sgRNAs 5 and 6), generating the AKRp-D9 and TEFp-D9 strains, respectively. The same strategy was used for D12FAD (sgRNAs 7 and 8), resulting in AKRp-D12 and TEFp-D12, respectively. b Fatty acid profile, c lipid contents and titres, and d growth obtained with the WT and engineered C. oleaginous strains in MNM + Glu in shake flasks after 96 h cultivation. All data and error bars represent average and standard deviation of biological triplicates. The WT yielded 9.2 ± 0.2 g/L biomass and 50 ± 1.5% [wlipid/dwbiomass] lipids. The biomass and lipid accumulated by D9OE, D12OE, and TEFp-D9 are comparable to the WT (p > 0.05). In Contrast, the AKRp-D9 exhibited lower growth rate (DCW at 5.6 ± 0.3 g/L) but maintained the cellular lipid accumulation levels after 96 h (47 ± 3% [wlipid/dwbiomass] (p > 0.05)). The D12FAD knockout and promoter exchange did not affect the ability of the strains to grow and accumulate lipid