# _Synechococcus elongatus_ PCC 7942 as a Platform for Bioproduction of Omega-3 Fatty Acids
## 3 Results
The effect of _fabG_ overexpression in ALA production was compared with two control strains: MSM_D\({}_{\text{AB}}\) and MSM_D\({}_{\text{AB}}\)_F\({}_{\text{F}}\) (Figure 4). Both strains are able to produce ALA since they express DesAB, but MSM_D\({}_{\text{AB}}\)_F\({}_{\text{F}}\) produces a large amount due to the positive effect of _fabF_[33]. Contrary to our expectations, we found that the combined overexpression of _fabG_ with _desA_ and _desB_ in MSM_D\({}_{\text{AB}}\)_F\({}_{\text{G}}\) strain did not lead to an increase in ALA levels, suggesting that FabG overproduction is detrimental to ALA production. In the same way, when _fabF_, _fabG_, _desA_, and _desB_ genes were combined in the strain MSM_D\({}_{\text{AB}}\)_F\({}_{\text{FG}}\), higher levels of ALA were obtained but still lower than the ones found in MSM_D\({}_{\text{AB}}\)_F\({}_{\text{F}}\). It might indicate that the positive effect due to _fabF_ overexpression was largely diminished by the overexpression of  
Figure 2: Genealogy of sequential modifications performed in Se7942 for omega-3 production. The complete description of each strain is shown in Supplementary Table S1. _fabG_. Interestingly, MSM_DAB_FFG showed a decrease in C16:0 contents and an increase in C18:0 levels compared to MSM_DAB_FG (Table 1). The increase in C18:0 could be caused for the overexpression of _fabG_ since this change was also detected in MSM_DAB_FG. On the other hand, the decrease in C16:0 was only detected in MSM_DAB_FFG, suggesting that this change is due to the effect of _fabF_ overexpression, as previously described [33]. Taking all these results together, we suggest that _fabG_ overexpression has a negative effect on ALA production in Se7942. Figure 3: Effect of _fabG_ and _fabZ_ overexpression on FA composition. (**A**) FA content of wt Se7942 (green bars) and the _fabG_ mutant derivative, MSM_F\({}_{\text{G}}\) (red bars). (**B**) FA content of wt Se7942 (green bars) and the _fabZ_ mutant derivative, MSM_F\({}_{\text{Z}}\) (yellow bars). In both graphs, the different FA species are shown along the \(x\)-axis. The \(y\)-axis shows the percentage of each FA with respect to the total amount of FAs analyzed. Data are the average of at least three independent biological replicates and are represented as the mean + SD. ns (not significant), \(p>0.05\); \(*\)\(p<0.05\), \(**\)\(p<0.01\), \(***\)\(p<0.001\), by unpaired Student’s \(t\)-test. Following the same strategy, the MSM_D\({}_{\text{AB}}\)_F\({}_{\text{FZ}}\) strain was developed to test the effect of _fabZ_ overexpression in ALA synthesis (Figure 2). This strain co-overexpresses _fabZ_ and _fabF_ genes from NS2. The co-overexpression of _fabZ_, _fabF_, and desaturase genes (_desA_ and _desB_) did not lead to an increase in ALA levels (Figure 4). In fact, the amount of ALA produced by MSM_D\({}_{\text{AB}}\)_F\({}_{\text{FZ}}\) was the lowest reported in all strains analyzed in this work (0.66%) (Figure 4). This result indicates that the overexpression of the _fabZ_ gene, as in the case of _fabG_, has a negative effect on ALA production. Similar to the MSM_D\({}_{\text{AB}}\)_F\({}_{\text{FG}}\) strain, the MSM_D\({}_{\text{AB}}\)_F\({}_{\text{FZ}}\) strain showed a decrease in C16:0 FA content and an increase in C18:0 FA levels compared to the MSM_D\({}_{\text{AB}}\)_F\({}_{\text{F}}\) strain, and an increase in C18:1 FA level was not detected in MSM_D\({}_{\text{AB}}\)_F\({}_{\text{FG}}\) (Table 1). Overproduction of FabF and DesABD in a Se7942 Derivative Strain Leads to High Steridonic Acid Yields  
To produce SDA in Se7942, three desaturases need to be heterologously expressed: DesA (\(\Delta\)12 desaturase), DesB (\(\Delta\)15 desaturase), and DesD (\(\Delta\)6 desaturase) [46] (Figure 1).