# _Synechococcus elongatus_ PCC 7942 as a Platform for Bioproduction of Omega-3 Fatty Acids
## 4 Discussion  
Cyanobacteria are promising microorganisms for sustainable bioproduction since they directly convert CO2 and light into fuels, chemicals, and other value-added products [20]. Moreover, these organisms naturally synthesize omega-3 FAs with different degrees of unsaturation, depending on the expression of the desaturale enzymes involved in the FA synthesis pathway. Biosynthesis of omega-3 FAs has been thoroughly studied in three models of unicellular cyanobacteria, S6803 [32, 47, 48, 49, 50], S87002 [29, 30, 31, 34], and _Synechococcus_ sp. NKBG 15041c [51]. Four desaturases are involved in the synthesis of omega-3 FAs in cyanobacteria: DesC (A9 desaturale), DesA (A12 desaturale), DesB (A15 or \(\omega\)-3 desaturale), and DesD (A6 desaturale) (Figure 1). In addition, it was demonstrated that only four cyanobacterial species naturally produce SDA (S6803, _Tylopthrix tennis_, _Lynghya_ sp. PCC 8106, and _Nodularia spunigena_[22]). Nevertheless, only in S6803, the activity of the enzymes was characterized [32, 47, 48, 49, 50]. Se7942 does not naturally produce omega-3 FAs since it lacks these three desaturases, although it was previously engineered with desaturases from S86803 [47] and from S87002 [33] to produce variable yields of ALA. To our knowledge, SDA production has not been reported in Se7942. In addition, there is little information available related to the FA synthesis and regulation in cyanobacteria in general, making it difficult to achieve higher levels of production. Overexpression of bottleneck enzymes and deletion of competing pathways are the most common strategies used to increase the production of valuable compounds in cyanobacteria [44]. The ALA production yield was improved in Se7942 by overexpression FabF, an enzyme that participates in the cycle of FA elongation, and by removing the activity of FadD, involved in the competitive FA degradation pathway [33]. Both modifications have in common that result in an increase in C18 species, as we report in this work with _fabG_ and _fabZ_ overexpression. However, these new genetic constructs did not improve  
Figure 6: FA profile of MSM_D_AB_F_FD strain at different post-induction times. Data are the average of at least three independent biological replicates and are represented as the mean + SD. Significance was calculated by one-way ANOVA followed by [PERSON]’s multiple comparison test. Bars labeled with different letters are significantly different (\(p<0.05\)). ALA synthesis (Figure 4 and Table 1). Whereas _fabF_ overexpression and _fudD_ deletion have in common a pronounced increase in C18:1 [33], this raise was not observed when _fabG_ and _fabZ_ were overexpressed (Figure 3). These results suggest that higher levels of C18:1 are required for an improvement in omega-3 FA production. In fact, a previously published report concluded that FabF, mainly involved in the extension of C16:1 into C18:1, is the key-limiting reaction in the synthesis of unsaturated fatty acids in this cyanobacterium [52]. In addition, SDA only improved when FabF was overexpressed (Figure 5 and Table 2), but not when FadD was eliminated (Table 2). In S6803, _fudD_ deletion results in down-regulation of DesD protein expression, but it did not have any effect on DesA and DesB protein levels [53]. Although we are expressing DesD from Ss6803 in Se7942, we speculate that this unknown mechanism of regulation could be maintained between cyanobacterial species. Taken together, our results indicate that the first step in the elongation reaction catalyzed by FabF seems to determine the ability of the cells to produce omega-3 FAs in Se7942. In addition, the reactions of reduction and dehydration catalyzed by FabG and FabZ, respectively, seem to have less influence on the biosynthesis of FAs. Production of SDA by overexpressing desaturases was previously used as a strategy in several cyanobacterial species, reaching SDA levels up to 12% of the total FA content in Ss7002 [20] and up to 13% of total FAs in Ss6803 [19].