# _Synechococcus elongatus_ PCC 7942 as a Platform for Bioproduction of Omega-3 Fatty Acids
## 3 Results  
Modification of the Lipid Profile of Se7942 by Overproduction of Enzymes Involved in the Elongation Cycle of the Fatty Acid Synthesis Pathway  
FabG, a \(\beta\)-ketoacyl-ACP reductase, catalyzes the first of the two reduction steps in the elongation cycle of the FA synthesis pathway (Figure 1). In this reversible reaction, \(\beta\)-ketoacyl-ACP is reduced to \(\beta\)-hydroxyacyl-ACP at the concomitant expense of NADPH [39]. Homologous expression of the _fabG_ gene in _E. coli_ increased the content of C16:0 and C18:0 by 2- or 3-fold, respectively [40]. To test the effect of _fabG_ overexpression in Se7942, MSM_F\({}_{\rm C}\) strain harboring Ptrc::fabG cassette installed in neutral site 1 (NS1) was constructed (Figure 2). The _fabG_ overexpression led to an increase in the content of C18:0 and C16:0 FAs and a decrease in C16:1 FA (Figure 3A). In _E. coli_, the reduction in the C16:1 level was not observed when fabG was overexpressed, whereas an increase in C18:0 was also detected [40]. Our results demonstrate that although the regulation of FA synthesis is different in these two bacteria, _fabG_ overexpression led to similar changes in the FA profile in both organisms. The next reaction in the FA elongation cycle is catalyzed by FabZ, a \(\beta\)-hydroxyacyl-ACP dehydatase (Figure 1), which performs the dehydration of \(\beta\)-hydroxyacyl-ACP to _trans_-2-enoyl-ACP [41]. In _E. coli_, two genes codify for two different \(\beta\)-hydroxyacyl-ACP dehydatases, _fabA_ and _fabZ_. In addition to its dehydration activity, FabA has a second function related to the isomerization of _trans_-2-decenoyl-ACP into _cis_-3-decenoyl-ACP, which is the first reaction toward the synthesis of unsaturated FAs [42]. Many cyanobacterial species lack a recognizable FabA homolog since the unsaturated FA synthesis is performed by desaturases [43]. The homologous overexpression of the _fabZ_ gene in _E. coli_ resulted in an about 2-fold increase in C16:0 and C18:0 levels [40]. The activity of FabZ is inhibited by high levels of (p)ppGpp in this organism [44; 45], but no regulators have been described for FabZ in cyanobacteria. To analyze the function of FabZ in Se7942, this gene was overexpressed using _trc_ promoter in NS1 of Se7942 in the strain MSM_F\({}_{\rm Z}\). This mutant showed a significant decrease in C14:0 and C16:1 FAs and a significant increase in C16:0, C18:0, and C18:1 FAs (Figure 3B). These results indicate that high levels of FabZ activity improved the transformation of C14:0 and C16:1 into C18 species. Moreover, FabZ in Se7942 seems to perform a similar function to the same enzyme in _E. coli_ since the FA profile was similar when FabZ activity was increased [40]. Overproduction of FabG or FabZ Has a Detrimental Effect in the Production of Alpha-Linoleic Acid in Se7942  
In cyanobacteria, three desaturation reactions convert oleic acid (OA) into ALA, catalyzed by DesC (\(\Delta\)9 desaturase), DesA (\(\Delta\)12 desaturase), and DesB (\(\Delta\)15 desaturase) [46] (Figure 1). Se7942 only possesses the _desC_ gene and lacks the remainders [22], not naturally producing omega-3 FA. We have previously demonstrated that the overexpression of _desA_ and _desB_ genes from Se7902 in Se7942 led to ALA production [33]. In addition, ALA yield was increased by _fabF_ overexpression and _fadD_ deletion, modifications that increased C18:0 FA levels. As we showed above, the individual overexpression of _fabG_ and _fabZ_ also led to increases in C18:0 species. Thus, we hypothesized that the combined overexpression of _fabG_ and _fabZ_ with _desAB_ might lead to an improvement in ALA yield. Therefore, considering these assumptions, we decided to co-overexpress _fabG_, _fabZ_, and _desAB_ desaturase genes. To examine the effect of _fabG_ overexpression in ALA synthesis, MSM_D\({}_{\text{AB}}\)_F\({}_{\text{G}}\) and MSM_D\({}_{\text{AB}}\)_F\({}_{\text{FG}}\) overproducing strains were developed (Figure 2).