Engineering the fatty acid synthesis pathway in Synechococcus elongatus PCC 7942 improves omega-3 fatty acid production

Abstract

Background: The microbial production of fatty acids has received great attention in the last few years as feedstock for the production of renewable energy. The main advantage of using cyanobacteria over other organisms is their ability to capture energy from sunlight and to transform CO₂ into products of interest by photosynthesis, such as fatty acids. Fatty acid synthesis is a ubiquitous and well-characterized pathway in most bacteria. However, the activity of the enzymes involved in this pathway in cyanobacteria remains poorly explored.

Results: To characterize the function of some enzymes involved in the saturated fatty acid synthesis in cyanobacteria, we genetically engineered Synechococcus elongatus PCC 7942 by overexpressing or deleting genes encoding enzymes of the fatty acid synthase system and tested the lipid profile of the mutants. These modifications were in turn used to improve alpha-linolenic acid production in this cyanobacterium. The mutant resulting from fabF overexpression and fadD deletion, combined with the overexpression of desA and desB desaturase genes from Synechococcus sp. PCC 7002, produced the highest levels of this omega-3 fatty acid.

Conclusions: The fatty acid composition of S. elongatus PCC 7942 can be significantly modified by genetically engineering the expression of genes coding for the enzymes involved in the first reactions of fatty acid synthesis pathway. Variations in fatty acid composition of S. elongatus PCC 7942 mutants did not follow the pattern observed in Escherichia coli derivatives. Some of these modifications can be used to improve omega-3 fatty acid production. This work provides new insights into the saturated fatty acid synthesis pathway and new strategies that might be used to manipulate the fatty acid content of cyanobacteria.

Keywords: Cyanobacteria; Fatty acid synthesis; Omega-3 fatty acids; Synechococcus elongatus PCC 7942; fab genes.

Fig. 1

FA synthesis in E. coli vs Se7942 (a). Main FA synthesis pathway. In the initiation step, acetyl-CoA is converted into malonyl-CoA by the acetyl-CoA–carboxylase complex (AccABCD). Malonyl-CoA is transferred to the acyl-carrier protein (ACP) by FabD. Malonyl-CoA is condensed with acetyl-CoA by the β-ketoacyl-ACP synthase III (FabH). The resulting β-ketoacyl-ACP enters the elongation cycle, leading to long-chain acyl-ACP by serial steps of reduction (catalyzed by FabG), dehydration (FabZ and FabA), reduction (FabI), and elongation by condensing additional malonyl-ACP molecules (FabB and FabF). Enzymes present in E. coli and absent in Se7942 are colored in red. b Scheme of fab pathway gene organization in E. coli (upper panel) and Se7942 (lower panel). Homologous genes (or predicted ORFs) are depicted by arrows in the same color. The white arrow represents a gene not included in the fab cluster. Double slashes represent DNA regions that are not shown

Fig. 2

Effect of fabD overexpression in FA composition. a Enzymatic reaction catalyzed by FabD. b FA content of wt Se7942 and the fabD mutant derivative, MSM24. 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. *p < 0.05, by unpaired Student’s t test

Fig. 3

Effects of FabH levels on Se7942 FA content. a Enzymatic reaction catalyzed by FabH. b Attempt to construct the Se7942 fabH-deficient mutant. Upper panel: scheme of the fabH vicinity in the wt and fabH mutant strains. Relevant genes are indicated by colored arrows, while upstream and downstream sequences flanking the deletion target (USDT and DSDT) by boxes. Primers used for PCR analysis are indicated by black arrows. Lower panel: PCR analysis of the fabH region of mutant strain MSM23. Lane 1, Hyperladder I (BioLine); lanes 2–7, MSM23 colonies; lane 8, wt Se7942 strain (WT). c FA content of wt Se7942 and the fabH merodiploid mutant (MSM23) and fabH-overexpressing derivative (MSM28). Data show the mean and + SD of at least three independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001, by unpaired Student’s t test

Fig. 4

Effect of the fabB overexpression on FA composition. a Enzymatic reaction catalyzed by FabB and FabF. The KASs type I and II, FabB and FabF, catalyze the Claisen condensation of FA-thioesters and malonyl-ACP to form a β-ketoacyl-ACP intermediate elongated by two carbon atoms (β-oxo-(C)-acyl-ACP). “R” represents the hydrocarbon chain and “(C)” the number of carbon present in the FA. b FA content of wt Se7942 (green bars) and the fabB overexpressing mutants MSM19 (blue) and MSM27 (purple). Data are the average of at least three independent biological replicates and are represented as the mean + SD. **p < 0.01, ***p < 0.001, by unpaired Student’s t test

Fig. 5

Effect of the fabF overexpression on FA composition. FA content of wt Se7942 (green bars) and the fabF mutant derivative, MSM29 (gray). Data are the average of at least three independent biological replicates and are represented as the mean + SD. ***p < 0.001, by unpaired Student’s t test

Fig. 6

Effect of the fadD deletion on FA composition. a Enzymatic reaction catalyzed by FadD. FadD is an acyl-CoA synthetase that loads free FAs on CoA. “R” represents the hydrocarbon chain. b FA content of wt Se7942 (green bars) and the fadD mutant derivative, MSM42 (cyan bars). Data are the average of at least three independent biological replicates and are represented as the mean + SD. *p < 0.05, **p < 0.01, by unpaired Student’s t test

Fig. 7

ALA production in Se7942. a Desaturation reactions needed to produce ALA in cyanobacteria. In bold, desaturase genes introduced in Se7942. b ALA content of wt Ss7002 (green bar), wt Se7942, desA and desB mutant derivatives, MSM17 (blue) and MSM26 (red), respectively. Data are the average of at least three independent biological replicates and are represented as the mean + SD. ***p < 0.001, by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test with a single control, Ss7002

Fig. 8

Effect of ALA production in the FA Profile. wt Se7942 and the ALA-producing mutant, MSM45, are represented by green and blue bars, respectively. Data are the average of at least three independent biological replicates and are represented as the mean + SD. ***p < 0.001, by unpaired Student’s t test