Fatty acid activation in cyanobacteria mediated by acyl-acyl carrier protein synthetase enables fatty acid recycling

Abstract

In cyanobacteria fatty acids destined for lipid synthesis can be synthesized de novo, but also exogenous free fatty acids from the culture medium can be directly incorporated into lipids. Activation of exogenous fatty acids is likely required prior to their utilization. To identify the enzymatic activity responsible for activation we cloned candidate genes from Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 and identified the encoded proteins as acyl-acyl carrier protein synthetases (Aas). The enzymes catalyze the ATP-dependent esterification of fatty acids to the thiol of acyl carrier protein. The two protein sequences are only distantly related to known prokaryotic Aas proteins but they display strong similarity to sequences that can be found in almost all organisms that perform oxygenic photosynthesis. To investigate the biological role of Aas activity in cyanobacteria, aas knockout mutants were generated in the background of Synechocystis sp. PCC 6803 and S. elongatus PCC 7942. The mutant strains showed two phenotypes characterized by the inability to utilize exogenous fatty acids and by the secretion of endogenous fatty acids into the culture medium. The analyses of extracellular and intracellular fatty acid profiles of aas mutant strains as well as labeling experiments indicated that the detected free fatty acids are released from membrane lipids. The data suggest a considerable turnover of lipid molecules and a role for Aas activity in recycling the released fatty acids. In this model, lipid degradation represents a third supply of fatty acids for lipid synthesis in cyanobacteria.

Figure 1.

A, Putative fatty acid-activating enzyme of Synechococcus was expressed in E. coli, purified, and analyzed by in vitro assays conducted in presence of radiolabeled palmitic acid, ATP, and either CoA or ACP as an acyl acceptor. Control measurements were performed either without protein (control I) or with protein denatured by boiling (control II). B, Substrate specificity of Aas of Synechococcus. Purified enzyme was tested in acyl-ACP activity assays using six different fatty acids. Each assay was performed in triplicate. The error bars represent the sd.

Figure 2.

Phylogenetic tree of acyl-ACP synthetases of photosynthetic organisms including two Lacs sequences of Arabidopsis as outgroup (clade V). The branch lengths of the tree are proportional to the calculated divergence. The 0.1 scale represents 10% sequence divergence, calculated as estimated numbers of replacement. The numbers indicate the confidence levels for various branches as determined by bootstrap analysis (Felsenstein, 1989). Abbreviations are given in Table I.

Figure 3.

Autoradiography of total lipid extracts of Synechocystis and Synechococcus wild-type cells (WT) and aas knockout mutant cells (ko) fed with radiolabeled oleic acid (18:1). Lipids were separated by TLC using acetone:toluene:water (91:30:8, v/v/v) as solvent system. The individual spots were identified by authentic standards as free fatty acids (FFAs), MGDG, digalactosyldiacylglycerol (DGDG), SQDG, and PG. Additional spots remained unidentified. In the extract of Synechocystis wild-type cells also monoglucosyldiacylglycerol being the precursor of MGDG is detectable as spot slightly above MGDG.

Figure 4.

Fatty acid profiles of cells and culture medium of wild-type and aas mutant strains. A, Free fatty acids in the culture media. B, Free fatty acids within the cells. C, Esterified fatty acids within the cells. Results are shown for Synechocystis wild type and aas knockout mutant (6803wt, 6803ko) as well as for the corresponding strains of Synechococcus (7942wt, 7942ko). For each strain three independent cultures were analyzed. The error bars represent the sd.

Figure 5.

Analysis by gas chromatography of methyl esters obtained from fatty acids secreted into the culture media by aas mutant cells of Synechocystis (A) and from the LPS fraction of Synechocystis (B). The presence of 3-hydroxymyristic acid (3-OH-14:0) in both extracts was confirmed by comigration with authentic standard (dotted line). Other fatty acids were identified by standards: 16:0 (a), 16:1 (b), internal standard 17:0 (c), 18:0 (d), 18:1 (e), 3-OH-14:0 (f), 18:2 (g), and 18:3 (h; A). Other peaks observed in the LPS fraction derived either from the extraction reagent or were not further investigated (B).

Figure 6.

Free fatty acids in cyanobacteria were released from complex lipids. aas mutant cells (A) and wild type (B) of Synechocystis were incubated with radiolabeled acetate to achieve labeling of fatty acids. The labeled fatty acids were subsequently incorporated into complex lipids. Following the addition of acetate, aliquots of the culture were removed at the times indicated. Lipid extracts of the cells were separated by TLC as described before. In aas mutant cells the label was clearly detectable in complex lipids after 10 min of incubation but did not yet accumulate in the pool of free fatty acids. In free fatty acids the label showed up with some delay, suggesting a release of fatty acids from lipid molecules. In wild-type cells label was incorporated into complex lipids but was detectable in only minor amounts in the pool of free fatty acids. The individual spots were identified as FFAs, monoglucosyldiacylglycerol (MGlucDG), MGDG (MGalDG), digalactosyldiacylglycerol (DGDG), SQDG, and PG. Analysis of the spot close to the solvent front (?) by two-dimensional TLC revealed a mixture of at least four different components that were not further investigated.

Figure 7.

Purified lipids labeled with ¹⁴C-oleic acid and ¹⁴C-palmitic acid were added individually to cultures of Synechocystis wild-type cells (WT) as well as to the corresponding aas mutant strain (ko). The cells were harvested after 20 h and the lipid extracts were analyzed by TLC as described before. In both strains the supplemented lipids were partially degraded to their corresponding lyso lipids and to free fatty acids but only the wild-type cells were able to utilize the released fatty acids to synthesize new lipid molecules. Upon supplementation with either SQDG (A) or PG (B), wild-type cells incorporated the released fatty acids into MGDG. The individual spots were identified by authentic standards as FFAs, MGDG, SQDG, PG, and lyso PG. The abbreviation of the supplemented lipid is highlighted in gray. Additional spots present in both strains could not be identified by available lipid standards.