Significance Cyanobacteria harbor a photosynthetic apparatus related to plant chloroplasts. The lipid compositions of the thylakoids that harbor the photosynthetic complexes in cyanobacteria and chloroplasts are highly similar. Chloroplasts contain triacylglycerol (storage oil) and wax esters; the latter are composed of phytol derived from chlorophyll and fatty acids (phytyl esters). However, the existence of these lipids in cyanobacteria in general remained unclear. Here we show that the cyanobacterium Synechocystis contains triacylglycerol and phytyl esters. A mutant, Δslr2103, was generated, which lacked these two lipids but showed no obvious growth defect. The slr2103 gene encodes a diacylglycerol acyltransferase different from known enzymes of triacylglycerol synthesis in bacteria. This pathway can be employed to produce oil for biotechnological applications in cyanobacteria.

Abstract Cyanobacteria are unicellular prokaryotic algae that perform oxygenic photosynthesis, similar to plants. The cells harbor thylakoid membranes composed of lipids related to those of chloroplasts in plants to accommodate the complexes of photosynthesis. The occurrence of storage lipids, including triacylglycerol or wax esters, which are found in plants, animals, and some bacteria, nevertheless remained unclear in cyanobacteria. We show here that the cyanobacterium Synechocystis sp. PCC6803 accumulates both triacylglycerol and wax esters (fatty acid phytyl esters). Phytyl esters accumulate in higher levels under abiotic stress conditions. The analysis of an insertional mutant revealed that the acyltransferase slr2103, with sequence similarity to plant esterase/lipase/thioesterase (ELT) proteins, is essential for triacylglycerol and phytyl ester synthesis in Synechocystis. The recombinant slr2103 enzyme showed acyltransferase activity with phytol and diacylglycerol, thus producing phytyl esters and triacylglycerol. Acyl-CoA thioesters were the preferred acyl donors, while acyl-ACP (acyl carrier protein), free fatty acids, or galactolipid-bound fatty acids were poor substrates. The slr2103 protein sequence is unrelated to acyltransferases from bacteria (AtfA) or plants (DGAT1, DGAT2, PDAT), and therefore establishes an independent group of bacterial acyltransferases involved in triacylglycerol and wax ester synthesis. The identification of the gene slr2103 responsible for triacylglycerol synthesis in cyanobacteria opens the possibility of using prokaryotic photosynthetic cells in biotechnological applications.

Triacylglycerol (TAG) is the most important storage lipid in many organisms. Plant TAG represents the largest source of oil for human consumption, biotechnological applications, and biofuels. Oleaginous eukaryotic microalgae are increasingly considered as feedstocks for the production of oils for food and industrial applications (1, 2). However, oil yield from microalgae is oftentimes low, and most strains accumulate oil only under specific stress conditions.

Oil is stored in lipid droplets in the cytosol of plants, animals, and fungi. Lipid droplets contain nonpolar lipids, in particular TAG, enclosed by a phospholipid monolayer membrane (3). In plant seeds, TAG is predominantly synthesized by the transfer of a fatty acyl group from acyl-CoA or from a phospholipid onto diacyl-glycerol by acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) or phospholipid:diacylglycerol acyltransferase (PDAT), respectively (4⇓⇓⇓–8). In addition to the storage in lipid droplets in the cytosol, plant chloroplasts accumulate nonpolar lipids in plastoglobules that are surrounded by a galactolipid monolayer (9). Plastoglobules contain TAG, carotenoids, tocopherol, and fatty acid phytyl esters (10). In Arabidopsis, phytyl esters, which are chloroplastic wax esters containing phytol, are synthesized during chlorotic stress (11, 12). Two acyltransferases (PES1, PES2) of the esterase/lipase/thioesterase (ELT) family were found to synthesize phytyl esters from phytol, which is derived from chlorophyll breakdown, and fatty acids from lipid turnover (13). The ELT enzymes PES1/PES2 from Arabidopsis and PYP1 from tomato show broad substrate specificities for the synthesis of phytyl esters and xanthophyll esters, respectively (13, 14).

According to the endosymbiont theory, plant chloroplasts are derived from an ancient cyanobacterium via endosymbiosis, suggesting that many molecular and structural characteristics of chloroplasts are of cyanobacterial origin (15, 16). For example, the cytosol of Synechocystis sp. PCC6803 and Nostoc punctiforme were shown to contain lipid droplets similar to plastoglobules in plant chloroplasts and lipid droplets in the cytosol of eukaryotic cells (17, 18). Additional evidence for the potential occurrence of TAG was obtained for filamentous cyanobacteria of the Nostocales. Nostoc commune is capable of producing a lipid comigrating with TAG after labeling with radioactive glycerol (19), and a lipid comigrating with TAG was identified in lipid droplets isolated from Nostoc punctiforme (18). TAGs were identified in the thermophilic Nostocales species Mastigocladus and Tolypothrix (20). However, evidence for the existence of TAG in nonfilamentous, nonthermophilic cyanobacteria such as Synechocystis is lacking (1). TAG accumulation has been reported for different nonphotosynthetic Gram-positive (Mycobacterium, Streptomycetes) and Gram-negative (Acinetobacter, Pseudomonas) bacteria (21). An acyltransferase essential for TAG and wax ester synthesis (WS/DGAT, AtfA-type) was isolated from Acinetobacter baylyi (22). Orthologs of AtfA represent the only known acyltransferases involved in TAG synthesis in bacteria (23).

To unravel whether cyanobacteria harbor a pathway for TAG synthesis, nonpolar lipids were isolated from Synechocystis and characterized by direct infusion mass spectrometry (MS). A candidate acyltransferase for the synthesis of TAG was identified based on sequence similarity with Arabidopsis PES1/PES2. Characterization of the corresponding Synechocystis mutant and of the recombinant gene product revealed that Synechocystis indeed contains bona fide TAG, and that a cyanobacterial PES1/PES2-like acyltransferase exists that establishes a different class of bacterial genes involved in phytyl/wax ester and TAG synthesis.

Discussion We show here that the nonfilamentous cyanobacterium Synechocystis contains an ORF, slr2103, encoding an acyltransferase capable of synthesizing phytyl esters and TAG. The slr2103 sequence is unrelated to AtfA-type acyltransferases, which is the only family of enzymes involved in TAG synthesis in bacteria known to date. Instead, slr2103 is related to the ELT family of acyltransferases from plants. ELT enzymes are involved in fatty acid phytyl ester and fatty acid xanthophyll ester synthesis in the chloroplasts of plants (13, 14). After the endosymbiont theory, the slr2103 sequence presumably represents the evolutionary origin for the ELT proteins. ELT proteins are specific for plants, green algae, and red algae (Fig. 1), but absent from animals and fungi. They consist of a hydrolase domain followed by an acyltransferase domain, and contain an N-terminal transit sequence for targeting to the chloroplast. The finding that the Synechocystis acyltransferase slr2103 harbors TAG and phytyl ester synthesis activities demonstrates that the hydrolase domain is not essential for activity. The acyl composition of phytyl esters and TAG in Synechocystis is dominated by the presence of high amounts of 18:1, 18:2, 18:3, and 16:0 (Figs. 2 and 3). This fatty acid pattern reflects the total fatty acid composition of the membrane lipids of Synechocystis, which mostly contains 16:0, 18:1, 18:2, and 18:3 (29). Therefore, it is likely that the fatty acids in TAG and phytyl esters are originally derived from membrane lipids. The amount of TAG is reduced to background levels in the Δslr2103 mutant under control or stress conditions, indicating that slr2103 is essential for TAG synthesis. On the other hand, the Δslr2103 mutant still contains about 50% of phytyl esters compared with WT, when grown under control conditions. This residual, low amount of phytyl esters might be derived from the activity of other acyltransferase-like enzymes, or even from chemical esterification of phytol with free fatty acids. The finding that the phytyl ester content during salt/dark stress or phytol supplementation is much more strongly decreased in Δslr2103 compared with WT indicates that slr2103 is the major acyltransferase involved in phytyl ester production under these conditions. In vitro acyltransferase activity of recombinant slr2103 protein with dioctanoin was much higher compared with phytol. We employed a detergent (CHAPS) in the enzyme assays, but it is still possible that phytol was poorly dissolved compared with dioctanoin. DGAT assays using dipalmitin (di16:0) instead of dioctanoin showed lower activity, in accordance with the scenario that lipids containing long chain acyl groups are poorly soluble. Therefore, from the enzyme activity data (Fig. 5), it is difficult to conclude whether phytol or diacylglycerol is the preferred substrate of slr2103. The acyltransferase slr2103 showed higher phytyl ester and TAG synthesis activity with acyl-CoA than with acyl-ACP (Fig. 5). Cyanobacteria harbor a type II fatty acid synthase, giving rise to the production of acyl-ACP thioesters similar to plant chloroplasts (30). The acyltransferases involved in membrane lipid synthesis in cyanobacteria prefer acyl-ACP, rather than acyl-CoA substrates (24, 30). In contrast, cyanobacteria also harbor acyl-CoA thioesters, as the initial steps of fatty acid synthesis are CoA-dependent and cyanobacteria contain a short-chain acyl-CoA-dependent pathway of polyhydroxyalcanoate synthesis (30, 31). Furthermore, cyanobacteria harbor CoA-dependent fatty acid modifying enzymes (e.g., the aldehyde-forming acyl-CoA reductase) (32). Therefore, in addition to the acyl-ACP pool used for membrane lipid synthesis, a separate acyl-CoA pool might exist in cyanobacteria important for the synthesis of low abundant nonpolar lipids such as phytyl esters and TAG. In plants, phytyl esters and TAG accumulate during stress, taking up fatty acids from membrane lipids and phytol from chlorophyll breakdown (13). Similarly, salt/dark stress results in the increase in phytyl esters, but not TAG, in Synechocystis (Fig. 2). It is possible that TAG production is increased in Synechocystis during other growth conditions. The finding that the gene slr2103, which is involved in phytyl ester and TAG ester synthesis in Synechocystis, is related to PES1/PES2 of plants indicates that the pathway of conversion of lipid and chlorophyll breakdown products into nonpolar lipids with subsequent storage in plastoglobules/lipid droplets is presumably derived from the cyanobacterial progenitor of chloroplasts. Plants are the largest source for global TAG production for human nutrition and biotechnological applications. In the past, numerous strategies were developed to produce TAG in eukaryotic microalgae, including green and red algae. The eukaryotic microalgae harbor a TAG synthesis pathway similar to plants, and TAG accumulation is most often dependent on chlorotic stress. The identification of the TAG synthesis pathway in cyanobacteria such as Synechocystis provides the means for developing a strategy of employing prokaryotic photosynthetic organisms for oil synthesis. The pathway of TAG synthesis in cyanobacteria is different from plants and eukaryotic algae. In addition, cyanobacteria are prokaryotic cells that can easily be grown and are amenable to genetic engineering to increase the capacity for oil production.

Materials and Methods Growth of Synechocystis and Generation of Δslr2103 Deletion Mutant. Synechocystis sp. PCC 6803 (glucose tolerant strain) was grown photomixotrophically in liquid or on solidified BG-11 medium (33) supplied with 5 mM glucose at 28 °C in incessant light (30 µmol m−2⋅s−1). Precultures of 50 mL were grown up to an OD 750 of 0.6 and used to inoculate 100-mL cultures in BG-11 medium. Cells were grown to an OD 750 of 0.6 and NaCl added to a final concentration of 0.5 M, and the cells grown in darkness for 3 d (salt/dark stress). For nitrogen deprivation, cells were harvested and resuspended in nitrogen-free medium to an OD 750 of 0.6 (or in nitrogen-replete medium for control), and the cells were grown for 3 d. Alternatively, 10 µL of a serial dilution of cells was spotted on solid BG-11 medium (control, in the light), BG-11 with 0.5 M NaCl (salt/dark stress, grown in darkness), or BG-11 lacking nitrogen (with light) and the cells grown for 10 d. The Δslr2103 deletion mutant of Synechocystis was generated by homologous recombination. The 5′ flanking sequence of slr2103 was amplified from genomic DNA, using oligonucleotides bn2263 and bn2264 (containing NcoI sites; SI Appendix, Table S1), and cloned into pJET1.2 (pJ-left-slr2103). In analogy, the 3′ flanking region was amplified with oligonucleotides bn2265 (harboring an MluI site) and bn2266 and ligated into pJET1.2 (pJ-right-slr2103). The construct pJ-nptII harbors the kanamycin resistance cassette nptII (amplified with oligonucleotides bn1116 and bn1117, introducing MluI and NcoI sites) cloned in pJET1.2. The 5′ flanking sequence and the nptII gene were released using NcoI/HindIII and NcoI (partial digestion)/MluI, respectively. HindIII is a restriction site in the pJET1.2 cloning vector. The two fragments were ligated in one step into the vector pJ-right-slr2103 (opened with MluI, HindIII), resulting in the knock-out construct pJ-Δslr2103-kan. The circular knock-out plasmid was transferred into Synechocystis PCC 6803 cells (34). Transformed cells were selected by restreaking on BG-11 medium with increasing kanamycin concentrations (30 µg⋅mL−1 final). The successful integration of the kanamycin cassette into the gene slr2103 was confirmed by PCR of genomic DNA. Expression of slr2103 in E. Coli and Substrate Feeding. The gene slr2103 was amplified by PCR using the primers bn3268 and bn3269, introducing SacI and PstI sites. The PCR product was ligated into the vector pQE-80L (Qiagen), and this construct was transferred into E. coli BL21(AI) cells (Thermo Fisher). E. coli cells were grown in LB medium at 37 °C to OD 600 of 0.6 and then cooled to 16 °C, and 0.5 mM isopropyl-β-D-thiogalactopyranoside and 0.1% (wt/vol) l-arabinose were added for induction of expression at 16 °C overnight. For substrate feeding experiments, the temperature was raised to 30 °C and the cells grown for 3 h in the presence of 3.3 mM phytol (Chemimpex, Wood Dale, IL) or 30 µM diacylglycerol (dioctanoin, di8:0, Larodan, Sweden) in the presence of 0.1% (wt/vol) CHAPS. Cells were harvested by centrifugation, the pellet washed once with water, and lipids extracted. Acyltransferase Assays. E. coli cells expressing slr2103 were harvested by centrifugation at 1,000 × g for 12 min. The pellet was washed and suspended in 4 mL buffer (1 mM EDTA, 200 mM sucrose, 100 mM Tris⋅HCl at pH 7.4). The cells were homogenized with a Precellys homogenizer (Bertin Technologies), using glass beads. The extract was centrifuged at 4,000 × g for 2 min. The supernatant was centrifuged at 35,000 × g for 45 min to obtain the membrane fraction. The pellet was resuspended and protein concentration measured with bicinchoninic acid. For enzyme assays (total volume, 200 µL), 400 µg membrane protein were incubated with 50 µM acyl donor (16:0-CoA, synthesized according to ref. 35; 16:0 free fatty acid [Merck KGaA, Darmstadt, Germany]; monogalactosyldiacylglycerol from spinach; Larodan) and 200 µM acyl acceptor (phytol, dioctanoin) in assay buffer (20 mM MgCl 2 , 0.1% CHAPS, 100 mM Tris⋅HCl at pH 7.4, 1.25 mg⋅mL−1 BSA, 10 mM Na orthovanadate). Lipid substrates were dissolved in a minimal volume of ethanol and directly added to the assay. After mixing, the assay was incubated for 20 min at 35 °C. The reaction was terminated and lipids extracted by adding 1 mL chloroform/methanol (2:1). Lipid Analysis. Cells were harvested from 100 mL Synechocystis culture by centrifugation and washed with BG-11 medium. The pellet was extracted three times with chloroform/methanol (1:2) (36). The extracts were combined and internal standards added. For fatty acid phytyl ester measurements, 17:0-phytol was used, which was synthesized from 17:0 and phytol (11). Tri-17:0 TAG and tri-17:1 TAG were used as internal standards for TAG quantification (Larodan). After addition of 1 mL of 300 mM ammonium acetate and 1 mL chloroform, extracts were centrifuged for phase separation. The organic phase was harvested and dried under a nitrogen stream. The lipids were dissolved in hexane and applied to silica solid phase extraction columns (Macherey & Nagel, Düren) equilibrated with hexane (37). After washing the column with hexane, phytyl esters and TAGs were eluted with hexane/diethyl ether (99:1) and hexane/diethyl ether (92:8), respectively. Synechocystis lipids were separated by TLC on silica plates (Silica 60 Durasil, Macherey & Nagel), using hexane/diethyl ether/acetic acid (70:30:1). The lipid bands were stained with primuline and observed under UV light. For lipid isolation, silica material from the plates was extracted with chloroform/methanol (2:1). Phytyl esters and TAG were measured by Q-TOF MS. Lipids were dissolved in chloroform/methanol/300 mM ammonium acetate (300:665:35) (38). The samples were infused at 1 µL⋅min−1 into the HPLC-Chip Cube MS interface of the Agilent 6530 Series Accurate-Mass Q-TOF mass spectrometer (Agilent, Böblingen). Lipids were quantified by neutral loss scanning, and the amounts calculated based on internal standards (13). Measurements of Chlorophyll and Photosynthetic Quantum Yield. Cell pellets of Synechocystis were extracted with 1 mL cold methanol. After centrifugation, the supernatant was collected. Methanol extraction was repeated until the pellet turned blue. The chlorophyll contents were calculated after measuring the absorbances at 470, 665, and 720 nm, according to the equation: chlorophyll a (µg⋅mL−1) = 12.9447 * (A665 – A720) (39). Chlorophyll fluorescence of Synechocystis cells in liquid BG-11 medium was measured by pulse-amplified modulation fluorometry (Junior PAM, Heinz Walz, Effeltrich, Germany). Synechocystis cells were dark adapted for 60 min before the measurements. Quantum yield of PSII was calculated according to the equation: (F m −F)/F m , where F m and F are the fluorescence emission of dark-adapted cells under measuring light and after applying a saturating light pulse, respectively (40). Transmission Electron Microscopy. For comparative ultrastructural analysis, cells of Synechocystis WT and Δslr2103 mutant were collected by centrifugation and resuspended in 8% agarose. After solidification, the agarose was cut into blocks of ∼1 mm3 and used for combined conventional and microwave-assisted fixation, dehydration, and resin embedding, as defined in SI Appendix, Table S2. Sectioning and ultrastructural analysis were performed as described (41). Data Availability Statement. All data discussed in the paper are available in the main text and SI Appendix.

Acknowledgments We thank Mathias Brands, Payal Patwari, and Jill Romer (Institute of Molecular Physiology and Biotechnology of Plants; University of Bonn) for their help with enzyme assays, and Claudia Riemey (IPK Gatersleben) for technical assistance in transmission electron microscopy. Funding was provided by University of Bonn.

Footnotes Author contributions: G.H. and P.D. designed research; M.A. and M.M. performed research; M.A., H.P., K.G., and M.M. contributed new reagents/analytic tools; M.A., H.P., K.G., G.H., and P.D. analyzed data; and M.A. and P.D. wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission.

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