Triacylglycerol and phytyl ester synthesis in Synechocystis sp. PCC6803 Mohammed Aizouqa, Helga Peiskera, Katharina Gutbroda, Michael Melzerb, Georg Hölzla, and Peter Dörmanna,1 aInstitute of Molecular Physiology and Biotechnology of Plants, University of Bonn, 53115 Bonn, Germany; and bDepartment of Physiology and Cell Biology, Leibniz Institute of Plant Genetics and Crop Plant Research, 06466 Seeland, OT Gatersleben, Germany Edited by Krishna K. Niyogi, University of California, Berkeley, CA, and approved February 3, 2020 (received for review September 16, 2019) Cyanobacteria are unicellular prokaryotic algae that perform According to the endosymbiont theory, plant chloroplasts are oxygenic photosynthesis, similar to plants. The cells harbor derived from an ancient cyanobacterium via endosymbiosis, thylakoid membranes composed of lipids related to those of suggesting that many molecular and structural characteristics of chloroplasts in plants to accommodate the complexes of photosynthesis. chloroplasts are of cyanobacterial origin (15, 16). For example, The occurrence of storage lipids, including triacylglycerol or wax esters, the cytosol of Synechocystis sp. PCC6803 and Nostoc punctiforme which are found in plants, animals, and some bacteria, nevertheless were shown to contain lipid droplets similar to plastoglobules in remained unclear in cyanobacteria. We show here that the cyanobac- plant chloroplasts and lipid droplets in the cytosol of eukaryotic terium Synechocystis sp. PCC6803 accumulates both triacylglycerol and cells (17, 18). Additional evidence for the potential occurrence wax esters (fatty acid phytyl esters). Phytyl esters accumulate in of TAG was obtained for filamentous cyanobacteria of the higher levels under abiotic stress conditions. The analysis of an Nostocales. Nostoc commune is capable of producing a lipid insertional mutant revealed that the acyltransferase slr2103, comigrating with TAG after labeling with radioactive glycerol with sequence similarity to plant esterase/lipase/thioesterase (19), and a lipid comigrating with TAG was identified in lipid (ELT) proteins, is essential for triacylglycerol and phytyl ester droplets isolated from Nostoc punctiforme (18). TAGs were synthesis in Synechocystis. The recombinant slr2103 enzyme showed identified in the thermophilic Nostocales species Mastigocladus acyltransferase activity with phytol and diacylglycerol, thus pro- and Tolypothrix (20). However, evidence for the existence of TAG in ducing phytyl esters and triacylglycerol. Acyl-CoA thioesters were the nonfilamentous, nonthermophilic cyanobacteria such as Synechocystis preferred acyl donors, while acyl-ACP (acyl carrier protein), free fatty is lacking (1). TAG accumulation has been reported for different acids, or galactolipid-bound fatty acids were poor substrates. The nonphotosynthetic Gram-positive (Mycobacterium, Streptomycetes) slr2103 protein sequence is unrelated to acyltransferases from bac- and Gram-negative (Acinetobacter, Pseudomonas) bacteria (21). An teria (AtfA) or plants (DGAT1, DGAT2, PDAT), and therefore estab- acyltransferase essential for TAG and wax ester synthesis (WS/ lishes an independent group of bacterial acyltransferases involved DGAT, AtfA-type) was isolated from Acinetobacter baylyi (22). in triacylglycerol and wax ester synthesis. The identification of the Orthologs of AtfA represent the only known acyltransferases in- gene slr2103 responsible for triacylglycerol synthesis in cyanobacteria volved in TAG synthesis in bacteria (23). opens the possibility of using prokaryotic photosynthetic cells in To unravel whether cyanobacteria harbor a pathway for TAG biotechnological applications. synthesis, nonpolar lipids were isolated from Synechocystis and characterized by direct infusion mass spectrometry (MS). A cyanobacteria | triacylglycerol | wax | acyltransferase candidate acyltransferase for the synthesis of TAG was identi- fied based on sequence similarity with Arabidopsis PES1/PES2. riacylglycerol (TAG) is the most important storage lipid in Tmany organisms. Plant TAG represents the largest source of Significance oil for human consumption, biotechnological applications, and biofuels. Oleaginous eukaryotic microalgae are increasingly Cyanobacteria harbor a photosynthetic apparatus related to plant considered as feedstocks for the production of oils for food and chloroplasts. The lipid compositions of the thylakoids that harbor industrial applications (1, 2). However, oil yield from microalgae the photosynthetic complexes in cyanobacteria and chloroplasts is oftentimes low, and most strains accumulate oil only under are highly similar. Chloroplasts contain triacylglycerol (storage oil) specific stress conditions. and wax esters; the latter are composed of phytol derived from Oil is stored in lipid droplets in the cytosol of plants, animals, chlorophyll and fatty acids (phytyl esters). However, the existence and fungi. Lipid droplets contain nonpolar lipids, in particular of these lipids in cyanobacteria in general remained unclear. Here TAG, enclosed by a phospholipid monolayer membrane (3). In we show that the cyanobacterium Synechocystis contains tri- plant seeds, TAG is predominantly synthesized by the transfer of a acylglycerol and phytyl esters. A mutant, Δslr2103, was gener- fatty acyl group from acyl-CoA or from a phospholipid onto diacyl- ated, which lacked these two lipids but showed no obvious glycerol by acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) or growth defect. The slr2103 gene encodes a diacylglycerol acyl- phospholipid:diacylglycerol acyltransferase (PDAT), respectively transferase different from known enzymes of triacylglycerol (4–8). In addition to the storage in lipid droplets in the cytosol, plant synthesis in bacteria. This pathway can be employed to produce chloroplasts accumulate nonpolar lipids in plastoglobules that are oil for biotechnological applications in cyanobacteria. surrounded by a galactolipid monolayer (9). Plastoglobules contain Author contributions: G.H. and P.D. designed research; M.A. and M.M. performed re- TAG, carotenoids, tocopherol, and fatty acid phytyl esters (10). In search; M.A., H.P., K.G., and M.M. contributed new reagents/analytic tools; M.A., H.P., Arabidopsis, phytyl esters, which are chloroplastic wax esters con- K.G., G.H., and P.D. analyzed data; and M.A. and P.D. wrote the paper. taining phytol, are synthesized during chlorotic stress (11, 12). Two The authors declare no competing interest. acyltransferases (PES1, PES2) of the esterase/lipase/thioesterase This article is a PNAS Direct Submission. (ELT) family were found to synthesize phytyl esters from phytol, Published under the PNAS license. which is derived from chlorophyll breakdown, and fatty acids from 1To whom correspondence may be addressed. Email: [email protected]. lipid turnover (13). The ELT enzymes PES1/PES2 from Arabidopsis This article contains supporting information online at https://www.pnas.org/lookup/suppl/ and PYP1 from tomato show broad substrate specificities for the doi:10.1073/pnas.1915930117/-/DCSupplemental. synthesis of phytyl esters and xanthophyll esters, respectively (13, 14). First published March 2, 2020. 6216–6222 | PNAS | March 17, 2020 | vol. 117 | no. 11 www.pnas.org/cgi/doi/10.1073/pnas.1915930117 Downloaded by guest on September 30, 2021 Characterization of the corresponding Synechocystis mutant and acyltransferase (DGAT1). Furthermore, the Acinetobacter wax of the recombinant gene product revealed that Synechocystis synthase/DGAT sequences (bacterial AtfA-type) and the re- indeed contains bona fide TAG, and that a cyanobacterial lated WSD1 sequence from Arabidopsis involved in wax ester PES1/PES2-like acyltransferase exists that establishes a different synthesis were included. The sequences were clustered into class of bacterial genes involved in phytyl/wax ester and TAG five groups: a plant-type ELT group containing the C-terminal synthesis. sequences of Arabidopsis PES1, PES2 and the related proteins from green and red algae, a group of ELT-like cyanobacterial Results acyltransferases including slr2103, a group of the three distantly Identification of an ELT-Like Acyltransferase in Cyanobacteria. ELT related LPAAT-like sequences from Synechocystis,andArabidopsis sequences of plants are characterized by the presence of an DGAT1 and the two AtfA-type sequences. The slr2103 sequences N-terminal hydrolase and a C-terminal acyltransferase domain are much closer related to the acyltransferase domain of Arabidopsis (13). ELT proteins with the two-domain structure are absent ELT sequences compared with Arabidopsis DGAT1 or the AtfA- from cyanobacterial genomes. Protein BLAST searches with the type sequences, indicating that they establish a different group of C-terminal, acyltransferase part of Arabidopsis PES2 (amino bacterial acyltransferases. acids 401 to 704) revealed the presence of one related sequence Generation of a Δslr2103 Deletion Mutant. A Synechocystis deletion (slr2103) and three less similar acyltransferase-like sequences Δ (sll1848, slr2060, sll1752) in the Synechocystis genome (Fig. 1). mutant ( slr2103) was generated by inserting a kanamycin re- sistance cassette into the ORF slr2103 (SI Appendix, Fig. S1). An Two of the sequences (sll1848, slr2060) were previously charac- isogenic mutant line was isolated after restreaking the cells on terized as lysophosphatidic acid acyltransferases (LPAAT) (24). kanamycin-containing medium. Growth of Δslr2103 mutant Further cyanobacterial slr2103-like sequences were