Triacylglycerol and Phytyl Ester Synthesis in Synechocystis Sp

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 identified 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 resistance 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 found by cells on BG-11 medium as well as on N-deficient medium was BLAST searches in the genomes of Cyanothece (Oscillatoriales) not compromised (SI Appendix,Fig.S2).Whengrowninliquid Anabaena Calothrix and and (Nostocales; Fig. 1). In contrast, BG-11 medium or under stress conditions (darkness and NaCl), three PES2-like sequences with two domains each were retrieved the chlorophyll a contents of Δslr2103 cells were slightly re- in green algae (Chlamydomonas reinhardtii), and one PES2-like duced, whereas the amounts of carotenoids remained similar sequence each in red algae (Galdieria and Chondrus). A phylo- to WT (SI Appendix,Fig.S3). The quantum yield of photo- genetic tree was constructed with the C-terminal acyltransferase system II determined by fluorescence measurements was de- portions (PES2 and related two-domain proteins) of the plant creased to 0.31 in Δslr2103 cells compared with 0.34 in WT (SI

and green and red algae, the cyanobacterial acyltransferases Appendix,Fig.S3). Therefore, the deletion of the gene slr2103 PLANT BIOLOGY (slr2103-like sequences), and with Arabidopsis diacylglycerol had only minor consequences for growth and photosynthesis in Synechocystis.

Synechocystis At-PES1 Fatty Acid Phytyl Esters Accumulate in in an slr2103- 0.20 100 Dependent Manner. Previously, fatty acid phytyl esters were ten- 60 At-PES2 tatively found in extremely low amounts in Synechococcus (25). Cre08g365950 To confirm their existence in cyanobacteria, phytyl esters were 88 93 Cre12g521650 ELT enriched by solid phase extraction and analyzed by quadrupole time-of-flight MS (Q-TOF MS). Different phytyl esters were 98 Galdieria clearly identified by the masses of their parental ammonium 55 Chondrus adduct ions, which gave rise to fatty acid ammonium ions after 89 Cre01g017100 fragmentation (SI Appendix, Fig. S4). The amounts of phytyl esters in Synechocystis WT cells are very low, with 16:0-phytol, Synechococcus 74 18:1-phytol, 18:2-phytol, and 18:3-phytol representing the most Syn-slr2103 abundant forms (Fig. 2). 100 cyanobacterial 54 Cyanothece It has previously been shown that phytyl esters accumulate in ELT-like 53 Anabaena plant chloroplasts during abiotic stress conditions, including N deprivation, drought, or extended darkness, as well as after 99 44 Calothrix feeding exogenous phytol to the plants (11, 12). Degradation of Syn-slr2060 chlorophyll and membrane lipids under stress results in the re- Syn-sll1848 LPAAT lease of free phytol and fatty acids, which are converted into 76 phytyl esters and TAG in plants. Growth of Synechocystis cells Syn-sll1752 under N deprivation did not result in the accumulation of phytyl At-DGAT1 DGAT1 type esters (Fig. 2). This can be explained because in contrast to Ab-WS/DGAT plants, cyanobacteria harbor light-harvesting complexes com- AtfA type posed of phycobilisomes that contain tetrapyrroles such as phyco- 93 At-WSD1 erythrobilin or phycocyanobilin instead of chlorophyll. Thus, Fig. 1. Phylogenetic relationship of lipid acyltransferases from cyanobac- degradation of phycobilisomes during N deprivation in Syn- teria, plants, and green and red algae. Protein sequences were aligned with echocystis does not cause phytol accumulation (26). We therefore ClustalW, and a phylogenetic tree constructed using the Neighbor Joining chose the combination of darkness and salt stress to stimulate method with 1,000 bootstrap repetitions (Mega 10.0.5). For plant ELT se- chlorophyll degradation. Exposure of Synechocystis to darkness quences, only the C-terminal acyltransferase-like parts were considered. A. results in the halt of chlorophyll synthesis (27). Salt stress dis- baylyi (ADP1) Ab-WS/DGAT, AF529086.1; Anabaena sp. (CA = ATCC 33047) turbs the ability of cyanobacteria to take up carbon from the – WP_066380359; Arabidopsis thaliana At-PES1 (amino acids 401 704), At- medium, forcing the cells to metabolize intracellular carbon (28). PES2 (401-701), At-DGAT1, At-WSD1; Calothrix brevissima WP_096644248; The combination of dark and salt stress is thus expected to cause C. reinhardtii Cre01g017100 (591-911), Cre08g365950 (411-692), Cre12g521650 (631-873); Chondrus crispus XP_005713590 (601-965); Cyanothece sp. (PCC the degradation of thylakoid lipids and chlorophyll. Indeed, 7425) WP_012629067; Galdieria sulphuraria XP_005702844 (561-921); Synechocystis cells accumulated fatty acid phytyl esters by more Synechococcus sp. (NKBG042902) WP_030007836; and Synechocystis sp. (PCC than twofold during exposure to darkness/high salt conditions 6803) slr2103, sll1848, slr2060, sll1752. (Fig. 2B). Next, we tested whether the cells can take up exogenous

Aizouq et al. PNAS | March 17, 2020 | vol. 117 | no. 11 | 6217 Downloaded by guest on September 30, 2021 to similar extents, and in addition, low amounts of 16:1-phytol A 12 # Total Phytyl Esters WT and 18:0-phytol accumulated. Δ 10 Δslr2103 The phytyl ester content in slr2103 cells grown under control −1 ) conditions was reduced to 1 to 2 nmol OD750 . The amount of

750 8 # phytyl esters in Δslr2103 did not increase during salt/dark stress 6 or after phytol feeding. Thus, phytyl esters were about eightfold or 10-fold higher in WT compared with Δslr2103 under salt/dark 4 stress or phytol feeding conditions, respectively. These results demonstrate that slr2103 represents the major enzyme for phytyl 2 * (nmol per OD * * ester synthesis in Synechocystis.

Fatty Acid Phytyl Esters 0 slr2103 Synechocystis Control Salt/Dark Nitrogen + Phytol The Gene Is Essential for TAG Synthesis in . Deprivation Previous studies on the accumulation of TAG in cyanobacteria B 4 were mostly inconclusive (1, 18, 19). TAG was only identified in Control WT filamentous, thermophilic cyanobacteria of the order Nostocales (20). To address the question of TAG accumulation in cyano- 3 Δslr2103 bacteria, nonpolar lipids were isolated from Synechocystis cells by solid phase extraction and subjected to Q-TOF MS analysis. 2 Several peaks with m/z values corresponding to ammonium ad- ducts of TAGs with different acyl groups were identified (Fig. 1 3A). MS/MS fragmentation resulted in the formation of diacyl- * glycerol ions with the neutral losses of fatty acid ammonia ad- 0 ducts (SI Appendix,Fig.S5). Scanning for different TAGs by 4 ∼ Salt/Dark MS/MS experiments revealed the presence of 14 molecular species, with tri-16:0 TAG and 16:0-16:0-18:3 TAG representing the 3 most abundant forms (Fig. 3A). To study the role of the gene slr2103 in TAG synthesis, non- 2 polar lipids from Synechocystis WT and the Δslr2103 mutant ) were analyzed by TLC or Q-TOF MS. TLC separation revealed 750 1 the presence of a lipid in WT comigrating with TAG. This band * 0 * * * * 4 Nitrogen Deprivation A B

(nmol per OD 3 5 Fatty Acid Phytyl Esters )

2 TAG 750 4

1 3 2 0 FFA

4 Triacylglycerols (nmol per OD 1 + Phytol * 3 0 WT Δslr 2 2103 TAG WT Δslr2103 1 C * * * * WT

* ) 1.0 0 * Δslr2103

12:0 14:0 16:0 16:1 16:2 16:3 16:4 18:0 18:1 18:2 18:3 18:4 750 0.8 Phytyl Ester 0.6 Fig. 2. Fatty acid phytyl esters in Synechocystis WT and Δslr2103 mutant. Synechocystis cells were grown in liquid BG-11 medium (control) or under 0.4

stress conditions (3 d of darkness/with 0.5 M NaCl; N deprivation) or in the Triacylglycerols (nmol per OD presence of phytol. Phytyl esters were quantified by Q-TOF MS. (A) Total 0.2 fatty acid phytyl esters. (B) Fatty acid phytyl ester composition. Acyl groups * are indicated as number of C atoms: number of double bonds. Means of 0.0 * * * * * * * * * * * ** three to four replicas ± SD. *Significant differences to the WT grown under 16:1 16:0 16:0 16:0 16:1 16:0 16:0 16:0 16:0 16:0 16:1 16:0 16:0 16:0 the same conditions. #Significant differences to the WT grown under control 16:1 16:1 16:0 16:0 16:1 16:1 16:0 16:0 16:0 16:0 18:3 18:3 18:2 18:1 conditions, respectively (Student t test, P < 0.05). 16:1 16:1 16:1 16:0 18:3 18:3 18:3 18:2 18:1 18:0 18:3 18:3 18:3 18:3

Fig. 3. Triacylglycerol accumulation in Synechocystis. (A) Separation of nonpolar lipids from Synechocystis WT and Δslr2103 mutant by TLC. Lipids phytol and use it for phytyl ester synthesis. Feeding of phytol to were stained with primuline. FFA, free fatty acids. The arrow indicates TAG in Synechocystis WT. (B) TAG content and (C) molecular species composition the cells stimulated an even higher increase in phytyl esters of of Synechocystis WT and Δslr2103 mutant. TAG was quantified by Q-TOF MS. up to fourfold (Fig. 2B). Therefore, under salt/dark treatment Means of three to four replicas ± SD. Asterisks indicate significant differ- and during phytol feeding, all forms of phytyl esters increased ences to the WT (Student t test, P < 0.05).

6218 | www.pnas.org/cgi/doi/10.1073/pnas.1915930117 Aizouq et al. Downloaded by guest on September 30, 2021 was absent from the Δslr2103 mutant. The total amount of TAG ∼ −1 in WT cells was 5 nmol OD750 , and it was about 15-fold lower WT −1 in Δslr2103 with ∼0.3 nmol OD750 (Fig. 3). All molecular species found in WT were reduced to background levels in the Δslr2103 mutant. TAG was also quantified in cells exposed to salt/dark stress or N deprivation. The amount of TAG in WT cells was strongly reduced to 26.5 ± 3.9% or 12.1 ± 2.0% under salt/dark stress and N deprivation, respectively. These results demonstrate that different molecular species of TAG are synthesized in Synechocystis, and that TAG production depends on slr2103.

Decrease in the Number of Lipid Droplets in Δslr2103 Mutant Cells. Plastoglobules in chloroplasts accumulate phytyl esters, TAG, carotenoids, and tocopherol (10, 13). Similarly, lipid droplets in cyanobacteria presumably also contain phytyl esters and TAG (18). To study the effect of the decrease in phytyl ester and TAG synthesis in Δslr2103 cells, lipid droplets were observed by transmission electron microscopy (Fig. 4A and SI Appendix, Fig. S6). The number of lipid droplets per cell cross section in the mutant was reduced to about 50% of WT (SI Appendix, Fig. S6). The residual number of lipid droplets in the mutant can be explained by the presence of other nonpolar lipids (carotenoids, tocopherols) and proteins in the lipid droplets (10). Therefore, deficiency in slr2103 affects phytyl ester and TAG accumulation and results in a reduced lipid droplet number per cell cross Δslr2103 section.

The slr2103 Protein Harbors Phytyl Ester Synthase Activity. The PLANT BIOLOGY slr2103 gene was introduced into Escherichia coli cells. After induction of protein expression, protein accumulation was observed by SDS gel electrophoresis. The Coomassie stained gel showed a strong band at ∼32 kDa, in the range of the calculated weight of the slr2103 His tag protein (34.0 kDa). Immunoblot analysis with anti-His tag antibodies revealed a clear band corresponding to the 32-kDa band observed by Coomassie staining (SI Appendix, Fig. S7). To study the capacity for phytyl ester synthesis, phytol was added to the slr2103-expressing cells. The synthesis of phytyl esters was followed by Q-TOF MS. While E. coli cells expressing slr2103 in the absence of phytol did not accumulate phytyl esters, phytol feeding resulted in the production of large amounts of phytyl esters. The most abundant phytyl esters were 18:1- phytol and 16:0-phytol, followed by 16:1-phytol, 17:0c-phytol, 14:0-phytol, and 19:0c-phytol (Fig. 5A). Therefore, the slr2103 250 nm gene encodes a protein with fatty acid phytyl ester synthesis activity. Next, membrane proteins were isolated from slr2103-expressing cells and employed for acyltransferase assays with phytol and Fig. 4. The number of lipid droplets in Synechocystis Δslr2103 is decreased. Δ different acyl donors. The phytyl ester synthase activity of Synechocystis WT and slr2103 mutant were grown in BG-11 medium and slr2103 with 16:0-CoA was higher than of the empty vector the cells observed by transmission electron microscopy. control, but activities with 16:0-ACP (acyl carrier protein) or 16:0 free fatty acids were very low (Fig. 5B). Furthermore, we tested the hypothesis of whether lipid-bound fatty acids (i.e., DGAT assays were performed with membrane proteins from 16:3 or 18:3 bound to spinach monogalactosyldiacylglycerol E. coli expressing slr2103 using dioctanoin and different acyl [MGDG]) can be directly transferred to phytol. However, the donors. 16:0-CoA was by far the best substrate of slr2103, with an − − phytyl ester synthase activity with MGDG-bound fatty acids activity of up to 300 nmol·min 1·mg 1 protein (Fig. 5D). Other was extremely low. Therefore, slr2103 showed phytyl ester synthase putative acyl donors (16:0-ACP, 16:0 free fatty acid, 16:3 or 18:3 activity with 16:0-CoA, while other potential acyl donors were poor derived from MGDG) showed activities similar to the empty substrates. vector control. To determine the capacity for TAG synthesis, slr2103- In conclusion, slr2103 shows highest acyltransferase activity expressing E. coli cells were incubated in the presence of dio- with acyl-CoAs. The acyl groups incorporated into phytyl esters ctanoin (di8:0). This short chain diacylglycerol was used because long chain diacylglycerols are barely water-soluble and are and TAGs after feeding of phytol or dioctanoin represent fatty therefore poor substrates. Dioctanoin was quickly taken up and acids typical for E. coli. Two different acyl acceptors are used for TAG synthesis in slr2103-expressing cells (Fig. 5C). The employed by slr2103 (i.e., long chain alcohols: phytol, wax ester E. coli fatty acids 14:0, 16:1, 16:0, 18:1, and 18:0 were pre- synthase activity) and diacylglycerol (dioctanoin, DGAT activity; dominantly employed for TAG synthesis by slr2103. Fig. 5E).

Aizouq et al. PNAS | March 17, 2020 | vol. 117 | no. 11 | 6219 Downloaded by guest on September 30, 2021 Discussion A 20 12 eV We show here that the nonfilamentous cyanobacterium Synechocystis slr2103 10 contains an ORF, slr2103, encoding an acyltransferase capable of ) 15 synthesizing phytyl esters and TAG. The slr2103 sequence is unre-

750 8 lated to AtfA-type acyltransferases, which is the only family of en- 10 6 zymes involved in TAG synthesis in bacteria known to date. Instead, slr2103 is related to the ELT family of acyltransferases from plants. 4 ELT enzymes are involved in fatty acid phytyl ester and fatty acid

Phytyl Esters 5 xanthophyll ester synthesis in the chloroplasts of plants (13, 14).

(nmol per OD 2 After the endosymbiont theory, the slr2103 sequence presumably 0 0 represents the evolutionary origin for the ELT proteins. ELT pro- eV slr 12:0 14:0 16:0 16:1 17:0c 18:0 18:1 19:0c teins are specific for plants, green algae, and red algae (Fig. 1), but 2103 Phytyl Ester absent from animals and fungi. They consist of a hydrolase domain B followed by an acyltransferase domain, and contain an N-terminal 8 transit sequence for targeting to the chloroplast. The finding that the Synechocystis acyltransferase slr2103 harbors TAG and phytyl ester

protein) 6

-1 synthesis activities demonstrates that the hydrolase domain is not essential for activity. mg 4 -1 The acyl composition of phytyl esters and TAG in Synecho- cystis is dominated by the presence of high amounts of 18:1, 18:2, 2 * 18:3, and 16:0 (Figs. 2 and 3). This fatty acid pattern reflects the

Phytyl Ester Synthesis total fatty acid composition of the membrane lipids of Syn- (nmol min 0 echocystis, which mostly contains 16:0, 18:1, 18:2, and 18:3 (29). 16:0- 16:0- 16:0 16:3 18:3 Therefore, it is likely that the fatty acids in TAG and phytyl CoA ACP FFA MGDG 25 10 esters are originally derived from membrane lipids. C * eV The amount of TAG is reduced to background levels in the Δ ) 20 * slr2103 8 slr2103 mutant under control or stress conditions, indicating

750 that slr2103 is essential for TAG synthesis. On the other hand, 15 * 6 the Δslr2103 mutant still contains about 50% of phytyl esters compared with WT, when grown under control conditions. This 10 4 residual, low amount of phytyl esters might be derived from the activity of other acyltransferase-like enzymes, or even from chem- Triacylglycerols (nmol per OD 5 * 2 ical esterification of phytol with free fatty acids. The finding that * * the phytyl ester content during salt/dark stress or phytol supple- 0 0 mentation is much more strongly decreased in Δslr2103 compared eV slr 8:0 8:0 8:0 8:0 8:0 2103 8:0 8:0 8:0 8:0 8:0 with WT indicates that slr2103 is the major acyltransferase involved 14:0 16:1 16:0 18:1 18:0 in phytyl ester production under these conditions. D 350 * In vitro acyltransferase activity of recombinant slr2103 protein 300 with dioctanoin was much higher compared with phytol. We 250 50 employed a detergent (CHAPS) in the enzyme assays, but it is

protein) still possible that phytol was poorly dissolved compared with -1 40 dioctanoin. DGAT assays using dipalmitin (di16:0) instead of

mg 30 dioctanoin showed lower activity, in accordance with the sce- -1 20 nario that lipids containing long chain acyl groups are poorly soluble. Therefore, from the enzyme activity data (Fig. 5), it is 10 difficult to conclude whether phytol or diacylglycerol is the Triacylglycerol Synthesis (nmol min 0 * preferred substrate of slr2103. 16:0- 16:0- 16:0 16:3 18:3 The acyltransferase slr2103 showed higher phytyl ester and CoA ACP FFA MGDG TAG synthesis activity with acyl-CoA than with acyl-ACP (Fig. E O O 5). Cyanobacteria harbor a type II fatty acid synthase, giving rise CoA-S O R to the production of acyl-ACP thioesters similar to plant chlo- Acyl-CoA O O R roplasts (30). The acyltransferases involved in membrane lipid OH synthesis in cyanobacteria prefer acyl-ACP, rather than acyl-CoA OH Phytol Diacylglycerol substrates (24, 30). In contrast, cyanobacteria also harbor acyl- CoA thioesters, as the initial steps of fatty acid synthesis are O O R CoA-dependent and cyanobacteria contain a short-chain acyl-CoA- O dependent pathway of polyhydroxyalcanoate synthesis (30, 31). O O R O Furthermore, cyanobacteria harbor CoA-dependent fatty acid

O O modifying enzymes (e.g., the aldehyde-forming acyl-CoA reductase) Fatty Acid Phytyl Ester Triacylglycerol

Fig. 5. The slr2103 protein harbors fatty acid phytyl ester and diacylglycerol with phytol and different acyl donors. (C) E. coli cells expressing slr2103 were acyltransferase synthase activity. (A) E. coli cells expressing Synechocystis grown with dioctanoin (di8:0). TAG was isolated and quantified by Q-TOF slr2103 were grown in the presence of phytol. Phytyl esters were purified MS. (Left) Total TAG. (Right) Molecular species. (D) Membrane proteins from and quantified by Q-TOF MS. (Left) Total phytyl esters. (Right) Molecular E. coli expressing slr2103 were used for DGAT assays with dioctanoin and species composition. 17:0c, cis-9,10-methylenehexadecanoic acid; 19:0c, cis- different acyl donors. (E) Acyltransferase reactions for phytyl ester and TAG 11,12-methyleneoctadecanoic acid. (B) Membrane proteins were isolated synthesis by Synechocystis slr2103. Mean and SD of three to four replicas from E. coli cells expressing slr2103 and employed for acyltransferase assays (Student t test, *P < 0.05).

6220 | www.pnas.org/cgi/doi/10.1073/pnas.1915930117 Aizouq et al. Downloaded by guest on September 30, 2021 (32). Therefore, in addition to the acyl-ACP pool used for mem- in the presence of 0.1% (wt/vol) CHAPS. Cells were harvested by centrifu- brane lipid synthesis, a separate acyl-CoA pool might exist in cya- gation, the pellet washed once with water, and lipids extracted. nobacteria important for the synthesis of low abundant nonpolar lipids such as phytyl esters and TAG. Acyltransferase Assays. E. coli cells expressing slr2103 were harvested by × In plants, phytyl esters and TAG accumulate during stress, centrifugation at 1,000 g for 12 min. The pellet was washed and sus- pended in 4 mL buffer (1 mM EDTA, 200 mM sucrose, 100 mM Tris·HCl at taking up fatty acids from membrane lipids and phytol from pH 7.4). The cells were homogenized with a Precellys homogenizer (Bertin chlorophyll breakdown (13). Similarly, salt/dark stress results in Technologies), using glass beads. The extract was centrifuged at 4,000 × g the increase in phytyl esters, but not TAG, in Synechocystis (Fig. for 2 min. The supernatant was centrifuged at 35,000 × g for 45 min to 2). It is possible that TAG production is increased in Synechocystis obtain the membrane fraction. The pellet was resuspended and protein during other growth conditions. The finding that the gene slr2103, concentration measured with bicinchoninic acid. For enzyme assays (total which is involved in phytyl ester and TAG ester synthesis in Syn- volume, 200 μL), 400 μg membrane protein were incubated with 50 μM echocystis, is related to PES1/PES2 of plants indicates that the acyl donor (16:0-CoA, synthesized according to ref. 35; 16:0 free fatty acid pathway of conversion of lipid and chlorophyll breakdown prod- [Merck KGaA, Darmstadt, Germany]; monogalactosyldiacylglycerol from spinach; Larodan) and 200 μM acyl acceptor (phytol, dioctanoin) in assay ucts into nonpolar lipids with subsequent storage in plastoglo- −1 buffer (20 mM MgCl2, 0.1% CHAPS, 100 mM Tris·HCl at pH 7.4, 1.25 mg·mL bules/lipid droplets is presumably derived from the cyanobacterial BSA, 10 mM Na orthovanadate). Lipid substrates were dissolved in a minimal progenitor of chloroplasts. volume of ethanol and directly added to the assay. After mixing, the assay Plants are the largest source for global TAG production for was incubated for 20 min at 35 °C. The reaction was terminated and lipids human nutrition and biotechnological applications. In the past, extracted by adding 1 mL chloroform/methanol (2:1). numerous strategies were developed to produce TAG in eukaryotic microalgae, including green and red algae. The eukaryotic Lipid Analysis. Cells were harvested from 100 mL Synechocystis culture by microalgae harbor a TAG synthesis pathway similar to plants, and centrifugation and washed with BG-11 medium. The pellet was extracted TAG accumulation is most often dependent on chlorotic stress. three times with chloroform/methanol (1:2) (36). The extracts were com- The identification of the TAG synthesis pathway in cyanobacteria bined and internal standards added. For fatty acid phytyl ester measure- such as Synechocystis provides the means for developing a strategy ments, 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 of employing prokaryotic photosynthetic organisms for oil syn- quantification (Larodan). After addition of 1 mL of 300 mM ammonium thesis. The pathway of TAG synthesis in cyanobacteria is different acetate and 1 mL chloroform, extracts were centrifuged for phase separa- from plants and eukaryotic algae. In addition, cyanobacteria are tion. The organic phase was harvested and dried under a nitrogen stream. prokaryotic cells that can easily be grown and are amenable to The lipids were dissolved in hexane and applied to silica solid phase ex- genetic engineering to increase the capacity for oil production. traction columns (Macherey & Nagel, Düren) equilibrated with hexane (37). After PLANT BIOLOGY washing the column with hexane, phytyl esters and TAGs were eluted with Materials and Methods hexane/diethyl ether (99:1) and hexane/diethyl ether (92:8), respectively. Growth of Synechocystis and Generation of Δslr2103 Deletion Mutant. Syn- Synechocystis lipids were separated by TLC on silica plates (Silica 60 echocystis sp. PCC 6803 (glucose tolerant strain) was grown photo- Durasil, Macherey & Nagel), using hexane/diethyl ether/acetic acid (70:30:1). mixotrophically in liquid or on solidified BG-11 medium (33) supplied with The lipid bands were stained with primuline and observed under UV light. 5 mM glucose at 28 °C in incessant light (30 μmol m−2·s−1). Precultures of 50 mL For lipid isolation, silica material from the plates was extracted with chloroform/methanol (2:1). were grown up to an OD750 of 0.6 and used to inoculate 100-mL cultures in Phytyl esters and TAG were measured by Q-TOF MS. Lipids were dissolved BG-11 medium. Cells were grown to an OD750 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 in chloroform/methanol/300 mM ammonium acetate (300:665:35) (38). The μ · −1 stress). For nitrogen deprivation, cells were harvested and resuspended in samples were infused at 1 L min into the HPLC-Chip Cube MS interface of the Agilent 6530 Series Accurate-Mass Q-TOF mass spectrometer (Agilent, nitrogen-free medium to an OD750 of 0.6 (or in nitrogen-replete medium for control), and the cells were grown for 3 d. Alternatively, 10 μL of a serial Böblingen). Lipids were quantified by neutral loss scanning, and the amounts dilution of cells was spotted on solid BG-11 medium (control, in the light), calculated based on internal standards (13). 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. Measurements of Chlorophyll and Photosynthetic Quantum Yield. Cell pellets of The Δslr2103 deletion mutant of Synechocystis was generated by ho- Synechocystis were extracted with 1 mL cold methanol. After centrifugation, mologous recombination. The 5′ flanking sequence of slr2103 was amplified the supernatant was collected. Methanol extraction was repeated until the from genomic DNA, using oligonucleotides bn2263 and bn2264 (containing pellet turned blue. The chlorophyll contents were calculated after measuring NcoI sites; SI Appendix, Table S1), and cloned into pJET1.2 (pJ-left-slr2103). In the absorbances at 470, 665, and 720 nm, according to the equation: chlo- −1 analogy, the 3′ flanking region was amplified with oligonucleotides bn2265 rophyll a (μg·mL ) = 12.9447 * (A665 – A720) (39). (harboring an MluI site) and bn2266 and ligated into pJET1.2 (pJ-right-slr2103). Chlorophyll fluorescence of Synechocystis cells in liquid BG-11 medium The construct pJ-nptII harbors the kanamycin resistance cassette nptII (ampli- was measured by pulse-amplified modulation fluorometry (Junior PAM, fied with oligonucleotides bn1116 and bn1117, introducing MluIandNcoI Heinz Walz, Effeltrich, Germany). Synechocystis cells were dark adapted for sites) cloned in pJET1.2. The 5′ flanking sequence and the nptII gene were 60 min before the measurements. Quantum yield of PSII was calculated − released using NcoI/HindIII and NcoI (partial digestion)/MluI, respectively. according to the equation: (Fm F)/Fm, where Fm and F are the fluorescence HindIII is a restriction site in the pJET1.2 cloning vector. The two fragments emission of dark-adapted cells under measuring light and after applying a were ligated in one step into the vector pJ-right-slr2103 (opened with MluI, saturating light pulse, respectively (40). HindIII), resulting in the knock-out construct pJ-Δslr2103-kan. The circular knock- out plasmid was transferred into Synechocystis PCC 6803 cells (34). Transformed Transmission Electron Microscopy. For comparative ultrastructural analysis, cells cells were selected by restreaking on BG-11 medium with increasing kanamycin of Synechocystis WT and Δslr2103 mutant were collected by centrifugation − concentrations (30 μg·mL 1 final). The successful integration of the kanamycin and resuspended in 8% agarose. After solidification, the agarose was cut into cassette into the gene slr2103 was confirmed by PCR of genomic DNA. blocks of ∼1mm3 and used for combined conventional and microwave-assisted fixation, dehydration, and resin embedding, as defined in SI Appendix,Table Expression of slr2103 in E. Coli and Substrate Feeding. The gene slr2103 was S2. Sectioning and ultrastructural analysis were performed as described (41). 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 Data Availability Statement. All data discussed in the paper are available in the this construct was transferred into E. coli BL21(AI) cells (Thermo Fisher). E. main text and SI Appendix. coli cells were grown in LB medium at 37 °C to OD600 of 0.6 and then cooled to 16 °C, and 0.5 mM isopropyl-β-D-thiogalactopyranoside and 0.1% (wt/vol) ACKNOWLEDGMENTS. We thank Mathias Brands, Payal Patwari, and Jill L-arabinose were added for induction of expression at 16 °C overnight. Romer (Institute of Molecular Physiology and Biotechnology of Plants; For substrate feeding experiments, the temperature was raised to 30 °C University of Bonn) for their help with enzyme assays, and Claudia Riemey and the cells grown for 3 h in the presence of 3.3 mM phytol (Chemimpex, (IPK Gatersleben) for technical assistance in transmission electron micros- Wood Dale, IL) or 30 μM diacylglycerol (dioctanoin, di8:0, Larodan, Sweden) copy. Funding was provided by University of Bonn.

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