High myristic acid content in the cyanobacterium Cyanothece sp. PCC 8801 results from substrate specificity of lysophosphatidic acid acyltransferase

Accepted Manuscript High myristic acid content in the cyanobacterium Cyanothece sp. PCC 8801 results from substrate specificity of lysophosphatidic acid acyltransferase

Masakazu Saito, Kaichiro Endo, Koichi Kobayashi, Mai Watanabe, Masahiko Ikeuchi, Akio Murakami, Norio Murata, Hajime Wada PII: DOI: Reference:

S1388-1981(18)30104-5 doi:10.1016/j.bbalip.2018.05.011 BBAMCB 58300

To appear in: Received date: Revised date: Accepted date:

7 November 2017 15 May 2018 19 May 2018

Please cite this article as: Masakazu Saito, Kaichiro Endo, Koichi Kobayashi, Mai Watanabe, Masahiko Ikeuchi, Akio Murakami, Norio Murata, Hajime Wada , High myristic acid content in the cyanobacterium Cyanothece sp. PCC 8801 results from substrate specificity of lysophosphatidic acid acyltransferase. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Bbamcb(2018), doi:10.1016/j.bbalip.2018.05.011

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High myristic acid content in the cyanobacterium Cyanothece sp. PCC 8801 results from substrate specificity of lysophosphatidic acid acyltransferase

Masakazu Saitoa, Kaichiro Endoa, Koichi Kobayashia, Mai Watanabea, Masahiko

a

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Ikeuchia, Akio Murakamib, Norio Muratac and Hajime Wadaa, *

Department of Life Sciences, Graduate School of Arts and Sciences, The University of

Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan

National Institute for Basic Biology, Myodaiji, Okazaki 444-8585, Japan

Hajime Wada

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*Corresponding author:

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c

Research Center for Inland Seas, Kobe University, Awaji, Hyogo 656-2401, Japan

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b

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Keywords: Cyanobacterium, Cyanothece sp. PCC 8801, Fatty acid, Glycerolipid,

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Lysophosphatidic acid acyltransferase

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Abbreviations: ACP, acyl-carrier protein; DG, diacylglycerol; DGDG, digalactosyldiacylglycerol; LPA, lysophosphatidic acid; MGDG, monogalactosyldiacylglycerol; MGlcDG, monoglucosyldiacylglycerol; PA, phosphatidic acid; PG, phosphatidylglycerol; SQDG, sulfoquinovosyldiacylglycerol; X:Y(Z), fatty acid containing X carbons with Y double bonds in the cis configuration at the Z position, as counted from the carboxy terminus

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ABSTRACT Analysis of fatty acids from the cyanobacterium Cyanothece sp. PCC 8801 revealed that this species contained high levels of myristic acid (14:0) and linoleic acid in its glycerolipids, with minor contributions from palmitic acid (16:0), stearic acid, and oleic

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acid. The level of 14:0 relative to total fatty acids reached nearly 50%. This 14:0 fatty acid was esterified primarily to the sn-2 position of the glycerol moiety of glycerolipids. This characteristic is unique because, in most of the cyanobacterial strains, the sn-2 position is esterified exclusively with C16 fatty acids, generally 16:0. Transformation of

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Synechocystis sp. PCC 6803 with the PCC8801_1274 gene for lysophosphatidic acid

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acyltransferase (1-acyl-sn-glycerol-3-phosphate acyltransferase) from Cyanothece sp. PCC 8801 increased the level of 14:0 from 2% to 17% in total lipids and the increase in

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the 14:0 content was observed in all lipid classes. These findings suggest that the high content of 14:0 in Cyanothece sp. PCC 8801 might be a result of the high specificity of

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this acyltransferase toward the 14:0-acyl-carrier protein.

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1. Introduction Cyanobacterial

cells

contain

monogalactosyldiacylglycerol

(MGDG),

digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), and phosphatidylglycerol (PG) as the major glycerolipids [1-3]. MGDG contributes about

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half of the total glycerolipids and the other three glycerolipids contribute to the remaining half to different degrees [1-3]. Cyanobacterial cells also contain a minor glycerolipid, monoglucosyldiacylglycerol (MGlcDG), which is a precursor for the biosynthesis of MGDG, at levels below 1% of total glycerolipids [4, 5], although some strains

contain

higher

levels

of

it

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unusual

[6].

Phosphatidylcholine

and

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phosphatidylethanolamine, which are abundant phospholipids in biomembranes such as the plasma membrane and endoplasmic reticulum membrane of plant and animal cells,

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are absent in cyanobacteria [1]. Cyanobacteria contain only PG as the phospholipid [1] and the genes required for biosynthesis of other phospholipids are not present in

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cyanobacterial genomes [2].

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Biosynthesis of glycerolipids in cyanobacteria has been studied extensively [1-3]. The first reaction of the biosynthetic pathway is the acylation of glycerol 3-phosphate at

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the sn-1 position to yield lysophosphatidic acid (LPA). This reaction is very important as the first step of glycerolipid biosynthesis, but the genes and enzymes involved in this reaction in cyanobacteria have not been characterized. The resultant LPA is acylated at the sn-2 position by LPA acyltransferase to yield phosphatidic acid (PA). In Synechocystis sp. PCC 6803 (hereafter, Synechocystis 6803), three genes for LPA acyltransferases (sll1848, sll1752, and slr2060) have been identified, and sll1848

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encodes a major LPA acyltransferase [7, 8]. PA is a common intermediate in the synthesis of all classes of glycerolipids. In the synthesis of MGDG, PA is converted to diacylglycerol (DG) by PA phosphatase [9], to MGlcDG by MGlcDG synthase [10], and finally to MGDG by

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MGlcDG epimerase [11]. A portion of the MGDG is converted to DGDG by the action of DGDG synthase [12, 13]. SQDG is synthesized by SQDG synthase [14], which transfers sulfoquinovose to DG from UDP-sulfoquinovose. In the synthesis of PG, PA is converted to CDP-DG by CDP-DG synthase [15], to PG phosphate by PGP synthase

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[16], and finally to PG by PGP phosphatase [17].

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For these glycerolipids in most strains of cyanobacteria, the C18 and C16 fatty acids are esterified at the sn-1 and sn-2 positions of the glycerol moiety, respectively

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[18-21]. These fatty acids are saturated, mono-unsaturated, di-unsaturated, tri-unsaturated, or tetra-unsaturated. Fatty-acid desaturases introduce double bonds into

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fatty acids at the specific position to produce these unsaturated fatty acids in

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cyanobacteria. Cyanobacterial desaturases belong to the acyl-lipid desaturase family,

[22-24].

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which introduces double bonds into fatty acids that have been esterified to glycerolipids

Previous studies have suggested that cyanobacterial strains can be classified into four groups in the modes of fatty-acid desaturation [18, 25] and the presence of particular fatty-acid desaturases [2]. Cyanobacterial strains in the group 1 are characterized by the presence of only saturated and mono-unsaturated fatty acids, and by the presence of genes for Δ9 desaturase, which introduces a double bond specifically

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at the Δ9 position of fatty acids bound at the sn-1 and sn-2 positions. Strains in other groups, groups 2, 3, and 4, contain poly-unsaturated fatty acids, which have more than two double bonds. In the strains of groups 2, 3, and 4, the C18 and C16 fatty acids are esterified to the sn-1 and sn-2 positions of the glycerol moiety, respectively. Strains in

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group 2 possess the genes for Δ9, Δ12, and ω3 desaturases, which introduce double bonds at the Δ9, Δ12, and Δ15 (ω3) positions, respectively, of C18 fatty acids bound to the sn-1 position, as well as at the Δ9 and Δ12 positions of C16 fatty acids bound to the sn-2 position. Strains in group 3 have the genes for Δ6, Δ9, and Δ12 desaturases, which

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introduce double bonds at the Δ6, Δ9, and Δ12 positions, respectively, of C18 fatty acids

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at the sn-1 position. Strains in group 4 possess the genes for Δ6, Δ9, Δ12, and ω3 desaturases, which introduce double bonds at the Δ6, Δ9, Δ12, and Δ15 (ω3) positions of

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C18 fatty acids, respectively, at the sn-1 position. In the present study, we searched CyanoBase

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(http://genome.microbedb.jp/cyanobase/) to identify cyanobacterial genes for fatty-acid

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desaturases and found that the cyanobacterium Cyanothece sp. PCC 8801 (hereafter abbreviated as Cyanothece 8801) has two genes for fatty-acid desaturases, which

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introduce double bonds at the Δ9 and Δ12 positions of fatty acids. The presence of only these two genes for acyl-lipid desaturase suggests that this strain does not belong to any of the aforementioned four groups of cyanobacteria. Therefore, we analyzed the glycerolipids and fatty acids of Cyanothece 8801. The analysis of fatty acids from Cyanothece 8801 indicated that this strain contained a high level of myristic acid (14:0). In addition, we identified the PCC8801_1274 gene of Cyanothece 8801 as LPA

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acyltransferase, which transfers an acyl group from acyl-acyl-carrier protein (acyl-ACP) to LPA for the synthesis of PA, a precursor of glycerolipids. Experiments in which the PCC8801_1274 gene of Cyanothece 8801 was expressed in Synechocystis 6803 indicated that the LPA acyltransferase of Cyanothece 8801 had high substrate specificity

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toward 14:0-ACP.

2. Materials and methods 2.1. Organisms

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The cyanobacterial strain Cyanothece 8801 was originally isolated from a rice field in

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Taiwan [26-29]. This strain is a unicellular diazotrophic cyanobacterium and the cells lack a sheath and divide in one plane [26, 27]. Based on its morphology and the pattern

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of cell division, it was originally identified as a Synechococcus and named Synechococcus RF-1 [26], and afterwards this strain was treated as a member of

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Cyanothece [30]. The Cyanothece 8801 strain was obtained from Dr. H. B. Pakrasi

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(Washington University).

Cells of this strain were grown photoautotrophically at 23, 30, or 38°C in BG-11

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medium [25] buffered with 4 mM Hepes-NaOH (pH 7.5) under conditions of continuous fluorescent white light at 20 μmol photons m−2 s−1 and aeration with air. The glucose-tolerant strain of Synechocystis 6803 [31] was originally obtained from Dr. Tatsuo Omata (Nagoya University). Cells of this strain were grown under the conditions described above at 30°C. Cells used for all experiments were in the exponential phase of growth.

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2.2. Isolation of genes potentially encoding LPA acyltransferases from Cyanothece sp. PCC 8801 Five Cyanothece 8801 genes, PCC8801_1274, PCC8801_2413, PCC8801_4209,

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PCC8801_0027, and PCC8801_3062, which are annotated as genes for acyltransferases in CyanoBase (http://genome.microbedb.jp/cyanobase/), were amplified using PCR. The primer sets were Nde-1274-F and Hpa-1274-R for PCC8801_1274, Nde-2413-F and EcoV-2413-R for PCC8801_2413, Nde-4209-F and Hpa-4209-R for

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PCC8801_4209, Nde-0027-F and EcoV-0027-R for PCC8801_0027, and Nde-3062-F

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and EcoV-3062-R for PCC8801_3062. The sequences of the primers used for PCR are listed in Supplementary Table S1. Chromosomal DNA from wild-type Cyanothece 8801

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cells was used as the template. Nucleotide sequences of the amplified genes matched sequences found in the database. The amplified PCC8801_1274 and PCC8801_4209

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genes were digested with NdeI and HpaI and inserted at the NdeI and HpaI sites of

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plasmid pTCP2031V (Supplementary Fig. S1), which was designed to incorporate a gene of interest at a neutral site (slr2031), along with the psbA2 (slr1311) promoter and

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a chloramphenicol-resistance gene cassette [32]. The amplified PCC8801_2413, PCC8801_0027, and PCC8801_3062 genes were digested with NdeI and EcoRV, and then inserted at the NdeI and HpaI sites of the plasmid described above.

2.3. Transformation of wild-type cells of Synechocystis sp. PCC 6803 with genes for acyltransferases from Cyanothece sp. PCC 8801

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Plasmids containing genes for acyltransferases from Cyanothece 8801 were used for the transformation of wild-type cells of Synechocystis 6803. Transformation was carried out as described by Golden et al. [33]. Chloramphenicol-resistant transformants were selected and streaked several times on BG-11 agar plates to segregate the

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transformant in which the Cyanothece 8801 gene had been integrated into all copies of the chromosomal DNA at a neutral site (slr2031). The segregation of genes was checked using PCR with the primer set up-f and down-r (Supplementary Fig. S1 and

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2.4. Analysis of lipids and fatty acids

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Table S1).

Lipids were extracted from collected cells using the method of Bligh and Dyer

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[34]. Extracted glycerolipids were separated via thin-layer chromatography with a solvent of chloroform:methanol:15 M ammonia (65:35:5 by vol) and visualized with

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0.01% (w/v) primuline in 80% (v/v) acetone under UV light. Each lipid class was

[35].

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analyzed and quantified using gas chromatography as described by Wada and Murata

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The distribution of fatty acids at the sn-position of the glycerol moiety of glycerolipids was analyzed through selective hydrolysis at the sn-1 position by Rhizopus delemar lipase [36], as described by Sato and Murata [37].

3. Results and discussion 3.1. Genes for fatty acid desaturases in Cyanothece sp. PCC 8801 The genome of Cyanothece 8801 contains four candidate genes for fatty acid 8

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desaturases, namely, PCC8801_0640, PCC8801_2805, PCC8801_3495, and PCC8801_1623 (CyanoBase, http://genome.microbedb.jp/cyanobase/). Using an ortholog search in Cyanobase, we found that PCC8801_0640 is homologous to the desC1 gene for Δ9 acyl-lipid desaturase (or Δ9 fatty acid desaturase), which acts on

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fatty acids esterified to the sn-1 position of the glycerol moiety of glycerolipids [2, 38]. Although its product is annotated as a stearoyl-CoA desaturase in CyanoBase, it is highly homologous to Δ9 acyl-lipid desaturases (DesC1) of Synechocystis 6803 (Score 417, Identity 71.3%, Positive 83.0%) and Synechococcus sp. PCC 7002 (Score 448,

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Identity 75.1%, Positive 86.3%), but not to the stearoyl-CoA desaturase of yeast (score

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135, identity 32.5%, positive 53.1%) or rat liver (score 142, identity 34.2%, positive 50.4%). Moreover, stearate is desaturated to oleate in the lipid-bound form by

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cyanobacteria, but not in the CoA-bound form [39]. PCC8801_2805 is homologous to the desA gene for Δ12 acyl-lipid desaturase (or

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Δ12 fatty acid desaturase; DesA) of Synechocystis 6803 (score 414, identity 56.9%,

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positive 75.5%) and Synechococcus sp. PCC 7002 (score 483, identity 65.0%, positive 77.5%), suggesting that this gene encodes Δ12 acyl-lipid desaturase in Cyanothece 8801.

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PCC8801_1623, which is also designated as a fatty acid desaturase gene in CyanoBase, likely encodes β-carotene hydroxylase based on amino acid sequence homology to β-carotene hydroxylases from other cyanobacterial strains. The product of PCC8801_3495, which is noted as a fatty acid desaturase gene in CyanoBase, is not homologous to any characterized fatty acid desaturases in terms of amino acid sequence and thus may not be a fatty acid desaturase. These results suggest that Cyanothece 8801

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has two genes for fatty acid desaturases, namely, Δ9 acyl-lipid desaturase (DesC1) and Δ12 acyl-lipid desaturase (DesA). The presence of only these two genes for acyl-lipid desaturase is peculiar among cyanobacteria and thus this strain may not belong to any of the four aforementioned groups of cyanobacteria. Therefore, we analyzed the

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glycerolipids and fatty acids of Cyanothece 8801 in relation to the fatty acid compositions of glycerolipids from the four groups of cyanobacteria.

3.2. Glycerolipids in Cyanothece sp. PCC 8801

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We analyzed glycerolipids from Cyanothece 8801 using thin-layer

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chromatography for comparison with those from Synechocystis 6803 (Supplementary Fig. S2). The major lipid components were MGDG, SQDG, DGDG, and PG. In

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addition, MGlcDG was detected as a minor component. We examined the effect of growth temperature on glycerolipid composition and observed only minor changes in

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glycerolipid composition due to changes in growth temperature (Fig. 1A).

3.3. Composition of fatty acids in glycerolipids: detection of myristic acid (14:0) as the

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major fatty acid

Fatty acid analysis of total glycerolipids revealed that myristic acid (14:0) and linoleic acid [18:2(9,12)] were the two major fatty acids, with minor contributions from palmitic acid (16:0), palmitoleic acid [16:1(9)], stearic acid (18:0), and oleic acid [18:1(9)] (Fig. 1B). Although the effect of growth temperature on fatty acid composition was minor, we observed that the content of 16:0 increased and 18:2(9,12) decreased

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upon an increase in growth temperature. The unique characteristic of Cyanothece 8801 with respect to fatty acid composition was its high content of 14:0, which reached almost 50% of total fatty acids in glycerolipids. Cyanothece 8801 grew well at the range of 28oC to 38oC, which is similar to Synechocystis sp. PCC 6803. It seems that

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temperature could not have great impact on the evolution of Cyanothece 8801 to develop high affinity to 14:0.

Several studies have reported that some strains of cyanobacteria contain relatively high levels of 14:0, making up 10%–30% of total fatty acids, for example, Chlorogloea

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(26%, [40]), Aphanizomenon flos-aquae (13%, [41]), Synechococcus sp. IPPAS B-266

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(20%, [42]), Nostoc spongiaeforme (18%, [43]), Phormidium corium (17%, [43]), and Cyanobacterium sp. IPPAS B-1200 (30%, [44]; 24%, [45]). Recently, Pittera et al. [46]

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reported that a marine picocyanobacterium Synechococcus WH7803 contained a high level of 14:0 similar to Cyanothece 8801 and it grew at between 16oC and 30oC. This

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strain preferred lower temperature for growth rather than high temperature as compared

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with Synechocystis sp. PCC 6803 and Cyanothece 8801. A high content of 14:0 might be preferable for growth at lower temperature because shorter-chain fatty acids have

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higher lateral mobility than longer ones. Nevertheless, most cyanobacterial strains contain 16:0 as the major component and the 14:0 content is less than 2% of total fatty acids in most strains [18, 25].

3.4. Fatty acid compositions of individual classes of glycerolipids Fatty acid analysis of individual glycerolipids revealed that MGDG and DGDG

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contained 14:0 and 18:2(9,12) as major fatty acids, with minor contributions from 16:0, 16:1(9), 18:0, and 18:1(9) (Figs. 2A and 2B). Although the effect of growth temperature on the fatty acid composition was relatively minor, the content of 16:0 increased and 18:2(9,12) decreased with increasing growth temperature.

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SQDG contained 14:0, 16:0, and 18:2(9,12) as major components and 16:1(9), 18:0, and 18:1(9) as minor components (Fig. 2C), whereas PG contained 14:0, 16:0, 18:1(9), and 18:2(9,12) as major components and 16:1(9) and 18:0 as minor components (Fig. 2D). In these two glycerolipids, higher growth temperature increased

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the relative content of 16:0 and decreased that of 18:2(9,12). By contrast, the contents of

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14:0 and other fatty acids were hardly affected by growth temperature. The fatty acid composition of MGDG and DGDG is different from that of SQDG

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and PG. This difference might be caused by the functional difference of these glycerolipids. MGDG and DGDG are galactolipids making the bulk of photosynthetic

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membranes and essential for development of thylakoid membrane [47]. MGDG is a

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non-bilayer lipid forming hexagonal II phase, whereas DGDG is a bilayer lipid forming lamellar phase [48, 49]. A dynamic feature of MGDG and DGDG is required for

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formation of thylakoid membrane and rapid adaptation to environmental changes, thus the content of unsaturated fatty acids such as 18:2 might be higher in MGDG and DGDG than that in SQDG and PG. SQDG and PG are anionic lipids having specific and overlapped functions, and are critical as the structural components of photosynthetic complexes in thylakoid membrane [50-54], thus the contents of saturated fatty acids, 14:0 and 16:0, might be higher than those in MGDG and DGDG.

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3.5. Distribution of fatty acids at sn-positions of glycerol moiety of glycerolipids The distribution of fatty acids in the sn-positions of the glycerol moiety in MGDG (Figs. 3A and 3B) indicated that, in MGDG, 18:2(9,12) and 14:0 were exclusively

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esterified at the sn-1 and sn-2 positions, respectively. In DGDG, the distribution of fatty acids in sn-positions was similar to that of MGDG (Figs. 3C and 3D). These results suggest that the main molecular species of these glycerolipids are sn-1-18:2(9,12)-sn-2-14:0 (Supplementary Fig. S3).

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Two fatty acids in SQDG, 16:0 and 18:2(9,12), were esterified at the sn-1 position

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(Fig. 3E), while 14:0 and 16:0 were at the sn-2 position (Fig. 3F). In PG, the distribution of fatty acids in sn-positions was similar to that of SQDG (Figs. 3G and 3H).

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These combinations of fatty acids at the two sn-positions suggest the presence of several molecular species of SQDG and PG, with more complex molecular species

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compositions than those of MGDG and DGDG (Supplementary Fig. S3).

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The high level of 14:0 in membrane lipids would make membrane thinner and transmembrane helices of membrane proteins would be shorter to cope with thinner

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membrane. However, the results obtained by the analysis of positional distribution of fatty acids indicated that 14:0 was exclusively esterified at the sn-2 position, whereas the sn-1 position was occupied with C16 and C18 fatty acids, thereby the content of 14:0/14:0 molecular species, expected to be deleterious, was kept in a low level. Pirerra et al. [46] have recently performed analysis of glycerolipids in a marine picocyanobacterium Synechococcus WH7803 containing a high level of 14:0. They

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observed that the sn-2 position of MGDG and DGDG was exclusively occupied with 14:0 and almost no 14:0 in the sn-1 position, and there was no 14:0 in PG and a moderate level of 14:0 at the sn-2 position of SQDG. The exclusive regioselective position of 14:0 at the sn-2 position of membrane lipids observed in Cyanothece PCC

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8801 and Synechococcus WH7803 would allow them to accommodate a high level of 14:0 in membrane glycerolipids.

3.6. Three predicted pathways of glycerolipid synthesis in Cyanothece sp. PCC 8801

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Three pathways of glycerolipid synthesis in Cyanothece 8801 (Fig. 4) could be

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predicted based on the molecular species of glycerolipids in Cyanothece 8801 (Supplementary Fig. S3) and the general scheme of glycerolipid synthesis and fatty acid

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desaturation in cyanobacteria [2, 39]. In the primary pathway (thick arrows in Fig. 4), 18:0 is esterified to the sn-1 position of glycerol 3-phosphate. To the resultant

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sn-1-18:0-glycerol-3-phosphate, 14:0 is esterified to the sn-2 position through the action

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of LPA acyltransferase [7, 8], resulting in the synthesis of sn-1-18:0-sn-2-14:0-PA. This species of PA is converted to MGDG, DGDG, SQDG, and PG. Then, 18:0 at the sn-1

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position is desaturated sequentially to 18:1(9) and 18:2(9,12) by DesC1 and DesA, respectively. The resultant sn-1-18:2-sn-2-14:0 species is the most abundant molecular species of glycerolipids. In the second pathway (thin arrows in Fig. 4), 18:0 is esterified to the sn-1 position of glycerol 3-phosphate as in the first pathway. To the resultant sn-1-18:0-glycerol-3-phosphate, 16:0 is esterified to the sn-2 position through the action

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of LPA acyltransferase, resulting in the synthesis of sn-1-18:0-sn-2-16:0-PA. This species of PA is converted to SQDG and PG, but not to MGDG or DGDG. Then, 18:0 at the sn-1 position is desaturated to 18:1(9) and 18:2(9,12) by DesC1 and DesA, resulting in the synthesis of sn-1-18:1(9)-sn-2-16:0 and sn-1-18:2(9,12)-sn-2-16:0 species of

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SQDG and PG. In the third pathway (also represented by thin arrows in Fig. 4), either 14:0 or 16:0 is esterified to the sn-1 position of glycerol 3-phosphate. Then, either 14:0 or 16:0 is esterified to the sn-2 position. The resultant PA is a mixture of

sn-1-14:0-sn-2-14:0, sn-1-14:0-sn-2-16:0, sn-1-16:0-sn-2-14:0, and sn-1-16:0-sn-2-16:0

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species. These species of PA are converted to SQDG and PG, but not to MGDG or

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DGDG. The fatty acids (14:0 and 16:0) esterified to these molecular species of SQDG and PG are not desaturated. A desaturation of 16:0 occurred, but this desaturation was

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relatively small, thus we omitted this desaturation in the scheme. The desaturation of 16:0 to 16:1(9) should be catalyzed by 9 desaturase that desaturates 18:0 to 18:1(9)

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because Cyanothece 8801 has only two desaturases, 9 and 12 desaturases.

3.7. High specificity of LPA acyltransferase toward 14:0-ACP

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As noted above, the high content of 14:0 at the sn-2 position of MGDG and DGDG is a unique characteristic of Cyanothece 8801. Because esterification of 14:0 to the sn-2 position is a result of the action of LPA acyltransferase, we hypothesized that the LPA acyltransferase of Cyanothece 8801 would have high specificity for 14:0-ACP, whereas the corresponding enzyme in most cyanobacteria has high specificity for 16:0-ACP [the reaction indicated by (2) in Fig. 4]. To examine this hypothesis, we

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isolated five genes from Cyanothece 8801 that might encode LPA acyltransferases: PCC8801_1274, PCC8801_2413, PCC8801_0027, PCC8801_4209, and PCC8801_3062. According to the annotation in CyanoBase, PCC8801_1274 is the gene that encodes 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPA acyltransferase),

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whereas the other four genes are assigned to phospholipid/glycerol acyltransferases. Homology searches indicated that PCC8801_1274 and PCC8801_2413 were similar to the sll1848 and sll1752 genes that encode the major and minor components of LPA acyltransferase in Synechocystis 6803 [7, 8]. We transformed these five genes from

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Cyanothece 8801 into Synechocystis 6803 cells, which contained 14:0 at a very low

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level (2% of total fatty acids).

The fatty acid compositions of total glycerolipids in wild-type and transformed

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cells of Synechocystis 6803 (Table 1) revealed that wild-type cells of Synechocystis 6803 did contain 16:0 at a high level (56%) and 14:0 at a very low level (2%), as

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demonstrated previously [21]. Transformation of Synechocystis 6803 with the

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PCC8801_1274 gene increased the level of 14:0 from 2% to 16% of total fatty acids and decreased 16:0 from 56% to 43%. In contrast, transformation with PCC8801_2413

16:0.

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and other candidate genes from Cyanothece 8801 did not affect the contents of 14:0 and

To see whether the higher amount of 14:0 in the transformed cells causes any changes in lipid biosynthesis we analyzed lipid composition, fatty acid composition of lipid classes and positional distribution of fatty acids in MGDG and SQDG of Synechocystis sp. PCC 6803 cells transformed with the empty vector (control) and the

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PCC8801_1274 gene. As shown in Fig. 5A, lipid composition was not changed by transformation with the PCC8801_1274 gene. The lipid composition of the transformed cells with the PCC8801_1274 gene was similar to that of the control cells transformed with empty vector, and also to that of the wild-type cells previously reported [21]. The

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content of 14:0 in the transformed cells with the PCC8801_1274 gene increased in parallel with a decrease in 16:0 content in all lipid classes as compared with the transformed cells with the empty vector (Figs. 5B to 5E). The relative distribution in the C18 fatty acids was also significantly changed with a decrease in the 18:2 content in

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MGDG and DGDG in the transformant with the PCC8801_1274 gene (Figs. 5B and

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5C). The fatty acid composition of lipid classes of the transformed cells with the empty vector was similar to that of the wild-type cells that was previously reported [21].

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We also analyzed the fatty acid distribution to the sn-1 and sn-2 positions of MGDG and SQDG in Synechocystis 6803 cells that had been transformed with the

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empty vector and the PCC8801_1274 gene. Fig. 6 shows that the change from 16:0 to

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14:0 occurred mainly at the sn-2 position of MGDG and SQDG, where only these two fatty acids are esterified. At the sn-1 position of MGDG, transformation with the

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PCC8801_1274 gene increased the levels of 14:0, 18:1(9), and 18:3(6,9,12), but decreased 16:0, 16:1(9), and 18:2(9,12). At the sn-1 position of SQDG, transformation with the PCC8801_1274 gene slightly increased the levels of 14:0, 16:0 and 18:1(9), but decreased 16:1(9). However, these changes in fatty acid levels were not as significant as those observed at the sn-2 position. Together, these results suggest that the LPA acyltransferase encoded by the PCC8801_1274 gene of Cyanothece PCC 8801 has

17

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high specificity toward 14:0-ACP and it is likely that this unique specificity results in the high 14:0 content of Cyanothece 8801. To find out the amino-acid residues and/or domains involved in the specificity toward 14:0-ACP we did basic sequence analysis and comparison of amino-acid

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sequence of LPA acyltransferase of Cyanothece 8801 to those of LPA acyltransferases of other organisms that are specific to 16:0-ACP. As shown in supplemental Fig. S4, sequence similarity was found among the LPA acyltransferases, but we were not able to find out the amino-acid residues, which are specific to the LPA acyltransferase of

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Cyanothece 8801 and might be involved in the specificity toward 14:0-ACP. To

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identify the amino-acid residues and/or domains involved in the specificity toward 14:0-ACP we need further analyses, for example, domain swapping analysis between

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14:0-specific LPA acyltransferases and 16:0-specific ones combined with site-directed mutagenesis.

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We searched Cyanothece 8801 LPA acyltransferase homologs in other

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cyanobacterial genomes and found homologs not only in 14:0-rich strains but also in other strains, which do not have a high content of 14:0. However, it was difficult to

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distinguish between 14:0-specific LPA acyltransferases and 16:0-specic ones based on the amino-acid sequences. A high content of 14:0 in membrane lipids in Cyanothece 8801 could be caused by the following two possibilities. One would be the existence of high level of 14:0-ACP pool in Cyanothece 8801 cells. To get a high level of 14:0-ACP pool fatty acid synthesis should be terminated at the level of 14:0. The other possibility is that the LPA

18

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acyltransferase (PCC8801_1274) of Cyanothece 8801 has a high affinity to 14:0-ACP and a high kcat that accelerates turnover of 14:0 and drives incorporation of 14:0 into glycerolipids. In the cells of Synechocystis 6803 transformed with the PCC8801_1274 gene for LPA acyltransferase of Cyanothece 8801, the level of 14:0 in total lipids was

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15% and much lower than that in Cyanothece 8801. This lower incorporation of 14:0 into glycerolipids might be caused by a lower level of 14:0-ACP pool in Synechocystis 6803 and competition of the LPA acyltransferase of Cyanothece 8801 with those of Synechocystis 6803. In Synechocystis 6803, three LPA acyltransferases (Sll1848,

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Sll1752 and Slr2060) are present and Sll1848 specific to 16:0-ACP is the major one [7,

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8]. Sll1848 and the LPA acyltransferase of Cyanothece 8801 use acyl-ACP and LPA as the substrates. Although the preference to 16:0-ACP and 14:0-ACP is different between

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the two LPA acyltransferases and the LPA acyltransferase of Cyanothece 8801 has higher specificity toward the 14:0-ACP than Sll1848, they use LPA as the common

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substrate. Thus, Sll1848 competes with the LPA acyltransferase of Cyanothece 8801

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and prevents the incorporation of 14:0 into glycerolipids in the transformed cells of Synechocystis 6803. It is also possible that Sll1848 would shift the equilibrium toward

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elongation of 14:0-ACP to 16:0-ACP by rapid utilization of 16:0-ACP. In these cases, a disruption of sll1848 in the transformed cells of Synechocystis 6803 would increase the content of 14:0.

4. Conclusions In this study, we found that Cyanothece 8801 has two genes for fatty acid

19

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desaturases, namely, Δ9 acyl-lipid desaturase (DesC1) and Δ12 acyl-lipid desaturase (DesA). This strain does not belong to any of the four groups of cyanobacteria that were previously described with respect to the presence or absence of genes for fatty-acid desaturases [2, 18]. The results of lipid and fatty acid analyses showed that the strain

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was also unique in terms of its fatty acid composition and positional distribution of fatty acids, particularly the high content of 14:0 in glycerolipids (almost 50% of the total fatty acids). The 14:0 fatty acid was esterified primarily at the sn-2 position of glycerolipids.

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In most cyanobacterial strains, the chain length of fatty acids bound to the sn-1

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position of glycerolipids is restricted to C16 and C18, whereas the sn-2 position is exclusively esterified with C16 fatty acids [1-3]. However, the reason why the chain

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length of fatty acids bound to the sn-2 position is restricted to C16 and whether the chain length has special importance for physiological activities, such as growth and

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photosynthesis, have not been clarified due to a lack of appropriate cyanobacterial

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strains. Cyanothece 8801 and Synechococcus WH7803 [46] are exceptional strains in which the sn-2 position of MGDG and DGDG is exclusively esterified by 14:0 fatty

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acids, and thus might be useful to address these issues. Comparison of the physiological activities of Cyanothece 8801 and Synechococcus WH7803 to other cyanobacterial strains, such as Synechocystis 6803 that have C16 fatty acids at the sn-2 position, might provide a better understanding of the importance of fatty acid chain length at the sn-2 position. We identified the PCC8801_1274 gene of Cyanothece 8801 as an LPA

20

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acyltransferase that has specificity toward 14:0-ACP. This gene might be a useful tool to manipulate fatty acids bound to the sn-2 position of glycerolipids. The introduction of this gene into other cyanobacterial strains will increase the content of 14:0 at the sn-2 position of glycerolipids, as we have shown in Synechocystis 6803 in this study. Such

length of fatty acids bound to the sn-2 position.

Acknowledgements

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gene-manipulated strains would also allow us to study the importance of the chain

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This research was supported by CREST (to HW and AM), Japan Science and

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Technology Agency.

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Legends for figures Fig. 1. Composition of glycerolipids and their fatty acids from Cyanothece sp. PCC 8801 cells grown at 23°C, 30°C, and 38°C. A, Glycerolipid composition. B, Fatty acid composition of total glycerolipids. Growth temperatures: hatched bar, 23°C; gray bar,

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30°C, white bar, 38°C. Three independent experiments were performed, and average values are presented with standard deviations. Abbreviations: 16:1, 16:1(9); 18:1, 18:1(9); 18:2, 18:2(9,12).

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Fig. 2. Fatty acid composition of individual glycerolipids from Cyanothece sp. PCC

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8801 cells grown at 23°C, 30°C, and 38°C. A, MGDG; B, DGDG; C, SQDG; D, PG. Growth temperatures: hatched bar, 23°C; gray bar, 30°C, white bar, 38°C. Three

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independent experiments were performed, and average values are presented with

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standard deviations. Abbreviations of fatty acids are the same as in Fig. 1.

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Fig. 3. Distribution of fatty acids at the sn-positions of individual glycerolipids from Cyanothece sp. PCC 8801 cells grown at 30°C. A, MGDG sn-1; B, MGDG sn-2; C,

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DGDG sn-1; D, DGDG sn-2; E, SQDG sn-1; F, SQDG sn-2; G, PG sn-1; H, PG sn-2. Three independent experiments were performed, and average values are presented with standard deviations. Abbreviations of fatty acids are the same as in Fig. 1.

Fig. 4. A schematic of the pathways predicted for the synthesis of glycerolipids in Cyanothece sp. PCC 8801. Thick and thin arrows indicate major and minor pathways,

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respectively. (1) Acylation of G3P at the sn-1 position to yield LPA by uncharacterized enzymes. (2) Acylation of LPA at the sn-2 position by LPA acyltransferase to yield PA. (3) MGDG is synthesized in three steps: the release of phosphate from PA by PA phosphatase to yield diacylglycerol (DG), the transfer of glucose to DG by MGlcDG

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synthase, and the conversion of glucose to galactose by MGlcDG epimerase. DGDG is synthesized through galactosylation of MGDG by DGDG synthase. SQDG is synthesized through release of phosphate from PA by PA phosphatase to yield DG and sulfoquinovosylation of DG by SQDG synthase. PG is synthesized in three steps: the

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conversion of PA to CDP-DG by CDP-DG synthase, the synthesis of PG phosphate

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from CDP-DG and G3P by PG phosphate synthase, and the release of phosphate from PG phosphate by PG phosphate phosphatase. (4) Desaturation at the Δ9 position by Δ9

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acyl-lipid desaturase (DesC1). (5) Desaturation at the Δ12 position by Δ12 acyl-lipid desaturase (DesA). Abbreviations: G3P, glycerol 3-phosphate; LPA, lysophosphatidic

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acid; PA, phosphatidic acid; ACP, acyl-carrier protein; Gal, galactosyl residue; Pg,

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phosphoglyceryl residue; Sq, sulfoquinovosyl residue.

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Fig. 5. Composition of glycerolipids and their fatty acids from the transformed cells of Synechocystis sp. PCC 6803 with the empty vector (control) and the PCC8801_1274 gene for lysophosphatidic acid (LPA) acyltransferase from Cyanothece sp. PCC 8801. A, Glycerolipid composition; B, fatty acid composition of MGDG; C, fatty acid composition of DGDG; D, fatty acid composition of SQDG; E, fatty acid composition of PG. White bar, control cells transformed with the empty vector; gray bar,

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PCC8801_1274-transformed cells. Three independent experiments were performed, and average values are presented with standard deviations. Abbreviations: 16:1, 16:1(9); 18:1a, 18:1(9); 18:1b, 18:1(11); 18:2, 18:2(9,12); γ-18:3, 18:3(6,9,12); α-18:3,

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18:3(9,12,15); 18:4, 18:4(6,9,12,15).

Fig. 6. Changes in fatty acid composition at the sn-1 and sn-2 positions of MGDG and SQDG from Synechocystis sp. PCC 6803 upon transformation with the PCC8801_1274 gene for lysophosphatidic acid (LPA) acyltransferase from Cyanothece sp. PCC 8801. A,

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sn-1 position of MGDG; B, sn-2 position of MGDG; C, sn-1 position of SQDG; D, sn-2

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position of SQDG. White bar, control cells transformed with the empty vector; gray bar, PCC8801_1274-transformed cells. Three independent experiments were performed, and

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average values are presented with standard deviations. Abbreviations of fatty acids are

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the same as in Fig. 5.

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Table 1. Changes in composition of fatty acids in glycerolipids from Synechocystis sp. PCC 6803 upon transformation with genes that were candidates for lysophosphatidic acid (LPA) acyltransferase from Cyanothece sp. PCC 8801. 16:

16:1(9

18:

18:1(9

18:1(11 18:2(9,12 γ-18:

α-18:

18:

0

0

)

0

)

)

)

3

3

4

55.

4.3 ±

2.6

8.6 ±

0.3 ±

13.3 ±

12.5

0.3 ±

0.3

2.2

7±

0.1

±

5.4

0.2

0.5

± 1.3

0.0

±

±

4.2

Wild type

1.3

0.3

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14:

T-127

15.

43.

3.5 ±

3.2

7.9 ±

0.3 ±

4

7±

1±

0.8

±

0.2

0.2

1.5

0.8 4.5 ±

1.9

5.7 ±

2.3

3±

0.3

±

0.1

±

0.3

0.5

0.2 7

61.

5.2 ±

1.7

1.9

3±

0.1

±

±

1.3

0.1

0.5

0.9

± 0.5

0.1

± 0.4

13.7

0.8 ±

0.6

± 0.3

0.1

± 0.4

8.4 ± 0.5

13.8

1.1 ±

0.7

± 0.5

0.2

±

PT

0.1

2.4

6.7 ±

0.5 ±

2.2

7±

0.4

±

0.6

0.2

±

1.4

9.6 ± 0.6

14.5

1.1 ±

0.6

± 1.0

0.0

±

0.3

0.2

58.

5.2 ±

1.1

6.0 ±

0.2 ±

2.4

9±

0.3

±

0.8

0.2

±

2.1

AC

9

0.4

3.7 ±

0.7 T-420

0.4 ±

58.

CE

2

5.7 ±

8.8 ±

0.9 ±

0.6

0.1 T-306

0.0

0.4

14.8

ED

T-002

1.4 ±

NU

3

60.

MA

T-241

1.8

9.7 ±

0.1

9.4 ± 0.7

15.5

0.8 ±

0.5

± 0.8

0.2

±

0.0

0.1

0.5 Abbreviations: γ-18:3, 18:3(6,9,12); α-18:3, 18:3(9,12,15); 18:4, 18:4(6,9,12,15); T-1274, transformant with PCC8801_1274; T-2413, transformant with PCC8801_2413; T-0027, transformant with PCC8801_0027; T-3062, transformant with PCC8801_3062; T-4209, transformant with PCC8801_4209.

33

ACCEPTED MANUSCRIPT

Highlights Cyanothece sp. PCC 8801 contains a very high level of myristic acid (14:0).



14:0 is exclusively present at the sn-2 position of glycerolipids.



A 14:0 specific LPA acyltransferase is identified in Cyanothece sp. PCC 8801.



Expression of the LPA acyltransferase in Synechocystis increases 14:0 content.

AC

CE

PT

ED

MA

NU

SC RI PT



34

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6