Characterisation of acyltransferases from Synechocystis sp. PCC6803

As phylogenetic ancestors of plant chloroplasts cyanobacteria resemble plastids with respect to lipid and fatty acid composition. These membrane lipids show the typical prokaryotic fatty acid pattern in which the sn-2 position is exclusively esterified by C(16) acyl groups. In the course of de novo glycerolipid biosynthesis this prokaryotic fatty acid pattern is established by the sequential acylation of glycerol-3-phosphate with acyl-ACPs by the activity of different acyltransferases. In silico approaches allowed the identification of putative Synechocystis acyltransferases involved in glycerolipid metabolism. Functional expression studies in Escherichia coli showed that sll1848 codes for a lysophosphatidic acid acyltransferase with a high specificity for 16:0-ACP, whereas slr2060 encodes a lysophospholipid acyltransferase, with a broad acyl-ACP specificity but a strong preference for lysophosphatidyglycerol especially its sn-2 acyl isomer as acyl-acceptor. The generation and analysis of the corresponding Synechocystis knockout mutants revealed that lysophosphatidic acid acyltransferase unlike the lysophospholipid acyltransferase is essential for the vital functions of the cells.     

集胞藻PCC6803 EGY同源基因破坏突变体的构建及表型分析

摘要：拟南芥中近来发现的定位于叶绿体的膜嵌合金属蛋白酶EGY1影响叶绿体发育与脂肪酸合成,经生物信息学分析,集胞藻PCC6803 (Synechocystis sp. PCC6803)中slr0643、sll0862基因编码同源蛋白。【目的】为了鉴定这两个基因的功能,【方法】本文通过同源重组插入卡那霉素抗性基因、切断目的基因,分别构建了slr0643::km和sll0862::km两种突变体,检测突变体的生理生化表型。【结果】在30℃,20 μE/m2s自养培养下,slr0643::km与野生型相比,早期     

The functional divergence of two glgP homologues in Synechocystis sp. PCC 6803

Glycogen phosphorylase (GlgP, EC 2.4.1.1) catalyzes the cleavage of glycogen into glucose-1-phosphate (Glc-1-P), the first step in glycogen catabolism. Two glgP homologues are found in the genome of Synechocystis sp. PCC 6803, a unicellular cyanobacterium: sll1356 and slr1367. We report on the different functions of these glgP homologues. sll1356, rather than slr1367, is essential for growth at high temperatures. On the other hand, when CO2-fixation and the supply of glucose are both limited, slr1367 is the key factor in glycogen metabolism. In cells growing autotrophically, sll1356 plays a more important role in glycogen digestion than slr1367. This functional divergence is also supported by a phylogenetic analysis of glgP homologues in cyanobacteria.     

Substrate specificity of lysophosphatidic acid acyltransferase beta -- evidence from membrane and whole cell assays

Membranes of mammalian cells contain lysophosphatidic acid acyltransferase (LPAAT) activities that catalyze the acylation of sn-1-acyl lysophosphatidic acid (lysoPA) to form phosphatidic acid. As the biological roles and biochemical properties of the six known LPAAT isoforms have yet to be fully elucidated, we have characterized human LPAAT-beta activity using two different assays. In a membrane-based assay, LPAAT-beta used lysoPA and lysophosphatidylmethanol (lysoPM) but not other lysophosphoglycerides as an acyl acceptor, and it preferentially transferred 18:1, 18:0, and 16:0 acyl groups over 12:0, 14:0, 20:0, and 20:4 acyl groups. The fact that lysoPM could traverse cell membranes permitted additional characterization of LPAAT-beta activity in cells: PC-3 and DU145 cells converted exogenously added lysoPM and (14)C-labeled 18:1 into (14)C-labeled phosphatidylmethanol (PM). The rate of PM formation was higher in cells that overexpressed LPAAT-beta and was inhibited by the LPAAT-beta inhibitor CT-32501. In contrast, if lysoPM and (14)C-labeled 20:4 were added to PC-3 or DU145 cells, (14)C-labeled PM was also formed, but the rate was neither higher in cells that overexpressed LPAAT-beta nor inhibited by CT-32501. We propose that LPAAT-beta catalyzes the intracellular transfer of 18:1, 18:0, and 16:0 acyl groups but not 20:4 groups to lysoPA.     

Cloning and characterization of unusual fatty acid desaturases from Anemone leveillei: identification of an acyl-coenzyme A C20 Delta5-desaturase responsible for the synthesis of sciadonic acid

The seed oil of Anemone leveillei contains significant amounts of sciadonic acid (20:3Delta(5,11,14); SA), an unusual non-methylene-interrupted fatty acid with pharmaceutical potential similar to arachidonic acid. Two candidate cDNAs (AL10 and AL21) for the C(20) Delta(5cis)-desaturase from developing seeds of A. leveillei were functionally characterized in transgenic Arabidopsis (Arabidopsis thaliana) plants. The open reading frames of both Delta(5)-desaturases showed some similarity to presumptive acyl-coenzyme A (CoA) desaturases found in animals and plants. When expressed in transgenic Arabidopsis, AL21 showed a broad range of substrate specificity, utilizing both saturated (16:0 and 18:0) and unsaturated (18:2, n-6 and 18:3, n-3) substrates. In contrast, AL10 did not show any activity in wild-type Arabidopsis. Coexpression of AL10 or AL21 with a C(18) Delta(9)-elongase in transgenic Arabidopsis plants resulted in the production of SA and juniperonic fatty acid (20:4Delta(5,11,14,17)). Thus, AL10 acted only on C(20) polyunsaturated fatty acids in a manner analogous to "front-end" desaturases. However, neither AL10 nor AL21 contain the cytochrome b(5) domain normally present in this class of enzymes. Acyl-CoA profiling of transgenic Arabidopsis plants and developing A. leveillei seeds revealed significant accumulation of Delta(5)-unsaturated fatty acids as acyl-CoAs compared to the accumulation of these fatty acids in total lipids. Positional analysis of triacylglycerols of A. leveillei seeds showed that Delta(5)-desaturated fatty acids were present in both sn-2 and sn-1 + sn-3 positions, although the majority of 16:1Delta(5), 18:1Delta(5), and SA was present at the sn-2 position. Our data provide biochemical evidence for the A. leveillei Delta(5)-desaturases using acyl-CoA substrates.     

Acyl specificity in triglyceride synthesis by lactating rat mammary gland.

We have studied the specificity of the acyl-CoA:diglyceride acyltransferase reaction in lactating rat mammary gland to provide a rational explanation at the enzyme level for the nonrandom distribution of fatty acids in milk fat triglycerides. Acyl-CoA:diglyceride acyltransferase activity was measured using various diglyceride and radioactive acyl-CoA substrates; products were identified as triglycerides by thin-layer and gas-liquid chromatography. Most of the enzymatic activity was located in the microsomal fraction and showed a broad specificity for the acyl donors tested C10, C12, C14, C16, C18, and C18:1 CoA esters). The acyltransferase activity was highly specific for sn-1,2-diglyceride enantiomers; rac-1,3- and sn-2,3-diglycerides were relatively inactive. The acyl-CoA specificity was not affected by the type of 1,2-diglyceride acceptor offered, although dilaurin was the best acceptor and sn-1,2-dilaurin greater than sn-1,2-dimyristin greater than sn-1,2-dipalmitin greater than sn-1,2-distearin. We have previously shown that in the microsomal fraction from lactating rat mammary gland, the acyltransferase activities concerned with the conversion of sn-glycero-3-phosphate to diacylglycerophosphate show a very marked specificity for long chain acyl-CoA's. Therefore, we conclude that the predominant localization of long chain fatty acids in the 1 and 2 positions, and of shorter chain fatty acids in the 3 position of the glycerol backbone, results at least in part from the specificities of the mammary gland acyltransferases.     

A novel Delta9 acyl-lipid desaturase, DesC2, from cyanobacteria acts on fatty acids esterified to the sn-2 position of glycerolipids

Acyl-lipid desaturases are enzymes that convert a C-C single bond into a C=C double bond in fatty acids that are esterified to membrane-bound glycerolipids. Four types of acyl-lipid desaturase, namely DesA, DesB, DesC, and DesD, acting at the Delta12, Delta15, Delta9, and Delta6 positions of fatty acids respectively, have been characterized in cyanobacteria. These enzymes are specific for fatty acids bound to the sn-1 position of glycerolipids. In the present study, we have cloned two putative genes for a Delta9 desaturase, designated desC1 and desC2, from Nostoc species. The desC1 gene is highly similar to the desC gene that encodes a Delta9 desaturase that acts on C18 fatty acids at the sn-1 position. Homologues of desC2 are found in genomes of cyanobacterial species in which Delta9-desaturated fatty acids are esterified to the sn-2 position. Heterologous expression of the desC2 gene in Synechocystis sp. PCC 6803, in which a saturated fatty acid is found at the sn-2 position, revealed that DesC2 could desaturate this fatty acid at the sn-2 position. These results suggest that the desC2 gene is a novel gene for a Delta9 acyl-lipid desaturase that acts on fatty acids esterified to the sn-2 position of glycerolipids.     

Hepatic sn-glycerol-3-phosphate acyltransferases: effect of monoacylglycerol analogs on mitochondrial and microsomal activities

The regulation of cellular diacylglycerol levels may have important consequences for protein kinase C activity. Because monoacylglycerols were said to inhibit the committed step of glycerolipid synthesis, the sn-glycerol-3-P acyltransferase (glycerol-P acyltransferase), we determined (1) whether both the mitochondrial and the microsomal glycerol-P acyltransferase isoenzymes were inhibited by 1- and 2-mono-18:1-glycerols, and their ether and amide analogs and (2) what the mechanism of inhibition was. 1- and 2-mono-18:1-glycerols, their ether and amide analogs, and 1-mono-18:1-glycerol 3-phosphate were all competitive inhibitors of the microsomal glycerol-P acyltransferase activity. The relative Ki values suggested that inhibition was strongest with the radyl group at the sn-1 position and that an oxygen bond is important at the sn-1 position. Although the monoacyl- and monoalkylglycerols were also competitive inhibitors of the mitochondrial glycerol-P acyltransferase, neither of the amide analogs was an inhibitor, suggesting that an oxygen bond is essential at both the sn-1 and sn-2 positions. Because monoradylglycerols inhibit several enzyme activities that contribute to the biosynthesis or the metabolism of diacylglycerol, these inhibitors may function within cells in part to regulate cellular diacylglycerol levels.     

Incorporation and remodeling of extracellular phosphatidylcholine with short acyl residues in Saccharomyces cerevisiae

The pem1/cho2 pem2/opi3 double mutant of Saccharomyces cerevisiae, which is auxotrophic for choline because of the deficiency in methylation activities of phosphatidylethanolamine, grew in the presence of 0.1 mM dioctanoyl-phosphatidylcholine (diC(8)PC). Analysis of the metabolism of methyl-(13)C-labeled diC(8)PC ((methyl-(13)C)(3)-diC(8)PC) by electrospray ionization tandem mass spectrometry (ESI-MS/MS) revealed that it was rapidly converted to (methyl-(13)C)(3)-PCs containing C16 or C18 acyl chains. (Methyl-(13)C)(3)-8:0-lyso-PC, (methyl-(13)C)(3)-8:0-16:0-PC and (methyl-(13)C)(3)-8:0-16:1-PC, which are the probable intermediate molecular species of acyl chain remodeling, appeared immediately after 5 min of pulse-labeling and decreased during the subsequent chase period. These results indicate that diC(8)PC was taken up by the pem1 pem2 double mutant and that the acyl chains of diC(8)PC were exchanged with longer yeast fatty acids. The temporary appearance of (methyl-(13)C)(3)-8:0-lyso-PC suggests that the remodeling reaction may consist of deacylation and reacylation by phospholipase activities and acyltransferase activities, respectively. The detailed analyses of the structures of (methyl-(13)C)(3)-8:0-16:0-PC and (methyl-(13)C)(3)-8:0-16:1-PC by MS/MS and MS(3) strongly suggest that most (methyl-(13)C)(3)-8:0-16:0-PCs have a C16:0 acyl chain at sn-1 position, whereas (methyl-(13)C)(3)-8:0-16:1-PCs have a C16:1 acyl chain at either sn-1 or sn-2 position in a similar frequency, implying that the initial C16:0 acyl chain substitution prefers the sn-1 position; however, the C16:1 acyl chain substitution starts at both sn-1 and sn-2 positions. The current study provides a pivotal insight into the acyl chain remodeling of phospholipids in yeast.     

Multidisciplinary Evidences that Synechocystis PCC6803 Exopolysaccharides Operate in Cell Sedimentation and Protection against Salt and Metal Stresses

Little is known about the production of exopolysaccharides (EPS) in cyanobacteria, and there are no genetic and physiological evidences that EPS are involved in cell protection against the frequently encountered environmental stresses caused by salt and metals. We studied four presumptive EPS production genes, sll0923, sll1581, slr1875 and sll5052, in the model cyanobacterium Synechocystis PCC6803, which produces copious amounts of EPS attached to cells (CPS) and released in the culture medium (RPS) as shown here. We show that sll0923, sll1581, slr1875 and sll5052 are all dispensable to the growth of all corresponding single and double deletion mutants in absence of stress. Furthermore, we report that sll0923, sll1581 and slr1875 unambiguously operate in the production of both CPS and RPS. Both sll1581 and slr1875 are more important than sll0923 for CPS production, whereas the contrary is true for RPS production. We show that the most EPS-depleted mutant, doubly deleted for sll1581 and slr1875, lacks the EPS mantle that surrounds WT cells and sorbs iron in their vicinity. Using this mutant, we demonstrate for the first time that cyanobacterial EPS directly operate in cell protection against NaCl, CoCl₂, CdSO₄ and Fe-starvation. We believe that our EPS-depleted mutants will be useful tools to investigate the role of EPS in cell-to-cell aggregation, biofilm formation, biomineralization and tolerance to environmental stresses. We also suggest using the fast sedimenting mutants as biotechnological cell factories to facilitate the otherwise expensive harvest of the producer cell biomass and/or its separation from products excreted in the growth media.     

Novel lipids in Myxococcus xanthus and their role in chemotaxis

Organisms that colonize solid surfaces, like Myxococcus xanthus, use novel signalling systems to organize multicellular behaviour. Phosphatidylethanolamine (PE) containing the fatty acid 16:1omega5 (Delta11) elicits a chemotactic response. The phenomenon was examined by observing the effects of PE species with varying fatty acid pairings. Wild-type M. xanthus contains 17 different PE species under vegetative conditions and 19 at the midpoint of development; 13 of the 17 have an unsaturated fatty acid at the sn-1 position, a novelty among Proteobacteria. Myxococcus xanthus has two glycerol-3-phosphate acyltransferase (PlsB) homologues which add the sn-1 fatty acid. Each produces PE with 16:1 at the sn-1 position and supports growth and fruiting body development. Deletion of plsB1 (MXAN3288) results in more dramatic changes in PE species distribution than deletion of plsB2 (MXAN1675). PlsB2 has a putative N-terminal eukaryotic fatty acid reductase domain and may support both ether lipid synthesis and PE synthesis. Disruption of a single sn-2 acyltransferase homologue (PlsC, of which M. xanthus contains five) results in minor changes in membrane PE. Derivatization of purified PE extracts with dimethyldisulfide was used to determine the position of the double bonds in unsaturated fatty acids. The results suggest that Delta5 and Delta11 desaturases may create the double bonds after synthesis of the fatty acid. Phosphatidylethanolamine enriched for 16:1 at the sn-1 position stimulates chemotaxis more strongly than PE with 16:1 enriched at the sn-2 position. It appears that the deployment of a rare fatty acid (16:1omega5) at an unusual position (sn-1) has facilitated the evolution of a novel cell signal.     

Molecular species of phosphatidylcholine in abetalipoproteinemia: effect of lecithin:cholesterol acyltransferase and lysolecithin acyltransferase

In order to study the role of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) in determining the molecular species composition of phosphatidylcholine (PC) and the specificity of lecithin:cholesterol acyltransferase (LCAT) in human plasma, we studied the PC species composition in plasma from abetalipoproteinemic (ABL) and control subjects before and after incubation at 37 degrees C. The ABL plasma contained significantly higher percentages of sn-2-18:1 species (16:0-18:1, 18:0-18:1, and 18:1-18:1) and lower percentages of sn-2-18:2 species (16:0-18:2, 18:0-18:2, and 18:1-18:2) as well as sn-2-20:4 species (16:0-20:4, 18:0-20:4, and 18:1-20:4). Similar abnormalities were found in the PC of ABL erythrocytes, while the PE of the erythrocytes was less affected. The relative contribution of various PC species towards LCAT reaction in ABL plasma was significantly different from that found in normal plasma. Thus, while 16:0-18:2 and 16:0-18:1 contributed, respectively, 43.8% and 15.9% of the total acyl groups used for cholesterol esterification in normal plasma, they contributed, respectively, 21.5% and 37.9% in ABL plasma. The relative contribution of 16:0-20:4 was also significantly lower in ABL plasma (4.7% vs. 9.0% in normal), while that of 16:0-16:0 was higher (6.4% vs. 0.5%). However, the selectivity factors of various species (percent contribution/percent concentration) were not significantly different between ABL and normal plasma, indicating that the substrate specificity of LCAT is not altered in the absence of VLDL and LDL. Incubation of ABL plasma in the presence of normal VLDL or LDL resulted in normalization of its molecular species composition and in the stimulation of its LCAT activity. Addition of LDL, but not VLDL, also resulted in the activation of lysolecithin acyltransferase (LAT) activity. The incorporation of [1-14C]palmitoyl lysoPC into various PC species in the presence of LDL was similar to that observed in normal plasma, with the 16:0-16:0 species having the highest specific activity. These results indicate that the absence of apoB-containing lipoproteins significantly affects the molecular species composition of plasma PC as well as its metabolism by LCAT and LAT reactions.     

Lysophosphatidic acid (LPA) receptors of the EDG family are differentially activated by LPA species. Structure-activity relationship of cloned LPA receptors

We examined the structure-activity relationship of cloned lysophosphatidic acid (LPA) receptors (endothelial cell differentiation gene (EDG) 2, EDG4, and EDG7) by measuring [Ca(2+)](i) in Sf9 insect cells expressing each receptor using LPA with various acyl chains bound at either the sn-1 or the sn-2 position of the glycerol backbone. For EDG7 the highest reactivity was observed with LPA with Delta9-unsaturated fatty acid (oleic (18:1), linoleic (18:2), and linolenic (18:3)) at sn-2 followed by 2-palmitoleoyl (16:1) and 2-arachidonoyl (20:4) LPA. In contrast, EDG2 and EDG4 showed broad ligand specificities, although EDG2 and EDG4 discriminated between 14:0 (myristoyl) and 16:0 (palmitoyl), and 12:0 (lauroyl) and 14:0 LPAs, respectively. EDG7 recognizes the cis double bond at the Delta9 position of octadecanoyl residues, since 2-elaidoyl (18:1, trans) and 2-petroselinoyl (18:1, cis-Delta12) LPA were poor ligands for EDG7. In conclusion, the present study demonstrates that each LPA receptor can be activated differentially by the LPA species.     

Loss of plastidic lysophosphatidic acid acyltransferase causes embryo-lethality in Arabidopsis

Phosphatidic acid is a key intermediate for chloroplast membrane lipid biosynthesis. De novo phosphatidic acid biosynthesis in plants occurs in two steps: first the acylation of the sn-1 position of glycerol-3-phosphate giving rise to lysophosphatidic acid; second, the acylation of the sn-2 position of lysophosphatidic acid to form phosphatidic acid. The second step is catalyzed by a lysophosphatidic acid acyltransferase (LPAAT). Here we describe the identification of the ATS2 gene of Arabidopsis encoding the plastidic isoform of this enzyme. Introduction of the ATS2 cDNA into E. coli JC 201, which is temperature-sensitive and carries a mutation in its LPAAT gene plsC, restored this mutant to nearly wild type growth at high temperature. A green-fluorescent protein fusion with ATS2 localized to the chloroplast. Disruption of the ATS2 gene of Arabidopsis by T-DNA insertion caused embryo lethality. The development of the embryos was arrested at the globular stage concomitant with a transient increase in ATS2 gene expression. Apparently, plastidic LPAAT is essential for embryo development in Arabidopsis during the transition from the globular to the heart stage when chloroplasts begin to form.     

Phosphatidyl choline fatty acid remodeling in the hepatic cell nuclei

This study was performed to determine whether fatty acids incorporated into liver cell nuclei phosphatidylcholine (PtdCho) could be remodeled in the isolated nuclear. For this reason, rat liver cell nuclei were incubated in vitro with [1-14C]20:4n-6-CoA. PtdCho molecular species with the highest specific activity had an unsaturated fatty acid at sn-1 and sn-2 positions (20:4-20:4>18:2-20:4>18:1-20:4). 16:0-20:4 and 18:0-20:4 PtdChos showed a minor specific activity. When labeled nuclei were reincubated in the absence of labeled substrate with the addition of cytosol, ATP and CoA, the specific activity of 20:4-20:4, 18:2-20:4 and 18:1-20:4 species decreased, while that of 16:0-20:4 and 18:0-20:4 increased. In conclusion, the asymmetric fatty acid distribution of saturated fatty acids at sn-1 position, and unsaturated fatty acids at sn-2 position of nuclear PtdCho molecular species was re-established by an acyl-CoA-dependent remodeling process.     

Regulation of triacylglycerol biosynthesis in embryos and microsomal preparations from the developing seeds of Cuphea lanceolata.

Embryos of Cuphea lanceolata have more than 80 mol% of decanoic acid ('capric acid') in their triacylglycerols, while this fatty acid is virtually absent in phosphatidylcholine (PtdCho). Seed development was complete 25-27 days after pollination, with rapid triacylglycerol deposition occurring between 9 and 24 days. PtdCho amounts increased until day 15 after pollination. Analysis of embryo lipids showed that the diacylglycerol (DAG) pool consisted of mainly long-chain molecular species, with a very small amount of mixed medium-chain/long-chain glycerols. Almost 100% of the fatty acid at position sn-2 in triacylglycerols (TAG) was decanoic acid. When equimolar mixtures of [14C]decanoic and [14C]oleic acid were fed to whole detached embryos, over half of the radioactivity in the DAG resided in [14C]oleate, whereas [14C]decanoic acid accounted for 93% of the label in the TAG. Microsomal preparations from developing embryos at the mid-stage of TAG accumulation catalysed the acylation of [14C]glycerol 3-phosphate with either decanoyl-CoA or oleoyl-CoA, resulting in the formation of phosphatidic acid (PtdOH), DAG and TAG. Very little [14C]glycerol entered PtdCho. In combined incubations, with an equimolar supply of [14C]oleoyl-CoA and [14C]decanoyl-CoA in the presence of glycerol 3-phosphate, the synthesized PtdCho species consisted to 95% of didecanoic and dioleic species. The didecanoyl-glycerols were very selectively utilized over the dioleoylglycerols in the production of TAG. Substantial amounts of [14C]oleate, but not [14C]decanoate, entered PtdCho. The microsomal preparations of developing embryos were used to assess the acyl specificities of the acyl-CoA:sn-glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15) and the acyl-CoA:sn-1-acyl-glycerol-3-phosphate acyltransferase (LPAAT, EC 2.3.1.51) in Cuphea lanceolata embryos. The efficiency of acyl-CoA utilization by the GPAT was in the order decanoyl = dodecanoyl greater than linoleoyl greater than myristoyl = oleoyl greater than palmitoyl. Decanoyl-CoA was the only acyl donor to be utilized to any extent by the LPAAT when sn-decanoylglycerol 3-phosphate was the acyl acceptor. sn-1-Acylglycerol 3-phosphates with acyl groups shorter than 16 carbon atoms did not serve as acyl acceptors for long-chain (greater than or equal to 16 carbon atoms) acyl-CoA species. On the basis of the results obtained, we propose a schematic model for triacylglycerol assembly and PtdCho synthesis in a tissue specialized in the synthesis of high amounts of medium-chain fatty acids.     

Exploring the photosynthetic production capacity of sucrose by cyanobacteria

Because cyanobacteria are photosynthetic, fast-growing microorganisms that can accumulate sucrose under salt stress, they have a potential application as a sugar source for the biomass-derived production of renewable fuels and chemicals. In the present study, the production of sucrose by the cyanobacteria Synechocystis sp. PCC6803, Synechococcus elongatus PCC7942, and Anabaena sp. PCC7120 was examined. The three species displayed different growth curves and intracellular sucrose accumulation rates in response to NaCl. Synechocystis sp. PCC6803 was used to examine the impact of modifying the metabolic pathway on the levels of sucrose production. The co-overexpression of sps (slr0045), spp (slr0953), and ugp (slr0207) lead to a 2-fold increase in intracellular sucrose accumulation, whereas knockout of ggpS (sll1566) resulted in a 1.5-fold increase in the production of this sugar. When combined, these genetic modifications resulted in a fourfold increase in intracellular sucrose accumulation. To explore methods for optimizing the transport of the intracellular sucrose to the growth medium, the acid-wash technique and the CscB (sucrose permease)-dependent export method were evaluated using Synechocystis sp. PCC6803. Whereas the acid-wash technique proved to be effective, the CscB-dependent export method was not effective. Taken together, these results suggest that using genetic engineering, photosynthetic cyanobacteria can be optimized for efficient sucrose production.     

Stereospecific analysis of major glycerolipids of Phycomyces blakesleeanus sporangiophores and mycelium.

The positional distribution of fatty acids was determined in the major groups of glycerolipids from the mycelium and sporangiophores of the fungus Phycomyces blakesleeanus. At the sn-1 positions of the triacylglycerols, in both regions of the fungus, greater than 65% of the fatty acids were 16:0 and 18:1. At the sn-2 positions of the triacylglycerols, 18:1, 18:2 and 18:3 comprised greater than 85% of the sporangial fatty acids and more than 90% of the mycelial fatty acids. Positions sn-3 of the triacylglycerols, from both regions of the fungus, contained approximately 40% of 16:0, approximately 30% of 18:2, and the largest proportions of 18:3 (21%) in the triacyglycerols. The major phosphoglycerides of P. blakesleeanus mycelium and sporangiophores are phosphatidylcholine and phosphatidylethanolamine, and more than 85% of the fatty acids at the sn-1 positions of these phosphatides consisted of 16:0, 18:2, and 18:3. The sn-2 positions of phosphatidylcholine and phosphatidylethanolamine contained approximately 98% unsaturated fatty acids. In the phosphoglycerides of both regions of the fungus, 18:2 and 18:3 constituted greater than 85% of the total fatty acids. Although the mycelium and sporangiophores of P. blakesleeanus had different morphological and physiological characteristics, the major glycerolipids of the two regions had similar stereospecific distributions of fatty acids.