Medium-chain fatty acid biosynthesis and utilization in Brassica napus plants expressing lauroyl-acyl carrier protein thioesterase

We have examined production of mediumchain fatty acids by Brassica napus L. plants transformed with a California bay (Umbellularia californica) medium-chain acyl-acyl carrier protein (ACP) thioesterase (UcFatB1) cDNA under the control of the constitutive cauliflower mosaic virus 35S promoter. These plants were found to accumulate medium-chain fatty acids in seeds but not in leaves or roots. Assay of thioesterase activity in extracts of leaves indicated that lauroyl-ACP thioesterase activity is comparable to oleoyl-ACP thioesterase (EC 3.1.2.14) activity in transformant leaves. Furthermore, leaf lauroyl-ACP thioesterase activity was in excess of that which produced a significant increase in the amount of laurate (12:0) in seed. Studies in which isolated chloroplasts were ¹⁴C-labelled were used to evaluate whether medium-chain fatty acids were produced in transformed leaves. Up to 34% of the fatty acids synthesized in vitro by isolated chloroplasts were 12:0. These results demonstrate that the normally seed-localized lauroyl-ACP thioesterase can be expressed in active form in leaves, imported into chloroplasts and can access acyl-ACP intermediates of leaf de-novo fatty acid synthesis. The most likely explanation for the lack of accumulation of 12:0 in transformed leaves is its rapid degradation by -oxidation. In support of this hypothesis, isocitrate lyase (EC 4.1.3.1) activity was found to be significantly increased in plants transformed with 35S-UcFatB1.Abbreviations ACP acyl carrier protein - CaMV cauliflower mosaic virus - control Brassica napus cultivar 212/86 - event 8 pCGN3831-212/86-8 - event 11 pCGN3831-212/86-11 - FAS fatty acid synthase - IL isocitrate lyase - KAS -keto-acyl ACP synthase - MS malate synthase - OTE oleoyl-ACP thioesterase - TAG triacylglycerol - UcFatB1 California bay medium-chain acyl-ACP thioesterase We are indebted to Calgene's Brossica-transformation, growth-chamber, greenhouse, and lipid-analysis personnel. Maelor Davies conducted the initial tranformant analysis. We thank Laura Olsen for IL and MS Western blot analysis and advice on IL and MS activity assays. This work was supported in part by a grant from the U.S. Department of Energy (No. DE-FG02-87ER12729). Acknowledgement is made to the Michigan Agricultural Experiment Station for its support of this research.     

Acyl carrier proteins from developing seeds of Cuphea lanceolata Ait

The structure of acyl carrier protein (ACP) may determine the fate of the acyl moieties linked to it in the course of de-novo fatty acid synthesis in higher plants. To investigate a possible correlation between the structure of ACP and the synthesis of medium-chain fatty acids, we isolated and characterized ACP from the seeds of Cuphea lanceolata Ait. (subgenus Eucuphea/Section Heterodon), an annual crop that contains up to 90% decanoic (capric) acid in seed triacylglycerols. After a cell-free extract prepared from developing seeds was treated to 65% saturation with ammonium sulfate, two ACP-isoforms (ACP 1 and ACP 2) were identified in the supernatant that could be purified to homogeneity by anion-exchange chromatography and subsequent reversed-phase high-performance liquid chromatography. The molecular mass determined by matrix-assisted ultraviolet-laser desorption ionization mass spectrometry of ACP 1 was 9315 Da, whereas further heterogeneity was observed for ACP 2 with molecular masses of 8598 and 8703 Da. Aminoterminal sequencing was performed showing a high homology in the primary structures of ACP 1 and ACP 2. Both isoforms were present in the embryo, whereas in the chloroplast-containing seed coat ACP 2 was found in minute amounts, if at all. The expression of ACP 2 correlated with the production of capric acid during the phase of storage-lipid accumulation. These data indicate that ACP 2 is part of the machinery for the synthesis of medium-chain fatty acids, whereas ACP 1 appears to be a constitutive protein.Abbreviations ACP acyl carrier protein - clACP acyl carrier protein from Cuphea lanceolata - 2D-PAOE two-dimensional polyacrylamide gel electrophoresis - DTT dithiothreitol - ecACP acyl carrier protein from Escherichia coli - FPLC fast protein liquid chromatography - HPLC high-performance liquid chromatography - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine This work was supported by a grant from the German Ministry of Research and Technology (BMFT). The authors wish to thank Professor Röbbelen, University of Göttingen, FRG, for kindly providing the plant material and A. Ingendoh, Department of Medical Physics of the University of Münster, FRG, for carrying out the mass-spectrometric analysis. Portions of this paper are part of the doctoral thesis of Markus Robers.     

The purification and function of acetyl coenzyme A:acyl carrier protein transacylase

When individual enzyme activities of the fatty acid synthetase (FAS) system were assayed in extracts from five different plant tissues, acetyl-CoA:acyl carrier protein (ACP) transacylase and beta-ketoacyl-ACP synthetases I and II had consistently low specific activities in comparison with the other enzymes of the system. However, two of these extracts synthesized significant levels of medium chain fatty acids (rather than C16 and C18 acid) from [14C]malonyl-CoA; these extracts had elevated levels of acetyl-CoA:ACP transacylase. To explore the role of the acetyl transacylase more carefully, this enzyme was purified some 180-fold from spinach leaf extracts. Varying concentrations of the transacylase were then added either to spinach leaf extracts or to a completely reconstituted FAS system consisting of highly purified enzymes. The results suggested that: (a) acetyl-CoA:ACP transacylase was the enzyme catalyzing the rate-limiting step in the plant FAS system; (b) increasing concentration of this enzyme markedly increased the levels of the medium chain fatty acids, whereas increase of the other enzymes of the FAS system led to increased levels of stearic acid synthesis; and (c) beta-ketoacyl-ACP synthetase I was not involved in the rate-limiting step. It is suggested that modulation of the activity of acetyl-CoA:ACP transacylase may have important implications in the type of fatty acid synthesized, as well as the amount of fatty acids formed.     

Regulated expression of the rat medium chain hydrolase gene in transgenic rape seed

Medium chain hydrolase (MCH) is an enzyme which regulates the chain length of fatty acid synthesis specifically in the mammary gland of the rat. During lactation, MCH interacts with fatty acid synthase (FAS) to cause premature release of acyl chains, thus providing medium chain fatty acids for synthesis of milk fat. In this study we have investigated the ability of rat MCH to interact with the phylogenetically more distant FAS structure present in plant systems and to cause a perturbation of fatty acid synthesis. Inin vitro experiments, addition of purified MCH to rapeseed homogenates was found to cause a significant perturbation of fatty acid synthesis towards medium chain length products. The rat MCH gene was expressed in transgenic oilseed rape using a seed specific rape acyl carrier protein (ACP) promoter and a rape ACP plastid targeting sequence. Western analysis showed MCH protein to be present in transgenic seed and for its expression to be developmentally regulated in concert with storage lipid synthesis. The chimaeric preprotein was correctly processed and immunogold labelling studies confirmed MCH to be localized within plastid organelles. However, fatty acid analysis of oil from MCH-expressing rape seed showed no significant differences to that from control seed.     

Redirection of metabolic flux for high levels of omega‐7 monounsaturated fatty acid accumulation in camelina seeds

Seed oils enriched in omega‐7 monounsaturated fatty acids, including palmitoleic acid (16:1?9) and cis‐vaccenic acid (18:1?11), have nutraceutical and industrial value for polyethylene production and biofuels. Existing oilseed crops accumulate only small amounts (<2%) of these novel fatty acids in their seed oils. We demonstrate a strategy for enhanced production of omega‐7 monounsaturated fatty acids in camelina (Camelina sativa) and soybean (Glycine max) that is dependent on redirection of metabolic flux from the typical ?9 desaturation of stearoyl (18:0)‐acyl carrier protein (ACP) to ?9 desaturation of palmitoyl (16:0)‐acyl carrier protein (ACP) and coenzyme A (CoA). This was achieved by seed‐specific co‐expression of a mutant ?9‐acyl‐ACP and an acyl‐CoA desaturase with high specificity for 16:0‐ACP and CoA substrates, respectively. This strategy was most effective in camelina where seed oils with ~17% omega‐7 monounsaturated fatty acids were obtained. Further increases in omega‐7 fatty acid accumulation to 60–65% of the total fatty acids in camelina seeds were achieved by inclusion of seed‐specific suppression of 3‐keto‐acyl‐ACP synthase II and the FatB 16:0‐ACP thioesterase genes to increase substrate pool sizes of 16:0‐ACP for the ?9‐acyl‐ACP desaturase and by blocking C18 fatty acid elongation. Seeds from these lines also had total saturated fatty acids reduced to ~5% of the seed oil versus ~12% in seeds of nontransformed plants. Consistent with accumulation of triacylglycerol species with shorter fatty acid chain lengths and increased monounsaturation, seed oils from engineered lines had marked shifts in thermotropic properties that may be of value for biofuel applications.     

In-vitro evidence for feed-back regulation of β-ketoacyl-acyl carrier protein synthase III in medium-chain fatty acid biosynthesis

The cerulenin-insensitive -ketoacyl-acyl carrier protein (ACP) synthase III (KAS III, EC 2.3.1.41) catalyzes the first condensing step of the fatty-acid synthase (FAS) reaction in plants and bacteria, using directly acetyl-CoA as substrate for condensation with malonyl-ACP. In order to identify a possible site for regulation of the biosynthesis of medium-chain fatty acids, the influence of acyl-ACPs of different chain-lengths (C₄,C₆,C₈ and C₁₀) on the activity of KAS III was investigated in vitro using an FAS preparation from seeds of Cuphea lanceolata Ait. (a crop accumulating up to 90% decanoic acid into triacylglycerols) that had been treated with 100 M cerulenin. All acyl-ACPs investigated led to a decrease in the activity of KAS III towards acetyl-CoA, an effect apparently related to the length of the acyl chain. Analysis of the reaction products of the assay revealed that short-chain acyl-ACPs elongated to a very small extent simultaneously with acetyl-CoA. This extent of elongation did not correlate with the decrease in KAS III-activity levels. These data excluded the possibility of competition between acetyl-CoA and acyl-ACPs, but indicated that acyl-ACPs inhibited the enzyme. Decanoyl-ACP caused the highest decrease in enzyme activity (IC₅₀ = 0.45 M), thus being a potent inhibitor of KAS III. Michaelis-Menten kinetics revealed that the inhibition of KAS III by decanoyl-ACP was non-competitive in relation to malonyl-ACP and uncompetitive in relation to acetyl-CoA. Moreover, our data indicate that KAS III has a strict specificity for the elongation of acetyl-CoA. An inhibition of KAS III by acyl-ACPs was observed in experiments using FAS preparations from rape seeds and spinach leaves, but the inhibition of KAS III from C. lanceolata seeds by decanoyl-ACP was approximately 1.5-fold higher. The data provide evidence that acyl-ACPs are involved in the modulation of plant fatty-acid biosynthesis by a feed-back mechanism.Abbreviations ACP acyl carrier protein - DTT dithiothreitol - TCA trichloroacetic acid - ecACP acyl carrier protein from Escherichia coli - FAS fatty-acid synthase - IC₅₀ concentration causing 50% inhibition - KAS -ketoacyl-ACP synthase - NEM N-ethylmaleimide In honour of Professor Hartmut K. Lichtenthaler's sixtieth birthdayThis work was supported by a grant from the German Ministry of Research and Technology (BMFT) and in part by the Fonds der Chemischen Industrie and the Ministry of Science and Research of the State Northrhine-Westfalia. The authors wish to thank Prof. G. Röbbelen (University of Göttingen, Göttingen, Germany) for kindly providing the plant material. This paper is part of the doctoral thesis of Fritzi Maike Brück.     

Xanthomonas campestris RpfB is a fatty Acyl‐CoA ligase required to counteract the thioesterase activity of the RpfF diffusible signal factor (DSF) synthase

In Xanthomonas campestris pv. campestris (Xcc), the proteins encoded by the rpf (regulator of pathogenicity factor) gene cluster produce and sense a fatty acid signal molecule called diffusible signalling factor (DSF, 2(Z)‐11‐methyldodecenoic acid). RpfB was reported to be involved in DSF processing and was predicted to encode an acyl‐CoA ligase. We report that RpfB activates a wide range of fatty acids to their CoA esters in vitro. Moreover, RpfB can functionally replace the paradigm bacterial acyl‐CoA ligase, Escherichia coli FadD, in the E. coli ß‐oxidation pathway and deletion of RpfB from the Xcc genome results in a strain unable to utilize fatty acids as carbon sources. An essential RpfB function in the pathogenicity factor pathway was demonstrated by the properties of a strain deleted for both the rpfB and rpfC genes. The ΔrpfB ΔrpfC strain grew poorly and lysed upon entering stationary phase. Deletion of rpfF, the gene encoding the DSF synthetic enzyme, restored normal growth to this strain. RpfF is a dual function enzyme that synthesizes DSF by dehydration of a 3‐hydroxyacyl‐acyl carrier protein (ACP) fatty acid synthetic intermediate and also cleaves the thioester bond linking DSF to ACP. However, the RpfF thioesterase activity is of broad specificity and upon elimination of its RpfC inhibitor RpfF attains maximal activity and its thioesterase activity proceeds to block membrane lipid synthesis by cleavage of acyl‐ACP intermediates. This resulted in release of the nascent acyl chains to the medium as free fatty acids. This lack of acyl chains for phospholipid synthesis results in cell lysis unless RpfB is present to counteract the RpfF thioesterase activity by catalysing uptake and activation of the free fatty acids to give acyl‐CoAs that can be utilized to restore membrane lipid synthesis. Heterologous expression of a different fatty acid activating enzyme, the Vibrio harveyi acyl‐ACP synthetase, replaced RpfB in counteracting the effects of high level RpfF thioesterase activity indicating that the essential role of RpfB is uptake and activation of free fatty acids.     

Synthesis of medium-chain fatty acids and their incorporation into triacylglycerols by cell-free fractions fromCuphea embryos

During their rapid maturation period, seeds of Cuphea wrightii A. Gray mainly accumulate medium-chain fatty acids (C₈ to C₁₄) in their storage lipids. The rate of lipid deposition (40–50 mg·d^–1·(g fresh weight)^–1) is fourfold higher than in seeds of Cuphea racemosa (L. f.) Spreng, which accumulate long-chain fatty acids (C₁₆ to C₁₈). Measurements of the key enzymes of fatty-acid synthesis in cell-free extracts of seeds of different maturities from Cuphea wrightii show that malonyl-CoA synthesis may be a triggering factor for the observed high capacity for fatty-acid synthesis. Experiments on the incorporation of [1-¹⁴C]acetate into fatty acids by purified plastid preparations from embryos of Cuphea wrightii have demonstrated that the biosynthesis of medium-chain fatty acids (C₈ to C₁₄) is localized in the plastid. Thus, in the presence of cofactors for lipid synthesis (ATP, NADPH, NADH, acyl carrier protein, and sn-glycerol-3-phosphate), purified plastid fractions predominantly synthesized free fatty acids, 30% of which were of medium chain length. Transesterification of the freshly synthesized fatty acids to coenzyme A and recombination with the microsomal fraction of the embryo homogenate induced triacylglycerol synthesis. It also stimulated fatty-acid synthesis by a factor 2–3 and increased the relative amount of medium-chain fatty acids bound to triacylglycerols, which corresponded to about 60–80% in this lipid fraction.Abbreviations ACP acyl carrier protein - FW fresh weight This work was supported by the Bundesminister für Forschung und Technologie. The authors thank S. Borchert for her suggestions for plastid preparation.     

A root acyl carrier protein-II from spinach is also expressed in leaves and seeds

During the synthesis of fatty acids and their utilization in plastids, fatty acyl moieties are linked to acyl carrier protein (ACP). In contrast to previously cloned organ-specific ACP isoforms, we have now isolated a cDNA clone for a potentially constitutive ACP isoform from a spinach root library. Identity between the amino acid sequence encoded by this cDNA and N-terminal sequence data for ACP-II protein from spinach leaf indicates that the root cDNA encodes ACP-II. The deduced amino acid sequence for ACP-II shows 62% identity with spinach leaf ACP-I. Southern analysis suggests that multiple ACP genes or pseudogenes occur in the spinach genome. High-stringency northern blot analysis and RNase protection studies confirm that, within the region encoding the mature ACP-II, the cloned ACP sequence is expressed in leaves and seeds as well as in roots. Quantitative RNase protection data indicate that the ratio of ACP-I and ACP-II mRNA sequences in leaf is similar to the ratio of the two proteins.     

Characterization of two acyl-acyl carrier protein thioesterases from developing Cuphea seeds specific for medium-chain- and oleoyl-acyl carrier protein

Two acyl-acyl carrier protein (ACP) thioesterases were partially purified from developing seeds of Cuphea lanceolata Ait., a plant with decanoic acid-rich triacylglycerols. The two enzymes differ markedly in their substrate specificity. One is specific for medium-chain acyl-ACPs, the other one for oleoyl-ACP. In addition, these enzymes are distinct with regard to molecular weight, pH optimum and sensitivity to salt. The thioesterases could be separated by Mono Q chromatography or gel filtration. The medium-chain acyl-ACP thioesterase and oleoyl-ACP thioesterase were purified from a crude extract 29- and 180-fold, respectively. In Cuphea wrightii A. Gray, which predominantly contains decanoic a nd lauric acid in the seeds, two different thioesterases were also found with a similar substrate specificity as in Cuphea lanceolata.     

A thioesterase bypasses the requirement for exogenous fatty acids in the plsX deletion of Streptococcus pneumoniae

PlsX is an acyl‐acyl carrier protein (ACP):phosphate transacylase that interconverts the two acyl donors in Gram‐positive bacterial phospholipid synthesis. The deletion of plsX in Staphylococcus aureus results in a requirement for both exogenous fatty acids and de novo type II fatty acid biosynthesis. Deletion of plsX (SP0037) in Streptococcus pneumoniae did not result in an auxotrophic phenotype. The ΔplsX S. pneumoniae strain was refractory to myristic acid‐dependent growth arrest, and unlike the wild‐type strain, was susceptible to fatty acid synthesis inhibitors in the presence of exogenous oleate. The ΔplsX strain contained longer chain saturated fatty acids imparting a distinctly altered phospholipid molecular species profile. An elevated pool of 18‐ and 20‐carbon saturated fatty acids was detected in the ΔplsX strain. A S. pneumoniae thioesterase (TesS, SP1408) hydrolyzed acyl‐ACP in vitro, and the ΔtesS ΔplsX double knockout strain was a fatty acid auxotroph. Thus, the TesS thioesterase hydrolyzed the accumulating acyl‐ACP in the ΔplsX strain to liberate fatty acids that were activated by fatty acid kinase to bypass a requirement for extracellular fatty acid. This work identifies tesS as the gene responsible for the difference in exogenous fatty acid growth requirement of the ΔplsX strains of S. aureus and S. pneumoniae.     

Photocontrol of gibberellin metabolism in situ in maize

Two forms of spinach acyl carrier protein (ACP-I and ACP-II) have recently been characterized and found to be expressed in a tissue-specific manner (JB Ohlrogge, TM Kuo, 1985 J Biol Chem 260: 8032). To examine possible different functions for these ACP isoforms, we have tested purified preparations of spinach leaf ACP-I and ACP-II and Escherichia coli ACP in several in vitro reactions of fatty acid metabolism. Total de novo fatty acid synthesis and malonyl-CoA:ACP transacylase do not appear to discriminate between acyl carrier protein isoforms. In contrast, the K_m of oleoyl-ACP thioesterase for oleoyl-ACP-II is 10-fold higher than for oleoyl-ACP-I, whereas the K_m of acyl-ACP glycerol-3-phosphate acyl transferase is 5-fold higher for oleoyl-ACP-I than for oleoyl-ACP-II. A characterization of these reactions and a possible role for ACP isoforms in regulation of fatty acid metabolism in plants are described.     

Synthesis in vitro of very long chain fatty acids in Vibrio sp. strain ABE-1

The activity of fatty acid synthetase (FAS) from Vibrio sp. strain ABE-1 required the presence of acyl carrier protein and was completely inhibited by thiolactomycin, an inhibitor specific for a type II FAS. These observations indicate that this enzyme is a type II FAS. Analysis by gas-liquid chromotography of the reaction products synthesized in vitro from [2-¹⁴C]malonyl-CoA by the partially purified FAS revealed, in addition to 16-and 18-carbon fatty acids which are normal constituents of this bacterium, the presence of fatty acids with very long chains. These fatty acids were identified as saturated and mono-unsaturated fatty acids with 20 up to as many as 30 carbon atoms. The longest fatty acids normally found in this bacterium contain 18-carbon atoms. These results suggest that the FAS from Vibrio sp. strain ABE-1 has potentially the ability to synthesize fatty acids with very long chains.Abbreviations ACP acyl carrier protein - FAME fatty acid methyl ester - FAS fatty acid synthetase - FID flame ionization detection - GLC gas-liquid chromatography - TLC thin-layer chromatography - In designations of fatty acids, such as 16:0, 16:1, etc the colon separates the number that denotes the number of carbon atoms and the number that denotes the number of double bonds, respectively, in the molecule - 16:0-CoA CoA ester of 16:0     

Reduced expression of FatA thioesterases in Arabidopsis affects the oil content and fatty acid composition of the seeds

Acyl–acyl carrier protein (ACP) thioesterases are enzymes that control the termination of intraplastidial fatty acid synthesis by hydrolyzing the acyl–ACP complexes. Among the different thioesterase gene families found in plants, the FatA-type fulfills a fundamental role in the export of the C18 fatty acid moieties that will be used to synthesize most plant glycerolipids. A reverse genomic approach has been used to study the FatA thioesterase in seed oil accumulation by screening different mutant collections of Arabidopsis thaliana for FatA knockouts. Two mutants were identified with T-DNA insertions in the promoter region of each of the two copies of FatA present in the Arabidopsis genome, from which a double FatA Arabidopsis mutant was made. The expression of both forms of FatA thioesterases was reduced in this double mutant (fata1 fata2), as was FatA activity. This decrease did not cause any evident morphological changes in the mutant plants, although the partial reduction of this activity affected the oil content and fatty acid composition of the Arabidopsis seeds. Thus, dry mutant seeds had less triacylglycerol content, while other neutral lipids like diacylglycerols were not affected. Furthermore, the metabolic flow of the different glycerolipid species into seed oil in the developing seeds was reduced at different stages of seed formation in the fata1 fata2 line. This diminished metabolic flow induced increases in the proportion of linolenic and erucic fatty acids in the seed oil, in a similar way as previously reported for the wri1 Arabidopsis mutant that accumulates oil poorly. The similarities between these two mutants and the origin of their phenotype are discussed in function of the results.     

Alteration of fatty acid chain length of Chlamydomonas reinhardtii by simultaneous expression of medium‐chain‐specific thioesterase and acyl carrier protein

Biofuel from fatty acids with chain lengths of 8–15 (C8–C15) have properties similar to those of conventional diesel and jet fuels, thus, can save time and reduce costs for the refurbishment of engines and maintenance of oiling facilities. Most oil‐producing algae yield C16–C18 fatty acids; however, the manipulation of algae using genetic engineering is a promising approach to obtain C8–C15 fatty acids. The introduction of a medium‐chain‐specific thioesterase (TE) is expected to effectively alter algae to produce medium‐chain fatty acids (MCFAs). TE is the main determinant of fatty acid chain length as it releases fatty acids from the acyl carrier protein (ACP) in the fatty acid elongation cycle. In a previous study, the introduction of heterologous C8–C12‐specific TEs into Chlamydomonas reinhardtii did not increase the yield of MCFAs. This effect was attributed to a low affinity of the heterologous TEs to C. reinhardtii ACP. Therefore, we introduced both the C10–C14‐specific TE gene and the ACP gene from the land plant Cuphea lanceolata into C. reinhardtii. We measured free fatty acids (FFAs) and triacylglycerols (TAGs) in the transformants using liquid chromatography–mass spectrometry. The production of C12:0 and C14:0, chain length 12 and 14 without unsaturation, FFAs was not significantly increased in any of the tested strains. However, we found a slight but significant increase in TAG‐containing MCFAs in both TE only and TE–ACP transformants. The increased production rate of C14:0‐containing TAGs ranged from 1.25‐ to 1.58‐fold, indicating the ability of medium‐chain‐specific TE to increase MCFAs. These results suggest that the selection of specific TEs is important when modifying eukaryotic algae to produce MCFAs.     

A specific acyl-ACP thioesterase implicated in medium-chain fatty acid production in immature cotyledons of Umbellularia californica

Umbellularia californica (California Bay) seeds accumulate 10:0 and 12:0 as principal reserve fatty acyl groups. An in vitro fatty acid synthesis system from the developing cotyledons produces chiefly 10:0 and 12:0, in approximately the same proportions as the intact tissue. The kinetics of acyl thioester and free fatty acid formation in this system suggest that a medium-chain specific acyl-acyl-carrier protein (ACP) hydrolysis mechanism is responsible for the preponderance of medium-chain products. A crude extract of the developing cotyledons exhibits hydrolytic activity toward acyl-ACPs, with marked preference for 12:0-ACP and 18:1-ACP in the test series 6:0, 8:0, 10:0, 11:0, 12:0, 14:0, 16:0, and 18:1-ACPs. Partial purification of the 12:0-ACP hydrolytic activity has resulted in its separation from the 18:1-ACP hydrolase(s) and the 12:0-coenzyme A hydrolase(s) that are also present, thereby demonstrating its specificity for the 12-carbon acyl chain length and the ACP derivative. During cotyledon development, as the proportion of medium-chain to other fatty acyl groups increases, the extractable yield of this activity also increases substantially. Collectively these results suggest a role for this 12-ACP thioesterase in medium-chain production in vivo.     

Expression of an active spinach acyl carrier protein-I/protein-A gene fusion

A synthetic gene encoding spinach acyl carrier protein I (ACP-I) was fused to a gene encoding the Fc-binding portion of staphylococcal protein A. This gene fusion, under the control of the P_R promoter, was expressed at high levels in Escherichia coli producing a 42 kDa fusion protein. This fusion protein was phosphopantethenylated in E. coli. In vitro the ACP portion of the fusion protein was able to participate in acyl ACP synthetase reactions, plant malonyl-CoA:ACP transacylase (MCT) reactions, and plant fatty acid synthetase (FAS) reactions. Inhibitory effects of high ACP concentrations on in vitro plant FAS were observed with the unfused ACP-1 but not with the fusion protein. As with unfused ACP-I, the fusion protein was a poor substrate for E. coli FAS reactions. When injected into rabbits, the fusion protein was also able to generate antiserum to spinach ACP-I.     

Immunogold localization of acyl carrier protein in plants and Escherichia coli: Evidence for membrane association in plants

Immunogold labelling was used to study the distribution of acyl carrier protein (ACP) in Escherichia coli and a variety of plant tissues. In E. coli, ACP is distributed throughout the cytoplasm, confirming the observation of S. Jackowski et al. (1985, J. Bacteriol., 162, 5–8_. In the mesocarp of Avocado (Persea americana) and maturing seeds of oil-seed rape (Brassica napus cv. Jet Neuf), over 95% of the ACP is localised to plastids. The protein is almost exclusively located in the chloroplasts of leaf material from oil-seed rape. Approximately 80% of the gold particles associated with the ACP were further localized to the thylakoid membrane of the chloroplast. Since acetyl-CoA carboxylase has been reported to be localized to the thylakoid membrane (C.G. Kannangara and C.J. Jensen, 1975, Eur. J. Biochem., 54, 25–30), these results are consistent with the view that the two sequential enzymes in fatty-acid synthesis are in close spacial proximity.Abbreviations ACC acetyl CoA carboxylase - ACP acyl carrier protein - FAS fatty-acid synthetase     

Identification and molecular characterization of the beta-ketoacyl-[acyl carrier protein] synthase component of the Arabidopsis mitochondrial fatty acid synthase

Substrate specificity of condensing enzymes is a predominant factor determining the nature of fatty acyl chains synthesized by type II fatty acid synthase (FAS) enzyme complexes composed of discrete enzymes. The gene (mtKAS) encoding the condensing enzyme, beta-ketoacyl-[acyl carrier protein] (ACP) synthase (KAS), constituent of the mitochondrial FAS was cloned from Arabidopsis thaliana, and its product was purified and characterized. The mtKAS cDNA complemented the KAS II defect in the E. coli CY244 strain mutated in both fabB and fabF encoding KAS I and KAS II, respectively, demonstrating its ability to catalyze the condensation reaction in fatty acid synthesis. In vitro assays using extracts of CY244 containing all E. coli FAS components, except that KAS I and II were replaced by mtKAS, gave C(4)-C(18) fatty acids exhibiting a bimodal distribution with peaks at C(8) and C(14)-C(16). Previously observed bimodal distributions obtained using mitochondrial extracts appear attributable to the mtKAS enzyme in the extracts. Although the mtKAS sequence is most similar to that of bacterial KAS IIs, sensitivity of mtKAS to the antibiotic cerulenin resembles that of E. coli KAS I. In the first or priming condensation reaction of de novo fatty acid synthesis, purified His-tagged mtKAS efficiently utilized malonyl-ACP, but not acetyl-CoA as primer substrate. Intracellular targeting using green fluorescent protein, Western blot, and deletion analyses identified an N-terminal signal conveying mtKAS into mitochondria. Thus, mtKAS with its broad chain length specificity accomplishes all condensation steps in mitochondrial fatty acid synthesis, whereas in plastids three KAS enzymes are required.     

Incorporation of extracellular fatty acids by a fatty acid kinase‐dependent pathway in Staphylococcus aureus

Acyl‐CoA and acyl‐acyl carrier protein (ACP) synthetases activate exogenous fatty acids for incorporation into phospholipids in Gram‐negative bacteria. However, Gram‐positive bacteria utilize an acyltransferase pathway for the biogenesis of phosphatidic acid that begins with the acylation of sn‐glycerol‐3‐phosphate by PlsY using an acyl‐phosphate (acyl‐PO₄) intermediate. PlsX generates acyl‐PO₄ from the acyl‐ACP end‐products of fatty acid synthesis. The plsX gene of Staphylococcus aureus was inactivated and the resulting strain was both a fatty acid auxotroph and required de novo fatty acid synthesis for growth. Exogenous fatty acids were only incorporated into the 1‐position and endogenous acyl groups were channeled into the 2‐position of the phospholipids in strain PDJ39 (ΔplsX). Extracellular fatty acids were not elongated. Removal of the exogenous fatty acid supplement led to the rapid accumulation of intracellular acyl‐ACP and the abrupt cessation of fatty acid synthesis. Extracts from the ΔplsX strain exhibited an ATP‐dependent fatty acid kinase activity, and the acyl‐PO₄ was converted to acyl‐ACP when purified PlsX is added. These data reveal the existence of a novel fatty acid kinase pathway for the incorporation of exogenous fatty acids into S. aureus phospholipids.