Gene cloning, expression and functional characterization of an acyl carrier protein AcpV from Vibrio anguillarum

Acyl carrier protein (ACP) is a small acidic protein that acts as an essential cofactor in many biosynthetic pathways depending on acyl transfer reactions. In this work, a Vibrio anguillarum ACP encoding gene, acpV, was first cloned from the chromosome of a virulent V. anguillarum strain MVM425. acpV was over-expressed in Escherichia coli and the resultant protein AcpV was purified. The purified AcpV was incubated with purified phosphopantetheinyl transferase (PPtase) in the presence of CoA to assay the 4′-phosphopantetheinylation of AcpV in vitro; and on the other hand, the acpV gene was co-expressed with PPtase-encoding gene in E. coli to examine the 4′-phosphopantetheinylation of AcpV in vivo. Our results suggested that acpV encoded a functional ACP of V. anguillarum, which can be 4′-phosphopantetheinylated well by AcpS-type PPtase (E. coli AcpS) both in vitro and in vivo, but cannot serve as a good substrate for Sfp-type PPtase (V. anguillarum AngD).     

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 role of acyl carrier protein isoforms from Cuphea lanceolata seeds in the de-novo biosynthesis of medium-chain fatty acids

To investigate the role of acyl carrier protein (ACP) in determining the fate of the acyl moieties linked to it in the course of de-novo fatty acid biosynthesis in higher plants, we carried out in vitro experiments to reconstitute the fatty acid synthase (FAS) reaction in extracts of spinach (Spinaciaoleracea L.) leaves, rape (Brassicanapus L.) seeds and Cuphea lanceolata Ait. seeds. The action of two major C. lanceolata ACP isoforms (ACP 1 and ACP 2) compared to ACP from Escherichia coli was monitored by saponification of the corresponding FAS products with subsequent analysis of the liberated fatty acids by high-performance liquid chromatography. In a second approach the preference of the medium-chain acyl-ACP-specific thioesterase (EC 3.1.2.14) of C. lanceolata seeds for the hydrolysis of acyl-ACPs prepared from the three ACP types was investigated. Both ACP isoforms from C. lanceolata seeds supported the synthesis of medium-chain fatty acids in a reconstituted FAS reaction of spinach leaf extracts. Compared to the isoform ACP 1, ACP 2 was more effective in supporting the synthesis of such fatty acids in the FAS reaction of rape seed extracts and caused a higher accumulation of FAS products in all experiments. No preference of the medium-chain thioesterase for one specific ACP isoform was observed. The results indicate that the presence of ACP 2 is essential for the synthesis of decanoic acid in C. lanceolata seeds, and its expression in the phase of accumulation of high levels of this fatty acid provides an additional and highly efficient cofactor for stimulating the FAS reaction. Received: 23 June 1997 / Accepted: 23 October 1997     

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.     

Partial molar volumes of acyl carrier proteins are related to their states of acylation

Acyl carrier protein (ACP), an abundant protein in every cell, plays a central role in a number of metabolic processes requiring acyl group transfer. Conformational flexibility while crucial for its function remains substantially unaddressed. By dual polarization interferometry we establish correlation between the chain length of aliphatic groups covalently linked to Escherichia coli and Plasmodium falciparum ACP and their respective partial molar volumes in solution which helps to subserve the aforesaid goal.     

Co-purification,co-imniunoprecipitation,and coordinate expression of acetyl-coenzyme A carboxylase activity,biotin carboxylase,and biotin carboxyl carrier protein of higher plants

Acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) is a regulatory enzyme of fatty acid synthesis, and in some higher-plant plastids is a multi-subunit complex consisting of biotin carboxylase (BC), biotin-carboxyl carrier protein (BCCP), and carboxyl transferase (CT). We recently described a Nicotiana tabacum L. (tobacco) cDNA with a deduced amino acid sequence similar to that of prokaryotic BC. We here provide further biochemical and immunological evidence that this higher-plant polypeptide is an authentic BC component of ACCase. The BC protein co-purified with ACCase activity and with BCCP during gel permeation chromatography of Pisum sativum L. (pea) chloroplast proteins. Antibodies to the Ricinus communis L. (castor) BC co-precipitated ACCase activity and BCCP. During castor seed development, ACCase activity and the levels of BC and BCCP increased and subsequently decreased in parallel, indicating their coordinate regulation. The BC protein comprised about 0.8% of the soluble protein in developing castor seed, and less than 0.05% of the protein in young leaf or root. Polypeptides cross-reacting with antibodies to castor BC were detected in several dicotyledons and in the monocotyledons Hemerocallis fulva L. (day lily), Iris L., and Allium cepa L. (onion), but not in the Gramineae species Hordeum vulgare L. (barley) and Panicum virgatum L. (switchgrass). The castor endosperm and pea chloroplast ACCases were not significantly inhibited by long-chain acyl-acyl carrier protein, free fatty acids or acyl carrier protein. The BC polypeptide was detected throughout Brassica napus L. (rapeseed) embryo development, in contrast to the multi-functional ACCase isoenzyme which was only detected early in development. These results firmly establish the identity of the BC polypeptide in plants and provide insight into the structure, regulation and roles of higherplant ACCases.Abbreviations ACCase acetyl-CoA carboxylase - ACP acyl carrier protein - BC biotin carboxylase - BCCP biotin carboxyl carrier protein - CT carboxyl transferase - MF multi-functional - MS multi-subunit We thank our colleagues Nicki Engeseth and Vicki Eccleston for advice on fatty acid analysis and Sarah Hunter for providing the developing Iris seed. This work was supported in part by grant MCB 9406466 from NSF. Acknowledgement is also made to the Michigan Agriculture Experiment Station for its support of this research.     

Application of neural networks to automated assignment of NMR spectra of proteins

Summary Simulated neural networks are described which aid the assignment of protein NMR spectra. A network trained to recognize amino acid type from TOCSY data was trained on 148 assigned spin systems from E. coli acyl carrier proteins (ACPs) and tested on spin systems from spinach ACP, which has a 37% sequence homology with E. coli ACP and a similar secondary structure. The output unit corresponding to the correct amino acid is one of the four most activated units in 83% of the spin systems tested. The utility of this information is illustrated by a second network which uses a constraint satisfaction algorithm to find the best fit of the spin systems to the amino acid sequence. Application to a stretch of 20 amino acids in spinach ACP results in 75% correct sequential assignment. Since the output of the amino acid type identification network can be coupled with a variety of sequential assignment strategies, the approach offers substantial potential for expediting assignment of protein NMR spectra.     

cDNA cloning and expression of Brassica napus enoyl-acyl carrier protein reductase in Escherichia coli

The onset of storage lipid biosynthesis during seed development in the oilseed crop Brassica napus (rape seed) coincides with a drastic qualitative and quantitative change in fatty acid composition. During this phase of storage lipid biosynthesis, the enzyme activities of the individual components of the fatty acid synthase system increase rapidly. We describe a rapid and simple purification procedure for the plastidlocalized NADH-dependent enoyl-acyl carrier protein reductase from developing B. napus seed, based on its affinity towards the acyl carrier protein (ACP). The purified protein was N-terminally sequenced and used to raise a potent antibody preparation. Immuno-screening of a seed-specific gt11 cDNA expression library resulted in the isolation of enoyl-ACP reductase cDNA clones. DNA sequence analysis of an apparently full-length cDNA clone revealed that the enoyl-ACP reductase mRNA is translated into a precursor protein with a putative 73 amino acid leader sequence which is removed during the translocation of the protein through the plastid membrane. Expression studies in Escherichia coli demonstrated that the full-length cDNA clone encodes the authentic B. napus NADH-dependent enoyl-ACP reductase. Characterization of the enoyl-ACP reductase genes by Southern blotting shows that the allo-tetraploid B. napus contains two pairs of related enoyl-ACP reductase genes derived from the two distinct genes found in both its ancestors, Brassica oleracea and B. campestris. Northern blot analysis of enoyl-ACP reductase mRNA steady-state levels during seed development suggests that the increase in enzyme activity during the phase of storage lipid accumulation is regulated at the level of gene expression.     

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.     

1H, 15N, 13C resonance assignment of the acyl carrier protein subunit of the Saccharomyces cerevisiae fatty acid synthase

Acyl carrier proteins participate in the synthesis of fatty acids. Here we report the NMR resonances assignment of the acyl carrier protein domain of the Saccharomyces cerevisiae fatty acid synthase which corresponds to the fragment 138A-302L in the primary structure. The assignment will allow performing NMR studies with the aim to investigate the intrinsic dynamics of this protein, and to study the structural changes upon apo-holo transformation in order to unveil the mechanism of binding of the growing acyl chain.     

Fatty-acid synthesis in plastids from maturing safflower and linseed cotyledons

Plastids isolated from maturing, nongreen safflower (Carthamus tinctorius L.) cotyledons yielded unesterified fatty acids as the predominant product of fatty-acid synthesis from [1-¹⁴C]acetate. Exogenous reduced pyridine nucleotides were not required for this synthesis, but [1-¹⁴C]acetate incorporation was absolutely dependent on addition of ATP. Linseed (Linum usitatissimum L.) cotyledons are green during development and plastids isolated from them resembled leaf chloroplasts with developed grana. In contrast to the safflower plastids, those from linseed were able to carry out fatty-acid synthesis at low irradiances without the addition of either pyridine nucleotides or ATP. Intact linseed cotyledons were capable of net photosynthesis at rates up to 95 mol·mg^-1 chlorophyll·h^-1. However, the low-light environment inside the linseed capsule (approx. 15% of external) means that photosynthesis will not contribute appreciably to the carbon economy of the developing seed and its main role may be to supply cofactors for fatty-acid synthesis.Abbreviations ACP acyl carrier protein - DHAP dihydroxyacetone phosphate - PC phosphatidylcholine - PEP phosphoenolpyruvate - UFA unesterified fatty acids     

Incorporation of [214C]malonyl CoA into fatty acids by a cell-free extract of Catharanthus roseus suspension culture cells

A cell-free extract containing the enzymes for de-novo synthesis, elongation and desaturation of fatty acids was prepared from cultured cells of Catharanthus roseus G. Don. ¹⁴C-Fatty acids synthesized by the extract from [2-¹⁴C]malonyl CoA substrate were palmitic (16:0), stearic (18:0) and oleic (18:1). Dialyzed extract was active and stable at room temperature and at 4° C, but was inactivated on boiling. There was an absolute requirement for NADPH for incorporation of [2-¹⁴C]malonyl CoA into total fatty acids. Escherichia coli acyl carrier protein stimulated total fatty-acid synthesis without affecting the relative ratio of individual fatty acids. Total fatty-acid synthesis at a rate of 45 nmol·mg^-1 protein·h^-1 occurred at a substrate level of 73 M malonyl CoA, cofactor levels of 500 M NADPH, 30 g·ml^-1 E. coli ACP, and 1.0 mg·ml^-1 extract protein. Total fatty acid synthesis was also sensitive to cerulenin and CoA levels. Variations in the relative abundance of individual ¹⁴C-fatty acids were regulated by concentrations of [¹⁴C]malonyl CoA. NADPH and ferredoxin, as well as by pH, temperature and length of incubation. Fatty-acid synthetase enzymes responsible for [¹⁴C]palmitic acid were rapidly saturated at a low substrate level (0.3 M malonyl CoA). Increasing the level of [2-¹⁴C]malonyl CoA permitted further synthesis of [¹⁴C]stearate and [¹⁴C]oleate. Desaturation of [¹⁴C]stearate to [¹⁴C]oleate was stimulated by increasing the levels of NADPH and ferredoxin. The desaturase and elongase enzymes were sensitive to acidic pH. The desaturase was also unstable at 41° C, although fatty acid synthetase and elongase were unaffected by this temperature.Abbreviation ACP Acyl carrier protein     

Mitochondrial acyl carrier proteins in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> are predominantly soluble matrix proteins and none can be confirmed as subunits of respiratory Complex I

Arabidopsis mitochondria are predicted to contain three acyl carrier proteins (ACPs). These small proteins are involved in fatty acid and lipoic acid synthesis in other organisms and have been previously reported to be subunits of respiratory Complex I in mitochondria in mammals, fungi and plants. Recently, the mammalian mitochondrial ACP (mtACP) has been shown to be largely a soluble matrix protein but also to be minimally associated with Complex I (Cronan et al. 2005), consistent with its involvement in synthesis of lipoic acid for TCA cycle decarboxylating dehydrogenases in the matrix but contrary to earlier claims it was primarily a Complex I subunit. We have investigated the localization of the ACPs in Arabidopsis mitochondria. Evidence is presented that mtACP1 and mtACP2 dominate the ACP composition in Arabidopsis mitochondria, and both are present in the mitochondrial matrix rather than in the membrane. No significant amounts of mtACPs were detected in Complex I isolated by blue native gel electrophoresis, rather mtACPs were detected at low molecular mass in the soluble fraction, showing that in A. thaliana mtACPs are predominately free soluble matrix proteins.     

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.     

Modification of Brassica napus seed oil by expression of the Escherichia coli fabH gene,encoding 3-ketoacyl-acyl carrier protein synthase III

The Escherichia coli fabH gene encoding 3-ketoacyl-acyl carrier protein synthase III (KAS III) was isolated and the effect of overproduction of bacterial KAS III was compared in both E. coli and Brassica napus. The change in fatty acid profile of E. coli was essentially the same as that reported by Tsay et al. (J Biol Chem 267 (1992) 6807–6814), namely higher C14:0 and lower C18:1 levels. In our study, however, an arrest of cell growth was also observed. This and other evidence suggests that in E. coli the accumulation of C14:0 may not be a direct effect of the KAS III overexpression, but a general metabolic consequence of the arrest of cell division. Bacterial KAS III was expressed in a seed- and developmentally specific manner in B. napus in either cytoplasm or plastid. Significant increases in KAS III activities were observed in both these transformation groups, up to 3.7 times the endogenous KAS III activity in mature seeds. Only the expression of the plastid-targeted KAS III gene, however, affected the fatty acid profile of the storage lipids, such that decreased amounts of C18:1 and increased amounts of C18:2 and C18:3 were observed as compared to control plants. Such changes in fatty acid composition reflect changes in the regulation and control of fatty acid biosynthesis. We propose that fatty acid biosynthesis is not controlled by one rate-limiting enzyme, such as acetyl-CoA carboxylase, but rather is shared by a number of component enzymes of the fatty acid biosynthetic machinery.     

Widening the bottleneck: Heterologous expression,purification, and characterization of the Ktedonobacter racemifer minimal type II polyketide synthase in Escherichia coli

Enzyme assemblies such as type II polyketide synthases (PKSs) produce a wide array of bioactive secondary metabolites. While the molecules produced by type II PKSs have found remarkable clinical success, the biosynthetic prowess of these enzymes has been stymied by 1) the inability to reconstitute the bioactivity of the minimal PKS enzymes in vitro and 2) limited exploration of type II PKSs from diverse phyla. To begin filling this unmet need, we expressed, purified, and characterized the ketosynthase chain length factor (KS-CLF) and acyl carrier protein (ACP) from Ktedonobacter racemifer (Kr). Using E. coli as a heterologous host, we obtained soluble proteins in titers signifying improvements over previous KS-CLF heterologous expression efforts. Characterization of these enzymes reveals that KrACP has self-malonylating activity. Sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis of holo-KrACP and KrKS-CLF indicates that these enzymes do not interact in vitro, suggesting that the acylated state of these proteins might play an important role in facilitating biosynthetically relevant interactions. These results lay important groundwork for optimizing the interaction between KrKS-CLF and KrACP and exploring the biosynthetic potential of other non-actinomycete type II PKSs.     

Oil synthesis in vitro in microsomal membranes from developing cotyledons of Linum usitatissimum L.

Microsomal preparations from developing linseed (Linum usitatissimum L.) cotyledons catalyzed i) acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine, ii) acylation of sn-glycerol 3-phosphate to yield phosphatidic acid, and iii) the utilisation of phosphatidic acid in the production of diacylglycerol and triacylglycerol. Selectivity studies for C₁₈ acyl species of acyl-CoA indicated a bias for the channelling of oleate to phosphatidylcholine for, presumably, its desaturation, and the utilisation of the polyunsaturated fatty-acid products in the acyl-CoA pool for phosphatidic acid and subsequent triacylglycerol synthesis. The microsomal preparations were capable of returning glycerol backbone with associated acyl components to phosphatidylcholine from diacylglycerol where it may be further enriched with polyunsaturated C₁₈ acids by desaturation. The acyl quality in linolenate-rich oilseeds appears to be under similar control to that found in linoleate-rich species. Present address: To whom the correspondence should be addressed     

Cloning and functional expression of an acyl-ACP thioesterase FatB type from Diploknema (Madhuca) butyracea seeds in Escherichia coli

A cDNA of fatty acyl-acyl carrier protein (ACP) thioesterase (Fat) from developing seed of Madhuca butyracea has been cloned. The deduced amino acid sequence of the cDNA corresponding to the mature polypeptide showed 30-40% and 60-75% identity to the reported FatA and FatB class of plant thioesterases, respectively. This gene, MbFatB, is present as a single copy in M. butyracea genome and the MbFatB protein was detected clearly in seed tissues of this plant but not in that of Indian mustard (Brassica juncea). Heterologous expression of the MbFatB gene driven by different promoters in E. coli wild type and fatty acid beta-oxidation mutant (fadD88) strains resulted production of the recombinant protein with various fusion tags either as biologically inactive (insoluble) or functionally active forms. Expression of functionally active recombinant MbFatB in E. coli affected bacterial growth and cell morphology as well as changed the fatty acid profiles of the membrane lipid and the culture supernatant. Alteration of the fatty acid composition was directed predominantly towards palmitate and to a lesser extent myristate and oleate due to acyl chain termination activity of plant thioesterase in bacteria. Thus, this new MbFatB gene isolated from a non-traditional oil-seed tree can be used in future for transgenic development of oil-seed Brassica, a widely cultivated crop that expresses predominantly oleoyl-ACP thioesterase (FatA) in its seed tissue and has high amount of unwanted erucic acid in edible oil in order to alter the fatty acid profile in a desirable way.     

<Emphasis Type="Italic">fabC</Emphasis> of <Emphasis Type="Italic">Streptomyces lydicus</Emphasis> involvement in the biosynthesis of streptolydigin

Streptolydigin, a secondary metabolite produced by Streptomyces lydicus, is a potent inhibitor of bacterial RNA polymerases. It has been suggested that streptolydigin biosynthesis is associated with polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS). Thus, there is great interest in understanding the role of fatty acid biosynthesis in the biosynthesis of streptolydigin. In this paper, we cloned a type II fatty acid synthase (FAS II) gene cluster of fabDHCF from the genome of S. lydicus and constructed the SlyfabCF-disrupted mutant. Sequence analysis showed that SlyfabDHCF is 3.7 kb in length and encodes four separated proteins with conserved motifs and active residues, as shown in the FAS II of other bacteria. The SlyfabCF disruption inhibited streptolydigin biosynthesis and retarded mycelial growth, which were likely caused by the inhibition of fatty acid synthesis. Streptolydigin was not detected in the culture of the mutant strain by liquid chromatography–mass spectrometry. Meanwhile, the streptolol moiety of streptolydigin accumulated in cultures. As encoded by fabCF, acyl carrier protein (ACP) and β-ketoacyl-ACP synthase II are required for streptolydigin biosynthesis and likely involved in the step between PKS and NRPS. Our results provide the first genetic and metabolic evidence that SlyfabCF is shared by fatty acid synthesis and antibiotic streptolydigin synthesis.     

Analysis of two linked genes coding for the acyl carrier protein (ACP) from Arabidopsis thaliana (columbia)

Two linked genes, A1 and A2, coding for nearly identical isoforms of the acyl carrier protein (ACP) were isolated from an Arabidopsis thaliana (columbia) genomic library and sequenced. The amino acids deduced from the nucleotide sequence of the two genes indicate they encode distinct transit peptides, but the mature proteins are the same except for residue 79. Both genes are predicted to contain three introns in similar positions, although they differ in sequence and length. The introns interrupt regions coding for a) the transit peptide, b) the junction of the transit peptide and mature protein, and c) the highly conserved domain surrounding serine 38 to which the phosphopantetheine is attached. Primer extension analysis indicates that at least A1 is active in young plants.