KAS IV: a 3-ketoacyl-ACP synthase from Cuphea sp . is a medium chain specific condensing enzyme

cDNA clones encoding a novel 3-ketoacyl-ACP synthase (KAS) have been isolated from Cuphea . The amino acid sequence of this enzyme is different from the previously characterized classes of KASs, designated KAS I and III, and similar to those designated as KAS II. To define the acyl chain specificity of this enzyme, we generated transgenic Brassica plants over-expressing the cDNA encoded protein in a seed specific manner. Expression of this enzyme in transgenic Brassica seeds which normally do not produce medium chain fatty acids does not result in any detectable modification of the fatty acid profile. However, co-expression of the Cuphea KAS with medium chain specific thioesterases, capable of production of either 12:0 or 8:0/10:0 fatty acids in seed oil, strongly enhances the levels of these medium chain fatty acids as compared with seed oil of plants expressing the thioesterases alone. By contrast, co-expression of the Cuphea KAS along with an 18:0/18:1-ACP thioesterase does not result in any detectable modification of the fatty acids. These data indicate that the Cuphea KAS reported here has a different acyl-chain specificity to the previously characterized KAS I, II and III. Therefore, we designate this enzyme KAS IV, a medium chain specific condensing enzyme.     

Identification of novel acyl-ACP thioesterase gene ClFATB1 from Cinnamomum longepaniculatum

A putative fatty acyl-acyl carrier protein (acyl-ACP) thioesterase (thioesterase) full-length cDNA sequence named as ClFATB1 was obtained from the seed cDNA library of Cinnamomum longepaniculatum by the SMART-RACE method. The novel gene encodes a protein of 382 amino acid residues with close homology to fatty acid thioesterase type B (FATB) enzymes of other plants, with two essential residues (His285 and Cys320) for thioesterase catalytic activity. The gene was transcribed in all tissues of C. longepaniculatum, the highest being in seeds. Recombinant ClFATB1 in Escherichia coli had higher specific activities against saturated 16:0- and 18:0-ACPs than on unsaturated 18:1-ACP. Overexpression of ClFATB1 in transgenic tobaccos upregulated thioesterase activities of crude proteins against 16:0-ACP and 18:0-ACP by 20.3 and 5.7%, respectively, and resulted in an increase in the contents of palmitic and stearic acids by 15.4 and 10.5%, respectively. However, ectopic expression of this gene decreased the substrate specificities of crude proteins to unsaturated 18:1-ACP by 12.7% in transgenic tobacco and lowered the contents of oleic, linoleic, and linolenic acids in transgenic leaves. So ClFATB1 would potentially upregulate the synthesis of saturated fatty acids and downregulate unsaturated ones in the fatty acid synthesis pathway of plants.     

Production of high levels of 8:0 and 10:0 fatty acids in transgenic canola by overexpression of Ch FatB2, a thioesterase cDNA from Cuphea hookeriana

The Mexican shrub Cuphea hookeriana accumulates up to 75% caprylate (8:0) and caprate (10:0) in its seed oil. An acyl-ACP thioesterase cDNA from C. hookeriana , designated Ch FatB2 , has been identified, which, when expressed in Escherichia coli , provides thioesterase activity specific for 8:0- and 10:0-ACP substrates. Expression of this clone in seeds of transgenic canola, an oilseed crop that normally does not accumulate any 8:0 and 10:0, resulted in a dramatic increase in the levels of these two fatty acids accompanied by a preferential decrease in the levels of linoleate (18:2) and linolenate (18:3). The Ch FatB2 differs from Ch FatB1 , another Cuphea hookeriana thioesterase reported recently, in both substrate specificity and expression pattern. The Ch FatB1 has a broad substrate specificity with strong preference for 16:0-ACP and is expressed throughout the plant; whereas Ch FatB2 is specific for 8:0/10:0-ACP and its expression is confined to the seed. It is proposed that the amplified expression of Ch FatB2 in the embryo provides the hydrolytic enzyme specificity determining the fatty acyl composition of Cuphea hookeriana seed oil.     

Deposition of stearate-oleate rich seed fat in Mangifera indica is mediated by a FatA type acyl-ACP thioesterase

Although the mechanism of accumulation of C8-C16 saturated fatty acids in seed oils has been well-studied, the control of stearic (C18:0) acid deposition in high stearate seed fat is still unclear. We investigated the mechanism that regulates high level of stearate and oleate (C18:1) accumulation in mango (Mangifera indica) seeds during its development, and examined the seed plastid extracts for induction of any specialized fatty acyl-ACP thioesterase (Fat) that may control this high level of deposition. Though the specificity of the Fat enzymes does not account directly for the fatty acid composition of mango seeds, our result suggested that an induced synthesis of a FatA type of thioesterase could be responsible for the high content of oleate and stearate in its seed fat. The major thioesterase from developing seed kernel was purified to near homogeneity, and characterized as a heat-labile, dimeric, neutral protein with relative substrate specificity of 100:35:1.8 towards oleoyl-, stearoyl- and palmitoyl-ACP, respectively. This enzyme was confirmed as Mi FatA by mass spectrometric analysis. Additionally, a heat-stable FatB type enzyme (Mi FatB) was also partially purified, with relative substrate specificity for the same substrates as 9:8.5:100, respectively. Mi FatA is an enzyme of great biotechnological interest because of its involvement in the regulation of stearate rich seed fat in mango.     

Characterization of acyl‐ACP thioesterases of mangosteen (Garcinia mangostana) seed and high levels of stearate production in transgenic canola

Acyl-acyl-carrier protein (ACP) thioesterases are, at least in part, responsible for the fatty acyl chain length composition of seed storage oils. Acyl-ACP thioesterases with specificity for each of the saturated acyl-ACP substrates from 8:0 through 16:0 have been cloned, with the exception of 18:0, and are members of the FatB class of thioesterases. The authors have determined that the tropical tree species mangosteen (Garcinia mangostana) stores 18:0 (stearate) in its seed oil in amounts of up to 56% by weight. Acyl-ACP thioesterase activity as measured in crude mangosteen seed extracts showed a preference for 18:1-ACP substrates, but had significant activity with 18:0 relative to that with 16:0-ACP, suggesting a thioesterase might be involved in the production of stearate. Three distinct acyl-ACP thioesterases were cloned from mangosteen seed cDNA; two representative of the FatA class and one representative of the FatB class. When expressed in vitro, the enzyme encoded by one of the FatAs (Garm FatA1) while preferring 18:1-ACP showed relatively low activity with 16:0-ACP as compared to 18:0-ACP, similar to the substrate preferences shown by the crude seed extract. Expression of Garm FatA1 in Brassica seeds led to the accumulation of stearate up to 22% in seed oil. These results suggest that Garm FatA1 is at least partially responsible for determining the high stearate composition of mangosteen seed oil and that FatA as well FatB thioesterases have evolved for specialized roles.     

Accumulation of palmitate in Arabidopsis mediated by the acyl-acyl carrier protein thioesterase FATB1

The acyl-acyl carrier protein thioesterase B1 from Arabidopsis (AtFATB1) was previously shown to exhibit in vitro hydrolytic activity for long chain acyl-acyl carrier proteins (P. D?rmann, T.A. Voelker, J.B. Ohlrogge [1995] Arch Biochem Biophys 316: 612-618). In this study, we address the question of which role in fatty acid biosynthesis this enzyme plays within the plant. Over-expression of the AtFATB1 cDNA under a seed-specific promoter resulted in accumulation of high amounts of palmitate (16:0) in seeds. RNA and protein-blot analysis in Arabidopsis and rapeseed (Brassica napus) showed that the endogenous AtFATB1 expression was highest in flowers and lower in leaves. All floral tissues of wild-type plants contained elevated amounts of 16:0, and in the polar lipid fraction of flowers close to 50 mol % of the fatty acids were 16:0. Therefore, flowers contain polar lipids with an unusually high amount of saturated fatty acids as compared to all other plant tissues. Antisense expression of the AtFATB1 cDNA under the cauliflower mosaic virus 35S promoter resulted in a reduction of seed and flower 16:0 content, but no changes in leaf fatty acids. We conclude that the AtFATB1 thioesterase contributes to 16:0 production particularly in flowers, but that additional factors are involved in leaves.     

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 CoA profiles of transgenic plants that accumulate medium-chain fatty acids indicate inefficient storage lipid synthesis in developing oilseeds

Several Brassica napus lines transformed with genes responsible for the synthesis of medium- or long-chain fatty acids were examined to determine limiting factor(s) for the subsequent accumulation of these fatty acids in seed lipids. Examination of a decanoic acid (10:0) accumulating line revealed a disproportionately high concentration of 10:0 CoA during seed development compared to long-chain acyl CoAs isolated from the same tissues, suggesting that poor incorporation of 10:0 CoA into seed lipids limits 10:0 fatty acid accumulation. This relationship was also seen for dodecanoyl (12:0) CoA and fatty acid in a high 12:0 line, but not for octadecanoic (18:0) CoA and fatty acid in a high 18:0 line. Comparison of 10:0 CoA and fatty acid proportions from seeds at different developmental stages for transgenic B. napus and Cuphea hookeriana, the source plant for the medium-chain thioesterase and 3-ketoacyl-ACP synthase transgenes, revealed that C. hookeriana incorporates 10:0 CoA into seed lipids more efficiently than transgenic B. napus. Furthermore, beta-oxidation and glyoxylate cycle activities were not increased above wild type levels during seed development in the 8:0/10:0 line, suggesting that lipid catabolism was not being induced in response to the elevated 10:0 CoA concentrations. Taken together, these data suggest that transgenic plants that are engineered to synthesize medium-chain fatty acids may lack the necessary mechanisms, such as specific acyltransferases, to incorporate these fatty acids efficiently into seed lipids.     

The distribution of caprylate, caprate and laurate in lipids from developing and mature seeds of transgenic Brassica napus L.

The composition and positional distribution of lipids in developing and mature transgenic Brassica napus seeds accumulating up to 7 mol% of caprylate (8:0), 29 mol% caprate (10:0) or 63 mol% of laurate (12:0) were examined. The accumulation of 8:0 and 10:0 resulted from over-expression of the medium-chain-specific thioesterase (Ch FatB2) alone or together with the respective chain-length-specific condensing enzyme (Ch KASIV). Seeds containing high levels of 12:0 were obtained from plants expressing bay thioesterase (BTE) alone or crossed with a line over-expressing the coconut lysophosphatidic acid acyltransferase (LPAAT), an enzyme responsible for the increase in acylation of 12:0 at the sn-2 position. In all instances, 10:0 and 12:0 fatty acids were present in substantial amounts in phosphatidylcholine during seed development with a drastic decrease of 80–90% in mature seeds. At all stages of seed development however, 8:0 was barely detectable in this membrane lipid. Altogether, these results indicate that these transgenic seeds exclude and/or remove the medium-chain fatty acids from their membrane and that this mechanism(s) is more effective with the shorter-chain fatty acids. Furthermore, seeds of 8:0- and 10:0-producing lines had only negligible levels of these fatty acids present in the sn-2 position of the triacylglycerols. In contrast, all 12:0-producing seeds had a substantial amount of this fatty acid in the sn-2 position of the triacylglycerols, suggesting that the endogenous LPAAT is able to acylate 12:0 if no other acyl-CoA species are available. Received: 11 February 2000 / Accepted: 2 May 2000     

Developmental induction, purification, and further characterization of 12:0-ACP thioesterase from immature cotyledons of Umbellularia californica

The fatty acyl content of developing cotyledons of Umbellularia californica (California Bay) changes from a long-chain composition to a predominance of 10:0 and 12:0 in just 4-5 days at the beginning of an approximately 100-day period of medium-chain deposition. This striking change occurs at the earliest appearance of 12:0-acyl-carrier protein (ACP) thioesterase activity. The coincidence of these rapid events is consistent with the hypothesis that the enzyme plays a major role in medium-chain biosynthesis. The 12:0-ACP thioesterase has been substantially purified; enzyme activity consistently comigrates in chromatographic and electrophoretic systems with a protein or pair of proteins having an apparent molecular weight of approximately 34 kDa. A native molecular weight of approximately 42 kDa has been estimated by gel filtration chromatography, suggesting that the enzyme is a monomer. Affinity chromatography on immobilized ACP is a critical step in the purification procedure, and resolves the 12:0-ACP and 18:1-ACP thioesterases sufficiently to confirm that the medium-chain enzyme has negligible action on 18:1-ACP.     

Structural and functional analyses of a saturated acyl ACP thioesterase, type B from immature seed tissue of Jatropha curcas

The distinguishing structural and functional domains of plant acyl-acyl carrier protein (ACP) thioesterases and their complex interaction with the ACP-linked fatty acid substrate complex have remained elusive. E. coli based heterologous expression and characterisation of many plant thioesterases reported so far have not been extended and linked to in silico modelling studies to explain the diversity in plant thioesterase substrate specificities. In this study, a thioesterase cDNA isolated from immature seed tissues of Jatropha curcas was found to be type B and specific to stearoyl acyl ACP when expressed in E. coli K27fadD88, a lipid utilisation mutant. Homology modelling and molecular docking of a selected region of the isolated JcFatB protein predicted that it had high affinity towards both stearate (18:0) and palmitate (16:0). Structural analysis of the sequence confirmed the presence of a transit peptide that is processed in multiple steps. The enzyme is localised in the chloroplasts and has an N-terminal inner chloroplast transmembrane domain characteristic of type B plant thioesterases. Docking of ligands with JcFatB and its comparison with a modelled Jatropha thioesterase type A provided further evidence for native substrate preferences of Jatropha thioesterases. This study provides essential clues to develop future methods for large-scale bacterial production of free fatty acids and for design of strategies to modulate the seed oil composition in this important non-edible, seed oil plant.     

Breeding high-stearic oilseed rape (Brassica napus) with high- and low-erucic background using optimised promoter-gene constructs

Seed lipids of oilseed rape (Brassica napus) usually contain small proportions (<3%) of stearic acid. The objective of this study was to increase the content of stearic fatty␣acid in rapeseed oil. An antisense down-regulation of the endogenous stearoyl-ACP desaturase (SAD) catalysing the reaction step from stearic to oleic acid in two different genetic backgrounds was studied. The result of down-regulation of the SAD yielded an about 10-fold increase of stearic acid from 3.7% up to 32% in single seeds of transgenic low-erucic acid rapeseed (LEAR), while high-erucic acid rapeseed (HEAR) showed a 4-fold increase of C18:0 from 1% up to 4%. It could be shown in pooled T2 seed material of LEAR rapeseed, that the stearic acid content is highly correlated with the down-regulation of SAD as indicated by the␣stearate desaturation proportion (SDP). The importance of the promoter strength for the alteration of a trait was confirmed in this study as no change in the fatty acid composition of transgenic plants was achieved with gene constructs controlled by the weak FatB4 seed-specific promoter from Cuphea lanceolata.Karim Zarhloul and Christof Stoll have contributed in equal parts to the present work     

Post-synthetic modification of plant cell walls by expression of microbial hydrolases in the apoplast

The systematic creation of defined cell wall modifications in the model plant Arabidopsis thaliana by expression of microbial hydrolases with known specific activities is a promising approach to examine the impacts of cell wall composition and structure on both plant fitness and cell wall recalcitrance. Moreover, this approach allows the direct evaluation in living plants of hydrolase specificity, which can differ from in vitro specificity. To express genes encoding microbial hydrolases in A. thaliana, and target the hydrolases to the apoplast compartment, we constructed an expression cassette composed of the Cauliflower Mosaic Virus 35S RNA promoter, the A. thaliana β-expansin signal peptide, and the fluorescent marker protein YFP. Using this construct we successfully introduced into Colombia-0 plants three Aspergillus nidulans hydrolases, β-xylosidase/α-arabinosidase, feruloyl esterase, acetylxylan esterase, and a Xanthomonas oryzae putative a-L: -arabinofuranosidase. Fusion with YFP permitted quick and easy screening of transformants, detection of apoplastic localization, and protein size confirmation. Compared to wild-type Col-0, all transgenic lines showed a significant increase in the corresponding hydrolytic activity in the apoplast and changes in cell wall composition. Examination of hydrolytic activity in the transgenic plants also showed, for the first time, that the X. oryzae gene indeed encoded an enzyme with α-L: -arabinofuranosidase activity. None of the transgenic plants showed a visible phenotype; however, the induced compositional changes increased the degradability of biomass from plants expressing feruloyl esterase and β-xylosidase/α-arabinosidase. Our results demonstrate the viability of creating a set of transgenic A. thaliana plants with modified cell walls to use as a toolset for investigation of how cell wall composition contributes to recalcitrance and affects plant fitness.     

Overproduction of alfalfa glutamine synthetase in transgenic tobacco plants

Summary We have obtained transgenic tobacco plants overexpressing the enzyme glutamine synthetase (GS) by fusing an alfalfa GS gene to the cauliflower mosaic virus 35S promotor and integrating it intoNicotiana tabacum var. W38 plants byAgrobacterium tumefaciens mediated gene transfer. The amount of RNA specific to alfalfa GS was about 10 times higher in transgenic tobacco plants than in alfalfa. The alfalfa GS produced by these transgenic plants was identified by Western blotting and represented 5% of total soluble protein in the transformed plants, amounting to a 5-fold increase in specific GS activity and in a 20-fold increase in resistance to the GS inhibitorl-phosphinothricin in vitro. Tissue from GS overproducing plants showed a sevenfold lower amount of free NH₃. The amino acid composition of the plant tissue was not altered significantly by GS overproduction. GS overproducing plants were fertile and grew normally. These data show that a high level of expression of a key metabolic enzyme such as glutamine synthetase does not interfere with growth and fertility of plants.     

Specificities of the Acyl-Acyl Carrier Protein (ACP) Thioesterase and Glycerol-3-Phosphate Acyltransferase for Octadecenoyl-ACP Isomers (Identification of a Petroselinoyl-ACP Thioesterase in Umbelliferae)

This study was designed to address the question: How specific for double bond position and conformation are plant enzymes that act on oleoyl-acyl carrier protein (ACP)? Octadecenoyl-ACPs with cis double bonds at positions [delta]6, [delta]7, [delta]8, [delta]9, [delta]10, [delta]11, or [delta]12 and elaidyl (18:1[delta]9trans)-ACP were synthesized and used to characterize the substrate specificity of the acyl-ACP thioesterase and acyl-ACP:sn-glycerol-3-phosphate acyltransferase. The two enzymes were found to be specific for the [delta]9 position of the double bond. The thioesterase was highly specific for the [delta]9 cis conformation, but the transferase was almost equally active with the cis and the trans isomer of 18:1[delta]9-ACP. In plants such as the Umbelliferae species coriander (Coriandrum sativum L.) that accumulate petroselinic acid (18:1[delta]6cis) in their seed triacylglycerols, a high petroselinoyl-ACP thioesterase activity was found in addition to the oleoyl-ACP thioesterase. The two activities could be separated by anion-exchange chromatography, indicating that the petroselinoyl-ACP thioesterase is represented by a distinct polypeptide.     

Isolation and functional characterisation of the genes encoding Δ(8)-Sphingolipid desaturase from Brassica rapa

△~8-Sphingohpid desaturase is the key enzyme that catalyses desaturation at the C8 position of the long-chain base of sphingolipids in higher plants.There have been no previous studies on the genes encoding△~8-sphingolipid desaturases in Brassica rapa.In this study,four genes encoding A -sphingohpid desaturases from B.rapa were isolated and characterised.Phylogenetic analyses indicated that these genes could be divided into two groups:BrD8A,BrD8C and BrD8D in groupⅠ,and BrD8B in groupⅡ.The two groups of genes diverged before the separation of Arabidopsis and Brassica.Though the four genes shared a high sequence similarity,and their coding desaturases all located in endoplasmic reticulum,they exhibited distinct expression patterns.Heterologous expression in Saccharomyces cerevisiae revealed that BrD8A/B/C/D were functionally diverse A -sphingohpid desaturases that catalyse different ratios of the two products 8(Z)- and 8(E)-C18-phytosphingenine.The aluminium tolerance of transgenic yeasts expressing BrD8A/B/C/D was enhanced compared with that of control cells.Expression of BrD8A in Arabidopsis changed the ratio of 8(Z):8(E)-C18-phytosphingenine in transgenic plants. The information reported here provides new insights into the biochemical functional diversity and evolutionary relationship of△~8-sphingolipid desaturase in plants and lays a foundation for further investigation of the mechanism of 8(Z)- and 8(E)-C18-phytosphingenine biosynthesis.     

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.