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.     

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.     

The 5[prime] Flanking Regions of Vicilin and Napin Storage Protein Genes Are Down-Regulated by Desiccation in Transgenic Tobacco

Drying of seeds, when imposed prematurely, elicits a switch in metabolism; events unique to development, such as synthesis of storage protein, are terminated, whereas syntheses associated with germination and growth are initiated. To determine the role of desiccation in down-regulating the expression of genes for storage proteins, the desiccation responsiveness of the 5[prime] and 3[prime] regulatory regions of the genes encoding the pea storage protein vicilin and the Brassica napus storage protein napin was tested in transgenic tobacco seed. Chimeric genes were introduced into tobacco; these genes consisted of the coding region of the reporter gene for [beta]-glucuronidase (GUS) and 5[prime] and/or 3[prime] regions from the vicilin or napin genes or, as controls, the same regions derived from constitutively expressed genes, presumed to be desiccation insensitive. In transgenic seed expressing the gene constructs containing the vicilin or napin promoters, GUS activities declined during late seed development, and more dramatically, after imbibition of mature dry seed or prematurely dried seed. In contrast, GUS activities increased after seed rehydration when the constitutive viral promoter replaced the storage-protein gene 5[prime] region. Transient expression assays support the hypothesis that premature drying down-regulates the expression of the storage-protein gene promoter. Following desiccation, this region may become insensitive to positive controlling factors; alternatively, changes to trans-acting factors may occur.     

Utility of the Arabidopsis FAE1 and yeast SLC1-1 genes for improvements in erucic acid and oil content in rapeseed

High-erucic acid (HEA) Brassica napus cultivars are regaining interest in industrial contexts. Erucic acid and its derivatives are important renewable raw materials utilized in the manufacture of plastic films, in the synthesis of Nylon 13,13, and in the lubricant and emollient industries. Theoretically, the highest level of erucic acid that can be achieved by means of classical breeding is 66 mol%; however, using new approaches on the basis of genetic engineering, it might be possible to develop a B. napus cultivar containing levels of erucic acid significantly above 66 mol% (>80 mol%). In an attempt to increase the amounts of very-long-chain fatty acids (VLCFAs), and erucic acid in particular, in Canadian HEA B. napus cultivars, we have focused on two targets using a transgenic approach. We examined both the role/function of the Arabidopsis thaliana FAE1 (fatty acid elongase) gene by expressing it under the control of the seed-specific napin promoter in B. napus germplasm with analysis of the changes in VLCFA content in the seed oil of transgenic lines, and the performance of the yeast SLC1-1 (sphingolipid compensation mutant) in B. napus cv. Hero transgenic progeny in the field. Here, we report analyses of the contents of 22:1, total VLCFAand oil in the seed oil, as well as seed yield of the field-grown FAE1 and SLC1-1 B. napus cv. Hero progeny.     

Fatty acid distribution and lipid metabolism in developing seeds of laurate-producing rape (Brassica napus L.)

The fatty acid composition and content of membrane and storage lipids of two transgenic laurate-producing rape (Brassica napus L.) lines were monitored during seed development. The two lines, the medium-laurate (ML) line and the high-laurate (HL) line, accumulated 34 mol% and 55 mol% of laurate in their seed triacylglycerols, respectively. The diacylglycerols contained about 17 and 33 mol% of laurate in the ML- and HL-lines, respectively, from the mid-stage of seed development up to seed maturity. The ML-line showed a maximal relative laurate content in phosphatidylcholine (17 mol%) at the mid-stage of seed development whereafter the content decreased to 2.7 mol% with seed maturity. The laurate content in phosphatidylcholine was observed to remain high (26 mol%) in the HL-line from the mid-stage to the end of triacylglycerol deposition. Thereafter, the relative content decreased and reached 6.6 mol% in the mature seeds. There was an enhanced activity of lauroyl-phosphatidylcholine- metabolizing enzymes in the seed membranes from laurate-producing lines compared with control lines, which might explain the decrease seen in laurate content in phosphatidylcholine during seed maturation. A comparison of the laurate distribution in the lipids from developing laurate-producing rape seeds and developing seeds from three species naturally accumulating laurate at similar levels revealed differences in laurate metabolism compared with these species. The results suggest that phospholipids and triacylglycerols are synthesized from the same diacylglycerol pool in rape seeds and that rape lysophosphatidic acid acyltransferase and diacylglycerol acyltransferase do not have the same preference for laurate substrates as the corresponding enzymes in seed tissues naturally accumulating this acyl group. In addition, the mechanisms that specifically remove or exclude laurate from membrane lipids appear less effective in rape seed than in tissues naturally evolved to synthesize laurate-rich oils. Received: 23 December 1996 / Accepted: 16 April 1997     

Expression of lauroyl-acyl carrier protein thioesterase in brassica napus seeds induces pathways for both fatty acid oxidation and biosynthesis and implies a set point for triacylglycerol accumulation

Expression of a California bay lauroyl-acyl carrier protein thioesterase (MCTE) in developing seeds of transgenic oilseed rape alters the fatty acid composition of the mature seed, resulting in up to 60 mol% of laurate in triacylglycerols. In this study, we examined the metabolism of lauric acid and 14C-acetate in developing seeds of oilseed rape that express high levels of MCTE. Lauroyl-CoA oxidase activity but not palmitoyl-CoA oxidase activity was increased several-fold in developing seeds expressing MCTE. In addition, isocitrate lyase and malate synthase activities were six- and 30-fold higher, respectively, in high-laurate developing seeds. Control seeds incorporated 14C-acetate almost entirely into fatty acids, whereas in seeds expressing MCTE, only 50% of the label was recovered in lipids and the remainder was in a range of water-soluble components, including sucrose and malate. Together, these results indicate that the pathways for beta-oxidation and the glyoxylate cycle have been induced in seeds expressing high levels of MCTE. Although a substantial portion of the fatty acid produced in these seeds is recycled to acetyl-CoA and sucrose through the beta-oxidation and glyoxylate cycle pathways, total seed oil is not reduced. How is oil content maintained if lauric acid is inefficiently converted to triacylglycerol? The levels of acyl carrier protein and several enzymes of fatty acid synthesis were increased two- to threefold at midstage development in high-laurate seeds. These results indicate that a coordinate induction of the fatty acid synthesis pathway occurs, presumably to compensate for the lauric acid lost through beta-oxidation or for a shortage of long-chain fatty acids.     

Characterization of recombinant thioesterase and acyl carrier protein domains of chicken fatty acid synthase expressed in Escherichia coli

Fatty acid synthase of animal tissue is a multifunctional enzyme comprised of two identical subunits, each containing seven partial activities and a site for the prosthetic group, 4'-phosphopantetheine (acyl carrier protein). We have recently isolated cDNA clones of chicken fatty acid synthase coding for the dehydratase, enoyl reductase, beta-ketoacyl reductase, acyl carrier protein, and thioesterase domains (Chirala, S.S., Kasturi, R., Pazirandeh, M., Stolow, D.T., Huang, W.Y., and Wakil, S.J. (1989) J. Biol. Chem. 264, 3750-3757). To gain insight into the structure and function of the various domains, the portion of the cDNA coding for the acyl carrier protein and thioesterase domains was expressed in Escherichia coli by using an expression vector that utilizes the phage lambda PL promoter. The recombinant protein was efficiently expressed and purified to near homogeneity using anion-exchange and hydroxyapatite chromatography. As expected from the coding capacity of the cDNA expressed, the protein has a molecular weight of 43,000 and reacts with antithioesterase antibodies. The recombinant thioesterase was found to be enzymatically active and has the same substrate specificity and kinetic properties as the native enzyme of the multifunctional synthase. Treatment of the recombinant protein with alpha-chymotrypsin results in the cleavage of the acyl carrier protein and thioesterase domain junction sequence at exactly the same site as with native fatty acid synthase. The amino acid composition of the purified recombinant protein revealed the presence of 0.6 mol of beta-alanine/mol of protein, indicating partial pantothenylation of the recombinant acyl carrier protein domain. These results indicate that the expressed protein has a conformation similar to the native enzyme and that its folding into functionally active domains is independent of the remaining domains of the multifunctional synthase subunit. These conclusions are consistent with the proposal that the multifunctional synthase gene has evolved from fusion of component genes.     

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.     

Natural variation of GmFATA1B regulates seed oil content and composition in soybean

Soybean (Glycine max) produces seeds that are rich in unsaturated fatty acids and is an important oilseed crop worldwide. Seed oil content and composition largely determine the economic value of soybean. Due to natural genetic variation, seed oil content varies substantially across soybean cultivars. Although much progress has been made in elucidating the genetic trajectory underlying fatty acid metabolism and oil biosynthesis in plants, the causal genes for many quantitative trait loci (QTLs) regulating seed oil content in soybean remain to be revealed. In this study, we identified GmFATA1B as the gene underlying a QTL that regulates seed oil content and composition, as well as seed size in soybean. Nine extra amino acids in the conserved region of GmFATA1B impair its function as a fatty acyl–acyl carrier protein thioesterase, thereby affecting seed oil content and composition. Heterogeneously overexpressing the functional GmFATA1B allele in Arabidopsis thaliana increased both the total oil content and the oleic acid and linoleic acid contents of seeds. Our findings uncover a previously unknown locus underlying variation in seed oil content in soybean and lay the foundation for improving seed oil content and composition in soybean.     

Vicilin and Napin Storage-Protein Gene Promoters Are Responsive to Abscisic Acid in Developing Transgenic Tobacco Seed but Lose Sensitivity following Premature Desiccation

In transgenic tobacco (Nicotiana tabacum L.) seed, expression of chimeric [beta]-glucuronidase (GUS) genes containing the vicilin or napin storage-protein gene promoters is responsive to premature drying and declines upon rehydration (L. Jiang, W.L. Downing, C.L. Baszczynski, A.R. Kermode [1995] Plant Physiol 107: 1439-1449). Desiccation may cause changes in the content of or sensitivity to abscisic acid (ABA), partially or wholly removing the effects of this "modulator" of developmental gene expression. We studied the enhancement of GUS reporter enzyme activities in transgenic tobacco by exogenous ABA before and after drying. A racemic mixture of ABA at 10 [mu]M caused a 2- to 3-fold increase in GUS activity in developing transgenic seed expressing chimeric genes containing the vicilin or napin gene promoters. However, when these seeds were prematurely dried prior to their culture on ABA medium, enhancement of GUS activity was virtually abolished. Use of optically pure ABAs revealed that the enhancement in GUS activity in developing fresh seed was due largely to the natural (+) form of ABA. Chimeric constructs containing a viral 35S promoter did not respond to ABA whether or not premature drying was applied. Thus, vicilin and napin chimeric genes, initially sensitive to ABA, become relatively insensitive to the hormone following drying. A decline in ABA sensitivity may be an important factor in the cessation of storage-protein gene expression.     

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.     

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.     

Stacking of a stearoyl‐ACP thioesterase with a dual‐silenced palmitoyl‐ACP thioesterase and ∆12 fatty acid desaturase in transgenic soybean

Soybean (Glycine max (L.) Merr) is valued for both its protein and oil, whose seed is composed of 40% and 20% of each component, respectively. Given its high percentage of polyunsaturated fatty acids, linoleic acid and linolenic acid, soybean oil oxidative stability is relatively poor. Historically food processors have employed a partial hydrogenation process to soybean oil as a means to improve both the oxidative stability and functionality in end‐use applications. However, the hydrogenation process leads to the formation of trans‐fats, which are associated with negative cardiovascular health. As a means to circumvent the need for the hydrogenation process, genetic approaches are being pursued to improve oil quality in oilseeds. In this regard, we report here on the introduction of the mangosteen (Garcinia mangostana) stearoyl‐ACP thioesterase into soybean and the subsequent stacking with an event that is dual‐silenced in palmitoyl‐ACP thioesterase and ?12 fatty acid desaturase expression in a seed‐specific fashion. Phenotypic analyses on transgenic soybean expressing the mangosteen stearoyl‐ACP thioesterase revealed increases in seed stearic acid levels up to 17%. The subsequent stacked with a soybean event silenced in both palmitoyl‐ACP thioesterase and ?12 fatty acid desaturase activity, resulted in a seed lipid phenotype of approximately 11%–19% stearate and approximately 70% oleate. The oil profile created by the stack was maintained for four generations under greenhouse conditions and a fifth generation under a field environment. However, in generation six and seven under field conditions, the oleate levels decreased to 30%–40%, while the stearic level remained elevated.     

napin基因启动子克隆及进化分析

napin是一个重要的种子贮藏蛋白,它以组织特异性方式表达形成。以甘蓝型油菜为材料,克隆了napin基因启动子,测序结果表明,该启动子全长1 135bp,含有启动子特有的TATA-box等调控元件。系统进化分析显示,拟南芥、萝卜、白菜型油菜和甘蓝型油菜napin基因启动子之间既具有较高的同源性,又存在一定的差别。通过进化分析,指导我们在油菜脂肪酸改良过程中,采用不同的启动子在脂肪酸改良方面可能起到不同的效果。     

Synthesis of the major oil-body membrane protein in developing rapeseed (Brassica napus) embryos. Integration with storage-lipid and storage-protein synthesis and implications for the mechanism of oil-body formation.

The synthesis of the major protein and lipid storage reserves during embryogenesis in oilseed rape (Brassica napus L., cv. Mikado) has been examined by biochemical, immunological and immunocytochemical techniques. The mature seeds contained about 45% (w/w) storage oil and 25% (w/w) protein. There were three major seed protein components, i.e. about 40-50% total protein was cruciferin, 20% was napin and 20% was a 18 kDa hydrophobic polypeptide associated with the proteinaceous membrane surrounding the storage oil bodies. Embryogenesis was divided into four overlapping stages with regard to the synthesis of these storage components: (1) for the first 3 weeks after flowering, little, if any, synthesis of storage components was observed; (2) storage-oil synthesis began at about week 3, and maximal rates were from weeks 4 to 7; (3) synthesis of the soluble storage proteins cruciferin and napin started at week 6 and rates were maximal between weeks 8 and 11; (4) the final stage was the synthesis of the 19 kDa oil-body polypeptide, which started at weeks 8-10 and was at a maximal rate between weeks 10 and 12. The synthesis of the 19 kDa oil-body protein therefore occurred independently of the synthesis of the soluble seed storage proteins. This former synthesis did not occur until shortly before the insertion of the 19 kDa polypeptide into the oil-body membrane. No evidence was found, either from sucrose-density-gradient-centrifugation experiments or from immunogold-labelling studies, for its prior accumulation in the endoplasmic reticulum. Conventional and immunogold-electron-microscopic studies showed that oil bodies were synthesized in the early to middle stages of seed development without a strongly electron-dense membrane. Such a membrane was only found at later stages of seed development, concomitantly with the synthesis of the 19 kDa protein. It is proposed that, in rapeseed embryos, oil bodies are initially formed with no proteinaceous membrane. Such a membrane is formed later in development after insertion by ribosomes of the hydrophobic 19 kDa polypeptide directly into the oil bodies.