Acyl-acyl Carrier Protein Thioesterase Research Articles

Molecular cloning and characterisation of an acyl carrier protein thioesterase gene (CocoFatB1) expressed in the endosperm of coconut (Cocos nucifera) and its heterologous expression in Nicotiana tabacum to engineer the accumulation of different fatty acids.

Coconut (Cocos nucifera L.) contains large amounts of medium chain fatty acids, which mostly recognise acyl-acyl carrier protein (ACP) thioesterases that hydrolyse acyl-ACP into free fatty acids to terminate acyl chain elongation during fatty acid biosynthesis. A full-length cDNA of an acyl-ACP thioesterase, designated CocoFatB1, was isolated from cDNA libraries prepared from coconut endosperm during fruit development. The gene contained an open reading frame of 1254 bp, encoding a 417-amino acid protein. The amino acid sequence of the CocoFatB1 protein showed 100% and 95% sequence similarity to CnFatB1 and oil palm (Elaeis guineensis Jacq.) acyl-ACP thioesterases, respectively. Real-time fluorescent quantitative PCR analysis indicated that the CocoFatB1 transcript was most abundant in the endosperm from 8-month-old coconuts; the leaves and endosperm from 15-month-old coconuts had ~80% and ~10% of this level. The CocoFatB1 coding region was overexpressed in tobacco (Nicotiana tabacum L.) under the control of the seed-specific napin promoter following Agrobacterium tumefaciens-mediated transformation. CocoFatB1 transcript expression varied 20-fold between different transgenic plants, with 21 plants exhibiting detectable levels of CocoFatB1 expression. Analysis of the fatty acid composition of transgenic tobacco seeds showed that the levels of myristic acid (14 : 0), palmitic acid (16 : 0) and stearic acid (18 : 0) were increased by 25%, 34% and 17%, respectively, compared with untransformed plants. These results indicated that CocoFatB1 acts specifically on 14 : 0-ACP, 16 : 0-ACP and 18 : 0-ACP, and can increase medium chain saturated fatty acids. The gene may valuable for engineering fatty acid metabolism in crop improvement programmes.

Comparative Transcriptome Analysis of Three Oil Palm Fruit and Seed Tissues That Differ in Oil Content and Fatty Acid Composition

Oil palm (Elaeis guineensis) produces two oils of major economic importance, commonly referred to as palm oil and palm kernel oil, extracted from the mesocarp and the endosperm, respectively. While lauric acid predominates in endosperm oil, the major fatty acids (FAs) of mesocarp oil are palmitic and oleic acids. The oil palm embryo also stores oil, which contains a significant proportion of linoleic acid. In addition, the three tissues display high variation for oil content at maturity. To gain insight into the mechanisms that govern such differences in oil content and FA composition, tissue transcriptome and lipid composition were compared during development. The contribution of the cytosolic and plastidial glycolytic routes differed markedly between the mesocarp and seed tissues, but transcriptional patterns of genes involved in the conversion of sucrose to pyruvate were not related to variations for oil content. Accumulation of lauric acid relied on the dramatic up-regulation of a specialized acyl-acyl carrier protein thioesterase paralog and the concerted recruitment of specific isoforms of triacylglycerol assembly enzymes. Three paralogs of the WRINKLED1 (WRI1) transcription factor were identified, of which EgWRI1-1 and EgWRI1-2 were massively transcribed during oil deposition in the mesocarp and the endosperm, respectively. None of the three WRI1 paralogs were detected in the embryo. The transcription level of FA synthesis genes correlated with the amount of WRI1 transcripts and oil content. Changes in triacylglycerol content and FA composition of Nicotiana benthamiana leaves infiltrated with various combinations of WRI1 and FatB paralogs from oil palm validated functions inferred from transcriptome analysis.

Modulating Membrane Composition Alters Free Fatty Acid Tolerance in Escherichia coli

Microbial synthesis of free fatty acids (FFA) is a promising strategy for converting renewable sugars to advanced biofuels and oleochemicals. Unfortunately, FFA production negatively impacts membrane integrity and cell viability in Escherichia coli, the dominant host in which FFA production has been studied. These negative effects provide a selective pressure against FFA production that could lead to genetic instability at industrial scale. In prior work, an engineered E. coli strain harboring an expression plasmid for the Umbellularia californica acyl-acyl carrier protein (ACP) thioesterase was shown to have highly elevated levels of unsaturated fatty acids in the cell membrane. The change in membrane content was hypothesized to be one underlying cause of the negative physiological effects associated with FFA production. In this work, a connection between the regulator of unsaturated fatty acid biosynthesis in E. coli, FabR, thioesterase expression, and unsaturated membrane content was established. A strategy for restoring normal membrane saturation levels and increasing tolerance towards endogenous production of FFAs was implemented by modulating acyl-ACP pools with a second thioesterase (from Geobacillus sp. Y412MC10) that primarily targets medium chain length, unsaturated acyl-ACPs. The strategy succeeded in restoring membrane content and improving viability in FFA producing E. coli while maintaining FFA titers. However, the restored fitness did not increase FFA productivity, indicating the existence of additional metabolic or regulatory barriers.

Improvement of free fatty acid production in Escherichia coli using codon-optimized Streptococcus pyogenes acyl-ACP thioesterase

Fatty acyl-acyl carrier protein (ACP) thioesterase (acyl-ACP TE) from Streptococcus pyogenes (strain MGAS10270) was codon-optimized and expressed in Escherichia coli K-12 W3110 and Escherichia coli K-12 MG1655. By employing codon-optimized S. pyogenes acyl-ACP TE to improve the total free fatty acids (FFAs) and to tailor the composition of FFAs, high-specificity production of saturated fatty acids (C12, C14) and unsaturated fatty acids (C18:1 C18:2) was achieved in recombinants. E. coli SGJS41 and SGJS46 (codon-optimized acyl-ACP TE of S. pyogenes) demonstrated the highest intracellular total FFA content (339mg/l vs 342mg/l); in particular, the content of C12 and C14 FFAs was about 3-5 fold, and the content of C18:1 and C18:2 FFAs was about 8-42 fold higher than that in the control E. coli and E. coli JES1017 (original acyl-ACP TE of S. pyogenes).

Molecular cloning and characterization of an acyl-ACP thioesterase gene (AhFatB1) from allotetraploid peanut (Arachis hypogaea L.)

Acyl-acyl carrier protein (ACP) thioesterase is a nuclear encoded plastid localized enzyme which plays an essential role in chain termination during de novo fatty acid synthesis in plant. FatB genes coding for this enzyme from a variety of plant species have been isolated and characterized. However, there are few reports on such genes in peanut ( Arachis hypogaea ), an important edible and oilseed crop. In this study, full-length cDNA of an acyl-acyl carrier protein thioesterase (EC 3.1.2.14), designated as AhFatB1 , was isolated from peanut cDNA libraries. The putative open reading frames consist of 1239 bp with five introns spliced from the corresponding genomic sequence, encoding a 413 amino acid protein, two homologous genes, AhFatB1A and AhFatB1B , with sequence difference at the 5’ non-coding regions were characterized at the nucleotide level from different cultivated peanut genotypes, and the two genes have their origin in different diploid progenitor which was evidenced by the characterization of AhFatB1 genes from Arachis duranensis and Arachis ipaensis , the putative A-genome donor and B-genome donor respectively. AhFatB1 genes are constitutively expressed in peanut tissues and the total FatB1 transcript accumulations are temporally regulated during peanut seed development. Keywords: Peanut thioesterase, palmitic acid oilseed

Heterologous Co-expression of accA, fabD, and Thioesterase Genes for Improving Long-Chain Fatty Acid Production in Pseudomonas aeruginosa and Escherichia coli

The goal of the present study was to increase the content of intracellular long-chain fatty acids in two bacterial strains, Pseudomonas aeruginosa PA14 and Escherichia coli K-12 MG1655, by co-overexpressing essential enzymes that are involved in the fatty acid synthesis metabolic pathway. Recently, microbial fatty acids and their derivatives have been receiving increasing attention as an alternative source of fuel. By introducing two genes (accA and fabD) of P. aeruginosa into the two bacterial strains and by co-expressing with them the fatty acyl-acyl carrier protein thioesterase gene of Streptococcus pyogenes (strain MGAS10270), we have engineered recombinant strains that are efficient producers of long-chain fatty acids (C16 and C18). The recombinant strains exhibit a 1.3-1.7-fold increase in the production of long-chain fatty acids over the wild-type strains. To enhance the production of total long-chain fatty acids, we researched the carbon sources for optimized culture conditions and results were used for post-culture incubation period. E. coli SGJS17 (containing the accA, fabD, and thioesterase genes) produced the highest content of intracellular total fatty acids; in particular, the unsaturated fatty acid content was about 20-fold higher than that in the wild-type E. coli.

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.

Engineering Escherichia coli for Biodiesel Production Utilizing a Bacterial Fatty Acid Methyltransferase

The production of low-cost biofuels in engineered microorganisms is of great interest due to the continual increase in the world's energy demands. Biodiesel is a renewable fuel that can potentially be produced in microbes cost-effectively. Fatty acid methyl esters (FAMEs) are a common component of biodiesel and can be synthesized from either triacylglycerol or free fatty acids (FFAs). Here we report the identification of a novel bacterial fatty acid methyltransferase (FAMT) that catalyzes the formation of FAMEs and 3-hydroxyl fatty acid methyl esters (3-OH-FAMEs) from the respective free acids and S-adenosylmethionine (AdoMet). FAMT exhibits a higher specificity toward 3-hydroxy free fatty acids (3-OH-FFAs) than FFAs, synthesizing 3-hydroxy fatty acid methyl esters (3-OH-FAMEs) in vivo. We have also identified bacterial members of the fatty acyl-acyl carrier protein (ACP) thioesterase (FAT) enzyme family with distinct acyl chain specificities. These bacterial FATs exhibit increased specificity toward 3-hydroxyacyl-ACP, generating 3-OH-FFAs, which can subsequently be utilized by FAMTs to produce 3-OH-FAMEs. PhaG (3-hydroxyacyl ACP:coenzyme A [CoA] transacylase) constitutes an alternative route to 3-OH-FFA synthesis; the coexpression of PhaG with FAMT led to the highest level of accumulation of 3-OH-FAMEs and FAMEs. The availability of AdoMet, the second substrate for FAMT, is an important factor regulating the amount of methyl esters produced by bacterial cells. Our results indicate that the deletion of the global methionine regulator metJ and the overexpression of methionine adenosyltransferase result in increased methyl ester synthesis.

Characterization of a novel thioesterase (PtTE) from Phaeodactylum tricornutum

The Phaeodactylum tricornutum (P. tricornutum) thioesterase PtTE was encoded by a 648 bp open reading frame. The deduced 216 amino acids showed no similarity with plant acyl-acyl carrier protein (ACP) thioesterases and bacterial thioesterases. Southern blot analysis revealed that one copy of PtTE was present in the P. tricornutum genome, and Real-time quantitative PCR showed that PtTE was up-regulated upon nitrogen deprivation. Thioesterase activity of PtTE was established by heterologous expression of PtTE cDNA in Escherichia coli (E. coli) XL1-Blue and K27fadD88, a mutant strain of fatty acid β-oxidation pathway. The substrate specificity of PtTE was determined by fatty acid profile analyses of the culture supernatant and membrane lipid of recombinant strains. Recombinant PtTE in E.coli enhanced total fatty acid content of XL1-Blue by 21%, and also changed the fatty acid compositions of membrane lipid and culture supernatant. These changes were directed predominantly towards C18:0 and C18:1 fatty acids. Overexpression of PtTE alone in P. tricornutum did not alter the fatty acid composition of P. tricornutum, but enhanced total fatty acid content by 72%. This novel thioesterase gene shows its potential in metabolic engineering for enhancing lipid yield of microalgae. This is so far the first report of thioesterase from eukaryotic microalgae.

Bacterial production of free fatty acids from freshwater macroalgal cellulose

The predominant strategy for using algae to produce biofuels relies on the overproduction of lipids in microalgae with subsequent conversion to biodiesel (methyl-esters) or green diesel (alkanes). Conditions that both optimize algal growth and lipid accumulation rarely overlap, and differences in growth rates can lead to wild species outcompeting the desired lipid-rich strains. Here, we demonstrate an alternative strategy in which cellulose contained in the cell walls of multicellular algae is used as a feedstock for cultivating biofuel-producing microorganisms. Cellulose was extracted from an environmental sample of Cladophora glomerata-dominated periphyton that was collected from Lake Mendota, WI, USA. The resulting cellulose cake was hydrolyzed by commercial enzymes to release fermentable glucose. The hydrolysis mixture was used to formulate an undefined medium that was able to support the growth, without supplementation, of a free fatty acid (FFA)-overproducing strain of Escherichia coli (Lennen et. al 2010). To maximize free fatty acid production from glucose, an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible vector was constructed to express the Umbellularia californica acyl-acyl carrier protein (ACP) thioesterase. Thioesterase expression was optimized by inducing cultures with 50 μM IPTG. Cell density and FFA titers from cultures grown on algae-based media reached 50% of those (∼90 μg/mL FFA) cultures grown on rich Luria-Bertani broth supplemented with 0.2% glucose. In comparison, cultures grown in two media based on AFEX-pretreated corn stover generated tenfold less FFA than cultures grown in algae-based media. This study demonstrates that macroalgal cellulose is a potential carbon source for the production of biofuels or other microbially synthesized compounds.

Development of Escherichia coli MG1655 strains to produce long chain fatty acids by engineering fatty acid synthesis (FAS) metabolism

The goal of this research was to develop recombinant Escherichia coli to improve fatty acid synthesis (FAS). Genes encoding acetyl-CoA carboxylase ( accA, accB, accC), malonyl-CoA-[acyl-carrier-protein] transacylase ( fabD), and acyl-acyl carrier protein thioesterase (EC 3.1.2.14 gene), which are all enzymes that catalyze key steps in the synthesis of fatty acids, were cloned and over-expressed in E. coli MG1655. The acetyl-CoA carboxylase (ACC) enzyme catalyzes the addition of CO 2 to acetyl-CoA to generate malonyl-CoA. The enzyme encoded by the fabD gene converts malonyl-CoA to malonyl-[acp], and the EC 3.1.2.14 gene converts fatty acyl-ACP chains to long chain fatty acids. All the genes except for the EC 3.1.2.14 gene were homologous to E. coli genes and were used to improve the enzymatic activities to over-express components of the FAS pathway through metabolic engineering. All recombinant E. coli MG1655 strains containing various gene combinations were developed using the pTrc99A expression vector. To observe changes in metabolism, the in vitro metabolites and fatty acids produced by the recombinants were analyzed. The fatty acids (C16) from recombinant strains were produced 1.23–2.41 times higher than that from the wild type.

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.

Acyl-ACP thioesterases from macadamia (Macadamia tetraphylla) nuts: Cloning, characterization and their impact on oil composition

The mechanisms by which macadamia nuts accumulate the unusual palmitoleic and asclepic acyl moieties, which constitute up to 20% of the fatty acids in some varieties, are still unknown. Acyl-acyl carrier protein (ACP) thioesterases (EC 3.1.2.14) are intraplastidial enzymes that terminate the synthesis of fatty acids in plants and that facilitate the export of the acyl moieties to the endoplasmic reticulum where they can be used in the production of glycerolipids. Here, we have investigated the possible role of acyl-ACP thioesterase activity in the composition of macadamia kernel oil. Accordingly, two acyl-ACP thioesterases were cloned from developing macadamia kernels, one of the FatA type and the other of the FatB type. These enzymes were heterologously expressed in Escherichia coli, and the recombinant thioesterases were purified, characterized kinetically and assayed with a variety of substrates, demonstrating the high specificity of macadamia FatA towards 16:1-ACP. Acyl-ACP thioesterase activity was also characterized in crude extracts from two different varieties of macadamia, Cate and Beaumont, which accumulate different amounts of n-7 fatty acids. The impact of acyl-ACP thioesterase activities on the oil composition of these kernels is discussed in the light of these results.

Stacking Multiple Transgenes at a Selected Genomic Site via Repeated Recombinase-Mediated DNA Cassette Exchanges

Recombinase-mediated DNA cassette exchange (RMCE) has been successfully used to insert transgenes at previously characterized genomic sites in plants. Following the same strategy, groups of transgenes can be stacked to the same site through multiple rounds of RMCE. A gene-silencing cassette, designed to simultaneously silence soybean (Glycine max) genes fatty acid ω-6 desaturase 2 (FAD2) and acyl-acyl carrier protein thioesterase 2 (FATB) to improve oleic acid content, was first inserted by RMCE at a precharacterized genomic site in soybean. Selected transgenic events were subsequently retransformed with the second DNA construct containing a Yarrowia lipolytica diacylglycerol acyltransferase gene (DGAT1) to increase oil content by the enhancement of triacylglycerol biosynthesis and three other genes, a Corynebacterium glutamicum dihydrodipicolinate synthetase gene (DHPS), a barley (Hordeum vulgare) high-lysine protein gene (BHL8), and a truncated soybean cysteine synthase gene (CGS), to improve the contents of the essential amino acids lysine and methionine. Molecular characterization confirmed that the second RMCE successfully stacked the four overexpression cassettes to the previously integrated FAD2-FATB gene-silencing cassette. Phenotypic analyses indicated that all the transgenes expressed expected phenotypes.

Production and secretion of fatty acids in genetically engineered cyanobacteria

Our purpose is to apply a fatty acid secretion strategy in photosynthetic microbial biofuel production, which will avoid the costly biomass recovery processes currently applied in algal biofuel systems. Starting with introducing acyl-acyl carrier protein thioesterases, we made five successive generations of genetic modifications into cyanobacterium Synechocystis sp. PCC 6803. The mutant strains were able to overproduce fatty acids (C10-C18) and secrete them into the medium at an efficiency of up to 133 +/- 12 mg/L of culture per day at a cell density of 1.5 x 10(8) cells/mL (0.23 g of dry weight/liter). Fatty acid secretion yields were increased by weakening the S layer and peptidoglycan layers. Although the fatty acid secreting strains had a long lag phase with many cells having damaged cell membranes when grown at low cell densities, these strains grew more rapidly in stationary phase and exhibited less cell damage than wild-type in a stationary culture. Our results suggest that fatty acid secreting cyanobacteria are a promising technology for renewable biofuel production.

Cloning and characterization of cDNAs encoding for long-chain saturated acyl-ACP thioesterases from the developing seeds of Brassica juncea

Four types of cDNAs corresponding to the fatty acyl-acyl carrier protein (ACP) thioesterase (Fat) enzyme were isolated from the developing seeds of Brassica juncea, a widely cultivated species amongst the oil-seed crops. The mature polypeptides deduced from the cDNAs showed sequence identity with the FatB class of plant thioesterases. Southern hybridization revealed the presence of at least four copies of BjFatB gene in the genome of this amphidiploid species. Western blot and RT-PCR analyses showed that the BjFatB class thioesterase is expressed poorly in flowers and leaves, but significantly in seeds at the mid-maturation stage. The enzymatic activities of different BjFatB isoforms were established upon heterologous expression of the four BjFatB CDSs in Escherichia coli K27fadD88, a mutant strain of fatty acid beta-oxidation pathway. The substrate specificity of each BjFatB isoform was determined in vivo by fatty acid profile analyses of the culture supernatant and membrane lipid of the recombinant K27fadD88 and E. coli DH10B (fadD(+)) clones, respectively. The BjFatB1 and BjFatB3 were predominantly active on C18:0-ACP substrate, whereas BjFatB2 and BjFatB4 were specific towards C18:0-ACP as well as C16:0-ACP. These novel FatB genes may find potential application in metabolic engineering of crop plants through their over-expression in seed tissues to generate stearate-rich vegetable fats/oils of commercial importance.

A process for microbial hydrocarbon synthesis: Overproduction of fatty acids in Escherichia coli and catalytic conversion to alkanes

The development of renewable alternatives to diesel and jet fuels is highly desirable for the heavy transportation sector, and would offer benefits over the production and use of short-chain alcohols for personal transportation. Here, we report the development of a metabolically engineered strain of Escherichia coli that overproduces medium-chain length fatty acids via three basic modifications: elimination of beta-oxidation, overexpression of the four subunits of acetyl-CoA carboxylase, and expression of a plant acyl-acyl carrier protein (ACP) thioesterase from Umbellularia californica (BTE). The expression level of BTE was optimized by comparing fatty acid production from strains harboring BTE on plasmids with four different copy numbers. Expression of BTE from low copy number plasmids resulted in the highest fatty acid production. Up to a seven-fold increase in total fatty acid production was observed in engineered strains over a negative control strain (lacking beta-oxidation), with a composition dominated by C(12) and C(14) saturated and unsaturated fatty acids. Next, a strategy for producing undecane via a combination of biotechnology and heterogeneous catalysis is demonstrated. Fatty acids were extracted from a culture of an overproducing strain into an alkane phase and fed to a Pd/C plug flow reactor, where the extracted fatty acids were decarboxylated into saturated alkanes. The result is an enriched alkane stream that can be recycled for continuous extractions. Complete conversion of C(12) fatty acids extracted from culture to alkanes has been demonstrated yielding a concentration of 0.44 g L(-1) (culture volume) undecane.

Cloning and functional characterization of an acyl-acyl carrier protein thioesterase (JcFATB1) from Jatropha curcas

A full-length cDNA of an acyl-acyl carrier protein (ACP) thioesterase (TE) (EC 3.1.2.14), named JcFATB1, was isolated from the woody oil plant Jatropha curcas L. The deduced amino acid sequence of the cDNA shares about 78% identity with FATB TEs, but only about 33% identity with FATA TEs from other plants. The deduced sequence also contains two essential residues (H(317) and C(352)) for TE catalytic activity and a putative chloroplast transit peptide at the N-terminal. Southern blot analysis revealed that a single copy of JcFATB1 is present in the J. curcas genome, and semi-quantitative PCR analysis showed that JcFATB1 was expressed in all tissues that were examined, most strongly in seeds, in which its expression peaked in late developmental stages. Seed-specific overexpression of the JcFATB1 cDNA in Arabidopsis resulted in increased levels of saturated fatty acids, especially palmitate, and in reduced levels of unsaturated fatty acids. The findings suggest that JcFATB1 from this woody oil plant can function as a saturated acyl-ACP TE and could potentially modify the seed oil of J. curcas to increase its levels of palmitate.

Characterization and cloning of a stearoyl/oleoyl specific fatty acyl-acyl carrier protein thioesterase from the seeds of Madhuca longifolia ( latifolia)

Deposition of oleate, stearate and palmitate at the later stages of seed development in Mahua ( Madhuca longifolia ( latifolia)), a tropical non-conventional oil seed plant, has been found to be the characteristic feature of the regulatory mechanism that produces the saturated fatty acid rich Mahua seed fat (commonly known as Mowrah fat). Although, the content of palmitate has been observed to be higher than that of stearate at the initial stages of seed development, it goes down when the stearate and oleate contents consistently rise till maturity. The present study was undertaken in order to identify the kind of acyl-ACP thioesterase(s) that drives the characteristic composition of signature fatty acids (oleate 37%, palmitate 25%, stearate 23%, linoleate 12.5%) in its seed oil at maturity. The relative Fat activities in the crude protein extracts of the matured seeds towards three thioester substrates (oleoyl-, stearoyl- and palmitoyl-ACP) have been found to be present in the following respective ratio 100:31:8. Upon further purification of the crude extract, the search revealed the presence of two partially purified thioesterases: a long-chain oleoyl preferring house-keeping LC-Fat and a novel stearoyl-oleoyl preferring SO-Fat. The characteristic accumulation of oleate and linoleate in the M. latifolia seed fat is believed to be primarily due to the thioesterase activity of the LC-Fat or MlFatA. On the other hand, the SO-Fat showed almost equal substrate specificity towards stearoyl- and oleoyl-ACP, when its activity towards palmitoyl-ACP compared to stearoyl-ACP was only about 12%. An RT-PCR based technique for cloning of a DNA fragment from the mRNA pool of the developing seed followed by nucleotide sequencing resulted in the identification of a FatB type of thioesterase gene ( MlFatB). This gene was found to exist as a single copy in the mother plant genome. Ectopic expression of this MlFatB gene product in E. coli strain fadD88 further proved that it induced a higher level of accumulation of both stearic and oleic acids when compared to the negative control line that did not contain this MlFatB gene. It also indicated that SO-Fat indeed is the product of the MlFatB gene present in the maturing seeds of M. latifolia in nature. Additionally, a predicted 3D-structure for MlFatB protein has been developed through use of bioinformatics tools.

Cloning and Expression Analysis of PtFATB Gene Encoding the Acyl-acyl Carrier Protein Thioesterase in Populus tomentosa Carr

Acyl-ACP thioesterases (FATs) terminates the fatty acid synthesis and allow the transport of fatty acids out of the plastids, which are the important determinants of cellular metabolism. FATB is a member of FAT enzymes that has been described previously in most of the plants. In silico cloning is a new method that utilizes the bioinformatics on the complete genome and available EST database. In this study, a full-length cDNA clone of PtFATB gene was isolated from Populus tomentosa using this approach. It is 1,450 bp in length and the open reading frame encodes a peptide of 421 amino acids. The predicted amino acid sequence shows significant homology with those from other plant species, which contain typical domains owned by FATB proteins. The transcripts of PtFATB were abundant in leaves, and less in roots detected by using semiquantitative RT-PCR. When the shoots were subjected to the stress treatments (cold, dry, NaCl) and ABA (Abscisic acid), the expression of PtFATB was only slightly reduced under the treatment of low temperature. This suggests that the expression of PtFATB is in a constitutive fashion. This study provides the basis not only for the identification and characterization of this gene but also for the improvement of cold tolerance by controlling the expression of the PtFATB gene in trees in near future.