Broad-range and binary-range acyl-acyl-carrier protein thioesterases suggest an alternative mechanism for medium-chain production in seeds.

In the current model of medium-chain (C8-14) fatty acid biosynthesis in seeds, specialized FatB acyl-acyl-carrier-protein (ACP) thioesterases are responsible for the production of medium chains. We have isolated and characterized FatB cDNAs from the maturing seeds of elm (Ulmus americana) and nutmeg (Myristica fragrans), which accumulate predominantly caprate (10:0)- and myristate (14:0)-containing oils, respectively. In neither species were we able to find cDNAs encoding enzymes specialized for these chain lengths. Nutmeg FatB hydrolyses C14-18 substrates in vitro and expression in Brassica napus seeds leads to an oil enriched in C14-18 saturates. Elm FatB1 displays a binary specificity: one activity is centered on 10:0-ACP, and a second is centered on palmitate (16:0)-ACP. After expression in B. napus seeds the oil is enriched in C10-18 saturates, predominantly 16:0, 14:0, and 10:0. The composition of free fatty acids produced by elm FatB1 in Escherichia coli shifts from C14-16 to mostly C8-10 by increasing the rate of chain termination by this enzyme. These results suggest the existence of an alternative mechanism used in the evolution of medium-chain production, a model of which is presented.     

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.     

A Determinant of Substrate Specificity Predicted from the Acyl-Acyl Carrier Protein Desaturase of Developing Cat''s Claw Seed

Cat's claw (Doxantha unguis-cati L.) vine accumulates nearly 80% palmitoleic acid (16:1Δ9) plus cis-vaccenic acid (18:1Δ11) in its seed oil. To characterize the biosynthetic origin of these unusual fatty acids, cDNAs for acyl-acyl carrier protein (acyl-ACP) desaturases were isolated from developing cat's claw seeds. The predominant acyl-ACP desaturase cDNA identified encoded a polypeptide that is closely related to the stearoyl (Δ9–18:0)-ACP desaturase from castor (Ricinis communis L.) and other species. Upon expression in Escherichia coli, the cat's claw polypeptide functioned as a Δ9 acyl-ACP desaturase but displayed a distinct substrate specificity for palmitate (16:0)-ACP rather than stearate (18:0)-ACP. Comparison of the predicted amino acid sequence of the cat's claw enzyme with that of the castor Δ9–18:0-ACP desaturase suggested that a single amino acid substitution (L118W) might account in large part for the differences in substrate specificity between the two desaturases. Consistent with this prediction, conversion of leucine-118 to tryptophan in the mature castor Δ9–18:0-ACP desaturase resulted in an 80-fold increase in the relative specificity of this enzyme for 16:0-ACP. The alteration in substrate specificity observed in the L118W mutant is in agreement with a crystallographic model of the proposed substrate-binding pocket of the castor Δ9–18:0-ACP desaturase.     

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.     

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.     

Structure-guided reshaping of the acyl binding pocket of ‘TesA thioesterase enhances octanoic acid production in E. coli

Medium-chain fatty acids (C6–C10) have attracted much attention recently for their unique properties compared to their long-chain counterparts, including low melting points and relatively higher carbon conversion yield. Thioesterase enzymes, which can catalyze the hydrolysis of acyl-ACP (acyl carrier protein) to release free fatty acids (FAs), regulate both overall FA yields and acyl chain length distributions in bacterial and yeast fermentation cultures. These enzymes typically prefer longer chain substrates. Herein, seeking to increase bacterial production of MCFAs, we conducted structure-guided mutational screening of multiple residues in the substrate-binding pocket of the E. coli thioesterase enzyme ‘TesA. Confirming our hypothesis that enhancing substrate selectivity for medium-chain acyl substrates would promote overall MCFA production, we found that replacement of residues lining the bottom of the pocket with more hydrophobic residues strongly promoted the C8 substrate selectivity of ‘TesA. Specifically, two rounds of saturation mutagenesis led to the identification of the ‘TesA^RD−2 variant that exhibited a 133-fold increase in selectivity for the C8-ACP substrate as compared to C16-ACP substrate. Moreover, the recombinant expression of this variant in an E. coli strain with a blocked β-oxidation pathway led to a 1030% increase in the in vivo octanoic acid (C8) production titer. When this strain was fermented in a 5-L fed-batch bioreactor, it produced 2.7 g/L of free C8 (45%, molar fraction) and 7.9 g/L of total free FAs, which is the highest-to-date free C8 titer to date reported using the E. coli type II fatty acid synthetic pathway. Thus, reshaping the substrate binding pocket of a bacterial thioesterase enzyme by manipulating the hydrophobicity of multiple residues altered the substrate selectivity and therefore fatty acid product distributions in cells. Our study demonstrates the relevance of this strategy for increasing titers of industrially attractive MCFAs as fermentation products.     

Acyl-ACP thioesterases from castor (Ricinus communis L.): An enzymatic system appropriate for high rates of oil synthesis and accumulation

Acyl–acyl carrier protein (ACP) thioesterases are enzymes that terminate the intraplastidial fatty acid synthesis in plants by hydrolyzing the acyl-ACP intermediates and releasing free fatty acids to be incorporated into glycerolipids. These enzymes are classified in two families, FatA and FatB, which differ in amino acid sequence and substrate specificity. In the present work, both FatA and FatB thioesterases were cloned, sequenced and characterized from castor (Ricinus communis) seeds, a crop of high interest in oleochemistry. Single copies of FatA and FatB were found in castor resulting to be closely related with those of Jatropha curcas. The corresponding mature proteins were heterologously expressed in Escherichia coli for biochemical characterization after purification, resulting in high catalytic efficiency of RcFatA on oleoyl-ACP and palmitoleoyl-ACP and high efficiencies of RcFatB for oleoyl-ACP and palmitoyl-ACP. The expression profile of these genes displayed the highest levels in expanding tissues that typically are very active in lipid biosynthesis such as developing seed endosperm and young expanding leaves. The contribution of these two enzymes to the synthesis of castor oil is discussed.     

A Cupheaβ‐ketoacyl‐ACP synthase shifts the synthesis of fatty acids towards shorter chains in Arabidopsis seeds expressing Cuphea FatB thioesterases

Acyl–acyl carrier protein (ACP) thioesterases with specificities on medium chain substrates (C8–C14) are requisite enzymes in plants that produce 8:0, 10:0, 12:0 and 14:0 seed oils, but they may not be the sole enzymatic determinants of chain length. The contribution to chain length regulation of a β-ketoacyl-ACP synthase, Cw KAS A1, derived from Cuphea wrightii, a species that accumulates 30% 10:0 and 54% 12:0 in seed oils, was investigated. Expression of Cw KAS A1 in Arabidopsis seeds reduced 16:0 from 8.2 to 6.2 mol%, suggesting a KAS II-type activity. In the presence of the KAS I inhibitor cerulenin, however, transgenic seed extracts extended 6:0- and 8:0-ACP at a rate four- to fivefold greater than extracts from untransformed plants, whereas no difference was observed in extension of 14:0- and 16:0-ACP. The effect of KAS A1 on seed oils was tested by combining it with the C. wrightii medium chain-specific thioesterases, Cw FatB1 and Cw FatB2, in crosses of transformed plants. Fatty acid synthesis shifted towards shorter chains in progeny expressing both classes of enzymes. KasA1/FatB1 homozygotes produced threefold more 12:0 than the FatB1 parent while 14:0 and 16:0 were reduced by one-third and one-half, respectively. F₂ progeny expressing KasA1 and FatB2 produced twofold more 10:0 and 1.4-fold more 12:0 than the FatB2 parent, and the double-transgenic progeny produced one-quarter less 14:0 and one-half less 16:0 than the FatB2 parent. It is hypothesized that the shift towards production of shorter chains resulted from increased pools of medium chain acyl-ACP resulting from KAS A1 activity. The combined activities of KAS A1 and FatB thioesterases appear to determine the C. wrightii phenotype.     

Characterization of substrate specificity of plant FatA and FatB acyl-ACP thioesterases

The specificity of plant acyl-acyl carrier protein (ACP) thioesterases is the major determinant of the chain length and level of saturated fatty acids found in most plant tissues. Although these enzymes have been previously characterized from a number of sources, information on kinetic parameters for a wide range of substrates with cloned enzymes is lacking. In the present study the substrate specificity of recombinant FatA thioesterase isoforms from Arabidopsis (AtFatA) and coriander (CsFatA) and FatB from Arabidopsis (AtFatB) have been re-examined with a comprehensive range of substrates including 14:1-ACP and 16:1-ACP. AtFatA displayed the highest catalytic efficiencies (kcat/Km) towards oleoyl-ACP with activities at least 20-fold lower for all other tested substrates and 75-fold lower with palmitoyl-ACP. Both chain length and double bond presence strongly influenced kcat of FatA with minor influence on Km. Arabidopsis FatB substrate specificity was found to differ from previous reports and this difference could be attributed to the influence of ACP structure. FatB activity with palmitoyl-ACP was 2.5-fold higher and the ratio of 16:0-ACP/14:0-ACP hydrolysis was 6.4-fold higher with spinach ACP compared to E. coli ACP. Additionally, the influence of amino acid domains from both AtFatA and AtFatB on their substrate specificity was studied by utilizing a domain-swapping approach. The characterization of the resulting chimeric enzymes pointed to the N-terminus as a determinant of the substrate specificity for both FatA and FatB acyl-ACP thioesterases.     

Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach

Background  

The large amount of available sequence information for the plant acyl-ACP thioesterases (TEs) made it possible to use a bioinformatics-guided approach to identify amino acid residues involved in substrate specificity. The Conserved Property Difference Locator (CPDL) program allowed the identification of putative specificity-determining residues that differ between the FatA and FatB TE classes. Six of the FatA residue differences identified by CPDL were incorporated into the FatB-like parent via site-directed mutagenesis and the effect of each on TE activity was determined. Variants were expressed in E. coli strain K27 that allows determination of enzyme activity by GCMS analysis of fatty acids released into the medium.     

Effects of genomic position and copy number of Acyl-ACP thioesterase transgenes on the level of the target fatty acids in Brassica napus L.

The effects of genomic position and copy number of acyl-acyl carrier protein (ACP) thioesterase (TE) transgenes on the major target fatty acid, either lauric acid (C12:0) or palmitic acid (C16:0) depending on the TE, in transgenic Brassica napus seed oil were investigated. Four transgenic parental lines, transformed individually with the bay-TE (Uc FatB1), elm-TE (Ua FatB1), nutmeg-TE (Mf FatB1) and Cuphea-TE (Ch FatB1) transgenes, were crossed with the non-transgenic recipient genotypes '212/86' or 'QO4'. Bay-TE and Cuphea-TE F₁ seeds, which carry half the number of the construct copies compared to the self-pollinated seeds of the transgenic parents, showed significantly lower levels of the target fatty acid. Doubled haploid (DH) lines were developed through microspore culture from F₁ hybrids with the elm-TE or the Cuphea-TE transgenes. DH lines carrying one to five copies of the Cuphea-TE transgene displayed a positive correlation between transgene copy number and the target fatty acid C16:0 level (r = 0.77**). DH lines with elm-TE transgene copies at four different loci showed different C16:0 levels, with one of the loci (E-II) leading to significantly higher C16:0 levels. This study supports the importance of the selection of high transgene copy number and/or the optimum genomic integration site in order to achieve maximum expression levels of the target fatty acid in transgenic oil quality modification.     

Cuphea wrightii thioesterases have unexpected broad specificities on saturated fatty acids

Cuphea wrightii A. Gray is an herbaceous annual that accumulates 30% caprate (10:0) and 54% laurate (12:0) in seed storage lipids. We investigated the role of acyl-acyl carrier protein (ACP) thioesterases (TE) in acyl chain-length regulation in C. wrightii. Two embryo-derived cDNAs, encoding the TEs Cw FatB1 and Cw FatB2, were isolated. Both proteins were detected in developing embryos and mature seeds but not in other tissues, suggesting involvement in seed oil synthesis. Although expected to be 10:0/12:0-ACP-specific, these genes produced a broad range of fatty acids (12:0, 14:0, and 16:0) in transgenic Arabidopsis with the greatest accumulation at 14:0. Cw FatB2 transformants also accumulated small amounts of 10:0. Because C. wrightii accumulates only ca. 5% 14:0 and ca. 2% 16:0, we tested the possibility that gene dosage effects might significantly alter the overall kinetics of the pathway. Phenotypic comparisons of progeny segregating for the transgenes individually and in a hybrid population demonstrated that increased enzyme pools in vivo had a minor effect on diverting fatty acid production to shorter chains. We propose that Cw FatB1 and Cw FatB2 may be necessary but not sufficient determinants of the C. wrightii phenotype.     

Cloning,characterization and structural model of a FatA-type thioesterase from sunflower seeds (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.)

The substrate specificity of acyl-acyl carrier protein (ACP) thioesterases (EC 3.1.2.14) determines the fatty acids available for the biosynthesis of storage and membrane lipids in seeds. In order to determine the mechanisms involved in the biosynthesis of fatty acids in sunflower seeds (Helianthus annuus L.), we isolated, cloned and sequenced a cDNA clone of acyl-ACP thioesterase from developing sunflower seeds, HaFatA1. Through the heterologous expression of HaFatA1 in Escherichia coli we have purified and characterized this enzyme, showing that sunflower HaFatA1 cDNA encodes a functional thioesterase with preference for monounsaturated acyl-ACPs. The HaFatA1 thioesterase was most efficient (kcat/K_m) in catalyzing oleoyl-ACP, both in vivo and in vitro. By comparing this sequence with those obtained from public databases, we constructed a phylogenetic tree that included FatA and FatB thioesterases, as well as related prokaryotic proteins. The phylogenetic relationships support the endosymbiotic theory of the origin of eukaryotic cells and the suggestion that eubacteria from the -subdivision were the guest cells in the symbiosis with archaea. These prokaryotic proteins are more homologous to plant FatB, suggesting that the ancient thioesterases were more similar to FatB. Finally, using the available structure prediction methods, a 3D model of plant acyl-ACP thioesterases is proposed that reflects the combined data from direct mutagenesis and chimera studies. In addition, the model was tested by mutating the residues proposed to interact with the ACP protein in the FatA thioesterase by site-directed mutagenesis. The results indicate that this region is involved in the stabilization of the substrate at the active site.     

A Stationary-Phase Acyl-Coenzyme A Synthetase of Streptomyces coelicolor A3(2) Is Necessary for the Normal Onset of Antibiotic Production

The fadD1 and macs1 genes of Streptomyces coelicolor are part of a two-gene operon. Both genes encode putative acyl coenzyme A synthetases (ACSs). The amino acid sequence of FadD1 has high homology with those of several ACSs, while MACS1 has the closest homology with medium-chain ACSs, broadly known as SA proteins. Like FadD of Escherichia coli, FadD1 also has a broad substrate specificity, although saturated long-chain fatty acids appears to be the preferred substrate. fadD1 is a growth-phase-regulated gene, and its mRNA is detected only during the stationary phase of growth. Interestingly, a mutation in fadD1 alters the levels of another ACS or ACSs, both at the stationary phase and at the exponential phase of growth, at least when glucose is used as a main carbon source. The mutant also shows a severe deficiency in antibiotic production, and at least for Act biosynthesis, this deficiency seems to be related to delayed expression of the Act biosynthetic genes. Antibiotic production is restored by the introduction of a wt fadD1 allele into the cell, demonstrating a strict link between ACS activity and the biosynthesis of secondary metabolites. The results of this study indicate that the ACSs may be useful targets for the design of rational approaches to improving antibiotic production.     

Cloning,characterization, and expression analysis of acyl–acyl carrier protein (ACP)-thioesterase B from seeds of Chinese Spicehush (Lindera communis)

Acyl–acyl carrier protein (ACP) thioesterases (TE EC 3.1.2.14) are fatty acid biosynthesis key enzymes that determine fatty acid carbon chain length in most plant tissues. A full-length cDNA corresponding to one of the fatty acyl–ACP thioesterase (Fat) genes, designated LcFatB, was isolated from developing Lindera communis seeds using PCR and RACE with degenerate primers based on conserved sequences of multiple TE gene sequences obtained from GenBank. The 1788 bp cDNA had an open reading frame (ORF) of 1260 bp encoding a protein of 419 amino acids. The deduced amino acid sequence showed 61–73% identity to proteins in the FatB class of plant thioesterases. Real-time quantitative PCR analysis revealed that LcFatB was expressed in all tissues of L. communis, with the highest expression in the developing seeds 75 days after flowering. Recombinant pET-MLcFatB was constructed using the pET-30a vector and transformed into Escherichia coli BL21(DE3)△ FadE, a strain that deleted the acyl-CoA dehydrogenase (FadE). SDS-PAGE analysis of proteins isolated from pET-MLcFatB E. coli cells after induction with IPTG revealed a protein band at ~ 40.5 kDa, corresponding to the predicted size of LcFatB mature protein. The decanoic acid and lauric acid contents of the pET-MLcFatB transformant were increased significantly. These findings suggest that an LcFatB gene from a non-traditional oil-seed tree could be used to function as a saturated acyl–ACP thioesterase and could potentially be used to modify the fatty acid composition of seed oil from L. communis or other species through transgenic approaches.     

An unusual seed-specific 3-ketoacyl-ACP synthase associated with the biosynthesis of petroselinic acid in coriander

Petroselinic acid (18:1⁶) is the major component of the seed oil of Umbelliferae species such as coriander (Coriandrum sativum) as well as Araliaceae and Garryaceae species. This unusual fatty acid is synthesized in plastids by the ⁴ desaturation of palmitoyl-acyl carrier protein (16:0-ACP) and subsequent elongation of ⁴-hexadecenoyl (16:1⁴)-ACP. To characterize the enzymatic nature of the elongation reaction, an in vitro assay was developed with 16:1⁴-ACP and 16:0-ACP as substrates. Extracts from developing coriander seeds elongated 16:1⁴-ACP in a competitive assay at rates ten-fold greater than that with 16:0-ACP. In contrast, extracts from castor seeds, which do not synthesize petroselinic acid, displayed a strong preference for the elongation of 16:0-ACP rather than 16:1⁴-ACP. In addition, the elongation of 16:1⁴-ACP and 16:0-ACP by coriander seed extracts was strongly inhibited by cerulenin at concentrations as low as 10 M. This finding suggested that the elongation of 16:1⁴-ACP and 16:0-ACP in coriander seed is catalyzed by a 3-ketoacyl-ACP synthase (KAS) I-type enzyme(s), rather than a KAS II-type activity that is typically associated with 16:0-ACP elongation. Consistent with this, a cDNA for a diverged form of KAS I was isolated from a cDNA library prepared from developing coriander seed. Using a variety of heterologous probing techniques, no KAS II-type cDNAs could be identified in this library. Multiple alignment of KAS amino acid sequences indicated that, although the polypeptide corresponding to the coriander cDNA is more closely related to KAS I, its active site motif deviates from those found in both KAS I and KAS II enzymes. Also suggestive of a possible role in petroselinic acid synthesis, antibodies raised to the recombinant protein recognize an abundant 45 kDa polypeptide in coriander endosperm that is not detected in coriander leaves. These antibodies also recognize a major band of similar size in developing seeds of English ivy (Hedera helix), an Araliaceae species that also accumulates petroselinic acid in a seed-specific manner.     

Cloning and Expression of Genes Encoding F107-C and K88-1NT Fimbrial Proteins of Enterotoxigenic Escherichia coli from Piglets

We cloned two genes coding F107-C and K88-1NT fimbrial subunits from strains E. coli C and 1NT isolated from Thua Thien Hue province, Vietnam. The mature peptide of faeG gene from strain E. coli 1NT (called faeG-1NT) is 100 % similarity with faeG gene, while the CDS of fedA gene from strain C (called fedA-C) has a similarity of 97 % with the fedA gene. Expression of the faeG-1NT and fedA-C genes in E. coli BL21 Star™ (DE3) produced proteins of ~31 and 22 kDa, respectively. The effect of IPTG concentration on the K88-1NT and F107-C fimbriae production was investigated. The results showed that 0.5 mM IPTG is suitable for higher expression of K88-1NT subunit, while 0.75 mM IPTG strongly stimulated expression of F107-C subunit. The optimal induction time for expression was also examined. Generally, highest expression of K88-1NT subunit occurred after 6 h of induction, while that of F107-C subunit is after 14 h.