Medium chain thioesterase

Knudsen and Grunnet (1982) have proposed an interesting system for the control of medium-chain fatty acid synthesis by ruminant mammary tissue. Their proposal is based on their observations that ruminant mammary tissue fatty acid-synthetase exhibits both medium-chain thioesterase (Grunnet and Knudsen 1978) and transacylase (Knudsen and Grunnet 1980) activity and that medium-chain fatty acids synthesized de novo can be incorporated into TG without an intermediate activation step (Grunnet and Knudsen 1981). They proposed that the synthesis of the medium-chain fatty acids is controlled by their incorporation into TG (Grunnet and Knudsen 1981). Further work will be needed to substantiate transacylation as a chain-termination mechanism in fatty acid synthesis by ruminant mammary tissue. 

The discovery of a novel pathway for biosynthesis of medium and short chain fatty acids in plants (a-keto acid elongation pathway, 1) raises the possibility (however unlikely) that medium-chain fatty acids (mcFAs) of certain oil seeds producing them may be derived by this pathway. Alternatively, these may be formed after release of elongating fatty acid chains from fatty acid synthase mediated biosynthesis (FAS) by specific medium chain thioesterases [2, 3,4]. Thus far the aKAE pathway is only known to occur in trichome glands of plants in the family Solanaceae. In the aKAE pathway, iso-, anteiso- or straight-chain keto acid products of branched-chain amino acid metabolism are elongated by one carbon (via acetate) per cycle. The final step is predicted to be oxidative decarboxylation to yield CoA activated acids. The mechanism that determines the chain length of aKAE products is not understood [1]. 

The absence of specialized, single-specificity medium-chain thioesterases in these two species shows a second evolutionary mechanism by which a normal long-chain fatty acid synthase can be modified to the production of medium chains. It also represents strong indirect evidence for the existence of modified elongation reactions in these seeds. 

We thank Toni Voelker, Maelor Davies and Vic Knauf of Calgene for providing seed material transformed with California bay acyl-ACP medium chain thioesterase. 

Voelker TA. Davies HM. Plant medium-chain thioesterases. International Patent Application 1992 WO 92/20236. 

Both goat and cow mammary tissue synthesize medium-chain fatty acids. However, attempts to isolate thioesterase II from the cytosol of ruminant mammary tissues have not been successful (Grunnet and Knudsen 1979). In contrast to the nonruminant, the fatty acid-... 

A type I thioesterase domain is present at the NHj-terminal of the animal FAS and is responsible for catalyzing hydrolysis of the completed fatty acyl chain from the enzyme. The active site contains both conserved serine and histidine residues [87] and is thought to function via a mechanism similar to that of the serine proteases [50] however, no conserved acidic residue is present to complete the charge relay/transfer. A second variety of thioesterase (type II) is encoded as a separate protein and interacts with the multifunctional FAS to release medium chain fatty acids [88, 89]. This enzyme has a weak sequence similarity to the type I thioesterase, which includes the conserved active site serine and histidine residues. These enzymes are also homologous to proteins encoded by genes involved in the synthesis of peptide antibiotics [90,91] (see below). 

One of the major non-food uses of vegetable oils (approximately 5(X) million pounds of oil per annum in the US) is the production of soaps, detergents, and other surfactants. The solubility and other physical properties of medium-chain fatty acids and their derivatives make them especially suited for surfactant manufacture. Coconut and palm kernel oils, which contain 40-60% lauric acid (12 0), are current major feedstocks for the surfactant industry. The mechanism of synthesis of lauric and other medium-chain fatty acids in plants involves the action of a medium-chain acyl-ACP thioesterase which terminates fatty acid synthesis after a 10 or 12 carbon chain has been assembled (M. Pollard,... 

Fig. 11. Genetic engineering of rapeseed oil. A high level of lauric acid was achieved by expressing a medium-chain acyl-ACP thioesterase (MCTE) from California Bay in the transgenic seeds. This enzyme intercepts the fatty acid synthesis pathway at 12 carbons and hydrolyzes the fatty acid from its ACP carrier. MoI% of major fatty acids in a typical canola cultivar are compared to the composition achieved through genetic engineering.

Eccleston VS, Ohlrogge JB (1998) Expression of lauroyl-acyl crirrier protein thioesterase in Brassica napus seeds induces pathways for both fatty acid oxidation and biosynthesis and imphes a set point for triacylglycerol accumulation. Plant Cell 10 613-621 Eccleston VS, Cranmer AM, Voelker TA, OUrogge JB (1996) Medium-chain fatty acid biosynthesis and utilization in Brassica napus plants expressing lauroyl-acyl crurier protein thioesterase. Planta 198 46-53... 

Weselake, RJ Taylor, DC Rahman, MH Shah, S Laroche, A MeVetty, P Harwood, J. Increasing the flow of carbon into seed oil. Biotechnol Adv., 2009,27, 866-78. Pollard, MR Anderson, L Fan, C Hawkins, D Davies, H. A speeific a( l-ACP thioesterase implicated in medium-chain fatty acid production in immature cotyledons of Umbellularia californica. Arch. Biochem. Biophys., 1991, 284, 306-312. 

Partial reactions for the yeast Type I synthetase were studied in a classic series of experiments by Lynen (1967). Peptides capable of partial reactions have been isolated from the yeast and chicken liver enzymes (cf. Wakil et ai, 1983). Particular interest has been focused on the thioesterase. This enzyme, more easily isolated by partial proteolysis than those for most of the other partial reactions, is important in at least partly regulating chain length termination. Thioesterases isolated from several animal fatty acid synthetases have very similar amino acid sequences around the active serine residue (Poulose et al, 1981). The medium-chain fatty acids produced by synthetases from some mammary glands appear to be due to thioesterase II (a second thioesterase) (Libertini and Smith, 1978). 

Chung AL, Zeng GL, Jin HL, Wu Q, Chen JC, Chen GQ. Production of medium-chain-length 3-hydroxyalkanoic acids by P-oxidation and phaC operon deleted Pseudomonas entomophila harboring thioesterase gene. Metah Eng 2013 17 23-9. 

Oleate desaturase expression by antisense RNA in soybean led to an oil containing over 80 % oleic acid as compared to a normal 23 %, with significant decrease in polyunsaturated fatty acids [155]. Ganoladoes not normally accumulate capric and caprylic acids. But the introduction of the Cuphea hookeriana acyl-AGP thioesterase cDNA in canola resulted in the accumulation of these two medium-chain fatty acids in the seeds [156]. In plants, 0)-6 desaturase-catalyzed pathway in the microsomes is a major source of polyunsaturated lipids, introduction of the... 

Dormann, P., Spener, F., and Ohlrogge, B. Characterization of two acyl-acyl carrier protein thioesterases from developing Cuphea seeds specific for medium-chain- and oleoyl-acyl carrier protein, Planta 189 (1993), 425-432. 

BROAD-RANGE AND BINARY-RANGE ACYL-ACP THIOESTERASES FROM MEDIUM-CHAIN PRODUCING SEEDS... 

Broad-Range and Binary-Range Acyl-ACP Thioesterases from Medium-Chain Producing Seeds. 

Thioesterase activity has also been targeted as a means of producing medium-chain saturated fatty acids (MCFA) in BOS. MCFA, such as lauric acid (12 0), are widely used for industrial applications, including cosmetics and surfactants, as well as for certain food applications such as confectionary. In most BOS, including canola, the endogenous acyl-ACP thioesterase exhibits a preference for 18-carbon FAs. Production of MCFA required the introduction of a thioesterase with appropriate medium-chain specificity, such as the 12 0 ACP-thioesterase from the California bay laurel (Umbellularia californica) (Voelker et al., 1996). Although the expression of this enzyme in canola resulted in a relatively high accumulation of lauric acid. 

Figure 2. Medium-chain-length (MCL) PHA production from non-related carbon sources. A. The fatty acid biosynthesis pathway. B. FabH and FabG mediated SCL-MCL PHA monomer supply. C. 3-hydroxyacyl-ACP CoA transacetylase (PhaG) mediated MCL PHA monomer supply. D. Thioesterase A (TesA) mediated MCL PHA monomer supply (via the P-oxidation pathway).

Schuch R, Brummel M, Spener F. Medium-chain acyl-ACP thioesterase is not the exclusive enzyme responsible for early chain-length termination in medium-chain fatty acid biosynthesis. Grasses y Aceites 1993 44 126-128. 

Effects of the constitutive expression of a medium chain acyl-ACP thioesterase from California Bay on leaf lipid composition of transformed Brassica plants... 

Constitutive expression of California bay medium chain acyl-ACP thioesterase in Brassica napus resulted in medium chain acyl-ACP thioesterase activity in transformed leaf tissue without alteration of leaf fatty acid composition. [ C] Labelling studies of intact leaf discs, chloroplasts and protoplasts were used to evaluate if medium chain acyl moieties are produced in transformed leaves. The possible metabolism of medium chain fatty acids in leaf via P-oxidation is discussed. 

Medium chain fatty acids (C,-Ci4) are unusual fetty adds found in the seed oils of several plant species such as Cuphea and California bay [1]. Medium chain fetty acids are produced by premature chain termination of fetty acid synthesis, mediated by a medium chain acyl-ACP thioesterase (MCTE) [2,3]. Since medium chain fetty acids are a valuable renewable resource, one goal of researchers has been to develop a medium chain-producing annual crop plant using genetic transformation [4]. Workers at Calgene transformed Brassica napus with California bay medium chain acyl-ACP thioesterase cDNA, testing the ability of a seed specific napin promote or the constitutive CaMV 35S promoter to alter seed oil fatty acid composition. We have used these transformants to evaluate possible roles for acyl-ACP chain length termination in the control of fatty acid synthesis. 

Pollard MR, Anderson L, Fan C, Hawkins DJ, Davies HM. A specific acyl-ACP thioesterase implicated in medium-chain fatty acid production in immature... 

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