MXPA99011166A - Engineering plant thioesterases and disclosure of plant thioesterases having novel substrate specificity - Google Patents

Abstract

Methods of altering substrate specificity of plant acyl-ACP thioesterases, and engineered plant acyl-ACP thioesterases so produced are provided. The C-terminal two thirds portion of plant thioesterases is identified as desirable for such modifications. DNA sequences and constructs for expression of engineered thioesterases, as well as the novel thioesterases produced therefrom are also provided. Such DNA sequences may be used for expression of the engineered thioesterases in host cells, particularly seed cells of oilseed crop plants, for the modification of fatty acid composition. A C12 preferring plant acyl-ACP thioesterase described herein may be altered to obtain a plant thioesterase having approximately equal activity on C14 and C12 substrates. Further modification of the C12 enzyme yields a thioesterase having greater activity on C14 as compared to C12 substrates. Of particular interest is a plant 18:1 thioesterase in which the relative 18:0 activity has been increased. Such FatA thioesterases find use for improved production of stearate in vegetable seed oils.

Description

TIOESTERASAS OF PLANTS FOR ENGINEERING AND DESCRIPTION OF TIOESTERASAS OF PLANTS THAT HAVE SPECIFICITY OF NOVEDOSA SUBSTRATE Technical Field The present invention is directed to proteins, sequences and constructions of nucleic acids and constructions and methods related thereto. INTRODUCTION Background Fatty acids are organic acids that have a hydrocarbon chain of approximately 4 to 24 carbons. Many different kinds of fatty acids are known, which differ from one another in chain length and in the presence, number and position of double bonds. In cells, there are normally fatty acids in covalently linked forms, the carboxyl portion being referred to as a fatty acyl group. The chain length and degree of saturation of these molecules are often described by the formula CX: Y, where "X" indicates the number of carbons and "Y" indicates the number of double bonds. The production of fatty acids in plants starts in the plastid with the reaction between acetyl-CoA and malonyl-ACP to produce butyryl-ACP catalyzed by the enzyme, β-ketoacyl-ACP synthase III. The elongation of acetyl-ACP to fatty acids of 16 and 18 carbons implies the cyclic action of the following sequence of reactions: condensation with a unit of two carbons of malonyl-ACP to form β-ketoacyl-ACP (β-ketoacyl-ACP synthase), reduction of the keto function to an alcohol (β-ketoacyl-ACP dehydrase), dehydration to form a enoyl-ACP (ß-hydroxyacyl-ACP to form saturated acyl-ACP (enoyl-ACP reductase). ß-ketoacyl-ACP synthase I, catalyses elongation to palmitoyl-ACP (C16: O), while ß-ketoacyl- ACP synthase II catalyses the final elongation to stearoyl-ACP (C18: 0) .The longer chain fatty acids produced by FAS, are usually 18 carbons long.An additional biochemical fatty acid step that occurs in the plastic, is the desaturation of stearoyl-ACP (C18: 0) to form oleoyl-ACP (C18: 0) in a reaction catalyzed by a desaturase 91-9 desaturase, also frequently referred to as a "stearoyl-ACP desaturase" due to its high activity to the stearate acyl-ACP of 18 carbons.The chain elongation of carbons in the plastids can be terminated by the transfer of the acyl group to glycerol 3-phosphate, with the resulting glycerol in the lipid biosynthesis pathway, "prokaryotic" plastids. Alternatively, specific thioesterases can intercept the prokaryotic path by hydrolyzing newly produced acyl-ACPs into free fatty acids and ACP. Subsequently, the free fatty acids are converted to fatty acyl-CoA in the plastid sheath and exported to the cytoplasm. Here, they were incorporated into the pathway of "eukaryotic" lipid biosynthesis in the endoplasmic reticulum that is responsible for the formation of phospholipids, triglycerides and other neutral lipids. After the transport of fatty acyl-CoA to the endoplasmic reticulum, subsequent sequential steps for the production of triglycerides can be presented. For example, polyunsaturated fatty acyl groups such as linoleoyl and α-linolenoyl, are produced as the result of sequential desaturation of oleoyl acyl groups by the action of membrane-bound enzymes. Triglycerides are formed by the action of enzymes 1-, 2- and 3-acyl-ACP transferase, glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase and diacylglycerol acyltransferase. The fatty acid composition of a plant cell is a reflection of the combination of free fatty acids and fatty acids (fatty acyl groups) incorporated into triglycerides as a result of acyltransferase activities. The properties of a given triglyceride will depend on various combinations of fatty acyl groups at different positions in the triglyceride molecule. For example, if fatty acyl groups are mostly saturated fatty acids, then the triglyceride will be solid at room temperature. However, in general, vegetable oils tend to be mixtures of different triglycerides that form the oil, which in turn are influenced by their respective fatty acyl compositions. Acyl vehicle protein thioesterases from acyl plants are of biochemical interest due to their roles in the synthesis of fatty acids and their utilities in seed bioengineering. plant oils. An acyl-ACP medium-chain thioesterase from the California laurel tree, Umbellularia californica, has been isolated (Davies et al., (1991) Arch. Biochem. Biophys. 290: 37-45), and its cDNA cloned and expressed in E. coli (Voelker et al. (1994) J. Bacterial 176: 7320-7327) and the seeds of Arabidopsis thaliana and Brassica napus (Voelker et al. (1992) Science 257: 72-74). In all cases, large amounts of laurate ((12: 0) and small amounts of myristate (14: 0) were accumulated.These results demonstrate the role of TE in determining chain length during the biosynthesis of the new fatty acid in plants and the utility of these enzymes for modifying seed oil compositions in higher plants Recently, a number of cDNAs encoding different acyl-ACP thioesterases from plants have been cloned (Knutzon et al. (1992) Plant Physiol. : 1751-1758; Voelker, et al. (1992) supra; Dorman et al., (993) Plant 189: 425-434: Dorman et al. (1994) The Plant Cell 7: 359-371). Sequence analyzes of these thioesterases show high homology, implying similarity in structure and function. Some of these thioesterase cDNAs have been expressed in E. coli and their substrate specificities determined by in vitro analysis. The fact that these enzymes share significant sequence homology, still showing different substrate specificities, indicates that impalpable changes may be sufficient to change the selectivity of the substrate.

There is little information available on the structural and functional divergence between these plant thioesterases, and the tertiary structure of any plant thioesterase has not yet been determined. The treatment of proteins can prove to be a useful tool to understand the mechanism of recognition of the thioesterase substrate and catalysis, and therefore leads to the rational design of new enzymes with suitable substrate specificities. Such novel enzymes could find use in plant bioengineering to provide various modifications of fatty acid compositions, particularly with respect to the production of vegetable oils which have important proportions of suitable fatty acyl groups, including medium chain fatty acyl groups (C8 or C 18). In addition, it is convenient to control the relative proportions of various fatty acid acyl groups that are present in oil storage seeds to provide a variety of oils for a wide variety of applications. Literature The strategy of using chimeric gene products has been applied to the study of the structure and function of phosphotransferases in yeast (Hjel mstad et al., (1 994) J. Biol .. Chem. 269: 20995-21002) and endon ucleases Restriction of Fla vobacterium Kim et al. (1994) Proc. Nati Acad. Sci. USA. 91: 883-887). Domain change has been used to redistribute protein functional domes in protein engineering (Hedstrom (1994) Biochemistry 33: 9382-9388). This thioesterase, like other bacterial or mammalian thioesterases, does not share sequence homology with plant thioesterases (Voelker et al. (1992) supra). DESCRIPTION OF THE FIGURES Figure 1. An alignment of the amino acid sequence of representative Class I thioesterases (FatA) and Class II (Fat B) is provided. UcFatBI (SEQ ID NO: 1) is C12 thioesterase from California laurel. CcFatBI (SEQ ID NO: 2) is a C14 thioesterase of camphor. CpFatBI (SEQ ID NO: 3) is a thioesterase of C8 and C10 of Cuphea palustris. CpFatB2 (SEQ ID NO: 4) is a thioesterase of C14 of Cuphea palustris C8 and C10. GarmFatAI (SEQ ID NO: 5) is an 18: 1 mangosteen thioesterase which also has considerable activity on C18: 0 acyl-ACP substrates. BrFatAI (SEQ ID NO: 6) is an 18: 1 thioesterase from Brassica rapa faka Brassica campestris). The amino acid sequences that are identical in all represented thioesterases are indicated by the bold shadows. Figure 2. Activity analysis results of wild type laurel thioesterases (Figure 2A) and wild-type camphor thioesterases (Figure 2B) under expression in £ are presented. coli Figure 3. The reduced nucleic acid and amino acid sequence of a PCR fragment (SEQ ID NO: 7) containing the coding region for the mature protein portion of an acyl-ACP thioesterase Class II camphor is provided.

Figure 4. The translated nucleic acid and amino acid sequence (SEQ ID NO: 8) of an acyl clone-ACP thioesterase class I mangosteen (GarmFatAI) is provided. GarmFatAI demonstrates the activity of primary thioesterase in acyl substrate-ACP 18: 1, but also demonstrates considerable activity in the substrate of 18: 0 (approximately 10-20% activity of 18: 1). Figure 5. The sequence of translated nucleic acids and amino acids (SEQ ID NO: 9) of the acyl clone-ACP Class I of mangosteen, GarmFatA2, is provided. GarmFatA2 has thioesterase activity mainly on the acyl-ACP substrate 18: 1 and activity is equally low in substrates 16: 0 and 18: 0. Figure 6. Transcriptional nucleic acid and amino acid sequence (SEQ ID NO: 10) of a Class II acyl-ACP thioesterase clone of Cuphea palustris (CpFatBI) having preferential activity on acyl-ACP substrates of C8 and C10 is provided. . Figure 7. A translated nucleic acid and amino acid sequence (SEQ ID NO: 11) of a Class II acyl-ACP thioesterase clone of Cuphea palustris (CpFatB29 having preferential activity on C14 acyl-AP substrates. A comparison of amino acid sequences of laurel thioesterases (C12) (SEQ ID NO: 1) and camphor (C14) (SEQ ID NO: 2) is provided.Amino acid residues differing between thioesterases are indicated by prominent shading.

Figure 9. Chimeric laurel / camphor, Ch-1 and Ch-2 constructions are shown, as mergers in the framework of the N and C terminal portions of the thioesterase (from left to right). The Kpnl site used to construct the chimeric constructions is shown. Figure 10. A comparison of amino acid sequences of acyl-ACP thioesterase from C. palustris CpFatBI is provided (C8 / C10) (SEQ ID NO: 3) and C. palustris CpFatB2 (C14) (SEQ ID NO: 4).

The amino acid residues that differ between thioesterases are indicated by prominent shading. Figure 11. Substrate specificities of the laurel / camphor chimeric enzymes and two thioesterases of laurel mutants (dark shaded columns) are provided. The control reinforcement activities (E. coli transformed with the vector alone) are indicated by the columns with light stripes. (A) Ch-1 (B) Ch-2 (C) laurel mutant M197R / R199H and (D) laurel mutant M197R / R199H / T231K. Figure 12. The relative thioesterase activity of wild type Garcinia mangifera / 5247) and mutant thioesterases (GarmFatAI) are provided on acyl-ACP substrates 18: 1, 18: 0 and 16: 0. Figure 13. A comparison of amino acid sequences of acyl-ACP thioesterase from B. rapa BrFatAI (C18: 1) is provided (SEQ ID NO: 6) and Garcinia mangifera GarmFatAI (C18: 1 / C18: 0) (SEQ ID NO: 5). The amino acid residues that differ between thioesterases are indicated by dark shading. Figure 14. Short domain replacement by RCP. The full-length gene is shown by two long parallel lines. The area striped represents the domain of interest. For each CPR initiator (a, b, c, and d), an arrow head is pointing towards the 3 'end. Initiators a and b are initiators forward and backward for the full length of DNA. The thin lines in the primers c and d represent sequences that exactly match the 3 'downstream of the domain. The glued tails of the c and d ini- tiators are the 5 'pendants corresponding to the new domain sequence. Figure 15. Replacement of long domain by CPR. Two RCP (RCP 1 and 2) with a gene I as a standard are carried out. A third RC P is carried out simultaneously with the I I gene as a standard. Initiators a and b are forward and backward primers for the full length I gene. The primer c equals the immediate sequence 3 'downstream of the original domain in gene I. The thin line in initiator d represents the sequence that equals 3 'downstream of the original domain in gene I, while the thick tail equals the 3' end sequence of the replacement domain in gene II, and initiates the endpoint 5. 'of the domain in gene II, while f initiates the other end. The thin tail in primer f represents the sequence that matches the 3 'downstream of the original domain in gene I. Figure 16. The relative changes in activity on the 18: 0 and 1 8: 1 substrates of the thioesterases of Garm Fat A 1 mutants compared to the wild-type Garm FatA 1 thioesterases are shown.

Figure 17. Specific activities of mutant thioesterase of Garm FatAI are provided in acyl substrates-ACP 16: 0, 18: 0 and 18: 1. Figure 18. An alignment of thioesterases from Garm FatAI and UcFatBI is generally provided as representatives of FatA and FatB. Unique, partially homologous and highly homologous regions are indicated in the two classes of thioesterase. Figure 19A. Wild type FatA and FatB mutants and chimeric mutants are represented. Results of activity analysis and specificity are provided. The interpretation of several stripes is in accordance with the key provided in Figure 18. Figure 19B. Recombination mutants are represented FatA and FatB. Results of activity analysis and specificity are provided. The interpretation of several stripes is in accordance with the key provided in Figure 18. Figure 20. Representations of fatty acid analysis histograms are provided from seeds of B. napus Quantum plants transformed with pCGN5255 and pCGN5274. Figure 21. Fatty acid composition analyzes of 5255 are provided in Figure 21A. The fatty acid composition analyzes of 5274 transgenics are provided in Figure 21B. Figure 22. Fatty acid composition analysis of 5290 is provided in Figure 22A. Analysis of fatty acid composition of transgenic 5291 in Figure 22B. SUMMARY OF THE INVENTION By this invention, methods for producing acyl-ACP thioesterases from engineered plants are provided, wherein acyl-ACP thioesterases from treated plants demonstrate altered substrate specificity with respect to acyl-ACP substrates hydrolyzed by the Plant thioesterase compared to acyl-ACP native thioesterase. Said methods comprise the steps of (1) modifying a gene sequence encoding already targeted plant thioesterase protein for modification in order to produce one or more sequences of modified thioesterase genes, wherein the modified sequences encode acyl thioesterases. Treated ACPs that have substitutions, insertions or deletions of one or more amino acid residues in the mature portion of the thioesterase of target plants, (2) express the modified coding sequences in a host cells, whereby the plant thioesterases are produced treated and (3) analyze the thioesterases of treated plants to detect those that have suitable alterations in substrate specificity.

Of particular interest for amino acid alterations is the two-thirds C-terminal portion of plant thioesterase and more particularly, the region corresponding to 229 to 285 amino acids (consensual numbering of the sequences) of the plant thioesterase sequences represented in the alignment of sequences of Figure 1. Addition- ally, the region of amino acids 285-312 of interest for modifying the specificity of the thioesterase substrate to the shorter chain fatty acids such as C8 and C 10. Useful information with respect to sites of potential modification in a thioesterase acylated to the site can be obtained by comparison of amyloid sequences of acyl thioesterase-AC P from related plants, where the compared thioesterases demonstrate different hydride activities. Comparisons of amino acid sequences of plant thioesterase at least a sequence identity of 75% in the region of mature proteins are particularly useful in this regard. In this way, the amino acid residues or peptide domains that are different in the related thioesterase can be selected for mutagenesis. Other methods for selecting amino acids or dominants of peptides for modification include analysis of thioesterase protein sequences for effects provided by substitutions, substitutions or deletions in flexility or secondary structure of the white thioesterase. In addition, useful thioesterase gene mutations can be discovered by random mutation of acyl thioesterase-ACP coding sequences of plants, following analysis of thioesterase activity or fatty acid composition to detect alterations in substrate specificity.

To produce a treated thioesterase, a DNA sequence encoding the thioesterase can be altered by domain exchange or mutagenesis, either random or site-directed, to introduce substitutions, insertions or deletions of amino acids. The DNA sequences can then be expressed in host cells for the production of treated thioesterases and for the analysis of resulting fatty acid compositions. The treated thioesterases produced in this way are also analyzed to determine the effects of amino acid sequence modifications on the specificity of thioesterase substrates. In this way, novel thioesterases can be discovered which demonstrate a variety of profiles with respect to the carbon chain lengths of acyl-ACP substrates that can be hydrolyzed or with respect to the relative activity of the thioesterase on different acyl substrates. ACP carbon chain length. Thus, DNA sequences and constructs for the expression of treated thioesterases, as well as the novel thioesterases produced therefrom are also considered within the scope of the invention described herein. Said DNA sequences can be used for the expression of the thioesterases treated in the host cells for the modification of the fatty acid composition. Of particular interest in the invention are DNA constructs for the expression of thioesterase treated in plant cells, especially in cells. of plant seeds of oily seed crop plants. As a result of the expression of said constructions, plant triglyceride oils can be produced, wherein the composition of the oil reflects the specificity of the altered substrate of the treated thioesterases. Therefore, the cells, plant seeds, and plants comprising the constructions provided herein are encompassed by the present invention, as well as novel plant oils that can be recovered from the seeds of plants. For example, an acyl-ACP plant preferably of C12 described herein, can be altered to obtain a plant thioesterase having approximately the same activity on the C14 and C12 substrates. Further modification of the C12 enzyme produces a thioesterase having higher activity on C14 compared to C12 substrates. Acyl-ACP thioesterase sequences from novel plants of Cuphea palustris and mangosteen (Garcinia magnifera) are also provided in the present invention. The sequence of C. palustris, CpFatBI, demonstrates substrate specificity towards fatty acyl-ACP of C8 and C10 with superior activity on C8. A mangosteen thioesterase gene, GarmFatAI, demonstrates primary activity on substrates of 18: 1-ACP, but also demonstrates substantial activity on 18: 0-ACP. Most importantly, this clone does not demonstrate specificity for 16: 0 substrates. Methods to modify the specificity of plant thioesterase compounds Novels of C8 / C10 and C18: 1 / C18: 0 are also provided in the present invention. In particular, mutations are provided that increase the 18: 0/18: 1 activity ratio of the mangosteen clone. The use of such mutated mangosteen thioesterase clones is provided for the improved production of 18: 0 fatty acids in seeds of transgenic plants. Such uses result in improved plants, seeds and oils. DETAILED DESCRIPTION OF THE INVENTION By this invention, methods of producing thioesterases from treated plants having altered substrate specificity are provided. A treated plant thioesterase of this invention may include any amino acid sequence, such as a protein, polypeptide or peptide fragment obtainable from a plant source demonstrating the ability to catalyze the production of free fatty acids from fatty acyl-ACP substrates under reactive conditions of plant enzymes. By "reactive enzyme conditions" it is meant that any necessary condition is available in an environment (i.e., such factors as temperature, pH, lack of inhibitory substances) that will allow the enzyme to function. The thioesterases from treated plants can be prepared by random or specific mutagenesis of a thioesterase encoding sequences to provide one or more amino acid substitutions in the translated amino acid sequence. Alternatively, a thioesterase from treated plants can be prepared by exchange of domain between the thioesterase of related plants, wherein the extensive regions of the native thioesterase encoding sequences are replaced with the corresponding region of a thioesterase from different plants. The targets for domino exchange may include peptides ranging from five or six to tens of amino acids in length. In an ideal case, this type of exchange can be achieved by the presence of unique restriction sites, conserved in exact exchange points in the genes that encode both proteins. Oligopeptide-based mutagenesis (cyclization) can be applied when convenient restriction sites are not available, although this process can be delayed when long domain sequences must be exchanged.

Alternatively, as described in the following examples, a rapid method for domain exchange can be used which is a modification of an overlap extension technique using polymerase chain reaction (PCR described by Horton et al. (BioTechniques (1990)). : 528-535) The entire procedure can be performed within six hours (time for two runs of CPR) without in vivo manipulation.The basis for the overlap extension method is that in a CPR, the initiators must be equal to their pattern sequence enough to start, but they do not need to be exactly matched, especially towards the 5 'end. In fact, PCR primers with 5 'pendants (unmatched sequences) are routinely used. The exchange of A domain based on RC P is designed for applications where the domain contains approximately six amino acids or less (short domain exchange) or where domains containing larger numbers of amino acids must be exchanged (long domain exchange). The specificities of its altered substrate of a treated thioesterase can be reflected by the presence of hydrolysis activity on an acyl-ACP substrate of a particular chain length that is not hydrolyzed by the enzyme of native thioesterases. The newly recognized acyl-ACP substrate. The newly recognized acyl-ACP substrate may differ from native substrates of the enzyme in various forms, such as having a shorter or longer carbon chain length (usually reflected by the addition or deletion of one or more 2-carbon units). ), having a greater or lesser degree of saturation, or by the presence of a methyl group, such as in certain fatty acids that are not commonly present in plant cells, ie, iso and anti-iso-fatty acids. Alternatively, the altered substrate specificity can be reflected by a modification of the relative hydrolysis activities in two or more acyl-ACP substrates of different chain lengths and / or saturation degree. Information on DNA and amino acid sequences for more than thirty acyl-AC plant thioesterases is now available and these sequences can be used in the methods of the present invention to identify regions suitable for modification in order to produce sequences for the expression of treated thioesterases. Plant thioesterases can be classified into two classes by sequence homology. All these plant thioesterases contain a temporary peptide, 60 to 80 amino acids in length, for the direction to the plastid. The temporal peptides have little homology between the species whereas the regions of mature proteins (minus the temporal peptides) show identification of important amino acid sequences. The first class, Class I (or FatA) includes acyl-ACP long chain thioesterases that have activity primarily in 18.1-ACP. 18: 1-ACP is the immediate precursor of most of the fatty acids found in phospholipids and triglycerides synthesized by the eukaryotic route. This class of thioesterase has been found essentially in all plant sources examined so far and is suggested to be an essential "host" enzyme (Jones et al., (Supra) required for membrane biosynthesis.) Examples of Calase I thioesterase Sunflower, Cuphea hookeriana and Brassica rapa (campestris), which has activity mainly in the substrate of 18: 1 ACOP, have been described (WO 92/20236 and WO 94/10288) Other thioesterases of 18: 0 have been reported in Arabidopsis thaliana (Dorman et al., (1995) arch. Bichem. Biohys .. 376: 612-618), Brassica napus (Loader et al. (1993) Plant Mol. Biol. 23: 769-778) and coriander (Dorman et al. (1994) Biochem Biophys, Acta 1212: 134-136) A specific Class I thioesterase of 18: 1-ACP Similar (GatA2) has been discovered to develop mangosteen (Garcinia mangifera) embryos and is described herein A Class I soybean thioesterase (WO 92/11373) was reported to provide 10- and 96-fold increases in 160-ACP activity and 18 1-ACP on expression in E coli, and a smaller increase (3-4 times) in activity of 18 0-ACP The mature protein regions of the thioesterases of Class I plants are highly homologous, demonstrating an identification of sequence greater than 80% In addition, another Class I mangosteen thioesterase (GatAI), also described herein, has been found to demonstrate thioesterase activity primarily on 18 1-ACP substrates (100-fold increase by E coli expression). ), but also demonstrates the selective activity in 180-ACP against 160-ACP The activity of 180 of GatAI is approximately 25% of the activity of 18 1, while in most of the Class I thioesterase tested to now, the activity of 18 1 is highly predominant, with the activity in the substrates of 160 and 180 detecting at least 5% of the activity levels of 18 1 A second class of plant thioesterases, the thioesterases Class II (or FatB), includes enzymes that use fatty acids with shorter chain lengths, from C80 to C140 (medium chain fatty acids) as well as C160 Class II thioesterases catalyze the hydrolysis of substrates containing saturated fatty acids. Class II thioesterase (or FatB) have been isolated of California Laurel, elm, Cuphea hookeriana, Cuphea palustris, Cuphea lanceolata, nutmeg, Arabidopsis thaliana, mango, leek and camphor. The mature protein regions of thioesterase from Class II plants are also highly homologous, demonstrating sequence identification of 70-80%. One of the characteristics of Class II thioesterase is the presence of a relatively hydrophobic region of approximately 40 amino acids in the N-terminal region of mature proteins. This hydrophobic region is not found in 18: 1-ACP thioesterases, and has no apparent effect on the activity of the enzyme. Recombinant expression of a Class II thioesterase from laurel cone without this region showed identical activity profiles in vitro (Jones et al. (Supra)). As more fully demonstrated in the following examples, the specificity of acyl-ACP substrates of plant thioesterases can be modified by various amino acid changes to the protein sequence, such as substitutions, insertions or deletions of amino acids, in the protein portion. Mature plant thioesterase. The specificity of modified substrates can be detected by the expression of thioesterases from plants treated in E. coli and analyzed for the activity of the enzyme. The specificity of the modified substrate can be indicated by a change in the preference of the acyl-ACP substrate so that the treated thioesterase is recently able to hydrolyze a Substrate not recognized by the native thioesterase. The newly recognized substrate can vary from the substrates of the native enzyme by the carbon chain length and / or degree of saturation of the fatty acyl portion of the substrate. Alternatively, the specificity of the modified substrate can be reflected by a change in the relative thioesterase activity in two or more substrates of the native thioesterase so that the treated thioesterase exhibits a different order of preference for acyl-ACP substrates. For example, a plant thioesterase having mainly hydrolysis activity on the C12 substrate and some minor activity on the C14 substrate can be modified to produce a treated thioesterase which exhibits increased activity at C14, for example, so that the treated thioesterase it has approximately equal activity in substrates of C12 and C14. Similarly, said plant C12 thioesterases can be further modified to produce a treated thioesterase having primary activity on C14 substrates and little or no activity on C12 substrates. Alternatively, a plant thioesterase can be modified so as to alter the relative activity towards a substrate having a higher or lower degree of saturation. For example, a Class I thioesterase (18: 1) can be modified to increase the relative activity in substrates of C18: 0 compared to the activity on other substrates of the enzyme, such as C18: 1 and C16: 0. Examples of these types of modifications of thioesterase are provided in the following examples. Further modification of plant thioesterases are also convenient and can be obtained using the methods and sequences provided herein. For example, plant thioesterases can be modified to change the enzymatic activity towards hydrolysis of shorter chain fatty acids such as C8 and C 10. the comparison of the closely related thioesterase sequences, such as the thioesterase C sequences. palustris C8 / C 10, C. palustris C 14 and C. hookeriana of C8 / C 10, provided in this, can be used to identify potential tt amino acid residues for the alteration of thioesterase specificity. In the initial experiments aided in altering the substrate specificity of plant thioesterase enzymes, two highly related Class I I thioesterases were studied, an aci lo-ACP thioesterase preferably of C 12 from California laurel. { Umbellularia californica) and an acyl thioesterase-ACP preferably of C 14, demonstrate an amino acid sequence identification of 90% in the region of mature proteins that still have different substrate specificities. Constructs for the expression of mature, non-ionic thioesterases were prepared which encode chimeric thioesterase enzymes containing the mature N-terminal protein region of camphor thioesterase or laurel and the C-terminal portion of the other thioesterase. The portion of the N-terminal thioesterase according to Coded in those constructs contains about a third of the mature thioesterase protein and the C-terminal spray contains the remaining two thirds of the mature thioesterase region. As described in more detail in the following examples, we have discovered that two-thirds of the C-terminal portion of these plant thioesterases is critical to determining substrate specificity. The chimeric enzyme containing the C-terminal portion of the camphor thioesterase (Ch-1) demonstrates the same activity profile as the native camphor thioesterase (specific for 14: 0) and the chimeric protein with the laurel thioesterase with termination in C (Ch-2) demonstrates the same activity profile as native bay laurel thioesterase (12: 0 specific). Additional studies of the C-terminal end of the protein were carried out to further localize regions of thioesterase proteins critical for substrate specificity. In one such study, the 13 consecutive C-terminal amino acids of laurel thioesterase were suppressed by the production of a mutant gene lacking the coding DNA for this region. The activity of the expressed mutant thioesterase was compared to an expressed wild type laurel thioesterase protein. The activity profiles of the expressed mutant. The activity profiles of the mutant terminating in 17 C wild-type laurel thioesterase proteins were equal, demonstrating that the C-terminal end of the thioesterase proteins is a critical region for substrate specificity.

Additional analysis of two thirds of the C-terminal portion of acyl laurel-AC P thioesterase was carried out, preferably of C 12, to identify particular amino acids involved in the specificity of the substrate. By examining sequence alignment of laurel and camphor thioesterases, less conservative amino acid substitutions were identified between the two thioesterases in the two-thirds C-terminal portion of the proteins. Substitutions of non-conservative amino acids include those in which the substituted amino acids have a different charge than the native amino acid residue. The amino acids considered to have side chains positively charged to p H7 are lysine and arginine. Histidine can also have a positively charged side chain under p-H acid conditions. The amino acids considered to have side chains negatively charged to p H 7 are aspartate and glutamate. Substitutions of non-conservative amino acids can also be indicated where amino acids substituted in size differ substantially from the size of the amino acid normally located in that position. Examples of unconserved amino acid differences between camphor thioesterases are M 197? R (Laurel TE? Camphor TE), R 199? H, T231? K, A293? D, R327? Q, P280-S and R381 - * S (the numbering of amino acid sequences for laurel and camphor thioesterases is shown in Figure 8). Secondary structure predictions can be used to identify amino acid substitutions by having similarly effects on the secondary structure of the thioesterase protein. For example, according to predictions of secondary structures using Cho and Fasman methods, the MRR tripeptide of laurel amino acids 197-199 and the corresponding camphor RRH are located behind the β-sheet and one spin anchored by two glycines highly preserved (G193 and G196). This region of plant thioesterase is highly conserved and the ß sheet and spin structure is also predicted in other flat thioesterase. As described in the following examples, when the tripeptide of M-R-R is changed to R-R-H, mimicking the sequence in camphor thioesterase, the activity of the mutant towards 12: 0, but not 14: 0 is reduced approximately 7 times compared to the wild type. This results in a treated thioesterase which has approximately equal specific activity with respect to the 12: 0 and 14: 0 substrates. A further modification of the treated laurel thioesterase M197R / R199H which converts the threonine residue at amino acid 231 to a lysine (T231K) alters the substrate specificity so that the treated thioesterase M197R / R199H / T321 K is highly specific for : 0-ACP. Interestingly, the T231K mutation alone does not affect the laurel thioesterase activity. The non-additive combinatorial effect of the substitution of T231K in thioesterase treated with M197R / R199H suggests that the amino acid sites altered closely duplicate each other (Sandberg, et al. (1993) Proc. Nati, Acad. Sci. 90: 8367-8371). As described in the following Examples, substitutions of amino acids near the active site (YREEC, amino acids 357-361 in the consensual numbering of Figure 1) of acyl-ACP plant thioesterases can result in large reductions in activity of thioesterase Modification of bay thioesterase to produce R327Q results in a 100-fold decrease in laurel thioesterase activity in Figure 8. Expression of treated thioesterases having altered substrate specificities in host cells and analysis of compositions of resulting fatty acids demonstrate that the specificities of altered substrates of the treated thioesterases are reflected in the fatty acid composition profiles of the host cells. It is important to note that the activity of the enzyme in vivo may have interactions or sequential parameters involved such as life time and bending / unfolding regimes that could not be reflected in in vitro activity analyzes. The major lipid components of E. coli membranes are phosphatidylethanolamine and phosphatidylglycerol, which contain portions of predominantly long chain fatty acyl. The major lipid components of E. coli membranes are phosphatidylethanolamine and phosphatidylglycerol, which contain fatty acyl portions of long chain. The recombinant expression of laurel thioesterase cDNA native to the fadD cells redirects the bacterial type II fatty acid synthase system of long chain to medium chain production and similar results are obtained by the expression of native laurel thioesterases in seeds of transgenic plants (Voelker et al. (1994) supra; Voelker et al. (1992) supra). Therefore, E. coli data in vivo can be used to predict the expression effects of thioesterase treated in transgenic plants. With native laurel thioesterase, E. coli fadD cells produce large amounts of free fatty acids 12: 0 and small amounts of 14: 0 (levels of approximately 5 to 10%) (Voelker et al. (1994) and Table I) . However, as demonstrated in the following examples, after two amino acid substitutions (M197R / R199H), the expression of a treated laurel thioesterase enzyme results in the accumulation of similar amounts of fatty acids of 12: 0 and 14. : 0 Similarly, the expression of laurel thioesterase treated with three amino acid substitutions (M197R / R199H / T231K) completely reverses the 12: 0/14: 0 ratio of fatty acids produced compared to the results with native bay thioesterase. The treatment of FatA thioesterases from plants is also described herein. In particular, mutations were found that provide Garm FatAl thioesterases from mutants that have a higher specific activity and a relative activity more convenient on substrates of 18: 0 against substrates of 18: 1. For example, a Garm Fat A1 D261K mutant having a substituted lysine residue (K) for the aspartate residue present in the wild type clone at position 261 (the numbering is as indicated in the consensual line on the sequences in the sequence comparison of Figure 1), has increased activity on substrates 18: 0 versus 18: 1. The double and triple mutants containing the D261K mutation have an even higher activity at 18: 0. Other mutations that increase the 18: 0 activity of mangosteen GarmFatAI thioesterase are described herein include S188A, S370A, G185A and V270A. These mutant thioesterases, as well as mutants having various combinations of these mutations, are of particular interest for use in plant genetic engineering applications to increase the fatty acid content of 818: 0 stearate in oilseed crop plants. For example, as described in more detail in the following examples, plants transformed to express a double mutant of GarmFatAI S188 / V270A, which has a substituted alanine residue for the serine residue at position 188 and for the valine residue in the position 270 (the numbering is as indicated in the consensual line on the sequences in the sequence comparison of Figure 1), have significantly increased levels of stearate. Transgenic plants with increased levels of C18: 0 fatty acid as a result of Garm FatA I thioesterase expression in Brassica napus seeds, it was reported in WO 97/12047, the description of which is incorporated herein by reference. The mutant thioesterases in the present invention can be used to provide even greater amounts of stearate content in seeds of transgenic plants as described in greater detail in the following examples. The vegetable-rich stearate oils are suitable for use in such applications as substitutes for margarines and non-hydrogenated cocoa butter ("trans-free") as described in WO 97/12047 or in fluid shortening applications, such as those described in copending application USSN 08/843, 400, entitled "Food Products Containing Structured Triglycerides" filed April 1, 1997. Therefore, as a result of modifications to the substrate specificity of plant thioesterases, it can be observed that the relative amounts of fatty acids produced in a cell can be altered where several substrates are available for hydrolysis.In addition, the molecules that are formed of available free fatty acids, such as triglycerides from seedlings of plants, can also be altered as a result of the expression of treated thioestases that have altered substrate specificities. known acyl-AC acrylateases and coding sequences, such as those provided herein, other acyl-ACP thioesterase sequences can be obtained from a variety of plant species and said thioesterase and coding sequences will find use in the methods of this invention. As noted above, the thioesterase coding sequences of plants are highly conserved, in particular for the thioesterase which are members of the same class of thioesterase, ie Class I or Class II. Therefore, to isolate additional thioesterases, a genomic or other appropriate bank from a candidate plant source of interest was tested with conserved sequences of one or more thioesterase sequences from Class I or Class II plants to identify homologously related clones. Positive clones were analyzed by digestion and / or sequencing of the restriction enzyme. The probes can also be considerably shorter than the entire sequence. Oligonucleotides can be used. For example, but at least they should be about 10, preferably at least about 15, more preferably at least 20 nucleotides in length. When regions of shorter length are used for comparison, a higher degree sequence identity is required than for longer sequences. Shorter probes are often particularly useful for polymerase chain reactions (PCR) (Gould, et al., PNAS USA (1989) 86: 1934-1938), especially for the isolation of plant thioesterases containing highly conserved sequences. . PCR that uses oligonucleotides for conserved regions of Plant thioesterases can also be used to generate homologous probes to screen banks. When longer nucleic acid fragments (> 100 bp) are used as probes, especially when full or long cDNA sequences are used, they can be sieved even with highly moderate restrictions (for example using formamide at 50 to 37 with minimal washing). in order to obtain signals from the target sample with a deviation of 20-50, that is, homologous sequences. (For additional information regarding screening techniques, see Beltz, et al., Methods in Enzymology (1983) 100: 266-285). The nucleic acid or amino acid sequences encoding an acyl-ACP thioesterase of treated plants of this invention will be combined with other non-native or "heterologous" sequences in a variety of ways. By "heterologous" sequences is meant any sequence that is not naturally bound to the acyl-ACP plant thioesterase, including, for example, combinations of nucleic acid sequences from the same plant that are not naturally bound. For expression in host cells, the sequence encoding a treated plant thioesterase is combined in a DNA construct having, in the 5 'to 3' direction of transcription, a transcription initiation control region capable of promoting transcription and translation in a cell host, the DNA sequence encoding the acyl-ACP plant thioesterase treated and a transcription and translation termination region. The DNA constructs may or may not contain pre-processing sequences, such as temporal peptide sequences. Temporary peptide sequences facilitate delivery of the protein to a given organelle and separate from the amino acid portion upon entering the organelle, releasing the "mature" sequence. The use of the acyl-ACP DNA sequence of precursor plants is preferred in expression cassettes of plant cells. Other sequences of temporary plastid peptides, such as a temporary peptide from the ACP seed, can also be used to translocate acyl-ACP plant thioesterases to various organelles of interest. Therefore, the thioesterase sequences of treated plants can be used in various constructs, such as for expression of the thioesterase of interest in a host cell to recover or study the enzyme in vitro or in vivo. Potential host cells include prokaryotic and eukaryotic cells. A host cell may be unicellular or be in a differentiated or undifferentiated multicellular organism depending on the intended use. The cells of this invention can be distinguished by having an acyl-ACP from plants treated herein. Depending on the host, the regulatory regions will vary, including regions of viral genes, of chromosomal plasmids, or Similar. For expression in prokaryotic or eukaryotic microorganisms, particularly unicellular hosts, a wide variety of constitutive or regulatory promoters may be employed. Expression in a microorganism can provide a ready source of the treated plant enzyme and is useful for identifying the particular characteristics of said enzymes. Among the transcriptional initiation regions that have been described are host regions of bacteria and yeasts, such as E. coli, B. subtilis, Saccharomyces cerevisiae, including genes such as beta-galactosidase, T7 polymerase, tryptophan E, and the like. For most of the plant, the constructions will involve functional regulatory regions in plants that provide expression of the acyl-ACP plant thioesterase and therefore result in the modification of the fatty acid composition in plant cells. The open reading frame coding for acyl-ACP plant thioesterase will bind at its 5 'end to a transcription initiation regulatory region such as the wild-type sequence found naturally upstream of the 5' to the thioesterase structural gene. Numerous other transcription initiation regions are available, which provide a wide variety of constitutive or regulatable, eg, inducible, transcription of structural gene functions. Among the transcriptional initiation regions used for plants, said regions are associated with structural genes such as for nopaline or manpina synthases or with napin, ACP promoters and Similar. The transcription / translation initiation regions corresponding to said structural genes are found immediately upstream of the respective initiation codons. In embodiments where the expression of the treated thioesterase protein is desired in a plant host, the use of part of the acyl-ACP thioesterase gene of native plants is considered. Namely, all or a portion of the regions without coding upstream of 5 '(promoter) can be used together with the regions without 3' downstream coding. If a different promoter is desired, said native promoter for the plant host of interest or a modified promoter, ie, having transcription initiation regions derived from a gene source and the translation initiation regions derived from a source of different genes (improved promoters), such as double 35S CaMV promoters, the sequences can be joined using normal techniques. For such applications, when regions without upstream coding 5 'of other regulated genes are obtained during seed maturation, those preferentially expressed in plant embryo tissues, such as ACP and transcriptional initiation control regions derived from napkin, are desired. . Said "seed-specific promoters" may be obtained and used in accordance with the teachings of the U.S.A. Series No. 07 / 147,781, filed on 1/25/88 (now E.U.A. Series No. 07 / 550,804, filed on 7/9/90) and E.U.A. Series No. 07 / 494,722 filed on or about March 16, 1990, which has a title "Novel Sequences Preferentially Expressed In Early Seed Development and Methods Related Thereto", the references of which are incorporated herein by reference. Transcription initiation regions that are expressed preferentially in seed tissues, ie, not detected in other parts of the plant, are considered suitable for fatty acid modifications in order to minimize any alteration or adverse effect of the gene product. The regulatory transcription termination regions can also be provided in the DNA constructs of this invention. The transcription termination regions may be provided by the DNA sequence encoding the acyl-ACP plant thioesterase or a convenient transcription termination region derived from a different gene source, eg, the transcription termination region that is associated naturally with the transcription initiation region. Where the transcription termination region is from a different gene source, it will contain at least about 0.5 kb, preferably about 1-3 kb of the 3 'sequence to the structural gene from which it is derived from the termination region. Expression or transcript constructions of plants having an acyl-ACP plant thioesterase as the DNA sequence of interest can be employed with a wide variety of plant life, particularly, the life of the plants involved in the production of vegetable oils for edible and industrial uses. The most especially preferred are oily seed oil cultures. Plants of interest include, but are not limited to, rapeseed (canola varieties and high erucic acid content), sunflower, safflower, cotton, Cuphea, soybean, peanut, coconut and oily palm and corn. Depending on the method for the introduction of the recombinant constructs into the host cell, other DNA sequences may be required. Most importantly, this invention applies to similar dicotyledonous and monocotyledonous species and will be readily applicable to new and / or improved transformation and regulation techniques. The method for transformation is not critical to the present invention, several plant transformation methods are currently available. As newer methods to transform crops are available, they can be applied directly later. For example, many species of plants naturally susceptible to Agrobacterium infection can be successfully transformed via tripartite or binary vector methods of transformation mediated by Agrobacterium. In addition, the techniques of microinjection, bombardment of DNA particles, electroporation, have been developed, which allow the transformation of several species of monocotyledonous and dicotyledonous plants. To develop the DNA construct, different construction components or fragments of it will normally be inserted into a convenient cloning vector which is capable of of replicating in a bacterial host, e.g., E. coli ,. There are numerous vectors that have been described in the literature. After each cloning, the plasmid can be isolated and subjected to further manipulation, such as restriction, insertion of new fragments, ligatures, deletions, insertion, resection, etc., so as to configure the components of the desired sequence. Once the construction is completed, it can be transferred to an appropriate vector for further manipulation according to the shape of the host cell transformation. Normally, within the DNA construct there will be a structural gene that has the regulatory regions necessary for expression in a host and that provides for the selection of transforming cells. In gene it can provide resistance to a cytotoxic agent, e.g., antibiotic, heavy metal, toxin, etc., the complementation that provides phototrophy to an auxotrophic host, viral immunity or the like. Depending on the number of different host species, the construction or expression components thereof are introduced, one or more markers may be used, where different conditions are used for selection for the different hosts. It is noted that the degeneracy of the DNA code provides that some codon substitutions are allowed from the DNA sequences without any corresponding modification of the amino acid sequence.

The manner in which the construction of DNA is introduced into the host of the plant is not critical to this invention. Any method that provides efficient transformation can be employed. Various methods for the transformation of plant cells include the use of Ti or Ri plasmids, microinjection, electroporation, bombardment of DNA particles, fusion of liposomes, bombardment of DNA or the like. In many cases, it will be convenient to bombard the construction on one or both sides by T-DNA, particularly having left and right edges, more particularly the right bank. This is particularly useful when the construction uses A. tumefaciens or A. rhizogenes as a mode of transformation, although the T-DNA borders can be used with other modes of transformation. When Agrobacterium is used for the transformation of plant cells, a vector can be used which can be introduced into the Agrobacterium host for homologous recombination with T-DNA or the Ti or Ri plasmid present in the Agrobacterium host. The Ti or Ri plasmid containing the T-DNA for recombination, can be armed (capable of causing scale formation) or disarmed (unable to cause embedding), the latter being permissible, while the vir genes are present in the Agrobacterium host. The armed plasmid can give a mixture of normal plant cells and embedding. In some cases where Agrobacterium is used as the vehicle to transform plant cells, the construction of Expression surrounded by T-DNA borders will be inserted into a broad-spectrum host vector described in the literature. PRK2 or derivatives thereof are commonly used. See, for example, Ditta et al., PNAS USA, (1980) 77: 7347-7351 and EPA 0 120 515, which are incorporated herein by reference. With the expression construct and the T-DNA there will be one or more markers, which allow the selection of transformed Agrobacterium cells and transformed plants. A number of markers have been developed for use with plant cells, such as resistance to chloramphenicol, aminoglycoside 418, hygromycin or the like. The particular marker employed is not essential for this invention, one or the other marker being preferred depending on the particular host and the form of construction. Once a transgenic plant is obtained which is capable of producing seeds having a modified fatty acid composition, traditional plant culture techniques, including mutagenesis methods, can be employed to further manipulate the fatty acid composition. It is noted that the transformation method is not critical to this invention. However, the use of methods of transforming genetically engineered plants, that is, the energy to insert a single desired DNA sequence, is critical. Hence, the ability to modify the fatty acid composition of plant oils was limited to the introduction of traits that could be sexually transferred during plant crossings or viable traits generated through mutagenesis.

Through the use of genetic engineering techniques that allow the introduction of genetic information between species and media to regulate the specific expression for tissues of endogenous genes, a new method for the production of seed oils is possible. of plants with modified fatty acid compositions. In addition, there is the potential for the development of seed oils of novel plants when applied to the tools described herein. One may choose to provide the transcription or transcription and translation of one or more sequences of interest in concert with the expression of an acyl thioesterase-AC P from plants treated in a plant host cell. In particular, the expression of a plant protein having medium chain or very long chain fatty acid activity in combination with the expression of an acyl-ACP thioesterase from treated plants may be preferred in some applications. See WO 95/27791 for LPAAT coding sequences. When it is desired to provide a transformed plant for the combined effect of more than one nucleic acid sequence of interest, each nucleic acid construction will normally be provided separately. The constructs, as described above, contain the regulatory transcriptional or transcriptional and translational regulatory regions. Someone skilled in the art will be able to determine the regulatory sequences to provide a desired time control and tissue specificity appropriate for the final product according to the above principles exhibited as regards the respective expression of constructions of contradictory. When two or more constructs will be employed, whether they are related or not to the same fatty acid modification sequence or a different fatty acid modification sequence, it may be convenient to use different regulatory sequences in each cassette to reduce recombination spontaneous homologation between the sequences, The constructions can be introduced into the host cells by the same or different methods, including the introduction of said characteristic crossing transgenic plants via the methods of traditional plant cultures while the resulting product is a plant having both characteristics integrated into its genome. The invention now generally described will be more readily understood by reference to the following examples which are included for the purpose of illustration only and are not intended to limit the present invention. EXAMPLES Example 1. Sequences of Acyl-ACP Plant Thioesterases A. California Laurel (Umbellularia california) The DNA sequence and translated amino acid sequence of the California laurel Class II thioesterase pCGN3822 clone is provided in Figure 1 of WO 92/20236. The expression of the mature portion of the laurel thioesterase protein in E. coli and analysis of thioesterase activity reveals a strong specificity of laurel thioesterase for substrate 12.0-ACP, although some activity towards 14: 0-ACP (Voelker et al. 81994) supra, and Figure 2A in the present is also observed). In addition, when laurel thioesterase is expressed in E. coli FadD cells, large amounts of laurate (more than 500 times above the control background) and small amounts of myristate (about 10% of laurate) are produced. The production of similar laurate and myristic relationships were also observed when expressing laurel thioesterase in seeds of Brassica napus or Arabidopsis thaliana (Voelker et al. (1992) supra). B. Camphor. { Cinnamomum camphora) The DNA sequence and translated amino acid sequence of an il-class camphor thioesterase encoding the region generated by PCR is given in Figure 5B of WO 92/20236. The sequence (SEQ ID NO: 7) of a DNA fragment obtained by reverse transcribed cDNA PCR and containing the mature protein region of the camphor clone is provided in Figure 3. The sequence starts at the Xba site located at the beginning of the mature protein budget that codes for the camphor thioesterase region. The camphor PCR fragment described above was cloned into the pAMP vector resulting in pCGN5219. pCGN5219 is digested with XbaI and SalI and the resulting camphor thioesterase fragment was cloned into pBCSK + digested from Xbal and SalI (Stratagene), resulting in pCGN5220. pCGN5220 was used to transform E. coli FadD for analysis of acyl thioesterase activity-ACP as described in Pollard et al. { Arch. Biochem & Biophys. (1991) 281: 306-312). The results of thioesterase activity analyzes on camphor thioesterase clones using acyl-ACP substrates of 8: 0, 10: 0, 12: 0, 14: 0, 16: 0, 18: 0 and 18: 1 , although a minor increase in hydrolysis activity of 12: 0 was also observed (Fig. 2B). C. Mangosteen. { Garcinia mangifera) A cDNA library was prepared from seeds extracted from mature mangosteen fruit using the methods as described in the Stratagene Zap / Stratagene cDNA synthesis kit; La Jolla, Ca). Oil analyzes of the mangosteen tissues used for RNA isolation reveal 18: 0 levels of approximately 60%. The analyzes of oils from less mature mangosteen fruit seeds reveal 18: 0 levels of 20-40%. Total RNA was isolated from the mangosteen seeds by modifying the CTAB DNA isolation method of Veb and Knapp (Plant Mol. Biol. Repórter (1990) 8: 180-195). Regulatory solutions include: REC: 50 mM TrisCI pH 9, 0.7 M NaCl, 10 mM EDTA, pH 8, 0.5% C . REC +: Add 1% B-mercaptoethanol immediately before use. RECP: 50 mM TrisCI pH 9, 10 mM EDTA pH 8, and 0.5% C . RECP +: Add B-mercaptoethanol to 1% immediately before use.

To extract 1 g of tissue, 10 ml of REC + and 0.5 g of PVPP were added to the tissue that has been immersed in liquid nitrogen and homogenized. The homogenized material was centrifuged for 10 min at 12,000 rpm. The supernatant was poured through a "Miracloth" cloth in 3 ml of cold chloroform and homogenized again. After centrifugation, 12,000 RPM for 10 minutes, the upper phase was taken and its volume determined. An equal volume of REPC + was added and the mixture was allowed to stand for 20 minutes at room temperature. The material was centrifuged for 20 minutes at 10,000 rpm twice and the supernatant discarded after each turn- The pellet was dissolved in 0.4 ml of 1M NaCl (dEPC) and extracted with an equal volume of phenol / chloroform. After precipitation with ethanol, the pellet was dissolved in 1 ml of REPC water. In summary, the cloning method for cDNA synthesis is as follows. The synthesis of the first cDNA strand is according to the Stratagene Manual of Instructions with some modifications according to Robinson, et al. (Methods in Molecular and Cellular Biology (1992) 3: 118-127). In particular, approximately 57 μg of total RNA precipitated with LiCl was used in place of 5 μg of poly (A) + RNA and the reaction was incubated at 45 ° C instead of at 37 ° C for 1 hour. Screening probes were prepared by mangosteen cDNA PCR using oligonucleotides for the acyl-ACP regions of conserved plants. The Garm 2 and Garm 106 probe prepared using the following oligonucleotides. The nucleotide base codes for oligo nucleotides below are as follows: A = adenine C = cytosine na T = thymine U = uracil G = guanine S = guanine or cytosine na K = guanine or thymine W = adenine or thymine M = adenine or cytosine R = adenine or guanine Y = cytosine na or thymus B = guanine, cytosine na or ty mine H = adenine, cytosine or thymine N = adenine, cytosine, guanine or thymine Garm 2 4874 : 5 'CUACUACUACUASYNTVNGYNTGATGAA 3' (SEQ ID NO: 12) 4875: 5 'CAUCAUCAUCAURCAYTCNCKNCKRTANTC 3' (SEQ ID NO: 13) Initiator 4874 is a sense initiator designed to correspond to the possible coding sequences for V / L / AW / S / YV / AMMN of conserved peptides, where a code of amino acids of a letter and a diagonal between the amino acids is used indicates that in that position it is possible that there is more than one amino acid. The initiator 4875 is a nonsense primer designed to correspond to possible coding sequences for the peptide D / E and R R E C.

Garm 106 5424: 5 'AUGGAGAUCUCUGAWCRBTAYCCTAMHTGGGGWGA 3' (SEQ ID NO: 14) 5577: 5 'ACGCGUACUAGUTTNKKNCKCCAYTCNGT 3' (SEQ ID NO: 15) Initiator 5424 is a sense primer designed to correspond to possible coding sequences for peptides E / D / RYPK / TWG D. Initiator 5577 is a nonsense primer designed to correspond to possible coding sequences for the TEWRK / PK peptide. The DNA fragments resulting from the above reactions are amplified for use as probes by cloning or by additional CPR and radiolabeled by random or specific initiation. Approximately 800,000 plates are prepared according to the manufacturer's instructions. For sieving, the plate filters are prehybridized at room temperature in 50% formamide, 5X SSC, 10X Denhardt, 0.1% (w / v) SDS, 5 mM Na2EDTA, 0.1 mg / ml sperm DNA from denatured salmon. Hybridization with a mixture of the Garm 2 and Garm 106 probes was carried out at room temperature in the same buffer as before with 10% (w / v) dextran sulfate and probe. Plaque purification and phagemid extirpation were carried out at room temperature in the same buffer as before with dextran sulphate added at 10% (w / v) and probed. Plaque purification and phagemid excision were performed as described in the instructions of the Stratagene Zap cDNA Synthesis Kit. Approximately, 90 clones of acyl-ACP thioesterase were identified and classified as to the type of thioesterase by DNA sequencing and / or PCR analysis. Of the clones analyzed, at least 28 were Class I (FatA) types, and 59 were Class II (FatB) types. Two subclasses of FatA type clones were observed, the most prominent type being called GarmFatAI and the only clone of the second subclass was called Garm FatA2. The DNA and translated amino acid sequence of the GarmFatAI clone C14-4 (pCGN5252) (SEQ ID NO: 8) was presented in Figure 4. The DNA sequence and translated amino acid sequence of the clone FatA2 C14-3 (SEQ ID NO: 9) was presented in Figure 5. Constructs for the expression of the Garm FatAI clone of Figure 4 in E. coli were prepared as follows. Restriction sites were inserted by PCR mutagenesis at amino acid 49 (Sacl), which is near the amino terminus of mature protein budget and following the stop codon for the protein coding region (Bam \). The mature protein coding region was inserted as the Sacl / SamHI fragment in pBC SK (Stratagene; La Jolla, CA) resulting in pCGN5247, which can be used to provide expression of mangosteen thioesterase as a lacZ fusion protein.

The results of thioesterase activity analysis in the Mangosteen Class I thioesterase clone GarmFatAI using acyl-ACP substrates 16: 0, 18: 0 and 18: 1 are shown below. Acyl-ACP activity Thioesterase (cpm / min) 16: 0 18: 0 18: 1 Control 1400 3100 1733 GarmFatAI 4366 23916 87366 Clone GarmFatAI demonstrates preferential activity on the acyl-ACP substrate C18: 1, and also demonstrates substantial activity (approximately 25% of the activity of 18: 1) in acyl substrates-ACP C18: 0. Only a small increase in C16: 0 activity represents only about 3% of the 18: 1 activity. The expression of thioesterase GarmFatA2 in E. coli and analysis of the resulting thioesterase activity demonstrates that C18: 1 is highly preferred as the acyl-ACP substrate. The activity of thioesterase in acyl substrates-ACP 16: 0 and 18: 0 are approximately equal and represent less than 5% of the observed 18: 1 activity. D. Brassica campestris (rapa) DNA sequence and amino acid sequence translated from an acyl-ACP thioesterase Class I of Brassica campestris are provided in WO 92/20236 (Figure 6). E. Cuphea palustris C8 / C10 The DNA sequence and translated amino acid sequence of an acyl-ACP thioesterase Class I of Brassica campestris are provided in WO 92/20236 (Figure 6). E. Cuphea palustris C8 / C10 Total RNA was isolated from developing seeds of C. palustris using the CTAB procedure described above A lambda ZipLox cDNA library (BRL; Gaithersburg, MD) containing approximately 6 x 106 pfu was constructed of total RNA. Approximately 500,000 plates from the unamplified bank were screened using a mixed probe containing the thioesterase coding regions of CUPH-1 clones (CMT-9) of Cuphea hookeriana Class II thioesterase, CUPH-2 (CMT-7) and CUPH- 5 (CMT-10). (The DNA sequences of these clones are provided in WO 94/10288). The low restriction hybridization conditions are used as follows: Hybridization was carried out at room temperature in a solution of 30% formamide and 2X SSC (1X SSC = 0.15 m NaCl, 0.015 M Na Citrate). We identified 82 putative positive clones, thirty of which were purified on plates. The nucleic acid sequence and translated amino acid sequence of a clone designated MCT29 (CpFatBI) (SEQ ID NO: 10) is provided in Figure 6. The translated amino acid sequence of this clone is approximately 83% identical to the sequence of a clone of CUPH-2 (CMT-7 in Figure 7 of WO 94/10288) of Cuphea hookeriana having primary thioesterase activity on fatty acyl substrates-ACP C8: 0 and C10: 0.

Constructs were prepared for the expression of MCT29 in E. coli. The Sphl and Stu sites are 5 'inserted at the mature protein N terminus budget located at amino acid 114 by PCR. The mature N termination provided by correspondence to Leu 84 originally identified as the N terminus of mature laurel thioesterase protein. The mature protein coding region was cloned as Stu \ J / Xba \ fragment in pUC118, resulting in clone MCT29LZ, to provide thioesterase expression of C. palustris in E. coli as a lacZ fusion protein. Lysates of transformed E. coli cells expressing the thioesterase MCT 29 protein were analyzed for acyl-ACP thioesterase activity. The results demonstrate that CpFatBI encodes a thioesterase enzyme that has activity mainly on substrates of C8- and C10-ACP, with a 50% higher activity in C8-ACP than in C10-ACP. The low activity on the C14-ACP substrate was also observed at levels of approximately 10% of the C8-ACP activity. MCT29LZ was also transformed into E. coli fadD, an E. coli mutant lacking acyl-CoA synthetase specific for medium chain (Overath et al., Eur. J. Biochem. (1969) 7: 559-574) for analysis of the composition of liquids. The results of these analyzes show a substantial increase in the production of fatty acids 8: 0 and 10: 0 in cells transformed with the clone MCT29LZ of C. palustris.

The closely related ChFatB2 C. hookeriana clone also demonstrates preferential activity in C8: 0 and C10: 0 acyl-ACP substrates, with 50% higher activity on C10: 0 substrates than C8: 0. The expression of the clone of ChFatB2 in seeds of transgenic Brassica plants results in the increased production of C8 and C10 fatty acids in the seeds, with C10 levels higher than the C8 levels. (See the co-pending application SN 08 / 261,695 filed on June 16, 1994). F. Cuphea palustris C14 The nucleic acid sequence and translated amino acid sequence of an additional C. palustris Class II thioesterase clone, MCT34 (CpFatB2), (SEQ ID NO: 11), is provided in Figure 7. The translated amino acid sequence of this clone is approximately 80% identical to the sequence of a Cuphea hookeriana clone of CUPH-4 (CMT-13 in Figure 8 of WO 94/10288). Constructs were prepared for the expression of MCT34 in E. coli. The Sph \ and Sftvl sites are 5 'inserted at the N terminus of the mature budget protein located at amino acid 108 by PCR. The mature protein coding region was cloned as a Stu [IXba] fragment in pUC118, resulting in the MCT34LZ clone, to provide for the expression of C. palustris thioesterase in E. coli as the LacZ fusion protein. The lysates of transformed E. coli cells expressing the thioesterase protein of MCT34 were analyzed for aci lo-ACP thioesterase activity. The results show that CpFatB2 encodes a thioesterase enzyme that has activity mainly on the substrate of C 14-ACP. Activity on the C 16-ACP substrate was also observed at levels of approximately 30% of the C 14 -ACP activity. MCT34LZ was also transformed into E. coli FadD, an E. coli mutant lacking medium-chain-specific acyl lo-CoA synthetase (Overath et al., Eur. J. Biochem. (1996) 7: 559-574) for the lipid composition analysis. The results of these analyzes show a substantial increase in the production of fatty acids of 14: 0 and 14: 1 in cells transformed with the clone of C. palustris MCT34 LZ. Example 2 Chimeric Thioesterase Constructs Both A DNc of laurel and camphor thioesterases contain open reading frames encoding 382 amino acids. Only 31 amino acids are different, among them, more than half are their conservative bitumens (Fig. 8). The use of the codon is conserved highly between the two genes, suggesting them the common origin. The plasmid pCG N3823 (WO 92/20236 and Voelker et al. (1994) supra) contains a fragment of Xba de 1.2 kb of a thioesterase cDNA preferably of C 12 of laurel in a structure of the base of the pBS plasmid (Stratagene, La Jolla, CA) and encodes the laurel madura thioesterase protein starting at the amino acid 84 (as numbered in Voelker et al. (1992) supra). The amino acid 84 of laurel thioesterase was initially identified as the amino terminus for the mature protein based on analysis of amino acid sequences of the purified protein. Comparison to amino acid sequences translated from other acyl-ACP medium-chain thioesterases of cloned plants, however, indicates that the amino terminus can also be located upstream of residue 84 (Jones et al. (1995) supra). The plasmid pCGN5220, described above, contains a fragment of Xba \ / Xho \ of thioesterase cDNA preferably of C14 of camphor inserted into the plasmid pBC + (Stratagene). The site X £ > al in the camphor cDNA is present in amino acid residue 84, a leucine, as in the laurel thioesterase coding region. There is a unique site of Kpn \ conserved in laurel and camphor cDNA clones in amino acid residue 177 of the coding sequence for the thioesterases of laurel and camphor precursors (Fig. 9). A second Kpnl site is located within polylinkers of the 3 'plasmids to the stop codons of the thioesterase sequences. The exchange of the two Kpnl fragments between pCGN3823 and pCGN5220 allows the fusion of the N-terminal region of one thioesterase to the C-terminal region of the other, forming two chimeric enzymes. To prepare the chimeric constructs, pCGN323 and pCGN5220 were digested with Kpnl and the resulting fragments gel-purified and ligated into the plasmid of the base structure of the opposite origin. DNA mini-preparations and restriction digestions were used to identify the correct fusion constructs. The chimeric constructs used for the expression and analysis of enzymes were also confirmed by DNA sequencing. The resulting chimeric enzymes contain 92 amino acids of the N-terminus of the thioesterase and 207 amino acids of the C-terminal portion of the other. The fusion protein containing the C-terminal portion of the camphor thioesterase is designated as Chimeric 1 (Ch-1) and the other fusion protein is called Chimeric 2 (Ch-2) (Fig. 9). Example 3 Flexibility and Secondary Structure Analysis The secondary acyl-ACP structures predicted from plants were determined by computer analysis. The predictions of secondary structures are based on Chou and Fasman methods (Chou et al. (1974) Biochem.13: 222-245; Prevelige et al. (1989) in Prediction of Protein Structure and the Principles of Protein Conformation (Fasman, GD ed. ) pp. 391-416, Plenum, New York); and Garnier et al. (1978) J. Mol. Biol .. 120_97-120). The flexibility of various regions of acyl-ACP regions of plants is predicted by computer analysis using MacVector (International Biotechnologies, Inc.), based on the flexibility prediction methods of Karplus and Schulz (Naturwiss. (1985) 72: 212-213).

Example 4 Treatment of FatB Thioesterase A. Laurel C12 Thioesterase Site-directed mutagenesis was used by PCR (Higuchi et al. (1988) Nucí. Acids Res. 16: 7351-7367) for amino acid replacements. The sense mutant primers used for mutagenesis are the following: M197R / R199H 5'-GGAAATAATGGCCGACGACATGATTTCCTTGTCC-3 '(SEQ ID NO: 16) R231K 5'-GGTTGTCCAAAATCCC-3' (SEQ ID NO: 17) R327Q 5'- GCGTGCTGCAGTCCCTGACC-3 '(SEQ ID NO: 18) R322M / R327Q 5'- GAGAGAGTGCACGAIGGATAGCGTGCTGCAGTCCCTGACC-3 '(SEQ ID NO: 19) where the bold letters M, R, H, T, K and Q are letter abbreviations for the amino acids methionine, arginine, histidine, threonine, lysine and glutamine, respectively, and the mutated nucleotides are underlined . The CPR conditions are the following: five cycles of CPR were programmed with denaturation for 1 minute at 94 ° C, renaturation for 30 seconds at 48 ° C, and elongation for 2 minutes at 72 ° C. These first five cycles were followed for 30 cycles with renaturation for 30 seconds at 60 ° C. The amplified DNA was recovered by ethanol precipitation and examined by gel electrophoresis. The DNA was then digested with Xibal and ßamHI, the ethanol was precipitated and ligated into pBC plasmid cut from Sba \ / Bam. The ligation mixture was used to transform Sure (Stratagene) cells by electroporation and the transformed cells were plated in the LB medium containing 50 mg / 1 chloramphenicol. Constructs containing the correct inserts were identified by DNA minipreparation and restriction digestion. The inserted DNA was sequenced to confirm the mutations. The designations noted above for the PCR primers were used for the mutant clones. As an example, M197R / R199H refers to a clone in which the methionine at residue 197 (from the precursor by laurel thioesterase) was changed to an arginine and wherein the arginine at residue 199 was changed to a histidine. Similarly, T231K indicates a mutant in which the threonine at residue 231 was changed to a lysine. B. C14 Thioesterase from Cuphea palustris To de-select the possible amino acid modifications for the alteration of substrate specificity of fatty acyl thioesterase-ACP of shorter chain length, the sequences for thioesterases preferably of C14: 0 can be compared with the sequences for thioesterases preferably of C8: 0 and C10: 0. A comparison of the amino acid sequences of thioesterase CpFatB2 (C14) to CpFatBI (C8 / C10) is shown in Figure 10. Most of the shock differences in these thioesterase sequences are found in amino acids 230 to 312. substitutions such as H2291, H241N, W253Y, E275-A, R290G, F292L, L295F and C3040R, can be made in a simple form or in combination. Alternatively, domain exchange clones can be prepared which provide exchange of portions of the C8 / 10 sequences and the C14 sequences. Of particular interest in this regard are the IEPQPV sequences that split into amino acids 274 and DRKFHKL starting at amino acid 289. EXAMPLE 5 Specificity of Chimeric Enzymes and Laurel Mutants E. coli cells transformed into the lacZ expression constructs are developed at 0.6 OD600 at 30 ° C, followed by the addition of 1 mM of IPTG and continued development at 30 ° C for 2 hours. The pelleted cells were resuspended and treated with sound in the analysis buffer and the acyl-ACP hydrolysis was measured as previously described (Davies, H.M. (1993) Phytochemistry 33, 1353-1356). Sure cells transformed with pCGN3823 and pBC served as positive and negative controls, respectively. Figure 11 shows the thioesterase specific activities of chimeric laurel / camphor enzymes when the E. coli cells transformed with Ch-1 and Ch-2 were induced and analyzed. For Ch-1 (Fig. 11A) the preferred substrate is 14.0-ACP, while for Ch-2 (Fig. 11B) it is 12: 0-CAP. These results indicate that the C-terminal portion of the thioesterase protein determines the specificity of the substrate. The enzyme specificities of two of the laurel mutants are shown in Figure 11C and 11D. A mutant in which Met197 becomes an arginine and arg199 becomes a histidine (M197R / R199H) results in altered specificity of laurel thioesterase so that the enzyme is equally specific toward both 12: 0-ACP substrates and 14: 0-ACP (Fig. 11C). Another mutant, T231K, gives an identical activity profile as the wild type (data not shown). However, the triple mutant M197R / R199H / T231 K, which combines all three mutations, demonstrates thioesterase activity specific for 14: 0-ACP (Fig. 11D). When this triple mutant enzyme is analyzed at a higher concentration, very low levels of 12: 0-ACP activity are detected. Two more mutants (R327Q and R322M / R327Q) were also tested for thioesterase activity. Both mutants show identical activity profiles and their specific activities towards 12: 0-ACP and 14_0-ACP decreases about 100 and 30 times, respectively, compared to the wild type laurel thioesterase. These data indicate that the R327Q mutation is responsible for the decreased activity. The decreased activity of R327Q is probably due to the fact that this amino acid position is located very close to the cysteine of the active site C320. The studies that demonstrated that the catalytic activity C320, demonstrated the catalytic activity of C320 where it was carried out in the following way. C320 was changed by site-directed mutagenesis to serian or alanine. The C320A mutant completely loses thioesterase activity, while C320S retained approximately 60% of the wild type activity. The exchange of cysteine and serine in the active site has also been demonstrated for animal thioesterases (Wilkowski et al. (1992) J. Biol. Chem. 267: 18488-18492). In animals, the active site is a serine and the change of this was serine and cysteine. EXAMPLE 6 Expression of Laurel Mutants in E. coli FadD Cells The mutant strain of fatty acid degradation of E. coli K27 (adD88), a strain lacking acyl-coenzyme A synthetase, can not use free fatty acids when supplied in the medium (Klein et al. (1971) Eur. J. Biochem. 19: 442-450). Therefore, it is an ideal host to observe the impact of recombinant thioesterases on the bacterial fatty acid tape without interference from fatty acid degradation. E. coli FadD was obtained from the E. coli Genetic Stock Center, Yale University (CGSC 5478). The fado cells were transformed with pBC, a wild-type laurel thioesterase gene or the mutant constructs and grown overnight at 30 ° C in LB medium containing 50 mg / l chloramphenicol and 1 mM IPTG. The total lipids were analyzed as previously described (Voelker et al. (1994) supra). The results of these analyzes are presented in the following Table I.

Table I Accumulation of Free Fatty Acids (nmol / ml of culture) Strain 12: 0 14: 0 Control * 0.3 1.6 Laurel thioesterase 505.5 39.0 M197R / R199H 123.5 181.1 M197R / R199H / T231K 35.4 352.9 * fadD cells transformed only with the pBC vector. When laurel thioesterase was expressed in fado cells, large amounts of laurate were produced (more than 500 times above the background control) and small amounts of myristate (about 10% of laurate) were produced (Table I) . This result is consistent with the previous report (Voelker et al. (1994) supra). When mutant M197R / R199H was expressed in fado cells, the ratio of the accumulation from 10: 0 to 14: 0 changes to 1: 1.5 (Table I), reflecting the thioesterase specificity of this mutant (Fig. 11C). When the mutant M197R / R199H / T231 K was expressed in fado cells, the ratio of 12: 0 to 14: 0 was completely reversed from that observed with the wild type laurel thioesterase. This result also agrees with the enzyme specificity of the mutant (Fig. 11D). Example 7 Kinetic Analysis In order to observe the impact of laurel thioesterase mutations, basic kinetics and studies were carried out. of inhibition. The progress curves of thioesterase activity were obtained by increasing the volume and sampling of 100 μl analysis in 5 minute intervals in 0.5 ml stop solution. Kinetic analyzes were carried out at 30 ° C in pH buffer containing 100mN of Tris-HCl, pH 8.0, 0.01% Triton X-100, 1 mM DTT, 10% glycerol. After extraction of each reaction mixture with 2.0 ml of dimethyl ether, the radioactivity in 900 μl of the organic fraction was determined by liquid scintillation counting. This procedure allows the accurate measurement of the extractable free fatty acid (marked with 14C) without interference from the interface between the organic and aqueous fractions. The production of laurate and myristate in this analysis was linear with respect to time for at least 30 minutes and with respect to enzyme concentrations up to 1 mU. All analyzes were performed in duplicate. Initial regime data was adapted for the following equations using kinetics software from Bio-Metallics, Inc. (Kcat): for competitive inhibition v = VmaxS / [Km, app (1 + l / Kls) + S (1 + l / KM)]; and for noncompetitive inhibition v = VmaxS / [Km, ap + S (1 + l / Kp)]; where v is speed; Vx is maximum speed; S is substrate concentration; Km, app is constant for Michaelis; K1S t K ,, are constants of inclination and inhibition of interception, respectively; I is concentration of inhibitor. The results of these analyzes are presented in the following Table II.

Table II Laurel TE Kinetics Constants Wild Type and Triple Mutant M197R / R199H / T231K Enzyme Km.app (μM) K¡ (μM) * 14: 0-ACP 12: 0-ACP 12: 0-ACP TE Laurel 6.4 + 1.9 1.9 + 0.5 10.2 + 1.2 (competitive * 'Mutant 2.3 ± 0.4 ND 11.6 ± 0.2 (competitive) * inhibition constants of 12: 0-ACP inclination with 1: 0-ACP as varied substrates ** competitive inhibition with respect to 14: 0-ACP. ND- Not determined Under the same experimental conditions, both the laurel thioesterase and the triple mutant M197R / R199H / T231 K have similar values of K m, app with respect to 14: 0-ACP. The specific activity of the mutant towards 12: 0 -ACP is very low to obtain any significant kinetic parameter under our analysis system. However, these results indicate that the mutations do not significantly increase the binding affinity of the substrate (14: 0-ACP) to the mutant enzyme. Inhibition analyzes were carried out under the conditions described above using cold 12: 0-ACP to compete with the substrate (14: 0-ACP marked with 14C). The results of these analyzes are presented in the following Table III. Table III Inhibition of Thioesterase Activity of 14: 0-ACP by 12: 0-ACP Enzyme Inhibitory Substrate Inhibition (12: 0- (14: 0-ACP) ACP) Concentration (μM) Concentration (μM) (%) Laurel TE 5 5 53 5 25 78 Mutant 5 5 48 5 25 76 In these inhibition assays, a very similar result is observed with the wild-type and mutant enzymes. When the equal amounts of inhibitor (12: 0-ACP) and substrate (14: 0-ACP) are reduced by approximately 50%. If the amount of 12: 0-ACP is 5 times that of 14: 0-ACP, the TE activity of 14: 0-ACP is reduced by more than 75%. Consistent with what was observed before (Pollard et al., Supra), a similar mechanism of kinetics was used by wild-type laurel TE, that is, both 12: 0 and 14: 0-ACP have similar km, but Vmax It is highly favorable for 12: 0-ACP. These data suggest that the specificity of the mutant enzyme was determined in the step of acyl hydrolysis, which is 12: 0 and 14: 0-ACP which can bind to the mutant enzyme with similar affinity, however, it was separated. : 0-ACP in a much higher regime. This conclusion was further supported by inhibition kinetics, which shows that 12: 0-ACP is a competitive inhibitor with respect to 14: 0-ACP (K values are 10.2 + 1.2 and 11.6 + 0.2 μM for wild-type enzymes and mutants, respectively (Table II)). Therefore, the amino acid substitutions described for laurel thioesterase apparently do not directly impact the substrate binding site, since 12: 0-ACP is a good competitive inhibitor for 14: 0-ACP in wild-type enzymes and mutants In fact, the Michaelis constants are similar and independent of the substrate length for bay thioesterases and the treated laurel enzyme, suggesting that the specificity should be determined largely in the acyl hydrolytic step, because the substrates (acyl) -ACP) are relatively long molecules (Mr of ACP is approximately 9 Kd), it is probably that the thioesterases have quite relaxed union bags. However, enzymes have high selectivities with a fatty acid chain length or structure (ie, the presence or absence of double bonds). In addition, the tripeptide Met-Arg-Arg of native laurel thioesterase is not the only determining factor for selectivity towards 12: 0-ACP, since this tripeptide was commonly found in the same location in other thioesterases specific for medium chain. Therefore, changes in treated bay thioesterases may only slightly alter certain secondary structures, similar to those observed when the Lactate dehydrogenase surface of Bacillus estearothermophilus were modified (Hawram et al. (1994) Trends in Biotech 12207-211) The change of the MRP tppypeptide to RRH, apparently reduced the flexibility of the β structure immediately after this tpppeptide, according to with predictions of chain flexibility in proteins (Jkarplus et al. (1985) Naturwiss 77, 212-213). This may lead to reduction of the flexibility of the substrate binding pocket and the active site. Example 8 Treatment by Engineering FatA thioesterase The alteration of the specificity of the thioesterase enzyme of a mangosteen Garm FatAI clone was provided as an example of the modification of FatA or Class I thioesterases. Suitable modifications with respect to FatA thioesterases include alteration in the substrate specificity. so that the activity in fatty acyl -ACP of C180 on activity in acyl substrates was increased Fatty-ACP of C18 1 or C160 For example, in order to increase relative activity in saturated fatty acids, such as mutations of C180 in regions of Class I thioesterases that differ from the corresponding regions in Class II thioesterase, which act Mainly in saturated fatty acids, may be useful Data from the laurel thioesterase engineering experiments indicate that the region of amino acids 225 to 285 (as numbered in the consensual sequence of the upper line in Figure 1), is important at the junction of the thioesterase substrate Comparison of the amino acid sequence of that region indicates that in the highly conserved region of amino acids 250-165, several charged amino acids are different in FatA compared to FatB thioesterases. In the FatA thioesterase, amino acid 261 is negatively charged with a few exceptions, whereas in the FatB clones analyzed so far, amino acid 261 is in most cases positively charged. Also in the FatA thioesterase, amino acid 254 is positively charged in all FatA thioesterases studied so far, whereas in the FatB clones analyzed so far, amino acid 254 in all cases is an uncharged amino acid. Therefore, alteration of the amino acid card in these positions can lead to the alteration of substrate preference. A FatA TE mutant at amino acid 261 (consensual numbering of Figure 1), D261K of mangosteen FatAI, is generated using site-directed mutagenesis by PCR similar to the methods described for the modification of laurel thioesterase sequences. Mutant D261K was measured for thioesterase activity as described above (Davies, H.M., (1993) supra). The results of these analyzes (Figure 12) show that the preference for 18: 0 versus 18: 1 was 35% (18: 0/10: 1) in the mutant FD561K compared to 25% in the Garm FatAI wild type. Both wild-type and Garm FatAI mutant clones demonstrate very low activity at 16: 0 and no activity on the chain length substrates by such as C10: 0 to C14: 0.

An additional Garm FatAI mutant was prepared using the D261K mutation indicated above, as well as a mutation to change amino acid 254 from lysine to valine. This mutant, K254V / D261K, demonstrates an increased ratio of 18: 0/18: 1 of 40%. These results again support the laurel evidence that indicates that the modification of this region can change the activity and specificity of the enzyme. A triple mutant, C249T / K254V / D261 K, is under construction to further modify the Garm Fat A1 clone towards the FatB thioesterase structure for the evaluation of additional specificity modification. Other suitable amino acid modifications of mangosteen Garm FatAI clones can be selected by comparison of the amino acid sequence of Garm FatAI thioesterase enriched from 18: 0 to the amino acid sequence for a FatA clone having activity primarily on 18: 1 substrates. , with little or no activity on substrates of 18: 0. A comparison of the amino acid sequences of Garm FatA11 and a thioesterase clone of preferably 18: 1 Brassica campestris (rapa), Br FatAI, is provided in Figure 13. In view of the alterations of binding substrates shown for the laurel thioesterase in the following region, of the ß-sheet and the turn (anchored by amino acids G169 and G172 of the comparison of mangosteen thioesterase and Brassica in Figure 13), this region is also a target for altering the specificity of the substrate of the Garm FatAI clone of mangosteen thioesterase. The structure analysis Secondary and the amino acid sequence comparison of Class I thioesterases from mangosteen and Brassica rapa, results in the identification of several target mutations to further alter the substrate specificity of the mangosteen thioesterase, Garm FatA1. White amino acids include Y182V, Q186E, D209S, V210D and H219F. Further analysis of alignments of plant thioesterase peptide sequences of the FatA type reveals a number of amino acid residues that are conserved within all thioesterases of Fat A type plants except the mangosteen Garm FatAI thioesterase. Five of these FatAI-specific amino acids are (using the consensual numbering of Figure 1) G185 (their consensus is D), S188 (their consensus is a), V270 (their consensus is D), H279 (their consensus is F) and S307 (your consensus is A). These five amino acids are of particular interest since they are non-conservative substitutions. Compared with conservative substitutions, for example S233 T, non-conservative substitutions tend more to cause structural or biochemical changes in the mangosteen enzyme and therefore contribute to its unique specificity towards 18: 0-ACP and extremely low activity towards 15: 0-ACP. These five identified amino acids have been mutagenized in Garm FatAI for the consensual amino acid or for alanine. For S188 and S307, only alanine substitutions were made since alanite is also the consensual amino acid for these residues. In addition, mutants containing several combinations of the amino acid substitutions above. A protein expression / purity affinity tag was used for the analysis of the above Garm FatA I mutants. Wild type and Garm FatA I mutant thioesterases were expressed in E. coli using the vector pQ E32 (Qiagen). The recombinant proteins containing an affinity tag consisting of six consecutive histidine residues were produced at high levels and purified using the N i -NTA resin from Qiagen following the manufacturer's instructions. To construct the expression plasmids, the mature portion of Garm FatA-1 (amino acid 65 to terminus C) was amplified by PCR and inserted into the vector pQ E between the Bam H I and SalI restriction sites in the polylinker. The DNA sequence was verified by sequencing and the plasmid was transformed into E. coli M15 cells (Qiagen). The cells were grown at 30 ° C in LB medium containing 100 mg / l of ampicillin and 30 mg / l of kanamycin, and the production of recombinant protein was induced by the addition of 2 mM I PTG and the cells were allowed to develop for an additional 4 hours. The cells were pelleted and used and the recombinant proteins were purified with N i -NTA resin following the manufacturer's instructions. The Garm FatA I enzymes were also expressed in E. coli from the pQE constructs, purified and analyzed for hydrolysis activity of 18: 0-AC P and 18: 1 -ACP. The results of these analyzes presented as a graph of change of quantity in specific activity are shown in Figure 16. The specific activity of the selected mutants is provided in Figure 17. The S188A mutant demonstrates a 4-fold increase in activity in 180-ACP and a 2-fold increase in activity of 18: 1. the selectivity of S307A increases the activity of 18: 0-ACP (2 times) without changing the activity of 18: 1-ACP. On the other hand, the modification of certain waste created negative effects. For example, G185D, V270D and H279F demonstrate greater double reduction in activities. However, in some cases, especially for small hydrophobic amino acids, the substitution of alanine (e.g., G185A and V270A) reverses the negative effects (G185A has a two-fold increase in activity). This is probably due to the neutral effect of alanine on the protein structure and its similarity to these small amino acids. This result also suggests that the alanine scanning of amino acids conserved in FatA could result in the production of mutants with increased hydrolysis activity. These studies also show that substitutions with positive impact on the activity of the enzyme are additive. When two mutations showing increased activity (e.g., S188A and V270A) were combined to form a double mutant, a further increase in enzyme activity was observed. The mutant S188A / V270A demonstrates a 13-fold increase in activity of 18: 0-ACP. In addition, when a positive mutation is combined with a mutation that shows selective increase in a substrate but not the other, the resulting mutant shows improved overall activity and a ratio of 19: 0/18: 1 ACP. For example, S188A / V270A / S307A shows a 7-fold increase in activity of 18: 0-ACP and a ratio of 18: 0/18: 1 of 0.8 / 1 compared to a ratio of 0.3 / 1 for Garm FatAI type wild. Example 9 Domain Exchange Methods are provided to prepare thioesterase domain exchange constructs when suitable restriction sites are not available. A. Short Domain Methods One method for short domain exchange was illustrated in Figure 14. Two separate PCRs result in the fragments (products of primers a + d, and primers b + c), which contains an overlapping sequence identical to the new domain. The primers c and d were synthesized to match the exact sequence at the 3 'end downstream of the original domain, plus a 5 'pendant corresponding to the new domain sequence. The length of the equalization sequence should be long enough to give a Tm of 50 ° C or more (calculated assuming a C or G = 4! C and a T or A = 2 ° C). Ideally, the length of 5 'pendants should not be greater than 18 bases (6 amino acids), although longer pendants may also work at lower efficiencies. The first two PCRs were carried out with approximately 0.2 μM of primers and 0.1 μg of the model DNA under CPR conditions described below. The second CPR run (CPR 3) was performed by mixing 10 μl of each PCR product 1 and 2 and initiators a and b were added to the final concentration of 0.2 μM. The resulting product is the gene targeted with the original domain replaced by a new domain sequence. The PCR product can be examined on an agarose gel before precipitation and restriction-digestion for subcloning. The modified A DN fragment can be sequenced to verify the desired measurement. B. Long Domination Methods To exchange longer dominions, as illustrated in the Figure 15, the exchange of a domino from gene I I to gene I can be achieved by first amplifying three fragments of PCR 1, 2, and 3. These overlapping fragments were partially mixed for the next PCR with the a and b ini- tiators. The conditions of the CPR are described below. The resulting full-length product is gene I with a new domain of the I I gene. By the same principle, two domains in the gene I can be changed simultaneously by an additional PCR in the first run, followed by the second PCR in the presence of four fragments (not shown). The conditions of RC P that have been used successfully are as follows: five cycles were programmed with denaturation for 1 min. At 94 ° C, renaturation for 30 seconds at 48 ° C, and elongation for 2 minutes at 72 ° C. The first five cycles were followed for 30 cycles using the same program except for renaturation during 30 seconds at 60 ° C. The rational for the first five cycles at lower temperature is to ensure the path of the CPR initiators with 5 'pendants. The increased temperature for the last cycles l imitates the additional amplification to amplified sequences during the first five cycles. The Tm for all primers should be designed around 60 ° C. For convenience of subsequent cloning, full-length anchor primers (a and b, Figure 14 and 15) usually include additional restriction sites and / or pendants for several PCR subcloning vectors. It is important to use the least possible amount of model DNA (usually less than 0. 1 μg) to reduce the non-mutagenized background. C. Exchange of FatA and FatB Domain The FatA and FatB thioesterase peptide sequences can be inear to show clear similarities and differences between the two enzyme classes (Voelker (1996) Genetic Engineering, Vo. 18, ed. K. Setlow, pp. 1 1 1 - 133, Figure 3). There is a hydrophobic region near the N terminus that is highly conserved in Fat B thioesterases and absent in Fat A thioesterases. However, there are five regions that are present in both classes of thioesterase that can be classified partially homogeneous, as well as an active site region around histidine and cysteine residues that is highly conserved among all members of FatA thioesterase. and FatB. Unlike the single rhombic h id section for different FatB, there are three additional unique regions that share little or no homology among them. A representation of these different peptide regimes describing the locations of the unique and conserved regions for Garm FatA I and UC FatB 1, are provided in Figure 18. The amino acid numbering is according to the number of the consensual sequence of the upper line in Figure 1. By deleting or exchanging these unique and conserved regions between Garm FatA I and UC FatB 1 using domain exchange techniques described above, mutants were generated and analyzed for the activity and specificity of the enzyme. The results of the analysis of this deletion and exchanged mutants are given in Figs. 19 A and B. These results demonstrate the following. The single h idrophobic section in FatB does not affect the activity or specificity of the thioesterase substrate FatB. The deletion of the single C-terminal section of FatB decreases the activity of the enzyme but does not alter the specificity of the substrate. The active site regions are interchangeable without altering the specificity of the substrate, indicating that the sequences outside the active site regions determine the specificity of the enzyme. Ichanging the unique sequence of residues 275 to 289 does not alter the substrate specificity but causes a decrease in the activity of the enzyme. A chimeric enzyme formed by the fusion of the N-terminal portion of FatB to residue 382 and the C-terminal portion of Fat A (382 to 430) is inactive, suggesting that the C-terminal portion of Fat B is critical to the overall activity of the enzyme. The chimeric enzymes formed by the fusion of the N and C terminals of Fat A and B (residue 275 as the cut-off point), are indicative, which show that sequences between 65 and 275 affect the overall structure of the entire enzyme. EXAMPLE 10 Transformation and Analysis of Plants Transgenic pans with increased levels of fatty acids of C18: 0 as a result of the expression of Garm FatAI thioesterase in Brassica napus seeds were reported in WO 97/12047. One construct, pCGN5255, comprising a Garm FatAI thioesterase gene under regulation of the 5 'and 3' napin regulatory regions was used for plant transformation. The fatty acid compositions in a high oleic acid line as high as 39%, compared to about 2% in non-transformed control plants, were reported in seeds in individual halves of a selected 5255 transgenic plant. Similar fatty acid levels of 18: 0 were reported in transgenic plants of a B. napus line transformed with a double Garm FatAI expression construct, pCGN5266. Stearate levels in seed samples combined from segregation of seed populations varied up to 14.2% in transformants of 5255 and 22% in transformants of 5266. For the analysis of stearate production in transgenic plants transformed with the Garm FatAI double mutant G185A / S188A, a napin expression construct was prepared which is identical to pCGN5255, but with the coding sequence of the G185A / S188A mutant encoding the substituted sequence for the wild-type Garm FatAI coding sequence. The double mutant construct, pCGN5274, was transformed into Agrobacterium tumefaciens and used to generate Quantum plants of the transgenic B. napus variety as a control for the 5274 plants. The combined segregation seeds of Quantum plants 5255 and 5274 were analyzed for determine the composition of fatty acids. A statistical analysis of these results is shown in the tabulated form below. Table IV Descriptive Statistics Plants 5255 Plants 5274 Average stearate% 4.59 7.31 Standard deviation 2.17 2.69 Number of samples 43 45% Stearate minimum 1.31 1.66% Stearate maximum 10.10 12.79 Variance 4.70 7.23 Scale 8.79 11.13 Average 4.13 7.45 Table IV Confidence Level of a Single Sample 955 for the Mean Average 95% lower 95% higher Plants 5,255 4,592 3,925 5,259 Plants 5,274 7,308 5,500 8,116 Histogram representations of the data of 5225 and 5274 are provided in Figure 20. The detailed results of the composition analyzes are provided in Figure 21. Additional constructs were prepared with a double Garm FatAI mutant, S188A / V270A. A napin expression construct, pCGN5290, was prepared which is identical to pCGN5255, except in the Garm FatAI coding region, the mutant S188A / V270A (described above) encoding the sequence was substituted. In addition, a double construct, pCGN5291, was prepared, which is identical to pCGN5266 except that the two wild type Garm FatAI coding sequences have been replaced with two S188A / V270A mutant coding sequences. Constructs pCGN5290 and pCGN5291 were transformed into Agrobacterium tumefaciens and used for the variety of transgenic B. napus generated Quantum. Approximately 25 transgenic plants for each construction were obtained. The mature seeds of each transgenic plant were cultivated and analyzed for fatty acid compositions.

The results of fatty acid composition analysis of combined seeds of T2 show that plants expressing the Garm FatAI S188A / V270A mutant produced significantly higher amounts of stearate (C18: 0) as a percentage of total fatty acids when compared to the transgenic plants that express pCGN5255 or pCGN5266 (Figure 22). More specifically, the stearate levels in seeds of plants expressing pCGN5290 and pCGN5291 are 68% and 57% higher than the levels obtained from the seeds of plants expressing pCGN5255 and pCGN5266 respectively. The stearate levels in the combined seed samples of the segregated seed populations varied up to 16.3% in 5290 transformants and 9.9% in 5255 control transformants. In addition, stearate levels in combined seed samples of seed populations in segregation varied up to 20.9% in 5291 transformants compared to 13% in control transformants 5266. The Brassica napus non-transformed control contains less than 3% stearate. A statistical analysis of these results is shown in the tabulated form below.

Table V Descriptive Statistics Plants 5255 Plants 5290 Plants 5266 Plants 5291 % Medium stearate 5.28 8.90 6.46 10.12 Desv. Norm 1.84 2.51 2.95 4.12 No. Samples 33 29 11 26% Min. Stearate 2.70 5.06 3.14 2.30% max. Stearate 9.86 16.30 13.00 20.30 Variance 3.38 6.29 8.71 16.99 Scale 7-16 11.24 9.86 18.59 Average 4.49 8.85 6.10 10.01 Table V Confidence Level of Single Sample 955 for Medium Media 95% Bottom 95% Superior Plants 5255 5,282 4,630 9,934 Plants 5290. 8,897 7,943 9,851 Plants 5266 6,461 4,478 8,444 Plants 5291 10,123 8,458 11,788 These results demonstrate that improved levels of stearate can be obtained in transgenic thioesterase plants by the expression of mutant thioesterases having increased C18: 0 activity relative to C18: 1 activity.

The above results demonstrate the ability to modify the acyl-ACP sequences of plants so that treated thioesterases having altered substrate specificity can be produced. Said thioesterase can be expressed in host cells to provide a supply of the treated thioesterase and modify the existing route of fatty acid synthesis so that novel compositions are obtained. In particular, the treated thioesterase can be expressed in the seeds of oily plants to provide a natural source of convenient TAG molecules. All publications and patent applications mentioned in this specification are indicative of the level of experience of those skilled in the art to which this invention pertains. All publications and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Although the above invention was described in some detail by way of illustration and example for the purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practical within the scope of the appended claims.

Claims (27)

1. CLAIMS 1. A method for obtaining an acyl-ACP thioesterase from treated plant having an altered substrate specificity with respect to acyl-ACP substrates hydrolyzed by said thioesterase, wherein the method comprises: (a) modifying a sequence of genes encoding a first plant thioesterase protein to produce one or more modified thioesterase gene sequences, wherein the modified sequences encode treated acyl thioesterases-ACP having substitutions, insertions or deletions of one or more amino acid residues in the portion mature of the first plant thioesterase, (b) expressing the sequences of modified genes in a host cell, whereby thioesterases from treated plants were produced and, (c) analyzing said thioesterases from treated plants to detect altered substrate specificity.
2. 2. A method according to claim 1, wherein the amino acid substitutions, insertions or deletions are in a two-thirds C-terminal portion of the first plant thioesterase.
3. 3. A method according to claim 1, wherein the substitutions, insertions or deletions of amino acids are in the region corresponding to amino acids 230 to 285 of the consensual numbering of the thioesterase amino acid sequences shown in Figure 1.
4. 4. A method according to claim 1, wherein the amino acid substitutions, insertions or deletions are in the region corresponding to amino acids 315 to 375 of the consensual numbering of the thioesterase amino acid sequences shown in Figure 1.
5. 5. A method according to claim 1, wherein one or more amino acid residues in the mature portion of said first plant thioesterase were substituted with the amino acids. corresponding to a second plant thioesterase, wherein the preferential ACP-acyl substrates for the first and second plant thioesterase are different with respect to carbon chain length and / or degree of saturation.
6. 6. A method according to claim 5, wherein the first thioesterase was modified by substitution of a peptide domain of the second thioesterase.
7. 7. A method according to claim 6, wherein the peptide domain comprises the active histidine and active cysteine residues of the second thioesterase protein.
8. 8. An acyl-ACP treated plant thioesterase, wherein the treated thioesterase demonstrates an altered substrate specificity with respect to the acyl-ACP substrates hydrolyzed by the thioesterase compared to the acyl-ACP wild type thioesterase in the plant.
9. 9. A treated thioesterase of claim 8, wherein the wild-type thioesterase is a FatB thioesterase.
10. 10. A treated thioesterase of claim 8, wherein the wild-type thioesterase is a FatA thioesterase.
11. 11. A treated thioesterase of claim 10, wherein the thiaesterase FatA is mangosteen Garm FatAI thioesterase.
12. 12. A treated thioesterase of claim 11, wherein the treated thioesterase contains the substitution S188A.
13. 13. A treated thioesterase of claim 11, wherein the treated thioesterase contains the S307A substitution.
14. 14. A treated thioesterase of claim 11, wherein the treated thioesterase contains the G185A substitution.
15. 15. A treated thioesterase of claim 11, wherein the treated thioesterase contains the V270A substitution.
16. 16. A treated thioesterase of any of claims 12-15, wherein the treated thioesterase is a double mutant.
17. 17. A treated thioesterase of any of claims 12-15, wherein the treated thioesterase is a triple mutant.
18. 18. A DNA sequence encoding an acyl-treated plant acyl thioesterase, wherein the treated thioesterase demonstrates an altered substrate specificity with respect to the acyl-ACP substrates hydrolyzed by the thioesterase compared to the acyl-ACP plant thioesterase wild type.
19. 19. A DNA sequence according to claim 18, wherein the wild-type thioesterase is a thioesterase FatB.
20. 20. A DNA sequence according to claim 18, wherein the wild-type thioesterase is a FatA thioesterase.
21. 21. A DNA sequence according to the claim 20, wherein the thioesterase FatA is a mangosteen Garm FatAI thioesterase.
22. 22. A DNA sequence according to the claim 21, wherein the DNA sequence encodes a mangosteen Garm FatAI thioesterase containing the amino acid substitution S188A.
23. 23. A DNA sequence according to claim 21, wherein the DNA sequence encodes a mangosteen Garm FatAI thioesterase containing the amino acid substitution S307A.
24. 24. A DNA sequence according to claim 21, wherein the DNA sequence encodes a mangosteen Garm FatAI thioesterase containing the amino acid substitution G185A.
25. 25. A DNA sequence according to the claim 21, wherein the DNA sequence encodes a mangosteen Garm FatAI thioesterase containing the amino acid substitution V270A.
26. 26. A DNA sequence of any of claims 22-25, wherein the DNA sequence encodes a double mangosteen Garm FatAI thioesterase.
27. 27. A DNA sequence of any of claims 22-25, wherein the DNA sequence encodes a triple mangosteen Garm FatAI thioesterase. RESU M EN Methods for altering the specificity of acyl substrate-ACP plant thioesterases and acyl-AC P thiostearases of treated plants thus produced are provided. Two thirds of the C-terminal portion of plant thioesterases are identified as being convenient for such modifications. DNA sequences and constructs are also provided for the expression of engineered thioesterases, as well as the novel thioesterases produced therefrom. Said DNA sequences can be used for the expression of thioesterases engineered in host cells, particularly those of seeds of oilseed crop plants, for the modification of fatty acid composition. The acyl-ACP plant thioesterase preferably of C 12, described herein, can be altered to obtain a plant thioesterase which has approximately equal activity in substrates of C 14 and C 12. The additional modification of the enzyme C 12 gives a thioesterase which has higher activity in C 14 compared to C 12 substrates. Of particular interest is a thioesterase plant of 18: 1 in which the relative 18: 0 activity was increased. Said FatA thioesterases are used for the improved production of stearate in vegetable seed oils.