BACKGROUND OF THE INVENTION

Field of the Invention

[001] The invention relates generally to phenylpropanoids, compositions and methods for producing chlorogenic and/or dactylifric acid species, and in particular to compositions and methods using plant HCT, HQT, and 4CL genes and proteins for making mono-, di-, and tri- and tetraesters and other complex chlorogenic and/or dactylifric acid species.

Description of Related Art

[002] Secondary metabolites in plants are not directly involved in primary cellular functions such as growth, photosynthesis or reproduction, but they are known to mediate plant- environment interactions and to play a major role in the plant's survival and adaptation to environmental changes. Three major classes of secondary metabolites, terpenoids, alkaloids, and phenolics, have been identified in plants.

[003] Structurally, the phenolics generally consist of a benzenoid ring bearing at least one hydroxyl substituent. As with all secondary metabolites the carbon skeletons can be modified by substrate-specific and/ or regio-specific biosynthetic enzymes (e.g. hydroxylases, methyltransferases, glycosylases etc.), resulting in a high structural diversity. More than 6,000 different phenolics have been identified. They serve a variety of important functions in plants, for example, pigmentation for the attraction of insect pollinators (anthocyanins), protection against solar radiation damage (hydroxy cinnamic acids and flavones), defence against pathogens (stilbenes), signalling (flavonoids), structural support and water transport (polymeric lignins).

[004] Table 1 shows the two main groups of phenolic compounds, flavonoids and nonflavanoids, with some of the classes within each group. The flavonoids, are characterised by a C6-C3-C6 structure and are classified as by the type of heterocycle (e.g. flavonols, flavones, flavanones, flavanols, anthocyanidins and isoflavones) they contain. The non-flavonoids, are more varied and include simple phenols, hydroxybenzoic acids, hydroxycinnamic acids (HCAs), coumarins, and tannins, among others. Table 1: Classification of the phenolic compounds

[005] HCAs feature a unique C6-C3 chemical structure and are rarely found as free acids in unprocessed plant material. They are generally identified in plant extracts after degradation of the soluble and insoluble-bound derivatives (Clifford, 2000). HCAs possess a phenol group and a carboxylic acid function, characteristic of the phenolic acids including benzoic and cinnamic acid derivatives. Both cis and trans forms of cinnamic acid compounds are common in nature, the latter being naturally derived from the biosynthetic precursor t-cinnamic acid. Isomerisation to cis derivatives may occur during extraction, processing, or exposure to light (Kahnt, 1967). The simple benzoic and cinnamic acid derivatives differ in the degree of hydroxylation and/ or methoxylation of the aromatic ring (Table 2). Coumaric, caffeic, ferulic and sinapic acids are amongst the most widely distributed phenolic compounds in plants. Three position isomers exist for coumaric acid: 2-, 3- and 4-coumaric acids, with the latter occurring most abundantly in nature.

Table 2: Substitution of aromatic ring in phenolic compounds Benzoic acids (C5-C1) Aromatic substitution Cinnamic acids (C6-C ₃ )

salicylic acid 2-OH 2-coumaric acid

4-hydroxybenzoic acid 4-OH (4-)coumaric acid

vanillic acid 3-OCH ₃ , 4-OH ferulic acid

syringic acid 3,5-di-OC¾, 4-OH sinapic acid

- 3-OCH ₃ , 4-OH, 5 -OH 5-hydroxyferulic acid

protocatechuic acid 3,4-di-OH caffeic acid

gentisic acid 2,5-di-OH - gallic acid 3,4,5-tri-OH -

[006] Other than gallic acid and its derivatives, all major groups of plant phenolics are synthesised by further metabolism of the simple HCAs. In many plants, hydroxycinnamoyl-CoA thioesters are the most common activated intermediates for phenylpropanoid biosynthesis (Ulbrich et al, 1979). Various hydroxycinnamoyl-CoA thioesters enter downstream pathways by taking part in different types of side-chain reactions including:

(a) side-chain elongation (condensation) with malonyl-CoA molecules leading to flavonoids, stilbenes, styrylpyrones, benzophenones, lignans and chain-elongated HCAs;

(b) NADPH-dependent reduction leading to dihydrocinnamic acids, hydroxycinnamyl alcohols (e.g. monolignols, precursors for lignins) and hydroxycinnamyl aldehydes;

(c) oxidative reactions resulting in side-chain shortening leading to benzoic acids;

(d) 2-hydroxylation and lactonisation leading to hydroxycoumarins; and

(e) conjugation with hydroxyacids or amino compounds leading to ester (e.g. CGAs) and amide conjugates, respectively.

[007] A schematic illustration of the reactions from which varied phenolic compounds are derived is presented in Figure 1.

[008] Chlorogenic acids (CGAs) are phenolic compounds which play a major role in the response to various biotic and abiotic stresses. CGAs are also important for their role as antioxidants in the diet of animals. Certain plants, such as coffee are rich in CGAs, and are among the most important dietary sources of this group of antioxidants. Hydroxy cinnamic acids are commonly found as soluble conjugates, esters, amides or glycosides, within the cytoplasm of plant cells. Among these conjugates, CGAs have been shown to account for up to 90 % of the total phenolic fraction of some plant species (see below). The generic name "CGA" used to refer to the single compound 5-O-caffeoylquinic acid (5-CQA), which was first detected in green coffee beans by Robiquet and Boutron in 1837 (Sondheimer, 1964). The definition was later extended to include all esters of quinic acid with a cinnamic acid derivative (Clifford, 2000). Quinic acid (1 ,3,4,5-tetrahydroxycyclohexane-l -carboxylic acid) is an alicyclic acid containing four readily accessible hydroxyl groups. CGAs are often grouped with esters of shikimic acid (3,4,5-trihydroxy-l -cyclohexene-l -carboxylic acid), which differ from quinic acid by the presence of a double bond at C-1 and only three readily accessible hydroxyl groups. CGAs are generally classified depending on the identity, number and position of the acyl group (Clifford, 1 999; Clifford, 2000) . The common CGA groups are caffeoylquinic acids (CQAs), coumaroylquinic acids, feruloylquinic acids (FQAs) and dicaffeoylquinic acids (diCQAs), the chemical structures of which are known to the skilled artisan.

Table 3: Quinic acid and representative chlorogenic acid (CGA) species

C = caffeic acid; F = ferulic acid

[009] Chlorogenic acids are formed between t-cinnamic acid derivatives and quinic acid., and are important intermediates for lignin biosynthesis in higher plants. CGA derivatives are abundant and diverse in the plant kingdom (Clifford, 1999; Clifford, 2000). They have been reported in several orders of mono- and dicotyledonous angiosperm species (Petersen et ah, 2009). The phylogenetically related Rubiaceae (e.g. plums, coffee), Solanaceae (e.g. tomato, tobacco, potato, eggplant), Asteraceae (e.g. artichoke, sunflower, chicory) and the more distant Rosaceae (e.g. apple, pears, peach) families possess most of the CGA-accumulating species. Monoesters of caffeic acid are relatively widespread, with 5-CQA being by far the most predominant isomer in green coffee beans (Clifford, 1979; Aerts et ah, 1994), potato tubers (Griffiths et al., 1997), tomato leaves (Niggeweg et al., 2004) and tobacco leaves (Hoffmann et ah, 2004). Some plant families such as Solanaceae and Rubiaceae also abound in diesters, such as diCQAs, diFQAs and mixed diesters (e.g. caffeoylferuloylquinic acids). Many plants, including coffee, produce CGAs derivatives esterified at C-3, C-4 and C-5, but not at C-l of the quinic acid moiety. This is in contrast with artichoke and some other Asteraceae plants, which abound in 1 ,3-diCQA (cynarin) and 1 ,5-diCQA, and also produce tri- and tetracaffeoylquinic acids.

[0010] Coffee plants contain the most diverse and highest amounts of CGAs reported thus far. C. arabica and C. canephora are the two Coffea species used extensively in the beverages known commercially as Arabica and Robusta coffee respectively. A total of 45 different CGAs have been identified in green Arabica coffee beans (Clifford et ah, 2006), while 69 CGAs have been reported in green Robusta coffee beans (Jaiswal, 2010). Quantitatively, the maj or coffee CGAs are the monocaffeoylquinic acids (3-CQA, 4-CQA and 5-CQA), the dicaffeoylquinic acids (3,4-diCQA, 3,5-diCQA and 4,5-diCQA) and the monoferuloylquinic acids (mainly 5-FQA). 5-CQA is the most abundant soluble ester, representing 45 to 50 % of the total CGAs in C. canephora seeds (Koshiro et ah, 2007). Other CGA compounds, such as coumaroylquinic and sinapoylquinic acids, as well as mixed diesters (e.g. caffeoylferuloylquinic acids) and 3,4,5-trimethoxycinnamoylquinic acid, can be found in low levels in Robusta coffee (Clifford, 2000; Jaiswal, 2010). C. canephora produces substantially more CGAs than C. Arabica.

[0011] The CGA level in green coffee grains varies between 7.9 and 14.4 % dry weight (DW) in C. canephora and 3.4 to 4.8 % DW in C. arabica (Ky et ah, 2001). (Lepelley et ah, 2007) reported lower but still relatively high CGA levels in C. canephora (6.6 % to 7.5 % DW). The content of diCQAs in C. canephora is also much higher than in C. arabica. CGA pools can vary with the coffee grain development (De Castro et ah, 2006; Lepelley et ah, 2007). While 5- CQA is the predominant molecule at all stages, 3-CQA and 4-CQA levels increase with seed growth. The 5-FQA level is relatively low in the early stage of grain development but rises 5 to 10 fold as the development progresses and reaches 22 % of total CGAs in ripe C. canephora. 3,5-diCQA is the major diCQA, especially in the early stages of fruit growth, but its level declines significantly during the development process.

[0012] Quinic acid is an abundant metabolite in young coffee grains, representing between 6 and 16 % DW. So, supply of this precursor may not restrict CGA biosynthesis as the grain develops. However, towards the end of grain development, quinic acid levels decrease below 1 % DW (Rogers et al, 1999; Lepelley et al, 2007). Considerable amounts of inositol (3- 4 % DW) are found in young coffee grains and other organic acids, such as citric and malic acids, are dominant in the mature coffee grain (Rogers et al, 1999). In Robusta coffee, caffeic and coumaric acids have also been found as conjugates of tryptophan (Murata et al, 1995).

[0013] Shikimate esters (also named dactylifric acids) have been reported in Palmae plants (e.g. palm, date, endive) (Maier et al., 1964; Harborne et al., 1974; Goupy et al, 1990; Ziouti et al, 1996). The shikimic acid precursor fails to accumulate to measurable levels in most of the important agronomic crops, although it is found in star anise (Illicium verum) and various species of evergreen trees. An ester of caffeic acid and 3,4-dihydroxyphenyllactic acid, commonly known as rosmarinic acid, is one of the active components of several medicinal plants and predominant in some Boraginaceae and Lamiaceae species, such as basil (Coleus blumei) and rosemary (Rosmarinus officinalis) (Petersen et al, 2003; Abdullah et al, 2008; Petersen et al, 2009). HCAs may also be conjugated to other acyl acceptors such as aliphatic acids (e.g. acetic, citric, malic, glucaric, tartric acids), sugar alcohols (e.g. arabinose, galactose, glucose, rhamnose), amino compounds (e.g. tryptophan, tyramine, octopamine, anthranilate, agmatine, choline, spermidine) and lipids (Clifford, 2000). The leaves of red clover (Trifolium pratense) accumulate phaselic acid (2-O-caffeoyl-L -malic acid), which prevents the breakdown of forage proteins during storage (Sullivan, 2009). Arabidopsis thaliana and other members of the Brassicaceae , such as radish (Raphanus sativus), accumulate hydroxycinnamoyl esters formed with aliphatic acids. These include sinapoyl -malic acid, a leaf-specific ester, as well as sinapoyl- choline, a seed-specific ester, and their common biosynthetic precursor sinapoyl-glucose (Chappie et al, 1992).

[0014] Besides the importance for the plant itself, HCAs and CGAs have attracted focus because they can influence food properties such as colour, taste and aroma. In general, phenolic compounds are considered to negatively influence food selection as they impart a bitter flavour (Drewnowski, 1997). CGAs, especially the diesters of caffeic acid, constitute a class of astringent compounds (Ohiokpehai et ah, 1983). In coffee beverages, (Farah et ah, 2006) reported that 3,4-diCQA levels in green and roasted coffee strongly correlate with satisfactory sensory attributes, but that higher levels of CQAs and 5-FQA are detrimental to beverage quality. A higher CGA content could be responsible for the perceived inferior quality of brews made with Robusta, which are occasionally described as bitter (Bertrand et ah, 2003).

[0015] CGAs and other HCA derivatives are significant components of diets rich in plant-based food such as cereals, legumes, oilseeds, fruits, vegetables and beverages. An increasing number of epidemiological studies have shown that people who consume higher quantities of plant-based food appear to have lower risks for significant health problems, such as cardiovascular diseases and certain cancers (Clifford, 2004; Finley, 2005). This health-promoting effect has been associated with the presence of phenolic phytonutrients. When compared to other beverages, coffee represents the richest source of CGAs and, interestingly, it has the highest antioxidant activity (Clifford, 1999; Wang et ah, 2009). The presence of conjugated ring structures and hydroxyl groups allows CGAs to actively scavenge free radicals generated in the aqueous phase (Rice-Evans et ah, 1996). This ability can prevent oxidative damage and lipid peroxidation in living tissues. While caffeic acid and 5-CQA have similar antioxidant capacities in vitro (Rice-Evans et ah, 1996), the number and nature of substitutions on the aromatic ring can influence the antioxidant properties. For instance, it has been shown in vitro that hydroxyl groups are more electron donating than methoxyl groups and that dihydroxyl-substituted caffeic acid shows a higher antioxidant activity than mono-substituted coumaric acid (Foley et ah, 1999; Shahidi et ah, 2010). Further studies demonstrate that CGAs can exert their health benefits through various other mechanisms, such as metal chelation and modification of redox potentials (Rice-Evans et ah, 1996).

[0016] CGAs have attracted much attention due to their exceptional antioxidant properties, as well as their high bioavailability and absorption in humans (Nardini et ah, 2002). Following coffee intake, CGAs are absorbed directly by the small intestine or hydrolysed by the large intestine microflora to release caffeic acid (Nardini et ah, 2002; Stalmach et ah, 2009; Stalmach et ah, 2010). This degradation product, which has same antioxidant capacities as 5- CQA in vitro (see above), is also absorbed and relatively stable in the gut (Scalbert et ah, 2002). CGAs have been attributed many other health benefits including anti-inflammatory, antibacterial, antiproliferative and anti carcinogenic properties (Rice-Evans et ah, 1997). CGAs have also been associated with reduced hepatic glucogenolysis and glucose absorption (Johnston et ah, 2003), inhibition of HIV-1 integrase (McDougall et ah, 1998), caffeine antagonistic cerebral effects (de Paulis et ah, 2004), reduced incidence of atherosclerosis, type 2 diabetes and various types of cancer (McCarty, 2005; Natella et ah, 2007). CGAs have been proposed to play a role in the prevention of neuro-degenerative diseases (Shahidi et ah, 2010).

[0017] Because CGAs and the activated CoA-thioesters needed to make them are difficult to synthesize, and nearly impossible to make in commercially useful quantities, there is a need for methods of producing chlorogenic and dactylifric acid species, as well as activated CoA-thioesters in vitro.

SUMMARY OF THE INVENTION

[0018] One aspect of the present invention features a recombinant Coffea wild-type HCT, HQT, and/or 4CL enzyme produced in a microbial cell and having enzyme activity substantially identical to a corresponding enzyme isolated from a Coffea spp. plant. Various alternative, independently selected embodiments of this aspect of the invention include the following: (1) the Coffea plant is C. canephora; (2) the microbial cell is E. coli, S. cerevisiae, or a food-grade lactic acid bacteria; (3) the recombinant enzyme is purified substantially to homogeneity; (4) the recombinant enzyme is immobilised on or covalently attached to a matrix; (5) the gene encoding the recombinant enzyme is engineered to provide more optimum codon usage for the microorganism in which it is to be expressed; (6) the gene encoding the recombinant enzyme is engineered to provide a purification tag on the N-terminus of the recombinant enzyme; (7) the enzyme is a 4CL enzyme capable of catalysing the production of a hydroxycinnamic acid (HCA)-CoA thioester from an HCA in the presence of coenzyme A (CoA), a divalent metal ion, and adenosine triphosphate (ATP); (8) the recombinant enzyme is catalytically active in vivo in the microorganism, or in vitro; (9) the recombinant enzyme catalyses the production of a CoA thioester from at least cinnamic acid, coumaric acid, caffeic acid, ferulic acid, and sinapic acid; (10) the recombinant enzyme is capable of converting at least about 80% of cinnamic acid, coumaric acid, caffeic acid, or ferulic acid to the corresponding HCA-CoA; (11) the recombinant enzyme is capable of producing in vitro a HCA-CoA thioester substantially free from the HCA acid precursor; (12) the recombinant enzyme is a HCT or HQT enzyme capable of catalysing in vivo in the microorganism or in vitro the production of a chlorogenic acid species or dactylifric acid species from a HCA-CoA thioester and an acyl acceptor molecule, and in particular wherein the HCA-CoA thioester comprises one or more of the thioesters coumaroyl-CoA, caffeoyl-CoA, cinnamoyl-CoA, feruloyl-CoA, or sinapoyl-CoA; and further wherein the acyl acceptor comprises quinic acid, shikimic acid, or a mixture thereof; (13) if the enzyme is recombinant HCT, it has a preference for shikimic acid as the acyl acceptor; (14) if the enzyme is recombinant HQT, it has a preference for quinic acid as the acyl acceptor; (15) if the enzyme is recombinant HCT or HQT, it is capable of catalysing the production in vitro of a HCA-CoA thioester from a chlorogenic acid or related ester in the presence of CoA in the temperature range of at least 22- 42 °C; (16) the recombinant enzyme is HCT and the HCA-CoA thioester formed is a di-, tri- or higher CGA or related ester; and (17) the recombinant enzyme has an amino acid of SEQ ID NO: 23 or 44 or 47.

[0019] Another aspect of the present invention features a mutant plant HCT enzyme comprising an amino acid sequence that is at least 80% identical to that of a corresponding wild- type HCT enzyme found in the same species of plant, and contains an altered amino acid residue in one or more positions relative to the wild-type HCT enzyme, wherein the mutant can bind at least one substrate or catalyze the formation of at least one product in either the forward or reverse direction. In various embodiments, the altered amino acid residue is at a position corresponding to an amino acid in a reference amino acid sequence that is the Coffea canephora HCT enzyme sequence (SEQ ID NO:23) and the amino acid is W23, N26, V27, D28, L29, V30, V31 , P32, N33, F34, H35, T36, P37, S38, V39, Y40, P1 10, R115, G143, G144, V149, G150, M151, R152, H153, H154, A155, A156, D157, G158, F159, S160, G161, L162, H163, F164, 1165, Y203, K210, K217, L231 , N248, Y252, Y255, L272, V274, D275, Q276, L280, Y281 , 1282, A283, T284, D285, R289, L294, N301, V302, 1303, F304, T305, L331, L346, K353, L355, V356, R357, G358, A359, H360, T361, F362, K363, C364, P365, N366, L367, G368, 1369, T370, S371 , W372, V373, R374, L375, P376, 1377, G383, M390, G391 , P392, G393, G394, 1395, A396, Y397, E398, G399, L400, S401 , or F402, or any combinations thereof. The alteration is a conservative substitution in one embodiment. In certain embodiments, the mutant HCT has improved ability relative to the wild-type HCT to bind at least one substrate, or to catalyze the formation of at least one product in either the forward or backward direction, or greater resistance to proteolysis. The mutant HCT may show a substrate preference for shikimic acid species relative to the wild-type enzyme. The mutant HCT may also or alternatively show an improved ability to catalyze the formation of diCQA relative to the wild-type enzyme. In certain embodiments, the mutant HCT comprises an alteration of the amino acid corresponding to H35, H153, H154, A155, A156, D157, K210, K217, Y252, Y255, or R374 of the reference HCT sequence (SEQ ID NO:23). It may comprise one or more of the mutations H35A, H153A, H 1 54A, A 1 55L, A 1 56 S , D 1 57A, Y252A, Y255 A, R374E, K21 0A/K21 7A, or H154N/A155L/A156S. The mutant HCT can be a mutant of C. canephora wild-type HCT enzyme. In certain embodiments, it has an amino acid sequence that is any of SEQ ID NOs:25, 27, 29, 31, 33, 35, 37, 39, or 41.

[0020] Another aspect of the invention features a mutant plant HQT enzyme comprising an amino acid sequence that is at least 80% identical to that of a corresponding wild-type HQT enzyme found in a plant, but which contains an altered amino acid residue in one or more positions relative to the wild-type HQT enzyme, wherein the mutant can bind at least one substrate or catalyze the formation of at least one product in either the forward or reverse direction. In various embodiments, the altered amino acid residue is at a position corresponding to an amino acid in a reference amino acid sequence that is the Coffea canephora HQT enzyme sequence (SEQ ID NO:44) and the amino acid is W23, N26, 127, D28, L29, L30, V31 , A32, R33, 134, H35, 136, L37, T38, V39, Y40, PI 10, R115, G143, A144, A149, G150, V151, Q152, H153, N154, L155, S156, D157, G158, V159, S160, S161, L162, H163, F164, 1165, Y203, K210, L217, 1231, N245, E246, G247, Y252, L272, N274, D275, Q276, L280, Y281 , V282, A283, T284, D285, R289, L294, N301 , V302, 1303, F304, T305, L331 , L346, S353, L355, V356, R357, G358, D359, R360, H361 , F362, A363, S364, P365, N366, L367, N368, 1369, N370, S371, W372, T373, R374, L375, P376, F377, G383, 1390, G391, P392, A393, 1394, 1395, L396, Y397, E398, G399, T400, V401 , or Y402. The alteration can be a conservative substitution. In certain embodiments, the mutant HQT has improved ability relative to the wild- type HQT to bind at least one substrate, or to catalyze the formation of at least one product in either the forward or backward direction, or greater resistance to proteolysis. The mutant HQT can show a substrate preference for quinic acid species relative to the wild-type enzyme. It can also or alternatively show an improved ability to catalyze the formation of diCQA relative to the wild-type enzyme. In certain embodiments, the mutant HQT comprises an alteration of the amino acid corresponding to H35, H153, H154, A155, A156, D157, K210, K217, Y252, Y255, or R374 of the reference HQT sequence (SEQ ID NO: 44). It may comprise one or more of the mutations H35A, H153A, N154A, L155A, S156A, D 157A, Y252A, R374E, K210A, or combinations thereof. The mutant HQT can be a mutant of C. canephora wild-type HQT enzyme. In one embodiment, it has an amino acid sequence that is SEQ ID NO: 46.

[0021] Another aspect of the invention features a composition comprising one or more of the above-described recombinant enzymes or mutants thereof, adapted for in vitro synthesis of one or more chlorogenic acid species or dactylifric acid species. The composition can further comprise substrates and cofactors sufficient for the production of one or more chlorogenic acid species or dactylifric acid species. In certain embodiments, one or more of the enzymes is immobilised on or covalently bound to a matrix. In certain embodiments, the substrates comprise one or more of coumaroyl-CoA, caffeoyl-CoA, cinnamoyl-CoA, feruloyl-CoA, or sinapoyl-CoA, and an acyl acceptor, the cofactors comprise Mg ^"1-1- , ATP, and optionally, CoA. The acyl acceptor can comprise quinic or shikimic acid species. In particular embodiments, the chlorogenic acid species or dactylifric acid species comprise caffeoylquinic acids (CQAs), coumaroylquinic acids, feruloylquinic acids (FQAs), caffeoylshikimic acids (CSAa), coumaroylshikimic acids, feruloylshikimic acids (FSAs), dicaffeoylquinic acids (diCQAs), or other di-, tri- or tetraesters, or combinations of any of the foregoing. The composition can be adapted for in vitro production of a HCA-CoA thioester catalysed by a 4CL enzyme, prior to in vitro production of the chlorogenic acid species or dactylifric acid species. In particular, the substrates for production of the HCA-CoA thioester can comprise one or more of cinnamic acid, coumaric acid, caffeic acid, or ferulic acid, and CoA.

[0022] Another aspect of the invention features a method of producing a chlorogenic acid or dactylifric acid species in vitro comprising the steps of: (a) providing an HCT or HQT or both; b) providing substrates and cofactors sufficient to form a chlorogenic acid or dactylifric acid species in the presence of an HCT or HQT or both; and (c) contacting the substrates and cofactors with the HCT or HQT, or both, under conditions permitting enzymatic activity, for a time sufficient to permit the formation of product that is a chlorogenic acid or dactylifric acid species. In certain embodiments, the substrates comprise a hydroxycinnamoyl-CoA thioester, and an acyl acceptor. In particular, the acyl acceptors include quinic acid, shikimic acid, or a combination thereof. In additional or altemative embodiments, the substrates comprise a mixture of coumaroyl-CoA, caffeoyl-CoA, cinnamoyl-CoA, feruloyl-CoA, or sinapoyl-CoA. In certain embodiments, the product comprises a mono-, di-, tri- or tetraester species of chlorogenic or dactylifric acid. In other embodiments of the method, the chlorogenic acid or dactylifric species comprise caffeoylquinic acids (CQAs), coumaroylquinic acids, feruloylquinic acids (FQAs), caffeoylshikimic acida (CSAa), coumaroylshikimic acids, feruloylshikimic acids (FSAs), dicaffeoylquinic acids (diCQAs), or other di-, tri- or tetraesters, or combinations of any of the foregoing. The method may further comprise an additional step wherein a HCA-CoA thioester is produced using a 4CL enzyme. In various embodiments of the method, the substrates comprise CoA, and one or more chlorogenic acid or dactylifric acid species. At least one of the HCT or HQT can be immobilised on or covalently bound to a matrix. The HCT or the HQT can be a Coffea HCT or HQT.

[0023] Another aspect of the invention features a method of producing a chlorogenic acid or dactylifric acid species in vitro comprising the steps of (a) providing an HCT, and optionally, an HQT or 4CL or both; (b) providing one or more HCA thioesters, an acyl acceptor, CoA, and a first chlorogenic acid or dactylifric acid species; and (c) contacting the one or more HCA thioesters, acyl acceptor, CoA, and first chlorogenic acid or dactylifric acid species with the HCT under conditions permitting enzymatic activity, and for a time sufficient to permit the formation of product that is a second chlorogenic acid or dactylifric acid species. In one embodiment, the first chlorogenic acid or dactylifric acid species is catalytically converted into a second chlorogenic acid or dactylifric acid species.

[0024] Another aspect of the invention features a food product comprising one or more exogenous chlorogenic acid or dactylifric acid species. The chlorogenic acid or dactylifric acid species in the food product can be produced in vitro. The exogenous chlorogenic acid or dactylifric acid species can be added to the food product as a mixture to the food product during processing. In one embodiment, the food product is a coffee product. The coffe product can be a soluble coffee product in which the exogenous chlorogenic acid or dactylifric acid species provide a functional ingredient that improves quality or provides a health benefit. In various embodiments, the exogenous chlorogenic acid or dactylifric acid species directly or indirectly provide a desirable flavour attribute, aroma attribute, or colour attribute to the product. Alternatively or additionally, the exogenous chlorogenic acid or dactylifric acid species can contribute antioxidant properties to the product. The exogenous chlorogenic acid or dactylifric acid species in the food product may comprise mono- and diester CGA or CSA species. In particular, the exogenous chlorogenic acid can comprise 3,4, caffeoylquinic acid at levels that improve the flavour and aroma of the food product as determined by sensory evaluation. In a specific instance, the exogenous chlorogenic acid comprises 3,4, caffeoylquinic acid but is substantially free of 5-feruloylquinic acid.

[0025] Another aspect of the invention features a recombinant microorganism comprising a gene for an expressible exogenous enzyme that is a Coffea HCT or HQT; wherein the microorganism expresses the enzyme. The recombinant microorganism can be a food-grade lactic acid bacterium. In certain embodiments, the recombinant microorganism produces a chlorogenic acid or dactylifric acid species in an amount not naturally produced in a comparable microorganism that does not express the enzyme.

[0026] Another aspect of the invention features a transgenic plant comprising a heterologous gene for an expressible exogenous enzyme that is a Coffea HCT or HQT; wherein the plant expresses the enzyme. In one embodiment, the transgenic plant produces less lignin than an equivalent plant that does not comprise the heterologous gene. In an additional or alternative embodiment, the transgenic plant comprises substantially equivalent biomass and growth characteristics as an equivalent plant that does not comprise the heterologous gene, under equivalent growth conditions.

[0027] Other and further objects, features, and advantages of the present invention will be readily apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] F ig . 1 : S id e-chain reactions involving hydroxycinnamoyl-CoA thioesters (Adapted from Strack et al, 1997).

[0029] Fig. 2: SDS-PAGE (Panel A) and western-blot (Panel B) analysis of Cc4CL2 following over-expression and purification. Lanes 1-6 as follows: Lane 1 : not induced; lane 2: induced; lane 3: insoluble cell extract; lane 4: soluble cell extract; lane 5: flow through; lane 6: elution fraction; M: MW marker (kDa).

[0030] Fig. 3 : SDS-PAGE analysis of the soluble fraction of the cell lysates following M4CL2 over-expression. Lanes 1 -6 are as follows: Lane 1 : BL21 (DE3); lane 2: BL21* (DE3); lane 3: BL21 (DE3) pLysS; lane 4: BL21* (DE3) pLysS; M: MW marker (kDa). [0031] Fig. 4: SDS-PAGE analysis of Nt4CL2 following purification by size-exclusion chromatography. Panel (A): Chromatogram. Panel (B): 12 % SDS-PAGE; lane 1 : purified Nt4CL2; M: MW marker (kDa).

[0032] Fig. 5: Analysis of GST-HQT following expression and affinity chromatography. Panel (A): 12 % SDS-PAGE. Panel (B): Western-blot against the GST-fusion. M: MW marker (kDa); Lanes were as follows: lanes 1-5: not induced, induced, lysate, soluble extracts 1 and 2; lanes 5-18: wash and elution fractions from the affinity column.

[0033] Fig. 6 : Agarose gel analysis of PCR products from Cchct and Cchqt amplification. Panel (A): Amplification of Cchct. Panel (B): Amplification of Cchqt. Lanes 1, 2, 3, 4: High Fidelity enzyme mix; lanes 5, 6, 7, 8: Pfu polymerase; M: MW marker.

[0034] Fig. 7: SDS-PAGE and western-blot of His6-CcHCT over-expression in BL21 * (DE3) pLysS cells. Panel (A): 12 % SDS-PAGE. Panel (B): Western-blot against the His6-tag. Lanes were as follows: 1 : non-induced cells; 2: soluble cell extract; 3: insoluble cell extract; 4: flow through; 5: wash; 6: elution; M: MW marker.

[0035] Fig. 8: Chromatogram and SDS-PAGE analysis of CcHCT from size-exclusion chromatography. Panel (A): Chromatogram for the size-exclusion step. Panel (B): 12% SDS- PAGE. Lanes were as follows: 1 : injected sample, M: MW marker (kDa); lanes 2-14: collected fractions n° 19-25 in the reverse order.

[0036] Fig. 9: SDS-PAGE analysis of HCT proteolysis. Panel (A): Lane 1 : purified CcHCT stored at -80 °C. M: MW marker (kDa); lane 2: same sample stored at 4 °C for several weeks. Panel (B): Limited proteolysis of with trypsin at a 1/500 (w/w) dilution on ice. M: MW marker; t0-t60: incubation time in min. (C) Limited proteolysis of the methylated derivative of CcHCT.

[0037] Fig. 10: SDS-PAGE analysis of the K-mutant HCT following limited proteolysis. Lanes were as follows: M: MW marker (kDa), lanes 1-5: trypsin digestion, lanes 6-10: chymotrypsin digestion, lanes 11-15: subtilisin digestion, respectively at t= 1, 5, 15, 60 min and overnight incubation with 1/1000 (w/w) protease at 4 °C.

[0038] Fig. 1 1 : SDS-PAGE analysis of His6-SUM03-CcHQT during the purification procedure. Panel (A): Lanes 1-4: cell lysate; lanes 5-8: fractions from the first affinity column, lanes 9-10: before and after SenP2 digestion, lanes 1 1-16: fractions from the ion-exchange column, M: MW marker (kDa). The band corresponding to His6-SUM03-HQT/ cleaved HQT is circled in red. Panel (B): size-exclusion chromatogram. Panel (C): Degradation pattern of CcHQT after storage at 4° C for several weeks.

[0039] Fig. 12: Overlap of the absorption spectra of CoA, 5-CQA and the reaction product caffeoyl-CoA. Reactions were set up using 5 mM CoA and 5 mM 5-CQA in 0. 1 M sodium phosphate pH 6.0. The reaction was started by adding 0.002 μΜ HQT and incubated at 34 °C. Samples taken at t= 0 (Panel A) and 30 min (Panel B) were analysed by HPLC with photodiode array (PDA) detector.

[0040] Fig. 13 : HPLC analysis of reactions comprising Nt4CL2 incubated with (hydroxy)cinnamic acids and CoA. Reactions were set up with 0.1 M Tris-HCl pH 7.5, 5 mM CoA and 1 mM of the various (hydroxy)cinnamic acids noted in each panel. Reactions were started by adding 1.6 μΜ of enzyme. Samples taken at the times indicated were analysed using HPLC-PDA with the methanol method. All chromatograms were extracted at λ= 325 nm except for cinnamic acid (Panel A), which was measured at λ= 300 nm. Panel (A): Cinnamic acid at t= 0 min (sample 598) and t= 20 min (sample 600); Panel (B) Coumaric acid at t= 0 min (sample 593) and t= 20 min (sample 594); Panel (C) Caffeic acid at t= 0 min (sample 590) and t= 20 min (sample 592); Panel (D) Ferulic acid at t= 0 min (sample 595) and t= 20 min (sample 597); and (E) Sinapic acid at t= 0 min (sample 601) and t= 20 min (sample 603).

[0041] Fig. 14: Absorption spectra of the CoA thioester products measured using HPLC- PDA. Panel (A): cinnamoyl-CoA; Panel (B) coumaroyl-CoA; Panel (C) caffeoyl-CoA; Panel (D) feruloyl-CoA; and Panel (E) sinapoyl-CoA.

[0042] Fig. 15: CoA thioester production by recombinant N/4CL2 with various HCAs and CoA in the presence of Mg-ATP. At t= 0, Enzyme (0.05 μΜ N/4CL2) was added to a 10 mL reaction mixture containing 0.8 mM coumaric/ caffeic or ferulic acid, 1 mM CoA, 2 mM MgC12 and 2 mM ATP in 0.1 M sodium phosphate pH 6.0. Samples (699-715) were taken at t= 0, 10, 60 and 150 min and analysed using HPLC-PDA with the methanol method. The conversion to CoA thioester in percentage was calculated from the HCA precursor peak area diminution.

[0043] Fig. 16: A representative reaction scheme showing forward and reverse reactions catalysed by HCT and/or HQT.

[0044] Fig. 17: HPLC profiles of HCT reactions with caffeoyl-CoA and quinic/ shikimic acid. Reactions were set up with 0.1 M sodium phosphate pH 6.5, 0.2 mM caffeoyl-CoA and 5 niM quinic / shikimic acid, or a mixture. Reactions were started by adding 1 μΜ enzyme. Samples taken at t= 5 and 60 min were analysed using HPLC-PDA with the methanol method. An overlap of the absorbance spectra for the putative CSA isomers is indicated in panel D (sample 1042). Panel (A): caffeoyl-CoA, t= 0 mm (sample 1023); Panel(B): Quinic acid, HCT t= 60 min (sample 1041); Panel (C): Shikimic acid, HCT t= 5 min (sample 1027); Panel (D): Shikimic acid, HCT t= 60 min (sample 1042); Panel (E): Quinic and shikimic acids, HCT t= 5 min (sample 1028); and Panel (F): Quinic and shikimic acids, HCT t= 60 min (sample 1043).

[0045] Fig. 18: HPLC profiles of HQT reactions with caffeoyl-CoA and quinic/ shikimic acid. Reactions were set up with 0.1 M sodium phosphate pH 6.5, 0.2 mM caffeoyl-CoA and 5 mM quinic/ shikimic acid or both. Reactions were started by adding 1 μΜ enzyme. Samples taken at t= 5 and 60 min were analysed using HPLC-PDA with the methanol method. See Fig. 17 Panel (A) for the control reaction. Panel (A): Quinic acid, HQT t= 60 min (sample 1044); Panel

(B) : Shikimic acid, HQT t= 60 min (sample 1045); Panel (C): Quinic and shikimic acids, HQT t= 5 min (sample 1031); and Panel (D): Quinic and shikimic acids, HQT t= 60 min (sample 1046).

[0046] Fig. 19: HPLC profiles of HCT reactions with coumaroyl-CoA and quinic/ shikimic acid. Reactions were set up with 0.2 mM coumaroyl-CoA and 10 mM quinic/ shikimic acid/ or a mixture of both in 0.1 M sodium phosphate pH 6.0. Reactions were started by adding 0.4 μΜ enzyme. Samples taken at t= 10 min were analysed using HPLC-PDA with the methanol method. Panel (A): coumaroyl-CoA, t= 0; Panel (B): Quinic acid, t= 10 min (sample 764); Panel

(C) : Shikimic acid, t= 10 min (sample 765); and Panel (D): Quinic and shikimic acids, t= 10 min (sample 766).

[0047] Fig. 20: HPLC profiles of HQT reactions with coumaroyl-CoA and quinic/ shikimic acid. The same conditions as Fig. 19 were applied. Panel (A): Quinic acid, t= 10 min (sample 767); Panel (B): Shikimic acid, t= 10 min (sample 768); and Panel (C): Quinic and shikimic acids, t= 10 min (sample 769).

[0048] Fig. 21 : HPLC profile of HCT reactions with feruloyl-CoA and quinic/ shikimic acid. Reaction mixtures containing 0.2 mM feruloyl-CoA, 10 mM quinic or shikimic acid or both and 0.1M sodium phosphate pH 6.0 were prepared in a 100 μL· final volume. The reaction was started by adding 0.01 μΜ HCT. Samples taken at t= 20 min were analysed using HPLC- PDA with the methanol method. Panel B shows the absorption spectrum of the putative feruloylshikimic acid peak. Panel (A): feruloyl-CoA t= 0; Panel (B): Shikimic acid t= 2 min (sample 717); Panel (C) Quinic acid t= 20 min (sample 722); and Panel (D) Quinic and shikimic acids t= 2 min (sample 718).

[0049] Fig. 22: HPLC profiles of HQT reactions with feruloyl-CoA and quinic/ shikimic acid. The same conditions as presented in Figure 21 were applied. Panel (A): Quinic acid t= 2 min (sample 719); Panel (B): Shikimic acid t= 2 min (sample 720); and Panel (C): Quinic and shikimic acids t= 2 min (sample 721).

[0050] Fig. 23: HPLC profiles of CQA standards upon heating. Samples containing 1 mM CQA isomer and 0.1M sodium phosphate pH 6.5 were incubated overnight and analysed using HPLC-PDA with the acetonitrile method. Panel (A): 5-CQA t= 0 (sample 1371); Panel (B): 4-CQA t= 0 (sample 1370); Panel (C): 3-CQA t= 0 (sample 1369); Panel (Α'): 5-CQA overnight at 34 °C (sample 1377); Panel (Β'): 4-CQA overnight at 34 °C (sample 1376); and Panel (C): 3-CQA overnight at 34 °C (sample 1375).

[0051] Fig. 24: HPLC profiles of HCT and HQT incubated with 5-CQA and CoA. Reactions were set up with 0.1 M sodium phosphate pH 6.0, 3.75 mM 5-CQA, 5 mM CoA, and milliQ. Reactions were started by adding 1 μΜ HCT or 1.0/ 0.01 μΜ HQT to the reaction mixture. Samples taken at t= 5, 15, 30 and 60 min were analysed using HPLC-PDA with the methanol method. The retention times here are different from earlier experiments due to the different chromatographic conditions. Panel (A): t= 0 (sample 427); Panel (B): 1 μΜ HCT t= 60 min (sample 425); Panel (C): 1 μΜ HQT t= 60 min (sample 419); and Panel (D): 0.01 μΜ HQT t= 60 min (sample 423).

[0052] Fig. 25: HPLC profiles of HCT and HQT incubated with 5-FQA and CoA. Reactions were set up with 0.1 M sodium phosphate pH 6.0, 3.75 mM 5-FQA, 5 mM CoA and milliQ water in a final volume of 100 μL. Reactions were started by adding 1 μΜ HCT/ HQT. Samples (395-428) taken at t= 0, 5, 15, 30 and 60 min were analysed using HPLC-PDA with the methanol method. Panel (A): t= 0 (sample 428); Panel (B): 1 μΜ HCT t= 60 min (sample 426); and Panel (C): 1 μΜ HQT t= 60 min (sample 420).

[0053] Fig. 26: HPLC profiles of CcHQT incubated with 3-, 4- and 5-CQA isomers and CoA. Reactions were set up with 1 mM CQA and 5 mM CoA in 0.1 M sodium phosphate pH 7.5. Reactions were started by adding 0.4 μΜ HQT. Samples taken at t= 0 and 30 min were analysed using HPLC-PDA with the methanol method. Panel (A): 3-CQA, t= 0 and t= 30 min (sample 612); Panel (B): 4-CQA, t= 0 and t= 30 mm (sample 611); and Panel (C) 5-CQA, t= 0 and t= 30 min (sample 610).

[0054] Fig. 27: HPLC profiles of HCT incubated with 3,5-diCQA and CoA. Reactions were set up containing 1 mM 3,5-diCQA and 5 mM CoA in 0.1 M sodium phosphate pH 6.0 to a final volume of 200 μ Reactions were started by adding 1 μΜ HCT/ HQT/ water as a control. Panel (A): Standards: 5-CQA and 3,5-diCQA; Panel (B): Control with no enzyme overnight (sample 882); and Panel (C): HCT overnight (sample 880).

[0055] Fig. 28: Influence of pH on caffeoyl-CoA production by HCT and HQT. Reactions were set up with 1 μΜ enzyme, 2 mM CoA and 2 mM 5-CQA in 0.1 M sodium phosphate at pH 4.6/ 6.0/ 7.6. Reactions were stopped at t= 300 min.

[0056] Fig. 29: Influence of pH on HCT and HQT activity towards 5-CQA and CoA. Reactions were set up with 2 mM CoA and 2 mM 5-CQA in 0.1 M sodium phosphate at pH 6.0, 7.0 and 8.0. Reactions were started by adding 1 μΜ HCT (A) or 0.1 μΜ HQT (B).Panel (A): HCT; Panel (B): HQT. Symbols: Squares: pH 6.0; Triangles: pH 7.0; Plain line: pH 8.0.

[0057] Fig. 30: Caffeoyl-CoA formation as a function of enzyme concentration. Reactions were set up with 5 mM CoA and 2 mM 5-CQA in 0.1 M sodium phosphate pH 6.0. The reaction was started by adding enzyme either HCT (Panel A) or HQT (Panel B). Enzyme concentration units tested are expressed in nM (HCT: 135, 271, 542, 813 nM; HQT: 8, 16, 33, 65, 130, 260, 521 nM).

[0058] Fig. 31 : Influence of 5-CQA concentration on caffeoyl-CoA formation catalysed by HQT. Reactions were set up with 0.08-10 mM 5-CQA and 2 mM CoA in 0.1 M sodium phosphate pH 6.0. Reactions were started by adding 0.1 μΜ HQT.

[0059] Fig. 32: Inhibitory effect of quinic acid on the level of caffeoyl-CoA formation by HQT. Reactions were set up with 5 mM 5-CQA, 5 mM CoA and 0-20 mM quinic acid in 0.1 M sodium phosphate pH 6.0. Reactions were started by adding 1 μΜ HQT. Samples taken at t= 0, 5 and 30 min (488-503) were analysed using HPLC-PDA with the methanol method.

[0060] Fig. 33: Influence of CoA concentration on the level of caffeoyl-CoA formed by HCT and HQT. Reactions were set up with 0.125-5 mM CoA and 1 mM 5-CQA in 0.1 M sodium phosphate pH 6.5. Reactions were started by adding 5 μΜ HCT or 0.5 μΜ HQT. Samples (1101 -1 145) taken at t= 0 and 180 min were analysed using HPLC-PDA with the acetonitrile method. Panel (A): Standard Curve for CoA concentration added; Panel (B): Effect of CoA concentration on the level of caffeoyl-CoA formation catalysed by HCT and HQT. Symbols: Diamonds: HCT; Triangles: HQT.

[0061] Fig. 34: HPLC profiles of HCT/ HQT incubated with 5-CQA and CoA at two different molar ratios. Reactions were set up with 1 mM 5-CQA and either 1 or 10 mM CoA in 0.1 M sodium phosphate pH 6.5. Reactions were started by adding 0.5 μΜ enzyme. Samples taken at t= 180 min were analysed using HPLC-PDA with the acetonitrile method. Panel (A): t= 0; Panel (B): Control 5-CQA and CoA no enzyme overnight; Panel (C): HCT 1/1 5-CQA/CoA ratio (sample 1085) and overlap of absorbance spectra of the diCQA peaks; Panel (D): HCT 1/10 5-CQA/CoA ratio (sample 1086); Panel (E): HQT 1/1 5-CQA/CoA ratio (sample 1089); and Panel (F): HQT 1/10 5-CQA/CoA ratio (sample 1090).

[0062] Fig. 35: Comparison of the activities of native and mutant HCTs in the forward and reverse reactions. Reaction mixtures are described below. Reactions were started by adding 5 μΜ enzyme. Samples taken at t= 210 min were analysed using HPLC-PDA with the acetonitrile method. Panels A, B, C, D, E, F, G, H: Reverse reaction: Reactions were set up with 1 mM 5-CQA and 1 mM CoA in 0.1 M sodium phosphate pH 6.5. Panels A'-K': Forward reaction: Reactions were set up with 0.1 mM caffeoyl-CoA and 1 mM quinic acid in 0.1 M sodium phosphate pH 6.5. Panels A / A': Control with no enzyme (samples 1321 / 1337); Panels B / B': native HCT (samples 1322 / 1338); Panels C / C: K-mutant (samples 1323 / 1339); Panels D / D': H35A mutant (samples 1326 / 1342); Panels E / Ε': Y255A mutant (samples 1329 / 1345); Panels F / F': D157A mutant (samples 1325 / 1341); Panels G / G: Y252A mutant (samples 1327 / 1343); Panels H / H: R374E mutant (samples 1328 / 1344); Panels I and Γ: H153A mutant (samples 1324 / 1340); Panels J and J': H154A mutant (samples 1330 / 1346); and Panels K / K': H154N/A155L/A156S mutant (samples 1331 / 1347).

[0063] Fig. 36: HPLC profiles of HCT HI 54 mutants incubated with 5-CQA and CoA at 1/1 and 1/10 molar ratios. Reactions were set up with 1 mM 5-CQA and 1 or 10 mM CoA in 0.1 M sodium phosphate pH 6.5. Reactions were started by adding 0.5 μΜ enzyme. Samples taken at t= 180 min were analysed using HPLC-PDA with the acetonitrile method. The t= 0 is the same as in Fig. 32. Panel (A): H154A 1/1 5-CQA/CoA (sample 1093); Panel B) H154A 1/10 5- CQA/CoA (sample 1094); Panel (C) H154N/A155L/A156S 1/1 5-CQA/CoA (sample 1097); and Panel (D) H154N/A155L/A156S 1/10 5-CQA/CoA (sample 1098). [0064] Fig. 37: HPLC profiles of reaction products generated by the H154A mutant HCTs from 5-CQA and various acyl donors. Reactions were set up with 10 mM 5-CQA and either 0.5 mM CoA/ caffeoyl-/ feruloyl-/ coumaroyl-CoA in 0.1 M sodium phosphate pH 6.5. The reaction was started by adding 0.5 μΜ enzyme. Samples were analysed after overnight incubation using HPLC-PDA with the acetonitrile method. Panel (A): Control with no enzyme 5- CQA + CoA (sample 1234); Panel (B) 5-CQA + CoA→ caffeoyl-CoA + diCQAs (sample 1258); Panel (C): 5-CQA+ caffeoyl-CoA→ diCQAs (sample 1259); Panel (D): 5-CQA + feruloyl-CoA→ diCQAs + mixed caffeoylferuloylquinic acids (*) (sample 1260); and Panel (E) 5-CQA + coumaroyl-CoA→ diCQAs + mixed coumaroylcaffeoylquinic acids (*) (sample 1261). Panels D and E also include an inset enlarging the di-ester HPLC profile.

[0065] Fig. 38: HPLC profiles of the reaction products synthesised by H154A mutant HCTs from 5-FQA and various acyl donors. Reactions were set up with 10 mM 5-FQA and either 0.5 mM CoA/ caffeoyl-/ feruloyl-/ coumaroyl-CoA in 0.1 M sodium phosphate pH 6.5. The reaction was started by adding 0.5 μΜ enzyme. Samples were analysed after overnight incubation using HPLC-PDA with the acetonitrile method. Panel (A): Control with no enzyme 5- FQA + CoA (sample 1238); Panel (B) Control with no enzyme 5-FQA + coumaroyl-CoA (sample 1241); Panel (C) 5-FQA + CoA→ new peaks = diFQAs (putative)(sample 1262); Panel (D) 5-FQA + caffeoyl-CoA→ new peaks = caffeoylferuloylquinic acids (putative)(sample 1263); Panel (E) 5-FQA + feruloyl-CoA→ new peaks = diFQAs (putative)(sample 1264); and Panel (F) 5-FQA + coumaroyl-CoA→ new peaks = coumaroylferuloylquinic acids (putative) (sample 1265).

[0066] Fig. 39: HPLC profiles of the reaction profiles synthesized by 4CL and HCT or HQT from coumaric or caffeic acid and various acyl donors. Reaction mixtures containing 0.5 mM coumaric/ caffeic acid, 0.5/ 10 mM quinic/ shikimic acid, 2.5 mM CoA, 2.5 mM ATP and 5 mM MgC12 in 0.1 M sodium phosphate pH 6.0 were prepared in a 500 μΕ final volume. Reactions were started by adding 0.4 μΜ Nt4CL2 and incubated at 35°C. 75 μΕ-νοηιιηβ samples were taken at t= 10 min and 5-fold dilutions were analysed using HPLC-PDA with the methanol method. A t= 15 min, 0.5 μΜ HCT/ HQT was added to the remaining 425 μΕ reaction mixture. Samples were taken at t= 30 min (15 min after adding HCT/ HQT), t= 105 min and overnight (data not shown for the latter two time points) were also analysed. Panel (A) control with no enzyme and 0.5 mM coumaric acid; Panel (B) 4CL and 0.5mM coumaric acid; Panel (C) consecutive reactions with 4CL and HQT with coumaric (0.5 mM) and quinic (0.5/ 10 mM) acids; Panel (D) consecutive reactions with 4CL and HCT with coumaric (0.5 mM) and shikimic (0.5/ 10 mM) acids; Panel (E) 4CL and 0.5mM caffeic acid; Panel (F) consecutive reactions with 4CL and HQT with caffeic (0.5 mM) and quinic (0.5/ 10 mM) acids; Panel (G) consecutive reactions with 4CL and HCT with caffeic (0.5 mM) and shikimic (0.5/ 10 mM) acids

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0067] The following abbreviations may be used herein:

4CL 4-coumarate CoA ligase

ACS acetyl-CoA synthase

AMP adenosine monophosphate

At Arabidopsis thaliana

ATP adenosine triphosphate

AU asymmetric unit

BME β-mercaptoethanol

bp base pair

C3H coumarate 3 -hydroxylase

C4H cinnamate 4-hydroxylase

CAD cinnamyl alcohol reductase

CAT chloramphenicol acetyltransferase

CBL chlorobenzoate CoA-ligase

Cc Coffea canephora

CCR cinnamoyl-CoA reductase

cDNA complementary deoxyribonucleic acid

CGA chlorogenic acids

CoA coenzyme A

CQA caffeoylquinic acid

CrAT carnitine acetyltransferase

CSA caffeoylshikimic acid

CV column volume A dactylifric acids

AHP 3-dehydroxy-d-arabino-heptulose-7 phosphate

AT deacetylvindoline 4-O-acetyltransferase

LS dynamic light scattering

^TP deoxynucleotide triphosphate

ON deoxynivalenol

TT dithiothreitol

W dry weight

MBL: European Molecular Biology Laboratory

PSP 5-enolpyruvate shikimate 3 -phosphate

ST expressed sequenced tag

PLC fast protein liquid chromatography

3 A feruloylquinic acid

ST glutathione S-transferase

CA hydroxycinnamic acid

CBT hydroxycinnamoyl/benzoyl-CoA anthranilate N-hydroxycinnamoyl/

benzoyltransferase

CT hydroxycinnamoyl-CoA shikimate/ quinate hydroxycinnamoyltransferase

PLC high-performance liquid chromatography

QT hydroxycinnamoyl-CoA quinate/ shikimate hydroxycinnamoyltransferase

TX high-throughput crystallography

'TG isopropyl β-d-l -thiogalactopyranoside

mutant K210A/K217 A mutant (CcHCT)

>a kilo Dalton (103 Da)

Luria-Bertani

T malonyltransferase

^' ,S 2-(N-morpholine)-ethane sulphonic acid

L molecular replacement

mass spectrometry

V molecular weight

macromolecular crystallography NCBI National Center for Biotechnology Information

NRPS non ribosomal peptide synthase

Nt Nicotiana tabacum

NTA nitriloacetic acid

OD optical density

PAGE polyacrylamide gel electrophoresis

PAL phenylalanine ammonia lyase

PapA5 polyketide-associated protein A5

PCR polymerase chain reaction

PDB protein data bank

PEG polyethylene glycol

PEP phosphoenolpyruvate

PMSF phenylmethanesulfonyl fluoride

PPi pyrophosphate

RMSD root mean square deviation

RT retention time

SBP substrate binding pocket

SDS sodium dodecylsulfate

SM secondary metabolites

SUMO small ubiquitin-related modifier

TRI101 trichothecene 3 -O-acetyltransf erase

UV ultraviolet

vs vinorine synthase

[0068] The terms "chlorogenic acid" or "chlorogenic acid species" or "CGA" are used herein essentially synonymously. Chlorogenic acids include all esters of quinic acid (1,3,4,5- tetrahydroxycyclohexane-l-carboxylic acid) with cinnamic acid or a cinnamic acid derivative. Common examples of chlorogenic acids include those shown in Table 2. For convenience, the expression "chlorogenic acids and related esters" is sometimes used herein. "Chlorogenic acids and related esters" includes chlorogenic acid species, as well as dactylifric acid species, and other closely related phenylpropanoid compounds found in plants. [0069] The terms "dactylifric acid" or "dactylifric acid species" or DA are also used synonymously herein. Dactylifric acids is used to refer to esters of shikimic acid (3,4,5- trihydroxy-l-cyclohexene-l-carboxylic acid) with cinnamic acid or a cinnamic acid derivative.

[0070] As used herein "a coffee product" is any product comprising any part of a coffee plant, preferably coffee beans or coffee cherries, and intended for oral consumption by a human or animal. A coffee product may be a pure soluble, or instant, coffee product, which is a dried coffee extract useful for preparing a coffee beverage by dissolution in water.

[0071] The term "food" or "food product" or "food composition" means a product or composition that is intended for ingestion by an animal, including a human, and provides nutrition to the animal.

[0072] As used herein, an amino acid residue can be determined to "correspond" to an amino acid in a related sequence, such as a reference sequence, when the amino acids sequences are first aligned so as to reflect what is known about the conserved motifs, secondary structures, catalytic residues or other features or putative features. Thus, gaps may be present in the alignments to allow for better fit. The skilled artisan will be familiar with various computer models and software packages that provide for aligning related sequences and allow determination of "corresponding" amino acids. Sequence alignments may also be adjusted on the basis of data such as 3-dimensional models, crystallization results, mutagenesis studies and more, either by a computer program, or after visual inspection by a skilled artisan familiar with such alignments.

[0073] The term "microorganism" encompasses at least bacteria, molds and other fungi, and yeasts. A "recombinant microorganism" for purposes herein is one expressing an exogenous gene encoding one or more proteins of interest, particularly an HCT, HQT, or 4CL enzyme.

[0074] As used throughout, ranges are used herein in shorthand, so as to avoid having to set out at length and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range.

[0075] As used herein and in the appended claims, the singular form of a word includes the plural, and vice versa, unless the context clearly dictates otherwise. Thus, the references "a", "an", and "the" are generally inclusive of the plurals of the respective terms. For example, reference to "an enzyme", "an amino acid residue", or "an alteration" includes a plurality of such "enzymes", "residues", or "alterations". Reference herein, for example to "an antioxidant" includes a plurality of such antioxidants, whereas reference to "pieces" includes a single piece. Similarly, the words "comprise", "comprises", and "comprising" are to be interpreted inclusively rather than exclusively. Likewise the terms "include", "including" and "or" should all be construed to be inclusive, unless such a construction is clearly prohibited from the context. Where used herein the term "examples," particularly when followed by a listing of terms is merely exemplary and illustrative, and should not be deemed to be exclusive or comprehensive.

[0076] The methods and compositions and other advances disclosed here are not limited to particular methodology, protocols, and reagents described herein because, as the skilled artisan will appreciate, they may vary. Further, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to, and does not, limit the scope of that which is disclosed or claimed.

[0077] Unless defined otherwise, all technical and scientific terms, terms of art, and acronyms used herein have the meanings commonly understood by one of ordinary skill in the art in the field(s) of the invention, or in the field(s) where the term is used. Although any compositions, methods, articles of manufacture, or other means or materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred compositions, methods, articles of manufacture, or other means or materials are described herein.

[0078] All publications and other references (including patents, published patent applications, publications, texts, and technical and/or scholarly articles) cited or referred to herein are in their entirety incorporated herein by reference to the extent permitted by applicable law. Full citations for publications not cited fully within the specification are set forth at the end of the specification. The discussion of any publication or reference is intended merely to summarize the assertions made therein. No admission is made that any such patents, patent applications, publications or references, or any portion thereof, are relevant, material, or prior art. The right to challenge the accuracy and pertinence of any assertion of such patents, patent applications, publications, and other references as relevant, material, or prior art is specifically reserved. The Invention

[0079] In one aspect, the invention provides at least one recombinant plant enzyme that is a wild-type HCT, HQT, or 4CL expressed in a microbial cell. The recombinant enzyme is particularly useful for producing commercially useful quantities of one or more chlorogenic acids species or related esters. The recombinant enzyme has enzyme activity substantially identical to a corresponding enzyme isolated from a Coffea spp. plant. The enzyme activity may be substantially identical in terms of substrate specificity and preferences, temperature stability and temperatures of activity, pH stability and pH activity profile, products produced, and the like. Presently preferred plants for HCT and HQT are Coffea canephora and Coffea arabica. For 4CL enzymes, Nicotiana tabacum is preferred in addition to Coffea spp.

[0080] The recombinant enzymes, singly or in combination with one another, are preferentially expressed in large quantities in E. coli, S. cerevisiae, or a food-grade lactic acid bacterium, such as a Lactobacillus. Food-grade lactic acid bacteria are useful for production of various food components, or for the production of fermented foods. Such organisms may be used to produce chlorogenic and dactylifric acids in vivo, for example in large-scale fermentations. They may also be used to produce a supply of enzymes for in vitro production of CGAs and DAs.

[0081] In one embodiment, the recombinant enzyme(s) is/are expressed in a probiotic microorganism. Probiotics include many types of bacteria but generally are selected from four genera of bacteria: Lactobacilllus (e.g. L. acidophilus), Bifidobacteria, Lactococcus, and Pediococcus. Other beneficial species include Enterococcus and Saccharomyces species. Probiotics are live microorganisms that have a beneficial effect in the prevention and treatment of specific medical conditions when ingested, and may colonize the gastrointestinal tract of an animal. The recombinant lactic acid bacteria or probiotic organism may produce useful or beneficial CGA or DA species, for example, in the gut of an animal consuming such microorganisms. Probiotics generally enhance systemic cellular immune responses and are useful as a dietary supplement to boost natural immunity in otherwise healthy adults. Including the recombinant enzymes HCT, HQT or 4CL in such organisms may provide another opportunity to increase functionality of useful probiotic organisms. [0082] One or more of the recombinant enzymes can be made in the microorganism in substantial quantities. The protein can be produced in the organism such that it is secreted, for example into an extracellular medium, or it can remain within and accumulate in the cell and obtained by disrupting the cells. The expression of the recombinant enzyme can be constitutive or inducible, depending for example, on the promoter used to drive the expression. In one embodiment, the recombinant enzyme can be partially or substantially purified from the microorganism with a convenient and preferably rapid method, for example using a simple chromatographic separation, affinity chromatography, or the like. The skilled artisan will appreciate that any of a wide variety of methods can be used for such purification steps. In a preferred embodiment, the recombinant enzyme can be purified to near-homogeneity or even to homogeneity as determined by gel filtrations or gel electrophoresis. The skilled artisan will also appreciate that the purification can be aided by including certain features in the expressed gene. In one embodiment, the gene encoding the recombinant enzyme is engineered to provide a purification tag on the N-terminus of the recombinant enzyme. Examples of such tags include hexahistidine (His ₆₎ tags which are well-known in the art.

[0083] The recombinant enzyme is readily adapted for in vitro production of chlorogenic and/or dactylifric acid species, preferably in commercially-useful quantities. In one embodiment, the enzyme is immobilised on or covalently attached to a matrix that is part of an in vitro production system for useful chlorogenic acid species and related esters. For example, CGA or DA compounds may be produced in one or more columns wherein 4CL is used to produce HCA- CoA compounds, and subsequently the HCA group is transferred to acyl acceptor such as quinic or shikimic acid. The production system can be run in a continuous fashion, or in semi- continuous or even batch mode. As another example, the recombinant enzymes, e.g., 4CL, HCT and/or HQT can be used in combination to produce useful chlorogenic acid species and related esters.

[0084] Preferably, the gene encoding the recombinant enzyme is engineered to provide more optimum codon usage for the microorganism in which it is to be expressed. Codon usage tables are available to show preferences for codon usage in various organisms. The skilled artisan will be familiar with such preferences and understand how to adapt a sequence for the microorganism selected to express the recombinant enzyme. [0085] In various embodiments, the recombinant enzyme is a 4CL enzyme capable of catalysing the production in vitro of a hydroxycinnamic acid (HCA)-CoA thioester from an HCA in the presence of coenzyme A (CoA), a divalent metal ion, and adenosine triphosphate (ATP). The recombinant enzyme preferably is catalytically active in vitro from at least pH 6.0 to pH 7.5.

[0086] The recombinant 4CL catalyses the production in vitro of a CoA thioester from at least cinnamic acid, coumaric acid, caffeic acid, ferulic acid, and sinapic acid, and under suitable condition is capable of converting at least about 80% of the cinnamic acid, coumaric acid, caffeic acid, or ferulic acid to the corresponding HCA-CoA. In one embodiment, an 80% conversion can be accomplished within about 10 to 20 minutes of incubation with recombinant enzyme. In other embodiments, the time for 80% conversion is 30, 40, 50, or 60 minutes. The skilled artisan will appreciate that time required to convert a given amount of substrate can be reduced by increasing the amount of catalyst (enzyme) present in the reaction. The amount of conversion of substrate to product in various embodiments is at least about 50%, 60%, 70%, 80%, 85% or even higher.

[0087] The recombinant enzyme can preferably produce a HCA-CoA thioester that is substantially free from the HCA acid precursor. The absence of the free acid form can improve the use of the HCA-CoA thioester produced as a substrate in a subsequent reaction, for example by an HCT or HQT enzyme. Typical HCA-CoA products for a recombinant 4CL enzyme include coumaroyl-CoA, caffeoyl-CoA, cinnamoyl-CoA, feruloyl-CoA, and sinapoyl-CoA, depending of course on the HCA substrate used, which can be determined based on the product desired.

[0088] The recombinant enzyme can also be a HCT or HQT enzyme capable of catalysing the production of a chlorogenic acid species or related ester from a HCA-CoA thioester and an acyl acceptor molecule. Preferred HCA-CoA thioesters comprise one or more of the thioesters coumaroyl-CoA, caffeoyl-CoA, cinnamoyl-CoA, feruloyl-CoA, or sinapoyl-CoA, depending on the desired CGA or DA to be produced. Presently preferred acyl acceptors comprise quinic acid, shikimic acid, or a mixture thereof. The skilled artisan will understand that while these acyl acceptors are presently preferred and known to perform well, other acyl acceptors that are synthetic or that are found in nature may be useful herein.

[0089] Recombinant HCTs generally have a preference for shikimic acid as the acyl acceptor, whereas recombinant HQTs generally have a preference for quinic acid as the acyl acceptor. [0090] The recombinant HCT or HQT enzymes are capable of catalysing the production in vitro of a HCA-CoA thioester from a chlorogenic acid or related ester, such as a dactylifric acid, in the presence of CoA. The recombinant enzymes preferably demonstrate similar catalytic activity over the temperature range of at least 22-42 °C, i.e. over that temperature range the catalytic activity does not vary in any substantial manner, despite the nearly 20 °C temperature range.

[0091] Surprisingly, the inventors have found that the recombinant enzymes can produce/synthesize in vitro one or more diester species of CGA and DA. This is believed to be the first such demonstration of in vitro biosynthesis of such compounds. In one embodiment, recombinant HCT can catalyse the production of a HCA-CoA thioester that is a di-, tri- or higher CGA or related ester. Examples of such compounds include 3,4 diCQA, 4,5-diCQA, 3,5-CQA, diferuloylquinic acid species, as well as mixed diesters such as caffeoylferuloylquinic acid species.

[0092] The inventors have also discovered a previously unknown reaction wherein HCT, at least in the presence of CoA, and/or one or more HCA-CoA, can rearrange or "remodel" CGA and DA species to form more complex (di-, tri-, tetraesters) CGAs of DA. This provides an opportunity to generate novel CGA and DA species that could possess heretofore unrecognized properties or benefits.

[0093] Published sequences useful for the amino acid sequence of the recombinant enzyme are available. In various embodiments, the recombinant enzyme has an amino acid sequence of SEQ ID NOs: 23, 44, or 47, or the sequence of any other published sequence of a suitable enzyme from a Coffea genome.

[0094] A related aspect of the invention features recombinant microorganisms comprising one or more genes for one or more expressible exogenous enzymes comprising Coffea HCT and/or HQT; wherein the microorganism expresses the enzyme(s). Th e recombinant microorganism is preferably a food-grade lactic acid bacterium.

[0095] In one embodiment, the recombinant microorganism can produce a chlorogenic acid or dactylifric acid species in an amount not naturally produced in a comparable microorganism that does not express the enzyme. [0096] The microorganism can conveniently be any microorganism, but particularly as described above with respect to the microorganisms, lactic acid bacteria, and probiotic organisms of the prior aspect of the invention.

[0097] In another of its several aspects, the invention provides mutant plant HCT and HQT enzymes. In one embodiment, the mutant HCT or HQT comprises an amino acid sequence that is at least 80% identical to that of a corresponding wild-type HCT or wild-type HQT enzyme found in a plant, but which contains an altered amino acid residue in one or more positions relative to corresponding the wild-type enzyme. In other embodiments, the mutant enzyme may be greater than about 80% identical, for example, at least about 85 or about 90% identical to the wild-type enzyme. In yet other embodiments, the amino acid sequences are at least 91 , 92, 93, 94, 95, 96, 97, 98, 99% or 100% identical. Preferably, the mutant can bind at least one substrate or can catalyse the formation of at least one product in either the forward or reverse direction of the reaction transferring an acyl moiety from a CoA thioester to an acceptor molecule to produce a CGA. For example, Figure 16 shows a typical reaction catalysed by the HCT and/or HQT enzymes.

[0098] The mutant enzymes generally have an alteration comprising a substitution, deletion, or addition of one or more amino acid residues. The alteration in some embodiments comprises a conservative substitution of one amino acid residue for a similar or related amino acid, for example a V residue could be conveniently substituted for an L or I residue, or vice versa. The skilled artisan will appreciate the generally-accepted notions of conservative amino acid substitutions and further details are available in standard Biochemistry, Molecular Biology or Protein Engineering texts.

[0099] In various embodiments, the mutant enzymes feature an altered amino acid residue at a position corresponding to an amino acid in a reference amino acid sequence. A preferred reference sequence for mutant HCTs is the Coffea canephora HCT amino acid sequence. The amino acid of C. canephora HCT is known in the art, and is set forth as SEQ ID NO: 23. In accordance with the present invention, the crystal structure of C. canephora HCT has been resolved, including co-crystallization with one or more substrates, and the substrate binding pocket(s) and other important features of the enzyme have been determined (data not shown). Based on these data, the inventors have been able to ascertain for the first time the amino acids responsible for binding substrates and for catalysis, as well as those residues closely associated with those important features. Thus, among presently preferred amino acid positions include those known to be involved with substrate binding, or located close to such residues, as well as residues likely involved with catalysis, or with important conformational features. In various embodiments, the mutant HCT contains an alteration at an amino acid that corresponds to one or more of the following positions in the reference sequence of C. canephora HCT (SEQ ID NO:23) (the letter is the one-letter code for the amino acid residue in the reference sequence and the number represents the position of that residue in the reference sequence): W23, N26, V27, D28, L29, V30, V31, P32, N33, F34, H35, T36, P37, S38, V39, Y40, PI 10, R115, G143, G144, V149, G150, M151, R152, H153, H154, A155, A156, D157, G158, F159, S160, G161, L162, H163, F164, 1165, Y203, K210, K217, L231 , N248, Y252, Y255, L272, V274, D275, Q276, L280, Y281, 1282, A283, T284, D285, R289, L294, N301, V302, 1303, F304, T305, L331, L346, K353, L355, V356, R357, G358, A359, H360, T361 , F362, K363, C364, P365, N366, L367, G368, 1369, T370, S371 , W372, V373, R374, L375, P376, 1377, G383, M390, G391 , P392, G393, G394, 1395, A396, Y397, E398, G399, L400, S401, or F402. It is contemplated that combinations of amino acids corresponding to these positions will be the subject of alteration in mutant HCTs.

[00100] A preferred reference sequence for mutant HQTs is a Coffea canephora

HQT amino acid sequence. The amino acid of C. canephora HQT is known in the art, and is set forth as SEQ ID NO:44. The skilled artisan will appreciate the close relationship among sequence for HQTs from different plants, as well as that between HQTs and HCTs from different plants. Based on these data, the inventors have been able to predict the amino acids responsible for docking/binding substrates and for catalysis of the CcHQT, as well as those residues closely associated with those important features.

[00101] In various embodiments, the mutant HQT contains an alteration at an amino acid that corresponds to one or more of the following positions in the reference sequence of C. canephora HQT (SEQ ID NO: 44) (the letter is the one-letter code for the amino acid residue in the reference sequence and the number represents the position of that residue in the reference sequence): W23, N26, 127, D28, L29, L30, V31, A32, R33, 134, H35, 136, L37, T38, V39, Y40, P1 10, R1 15, G143, A144, A149, G150, V151 , Q152, H153, N154, L155, S 156, D157, G158, V159, S160, S161, L162, H163, F164, 1165, Y203, K210, L217, 1231, N245, E246, G247, Y252, L272, N274, D275, Q276, L280, Y281 , V282, A283, T284, D285, R289, L294, N301 , V302, 1303, F304, T305, L331 , L346, S353, L355, V356, R357, G358, D359, R360, H361, F362, A363, S364, P365, N366, L367, N368, 1369, N370, S371 , W372, T373, R374, L375, P376, F377, G383, 1390, G391, P392, A393, 1394, 1395, L396, Y397, E398, G399, T400, V401, or Y402.

[00102] In some embodiments, the mutant HCT or HQT has improved ability to bind at least one substrate or to catalyze the formation of at least one product in either the forward or backward direction, relative to the corresponding wild-type HCT or HQT. In other embodiments, the mutant enzymes feature other properties that make them advantageous relative to the wild-type enzymes on which they are based. For example, the mutant may provide stability over a wider pH, or improved thermostability, or may preferentially catalyse formation of a desired product, or allow a reaction to proceed fully in one direction. In one embodiment, the mutant, preferably a mutant HCT, is useful for producing di- or triesters of CGS or DA species, or even tetraesters of these acids (e. g. quinic or shikimic acids). In yet other embodiments, the mutant is more readily produced as a soluble, active enzyme in a recombinant system, or is more amenable to immobilization on a matrix for use in in vitro synthesis systems. In yet other another embodiment, the mutant enzyme is more resistant to proteolysis than the wild-type enzyme, particularly in a recombinant expression system, such as a recombinant microorganism.

[00103] The wild-type HCT or HQT is preferably from a plant that is relatively abundant in chlorogenic acids or dactylifric acid species in nature, including mono-, di-, tri-, tetraesters, or other complex esters. Many monocot and dicots have been found to contain chlorogenic acid and dactylifric acid species and the HCTs and HQTs found in them are contemplated for use herein. Examples of such plants include the Rubiaceae (e.g. coffee, plums), Rosaceae (e.g. apple, pear, peach), Solanaceae (e.g. tobacco, tomato, potato, eggplant), and Asteraceae (e.g. artichoke, sunflower, chicory), as well as the model plant Arabidopsis. Among presently preferred plant HCTs are those from Coffea canephora, Coffea arabica, Solarium lycopersicum, Nicotiana tabacum, Cynara cardunculus, Trifolium pratense, and Arabidopsis thaliana. Coffea HCT sequences are particular preferred as wild-type HCT sequence from which to make mutant HCTs because coffee plants tend to among the highest concentrations of CGA species and related compounds, such as DAs, known. Nicotiana tabacum, and Coffea spp are among the preferred plants for wild-type HQT at present. [00104] The skilled artisan will appreciate that public databases such as those provided by NCBI and EMBL make amino acid sequences readily available, and are good sources for suitable sequences for HCT and HQT enzymes. Particular HCT amino acid sequences contemplated for use herewith include those with database Accession Numbers ABO47805 (C. canephora), CAJ40778 (C. arabica), ABO40491 (C. arabica), CAD47830 (N. tabacum), AAZ80046 (C. cardunculus), NP_199704 (A. thaliana), ACI28534 (T. pratense), and AB052899 (P. radiata). HQT sequences contemplated for use herein include CAE46933 (S. lycospersicum) , DQ20063 (S. tuberosum), CAE46932 (N. tabacum), AB077956 (C. canephora), ABK79690 (C. cardunculus), and ABK79689 (C. scolimus).

[00105] The skilled artisan will also appreciate that in some cases an amino acid sequence has not been identified, or fully sequenced, and in other cases only a gene sequence encoding a HCT or HQT is available. It is routine to use a deduced amino acid sequence, or to use a partial sequence to obtain a full-length clone for a sequence of interest. Thus obtaining the required information for producing a mutant HCT or HQT enzyme from a large variety of wild- type HCT or HQT sequences is either available or can be obtained using standard methodologies.

[00106] In some embodiments the mutant enzyme may have no particular substrate preference with respect to quinic acid or shikimic acid, for example the enzyme will bind both substrates with similar avidity or affinity and/or will catalyze reactions using either substrate at similar rates, and make similar amounts of product from each substrate. However, the mutant enzymes do show a substrate preference for quinic acid species or shikimic acid species relative to the wild-type enzyme in some embodiments. The enzyme may preferentially bind one substrate over the other, or may catalyze product formation more quickly or more completely with one substrate over the other under similar conditions. In one embodiment, the mutant HCT has a preference for shikimic acid species as a substrate relative to the wild-type HCT. In another embodiment, a mutant HQT prefers quinic over shikimic acid as an acyl acceptor. In other embodiments, the preference may be reversed.

[00107] The mutant enzymes can generally catalyze the formation of mono-, di-, tri, or tetraesters with similar proficiency as the wild-type in which it is based. In various embodiments the mutant HCT in particular provides an improved ability to catalyze to formation of di-, tri-, and higher esters relative to the wild-type enzyme. In one embodiment, the mutant HCT more proficiently produces diCQA than the wild-type HCT from which it is derived.

[00108] The mutant HCT comprises an alteration of the amino acid corresponding to H35, H153, H154, A155, A156, D157, K210, K217, Y252, Y255, or R374 of the reference HCT sequence (SEQ ID NO:23) in various embodiments. The mutant may comprise any one or more of the aforementioned alterations, and preferably each alteration is a conservative substitution. The specific mutations H35A, HI 53 A, H154A, A155L, A156S, D157A, Y252A, Y255A, R374E, K210A/K217A, or H154N/A155L/A156S, or combinations thereof may also be useful. The mutant HCT in one embodiment has an amino acid sequence that is any of SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, or 41.

[00109] In other embodiments, the mutant HQT comprises an alteration of the amino acid corresponding to H35, H153, H154, A155, A156, D157, K210, K217, Y252, Y255, or R374 of the reference HQT sequence (SEQ ID NO:44). The mutant may comprise any one or more of the aforementioned alterations, and preferably each such alteration is a conservative substitution. The specific mutations 41H35A, H153A, N154A, L155A, S156A, D157A, Y252A, R374E, K210A, or combinations thereof may also be useful. The mutant HQT in one embodiment has an amino acid sequence that is SEQ ID NO: 46.

[00110] In yet another aspect of the invention, coffee products containing synthesized chlorogenic acid species and methods for making such coffee products are provided herein. As used herein "a coffee product" may be any product based on any part of a coffee plant, e.g. based on coffee beans or coffee cherries, and intended for oral consumption by a human or animal. A coffee plant may be any Coffea species of, preferably C. arabica or C. canephora. Coffee cherry is the fruit of the coffee plant containing the seeds, called coffee beans. Coffee beans are typically obtained by removal of the fruit pulp from the coffee cherries, e.g. after fermenting the cherries in water or drying the cherries in the sun to facilitate the release of the pulp from the beans. Coffee beans may be used as so-called "green", or raw, coffee beans for production of coffee products, or they may be roasted. Roasting is a heating process that results in a darker colour and formation of the aroma compounds that gives roasted coffee its typical aroma. Coffee beans, green or roasted, may be ground and sold as such for brewing of coffee, e.g. in multi-serve packs for use in conventional home brewers, or in single-serve capsules for use in dedicated brewing machines. Coffee beans may be decaffeinated by methods known in the art if a decaffeinated coffee product is desired.

[00111] A coffee product may be based on an extract of coffee beans, either whole or ground. Coffee beans to be extracted may be green (raw) coffee beans or roasted coffee beans, or a mixture thereof. Extraction of coffee beans with water and/or steam is well known in the art, e.g. from EP 0916267. The most volatile aroma components may be stripped from the beans before extraction, e.g. if the extract is to be used for the production of pure soluble coffee. Methods for stripping of volatile aroma components are well known in the art, e.g. from EP 1078576. A coffee extract may be sold and used as such, e.g. in the form of a ready-to-drink (RTD) coffee beverage, or it may be concentrated, e.g. by evaporation or membrane filtration, and sold as a liquid concentrate useful as an ingredient in food or beverage products, or for direct use as a coffee beverage with or without dilution. An RTD coffee product may contain additional ingredients, such as e.g. a creamer, milk, milk protein, milk fat, vegetable fat, vegetable protein, sugar, artificial sweetener, stabilizer, and/or other ingredients conventionally used in coffee products. A coffee product may be a pure soluble, or instant, coffee product. A pure soluble coffee product is a dried coffee extract useful for preparing a coffee beverage by dissolution in water. A pure soluble coffee product may be produced by drying a coffee extract obtained as described above. Often the coffee extract will be concentrated, e.g. by evaporation, before drying, e.g. by spray drying or freeze drying. If volatile aroma has been stripped from the coffee beans before extraction, this aroma may be added back to the extract, e.g. before and/or after drying, to improve the aroma of the soluble coffee product. A pure soluble coffee product may be mixed with additional dry ingredients, such as e.g. a creamer, milk, milk protein, milk fat, vegetable fat, vegetable protein, sugar, artificial sweetener, stabilizer, and/or other ingredients conventionally used in coffee products, to produce an instant beverage product.

[00112] In various embodiments, the coffee product additionally comprises one or more exogenous or added chlorogenic acid and/or dactylifric acid species produced or synthesized in accordance with the methods provided herein. Preferably the chlorogenic acid species provides an improved flavour profile, or has been associated with improved flavours in coffee. Examples of such chlorogenic acid species include diCGA species such as 3 ,4 dicaffeoylquinic acid. Moreover, the exogenous CGA should contain low amounts of compounds that have been associated with undesirable flavours, for example bitternness or excess astringency, such as 5-feruloylquinic acid (5-FQA).

[00113] In one embodiment, a composition comprising a synthetic chlorogenic acid species is added to a coffee product before the completion of manufacture. Preferably, the chlorogenic acid species is at least partially purified, although in some cases a complex mixture comprising one or more chlorogenic acids synthetically produced, and one or more precursors, substrates, or other products may be present, provided that all such compounds, precursors, or products are food-grade and/or approved for use in foods. In one embodiment, the chlorogenic acid species is synthesized completely in vitro, for example in an enzymatic production system, such as an immobilised enzyme system comprising one or more of the enzymes disclosed herein.

[00114] In another embodiment, a recombinant organism that has been modified to contain the one or more of the enzymes disclosed herein is used to at least partially synthesize the chlorogenic acid species or a precursor thereto. A single chlorogenic acid species may be useful in some instances, for example where it contributes to a particularly advantageous flavour profile, or provides a desirable note or other attribute to the product. In other embodiments, a blend of two or more chlorogenic acid species may be more useful. In such cases, the chlorogenic acid and related species may be synthesized separately and subsequently blended, or they by co-synthesized in an in vitro system, or in a recombinant organism adapted for such synthesis by the inclusion of one or more of the enzymes disclosed herein.

[00115] Preferably, the chlorogenic acid species included with the coffee product are products that are not readily available from natural sources and which cannot be readily (or affordably) synthesized by other means in commercially useful quantities. In other embodiments, the chlorogenic acid species have been associated with one or more beneficial effects on human health. The chlorogenic acid may be monomeric chlorogenic acid species, or di-, tri- or more complex chlorogenic acid species. Examples of preferred chlorogenic acid and dactylifric species include caffeoylquinic acids (CQAs), coumaroylquinic acids, feruloylquinic acids (FQAs), caffeoylshikimic acids (CSAs), coumaroylshikimic acids, feruloylshikimic acids (FSAs), dicaffeoylquinic acids (diCQAs), or other di-, tri- or tetraesters, or combinations thereof.

[00116] In a related aspect, the invention also provides a food product comprising one or more synthetic chlorogenic acid species made in accordance with the methods disclosed herein. The chlorogenic acid species may be added to a product that benefits from the addition of one or more chlorogenic acid species. In one embodiment, the food product may be a coffee- flavoured product, even if the product contains no coffee per se. In another embodiment, the chlorogenic acid species may help to form the flavour profile for a simulated coffee flavouring or other artificial flavouring. In another embodiment, the chlorogenic acid species may be added as a functional food component, for example as an antioxidant, or as an aid to preventing, reversing, or minimizing health problems, such as those associated with cardiovascular disease or cancer.

[00117] Preferably the food product comprising exogenous one or more chlorogenic acid or dactylifric acid species. In one embodiment, the chlorogenic acid or dactylifric acid species are produced in vitro.

[00118] The food product is a coffee product as described above in certain embodiments. Presently preferred are soluble coffee product wherein the exogenous chlorogenic acid or dactylifric acid species provide a functional ingredient that improves quality or provides a health benefit, or directly or indirectly provide a desirable flavour attribute, aroma attribute, or colour attribute.

[00119] The exogenous chlorogenic acid or dactylifric acid species are added to the food product as a mixture to the food product during processing in one embodiment. In other embodiments, individual CGA or DA species are added to the food product. Exogenous chlorogenic acid or dactylifric acid species contribute antioxidant properties useful to the food and/or to the person consuming the food in some embodiments. Mono- and diester CGA or CSA species are useful herein, particularly where those compounds have been synthesized or produced in accordance with this disclosure. 3,4, caffeoylquinic acid can be added to a food product at levels that improve the flavour and aroma of the food product as determined by sensory evaluation. In most cases, to avoid low acceptability by a consumer, the CGA and or DA added are substantially free of 5-feruloylquinic acid, although in some foods and beverages additional bitterness or astringency may be desirable.

[00120] In another aspect of the invention compositions are provided comprising one or more recombinant enzymes (as described above), one or more mutant HCTs or mutant HQTs (as described above). The compositions are adapted for in vitro synthesis of one or more chlorogenic acid species or dactylifric acid species. The compositions may be provided as kits providing two or more separate packages such that the final composition is mixed by the end- user in order to use the compositions. [00121] The compositions comprise substrates and cofactors sufficient for the production of one or more chlorogenic acid species or dactylifric acid species. For example, the substrates may comprise coumaroyl-CoA, caffeoyl-CoA, cinnamoyl-CoA, feruloyl-CoA, sinapoyl-CoA, or combinations thereof, and an acyl acceptor. The cofactors comprise a catalytically useful divalent cation, preferably Mg ⁺⁺ , ATP, and optionally, CoA.

[00122] The acyl acceptor generally comprises quinic or shikimic acid species, or a combination thereof. Other acyl acceptors may be used as well.

[00123] In a presently preferred embodiment, one or more of the enzymes is immobilised on or covalently bound to a matrix. Such immobilised enzymes can conveniently be included with a kit for assembly of the final composition by the end user.

[00124] The chlorogenic acid species or dactylifric acid species that can be produced by the composition include caffeoylquinic acids (CQAs), coumaroylquinic acids, feruloylquinic acids (FQAs), caffeoylshikimic acida (CSAa), coumaroylshikimic acids, feruloylshikimic acids (FSAs), dicaffeoylquinic acids (diCQAs), or other di-, tri- or tetraesters, or combinations thereof. The di-, tri-, and higher esters may be mixed in having different HCAs attached to a single acyl acceptor.

[00125] The composition can also be adapted for in vitro production of a HCA-

CoA thioester catalysed by a 4CL enzyme, prior to in vitro production of the chlorogenic acid species or dactylifric acid species. In this manner, one of the substrates can be produced in the enzyme composition to provide further ease of use for making compounds that are otherwise difficult or impossible to synthesize. The substrates for production of the HCA-CoA thioester generally comprise one or more of cinnamic acid, coumaric acid, caffeic acid, or ferulic acid, and CoA

[00126] In yet another of its several aspects, the invention provides methods of producing a chlorogenic acid or dactylifric acid species in vitro.

[00127] The methods comprise the steps of: a) providing an HCT or HQT or both; b) providing substrates and cofactors sufficient to form a chlorogenic acid or dactylifric acid species in the presence of an HCT or HQT or both; and c) contacting the substrates and cofactors with the HCT or HQT, or both. The contacting step is done under conditions permitting enzymatic activity. The enzyme and substrate are maintained in contact under reaction conditions maintained for enough time to form CGA or DA species product(s). he product comprises a mono-, di-, tri- or tetraester species of chlorogenic or dactylifric acid. The product can be a mono-, di-, tri- or tetraester species of chlorogenic or dactylifric acid. Coffea HCTs are particularly useful herein.

[00128] Preferred substrates include at least one hydroxycinnamoyl-CoA thioester, such as coumaroyl-CoA, caffeoyl-CoA, cinnamoyl-CoA, feruloyl-CoA, or sinapoyl-CoA. Substrates also include an acyl acceptor, such as quinic acid, shikimic acid, or a combination thereof.

[00129] The chlorogenic acid or dactylifric species preferred as products include caffeoylquinic acids (CQAs), coumaroylquinic acids, feruloylquinic acids (FQAs), caffeoylshikimic acida (CSAa), coumaroylshikimic acids, feruloylshikimic acids (FSAs), dicaffeoylquinic acids (diCQAs), or other di-, tri- or tetraesters, or combinations, as discussed above.

[00130] In one embodiment, the method includes an additional step wherein a HCA-CoA thioester is produced using a 4CL enzyme.

[00131] The method can also include additional substrates such as CoA, and one or more chlorogenic acid or dactylifric acid species. As discussed above, HCT can remodel CGAs or DAs in the presence of HCA-CoA and/or CoA to form further CGAs and/or DAs.

[00132] As with other aspects of the invention involving enzymes, at least one of the HCT, HQT, or 4CL are preferably immobilised on, or covalently bound to, a matrix. The matrix can be any matrix used for enzyme immobilisation, such as a polymer with desirable properties, for example a complex polysaccharide or dextrin, a silica resin, a glass bead, and the like. The skilled artisan is familiar with such matrices and will appreciate how to employ them, and further understand such an immobilisation is only useful where the enzyme retains a significant portion of its activity.

[00133] In a further aspect of the invention, methods of producing a chlorogenic acid or dactylifric acid species in vitro are provided. The methods disclosed in this aspect of the invention comprise the steps of: a) providing an HCT, and optionally, an HQT or 4CL or both; b) providing one or more HCA thioesters, an acyl acceptor, CoA, and a first chlorogenic acid or dactylifric acid species; c) contacting the one or more HCA thioesters, acyl acceptor, CoA, and first chlorogenic acid or dactylifric acid species with the HCT under conditions permitting enzymatic activity, and for a time sufficient to permit the formation of product that is a second chlorogenic acid or dactylifric acid species. The method provides that the first chlorogenic acid or dactylifric acid species is catalytically converted into a second chlorogenic acid or dactylifric acid species by the action of the HCT, or the HCT in combination with an HQT and/or 4CL enzyme. The HCT enzyme is preferably a Coffea HCT, particularly a C. canephora HCT, and may comprise SEQ ID NO:23, for example. The HQT enzyme is preferably a Coffea HQT, particularly a C. canephora HQT, and may comprise SEQ ID NO:44, for example. The 4CL enzyme can be a 4CL from tobacco (Nicotiana tabacum), examples of which are known in the art. The 4CL enzyme can also be a Coffea ACL, particularly a C. canephora 4CL2, and may comprise SEQ ID NO: 47.

[00134] Another aspect of the invention features methods of modifying the lignin content and profile in plants. It is expected that reducing lignin in plant material will make this material more useful for many applications, including in biofuel production and plant feed because the biomass will be easier to solubilize and to degrade. By way of background, it has been shown previously that lignin levels can be reduced in plants by decreasing the expression of genes encoding proteins involved in making lignin precursors, particularly the phenylpropanoid pathway enzymes 4CL, HCT and HQT. However, improvements in forage digestibility or saccharification efficiency were offset by poor growth and reduced biomass production. Thus, studies to date have led to the conclusion that reducing lignin via a simple "knock down" of one or more genes of the phenylpropanoid pathway will not generate plants that have both reduced levels of lignin (with better utility for animal fodder and easier enzymatic degradation for biofuels etc) and normal biomass production.

[00135] However, the use of enzymes that favour the production of mono-, di-, and/or triesters of chlorogenic acid and the related shikimate containing compounds opens the opportunity to create plants that have smaller reductions of lignin but identical, or better overall biomass productivity, thereby improving the utility of the plants for important biomass related applications such as use for biofuels or animal fodder. In accordance with the present invention, this can be accomplished by expressing the coffee HCT or HQT enzymes, or the mutants of HCT in the cells of plants in order to shunt some of the phenylpropanoid precursors into mono, di, and tri CGA and related shikimate containing compounds. In addition to having slightly lower levels of lignin, and/or less complex lignin due to the interference of the stored CGA type compounds in lignin formation, it is expected that plant materials with higher CGA will have increased levels of relatively easily extractable "phenolic related matter" (i.e., the increased levels of soluble CGA). Additional benefits that may be associated with plants with increased CGA and related compounds include increased protection of these plants from oxidative stress-related damage, as well as protection from pathogens such as bacteria and insects.

[00136] The coffee HCT, HQT and mutants thereof described herein can be over- expressed in plants in accordance with methods known in the art, for instance as described in US Patent Pub. No. 20090158456 (describing the use of Coffea HCT and HQT genes in the engineering of plants). Transformation protocols may utilize constitutive promoters such as the 35S promoter, or other green tissue specific strong promoters, such as the coffee rbcs promoter described in U.S. Patent No. 7,153,953. Alternatively, tissue specific promoters can be used to increase the CGA production in specific parts of the plant (in tubers for example, or in seeds using seed specific promoters such as the oleosin promoter described in U.S. Patent Publication No. 20090229007. It is also advantageous, for example in crops for forage, to express higher levels of coffee HCT, HQT, or related mutants either late in leaf maturation, or only in the mature, early senescing leaves. One promoter for these late stages of leaf maturation is the coffee cysteine proteinase inhibitor 4 promoter.

[00137] As it is clear that the high levels of chlorogenic acids in the coffee grain are also driven in part by the high level of quinic acid found early during grain maturation, a further improvement in CGA accumulation and lignin amount/structure modifications in plants can be achieved by additionally engineering the plants with coffee HCT/HQT and related mutants, with genes that lead to elevated levels of quinic acid (or shikimic acid). Alternatively, plants to be first used for reduced lignin and increased CGA and related shikimate compounds would be species that have relatively high levels of quinic acid or shikimate in the tissues where the coffee HCT/HQT and coffee HCT mutants are designed to be expressed.

[00138] These and other aspects of the invention will be further understood by reference to the Examples that follow. EXAMPLES

[00139] The invention can be further illustrated by the following examples, although it will be understood that the examples are included merely for purposes of illustration and are not intended to limit the scope of the invention unless otherwise specifically indicated.

I. Cloning, Expression, and Purification

Introduction

[00140] A protein difficult to isolate in adequate quantities from its natural source can often be produced in sufficient quantities using recombinant expression in bacterial, yeast, insect or mammalian cells (Baneyx, 1999; Kost et ah, 1999; Malissard et ah, 1999; Hunt, 2005; Baldi et ah, 2007). Expression in E. coli is generally the easiest, quickest, and cheapest method currently available, however, many eukaryotic proteins do not fold properly in prokaryotic systems, instead forming insoluble aggregates or inclusion bodies. In some cases, the solubility of such proteins can be improved by: (a) decreasing expression temperature; (b) using a fusion partner/ polypeptide tag (Esposito et ah, 2006); or (c) refolding (Li et ah, 2004). The use of cleavable polypeptide/ protein tags permits simplified affinity purification procedures to obtain sufficient amounts of protein for subsequent characterisation.

Preliminary Cloning

[00141] Cornell University and Nestle S.A. released 47,000 expressed sequence tag (EST) sequences from Coffea canephora (Lin et ah, 2005). The EST databank (http://www.sgn.cornell.edu) was used for a BLAST search (Basic Alignment Search Tool, http://www.ncbi.nlm.nih.gov/blast/) against Nicotiana tabacum hct (AJ507825) and hqt (AJ582651) gene sequences (Hoffmann et ah, 2003; Hoffmann et ah, 2004). This search resulted in two hits: the unigenes CGN-U123197 and CGN-U125212, corresponding to the partial open reading frames (ORF) for putative Coffea canephora hydroxycinnamoyltransf erases. The full length cDNAs were isolated by 5 -RACE-PCR for rapid amplification of cDNA ends followed by the cloning of Cchct and Cchqt genes (Lepelley et ah, 2007). A multiple sequence alignment was generated with ClustalW (Larkin et ah, 2007) using acyltransferase nucleotide sequences from different plant species. Sequence comparisons confirmed that one of the cloned sequences lies on a branch containing hqt gene sequences, the other in a branch clustering hct gene sequences from different plant species (not shown). Cchct and Cchqt exhibit 66 % sequence identity at the nucleotide level. The translated sequences contain both the HX3D and DFGWG motifs, which are characteristic of BAHD acyltransferase amino acid sequences.

Production of recombinant 4CL2:

Coffea canephora 4-coumarate CoA ligase isoform 2

[00142] A plasmid provided by Nestle contained the Cc4cl2 gene (1626 bp) inserted into the inverted pET28a(+) vector with BamHI (5') and Hindlll (3') restriction sites. This plasmid allows for expression of Cc4CL2 with an N-terminal His ₆ -tag. BL21 (DE3) cells were transformed with the plasmid for standard expression tests at 37 and 20 °C. A band corresponding to the expected size of His ₆ -Cc4CL2 with a 24 amino acid linker (62.6 kDa) was detected in the cell extracts using SDS-PAGE and westem-blot analysis against the polyhistidine tag (Fig. 2).

[00143] Expression was scaled-up in flasks containing 1 L of Luria-Bertani (LB) medium supplemented with 30 μg/mL kanamycin, which were inoculated with a 1/100 (v/v) overnight starter culture. Cells were grown at 37 °C until the optical density (OD) at λ= 600 nm reached 0.6. After cooling to room temperature, over-expression was induced with 1 mM IPTG and cells were grown overnight at 20 °C. The cells were harvested by centrifugation at 5,000 x g and resuspended in a solution composed of 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 2.5 mM MgCl _2j 10 % glycerol and 5 mM β-mercaptoethanol (BME) (buffer A). The cells then were flash-frozen and stored at -80 °C until purification. EDTA-free protease inhibitors tablets, lysozyme and DNasel were added prior to cell lysis using a French press (2 cycles at 10 kPSI). The lysate was centrifuged for 30 min at 50,000 x g and 4 °C and the supernatant loaded on to a 5 mL HisTrap column (GE Healthcare) using an Akta Prime system. After washing the column with 10 CV of buffer A and 5 CV of 5 % buffer B (buffer A supplemented with 500 mM imidazole), an elution gradient of 5-100 % buffer B was applied. 1 mL fractions were collected and analysed on 12 % SDS-PAGE gel stained with Coomassie blue. A SDS-PAGE band corresponding to the size of His ₆ -Cc4CL2 was observed in the elution fractions collected from the affinity column (Fig. 2A). Over-expression of the recombinant protein was confirmed by Western-blot analysis against the hexahistidine tag. The fractions containing the protein were pooled and dialysed against 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2.5 mM MgCl ₂ and 1 mM DTT. A tag cleavage test was carried out with thrombin protease (Sigma) after dialysing the protein into a buffer composed of 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 % glycerol, 2.5 mM MgCl ₂ , 2.5 mM CaCl ₂ and 5 mM BME. Cleavage was successful at both room temperature and at 4 °C, but some precipitation occurred. The cleaved Cc4CL2 was recovered with subtractive affinity chromatography. Dynamic Light Scattering (DLS) analysis performed at 20 °C indicated that coffee 4CL2 with or without the His ₆ -tag was highly polydisperse. The over- expression procedure was repeated with Rosetta 2 (DE3) cells (Novagen) to overcome the codon bias for expression in E. coli. However, the resulting protein was still aggregated.

Nicotiana tobacco 4-coumarate CoA ligase isoform 2

[00144] Because Cc4CL2 required more exploratory work to obtain a fully soluble, monodisperse protein, a synthetic gene encoding Nicotiana tabacum 4CL2 (GenBank accession number: U50846) was purchased from Geneart (Regensburg, Germany). This protein had previously been used for the enzymatic synthesis of various hydroxycinnamoyl-CoA thioesters (Beuerle et ah, 2002). The codon-optimised gene (1653 bp) was subcloned into the pET21 d vector (Novagen) digested with BamHI (5') and Xhol (3') for expression with an uncleavable C-terminal His ₆ -tag and a two-amino acid (Lys-Glu) linker (60.5 kDa).

[00145] Several E. coli strains, namely BL21 (DE3), BL21 * (DE3), BL21 (DE3) pLysS and BL21 * (DE3) pLysS, were transformed with the pET21d_M c/2 plasmid for small- scale expression tests. The soluble extracts were analysed by SDS-PAGE (Fig. 3). The highest soluble expression level was obtained with BL21 * (DE3) cells, which were subsequently used for large scale production and purification of the recombinant N/4CL2-His6. Four (4) L of LB medium containing 100 mg/L ampicillin were inoculated with a 1/100 (v/v) overnight starter culture and grown at 37 °C until the OD at λ= 600 nm reached 0.6. The temperature was lowered to 20 °C before inducing the expression with 1 mM IPTG, and then the cells were incubated overnight. After centrifugation for 15 min at 5,000 x g, the supernatant was discarded and the bacterial pellet resuspended on ice in a lysis buffer composed of 50 mM bis-Tris pH 7.0, 10 % glycerol and 10 mM NaCl according to the previously described procedure (Beuerle et ah, 2002). In contrast with the authors however, a later comparison with a standard buffering agent (50 mM Tris-HCl pH 8.0) did not show any difference in the protein stability, so this standard buffer was subsequently used. [00146] Cell lysis was achieved using one cycle at 2.0 kPSI in the One Shot cell disrupter (Constant Systems). The lysate was centrifuged for 30 min at 50,000 x g and 4 °C. The supernatant was then loaded on a 2 mL nickel-nitriloacetic acid (NTA) gravity column (Qiagen). After washing with 10 CV of lysis buffer (see above) and 10 CV of 20 mM Tns-HCl pH 8.0 and 500 mM NaCl, the protein was eluted with 20 mM Tris-HCl pH 8.0, 500 mM NaCl and 500 mM imidazole. The fractions containing the target protein were pooled and dialysed overnight against 20 mM Tris-HCl pH 8.0 and 50 mM NaCl. The protein was further purified by anion-exchange chromatography (MonoQ GL 5/50, GE Healthcare) using a gradient from 50 mM to 1 M NaCl on a Akta Explorer system at 4 °C. The fractions containing the recombinant protein were pooled, concentrated and loaded on a HiLoad 16/60 Superdex 200 (GE Healthcare) for size- exclusion chromatography in 20 mM Tris-HCl pH 7.5 and 50 mM NaCl (Fig. 4A). The purest fractions from the size-exclusion chromatography were pooled and concentrated to 15 mg/mL in 10 mM Tris-HCl pH 7.5 and 50 mM NaCl (Fig. 4B). The protein was stable in a limited proteolysis experiment on ice with trypsin 1/1000 (w/w) for at least 1 h. Aliquots were flash- frozen and stored at -80 °C for biochemical assays. The remaining protein solution was incubated with 2 mM ATP, 2 mM MgCl ₂ , 1 mM coumaric acid and 1 mM CoA for subsequent crystallisation studies.

Production of recombinant HCT and HQT

HCT and HQT GST-fusion as a starting material

[00147] The cDNAs encoding Coffea canephora HCT and HQT (GenBank accession numbers: EF137954 and EF153931 respectively) were provided by Nestle R&D centre in Tours. They were inserted in the pGTPcl03a vector (GTP Technologies), which produces a recombinant protein in fusion with N-terminal glutathione S-transferase (GST). BL21 (DE3) cells were transformed with pGTPcl 03 a Cc zci and pGTPcl03a_Cc/z</i plasmids and plated on LB-agar medium containing 30 μg/mL kanamycin. Cultures of 100 mL LB medium were grown until the OD at λ= 600 nm reached 0.6 and expression was induced with 1 mM IPTG. Cells were grown for 3 h at 37 °C or overnight at 20 °C. No band corresponding to the size of the full-length GST-HCT and GST-HQT proteins (75.7 and 75.3 kDa respectively) could be detected on a 12 % SDS-PAGE gel stained with Coomassie blue, where the non-induced and induced cell fractions were loaded. [00148] Like most eukaryotic genes, coffee hct and hqt display an unfavourable codon distribution in E. coli that can limit expression yields (Kane, 1995). Consequently, expression in a strain that co-expresses the transfer RNAs for these rare codons was considered. Rosetta 2(DE3) cells (Novagen) were transformed with each plasmid and plated on LB-agar medium containing 30 μg/mL kanamycin and 34 μg/mL chloramphenicol. The full-length GST- HCT and GST-HQT proteins could not be detected on SDS-PAGE, while only a faint band was detected on western-blot together with N-terminal fragments of 25-30 kDa. An affinity chromatography purification step was carried out for GST-HQT over-expressed in Rosetta 2(DE3) at 37 °C for 3 h. The bacterial pellet was resuspended in phosphate buffered saline (PBS) buffer pH 7.4 containing 1 mM DTT. The cells were subjected to three cycles of freezing in liquid nitrogen and thawing in a 25 °C water-bath. The cell lysate was centrifuged for 30 min at 50,000 x g and 4 °C. The supernatant was loaded onto a 5 mL Glutathione Sepharose 4 Fast Flow column (GE Healthcare) equilibrated with PBS buffer pH 7.4. After washing with 5 CV of buffer, the protein was eluted in 50 mM Tris-HCl pH 8.0, 10 mM reduced glutathione and 1 mM DTT. The fractions were analysed by SDS-PAGE with Coomassie blue staining. The elution fractions contained several bands, among which was a band at 75 kDa, corresponding to the expected size of GST-HQT. Western-blot analysis confirmed the production of GST-HQT by detecting the 75 kDa polypeptide, as well as a band at 60 kDa and smeared bands around 25-30 kDa, probably corresponding to truncated N-terminal fragments including the GST fusion (Fig. 5).

HCT and HQT sub-cloning for expression with a hexahistidine-tag

[00149] To improve the expression levels of the full-length proteins, both Cchct and Cchqt genes were sub-cloned into the pPROEX HTb vector (Life Technologies) for expression with a cleavable hexahistidine (His ₆ )-tag at the N-terminus. The target nucleotides were amplified by polymerase chain reaction (PCR) using specifically designed oligonucleotide primers flanked with the appropriate cloning sites (Table 4). The reaction mixtures were made in a final volume of 50 μΕ as follows: 1 μΕ of plasmid DNA template, 5 μΕ of PCR buffer (lOx), 1 μΕ of forward and reverse primers (10 pmol/μΕ), 200 μΕ dNTP mix (10 mM) and 0.3 μΕ (2.5 u) of High Fidelity PCR enzyme mix (Fermentas) or Pfii DNA polymerase (Stratagene). A standard PCR program was run with an annealing temperature of 55 °C and an elongation time of 4 min. The PCR products were analysed using 1 % agarose gel electrophoresis stained with SYBR Safe (Fig. 6). The PCR products, obtained with Pfii DNA polymerase, were double digested with the appropriate restriction enzymes and purified by gel extraction (Qiaquick kit, Qiagen). The DNA fragments were ligated into the linearised and dephosphorylated pPROEX HTb vector. TOP10 cells (Invitrogen) were transformed for plasmid DNA preparation and the insert sequence was verified by DNA sequencing (Genecore, Germany).

Table 4: Oligonucleotide primers used to amplify Cchct and Cchqt

Amplicon 'chct

Template GTPcl03a_Cc/?cf

Primer 5 ' agcactagggatccatgaaaatcgaggtgaaggaatcg (BamHI) (SEQ ID NO: l)

Primer 3 ' cgtcgatcactcgagtcaaatgtcatacaagaaactctggaa (Xhol) (SEQ ID NO:2)

Amplicon 'chqt

Template GTPcl03a_Cc f

Primer 5 ' agcactagggatccatgaagataaccgtgaaggaaaca (BamHI) (SEQ ID NO:3)

Primer 3 ' cgtcgatcatctagatcagaaatcgtacaggaacctttg (Xbal) (SEQ ID NO:4)

Expression and buffer tests for hexahistidine-tagged HCT

[00150] Several E. coli strains were transformed with the new pPROEX HTb zci construct and small-scale expression tests performed at 37 °C and 20 °C. The bacterial pellets were resuspended in BugBuster protein extraction reagent (Novagen) for chemical lysis. The soluble cell extracts were loaded onto single-use His-Trap Spin columns (GE Healthcare) containing 100 μΕ Ni-NTA agarose beads for affinity purification. The fractions were eluted with buffer containing 500 mM imidazole and analysed by SDS-PAGE and western-blot against the His ₆ -tag. HCT expression levels were highest in BL21 * (DE3) pLysS cells grown overnight at 20 °C. Two truncated N-terminal fragments (-25-30 kDa) remained in the insoluble fraction of the cell lysate (Fig. 7).

[00151] To optimise the purification conditions for HCT, 100 mL LB cultures of

BL21 * (DE3) pLysS were prepared. Expression was induced when the OD reached 0.6/0.9 with 0.5/1 mM IPTG and cells incubated overnight at 20 °C. The pellets were resuspended in different lysis buffers (50 mM Tns-HCl pH 7.4, 300/500 mM NaCl, 20 mM imidazole, 2 mM BME, 0/10 % glycerol, 0/1 % Tween 20) supplemented with lysozyme and DNasel. The cells were lysed with 4 cycles of freeze and thaw using liquid nitrogen and a water-bath at 25 °C. A similar purification step using His-Trap Spin columns was then performed. No major difference was detected for induction at 0.5 or 1 mM IPTG and at OD at λ= 600 nm of 0.6 or 0.9. The best buffer composition was 50 mM Tris-HCl pH 7.4, 500 mM NaCl, 20 mM imidazole, 2 mM BME and 10 % glycerol.

HCT codon-optimised genes for expression in E. coli

[00152] A synthetic cDNA with improved codon usage was later obtained from

Geneart (Regensburg, Germany) to allow a high and stable expression rate of the heterologous protein. The optimised genes for Cchct and Cchqt were cloned in the pPROEX HTb expression vector. Similar protein expression levels were obtained with the wild-type and the codon- optimised hct genes. The synthetic gene was used in all the following experiments.

Large scale expression and purification of HCT

[00153] The synthetic pPROEX HTb zci construct was used to over-express

CcHCT with an N-terminal His ₆ -tag in BL21 * (DE3) pLysS strain of E. coli. A single isolated bacterial colony grown on a freshly streaked plate of LB-agar medium containing 100 μg/mL ampicillin and 34 μg/mL chloramphenicol was used to inoculate a starter culture for overnight growth at 37 °C. Cultures of 1 L LB medium were then inoculated with a 1/100 (v/v) starter culture and incubated at 37 °C until the OD at λ= 600 nm reached 0.6. The flasks were cooled in ice-water and 1 mM IPTG was added for overnight expression at 20 °C. The cultures were centrifuged for 20 min at 5,000 x g and 4 °C and the bacterial pellet produced from a 1 L culture was resuspended in 25 mL buffer composed of 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 10 % glycerol, 20 mM imidazole and 5 mM BME (buffer A). The cell stock was flash-frozen and stored at -80 °C.

[00154] All subsequent operations were carried out at 4 °C. Prior to cell lysis, lysozyme (-200 μg/mL), DNasel (-20 μg/mL) and EDTA-free Complete Protease Inhibitor Cocktail Tablets (Roche) were added to the thawing cells and stirred for 15 min. The resuspended cells were ruptured by passage through a French pressure cell (2 cycles at 10,000 PSI). The lysate was then centrifuged at 50,000 x g for 30 min to remove the cell debris. A 5 mL His-Trap HP column (GE Healthcare) was equilibrated with 10 % (v/v) buffer B (buffer A supplemented with 500 mM imidazole). The soluble fraction of the cell lysate was loaded and a gradient from 10 to 100 % (v/v) buffer B imidazole was applied. The His ₆ -tagged protein eluted with 250 mM imidazole. The fractions containing the recombinant protein were pooled, incubated with 1/100 (w/w) Tobacco Etch Virus (TEV) protease, and dialysed overnight in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 % glycerol, 1 mM DTT and 0.5 mM EDTA. The TEV cleavage leaves only three residues (Gly-Ala-Met) attached to the target protein. Cleaved HCT was recovered by subtractive chromatography using the same nickel column. The protein sample was concentrated to 500 μΕ and injected on a Superdex 200 10/300 GL size-exclusion column (GE Healthcare) with 20 mM Tris-HCl pH 7.5, 150 mM NaCl and 5 mM BME. HCT eluted at 14.5 mL, which corresponds to the expected size for a monomer of 50 kDa, according to the column calibration curve. The peak fractions were collected and analysed on a 12 % SDS-PAGE with Coomassie blue staining (Fig. 8). Only the purest fractions were pooled, concentrated by centrifugation and flash-frozen at -80 °C. A mean yield of 4.5 mg/L culture was obtained.

Degradation and limited proteolysis of HCT

[00155] A comparison of protein samples produced from batch to batch and stored at different temperatures (-80, -20 and 4 °C) was carried out. It was observed that the purified protein sample usually contained two additional bands around 25-30 kDa that were amplified with time (Fig. 9A). This indicates that the native enzyme is partially degraded into two major fragments by an endogenous endopeptidase activity, probably originating from the crude E. coli extract. Size-exclusion chromatography of the degraded sample showed only one symmetric peak of elution corresponding to a full-length protein size (data not shown). This indicates that the fragments remained physically associated throughout the purification procedure. N-terminal sequencing of CcHCT fragments after storage resulted with a limited confidence level in the sequence DSVPE, which maps on Ser218.

[00156] The degradation pattern was reproduced by incubating freshly purified

His ₆ -CcHCT with several proteases. Reaction samples were taken at several time points and supplemented with 1 mM phenylmethanesulfonyl fluoride (PMSF) to stop the reaction. 12 % SDS-PAGE and western-blot (anti-Hiss) analysis revealed two bands of approximately 25 kDa appearing after a few minutes incubation with trypsin, thermolysin or subtilisin. The band of the full-length protein was barely visible after 10 min incubation (Fig. 9B). The protein was intact in the reactant mixture where no protease was added. The fragment bands were extracted from the polyacrylamide gel and subjected to N-terminal sequencing. This resulted in the identification of the sequence SDSVPETA, which maps after Lys210 that is predicted to be part of the inter- domain loop region.

Chemical modification of CcHCT by reductive methylation

[00157] The reductive methylation of solvent-accessible lysine residues is a simple and inexpensive method to aid in the crystallisation of some proteins. The protocol used was adapted from (Walter et ah, 2006) and applied to CcHCT, which contains 20 potential methylation sites (19 Lys and the N-terminal amino group). The reaction was performed at 4 °C with 30 mg of pure protein at 0.65 mg/mL in 50 mM sodium HEPES pH 7.5, 250 mM NaCl and 5 mM BME. 20 μΕ of freshly prepared 1 M dimethylamine-borane complex (ABC) (Fluka) and 40 μΕ of 1 M formaldehyde per mg of protein were added to the protein sample and incubated for 1 h. The procedure was repeated once before leaving overnight under gentle agitation. The precipitated material formed during the methylation reaction was removed prior to concentration of the remaining protein sample (76 %). The buffer was exchanged to 20 mM Tris-HCl pH 7.5, 200 mM NaCl and 5 mM BME for size-exclusion chromatography using a Superdex 200.

[00158] Limited proteolysis was then carried out on the native and methylated

HCTs. The proteins were incubated at 30 °C with trypsin at a 1/100 (w/w) ratio in a buffer composed of 20 mM Tris-HCl pH 7.5. The reaction was monitored by taking aliquots from t= 0 to 120 min and stopped by the addition of 1 mM PMSF prior to loading the samples on a 12 % SDS-PAGE gel. As expected, the results showed that, contrary to native CcHCT, the methylated derivative was resistant to trypsin proteolysis (Fig. 9C). From these observations and the N- terminal sequencing results (above), it was postulated that the solvent-accessible Lys residues part of the predicted inter-domain loop could have an influence on the stability of CcHCT. Once cleaved, the loose parts may hinder the crystal formation. The two charged amino acids, Lys210 and Lys217, which are characterised by a high conformational energy, were consequently mutated to Ala to engineer a trypsin-resistant protein.

K210A/K217A (K) mutant CcHCT [00159] K-mutant CcHCT was produced using expression and purification procedures similar to that for the native protein (above). Limited proteolysis with trypsin and chymotrypsin had no or little effect on the K-mutant HCT, while subtilisin could still cut the protein into two stable fragments (Fig. 10).

Hise-HQT expression

[00160] A similar expression protocol was applied to pPROEX_HTb_Cc z<//. No band corresponding to the correct size of the His ₆ -CcHQT could be detected in the elution fractions loaded on polyacrylamide gel. However, multiple bands at lower molecular weight (25- 30 kDa) were observed in the insoluble and soluble fractions, similar to the expression pattern for GST-HQT (Fig. 5). These bands probably correspond to N-terminal truncated fragments of CcHQT. No further work was carried out with this expression vector as no satisfactory expression conditions were found. A synthetic Cchqt gene (Geneart) was also tested, but did not result in any improvement in the expression level of the full-length CcHQT.

Cchqt subcloning in pET-28M-SUM03

[00161] As CcHQT was difficult to over-express with previous systems, the synthetic Cchqt gene was sub-cloned into pET-28M-SUM03 expression vector (EMBL Heidelberg) using the BamHI (5') and Xhol (3') restriction sites. The new construct was designed to enhance the expression level and solubility of CcHQT with a N-terminal His6-SUM03 (small ubiquitin-related modifier) fusion tag (-100 amino acid residues, 11.5 kDa) that has been shown to provide a chaperoning effect (Malakhov et al, 2004). The fusion tag is cleaved off with 1/100 (w/w) SenP2 protease incubated overnight at 4 °C using the elution fraction from the first affinity chromatography step. The robust and specific SenP2 protease is known to recognise the 3 -dimensional structure of human SUM03 rather than a short, but specific, amino acid sequence (Reverter et al., 2006). This procedure introduces only one residue (Ser) at the N-terminus of the expressed protein.

Expression and purification of His ₆ -SUM03-CcHQT

[00162] Following the recommended procedure of the Protein Expression and

Purification core facility of EMBL-Heidelberg, BL21 (DE3) cells were transformed with pET- 28M-SUM03 -Cchqt and grown at 37 °C in LB medium containing 30 μg/mL kanamycin and 34 μg/mL chloramphenicol to an OD at λ= 600 nm of 0.5. Expression was induced with 0.2 mM IPTG and cells grown overnight at 18 °C. The cells were harvested by centrifugation and flash- frozen in liquid nitrogen. The bacterial pellets were thawed and resuspended to a final volume of 25 mL/L culture in a lysis buffer composed of 50 mM Tris-HCl pH 8.0, 250 mM NaCl, 20 mM imidazole, Complete Protease Inhibitor, DNasel and lysozyme. The cells were maintained on ice and ruptured by sonication (Vibra Cell, Bioblock Scientific) with 6 x 25 sec pulses at 60 % intensity and 30 sec breaks. The cell lysate was cleared by centrifugation and the supernatant loaded on to a 5 mL Ni-NTA column (Qiagen). After washing to eliminate unbound contaminants, His6-SUM03-HQT was eluted using a gradient of 20 to 300 mM imidazole in the buffer. A protein of 70 kDa that co-elutes with CcHQT from the affinity column may correspond from size comparison to E. coli chaperone DnaK (Fig. 11 A). The purest fractions were dialysed against 50 mM Tris-HCl pH 8.0 and 250 mM NaCl and digested with 1/100 (w/w) SenP2 overnight at 4 °C. The following day, the digestion mixture was centrifuged and loaded again on the Ni-NTA column. The cleaved CcHQT was found in the flow through and remained stable in solution, while the His ₆ -SUM03 fusion was retained on the column. The sample buffer was exchanged for 50 mM Tris-HCl pH 8.0 and 100 mM NaCl, filtered and loaded on a 5 mL HiTrap Q anion-exchange column using an Akta purifier system. CcHQT was eluted in the flow through (Fig. 1 IB), while the contaminants remained bound to the MonoQ column. Finally, HQT was analysed by size-exclusion chromatography and eluted as a monomer. The mean yield was 1 mg/L culture of pure CcHQT. The protein was also sensitive to proteolysis and showed a degradation profile similar to that of CcHCT (Fig. 1 1 C). N-terminal sequencing of truncated fragments from HQT degradation gave two sequences: TGPRA and GPRAS, consistent with a cleavage at Lys219 and Thr220. A similar mutant as for HCT, K210A/K219A HQT, was produced and purified using a similar protocol to the native HQT.

HCT and HQT site-directed mutagenesis

[00163] The ability to change specific residues or regions of proteins by site- directed mutagenesis allows a better understanding of structure-function relationships in proteins. Prior to structure determination of the native HCT, several residues potentially involved in catalysis and/ or substrate binding were selected from sequence and structure comparisons with VS, MaT and TRII OI . HCT mutants were designed and generated by site-directed mutagenesis on the Cchct gene template to produce H35A, HI 53 A (HX ₃ D), H154A (HXX ₂ D), D157A (HX ₃ D), Y252A, Y255A, R374E and H154N/A155L/A156S (HX ₃ D) mutant HCTs.

Table 5: Sequences of the oligonucleotide primers used for the production of mutant HCTs

[00164] The QuikChange site-directed mutagenesis kit (Stratagene) was used to generate single site mutations in the codon-optimised hct. The double mutants were produced by using the template containing the first mutation and primers corresponding to the second mutation to be introduced. The gene encoding the triple H154N/A155L/A156S mutant HCT was constructed in a single step as the target bases were adjacent. According to the manufacturer's specifications, the reaction mixture was composed of 0.25 iL DNA template, 2.5 fL lOx DNA polymerase buffer, 0.5 μΙ_, dNTP, 1 μΙ_, forward (F) and 1 iL reverse (R) mutagenic primers at 15 pmol^L (Table 6), 0.5 μΙ_, Pfu Turbo polymerase and completed to 25 fL with milliQ water. After denaturing at 95 °C for 1 min, amplification consisted of 18 cycles of 1 min at 95 °C, 1 min at 60 °C for annealing and 8 min at 68 °C for elongation (1 min/kb of plasmid length). An additional final step of elongation was carried out at 68 °C for 8 min. The PCR products were digested with 0.5 μΙ_, Dpnl for 1 h at 37 °C. XL-1 Blue super competent cells (Stratagene) were transformed with 5 μΙ_, of the digest. The cells were plated on LB-agar containing the appropriate antibiotic to select colonies containing the mutant plasmid. A few clones were selected on each plate for plasmid DNA preparation and sequencing (Genecore, EMBL-Heidelberg) to identify one that contained the desired mutation. Each mutant was then over-expressed and purified as previously described for the wild-type enzymes. All mutant proteins were successfully produced and purified to homogeneity.

Expression Results

[00165] The production and purification of sufficient quantities of CcHCT,

CcHQT, Cc4CL2 and N/4CL2 allowed for a detailed characterisation of these acyltransferases and CoA ligases. Some results of biochemical assays performed on CcHCT, CcHQT, N/4CL2 and some of the CcHCT mutants are presented here. Crystallisation trials resulted in crystals of the native and K-mutant HCTs and native N/4CL2. These crystal structures were then solved by MR. N/4CL2 was crystallised in two forms, an apo form and a ternary complex with CoA and AMP bound. Docking of HCAs was carried out in the predicted SBP of the N/4CL2-CoA-AMP ternary complex crystal structure to identify the potential residues involved in catalysis and/ or substrate binding. The overall structure of CcHCT was determined and the native and double mutant structures compared. This was followed by a comparison with the crystal structures of other BAHD acyltransferases. As crystals of CcHCT in complex with its biological substrates could not be obtained, the results of ligand docking experiments using AutoDock Vina are described. A homology-model based on the crystal structure of native CcHCT was constructed and used to compare CcHCT and CcHQT active sites.

Protein over-expression, purification and characterisation

Coffee and tobacco 4-coumarate CoA ligases

[00166] During these investigations of CGA biosynthesis in coffee, the need to generate hydroxycinnamoyl-CoA thioesters was encountered. In a number of biological systems, CoA activation can facilitate the transfer of acylated moieties by acyltransferases. Due to their limited stability, hydroxycinnamoyl-CoA thioesters are not commercially available. Initially, caffeoyl-CoA was synthesised from 5-CQA and CoA using CcHQT in the reverse reaction. But due to the reversibility of the acyltransfer reaction, only a small amount of CoA thioester was produced. As this procedure was material- and time-consuming, using a 4-coumarate CoA ligase (EC 6.2.1.12) that efficiently catalysed the formation of hydroxy cinnamoyl-CoA thioesters was considered. The complete cDNA encoding Cc4CL2 was obtained from Nestle R&D Tours and the recombinant protein produced and purified. It was most likely incorrectly folded as aggregation occurred, and it was inactive towards the biological substrate, coumaric acid. A synthetic gene encoding M4CL2 was then ordered (Beuerle et ah, 2002). This protein was purified using several chromatographic steps with typical yields of 10 mg/L culture and used for further biochemical and structural studies.

Coffee HCT and HQT hydroxycinnamoyltransferases

[00167] Over-expression of these target proteins required the sub-cloning from the pGTP vector into pPROEX HTb for hct and pET-28M-SUM03 for hqt, as well as the screening of a number of different a, coli host strains. High yields of N-terminal His ₆ -tagged CcHCT were obtained with BL21 * (DE3) pLysS induced at 20 °C (4.5 mg L culture). Coffea canephora cDNAs contain a high number of rare E. coli codons. However, a comparison between the wild- type Cchct gene and a codon-optimised gene indicated that the protein expression levels were comparable. The synthetic gene was used in all subsequent protein production, for which an optimised protocol is described above. CcHQT was expressed in BL21 (DE3) cells in fusion with a His6-SUMO partner, but lower yields were obtained (~1 mg/L culture). HCT and HQT were purified by immobilised metal affinity chromatography (IMAC). The fusion partner or His6-tag were subsequently cleaved using specific proteases. CcHCT (residues 1-434) plus a five amino acid N-terminal extension (GAMGS) and HQT (1-430) plus a serine introduced at its N- terminus were recovered by subtractive chromatography. Ion-exchange and size-exclusion chromatography were carried out to remove all remaining contaminants. Both CcHCT and CcHQT exist as monomers of 48 kDa in solution and can be concentrated up to 50 mg/mL.

[00168] The native enzymes were partially degraded into two proteolytic fragments of -25 kDa that appeared during storage at 4 °C and amounts of which increased with time. This may be due to protease contamination from the E. coli extract. Limited proteolysis experiments also clearly showed two similar-sized fragments on SDS-PAGE although the fragments remain physically associated throughout size-exclusion chromatography. Mass spectrometry and N-terminal sequencing analysis showed that the protease cleavage site was located in the predicted cross-over loop region near two lysine residues. To increase the stability of the recombinant CcHCT and CcHQT, trypsin-proteolysis resistant mutants, K210A/K217A CcHCT and K210A/K219A CcHQT, were produced.

II. Biochemical Characterisation

Introduction

[00169] Nicotiana tabacum 4-coumarate CoA ligase (M4CL2) and Coffea canephora hydroxycinnamoyl-CoA shikimate/ quinate hydroxycinnamoyltransferases (CcHCT/ CcHQT) were over-expressed in E. coli and purified to homogeneity using several chromatographic steps. The following section presents the experimental conditions used to investigate the in vitro activity of these enzymes, which are essential components of the biochemical pathway leading to CGAs and other phenolic compounds in plants. N/4CL2 is involved in the synthesis of hydroxycinnamoyl-CoA thioesters, while CcHCT and CcHQT catalyse the transfer of hydroxy cinnamoyl moieties between CoA and quinic and shikimic acids. Mutants, notably targeting the conserved HX3D motif (see below), were also assayed. The acyltransfer leading to CGA biosynthesis is designated as the "forward" reaction, while the "reverse" reaction involves the conversion of a CGA molecule to the corresponding hydroxycinnamoyl-CoA thioester (Fig. 16). The reverse reactions using 5-CQA 5-FQA and CoA as substrates were studied first because the CoA thioesters were not commercially available. To study the forward reaction catalysed by CcHCT and CcHQT, these substrates were synthesised enzymatically using N/4CL2 and purified based on a previously published procedure (Beuerle et al, 2002).

Chromatographic Analysis

[00170] To study an enzyme-catalysed reaction, one generally needs to measure substrate depletion and/or product formation. Spectrophotometric methods are useful where either a substrate or a product in the reaction absorb light in a spectral region where other substrates or products do not. In the reactions under study, CoA thioesters and quinate/ shikimate esters have distinct maximum absorbance values, but present overlapping absorption spectra (Fig. 12). Liquid chromatography is more informative about the chemical entities present in the reaction mixtures under study. It is also more accurate as the peak area of the absorbing species is directly proportional to its concentration. Therefore, high-performance liquid chromatography (HPLC) was preferred for the studies carried out here. Quinic acid is not detectable in the range of 210 to 400 nm wavelength, while shikimic acid can be measured at λ= 210 nm due to the double bond within the cyclohexene ring. High-performance anion-exchange chromatography coupled to pulsed electrochemical detection (HPAE-PED) is another useful technique for the analysis of these acids (Rogers et ah, 1999).

[00171] In this study, the analytes were resolved according to their relative hydrophobicity by HPLC on a Ci ₈ -bonded reverse-phase column (octadecyl hydrocarbon chains anchored to non-polar silica gel particles). A Novapak column (4 μηι, 4.6x 250 mm, Waters) or a Nucleosil column (5 μιη, 4x 250 mm, Macherey-Nagel) were used in various experiments. Before injection, samples were filtered using a 1 ml syringe and a 13 mm Acrodisc filter with a 0.2 μιη membrane (Pall Corporation). A pre-column was used to eliminate compounds that irreversibly bind the C ₁₈ column and to filter any precipitation that developed between the time samples were loaded for analysis and the actual time of injection by the automatic sampler. Samples were injected into the mobile phase (injection volume of 10 μΕ, unless otherwise specified) with a flow rate of 0.8 mL/min. The mobile phase consisted of two solvents (A and B) whose relative percentages varied according to the programmed elution gradient. The percentage of organic solvent in the mobile phase was incrementally increased to elute the compounds off the column, the more hydrophobic component eluting last (Table 6). The mobile phase was acidified with 0. 1 % phosphoric acid (0. 1 % formic acid when coupling with a mass spectrometer) to better separate the ionic and ionisable compounds. The HPLC system used comprised an autosampler (Waters 717), high precision pumps (Waters 600E System Controller), an oven where the column was kept at 30 °C and a Waters photodiode array (PDA) UV detector, scanning wavelengths in the range 210 to 400 nm wavelength. In some experiments, a simpler system equipped with a dual absorbance (DA) detector, but without a column incubator, was used to record the absorbance at two particular wavelengths. Data were processed with the Waters Empower software. Peak assignments were made by comparison with available standards using the peak retention time (RT) and with UV absorbance spectra recorded with the PDA detector. [00172] The elution gradient used in the initial studies employed acetonitrile. Due to a global acetonitrile shortage in the first half of 2009 (Lowe, 2009), an alternative methanol- based method was designed. Because acetonitrile and methanol differ in their relative eluotropic strength, the elution gradient was modified so as to maintain a good separation of coffee CGA isomers (Table 6).

Table 6: Mobile phase gradients used for HPLC analysis of coffee CGAs

[00173] 5-CQA and rosmarinic acid were available as commercial standards. 5-

FQA was provided in limited amounts by Nestle. A kit of mono- and diCQAs purified from plant extracts was from Biopurify (Chengdu, China). All other chemicals were obtained in a lyophilised form and stored as specified in accordance with recommendations. MilliQ-purified water was used for all reagent dilutions and buffer preparation. Stock solutions were freshly prepared and stored on ice for a maximum of one day, or frozen at -80 °C prior to use. CoA and 5-CQA stock solutions were prepared at 100 mM and 20 mM, respectively. The HPLC-grade solvents (methanol, acetonitrile), as well as the additives (phosphoric and formic acids), were filtered before use. The reaction buffer was prepared using monobasic (0.5 M NaI¾PC)4 at pH 4.0) and dibasic (0.5 M Na ₂ HP0 ₄ at pH 9.0) solutions mixed to obtain the appropriate pH value and stored at room temperature.

Table 7: Information on the substrate compounds used in the biochemical assays Compound Formula MW (g/mol) Storage (°C) Min. purity (%) Brand

99.1 Biopurify

5-CQA CieHigOg 354.31 20

98 Fluka

3-CQA CieHigOg 354.31 4 99.4 Biopurify

4-CQA CieHigOg 354.31 4 99.6 Biopurify

5-FQA C17H20O9 368 4 98 Nestle

3,5-diCQA C25H24O12 516.46 4 99.3 Biopurify

3,4-diCQA C25H24O12 516.46 4 99.4 Biopurify

4,5-diCQA C25H24O12 516.46 4 99.1 Biopurify caffeic acid CgHgC 180.16 20 98 Sigma ferulic acid C ₁₀ H ₁₀ O ₄ 194.19 20 99 Sigma f-cinnamic acid 148.16 20 99 Sigma coumaric acid CgHg0 ₃ 164.16 20 98 Sigma sinapic acid C ₁₁ H ₁₂ O ₅ 224.21 20 98 Sigma

85 Sigma coenzyme A C ₂₁ H ₃₆ N ₇ 0 ₁₆ P ₃ S 767 -20

96 BioChemika quinic acid C7H12O6 192 20 98 Fluka shikimic acid C ₇ H ₁₀ O ₅ 174 20 99 Sigma rosmarinic acid CigHieOg 360.3 20 97 Aldrich spermidine C ₇ H ₁₉ N ₃ 145.25 20 99.5 Sigma myo-inositol C ₆ Hi ₂ 06 180.2 20 99.5 Sigma pHPL C ₉ H ₁₀ O ₄ 182.2 20 98 Aldrich

[00174] The retention time (RT) and absorbance spectra was determined for each of the HCAs that serve as substrates for 4CL2 (Table 8). Caffeic, coumaric, ferulic and sinapic acids were injected individually and analysed by HPLC with either methanol or acetonitrile as the organic solvent.

Table 8.-Typical retention times and absorbance maxima of the HCA standards

[00175] CoA eluted close to the void volume and was best measured at λ= 256 nm

(Table 9). As can be seen in Table 9, the CGA elution sequence, which was driven by an increase in hydrophobicity, did not change in the chromatographic systems used (3-CQA< 4- CQA< 5-CQA< 5-FQA< 3,4-diCQA< 3,5-diCQA< 4,5-diCQA). The UV spectrum of each CQA isomer was very similar. RTs for each compound could vary depending on the mobile phase (acetonitrile/ methanol), HPLC device (photodiode array or dual absorbance detector), or column (Nucleosil/ Novapak) used (Table 9). Pure CGAs were systematically injected as standards either individually or in a mixture before each reaction series. As an alternative to pure commercial standards, methanolic extracts of green coffee beans proved to be a convenient source of the major coffee CGAs. Typically, the beans were frozen in liquid nitrogen then ground. Phenolic compounds were extracted with 70 % methanol for 1 h at 40 °C in shaking flasks. The methanolic extract was filtered (0.2 μπι) and a 10-fold dilution was analysed by HPLC-PDA (data not shown).

Table 9: Relative retention times and absorbance maxima of the CGA standards in the HPLC system

[00176] A calibration of peak area against concentration was obtained for 5-CQA at λ= 325 nm. Linearity was respected in the range 0 to 10 mM (data not shown). To quantify CGAs in a coffee methanolic extract, a reference solution of 5-CQA at a known concentration was analysed as an external standard before the sample of interest was tested. The concentration of CGAs in the sample was determined from the corresponding peak area at λ= 325 nm and the values for the 5-CQA standard (Equation 1). The relative response factor (RRF) was taken into account as a correction for molecular weight differences with the 5-CQA standard (Lepelley et Equation 1: Quantification of CGA compounds with 5-CQA as an external standard.

where A: absorbance; C: concentration; RRF: relative response factor: RRF= 1 for mono-CQAs, 0.96 for 5-FQA and 1.36 for diCQAs.

[00177] The stability of CGA compounds was verified by heating 10 mM 5-CQA/

5-FQA in 0.1 M sodium phosphate pH 6.0/ 7.0 at 90 °C. Samples taken at t= 0, 30 and 60 min were analysed by HPLC-DA. Similar experiments were performed with 1 mM 3-/ 4-/ 5-CQA, 3,4-/ 3,5-/ 4,5-diCQA in 0.1 M sodium phosphate pH 6.5 incubated overnight (-15 h) at 34/ 25 °C. Samples (1369-1386) were analysed using HPLC -PDA with acetonitrile.

[00178] Stock solutions of substrates and buffer were mixed with MilliQ water to reach the desired concentration and final volume. Reactions were started by adding the enzyme (or water for the control reaction) and solutions incubated in a 34 °C water-bath during sampling, unless otherwise stated. Typically, a 40 μΐ. aliquot was taken and diluted with 210 μΐ. HPLC solvent A. After filtration, the automated system injected a 10 μL sample into the HPLC system. The following sections describe the enzymatic reaction details. The figure legends in the Results section of this thesis also contain reaction details and sample numbers. All chromatograms reported represent, as a function of time, the absorbance value measured at λ= 325 nm, except where otherwise stated.

Enzymatic synthesis and purification of the CoA thioesters

Coffee and tobacco 4-coumarate CoA ligases

[00179] To assay the activity of Cc4CL2, a pure fraction of the recombinant protein (5 μΜ) was incubated with 0.2 mM coumaric acid, its major biological substrate, in the presence of 1 mM CoA, 5 mM MgCl ₂ and 5 mM ATP in 0.1 M sodium phosphate pH 7.5. Samples (876-879) taken at t= 0, 15 min and after overnight incubation at 34 °C were analysed using HPLC-PDA with methanol.

[00180] The activity of M4CL2 was tested with cinnamic acid and its common derivatives coumaric, caffeic, ferulic and sinapic acids. Reaction mixtures containing 1 mM (hydroxy)cinnamic acid, 5 mM CoA, 2.5 mM MgCl ₂ and 2.5 mM ATP in 0.1 M Tris-HCl pH 7.5 were prepared in a 400 μL final volume. Reactions were started by adding 1.6 μΜ recombinant enzyme. Samples (590-603) taken at t= 0, 2 and 20 min were analysed using HPLC-PDA with methanol. Similar reaction mixtures were made up in three different buffers: 0.1 M sodium phosphate pH 6.0/ 7.5 and 0.1 M Tris-HCl pH 7.5. Samples (634-683) taken at t= 0, 5, 25 min and after overnight incubation were analysed using HPLC -PDA using methanol.

[00181] Because commercial CoA thioesters were not available, coumaroyl-CoA, caffeoyl-CoA and feruloyl-CoA were synthesised enzymatically as follows: 10 mL reaction mixtures composed of 0.8 mM coumaric/ caffeic/ ferulic acid, 1 mM CoA, 2 mM MgCl ₂ and 2 mM ATP were prepared in 0.1 M sodium phosphate pH 6.0. Reactions were started by adding 0.05 μΜ Μ40.2. Samples (699-715) taken at t= 0, 10, 60 and 150 min were filtered through a 50 kDa cut-off membrane to remove the enzyme. The filtrates were analysed using HPLC -PDA with the methanol method. The reaction was complete after 150 min and all subsequent reactions to produce the hydroxycinnamoyl-CoA thioesters were stopped at this time. The final concentration of the CoA thioester was deduced from the initial amount of HCA supplied in the reaction.

Purification of CoA thioesters

[00182] Successive injections of reaction volumes of up to 150 μL, corresponding to the maximum loop capacity, were carried out to purify caffeoyl-CoA by preparative HPLC using either acetonitrile or methanol as described above. The reaction product was subsequently collected manually as it eluted off the column.

[00183] Based on a previously described procedure (Beuerle et ah, 2002), a simpler method to purify CoA thioesters from a reaction mixture was developed. A 1 mL solid- phase extraction (SPE) Sep-Pak Cis cartridge (Waters) was activated with 10 column volumes (CV) of methanol, washed with 10 CV milliQ H ₂ 0 and equilibrated with 10 CV of 4 % ammonium acetate buffer. Ammonium acetate was added to the reaction mixture to a final concentration of 4 %, before loading onto the cartridge using a syringe. The column was subsequently washed with 10 CV of 4 % ammonium acetate and 10 CV of milliQ water. The CoA thioesters were then eluted with several 500 iL fractions of 100 % methanol, which were analysed by HPLC.

[00184] The fractions containing the CoA thioester collected from preparative

HPLC or eluted from the SPE column were placed in 1.5 mL Eppendorf tubes and evaporated to near dryness using a vacuum concentrator. The residue was resuspended in milliQ-purified water before further HPLC analysis or use in enzymatic reactions.

Comparison of the HCT and HQT-catalysed forward reactions

HCT and HQT acyl acceptor specificity

[00185] In the forward direction, HCT and HQT catalyse the transfer of hydroxycinnamoyl moieties from CoA to quinic or shikimic acid. To verify the substrate preferences, reaction mixtures containing 5 mM quinic/ shikimic acid or both and 0.2 mM caffeoyl-CoA in 0.1 M sodium phosphate pH 6.5 were prepared in a final volume of 100 μ Reactions were started by adding 1 μΜ HCT/ HQT. Samples (1023-1052) taken at t= 0, 5 and 60 min were analysed using HPLC -PDA with the methanol method.

[00186] To test the reactivity of HCT/ HQT towards other acyl acceptor molecules, reactions containing 10 mM quinic/ shikimic/ 4-hydroxyphenyllactic acid/ spermidine or myoinositol and 0.2 mM caffeoyl-CoA in 0.1 M sodium phosphate pH 6.5 were prepared in a 100 μΙ_, final volume. Reactions were started by adding 0.5 μΜ HCT/ HQT. Samples (1146-1163) taken at t= 0, 15 min and after overnight incubation were analysed using HPLC-PDA with the methanol method.

HCT and HQT phenolic donor specificity

[00187] To compare the phenolic donor preference of HCT and HQT in the forward reaction, the following assays were set up. Reaction mixtures containing 0.2 mM caffeoyl-CoA/ coumaroyl-CoA/ feruloyl-CoA and 10 mM quinic/ shikimic acid in 0.1 M sodium phosphate pH 6.0 were prepared in a 500 μΙ_, final volume. Reactions were started by adding 0.4/ 0.004 μΜ HCT/ HQT. Samples (716-727, 764-775 and 800-811) taken at t= 0, 10, and 75 min were analysed with the methanol method.

Comparison of the HCT and HQT-catalysed reverse reactions

Characterisation of the products obtained in the reverse reaction

[00188] The activity of recombinant HCT and HQT was first tested towards the

CGAs available, 5-CQA and 5-FQA, with CoA as the cofactor. Reaction mixtures containing 3.75 mM 5-CQA/ 5-FQA and 5 mM CoA in 0.1 M sodium phosphate pH 6.0 were prepared in a final volume of 100 μΐ,. Reactions were started by adding 1 uM HCT or 1/ 0.1/ 0.01 μΜ HQT. Samples (395-428) taken at t= 0, 5, 15, 30, and 60 min were analysed using HPLC-PDA with the methanol method.

[00189] To confirm the formation of caffeoyl- and feruloyl-CoA, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed. For this, reaction mixtures made up of 2 mM 5-CQA 5-FQA and 2 mM CoA in 0.1 M sodium phosphate buffer pH 6.0 were prepared to a final volume of 500 μ Reactions were started by adding 0.04 μΜ HQT and incubated at 25 °C. Samples taken at t= 0 and 45 min for 5-CQA (sample 3) and t= 0 and 60 min for 5-FQA (sample 4) were analysed using HPLC-PDA with the acetonitrile method. These samples were also sent to the Nestle Research Centre, (Lausanne) for LC-MS/MS analysis. The negative detection mode was adopted because phenolic acids ionise better in this mode.

[00190] The Biopunfy kit enabled the testing of the activity of HCT and HQT towards other CGA monoesters (3-CQA and 4-CQA) and diesters (3,4-diCQA, 3,5-diCQA and 4,5-diCQA). Reaction mixtures comprised 1 mM CGA and 1/ 5 mM CoA in 0.1 M sodium phosphate pH 6.0/ 7.5. Reactions were started by adding 0.4 μΜ HCT/ HQT. Samples (604-633 and 684-698) were taken at t= 0, 30 min and after overnight incubation and analysed using HPLC-PDA with the methanol method.

[00191] Rosmarinic acid is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid, similar in nature to CGAs. The reactivity of HCT and HQT was therefore tested towards this potential substrate. A reaction mixture composed of 1 mM rosmarinic acid and 1 mM CoA in 0.1 M sodium phosphate pH 6.5 was prepared in a 100 μL final volume. Reactions were started by adding 1 μΜ HCT/ HQT. Samples (978-987) were taken at t= 0 and after overnight incubation and analysed using HPLC-PDA with the methanol method.

Temperature and pH influence

[00192] Initial characterisation of the optimal pH and temperature conditions was carried out with a reaction mixture composed of 2 mM 5-CQA, 2 mM CoA and 1 μΜ HCT/ HQT in 0.1 M sodium phosphate buffer. The temperature (22, 34 and 42 °C at pH 6.0) and pH values (4.6, 6.0 and 7.6 at 34 °C) were varied. Samples (36-47 and 49-58) were analysed by HPLC-PDA using methanol. Influence of enzyme, 5-CQA, CoA and quinic acid concentrations

[00193] To study the influence of 5-CQA concentration, reaction mixtures containing 0.02 to 10 mM 5-CQA and 2 mM CoA in 0.1 M sodium phosphate pH 7.0 were prepared in a 500 final volume. Reactions were started by adding 0.1 μΜ HQT and incubated at 30 °C. Samples taken at t= 0 to 30 min were analysed using HPLC-DA with acetonitrile.

[00194] To determine the influence of enzyme concentration, reaction mixtures containing 2 mM 5-CQA and 5 mM CoA in 0.1 M sodium phosphate pH 6.0 were prepared in a 500 final volume. Reactions were started by adding various concentrations of HCT/ HQT and incubated at 30 °C. Samples taken at t= 0 to 60 min were analysed using HPLC-DA with acetonitrile.

[00195] The influence of quinic acid concentration was studied using reaction mixtures containing 5 mM CoA, 5 mM 5-CQA and 0/ 1/ 5/ 20 mM quinic acid in 0.1 M sodium phosphate pH 6.0 in a 100 final volume. Reactions were started by adding 1 μΜ HQT. Samples (488-503) taken at t= 0, 5, and 30 min for were analysed using HPLC-PDA with methanol.

[00196] To study the influence of CoA, varied concentrations of CoA (0.125, 0.25,

0.5, 1 and 5 mM) were added to 1 mM 5-CQA in 0.1 M sodium phosphate pH 6.5 to a 500 final volume. Reactions were started by adding 0.5 μM HCT/ HQT. Samples (1101 -1145 and 1164-1168) taken at t= 0, 3, 30, 180 min, and after overnight incubation were analysed using HPLC-PDA with acetonitrile.

Comparison of the activity of native and mutant HCTs

Activity assay in the forward and reverse reactions

[00197] To determine the influence of point mutations on HCT enzymatic activity,

0.1 mM caffeoyl-CoA, 1 mM quinic/ shikimic acid or both were mixed in 0. 1 M sodium phosphate pH 6.5 to a 100 μL final volume. Reactions were started by adding 5 μΜ native, K21 0A/K217A (K), H 1 53 A, H35A, D 1 57A, Y252A, Y255A, R374E, H 1 54A and H154N/A155L/A156S mutant HCTs. Samples (1321 -1350) taken at t= 0 and 210 min were analysed using HPLC-PDA with methanol as the organic solvent. Additional analysis of the H154A and H154N/A155L/A156S mutants

[00198] To study in more detail the activity of the mutants targeting HI 54, reaction mixtures containing 5 mM 5-CQA and 5 mM CoA in 0.1 M sodium phosphate pH 6.0 were prepared in a 100 final volume. Reactions were started by adding 1 μΜ native, H154A or H154N/A155L/A156S mutant HCTs. Samples (326-349, 464-487, 956-977 and 1023-1052) were taken at various time points and analysed using HPLC-DA/ PDA with methanol or acetonitrile.

[00199] Caffeoyl-CoA (0.01-0.2 mM) was similarly incubated with 1 mM 5-CQA and the native or H154A mutant HCTs. Samples (1 169-1 193) taken at t= 0, 15 min and after overnight incubation were analysed using HPLC-PDA with the acetonitrile method.

[00200] In addition to the assays above, the H154A and H154N/A155L/A156S HCT mutants were incubated with various acyl donor and acceptor moieties. In this case, reaction mixtures consisting of 10 mM 5-CQA/ 5-FQA and 0.2 mM CoA/ caffeoyl-CoA/ feruloyl-CoA/ coumaroyl-CoA in 0.1 M sodium phosphate pH 6.5 were prepared in a 200 μΙ_, final volume. Reactions were started by adding 0.5 μΜ native or variant HCT. Samples (1194- 1273) taken at t= 0, 60 min and after overnight incubation were analysed using HPLC-PDA with acetonitrile.

Characterization of Substrate Binding Pockets (SBP) for 4CL2, HCT, and HCQ.

[00201] Substrate docking studies revealed that the substrate binding pocket (SBP) of M4CL2 could be clearly defined as part of the N-terminal domain with CoA thiol and AMP phosphate group at the entry of the pocket. Amino acid residues within 4 A of the predicted SBP are: Gln213, Leu221, Ile238, Tyr239, Ser243, Val244, Pro279, Met306-Ala309, Gly330-Thr336, Pro340, Val341 and Phe348. (Data not shown). These residues in M4CL2, and corresponding residues in related 4CL enzymes may be useful targets for altering the enzymatic activity with respect to one or more substrates or products.

[00202] Pocket-Finder (Hendlich et ah, 1997) was used to predict the location of the SBP in the native CcHCT crystal structure chain A, which most likely adopts a functional conformation (see Results section). The 1.6 A probe radius used found a major site (volume- 1850 A ³ ) corresponding to the solvent channel located at the domain interface (data not shown). The residues lining the solvent channel in HCT were identified. They belong to the following sequence regions: Asn26-Tyr40, Prol l O, Argl l 5, Vall 49-Ilel 65, Tyr203, Tyr255, Tyr281 - Asp285, Arg289, Asn301 -Thr305, Leu331 , Leu346, Leu355-Phe362, Gly368-Ile377, Met390- Tyr397 and Leu400-Phe402. A number of these residues are conserved between the HCT and HQT enzymes, while some are not. These differences may contribute to the different substrate specificities of HCT and HQT.

Characterisation Results

[00203] Reverse-phase HPLC using an elution gradient with either acetonitrile or methanol was the preferred technique for the characterisation and analysis of plant secondary metabolites in the biochemical characterisation of the enzymes produced herein. The HPLC were adjusted to optimise CGA separation. A preparative HPLC procedure was developed for the purification of caffeoyl-CoA from enzymatic reactions. However, a simpler SPE method was ultimately used for the purification of coumaroyl-CoA, caffeoyl-CoA and feruloyl-CoA esters, which are substrates for HCT and HQT in the forward reactions leading to CGA biosynthesis.

Enzymatic synthesis and purification of CoA thioesters

Coffee and tobacco 4-coumarate CoA ligases

[00204] Enzymatic synthesis was found to be an efficient way of producing large amounts of CoA-activated HCAs that are not commercially available. The well-characterised M4CL2 was produced using a published procedure (Beuerle et al, 2002). With all substrates used, apart from sinapic acid, there is a high conversion of HCA to the relevant CoA thioester. The retention time and maximum absorbance values in absorbance spectra are presented in Table 10. Fig.13 shows the activity of purified recombinant 4CL2 towards cinnamic, coumaric, caffeic, ferulic and sinapic acids in the presence of CoA and Mg-ATP required for catalysis.

Table 10: Relative retention times and absorbance maxima of CoA thioester products

[00205] Hydroxycinnamoyl-CoA thioesters were characterised by their UV spectra

(Fig. 14). Later experiments show that, with the exception of sinapic acid, the conversion by M4CL2 of these substrates reached over 80 % after 60 min (Fig. 15). During optimisation of the reaction conditions, no major difference was observed in product levels obtained at pH 6.0 or 7.5, or with sodium phosphate compared to Tris-HCl (data not shown). Therefore all subsequent reactions were carried out in 0.1 M sodium phosphate pH 6.0. The CoA esters could be produced almost free of their acid precursors by the addition of more enzyme. This further enabled the quantification of the CoA thioester product, assuming that one mole of HCA supplied in the reaction generated one mole CoA thioester.

Purification of CoA thioesters by preparative HPLC or SPE

[00206] The coumaroyl-, caffeoyl- and feruloyl-CoA thioesters produced were purified by preparative HPLC or solid phase extraction (SPE). Initially, preparative HPLC was used for purification. A 100-150 iL volume of reaction mixture was injected and the fractions containing the CoA ester were collected manually. This purification method was abandoned because it was time-consuming and the HPLC profiles obtained indicated a partial degradation of caffeoyl-CoA to caffeic acid. This was most likely due to the increase in relative amounts of phosphoric acid during concentration. A simpler Sep-Pak purification procedure was eventually devised. The enzyme was removed from the reaction mixture by filtration through a 50 kDa membrane before loading onto a Sep-Pak column. The CoA thioesters were bound to the C ₁₈ column using a 4 % ammonium acetate buffer, washed with milliQ water and eluted with methanol. The elution fractions were collected and analysed by HPLC. The fractions containing the CoA ester were lyophilised and resuspended in milliQ water before use.

Comparison of the HCT and HQT-catalysed forward reactions

Test of the acyl acceptor specificity

[00207] Coumaroyl-, caffeoyl- and feruloyl-CoA thioesters were produced using

M4CL2, and then purified. The purified thioesters were substantially free from their acid precursors. The reactivity of CcHCT and CcHQT towards these activated substrates was tested. HCT and HQT can transfer the acyl moiety between CoA and acceptor molecules such as quinic and shikimic acids. The precursor molecule for the major CGA compound, 5-CQA, is caffeoyl- CoA, which may be an important precursor in mono- and dicaffeoylquinic acid biosynthesis. Little was known about the synthesis of coumaroylquinic or feruloylquinic acids in plants. However, coumaroyl-CoA is the precursor for coumaroylquinic acids, which are present only in trace amounts in coffee. Feruloyl-CoA appears to be the precursor for 5 -feruloylquinic acid (5- FQA).

Activity of CcHCT and CcHQT towards caffeoyl-CoA

[00208] The results concerning the reactions containing caffeoyl-CoA and catalysed by HCT are presented in Fig. 17. When CcHCT was incubated with caffeoyl-CoA and quinic acid, 5-CQA (RT= 17.6 min) was formed. 76 % of the caffeoyl-CoA substrate had been consumed after 60 min respectively (Panel B). With shikimic acid as the acyl acceptor, all the caffeoyl-CoA present was transformed after only 5 min (Panel C) to a product (RT= 35.4 min) with a typical CGA absorbance spectrum (Panel D). The UV spectrum showed a maximum at λ= 322.6 nm and is similar to that of 5-CQA. As caffeoyl-CoA was used as a precursor, and the absorption spectrum of the novel compound shared some properties with that of 5-CQA, it was hypothesised that the novel compound resulted from a coupling of the caffeoyl moiety with shikimic acid via an ester linkage, presumably caffeoylshikimic acid (CSA). After 60 min, a major peak representing the same ester and two other peaks, which may correspond to the two other CSA isomers (RT= 25.9 and 32.2 min) were present (Panel D). When both quinic and shikimic acids were supplied to CcHCT at the same concentration, a caffeoylshikimic acid isomer was formed after 5 min (Panel E); after 60 min, small peaks corresponding to the two other CSA isomers were detected as well as a small peak for 5-CQA (Panel F). This result clearly demonstrates that CcHCT strongly favours shikimic over quinic acid as the acyl acceptor molecule and is capable of producing more than one CSA isomer.

[00209] CcHQT was incubated with caffeoyl-CoA and quinic or shikimic acid

(Fig. 18). With quinic acid as a substrate, caffeoyl-CoA was converted to 5-CQA (RT= 17.5 min). As for CcHCT, incubation with caffeoyl-CoA and shikimic acid resulted in the accumulation of a CSA (RT= 35.5 min) (Fig. 18B). Conversely, when both quinic and shikimic acids were supplied to CcHQT, the enzyme mainly formed 5-CQA and only a small amount of CSA after t= 5 min (Fig. 18C). After 60 min, 5-CQA and CSA are present in much closer ratios, 57 and 43 % respectively (Fig. 18D). This indicates that CcHQT prefers quinic acid, but is also capable of forming CSA.

[00210] The activity of CcHCT and CcHQT towards other potential substrates (4- hydroxyphenyllactic acid, spermidine and wryo-inositol) was also assayed. These acceptor molecules were incubated with caffeoyl-CoA and the enzymes, however no activity was observed (data not shown). CcHCT and CcHQT are therefore active only towards the alicyclic quinic and shikimic acids.

Activity of HCT and HQT towards coumaroyl-CoA

[00211] The incubation of coumaroyl-CoA and quinic acid with CcHCT resulted in a peak (RT= 27.2 min) presumably corresponding to a coumaroylquinic acid (Fig. 19B). With shikimic acid, three peaks were detected at RT= 33.2, 38.5 and 42. 1 min, presumably representing 3-, 4- and 5-coumaroylshikimic acids (Fig. 19C). When both acyl acceptors were present, a mixture of the three coumaroylshikimic acids plus a small amount of coumaroylquinic acid was observed (Fig. 19D). So with coumaroyl-CoA, CcHCT appears to prefer shikimic acid as an acyl acceptor and produces more than one coumaroylshikimic acid isomer.

[00212] CcHQT formed a coumaroylquinic acid from coumaroyl-CoA and quinic acid (Fig. 20A). Coumaroyl-CoA and shikimic acid incubated with CcHQT resulted in only one coumaroylshikimic acid isomer at RT= 41.9 min (Fig. 20B). When both quinic and shikimic acids were supplied, HQT synthesised a mixture of presumably coumaroylquinic and coumaroylshikimic acid in the ratio of 85 and 15 % respectively at t= 10 min (Fig. 20C). This ratio shifted to 78 and 22 % at t= 75 min (data not shown). As seen when using caffeoyl-CoA as the forward substrate, it is again noted that CcHQT prefers quinic acid and produces only one coumaroylshikimic acid isomer.

Activity of CcHCT and CcHQT towards feruloyl-CoA

[00213] CcHCT can synthesise a shikimate ester, presumably a feruloylshikimic acid (RT= 45.2 min) from feruloyl-CoA and shikimic acid and the reaction was complete after 2 min. The new compound has a typical CGA absorbance spectrum (Fig. 2 IB). However, only a small peak of 5-FQA (RT= 30.5 min) is formed in the presence of quinic acid at t= 20 min (Fig. 21 C). When both acyl acceptors were supplied, CcHCT catalyses the synthesis of a feruloylshikimic acid corresponding to the RT for the 5-acyl isomer (Fig. 21D). This demonstrates the preference of CcHCT for shikimic acid and its much lower efficiency towards 5-FQA formation.

[00214] The incubation of feruloyl-CoA with HQT and quinic acid leads to 5-FQA

(RT= 30.5 min) (Fig. 22A), while with shikimic acid it leads to a feruloylshikimic acid (RT= 45.1 min) (Fig. 22B). The conversion was complete after t= 2 min and 20 min respectively. As expected, when both quinic acid and shikimic acid were supplied, HQT preferentially synthesised 5-FQA (Fig. 22C).

[00215] In summary, in the presence of the same CoA thioester (caffeoyl-/ coumaroyl-/ feruloyl-CoA), CcHCT clearly prefers shikimic over quinic acid. CcHCT is also much less efficient with feruloyl-CoA in the presence of quinic acid. In contrast, CcHQT is able to use all three CoA thioesters equally with both quinic and shikimic acids. It is also interesting to note that HCT can form three probable shikimate esters of caffeic and coumaric acids, while with feruloyl-CoA only a single shikimate ester is formed. In contrast, CcHQT forms only a single shikimate ester with all CoA thioesters tested.

Comparison of the HCT and HQT-catalysed reverse reactions

Stability of chlorogenic acids

[00216] CGAs are known to be unstable at basic pH and stable at acidic pH

(Friedman, 2000). This was verified by heating buffered solutions of 5-CQA and 5-FQA to 90 °C. At pH 6.0, significant amounts of 3- and 4-CQA were produced from the 5-CQA isomer. At neutral pH, 5-CQA was converted to approximately equivalent levels of 3- and 4-CQA isomers, resulting in an equimolar mixture of the three caffeoylquinic acid isomers (Table 11). No major difference was observed between 30 min and 60 min incubation times (data not shown). 5-FQA behaved very similarly to 5-CQA by partial isomerisation to 3- and 4-FQA (data not shown). Further experiments performed at 34 and 25 °C on the mono- and diCQAs from Biopurify showed that, even though the enzymatic reactions were carried out at 34 °C and pH 6.5, 5-CQA partially isomerised into 4-CQA and to a lesser extent into 3-CQA (Fig. 23C and C). In a 4-CQA solution, the acyl migrated to the 3-hydroxyl position and to a lesser extent to the 5-hydroxyl position (Fig. 23B, B'). 3-CQA isomerised into 4-CQA and, to a lesser extent, into 5-CQA (Fig. 23 A, A'). Similarly, 3,5-diCQA isomerised into 3,4- and 4,5-diCQA when incubated overnight at 25 °C and this isomerisation increased when the sample was kept at 34 °C (data not shown).

Table 11 : Effect of temperature and pH on the isomerisation of 5-CQA 10 mM 5-CQA in 0.1 M sodium phosphate ph 6.0 or 7.0.

[00217] This isomerisation most probably occurs via the acyl transfer of the hydroxycinnamoyl moiety between the three readily accessible hydroxyl positions of quinic acid (hydroxyl at C-l is not involved). In some of the enzymatic reactions that will be described later, a significant amount of 3- and 4-CQA isomers was derived from solutions containing 5-CQA, especially after overnight incubation. It is assumed that this occurred not from enzymatic synthesis but from chemical isomerisation.

Characterisation of the reverse reaction products formed by HCT and HQT

[00218] When CoA was added as a substrate with 5-CQA in the presence of CcHCT or CcHQT, there was the emergence of a peak at RT= 27.5 min showing a characteristic CoA thioester UV spectrum (Fig. 24B, C), presumably corresponding to caffeoyl-CoA. However, under identical reaction conditions and enzyme concentrations, CcHCT (Fig. 24B) produces significantly less caffeoyl-CoA than HQT (Fig. 24C). A 100-fold dilution of HQT can synthesise the same amount of caffeoyl-CoA as HCT at a 1 μΜ concentration (Fig. 24B, D). This is further evidence that CcHQT has a greater preference for reactions involving quinate moieties than does CcHCT.

[00219] Further evidence of this is that no product formation was observed when

CcHCT was incubated with 5-FQA and CoA under the conditions tested (Fig. 25A, B) and that, in contrast, CcHQT was with the same substrates able to synthesise a product at RT= 39.8 min, which probably corresponds to feruloyl-CoA, as it shows a UV spectrum characteristic of a CoA thioester (Fig. 25C).

[00220] The main reaction products when using 5-CQA or 5-FQA as substrates were presumed to be CoA thioesters. The absorption spectra of the compounds contained in the HPLC peaks measured using HPLC-PDA present two maxima around 256 and 346 nm, the latter being characteristic of the presence of a thioester bond. The compounds were subjected to a LC- MS/MS analysis to confirm their identity. An m/z value of 928 for the reaction product synthesised by CcHQT from 5-CQA and CoA corresponds to caffeoyl-CoA (MW= 930 g/mol). An m/z value of 942 for the reaction product synthesised by HQT from 5-FQA and CoA corresponds to feruloyl-CoA (MW= 944 g/mol).

Substrate specificity of HCT and HQT in the reverse reaction

[00221] For preliminary experiments testing CcHCT and CcHQT specificity (data not shown), various CGA isomers were generated by heating solutions of 10 mM 5-CQA/ 5-FQA buffered at pH 7.0. Only the 5-CQA peak was clearly decreased by conversion to caffeoyl-CoA with CcHCT and CcHQT in the presence of CoA. No activity towards 3 -CQA and 4-CQA was detected. Similarly, with a FQA mixture only the 5-FQA isomer peak was decreased in the presence of CcHQT. HCT was found to be less efficient towards this substrate. When the CQA kit was obtained from Biopurify, reaction mixtures were prepared with each monoester individually and CoA. Again, only 5-CQA was converted by CcHCT and CcHQT to caffeoyl- CoA in the presence of CoA (Fig. 26), confirming the results obtained with the CQA isomer mixture. Rosmarinic acid was also incubated overnight with CoA, but no reaction product was observed with CcHCT or CcHQT (data not shown).

[00222] CcHCT and CcHQT were also incubated with 3,5-diCQA and CoA. In the control with no enzyme, 3,5-diCQA isomerises into 3,4-diCQA and possibly 4,5-diCQA, however, this peak could not be resolved from the 3,5-diCQA peak at RT= 47.2 min (Fig. 27B). In the presence of CcHCT, 5-CQA and caffeoyl-CoA are detected as indicated from the 5-CQA standard and the absorbance spectra (Fig. 27A, C). In the presence of CcHQT, no reaction product was observed after overnight incubation (data not shown).

Additional enzymatic properties of HCT/ HQT

[00223] Additional experiments were conducted to further study the reaction properties of CcHCT and CcHQT. Temperature variation (22 °C, 34 °C and 42 °C) did not significantly alter the activity of CcHCT and CcHQT in the reverse reaction with 5-CQA and CoA. All subsequent enzymatic reactions were therefore carried out in a 34 °C water-bath. [00224] To address the effects of pH on the reverse reactions of CcHCT and CcHQT, several experiments were carried out. In the first set of reactions performed, CcHQT and CcHCT were not active at pH 4.6 (Fig. 28). The results from a more extensive analysis on the effects of pH on the reverse reaction confirmed that the CcHQT optimal pH value is close to 6.0, whereas CcHCT seemed to be more active towards 5-CQA at pH 8.0 (Fig. 29). pH values higher than pH 8.0 have not yet been tested with either enzyme because CGA compounds are not stable at this pH. These results also highlight the remarkable difference in the catalysis efficiency of CcHCT and CcHQT with similar levels of 5-CQA and CoA substrates and confirm the preference of CcHQT for quinate substrates.

Effect of enzyme and substrate concentrations on product formation

[00225] As seen above, the production of the same amount of caffeoyl-CoA (at constant 5- CQA and CoA concentrations) requires very different amounts of HCT and HQT. The reaction steady-state persists for many minutes with HCT in the range of concentrations that were used (0.1-0.8 μΜ) (Fig. 30A). Under these conditions, the Michaelis-Menten law may be applicable (Bisswanger, 2002). The reaction velocity as a function of substrate and enzyme concentrations can yield the Michaelis and catalytic constants. However, lower enzyme concentrations are required in the case of HQT (Fig. 30B).

[00226] When serial dilutions of 5-CQA were incubated with CoA and CcHQT, a maximal conversion to caffeoyl-CoA production is reached very rapidly, which corresponds to the equilibrium attained between substrates and products (Fig. 31).

[00227] To study the potential effect of quinic acid concentration on the CcHQT-catalysed reverse reaction, initial quinic acid concentrations were varied from 0 to 20 mM in the presence of constant levels of 5-CQA (5 mM) and CoA (5 mM). The results show that increasing quinic acid concentration produces a significant inhibitory effect on the reverse reaction (Fig. 32). It would be interesting in future experiments to compare this with CcHCT.

[00228] The effect of varying the CoA concentration in the reverse reaction was also explored. As expected, increasing the CoA concentration while maintaining 5-CQA levels constant pushes the reverse reaction towards the formation of more caffeoyl-CoA (Fig. 33).

[00229] Thus, caffeoyl-CoA formation depends on the initial concentration of 5-CQA, CoA and quinic acid. It should be noted that the production of caffeoyl-CoA by CcHQT was initially used for the production of this compound. However, as this reaction reaches equilibrium well below the 100 % conversion rate due to the forward reaction, it is not efficient. The preliminary data on the properties of CcHCT and CcHQT enzymes presented above provides a basis for an understanding of the kinetics of these enzymes.

Formation of diCQA in the reverse reaction

[00230] In reactions containing 5-CQA and CoA catalysed by CcHCT, unknown peaks were observed at later retention times (Fig. 34C). To confirm the identity of these peaks, CGA standards were run, confirming their assignments as 3,4-, 3,5- and 4,5-diCQA. DiCQA formation was observed with CcHCT when 5-CQA and CoA were supplied in a 1 : 1 molar ratio but not when it was 1 : 10 (Fig. 34C, D). DiCQAs were not observed when using CcHQT (Fig. 34E, F).

Comparison of the activity of native and mutant HCTs

Comparison of native and mutant HCT in the forward and reverse reactions

[00231] To determine the importance of specific residues on the enzymatic activity of CcHCT, point amino acid mutations were introduced. The activities of the mutant HCTs tested (K, H35A, H153A, H154A, D157A, Y255A, Y252A and R374E) were compared to that of the native CcHCT. All mutants were incubated with 1 mM CoA and 1 mM 5-CQA (reverse reaction), or with 0.2 mM caffeoyl-CoA and 1 mM quinic/ shikimic acid (forward reaction). Fig. 35 shows a summary of results when either 5-CQA and CoA or caffeoyl-CoA and quinic acid were used.

[00232] In the forward reaction, both the native and the K-mutant HCTs used caffeoyl- CoA and quinic acid to form 5-CQA and, to a lesser extent, free caffeic acid. For both proteins there appears to be a complete conversion of the caffeoyl-CoA substrate. When using caffeoyl- CoA and shikimic acid as substrates, the K-mutant HCT catalysed the formation of all three caffeoylshikimic acids produced by native CcHCT (data not shown). In the reverse reaction using 5-CQA and CoA, both the native and K-mutant HCTs produced low levels of caffeoyl- CoA and free caffeic acid, indicating only a partial conversion of 5-CQA to the thioester product.

[00233] In the forward reaction with caffeoyl-CoA and quinic acid, the mutants H35A and Y255A showed a relatively weak catalytic activity, with only a partial conversion of caffeoyl- CoA into 5-CQA, and caffeic acid. With shikimic acid, the H35A and Y255A mutants were also weakly catalytically active (data not shown). Neither the H35A nor the Y255A mutant HCTs showed any detectable activity in the reverse reaction.

[00234] In the forward reaction, the mutants D157A and R374E only produced caffeic acid in the presence of caffeoyl-CoA and quinic acid. The Y252A mutant produced caffeic acid and a small amount of 5-CQA. In comparison to the H35A and Y255A mutants, these mutants fully transformed the caffeoyl-CoA substrate provided into caffeic acid. In the presence of caffeoyl-CoA and shikimic acid, the D157A mutant HCT exclusively formed caffeic acid (data not shown). The Y252A and R374E mutants were not tested with caffeoyl-CoA and shikimic acid. In the reverse reaction, D157A, Y252A and R374E mutants showed no activity towards 5- CQA and CoA as no peak of caffeoyl-CoA was detected. It is noted however that Y252A did produce caffeic acid. These results suggest that these mutants are not effective in the reverse reaction involving the quinate ester. Most probably, the Y252A mutant is capable of making caffeoyl-CoA, which is subsequently degraded into caffeic acid via a putative lyase activity.

[00235] In accordance with its proposed role in catalysis, the HI 53 A mutant was inactive in the forward reaction involving caffeoyl-CoA and quinic or shikimic acid. As expected, this mutant is also inactive in the reverse direction with 5-CQA and CoA. No caffeic acid was detected. Most interesting are the H154A and H154N/A155L/A156S mutants, which formed 5- CQA and diCQAs from caffeoyl-CoA and quinic acid with a major peak corresponding to the 3,5-diester. The triple mutant also formed a small amount of free caffeic acid. In the reverse direction with 5-CQA and CoA, the two mutants formed a low level of caffeoyl-CoA and a relatively significant amount of diCQAs, again suggesting the importance of the 5-CQA/CoA molar ratio. The major diCQA peak corresponds to the 3,5-diester from which low amounts of the other two peaks, the 3,4- and 4,5-diCQA isomers, were presumably derived by chemical isomerisation. Again, the triple mutant also produced a small amount of free caffeic acid.

Additional analysis of the H154 mutants in the reverse reaction

[00236] The results presented in Fig. 35 suggest that the mutants targeting His 154 favour the synthesis of diCQAs when compared to native CcHCT. The two mutants involving HI 54 were thus further studied by doing a new comparison between the native HCT and the H154A and H154N/A155L/A156S mutants. In the forward reaction, caffeoyl-CoA and quinic or shikimic acid or both were supplied. The results showed that the HI 54 mutants have comparable activities to the native enzyme, namely they both continue to show a clear preference for shikimic over quinic acid with caffeoyl-CoA as the second substrate. Caffeic acid formation was observed in the triple mutant only. In the forward reaction with caffeoyl-CoA and shikimic acid, a major peak was detected that is thought to correspond to a caffeoylshikimic acid isomer and two other smaller peaks that are thought to be the two other CSA isomers were seen (data not shown). These results are similar to results obtained with native CcHCT (Fig. 17). The effect of different 5-CQA/CoA molar ratios (1/1 and 1/10) supplied in the reaction was also tested. This experiment confirmed that both mutants again produced diCQAs. Fig. 36 also shows that adding more CoA slightly increased the amount caffeoyl-CoA produced, and that the level of diCQA decreases.

Formation of mixed diesters

To determine if the native and HI 54 mutant HCTs could form mixed diesters of quinic acid (e.g. caffeoylferuloylquinic acids found in coffee extracts), the enzymes were incubated with 5-CQA/ 5-FQA and CoA/ caffeoyl-/ feruloyl-/ coumaroyl-CoA thioesters. As in the previous experiment, the HI 54 mutants formed higher levels of these diesters. The results concerning the H154A mutant with 5-CQA and CoA or diverse acyl-CoA thioesters are presented in Fig. 37. The control with no enzyme (Panel A) and all enzymatic reactions showed a partial isomerisation of 5-CQA (RT= 15.9 min) to the 3- and 4-CQA isomers (RT= 7.4 and 14.5 min) after overnight incubation in 0.1 M sodium phosphate pH 6.5. In the presence of 10 mM 5-CQA and 0.5 mM CoA (Panel B), as well as 10 mM 5-CQA and 0.5 mM caffeoyl-CoA (Panel C), three peaks corresponding to 3,4-, 3,5- and 4,5-diCQAs (RT= 41.4, 42.4 and 43.6 min respectively) were formed with native HCT. Interestingly, when 5-CQA and feruloyl-CoA are incubated with H154A mutant, all three diCQAs are synthesised. Three additional peaks formed at RT= 43.9, 45.0 and 45.9 min may correspond to mixed caffeoylferuloylquinic acids. When 5-CQA and coumaroyl-CoA are incubated with the H154A mutant, the three diCQAs are formed (RT= 41.4, 42.4 and 43.6 min), plus two additional peaks at RT= 44.6 and 45.7 min that may correspond to mixed caffeoylcoumaroylquinic acids (Panel E). Similarly, the H154A mutant was incubated with 5-FQA and CoA or diverse acyl-CoA thioesters (Fig. 38). The control with no enzyme (Panel A) and all enzymatic reactions showed a partial isomerisation of 5-FQA (RT= 32.3 min) to the 3- and 4-FQA isomers (RT= 13.7 and 28.2 min) after overnight incubation in 0.1 M sodium phosphate pH 6.5. Additionally, 4 unknown peaks (RT= 43.7, 44.5, 47.7 and 48.4 min) were observed at later retention times, but these showed a maximum absorbance value at 256 nm, so they were not CGA molecules. Consequently, these peaks are not described in the following enzymatic reactions. With 5-FQA and either CoA or feruloyl-CoA, a new peak appeared at RT= 46.3 min that may correspond to a diferuloylquinic acid (Panels B and D). When 5-FQA and caffeoyl-CoA were incubated, 4 new peaks at RT= 44.2, 45.3, 46.1 and 46.3 min were observed. These were presumed to be the same diFQA (RT= 46.3 min) and mixed caffeoylferuloylquinic acids. When 5-FQA and coumaroyl-CoA were incubated, the same diferuloylquinic acid (RT= 46.3 min) and presumably mixed coumaroylferuloylquinic acids were obtained (RT= 45.4 and 47.3 min).

[00237] In summary, the HI 54 mutants seem to favour diCQA formation. When feruloyl- or coumaroyl-CoA thioesters were supplied with 5-CQA/ 5-FQA, new peaks with a typical quinate ester absorbance spectrum were detected at late retention times. They were presumed to be new esters of quinic acid. New peaks were observed and putatively attributed to either diferuloylquinic acids (diFQAs) (RT= 45.5, 46.3 , and 47.5 min) or mixed diesters, feruloylcaffeoylquinic (RT= 44.2, 45.3, and 46.1 min) and feruloylcoumaroylquinic acids (RT= 45.4, 46.3, and 47.3 min), their identity should to be confirmed by mass spectrometry analysis. These assays show that these enzymes may be involved in the biosynthesis of not only caffeoylquinic, feruloylquinic and coumaroylquinic acids but also mixed diesters that are present in coffee (e.g. caffeoylferuloylquinic acids). The identity of the new products that were formed must be confirmed by comparison with authentic standards or by mass spectrometry analysis.

Production of Chlorogenic or Dactyilfric Acids in vitro Using Combinations of 4CL, HCT and/or HOT

[00238] Reaction mixtures containing 0.5 mM coumaric/ caffeic acid, 0.5/ 10 mM quinic/ shikimic acid, 2.5 mM CoA, 2.5 mM ATP and 5 mM MgC12 in 0.1 M sodium phosphate pH 6.0 were prepared in a 500 μΙ_, final volume. Reactions were started by adding 0.4 μΜ Nt4CL2 and incubated at 35°C. 75 μΐ,-νοΐυηιβ samples were taken at t= 10 min and 5-fold dilutions (824- 833) were analysed using HPLC-PDA with the methanol method. A t= 15 min, 0.5 μΜ HCT/ HQT was added to the remaining 425 μΙ_, reaction mixture. Samples (834-863) taken at t= 30 min (15 min after adding HCT/ HQT), t= 105 min and overnight (data not shown for the latter two time points) were also analysed. The experimental setup is shown below in Table 12.

Table 12.

[00239] Results are shown in Fig. 39. Panel A shows results of a reaction with no enzyme and 0.5 mM coumaric acid as substrate. Panel B shows results of a reaction with 4CL alone and 0.5mM coumaric acid as substrate. As can be seen, in the presence of coumaric acid, Co A, ATP and Mg2+, 4CL forms coumaroyl-CoA. Panel C shows the results of consecutive reactions with 4CL and HQT with coumaric (0.5 mM) and quinic (0.5/ 10 mM) acids as substrates. Here, HQT converts coumaroyl-CoA produced by 4CL to a coumaroylquinic acid. The conversion is more efficient with lOmM (samples 834, 844, 854) than 0.5mM (samples 835, 845, 855) quinic acid.. Panel D shows consecutive reactions with 4CL and HCT with coumaric (0.5 mM) and shikimic (0.5/ 10 mM) acids as substrates. In this reaction, HCT converts coumaroyl-CoA produced by 4CL to a coumaroylshikimic acid after 30 min. The conversion is more efficient with lOmM (samples 836, 846, 856) than 0.5mM (samples 837, 847, 857) quinic acid, as a significant amount of coumaroyl-CoA remains in the latter. [00240] Panel E shows results of an initial reaction with 4CL alone and 0.5mM caffeic acid as substrate. As can be seen, caffeic acid is converted to caffeoyl-CoA by 4CL in the presence of CoA, ATP and Mg2+. Panel F shows results of consecutive reactions with 4CL and HQT with caffeic (0.5 mM) and quinic (0.5/ 10 mM) acids as substrates. Here, HQT converts caffeoyl-CoA (produced by 4CL) to 5-CQA. The conversion is more efficient with 10 mM than 0.5 mM quinic acid. The conversion is complete with 10 mM quinic acid and almost no caffeoyl- CoA remains after 15 min (samples 838, 848, 858). Caffeic acid is still present in sample 839, so 4CL has not converted all the precursor to caffeoyl-CoA yet. Panel G shows the results of consecutive reactions with 4CL and HCT with caffeic (0.5 mM) and shikimic (0.5/ 10 mM) acids as substrates. In this reaction, HCT converts caffeoyl-CoA produced by 4CL to a caffeoylshikimic acid after l Omin. The conversion is more efficient with 10 mM than 0.5 mM shikimic acid as some caffeoyl-CoA remains in the latter.

[00241] The experimental results set forth above demonstrate that chlorogenic acids or dactylifric acids can be produced in a single in-vitro reaction. The chlorogenic or dactyfrilic acids are generated using either caffeic acid or coumaric acid and either quinic acid or shikimic acid, with the inclusion of recombinant 4CL and either recombinant HCT or recombinant HQT and the appropriate cofactors and buffer conditions. In the experiements described above, the reactions were carried out as partially consecutive steps, i.e., 4CL was added before HCT or HQT. However, it is expected that the same or similar results would be observed if the enzymes were added together. This makes feasible the performance of such reactions in situ, in recombinant microbial or plant cells co-expressing the enzymes. The data shown above also suggest that such co-reactions may "push" the reaction towards the production of more chlorogenic acids or dactylifric acids, in particular when the quinic acid and shikimic acids are in high concentrations.

III. Use of Synthesized Chlorogenic Acid Species in the Production of a Coffee Product

Hypothetical Production of a Coffee Product

[00242] A immobilised enzyme system will be constructed to produce commercially useful quantities of CGAs and DAs. The enzymes will be cloned CcHCT and CcHQT which are over-expressed in food-grade lactic acid bacteria, and purified to homogeneity (as seen on SDS gel electrophoresis) with a single-step column separation.

[00243] The enzymes will be used to catalyze the production of CGA and DA species. Preferably caffeic acid is activated to the -CoA thioester using a 4CL enzyme in a separate preparatory step that proceeds nearly completely to product formation. The caffeoyl-CoA will be used as the substrate and is to be combined with quinic acid and/or a combination of quinic and shikimic acid in a semi-continuous process to yield caffeoylquinic and shikimic esters, as well as diesters. The conditions are optimized for the production of 3,4, caffeoylquinic acid as a major product. The product of the immobilised enzyme system is a food-grade mixture of CGA and DA species that includes some remaining caffeoyl-CoA.

[00244] A food product in the form of a soluble coffee product is to be manufactured according to procedures described above. Prior to completing the product and packaging the product for consumers, a mixture of exogenous chlorogenic acids and/or dactylifric acid species will be added to the coffee product to improve the flavour profile and aroma. The mixture is preferably enriched in 3,4 dicaffeoylquinic acid, and relatively deficient in 5-feruloylquinic acid. The addition of the exogenous mixture will provide several advantages to the soluble coffee product. The flavour of the product will be improved. Consumers will note that there is slight but desirable bitter taste, and a pleasant roasted aroma and flavour. Consumers will also note a degree of astringency that is not unpleasant or overpowering.

[00245] In the specification, there have been disclosed typical preferred embodiments of the invention. Although specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limiting the claims. The scope of the invention is set forth in the claims. In light of the above teachings, many modifications and variations of the invention are possible and may be apparent to the skilled artisan. It is therefore to be understood that within the scope of the appended claims the invention may be practiced otherwise than as specifically described.

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Sequences:

SEQ ID NO: 1

DNA

GTPcl03a_Cchct Primer 5'

cagcactagggatccatgaaaatcgaggtgaaggaatcg

SEQ ID NO: 2

DNA

GTPcl03a_Cchct Primer 3'

tcgtcgatcactcgagtcaaatgtcatacaagaaactctggaa

SEQ ID NO: 3

DNA

pGTPcl03a_Cchqt Primer 5'

cagcactagggatccatgaagataaccgtgaaggaaaca SEQ ID NO: 4

DNA

pGTPcl03a_Cchqt Primer 3'

tcgtcgatcatctagatcagaaatcgtacaggaacctttg

SEQ ID NO: 5

DNA

cl03g/al04c (H35A)

Forward Primer: atctggtggtgccgaactttgctaccccgagcg

SEQ ID NO: 6

DNA

Reverse Primer: cgctcggggtagcaaagttcggcaccaccagat

SEQ ID NO: 7

DNA

c457g/a458c (H153A)

Forward Primer: ggtgtgggcatgcgtgctcatgcggcgga

SEQ ID NO: 8

DNA

Reverse Primer: tccgccgcatgagcacgcatgcccacacc

SEQ ID NO: 9

DNA

c460a/t462t (H154A)

Forward Primer: ggtgtgggcatgcgtcataatgcggcgga

SEQ ID NO: 10

DNA

Reverse Primer: tccgccgcattatgacgcatgcccacacc

SEQ ID NO: 11

DNA

a470c (D157A)

Forward Primer: catcatgcggcggctggctttagcggc

SEQ ID NO: 12

DNA

Reverse Primer: gccgctaaagccagccgccgcatgatg

SEQ ID NO: 13

DNA

t754g/a755c (Y252A)

Forward Primer: aagatggcaacaccattagcgctagcagctatgaaatgctgg

SEQ ID NO: 14

DNA

Reverse Primer: ccagcatttcatagctgctagcgctaatggtgttgccatctt

SEQ ID NO: 15

DNA

t763a (Y255A)

Forward Primer: ccattagctatagcagcaatgaaatgctggccggc

SEQ ID NO: 16

DNA Reverse Primer: gccggccagcatttcattgctgctatagctaatgg

SEQ ID NO: 17

DNA

cll20g/gll21a/tll22a (R374E)

Forward Primer: gcattaccagctgggtggaactgccgatccatgatgc

SEQ ID NO: 18

DNA

Reverse Primer: gcatcatggatcggcagttccacccagctggtaatgc

SEQ ID NO: 19

DNA

C460a/g463c/c464t/g466a/c467g/g468c (H154N/A155L/A156S)

Forward Primer : gggtgtgggcatgcgtcataatctgagcgatggctttagcggcctgcat

SEQ ID NO: 20

DNA

Reverse Primer: atgcaggccgctaaagccatcgctcagattatgacgcatgcccacaccc

SEQ ID NO: 21

DNA

>Cchct_pMLl

atgaaaatcgaggtgaaggaatcgactatggtgagacctgcccaagaaactcctggg aggaacttgtggaa ctcgaacgtggacttggtggtgccaaatttccacacccctagcgtctacttctataggcc gacgggatcat cgaatttctttgatgccaaagtgctaaaggacgctttgagccgagcccttgtcccgttct acccaatggct ggtaggttaaagagagacgaagatgggcggattgagattgagtgcaatggtgaaggcgtg cttttcgtgga ggccgagtctgatggagtagttgatgatttcggtgactttgcaccaactttagagcttcg tagactcattc ctgcagttgattattctcaaggaatatcaagctacgctctcctagtgctgcaggtgacat atttcaagtgt ggtggagtctctcttggtgttggcatgcgacatcatgcagctgatggattttcaggtctt catttcatcaa ttcatggtctgatatggcccgtggccttgatgtgaccctgccaccattcatagaccgcac cctcctccgtg cccgcgatccgccccagcctcaattccagcacattgagtaccaacctcctccagccttaa aagtttccccg caaactgcaaaatctgactcagttcctgaaactgcggtgtccatcttcaagttaaccagg gagcaaatcag tgccctgaaagccaagtccaaggaagatggaaacaccattagctatagctcctacgaaat gttggcaggcc atgtatggcgctgtgcttgcaaggcacgaggactcgaagttgatcaaggaacaaaattgt acattgctact gatggacgggcaagacttaggccttcgctcccacctggctattttggcaatgtcatcttc acggcaacccc gatagctatagctggtgaccttgaattcaagccagtctggtatgctgccagtaaaatcca cgatgcattgg cgaggatggacaacgactacttaaggtcagctcttgattacttggaattacagcctgatt tgaaggccctg gttcgtggtgcccatactttcaagtgtccaaatctggggatcacaagctgggtaaggtta cccatccatga tgctgattttggctggggtcggcccatatttatgggtcctggtggcatcgcttatgaagg tttaagcttta tattgcctagtccaaccaatgatggaagcatgtcagtagctatttcattgcagggcgagc acatgaaactc ttccagagtttcttgtatgacatttga

SEQ ID NO: 22

DNA

>CcHCT (Geneart synthetic gene with codons optimised for expression in E. coli)

atgaaaatcgaagtgaaagaaagcaccatggttcgtccggcgcaggaaaccccgggt cgtaacctgtggaa cagcaacgtggatctggtggtgccgaactttcataccccgagcgtgtatttttatcgtcc gaccggcagca gcaacttttttgatgcgaaagtgctgaaagatgccctgagccgtgcgctggtgccgtttt atccgatggcg ggtcgtctgaaacgtgatgaagatggccgcattgaaattgaatgcaacggcgaaggcgtg ctgtttgtgga agcggaaagcgatggcgtggtggatgattttggcgattttgcgccgaccctggaactgcg tcgtctgattc cggcggtggattatagccagggcattagcagctatgcgctgctggtgctgcaggtgacct attttaaatgc ggcggtgtgagcctgggtgtgggcatgcgtcatcatgcggcggatggctttagcggcctg cattttattaa cagctggtctgatatggcgcgtggcctggatgttaccctgccgccgtttattgatcgtac cctgctgcgtg cgcgtgatccgccgcagccgcagtttcagcatattgaatatcaaccgccgccggcactga aagttagcccg cagaccgcgaaaagcgatagcgtgccggaaaccgcggtgagcatttttaaactgacccgc gaacaaattag cgcgctgaaagcgaaaagcaaagaagatggcaacaccattagctatagcagctatgaaat gctggccggcc atgtgtggcgttgcgcgtgcaaagcgcgtggtctggaagtggatcagggcaccaaactgt atattgcgacc gatggccgtgcgcgtctgcgtccgagcctgccgccgggttattttggcaacgtgattttt accgcgacccc gattgcgattgcgggcgatctggaatttaaaccggtgtggtatgcggcgagcaaaattca tgatgcgctgg cccgtatggataacgattatctgcgtagcgcgctggattatctggaactgcagccggatc tgaaagcgctg gtgcgtggcgcgcatacctttaaatgcccgaacctgggcattaccagctgggtgcgtctg ccgatccatga tgcggattttggctggggccgtccgatttttatgggtccgggcggtattgcgtatgaagg cctgagcttta ttctgccgagcccgaccaacgatggcagcatgagcgtggcgattagcctgcagggcgaac atatgaaactg tttcagagcttcctgtatgatatctaataa

SEQ ID NO: 23

PRT

CcHCT

MKIEVKESTMVRPAQETPGRNLWNSNVDLWPNFHTPSVYFYRPTGSSNFFDAKVLKDALS PALVPFYPMA GRLKRDEDGRIEIECNGEGVLFVEAESDGWDDFGDFAPTLELRRLIPAVDYSQGISSYAL LVLQVTYFKC GGVSLGVGMRHHAADGFSGLHFINSWSDMARGLDVTLPPFIDRTLLRARDPPQPQFQHIE YQPPPALKVSP QTAKSDSVPETAVSI FKLTREQISALKAKSKEDGNTI SYSSYEMLAGHVWRCACKARGLEVDQGTKLYIAT DGRARLRPSLPPGYFGNVIFTATPIAIAGDLEFKPVWYAASKIHDALARMDNDYLRSALD YLELQPDLKAL VRGAHTFKCPNLGITSWVRLPIHDADFGWGRPIFMGPGGIAYEGLSFILPSPTNDGSMSV AI SLQGEHMKL FQSFLYDI—

SEQ ID NO: 24

DNA

>gene encoding K-mutant HCT

atgaaaatcgaagtgaaagaaagcaccatggttcgtccggcgcaggaaaccccgggt cgtaacctgtggaa cagcaacgtggatctggtggtgccgaactttcataccccgagcgtgtatttttatcgtcc gaccggcagca gcaacttttttgatgcgaaagtgctgaaagatgccctgagccgtgcgctggtgccgtttt atccgatggcg ggtcgtctgaaacgtgatgaagatggccgcattgaaattgaatgcaacggcgaaggcgtg ctgtttgtgga agcggaaagcgatggcgtggtggatgattttggcgattttgcgccgaccctggaactgcg tcgtctgattc cggcggtggattatagccagggcattagcagctatgcgctgctggtgctgcaggtgacct attttaaatgc ggcggtgtgagcctgggtgtgggcatgcgtcatcatgcggcggatggctttagcggcctg cattttattaa cagctggtctgatatggcgcgtggcctggatgttaccctgccgccgtttattgatcgtac cctgctgcgtg cgcgtgatccgccgcagccgcagtttcagcatattgaatatcaaccgccgccggcactgg cggttagcccg cagaccgcggcgagcgatagcgtgccggaaaccgcggtgagcatttttaaactgacccgc gaacaaattag cgcgctgaaagcgaaaagcaaagaagatggcaacaccattagctatagcagctatgaaat gctggccggcc atgtgtggcgttgcgcgtgcaaagcgcgtggtctggaagtggatcagggcaccaaactgt atattgcgacc gatggccgtgcgcgtctgcgtccgagcctgccgccgggttattttggcaacgtgattttt accgcgacccc gattgcgattgcgggcgatctggaatttaaaccggtgtggtatgcggcgagcaaaattca tgatgcgctgg cccgtatggataacgattatctgcgtagcgcgctggattatctggaactgcagccggatc tgaaagcgctg gtgcgtggcgcgcatacctttaaatgcccgaacctgggcattaccagctgggtgcgtctg ccgatccatga tgcggattttggctggggccgtccgatttttatgggtccgggcggtattgcgtatgaagg cctgagcttta ttctgccgagcccgaccaacgatggcagcatgagcgtggcgattagcctgcagggcgaac atatgaaactg tttcagagcttcctgtatgatatctaataa

SEQ ID NO: 25

>K-mutant_HCT (K210A/K217A)

MKIEVKESTMVRPAQETPGRNLWNSNVDLWPNFHTPSVYFYRPTGSSNFFDAKVLKDALS PALVPFYPMA GRLKRDEDGRIEIECNGEGVLFVEAESDGWDDFGDFAPTLELRRLIPAVDYSQGISSYAL LVLQVTYFKC GGVSLGVGMRHHAADGFSGLHFINSWSDMARGLDVTLPPFIDRTLLRARDPPQPQFQHIE YQPPPAIAVSP QTAASDSVPETAVSI FKLTREQISALKAKSKEDGNTISYSSYEMIAGHVWRCACKARGLEVDQGTKLYIAT DGRARLRPSLPPGYFGNVIFTATPIAIAGDLEFKPVWYAASKIHDALARMDNDYLRSALD YLELQPDLKAL VRGAHTFKCPNLGITSWVRLPIHDADFGWGRPIFMGPGGIAYEGLSFILPSPTNDGSMSV AI SLQGEHMKL FQSFLYDI—

SEQ ID NO: 26

DNA

>gene encoding HCT_H35A (c!03g/al04c) atgaaaatcgaagtgaaagaaagcaccatggttcgtccggcgcaggaaaccccgggtcgt aacctgtggaa cagcaacgtggatctggtggtgccgaactttgctaccccgagcgtgtatttttatcgtcc gaccggcagca gcaacttttttgatgcgaaagtgctgaaagatgccctgagccgtgcgctggtgccgtttt atccgatggcg ggtcgtctgaaacgtgatgaagatggccgcattgaaattgaatgcaacggcgaaggcgtg ctgtttgtgga agcggaaagcgatggcgtggtggatgattttggcgattttgcgccgaccctggaactgcg tcgtctgattc cggcggtggattatagccagggcattagcagctatgcgctgctggtgctgcaggtgacct attttaaatgc ggcggtgtgagcctgggtgtgggcatgcgtcatcatgcggcggatggctttagcggcctg cattttattaa cagctggtctgatatggcgcgtggcctggatgttaccctgccgccgtttattgatcgtac cctgctgcgtg cgcgtgatccgccgcagccgcagtttcagcatattgaatatcaaccgccgccggcactga aagttagcccg cagaccgcgaaaagcgatagcgtgccggaaaccgcggtgagcatttttaaactgacccgc gaacaaattag cgcgctgaaagcgaaaagcaaagaagatggcaacaccattagctatagcagctatgaaat gctggccggcc atgtgtggcgttgcgcgtgcaaagcgcgtggtctggaagtggatcagggcaccaaactgt atattgcgacc gatggccgtgcgcgtctgcgtccgagcctgccgccgggttattttggcaacgtgattttt accgcgacccc gattgcgattgcgggcgatctggaatttaaaccggtgtggtatgcggcgagcaaaattca tgatgcgctgg cccgtatggataacgattatctgcgtagcgcgctggattatctggaactgcagccggatc tgaaagcgctg gtgcgtggcgcgcatacctttaaatgcccgaacctgggcattaccagctgggtgcgtctg ccgatccatga tgcggattttggctggggccgtccgatttttatgggtccgggcggtattgcgtatgaagg cctgagcttta ttctgccgagcccgaccaacgatggcagcatgagcgtggcgattagcctgcagggcgaac atatgaaactg tttcagagcttcctgtatgatatctaataa

SEQ ID NO: 27

PRT

>HCT_H35A

MKIEVKESTMVRPAQETPGRNLWNSNVDLWPNFATPSVYFYRPTGSSNFFDAKVLKDALS PALVPFYPMA GRLKRDEDGRIEIECNGEGVLFVEAESDGWDDFGDFAPTLELRRLIPAVDYSQGISSYAL LVLQVTYFKC GGVSLGVGMRHHAADGFSGLHFINSWSDMARGLDVTLPPFIDRTLLRARDPPQPQFQHIE YQPPPALKVSP QTAKSDSVPETAVSI FKLTREQISALKAKSKEDGNTI SYSSYEMLAGHVWRCACKARGLEVDQGTKLYIAT DGRARLRPSLPPGYFGNVIFTATPIAIAGDLEFKPVWYAASKIHDALARMDNDYLRSALD YLELQPDLKAL VRGAHTFKCPNLGITSWVRLPIHDADFGWGRPIFMGPGGIAYEGLSFILPSPTNDGSMSV AI SLQGEHMKL FQSFLYDI—

SEQ ID NO: 28

DNA

>gene encoding HCT_H153A (c457g/a458c)

atgaaaatcgaagtgaaagaaagcaccatggttcgtccggcgcaggaaaccccgggt cgtaacctgtggaa cagcaacgtggatctggtggtgccgaactttcataccccgagcgtgtatttttatcgtcc gaccggcagca gcaacttttttgatgcgaaagtgctgaaagatgccctgagccgtgcgctggtgccgtttt atccgatggcg ggtcgtctgaaacgtgatgaagatggccgcattgaaattgaatgcaacggcgaaggcgtg ctgtttgtgga agcggaaagcgatggcgtggtggatgattttggcgattttgcgccgaccctggaactgcg tcgtctgattc cggcggtggattatagccagggcattagcagctatgcgctgctggtgctgcaggtgacct attttaaatgc ggcggtgtgagcctgggtgtgggcatgcgtgctcatgcggcggatggctttagcggcctg cattttattaa cagctggtctgatatggcgcgtggcctggatgttaccctgccgccgtttattgatcgtac cctgctgcgtg cgcgtgatccgccgcagccgcagtttcagcatattgaatatcaaccgccgccggcactga aagttagcccg cagaccgcgaaaagcgatagcgtgccggaaaccgcggtgagcatttttaaactgacccgc gaacaaattag cgcgctgaaagcgaaaagcaaagaagatggcaacaccattagctatagcagctatgaaat gctggccggcc atgtgtggcgttgcgcgtgcaaagcgcgtggtctggaagtggatcagggcaccaaactgt atattgcgacc gatggccgtgcgcgtctgcgtccgagcctgccgccgggttattttggcaacgtgattttt accgcgacccc gattgcgattgcgggcgatctggaatttaaaccggtgtggtatgcggcgagcaaaattca tgatgcgctgg cccgtatggataacgattatctgcgtagcgcgctggattatctggaactgcagccggatc tgaaagcgctg gtgcgtggcgcgcatacctttaaatgcccgaacctgggcattaccagctgggtgcgtctg ccgatccatga tgcggattttggctggggccgtccgatttttatgggtccgggcggtattgcgtatgaagg cctgagcttta ttctgccgagcccgaccaacgatggcagcatgagcgtggcgattagcctgcagggcgaac atatgaaactg tttcagagcttcctgtatgatatctaataa

SEQ ID NO: 29

PRT

>HCT H153A MKIEVKESTMVRPAQETPGRNLWNSNVDLWPNFHTPSVYFYRPTGSSNFFDAKVLKDALS PALVPFYPMA GRLKRDEDGRIEIECNGEGVLFVEAESDGWDDFGDFAPTLELRRLIPAVDYSQGISSYAL LVLQVTYFKC GGVSLGVGMRAHAADGFSGLHFINSWSDMARGLDVTLPPFIDRTLLRARDPPQPQFQHIE YQPPPALKVSP QTAKSDSVPETAVSI FKLTREQISALKAKSKEDGNTI SYSSYEMLAGHVWRCACKARGLEVDQGTKLYIAT DGRARLRPSLPPGYFGNVIFTATPIAIAGDLEFKPVWYAASKIHDALARMDNDYLRSALD YLELQPDLKAL VRGAHTFKCPNLGITSWVRLPIHDADFGWGRPIFMGPGGIAYEGLSFILPSPTNDGSMSV AI SLQGEHMKL FQSFLYDI—

SEQ ID NO: 30

DNA

>gene encoding HCT_H154N (c460a)

atgaaaatcgaagtgaaagaaagcaccatggttcgtccggcgcaggaaaccccgggt cgtaacctgtggaa cagcaacgtggatctggtggtgccgaactttcataccccgagcgtgtatttttatcgtcc gaccggcagca gcaacttttttgatgcgaaagtgctgaaagatgccctgagccgtgcgctggtgccgtttt atccgatggcg ggtcgtctgaaacgtgatgaagatggccgcattgaaattgaatgcaacggcgaaggcgtg ctgtttgtgga agcggaaagcgatggcgtggtggatgattttggcgattttgcgccgaccctggaactgcg tcgtctgattc cggcggtggattatagccagggcattagcagctatgcgctgctggtgctgcaggtgacct attttaaatgc ggcggtgtgagcctgggtgtgggcatgcgtcataatgcggcggatggctttagcggcctg cattttattaa cagctggtctgatatggcgcgtggcctggatgttaccctgccgccgtttattgatcgtac cctgctgcgtg cgcgtgatccgccgcagccgcagtttcagcatattgaatatcaaccgccgccggcactga aagttagcccg cagaccgcgaaaagcgatagcgtgccggaaaccgcggtgagcatttttaaactgacccgc gaacaaattag cgcgctgaaagcgaaaagcaaagaagatggcaacaccattagctatagcagctatgaaat gctggccggcc atgtgtggcgttgcgcgtgcaaagcgcgtggtctggaagtggatcagggcaccaaactgt atattgcgacc gatggccgtgcgcgtctgcgtccgagcctgccgccgggttattttggcaacgtgattttt accgcgacccc gattgcgattgcgggcgatctggaatttaaaccggtgtggtatgcggcgagcaaaattca tgatgcgctgg cccgtatggataacgattatctgcgtagcgcgctggattatctggaactgcagccggatc tgaaagcgctg gtgcgtggcgcgcatacctttaaatgcccgaacctgggcattaccagctgggtgcgtctg ccgatccatga tgcggattttggctggggccgtccgatttttatgggtccgggcggtattgcgtatgaagg cctgagcttta ttctgccgagcccgaccaacgatggcagcatgagcgtggcgattagcctgcagggcgaac atatgaaactg tttcagagcttcctgtatgatatctaataa

SEQ ID NO: 31

PRT

>HCT_H154N

MKIEVKESTMVRPAQETPGRNLWNSNVDLWPNFHTPSVYFYRPTGSSNFFDAKVLKDALS PALVPFYPMA GRLKRDEDGRIEIECNGEGVLFVEAESDGWDDFGDFAPTLELRRLIPAVDYSQGI SSYALLVLQVTYFKC GGVSLGVGMRHNAADGFSGLHFINSWSDMARGLDVTLPPFIDRTLLRARDPPQPQFQHIE YQPPPALKVSP QTAKSDSVPETAVSI FKLTREQISALKAKSKEDGNTI SYSSYEMIAGHVWRCACKARGLEVDQGTKLYIAT DGRARLRPSLPPGYFGNVIFTATPIAIAGDLEFKPVWYAASKIHDALARMDNDYLRSALD YLELQPDLKAL VRGAHTFKCPNLGITSWVRLPIHDADFGWGRPI FMGPGGIAYEGLSFILPSPTNDGSMSVAISLQGEHMKL FQSFLYDI—

SEQ ID NO: 32

DNA

>gene encoding HCT_D157A (a470c)

atgaaaatcgaagtgaaagaaagcaccatggttcgtccggcgcaggaaaccccgggt cgtaacctgtggaa cagcaacgtggatctggtggtgccgaactttcataccccgagcgtgtatttttatcgtcc gaccggcagca gcaacttttttgatgcgaaagtgctgaaagatgccctgagccgtgcgctggtgccgtttt atccgatggcg ggtcgtctgaaacgtgatgaagatggccgcattgaaattgaatgcaacggcgaaggcgtg ctgtttgtgga agcggaaagcgatggcgtggtggatgattttggcgattttgcgccgaccctggaactgcg tcgtctgattc cggcggtggattatagccagggcattagcagctatgcgctgctggtgctgcaggtgacct attttaaatgc ggcggtgtgagcctgggtgtgggcatgcgtcatcatgcggcggctggctttagcggcctg cattttattaa cagctggtctgatatggcgcgtggcctggatgttaccctgccgccgtttattgatcgtac cctgctgcgtg cgcgtgatccgccgcagccgcagtttcagcatattgaatatcaaccgccgccggcactga aagttagcccg cagaccgcgaaaagcgatagcgtgccggaaaccgcggtgagcatttttaaactgacccgc gaacaaattag cgcgctgaaagcgaaaagcaaagaagatggcaacaccattagctatagcagctatgaaat gctggccggcc atgtgtggcgttgcgcgtgcaaagcgcgtggtctggaagtggatcagggcaccaaactgt atattgcgacc gatggccgtgcgcgtctgcgtccgagcctgccgccgggttattttggcaacgtgattttt accgcgacccc gattgcgattgcgggcgatctggaatttaaaccggtgtggtatgcggcgagcaaaattca tgatgcgctgg cccgtatggataacgattatctgcgtagcgcgctggattatctggaactgcagccggatc tgaaagcgctg gtgcgtggcgcgcatacctttaaatgcccgaacctgggcattaccagctgggtgcgtctg ccgatccatga tgcggattttggctggggccgtccgatttttatgggtccgggcggtattgcgtatgaagg cctgagcttta ttctgccgagcccgaccaacgatggcagcatgagcgtggcgattagcctgcagggcgaac atatgaaactg tttcagagcttcctgtatgatatctaataa

SEQ ID NO: 33

PRT

>HCT_D157A

MKIEVKESTMVRPAQETPGRNLWNSNVDLWPNFHTPSVYFYRPTGSSNFFDAKVLKDALS PALVPFYPMA GRLKRDEDGRIEIECNGEGVLFVEAESDGWDDFGDFAPTLELRRLIPAVDYSQGISSYAL LVLQVTYFKC GGVSLGVGMRHHAAAGFSGLHFINSWSDMARGLDVTLPPFIDRTLLRARDPPQPQFQHIE YQPPPALKVSP QTAKSDSVPETAVSI FKLTREQISALKAKSKEDGNTI SYSSYEMIAGHVWRCACKARGLEVDQGTKLYIAT DGRARLRPSLPPGYFGNVIFTATPIAIAGDLEFKPVWYAASKIHDAIARMDNDYLRSALD YLELQPDLKAL VRGAHTFKCPNLGITSWVRLPIHDADFGWGRPIFMGPGGIAYEGLSFILPSPTNDGSMSV AI SLQGEHMKL FQSFLYDI—

SEQ ID NO: 34

DNA

>gene encoding HCT_Y252A ( t754g/a755c )

atgaaaatcgaagtgaaagaaagcaccatggttcgtccggcgcaggaaaccccgggt cgtaacctgtggaa cagcaacgtggatctggtggtgccgaactttcataccccgagcgtgtatttttatcgtcc gaccggcagca gcaacttttttgatgcgaaagtgctgaaagatgccctgagccgtgcgctggtgccgtttt atccgatggcg ggtcgtctgaaacgtgatgaagatggccgcattgaaattgaatgcaacggcgaaggcgtg ctgtttgtgga agcggaaagcgatggcgtggtggatgattttggcgattttgcgccgaccctggaactgcg tcgtctgattc cggcggtggattatagccagggcattagcagctatgcgctgctggtgctgcaggtgacct attttaaatgc ggcggtgtgagcctgggtgtgggcatgcgtcatcatgcggcggatggctttagcggcctg cattttattaa cagctggtctgatatggcgcgtggcctggatgttaccctgccgccgtttattgatcgtac cctgctgcgtg cgcgtgatccgccgcagccgcagtttcagcatattgaatatcaaccgccgccggcactga aagttagcccg cagaccgcgaaaagcgatagcgtgccggaaaccgcggtgagcatttttaaactgacccgc gaacaaattag cgcgctgaaagcgaaaagcaaagaagatggcaacaccattagcgctagcagctatgaaat gctggccggcc atgtgtggcgttgcgcgtgcaaagcgcgtggtctggaagtggatcagggcaccaaactgt atattgcgacc gatggccgtgcgcgtctgcgtccgagcctgccgccgggttattttggcaacgtgattttt accgcgacccc gattgcgattgcgggcgatctggaatttaaaccggtgtggtatgcggcgagcaaaattca tgatgcgctgg cccgtatggataacgattatctgcgtagcgcgctggattatctggaactgcagccggatc tgaaagcgctg gtgcgtggcgcgcatacctttaaatgcccgaacctgggcattaccagctgggtgcgtctg ccgatccatga tgcggattttggctggggccgtccgatttttatgggtccgggcggtattgcgtatgaagg cctgagcttta ttctgccgagcccgaccaacgatggcagcatgagcgtggcgattagcctgcagggcgaac atatgaaactg tttcagagcttcctgtatgatatctaataa

SEQ ID NO: 35

PRT

>HCT_Y252A

MKIEVKESTMVRPAQETPGRNLWNSNVDLWPNFHTPSVYFYRPTGSSNFFDAKVLKDALS PALVPFYPMA GRLKRDEDGRIEIECNGEGVLFVEAESDGWDDFGDFAPTLELRRLIPAVDYSQGI SSYALLVLQVTYFKC GGVSLGVGMRHHAADGFSGLHFINSWSDMARGLDVTLPPFIDRTLLRARDPPQPQFQHIE YQPPPALKVSP QTAKSDSVPETAVSI FKLTREQISALKAKSKEDGNTI SASSYEMLAGHVWRCACKARGLEVDQGTKLYIAT DGRARLRPSLPPGYFGNVIFTATPIAIAGDLEFKPVWYAASKIHDALARMDNDYLRSALD YLELQPDLKAL VRGAHTFKCPNLGITSWVRLPIHDADFGWGRPIFMGPGGIAYEGLSFILPSPTNDGSMSV AI SLQGEHMKL FQSFLYDI—

SEQ ID NO: 36

DNA

>gene encoding HCT_Y255N (t763a) atgaaaatcgaagtgaaagaaagcaccatggttcgtccggcgcaggaaaccccgggtcgt aacctgtggaa cagcaacgtggatctggtggtgccgaactttcataccccgagcgtgtatttttatcgtcc gaccggcagca gcaacttttttgatgcgaaagtgctgaaagatgccctgagccgtgcgctggtgccgtttt atccgatggcg ggtcgtctgaaacgtgatgaagatggccgcattgaaattgaatgcaacggcgaaggcgtg ctgtttgtgga agcggaaagcgatggcgtggtggatgattttggcgattttgcgccgaccctggaactgcg tcgtctgattc cggcggtggattatagccagggcattagcagctatgcgctgctggtgctgcaggtgacct attttaaatgc ggcggtgtgagcctgggtgtgggcatgcgtcatcatgcggcggatggctttagcggcctg cattttattaa cagctggtctgatatggcgcgtggcctggatgttaccctgccgccgtttattgatcgtac cctgctgcgtg cgcgtgatccgccgcagccgcagtttcagcatattgaatatcaaccgccgccggcactga aagttagcccg cagaccgcgaaaagcgatagcgtgccggaaaccgcggtgagcatttttaaactgacccgc gaacaaattag cgcgctgaaagcgaaaagcaaagaagatggcaacaccattagctatagcagcaatgaaat gctggccggcc atgtgtggcgttgcgcgtgcaaagcgcgtggtctggaagtggatcagggcaccaaactgt atattgcgacc gatggccgtgcgcgtctgcgtccgagcctgccgccgggttattttggcaacgtgattttt accgcgacccc gattgcgattgcgggcgatctggaatttaaaccggtgtggtatgcggcgagcaaaattca tgatgcgctgg cccgtatggataacgattatctgcgtagcgcgctggattatctggaactgcagccggatc tgaaagcgctg gtgcgtggcgcgcatacctttaaatgcccgaacctgggcattaccagctgggtgcgtctg ccgatccatga tgcggattttggctggggccgtccgatttttatgggtccgggcggtattgcgtatgaagg cctgagcttta ttctgccgagcccgaccaacgatggcagcatgagcgtggcgattagcctgcagggcgaac atatgaaactg tttcagagcttcctgtatgatatctaataa

SEQ ID NO: 37

PRT

>HCT_Y255N

MKIEVKESTMVRPAQETPGRNLWNSNVDLWPNFHTPSVYFYRPTGSSNFFDAKVLKDALS PALVPFYPMA GRLKRDEDGRIEIECNGEGVLFVEAESDGWDDFGDFAPTLELRRLIPAVDYSQGISSYAL LVLQVTYFKC GGVSLGVGMRHHAADGFSGLHFINSWSDMARGLDVTLPPFIDRTLLRARDPPQPQFQHIE YQPPPALKVSP QTAKSDSVPETAVSI FKLTREQISALKAKSKEDGNTI SYSSNEMLAGHVWRCACKARGLEVDQGTKLYIAT DGRARLRPSLPPGYFGNVIFTATPIAIAGDLEFKPVWYAASKIHDALARMDNDYLRSALD YLELQPDLKAL VRGAHTFKCPNLGITSWVRLPIHDADFGWGRPIFMGPGGIAYEGLSFILPSPTNDGSMSV AI SLQGEHMKL FQSFLYDI—

SEQ ID NO: 38

DNA

>gene encoding HCT_R374E (cll20g/gll21a/tll22a )

atgaaaatcgaagtgaaagaaagcaccatggttcgtccggcgcaggaaaccccgggt cgtaacctgtggaa cagcaacgtggatctggtggtgccgaactttcataccccgagcgtgtatttttatcgtcc gaccggcagca gcaacttttttgatgcgaaagtgctgaaagatgccctgagccgtgcgctggtgccgtttt atccgatggcg ggtcgtctgaaacgtgatgaagatggccgcattgaaattgaatgcaacggcgaaggcgtg ctgtttgtgga agcggaaagcgatggcgtggtggatgattttggcgattttgcgccgaccctggaactgcg tcgtctgattc cggcggtggattatagccagggcattagcagctatgcgctgctggtgctgcaggtgacct attttaaatgc ggcggtgtgagcctgggtgtgggcatgcgtcatcatgcggcggatggctttagcggcctg cattttattaa cagctggtctgatatggcgcgtggcctggatgttaccctgccgccgtttattgatcgtac cctgctgcgtg cgcgtgatccgccgcagccgcagtttcagcatattgaatatcaaccgccgccggcactga aagttagcccg cagaccgcgaaaagcgatagcgtgccggaaaccgcggtgagcatttttaaactgacccgc gaacaaattag cgcgctgaaagcgaaaagcaaagaagatggcaacaccattagctatagcagctatgaaat gctggccggcc atgtgtggcgttgcgcgtgcaaagcgcgtggtctggaagtggatcagggcaccaaactgt atattgcgacc gatggccgtgcgcgtctgcgtccgagcctgccgccgggttattttggcaacgtgattttt accgcgacccc gattgcgattgcgggcgatctggaatttaaaccggtgtggtatgcggcgagcaaaattca tgatgcgctgg cccgtatggataacgattatctgcgtagcgcgctggattatctggaactgcagccggatc tgaaagcgctg gtgcgtggcgcgcatacctttaaatgcccgaacctgggcattaccagctgggtggaactg ccgatccatga tgcggattttggctggggccgtccgatttttatgggtccgggcggtattgcgtatgaagg cctgagcttta ttctgccgagcccgaccaacgatggcagcatgagcgtggcgattagcctgcagggcgaac atatgaaactg tttcagagcttcctgtatgatatctaataa

SEQ ID NO: 39

PRT

>HCT R374E MKIEVKESTMVRPAQETPGRNLWNSNVDLWPNFHTPSVYFYRPTGSSNFFDAKVLKDALS PALVPFYPMA GRLKRDEDGRIEIECNGEGVLFVEAESDGWDDFGDFAPTLELRRLIPAVDYSQGISSYAL LVLQVTYFKC GGVSLGVGMRHHAADGFSGLHFINSWSDMARGLDVTLPPFIDRTLLRARDPPQPQFQHIE YQPPPALKVSP QTAKSDSVPETAVSI FKLTREQISALKAKSKEDGNTI SYSSYEMLAGHVWRCACKARGLEVDQGTKLYIAT DGRARLRPSLPPGYFGNVIFTATPIAIAGDLEFKPVWYAASKIHDALARMDNDYLRSALD YLELQPDLKAL VRGAHTFKCPNLGITSWVELPIHDADFGWGRPIFMGPGGIAYEGLSFILPSPTNDGSMSV AI SLQGEHMKL FQSFLYDI—

SEQ ID NO: 40

DNA

>gene encoding HCT_H154N/A155L/A156S

( c460a/g463c/c464t/g466a/c467g/g468c )

atgaaaatcgaagtgaaagaaagcaccatggttcgtccggcgcaggaaaccccgggt cgtaacctgtggaa cagcaacgtggatctggtggtgccgaactttcataccccgagcgtgtatttttatcgtcc gaccggcagca gcaacttttttgatgcgaaagtgctgaaagatgccctgagccgtgcgctggtgccgtttt atccgatggcg ggtcgtctgaaacgtgatgaagatggccgcattgaaattgaatgcaacggcgaaggcgtg ctgtttgtgga agcggaaagcgatggcgtggtggatgattttggcgattttgcgccgaccctggaactgcg tcgtctgattc cggcggtggattatagccagggcattagcagctatgcgctgctggtgctgcaggtgacct attttaaatgc ggcggtgtgagcctgggtgtgggcatgcgtcataatctgagcgatggctttagcggcctg cattttattaa cagctggtctgatatggcgcgtggcctggatgttaccctgccgccgtttattgatcgtac cctgctgcgtg cgcgtgatccgccgcagccgcagtttcagcatattgaatatcaaccgccgccggcactga aagttagcccg cagaccgcgaaaagcgatagcgtgccggaaaccgcggtgagcatttttaaactgacccgc gaacaaattag cgcgctgaaagcgaaaagcaaagaagatggcaacaccattagctatagcagctatgaaat gctggccggcc atgtgtggcgttgcgcgtgcaaagcgcgtggtctggaagtggatcagggcaccaaactgt atattgcgacc gatggccgtgcgcgtctgcgtccgagcctgccgccgggttattttggcaacgtgattttt accgcgacccc gattgcgattgcgggcgatctggaatttaaaccggtgtggtatgcggcgagcaaaattca tgatgcgctgg cccgtatggataacgattatctgcgtagcgcgctggattatctggaactgcagccggatc tgaaagcgctg gtgcgtggcgcgcatacctttaaatgcccgaacctgggcattaccagctgggtgcgtctg ccgatccatga tgcggattttggctggggccgtccgatttttatgggtccgggcggtattgcgtatgaagg cctgagcttta ttctgccgagcccgaccaacgatggcagcatgagcgtggcgattagcctgcagggcgaac atatgaaactg tttcagagcttcctgtatgatatctaataa

SEQ ID NO: 41

PRT

>HCT_H154N/A155L/A156S

MKIEVKESTMVRPAQETPGRNLWNSNVDLWPNFHTPSVYFYRPTGSSNFFDAKVLKDALS PALVPFYPMA GRLKRDEDGRIEIECNGEGVLFVEAESDGWDDFGDFAPTLELRRLIPAVDYSQGISSYAL LVLQVTYFKC GGVSLGVGMRHNLSDGFSGLHFINSWSDMARGLDVTLPPFIDRTLLRARDPPQPQFQHIE YQPPPALKVSP QTAKSDSVPETAVSI FKLTREQISALKAKSKEDGNTI SYSSYEMIAGHVWRCACKARGLEVDQGTKLYIAT DGRARLRPSLPPGYFGNVIFTATPIAIAGDLEFKPVWYAASKIHDALARMDNDYLRSALD YLELQPDLKAL VRGAHTFKCPNLGI TSWVRLPIHDADFGWGRPIFMGPGGIAYEGLSFILPSPTNDGSMSVAISLQGEHMKL FQSFLYDI—

SEQ ID NO: 42

DNA

>Cchqt_pML2

atgaagataaccgtgaaggaaacagcaatggttcgtccagcccaaccaacacccaca aagaggctatggaa ctcgaatttggatcttctggtagcaagaattcatatcttgaccgtctacttctacaagcc taatggttctg ctaatttctttgatacccgtgtgctcaaagaagcattgagcaatgttcttgtctctttct accccatggct gggagattagcaagagatgaagaaggaaggattgagatcgactgtaacggtgaaggtgta ctctttgttga ggctgaatcagactcgagtgtcgatgattttggtgattttgcaccaagtttggagctcag gaggctcatcc cgaccgtcgattgttctggtgacatttcttcttaccccctcgttattttccaggtaactc gtttcaagtgt ggagcagcagctcttggagctggggtgcagcacaatttatcagatggcgtttcatctctg cacttcatcaa tacatggtcagacatagctcgtggcctcaccattgctgtccctccgttcattgaccggac acttatccgtg ctagggaccctcccgtgcctgtattcgaacatgtcgaatattatccgccccctcaactaa aatcagattca agaattcaagaactcaaaacaggccctagggcaagtaccacagctgtactaaaaat cacacctgaacaact tagccagcttaaagctaaggccaacaatgagggcagcacttatgagatcttagcagcaca tatatggcgta ctgcatgcaaagcacgtggcctcaccaatgatcaatccaccaagctgtacgttgccactg atggccggtct aggctgattcctcccctgcctcctggctatctaggtaatgtggtcttcactgcaactcca attgctgaatc cagtaatcttcaatcagagcccttgaccaattctgcaaaaagaatccacaacgccttggc aagaatggata atgattacttaagatcagccctggattatcttgaaattcaacctgatttgtctgctttgg ttagagggcct cggcattttgctagtcctaatttgaatatcaatagttggacaaggcttccgtttcatgat gcagactttgg ctggggcagacctattcatatagggccggcaatcattctttatgagggtacggtatatgt attgccaagtc caggcaaagacaggactttatcgttagctgtgtgcttagatgccgatcacatgccactct tccaaaggttc ctgtacgatttctga

SEQ ID NO: 43

DNA

>Cchqt (Geneart synthetic gene with codons optimised for expression in E. coli)

atgaaaatcaccgtgaaagaaaccgcgatggtgcgtccggcgcagccgaccccgacc aaacgtctgtggaa cagcaacctggatctgctggttgcgcgtattcatattctgaccgtgtacttctataaacc gaacggcagcg cgaacttttttgatacccgcgtgctgaaagaagccctgagcaacgttctggtgagctttt atccgatggcg ggtcgtctggcccgtgatgaagaaggccgtattgaaattgattgcaacggcgaaggcgtg ctgtttgtgga agcggaaagcgatagcagcgtggatgattttggcgattttgcgccgagcctggaactgcg tcgtctgattc cgaccgtggattgcagcggcgacattagcagctatccgctggtgatttttcaggtgaccc gttttaaatgc ggcgcagcagcgctgggtgcgggtgtgcagcataacctgagcgatggcgtgagcagcctg cattttattaa cacctggtctgatattgcgcgtggcctgaccattgcggtgccgccgtttattgatcgtac cctgattcgtg cgcgtgatccgccggtgccggtgtttgaacatgtggaatattatccgccgccgcagctga aaagcgatagc cgtattcaggaactgaaaaccggtccgcgtgcgagcaccaccgcggtgctgaaaattacc ccggaacagct gtctcagctgaaagcgaaagcgaacaacgaaggcagcacctatgaaattctggccgcgca tatttggcgta ccgcgtgcaaagcgcgcggtctgaccaacgatcagagcaccaaactgtatgtggcgaccg atggccgtagc cgcctgattccgccgctgccgccgggttatctgggcaacgtggtgtttaccgcgaccccg attgcggaaag cagcaacctgcagagcgaaccgctgaccaacagcgcgaaacgtattcataacgcgctggc ccgtatggata acgattatctgcgtagcgcgctggattatctggaaattcagccggatctgagcgcgctgg ttcgtggcccg cgtcattttgcgagcccgaacctgaacattaacagctggacccgtctgccgtttcatgat gcggattttgg ctggggccgtccgattcatattggtccggcgattattctgtatgaaggcaccgtgtatgt gctgccgagcc cgggcaaagatcgcaccctgagcctggccgtgtgcctggatgcggatcatatgccgctgt ttcagcgtttt ctgtatgatttctaa

SEQ ID NO: 44

PRT

>CcHQT

MKITVKETAMVRPAQPTPTKRLWNSNLDLLVARIHILTVYFYKPNGSANFFDTRVLKEAL SNVLVSFYPMA GRIARDEEGRIEIDCNGEGVLFVEAESDSSVDDFGDFAPSLELRRLIPTVDCSGDI SSYPLVIFQVTRFKC GAAALGAGVQHNLSDGVSSLHFINTWSDIARGLTIAVPPFIDRTLIRARDPPVPVFEHVE YYPPPQLKSDS RIQELKTGPPASTTAVLKITPEQLSQLKAKANNEGSTYEIIAAHIWRTACKARGLTNDQS TKLYVATDGRS RLIPPLPPGYLGNWFTATPIAESSNLQSEPLTNSAKRIHNAIARMDNDYLRSALDYLEIQ PDLSALVRGP RHFASPNLNINSWTRLPFHDADFGWGRPIHIGPAIILYEGTVYVLPSPGKDRTLSIAVCL DADHMPLFQRF LYDF

SEQ ID NO: 45

DNA

>gene encoding K-mutant HQT (Geneart)

atgaaaatcaccgtgaaagaaaccgcgatggtgcgtccggcgcagccgaccccgacc aaacgtctgtggaa cagcaacctggatctgctggttgcgcgtattcatattctgaccgtgtacttctataaacc gaacggcagcg cgaacttttttgatacccgcgtgctgaaagaagccctgagcaacgttctggtgagctttt atccgatggcg ggtcgtctggcccgtgatgaagaaggccgtattgaaattgattgcaacggcgaaggcgtg ctgtttgtgga agcggaaagcgatagcagcgtggatgattttggcgattttgcgccgagcctggaactgcg tcgtctgattc cgaccgtggattgcagcggcgacattagcagctatccgctggtgatttttcaggtgaccc gttttaaatgc ggcgcagcagcgctgggtgcgggtgtgcagcataacctgagcgatggcgtgagcagcctg cattttattaa cacctggtctgatattgcgcgtggcctgaccattgcggtgccgccgtttattgatcgtac cctgattcgtg cgcgtgatccgccggtgccggtgtttgaacatgtggaatattatccgccgccgcagctgg cgagcgatagc cgtattcaggaactggcgaccggtccgcgtgcgagcaccaccgcggtgctgaaaattacc ccggaacagct gtctcagctgaaagcgaaagcgaacaacgaaggcagcacctatgaaattctggccgcgca tatttggcgta ccgcgtgcaaagcgcgcggtctgaccaacgatcagagcaccaaactgtatgtggcgaccg atggccgtagc cgcctgattccgccgctgccgccgggttatctgggcaacgtggtgtttaccgcgaccccg attgcggaaag cagcaacctgcagagcgaaccgctgaccaacagcgcgaaacgtattcataacgcgctggc ccgtatggata acgattatctgcgtagcgcgctggattatctggaaattcagccggatctgagcgcgctgg ttcgtggcccg cgtcattttgcgagcccgaacctgaacattaacagctggacccgtctgccgtttcatgat gcggattttgg ctggggccgtccgattcatattggtccggcgattattctgtatgaaggcaccgtgtatgt gctgccgagcc cgggcaaagatcgcaccctgagcctggccgtgtgcctggatgcggatcatatgccgctgt ttcagcgtttt ctgtatgatttctaa

SEQ ID NO: 46

PRT

>K-mutant_HQT (K210A/K219A)

MKITVKETAMVRPAQPTPTKRLWNSNLDLLVARIHILTVYFYKPNGSANFFDTRVLKEAL SNVLVSFYPMA GRIARDEEGRIEIDCNGEGVLFVEAESDSSVDDFGDFAPSLELRRLIPTVDCSGDI SSYPLVIFQVTRFKC GAAALGAGVQHNLSDGVSSLHFINTWSDIARGLTIAVPPFIDRTLIRARDPPVPVFEHVE YYPPPQIASDS RIQEIA.TGPPA.STTAVLKITPEQLSQLKAKANNEGSTYEIIAAHIWRTACKARGLTND QSTKLYVATDGRS RLIPPLPPGYLGNWFTATPIAESSNLQSEPLTNSAKRIHNAIARMDNDYLRSALDYLEIQ PDLSALVRGP RHFASPNLNINSWTRLPFHDADFGWGRPIHIGPAIILYEGTVYVLPSPGKDRTLSIAVCL DADHMPLFQRF LYDF-

SEQ ID NO: 47

PRT

>Cc4CL2

MILLPNSPEFVFAFLGASFRGAISTMANPYFTSAEVIKQAKASNAKLIITQGWYVEKVMD YACENGVKWC IDSAPEGCLRFSELTEADEREMLDVEISPEDWALPYSSGTTGLPKGVMLTHKGLVTSVAQ QVDGENPNFY IHNQVMMCVLPLFHIYSLNSILLCGLPAGTTILIMQKFDIIPFLELIQKYKVTTGPFVPP IVLAIAKSPEV DKYDLSSVKTVMSGAAPLGKELEDAVRTKFPKAKLGQGYGMTEAGPVLAMCSAFAKDPFE VKSGGCGSWR NAEMKIVDPETGSSLPRNQPGEICIRGDQIMKGYLDDPEATKATIDEDGWLHTGDVGYID EDDELFIVDRL KELIKYKGFQVAPAELEALLLAHSDISDAAWPMKDDAAGEVPVAFWKSKDSNITEDEIKE YIKKQVIFY KRINRVFFVDAIPKSPSGKILRKDLRARLAAGVPK-