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WO2012126789A1 - Production de formes d'acide chlorogénique et d'acide dactylifrique - Google Patents

Production de formes d'acide chlorogénique et d'acide dactylifrique Download PDF

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Publication number
WO2012126789A1
WO2012126789A1 PCT/EP2012/054506 EP2012054506W WO2012126789A1 WO 2012126789 A1 WO2012126789 A1 WO 2012126789A1 EP 2012054506 W EP2012054506 W EP 2012054506W WO 2012126789 A1 WO2012126789 A1 WO 2012126789A1
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Prior art keywords
acid
coa
hct
enzyme
hqt
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James Mccarthy
Laura LALLEMAND
Andrew Mccarthy
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Nestec SA
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Nestec SA
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine

Definitions

  • 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.
  • 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.
  • the phenolics generally consist of a benzenoid ring bearing at least one hydroxyl substituent.
  • 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.
  • 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
  • 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.
  • vanillic acid 3-OCH 3 4-OH ferulic acid
  • 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:
  • ester e.g. CGAs
  • amide conjugates respectively.
  • Chlorogenic acids 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).
  • CGA 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.
  • C caffeic acid
  • F ferulic acid
  • 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
  • Rosaceae e.g.
  • 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).
  • 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.
  • 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.
  • 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).
  • Shikimate esters also named dactylifric acids
  • Palmae plants e.g. palm, date, endive
  • 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 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.
  • HCAs and CGAs have attracted focus because they can influence food properties such as colour, taste and aroma.
  • 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).
  • 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).
  • CGAs and other HCA derivatives are significant components of diets rich in plant-based food such as cereals, legumes, oilseeds, fruits, vegetables and beverages.
  • 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.
  • coffee represents the richest source of CGAs and, interestingly, it has the highest antioxidant activity (Clifford, 1999; Wang et ah, 2009).
  • 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).
  • 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.
  • 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 is catalytically active in vivo in the microorgan
  • 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • compositions 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.
  • one or more of the enzymes is immobilised on or covalently bound to a matrix.
  • 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.
  • 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.
  • CQAs caffeoylquinic acids
  • FQAs coumaroylquinic acids
  • FQAs caffeoylshikimic acids
  • CSAa coumaroylshikimic acids
  • FSAs coumaroylshikimic acids
  • FSAs feruloylshikimic acids
  • diCQAs dicaffeoylquinic acids
  • 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.
  • 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.
  • 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.
  • the substrates comprise a hydroxycinnamoyl-CoA thioester, and an acyl acceptor.
  • the acyl acceptors include quinic acid, shikimic acid, or a combination thereof.
  • the substrates comprise a mixture of coumaroyl-CoA, caffeoyl-CoA, cinnamoyl-CoA, feruloyl-CoA, or sinapoyl-CoA.
  • the product comprises a mono-, di-, tri- or tetraester species of chlorogenic or dactylifric acid.
  • 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.
  • 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.
  • 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.
  • the first chlorogenic acid or dactylifric acid species is catalytically converted into a second chlorogenic acid or dactylifric acid species.
  • 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.
  • 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.
  • the exogenous chlorogenic acid or dactylifric acid species directly or indirectly provide a desirable flavour attribute, aroma attribute, or colour attribute to the product.
  • 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.
  • 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.
  • the exogenous chlorogenic acid comprises 3,4, caffeoylquinic acid but is substantially free of 5-feruloylquinic acid.
  • 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.
  • 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.
  • transgenic plant comprising a heterologous gene for an expressible exogenous enzyme that is a Coffea HCT or HQT; wherein the plant expresses the enzyme.
  • the transgenic plant produces less lignin than an equivalent plant that does not comprise the heterologous gene.
  • 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.
  • F ig . 1 S id e-chain reactions involving hydroxycinnamoyl-CoA thioesters (Adapted from Strack et al, 1997).
  • 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).
  • 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).
  • Fig. 5 Analysis of GST-HQT following expression and affinity chromatography.
  • Panel (A) 12 % SDS-PAGE.
  • 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.
  • 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.
  • 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.
  • Fig. 8 Chromatogram and SDS-PAGE analysis of CcHCT from size-exclusion chromatography.
  • Fig. 9 SDS-PAGE analysis of HCT proteolysis.
  • C Limited proteolysis of the methylated derivative of CcHCT.
  • Fig. 1 1 SDS-PAGE analysis of His6-SUM03-CcHQT during the purification procedure.
  • PDA photodiode array
  • Fig. 14 Absorption spectra of the CoA thioester products measured using HPLC- PDA.
  • Fig. 15 CoA thioester production by recombinant N/4CL2 with various HCAs and CoA in the presence of Mg-ATP.
  • Enzyme 0.05 ⁇ N/4CL2
  • 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.
  • Fig. 16 A representative reaction scheme showing forward and reverse reactions catalysed by HCT and/or HQT.
  • 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);
  • Panel (C): Quinic and shikimic acids, t 10 min (sample 769).
  • Fig. 21 HPLC profile of HCT reactions with feruloyl-CoA and quinic/ shikimic acid.
  • Panel B shows the absorption spectrum of the putative feruloylshikimic acid peak.
  • 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);
  • Panel (C): Quinic and shikimic acids t 2 min (sample 721).
  • 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.
  • 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 ⁇ Reactions were started by adding 1 ⁇ HCT/ HQT/ water as a control.
  • 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 ⁇ HCT (A) or 0.1 ⁇ HQT (B).Panel (A): HCT; Panel (B): HQT. Symbols: Squares: pH 6.0; Triangles: pH 7.0; Plain line: pH 8.0.
  • 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).
  • 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 ⁇ HQT.
  • 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).
  • 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 / ⁇ ': 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 ⁇ : H153A mutant (samples 1324 / 1340); Panels J and J': H154A mutant (samples 1330 / 1346); and Panels K / K': H154N/A155L
  • 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 ⁇ enzyme. Samples were analysed after overnight incubation using HPLC-PDA with the acetonitrile method.
  • 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 ⁇ enzyme. Samples were analysed after overnight incubation using HPLC-PDA with the acetonitrile method.
  • Fig. 39 HPLC profiles of the reaction profiles synthesized by 4CL and HCT or HQT from coumaric or caffeic acid and various acyl donors.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Nicotiana tabacum is preferred in addition to Coffea spp.
  • the recombinant enzymes 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the gene encoding the recombinant enzyme is engineered to provide a purification tag on the N-terminus of the recombinant enzyme.
  • tags include hexahistidine (His 6) tags which are well-known in the art.
  • the recombinant enzyme is readily adapted for in vitro production of chlorogenic and/or dactylifric acid species, preferably in commercially-useful quantities.
  • 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.
  • 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.
  • the recombinant enzymes e.g., 4CL, HCT and/or HQT can be used in combination to produce useful chlorogenic acid species and related esters.
  • 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.
  • 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).
  • HCA hydroxycinnamic acid
  • CoA coenzyme A
  • ATP adenosine triphosphate
  • the recombinant enzyme preferably is catalytically active in vitro from at least pH 6.0 to pH 7.5.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • recombinant HCT can catalyse the production of a HCA-CoA thioester that is a di-, tri- or higher CGA or related ester.
  • 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.
  • HCT at least in the presence of CoA, and/or one or more HCA-CoA
  • HCT 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the invention provides mutant plant HCT and HQT enzymes.
  • 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.
  • 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.
  • the amino acid sequences are at least 91 , 92, 93, 94, 95, 96, 97, 98, 99% or 100% identical.
  • 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.
  • Figure 16 shows a typical reaction catalysed by the HCT and/or HQT enzymes.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • amino acids responsible for binding substrates and for catalysis as well as those residues closely associated with those important features.
  • 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.
  • 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, R
  • 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.
  • 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, A
  • 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.
  • the mutant enzymes feature other properties that make them advantageous relative to the wild-type enzymes on which they are based.
  • 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.
  • 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.
  • 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.
  • the mutant enzyme is more resistant to proteolysis than the wild-type enzyme, particularly in a recombinant expression system, such as a recombinant microorganism.
  • 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.
  • Rubiaceae e.g. coffee, plums
  • Rosaceae e.g. apple, pear, peach
  • Solanaceae e.g. tobacco, tomato, potato, eggplant
  • Asteraceae e.g.
  • HCTs 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.
  • 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).
  • 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.
  • 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.
  • the mutant HCT has a preference for shikimic acid species as a substrate relative to the wild-type HCT.
  • a mutant HQT prefers quinic over shikimic acid as an acyl acceptor. In other embodiments, the preference may be reversed.
  • 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.
  • 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.
  • the mutant HCT more proficiently produces diCQA than the wild-type HCT from which it is derived.
  • 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.
  • 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.
  • 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.
  • 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.
  • RTD ready-to-drink
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • a composition comprising a synthetic chlorogenic acid species is added to a coffee product before the completion of manufacture.
  • 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.
  • 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.
  • 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.
  • a blend of two or more chlorogenic acid species may be more useful.
  • 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.
  • 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.
  • 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.
  • CQAs caffeoylquinic acids
  • FQAs coumaroylquinic acids
  • FQAs caffeoylshikimic acids
  • FSAs coumaroylshikimic acids
  • FSAs feruloylshikimic acids
  • diCQAs dicaffeoylquinic acids
  • 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.
  • the food product may be a coffee- flavoured product, even if the product contains no coffee per se.
  • the chlorogenic acid species may help to form the flavour profile for a simulated coffee flavouring or other artificial flavouring.
  • 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.
  • the food product comprising exogenous one or more chlorogenic acid or dactylifric acid species.
  • the chlorogenic acid or dactylifric acid species are produced in vitro.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions 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.
  • the compositions comprise substrates and cofactors sufficient for the production of one or more chlorogenic acid species or dactylifric acid species.
  • 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.
  • the acyl acceptor generally comprises quinic or shikimic acid species, or a combination thereof. Other acyl acceptors may be used as well.
  • one or more of the enzymes is immobilised on or covalently bound to a matrix.
  • immobilised enzymes can conveniently be included with a kit for assembly of the final composition by the end user.
  • 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.
  • composition can also be adapted for in vitro production of a HCA-
  • 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
  • the invention provides methods of producing a chlorogenic acid or dactylifric acid species in vitro.
  • 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.
  • 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.
  • 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.
  • CQAs caffeoylquinic acids
  • FQAs coumaroylquinic acids
  • FQAs caffeoylshikimic acida
  • FSAs coumaroylshikimic acids
  • FSAs coumaroylshikimic acids
  • diCQAs dicaffeoylquinic acids
  • the method includes an additional step wherein a HCA-CoA thioester is produced using a 4CL enzyme.
  • the method can also include additional substrates such as CoA, and one or more chlorogenic acid or dactylifric acid species.
  • additional substrates such as CoA, and one or more chlorogenic acid or dactylifric acid species.
  • HCT can remodel CGAs or DAs in the presence of HCA-CoA and/or CoA to form further CGAs and/or DAs.
  • 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.
  • 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.
  • methods of producing a chlorogenic acid or dactylifric acid species in vitro 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.
  • 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.
  • 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.
  • improvements in forage digestibility or saccharification efficiency were offset by poor growth and reduced biomass production.
  • 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.
  • 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.
  • tissue specific promoters can be used to increase the CGA production in specific
  • One promoter for these late stages of leaf maturation is the coffee cysteine proteinase inhibitor 4 promoter.
  • 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.
  • 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).
  • a fusion partner/ polypeptide tag Esposito et ah, 2006
  • a refolding Li et ah, 2004.
  • 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).
  • 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 ⁇ -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.
  • 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).
  • 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.
  • Coffea canephora HCT and HQT 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).
  • GTP Technologies GTP Technologies
  • 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 ⁇ g/mL kanamycin.
  • Rosetta 2(DE3) cells (Novagen) were transformed with each plasmid and plated on LB-agar medium containing 30 ⁇ g/mL kanamycin and 34 ⁇ g/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.
  • PBS phosphate buffered saline
  • 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).
  • 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).
  • reaction mixtures were made in a final volume of 50 ⁇ as follows: 1 ⁇ of plasmid DNA template, 5 ⁇ of PCR buffer (lOx), 1 ⁇ of forward and reverse primers (10 pmol/ ⁇ ), 200 ⁇ dNTP mix (10 mM) and 0.3 ⁇ (2.5 u) of High Fidelity PCR enzyme mix (Fermentas) or Pfii DNA polymerase (Stratagene).
  • Fermentas High Fidelity PCR enzyme mix
  • 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).
  • 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).
  • Primer 5 ' agcactagggatccatgaaaatcgaggtgaaggaatcg (BamHI) (SEQ ID NO: l)
  • 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.
  • 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
  • the best buffer composition was 50 mM Tris-HCl pH 7.4, 500 mM NaCl, 20 mM imidazole, 2 mM BME and 10 % glycerol.
  • 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.
  • 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.
  • TEV Tobacco Etch Virus
  • HCT Cleaved HCT was recovered by subtractive chromatography using the same nickel column.
  • the protein sample was concentrated to 500 ⁇ 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.
  • 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 K210A/K217A (K) mutant CcHCT
  • 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).
  • 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 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).
  • 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.
  • 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 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.
  • 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.
  • the reaction mixture was composed of 0.25 iL DNA template, 2.5 fL lOx DNA polymerase buffer, 0.5 ⁇ _, dNTP, 1 ⁇ _, forward (F) and 1 iL reverse (R) mutagenic primers at 15 pmol ⁇ L (Table 6), 0.5 ⁇ _, Pfu Turbo polymerase and completed to 25 fL with milliQ water.
  • 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).
  • 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.
  • CcHCT 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.
  • 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.
  • 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.
  • 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.
  • McHCT/ CcHQT Coffea canephora hydroxycinnamoyl-CoA shikimate/ quinate hydroxycinnamoyltransferases
  • 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,
  • High-performance anion-exchange chromatography coupled to pulsed electrochemical detection is another useful technique for the analysis of these acids (Rogers et ah, 1999).
  • 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 ⁇ , 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.
  • 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.
  • DA dual absorbance
  • 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.
  • 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 NaI3 ⁇ 4PC)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.
  • methanolic extracts of green coffee beans proved to be a convenient source of the major coffee CGAs.
  • 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 ⁇ ) and a 10-fold dilution was analysed by HPLC-PDA (data not shown).
  • A absorbance
  • C concentration
  • 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.
  • reaction volumes of up to 150 ⁇ L, 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.
  • 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.
  • HCT and HQT catalyse the transfer of hydroxycinnamoyl moieties from CoA to quinic or shikimic acid.
  • 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).
  • 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.
  • 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 ⁇ 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.
  • SBP Substrate Binding Pockets
  • 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.
  • 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.
  • 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.
  • 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).
  • CSA caffeoylshikimic acid
  • CcHQT was incubated with caffeoyl-CoA and quinic or shikimic acid
  • CcHCT and CcHQT are therefore active only towards the alicyclic quinic and shikimic acids.
  • the new compound has a typical CGA absorbance spectrum (Fig. 2 IB).
  • 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.
  • CcHCT clearly prefers shikimic over quinic acid.
  • CcHCT is also much less efficient with feruloyl-CoA in the presence of quinic acid.
  • CcHQT is able to use all three CoA thioesters equally with both quinic and shikimic acids.
  • HCT can form three probable shikimate esters of caffeic and coumaric acids, while with feruloyl-CoA only a single shikimate ester is formed.
  • CcHQT forms only a single shikimate ester with all CoA thioesters tested.
  • CGAs are known to be unstable at basic pH and stable at acidic pH
  • CcHCT and CcHQT were also incubated with 3,5-diCQA and CoA.
  • 5-CQA and caffeoyl-CoA are detected as indicated from the 5-CQA standard and the absorbance spectra (Fig. 27A, C).
  • no reaction product was observed after overnight incubation (data not shown).
  • 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.
  • 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.
  • both proteins there appears to be a complete conversion of the caffeoyl-CoA substrate.
  • the K-mutant HCT catalysed the formation of all three caffeoylshikimic acids produced by native CcHCT (data not shown).
  • 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.
  • 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.
  • 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.
  • 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.
  • these mutants fully transformed the caffeoyl-CoA substrate provided into caffeic 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.
  • 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.
  • 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.
  • 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.
  • the triple mutant also produced a small amount of free caffeic acid.
  • the enzymes were incubated with 5-CQA/ 5-FQA and CoA/ caffeoyl-/ feruloyl-/ coumaroyl-CoA thioesters.
  • 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 H154A mutant was incubated with 5-FQA and CoA or diverse acyl-CoA thioesters (Fig. 38).
  • the HI 54 mutants seem to favour diCQA formation.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Panel E shows results of an initial reaction with 4CL alone and 0.5mM caffeic acid as substrate.
  • 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.
  • HQT converts caffeoyl-CoA (produced by 4CL) to 5-CQA. The conversion is more efficient with 10 mM than 0.5 mM quinic acid.
  • 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.
  • 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.
  • 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.
  • the enzymes will be used to catalyze the production of CGA and DA species.
  • 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.
  • a food product in the form of a soluble coffee product is to be manufactured according to procedures described above.
  • 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.
  • Crystallography 65 500-509. Cle, C, L. M. Hill, R. Niggeweg, C. R. Martin, et al. (2008). "Modulation of chlorogenic acid biosynthesis in Solanum lycopersicum; consequences for phenolic accumulation and UV- tolerance.” Phytochemistry 69(11): 2149-2156.
  • Arabidopsis thaliana comprises one rare, sinapate-activating and three commonly occurring isoenzymes.” Proc Natl Acad Sci U S A 101(7): 2209-2214.
  • dicaffeoyltartaric acids are selective inhibitors of human immunodeficiency virus type 1 integrase.
  • thaliana is a 3 '-hydroxylase of phenolic esters, a missing link in the phenylpropanoid pathway.” Journal of Biological Chemistry 276(39): 36566-36574.

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Abstract

L'invention concerne des compositions et des procédés de préparation de formes d'acide chlorogénique et d'acide dactylifrique ainsi que des précurseurs thioester de CoA activés. L'invention concerne également des enzymes HCT, HQT et 4CL de plantes recombinantes et mutantes pour la préparation de tels composés in vitro ou in vivo. Des produits alimentaires, tels que des produits à base de café, comprenant des formes d'acide chlorogénique et d'acide dactylifrique exogènes sont également décrits.
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US10442610B2 (en) 2014-03-11 2019-10-15 Starbucks Corporation Pod-based restrictors and methods
CN116640786A (zh) * 2023-05-22 2023-08-25 湖南农业大学 一种绿原酸合成关键酶基因hct-45178及其应用
WO2024182557A3 (fr) * 2023-02-28 2025-01-09 Compound Foods Inc. Boissons de type café alternatives

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EP0916267A2 (fr) 1997-11-03 1999-05-19 Societe Des Produits Nestle S.A. Produit et procédé d'extraction
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