Comparative metabolic activity related to flavonoid synthesis in leaves and flowers of Chrysanthemum morifolium in response to K deficiency

K limitation could decrease the flavonoids content in many Chinese traditional herbs. These results may be caused by the influence of K deficiency on the synthesis pathway of flavonoids. In this paper, we aim to study the influence of K deficiency on secondary metabolites and activities of phenylalanine ammonia lyase (PAL), 4-coumarate coenzyme A ligase (4CL) and cinnamate 4-hydroxylase (C4H) enzymes in the process of flavonoid synthesis in Chrysanthemum morifolium Ramat. The results show that K deficiency decreased the flavonoid and chlorogenic acid contents slightly in flower of C. morifolium while the same effect was obtained on C4H and PAL. Total flavonoids content increased with the content of cinnamic acid and p-coumaric acid in the plant under K deficiency, respectively. The regression equations between content of flavonoids and cinnamic acid/ p-coumaric acid should be expressed in term of linear effects (R²?=?0.9809, P?<?0.001 for cinnamic acid and R²?=?0.9929, P?<?0.0001 for p-coumaric acid, respectively). But the effect of phenylalanine on flavonoids should be expressed in term of quadratic pattern (R²?=?0.9375, P?<?0.05). There were two kinds principal components from chorismate to coumaryl CoA synthesis process, principal 1 was the substrate (phenylalanine, cinnamic acid, p-coumaric acid and PAL) and principal 2 was the enzyme (4CL, C4H), the principal 1 was the domination principal in both K deficient (88.36% and 10.57%) and sufficient treatments (88.17% and 9.64%), however, the factor loading (correlation coefficient of measured targets and the corresponding principal component) in principal 1 was opposite under different K application.     

Bioconversion of Pinoresinol Diglucoside and Pinoresinol from Substrates in the Phenylpropanoid Pathway by Resting Cells of Phomopsis sp.XP-8

Pinoresinol diglucoside (PDG) and pinoresinol (Pin) are normally produced by plant cells via the phenylpropanoid pathway. This study reveals the existence of a related pathway in Phomopsis sp. XP-8, a PDG-producing fungal strain isolated from the bark of the Tu-chung tree (Eucommiaulmoides Oliv.). After addition of 0.15 g/L glucose to Phomopsis sp. XP-8, PDG and Pin formed when phenylalanine, tyrosine, leucine, cinnamic acid, and p-coumaric acid were used as the substrates respectively. No PDG formed in the absence of glucose, but Pin was detected after addition of all these substrates except leucine. In all systems in the presence of glucose, production of PDG and/or Pin and the accumulation of phenylalanine, cinnamic acid, or p-coumaric acid correlated directly with added substrate in a time- and substrate concentration- dependent manner. After analysis of products produced after addition of each substrate, the mass flow sequence for PDG and Pin biosynthesis was defined as: glucose to phenylalanine, phenylalanine to cinnamic acid, then to p-coumaric acid, and finally to Pin or PDG. During the bioconversion, the activities of four key enzymes in the phenylpropanoid pathway were also determined and correlated with accumulation of their corresponding products. PDG production by Phomopsis sp. exhibits greater efficiency and cost effectiveness than the currently-used plant-based system and will pave the way for large scale production of PDG and/or Pin for medical applications.     

Heterologous production of flavanones in<Emphasis Type="Italic"> Escherichia coli</Emphasis>: potential for combinatorial biosynthesis of flavonoids in bacteria

Chalcones, the central precursor of flavonoids, are synthesized exclusively in plants from tyrosine and phenylalanine via the sequential reaction of phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate:coenzyme A ligase (4CL) and chalcone synthase (CHS). Chalcones are converted into the corresponding flavanones by the action of chalcone isomerase (CHI), or non-enzymatically under alkaline conditions. PAL from the yeast Rhodotorula rubra, 4CL from an actinomycete Streptomyces coelicolor A3(2), and CHS from a licorice plant Glycyrrhiza echinata, assembled as artificial gene clusters in different organizations, were used for fermentation production of flavanones in Escherichia coli. Because the bacterial 4CL enzyme attaches CoA to both cinnamic acid and 4-coumaric acid, the designed biosynthetic pathway bypassed the C4H step. E. coli carrying one of the designed gene clusters produced about 450 μg naringenin/l from tyrosine and 750 μg pinocembrin/l from phenylalanine. The successful production of plant-specific flavanones in bacteria demonstrates the usefulness of combinatorial biosynthesis approaches not only for the production of various compounds of plant and animal origin but also for the construction of libraries of "unnatural" natural compounds. Dedicated to Professor Sir David Hopwood.     

Role de la phenylalanine dans la biosynthese des flavonoides et acides “cinnamique” dans les tiges volubiles de Periploca Graeca

Phenylalanine is the precursor of the cinnamic acids and coumarins in the stems and leafs of P. graeca L. Esterification of p-coumaric acid by quinic acid is required before oxidation to chlorogenic acid. In our experiments, we did not obtain radioactive flavonols from ¹⁴C phenylalanine. PAL activity varies as a result of light and temperature in the same manner as the level of flavonoids (especially the phenolic acids). This enzyme, therefore, plays a regulatory role in the synthesis of these phenolic substances. The variation in PAL activity during illumination does not follow the same course as described for other plants.     

Production of plant-specific flavanones by Escherichia coli containing an artificial gene cluster

In plants, chalcones are precursors for a large number of flavonoid-derived plant natural products and are converted to flavanones by chalcone isomerase or nonenzymatically. Chalcones are synthesized from tyrosine and phenylalanine via the phenylpropanoid pathway involving phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate:coenzyme A ligase (4CL), and chalcone synthase (CHS). For the purpose of production of flavanones in Escherichia coli, three sets of an artificial gene cluster which contained three genes of heterologous origins--PAL from the yeast Rhodotorula rubra, 4CL from the actinomycete Streptomyces coelicolor A3(2), and CHS from the licorice plant Glycyrrhiza echinata--were constructed. The constructions of the three sets were done as follows: (i) PAL, 4CL, and CHS were placed in that order under the control of the T7 promoter (P(T7)) and the ribosome-binding sequence (RBS) in the pET vector, where the initiation codons of 4CL and CHS were overlapped with the termination codons of the preceding genes; (ii) the three genes were transcribed by a single P(T7) in front of PAL, and each of the three contained the RBS at appropriate positions; and (iii) all three genes contained both P(T7) and the RBS. These pathways bypassed C4H, a cytochrome P-450 hydroxylase, because the bacterial 4CL enzyme ligated coenzyme A to both cinnamic acid and 4-coumaric acid. E. coli cells containing the gene clusters produced two flavanones, pinocembrin from phenylalanine and naringenin from tyrosine, in addition to their precursors, cinnamic acid and 4-coumaric acid. Of the three sets, the third gene cluster conferred on the host the highest ability to produce the flavanones. This is a new metabolic engineering technique for the production in bacteria of a variety of compounds of plant and animal origin.     

Soybean root growth inhibition and lignification induced by p-coumaric acid

The effects of 0.25–2 mM p-coumaric acid, a phenylpropanoid metabolite with recognized allelopathic properties, were tested on root growth, cell viability, phenylalanine ammonia-lyase (PAL) activities, soluble and cell wall-bound peroxidase (POD) activities, hydrogen peroxide (H₂O₂) level and lignin content and its monomeric composition in soybean (Glycine max (L.) Merr.) roots. At ≥0.25 mM, exogenously supplied p-coumaric acid induced premature cessation of root growth, increased POD activity and lignin content and decreased the H₂O₂ content. At ≥0.5 mM, the allelochemical decreased the cell viability and PAL activity. When applied jointly with PIP (an inhibitor of the cinnamate 4-hydroxylase, C4H), 1 mM p-coumaric acid increased lignin content. In contrast, the application of MDCA (an inhibitor of the 4-coumarate:CoA ligase, 4CL) with p-coumaric acid did not increase lignin content. The lignin monomeric composition of p-coumaric acid-exposed roots revealed a significant increase of p-hydroxyphenyl (H) and guaiacyl (G) units. Taken together, these results suggest that p-coumaric acid's mode of action is entry via the phenylpropanoid pathway, resulting in an increase of H and G lignin monomers that solidify the cell wall and restrict soybean root growth.     

Production of Plant-Specific Flavanones by Escherichia coli Containing an Artificial Gene Cluster

In plants, chalcones are precursors for a large number of flavonoid-derived plant natural products and are converted to flavanones by chalcone isomerase or nonenzymatically. Chalcones are synthesized from tyrosine and phenylalanine via the phenylpropanoid pathway involving phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate:coenzyme A ligase (4CL), and chalcone synthase (CHS). For the purpose of production of flavanones in Escherichia coli, three sets of an artificial gene cluster which contained three genes of heterologous origins—PAL from the yeast Rhodotorula rubra, 4CL from the actinomycete Streptomyces coelicolor A3(2), and CHS from the licorice plant Glycyrrhiza echinata—were constructed. The constructions of the three sets were done as follows: (i) PAL, 4CL, and CHS were placed in that order under the control of the T7 promoter (P_T7) and the ribosome-binding sequence (RBS) in the pET vector, where the initiation codons of 4CL and CHS were overlapped with the termination codons of the preceding genes; (ii) the three genes were transcribed by a single P_T7 in front of PAL, and each of the three contained the RBS at appropriate positions; and (iii) all three genes contained both P_T7 and the RBS. These pathways bypassed C4H, a cytochrome P-450 hydroxylase, because the bacterial 4CL enzyme ligated coenzyme A to both cinnamic acid and 4-coumaric acid. E. coli cells containing the gene clusters produced two flavanones, pinocembrin from phenylalanine and naringenin from tyrosine, in addition to their precursors, cinnamic acid and 4-coumaric acid. Of the three sets, the third gene cluster conferred on the host the highest ability to produce the flavanones. This is a new metabolic engineering technique for the production in bacteria of a variety of compounds of plant and animal origin.     

Photocontrol of chlorogenic acid biosynthesis in potato tuber discs

The appearance of phenylalanine ammonia-lyase activity and the accumulation of chlorogenic acid in potato tuber discs are stimulated by illumination with white light, whereas the appearance of cinnamic acid 4-hydroxylase activity is unaffected by illumination. The photosensitive step in chlorogenic acid biosynthesis may be by-passed by treatment of discs with exogenous supplies of cinnamic acid, whereas treatment of discs with phenylalanine does not isolate the photosensitive step. Therefore, the site of photocontrol of chlorogenic acid biosynthesis in potato tuber discs is the reaction catalysed by phenylalanine ammonia-lyase. Cinnamic acid 4-hydroxylase activity in vitro is unaffected by p-coumaric acid, caffeic acid or chlorogenic acid. Phenylalanine ammonia-lyase activity in vitro is sensitive to inhibition by cinnamic acid. The in vitro properties of the two enzymes are also consistent with the hypothesis that phenylalanine ammonia-lyase rather than cinnamic acid 4-hydroxylase is important in the regulation of chlorogenic acid biosynthesis in potato tuber discs.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> modular coculture system for resveratrol glucosides production

In bio-based fermentation, the overall bioprocess efficiency is significantly affected by the metabolic burden associated with the expression of complete biosynthetic pathway as well as precursor and cofactor generating enzymes into a single microbial cell. To attenuate such burden by compartmentalizing the enzyme expression, recently synthetic biologists have used coculture or poly-culture techniques for biomolecules synthesis. In this paper, coculture system of two metabolically engineered Escherichia coli populations were employed which comprises upstream module expressing two enzymes converting para-coumaric acid into resveratrol and the downstream module expressing glucosyltransferase to convert the resveratrol into its glucosidated forms; polydatin and resveratroloside. Upon optimization of the initial inoculum ratio of two E. coli populations, 92 mg resveratrol glucosides/L (236 µM) was produced i.e. achieving 84% bioconversion from 280 µM of p-coumaric acid in 60 h by 3 L fed batch fermentor. This is the report of applying coculture system to produce resveratrol glucosides by expressing the aglycone formation pathway and sugar dependent pathway into two different cells.     

Phenylalanine ammonia lyase and cinnamic acid hydroxylases as assembled consecutive enzymes on microsomal membranes of cucumber cotyledons: Cooperation and subcellular distribution

1. Cooperation between phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and cinnamic acid hydroxylases was investigated using microsomal fractions from cotyledons of cucumber (Cucumis sativus L.). The interpretations were based on experiments which demonstrate a limited exchange between the pool of cinnamic acid formed by the membrane-bound phenylalanine ammonia-lyase and the cinnamic acid pool external to the enzyme-membrane system. 2. The extent of cooperation between the microsomal enzymes was proved to be influenced by treatment of the cotyledons with light. On exposure to UV-light, which is known to enhance greatly the soluble phenylalanine ammonia-lyase activity in cell cultures, differential effects on the levels of microsomal and soluble phenylalanine ammonia-lyase, and of cinnamic acid hydroxylases, were observed. The time course of the enzyme activities and their cooperation in vitro after treatment of the cotyledons with light were studied. 3. The extent of cooperation in vitro was found to vary depending on the concentration of L-phenylalanine. 4. Homogenates obtained from etiolated cotyledons of Cucumis sativus in the absence of Mg²⁺ were fractionated by sucrose density gradient centrifugation and examined for phenylalanine ammonia-lyase, cinnamic acid o-hydroxylase, cinnamic acid o-hydroxylase, and several marker enzymes. Ammonia-lyase activity was highest in fractions with 25% sucrose, in which primarily smooth endoplasmic reticulum is localized. Hydroxylase activities co-occur with phenylalanine ammonia-lyase in these fractions (density=1.100 g/cm³), and also in fractions at higher densities (d=1.12–1.13 and 1.15 g/cm³).Abbreviations PAL L-phenylalanine ammonia-lyase - Tris tris-(hydroxymethyl)aminomethane - EDTA ethylenediamine tetraacetic acid - ATPase ATP phosphohydrolase     

Metabolism of aromatic acids by Polyporus hispidus

When grown on glucose as principal carbon source the culture medium of Polyporus hispidus was found to contain phenolic acids, including p-coumaric and caffeic acids. ¹⁴C-Studies indicated that phenylalanine is converted to cinnamic acid as well as to phenylpyruvic acid and tyrosine in cultures. Cell-free preparations of mycelium contained phenylalanine and tyrosine ammonia-lyse activities and were capable of effecting the hydroxylation of cinnamic, p-coumaric and benzoic acids.     

Exogenous application of L-phenylalanine and ferulic acid enhance phenylalanine ammonia lyase activity and accumulation of phenolic acids in pea (Pisum sativum) to offer protection against Erysiphe pisi

Phenylalanine ammonia lyase (PAL) activity was measured using HPLC in pea leaves following exogenous application of L-phenylalanine and ferulic acid. Treatment with different concentrations (50, 100, 150 ppm) of L-phenylalanine caused increased activity of PAL activity in comparison to control. In pea leaves treated with 50 ppm L-phenylalanine, maximum PAL activity was observed after 72 h of treatment. Application of ferulic acid first reduced PAL activity at lower concentration (50 ppm) but it further increased at higher concentrations of the compound (100 and 150 ppm) in pea leaves compared to control. Minimum PAL activity was 0.19 nM cinnamic acid/min/g fresh wt after 24 h at 50 ppm and then increased with time. Treatment with both compounds significantly increased the accumulation of phenolic acids and salicylic acid and reduced conidial germination of Erysiphe pisi on pea leaves. They were equally effective at 100 and 150 ppm in reducing conidial germination. Conidial germination on L-phenylalanine-treated leaves was 26% after 24 h and that on ferulic acid treated leaves 34% compared to control (46%). Foliar application of different concentrations of L-phenylalanine increased the level of ferulic acid in the leaves of pea plants. Maximum enzyme activity in terms of the accumulation of cinnamic acid (79.3 and 83.5 μg/g fresh wt) was observed following the application of L-phenylalanine after 24 and 48 h respectively. At 50 ppm, cinnamic acid accumulation in pea leaves was 35.6 and 39.4 μg/g fresh wt and 74.3 and 86.5 μg/g fresh wt at 100 ppm.     

Cinnamic acid production using <Emphasis Type="Italic">Streptomyces lividans</Emphasis> expressing phenylalanine ammonia lyase

Cinnamic acid production was demonstrated using Streptomyces as a host. A gene encoding phenylalanine ammonia lyase (PAL) from Streptomyces maritimus was introduced into Streptomyces lividans, and its expression was confirmed by Western blot analysis. After 4 days cultivation using glucose as carbon source, the maximal level of cinnamic acid reached 210 mg/L. When glycerol (30 g/L) was used as carbon source, the maximal level of produced cinnamic acid reached 450 mg/L. In addition, using raw starch, xylose or xylan as carbon source, the maximal level of cinnamic acid reached 460, 300, and 130 mg/L, respectively. We demonstrated that S. lividans has great potential to produce cinnamic acid as well as other aromatic compounds.     

Properties of phenylalanine ammonia-lyase extracted from Cucumis sativus hypocotyls

Some of the in vitro properties of PAL from gherkin hypocotyls were investigated. No metal ion requirement for this enzyme could be demonstrated but there were indications that PAL was a sulphydryl enzyme. Kinetic analysis suggested that PAL exhibited negative homotropic cooperativity. Two K_m values were determined, these were K_m^H, 2·9 × 10^?4 M and K_m^L, 4·3 × 10^?5 M. Strong inhibition of the enzyme was exerted by d-phenylalanine, trans-cinnamic acid, o-coumaric acid, gallic acid, quercetin and kaempferol. Kinetic studies on the inhibition patterns of these compounds established that d-phenylalanine linearized the curvilinear kinetics, trans-cinnamic acid and o-coumaric acid acted as competitive inhibitors whilst gallic acid, quercetin and kaempferol acted as mixed inhibitors. Using a number of desensitization techniques PAL was partially desensitized to inhibition by the mixed inhibitors. These results led to the conclusion that PAL may have a regulatory role in phenol, coumarin and flavonoid biosynthesis.     

Modulation of L-phenylalanine ammonia-lyase by pathway intermediates in cell suspension cultures of dwarf French bean (Phaseolus vulgaris L.)

The increase in extractable phenylalanine ammonia-lyase (PAL;EC 4.3.1.5.) activity induced in French bean cell suspension cultures in response to treatment with autoclaved ribonuclease A was inhibited by addition of the phenylpropanoid pathway intermediates cinnamic acid, 4-coumaric acid or ferulic acid. The effectiveness of inhibition was in the order cinnamic acid>4-coumaric acid>ferulic acid. Cinnamic acid also inhibited the PAL activity increase induced by dilution of the suspensions into an excess of fresh culture medium. Addition of low concentrations (<10^-5M) of the pathway intermediates to cultures at the time of application of ribonuclease gave variable responses ranging from inhibition to 30–40% stimulation of the PAL activity measured at 8 h. Following addition of pathway intermediates to cultures 4–5 h after ribonuclease treatment, rapid increases followed by equally rapid declines in PAL activity were observed. The cinnamic acid-stimulated increase in enzyme activity was unaffected by treatment with cycloheximide at a concentration which gave complete inhibition of the ribonuclease-induced response. However, cycloheximide completely abolished the subsequent decline in enzyme activity. Treatment of induced cultures with -aminooxy--phenylpropionic acid (AOPPA) resulted in increased but delayed rates of enzyme appearance when compared to controls not treated with the phenylalanine analogue. The results are discussed in relation to current views on the regulation of enzyme levels in higher plants.Abbreviations AOPPA -aminooxy--phenylpropionic acid - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5) - AOA -aminooxyacetic acid     

Cloning, expression and characterization of phenylalanine ammonia-lyase from Rhodotorula glutinis

The industrial-scale production of phenylalanine ammonia-lyase (PAL) mainly uses strains of Rhodotorula. However, the PAL gene from Rhodotorula has not been cloned. Here, the full-length gene of PAL from Rhodotorula glutinis was isolated. It was 2,121 bp, encoding a polypeptide with 706 amino acids and a calculated MW of 75.5 kDa. Though R. glutinis is an anamorph of Rhodosporium toruloides, the amino acid sequences of PALs them are not the same (about 74 % identity). PAL was expressed in E. coli and characterized. Its specific activity was 4.2 U mg^?1 and the k _cat/K _m was 1.9 × 10⁴ mM^?1 s^?1, exhibiting the highest catalytic ability among the reported PALs. The genetic and biochemical information reported here should facilitate future application in industry.     

Construction of a Corynebacterium glutamicum platform strain for the production of stilbenes and (2S)-flavanones

Corynebacterium glutamicum is an important organism in industrial biotechnology for the microbial production of bulk chemicals, in particular amino acids. However, until now activity of a complex catabolic network for the degradation of aromatic compounds averted application of C. glutamicum as production host for aromatic compounds of pharmaceutical or biotechnological interest. In the course of the construction of a suitable C. glutamicum platform strain for plant polyphenol production, four gene clusters comprising 21 genes involved in the catabolism of aromatic compounds were deleted. Expression of plant-derived and codon-optimized genes coding for a chalcone synthase (CHS) and a chalcone isomerase (CHI) in this strain background enabled formation of 35 mg/L naringenin and 37 mg/L eriodictyol from the supplemented phenylpropanoids p-coumaric acid and caffeic acid, respectively. Furthermore, expression of genes coding for a 4-coumarate: CoA-ligase (4CL) and a stilbene synthase (STS) led to the production of the stilbenes pinosylvin, resveratrol and piceatannol starting from supplemented phenylpropanoids cinnamic acid, p-coumaric acid and caffeic acid, respectively. Stilbene concentrations of up to 158 mg/L could be achieved. Additional engineering of the amino acid metabolism for an optimal connection to the synthetic plant polyphenol pathways enabled resveratrol production directly from glucose. The construction of these C. glutamicum platform strains for the synthesis of plant polyphenols opens the door towards the microbial production of high-value aromatic compounds from cheap carbon sources with this microorganism.     

Engineering the oleaginous yeast Yarrowia lipolytica for high-level resveratrol production

Resveratrol is a plant secondary metabolite with multiple health-beneficial properties. Microbial production of resveratrol in model microorganisms requires extensive engineering to reach commercially viable levels. Here, we explored the potential of the non-conventional yeast Yarrowia lipolytica to produce resveratrol and several other shikimate pathway-derived metabolites (p-coumaric acid, cis,cis-muconic acid, and salicylic acid). The Y. lipolytica strain expressing a heterologous pathway produced 52.1 ± 1.2 mg/L resveratrol in a small-scale cultivation. The titer increased to 409.0 ± 1.2 mg/L when the strain was further engineered with feedback-insensitive alleles of the key genes in the shikimate pathway and with five additional copies of the heterologous biosynthetic genes. In controlled fed-batch bioreactor, the strain produced 12.4 ± 0.3 g/L resveratrol, the highest reported titer to date for de novo resveratrol production, with a yield on glucose of 54.4 ± 1.6 mg/g and a productivity of 0.14 ± 0.01 g/L/h. The study showed that Y. lipolytica is an attractive host organism for the production of resveratrol and possibly other shikimate-pathway derived metabolites.     

The effects of infection by Phytophthora infestans on the control of phenylpropanoid metabolism in wounded potato tissue

During the first 24 h of in vitro incubation of excised potato tuber (Solanum tuberosum L.) discs, the appearance of phenylalanine ammonia-lyase (PAL; EC 3.4.1.5) and the accumulation of chlorogenic acid are both stimulated by infection with Phytophthora infestans (Mont.) de Bary. Whereas in control tissue the level of PAL reached a stable plateau value after 40 h, in infected tissue it subsequently rose again, in one experiment, as the fungal mycelium developed. In the infected but not the control tissue, the level of chlorogenic acid subsequently fell to about to about 20% of its maximum after 50 h. The time courses of increases in cinnamic acid 4-hydroxylase (CA4H; EC 1.14.13.11; 0–60 h) and of caffeic acid acid o-methyltransferase (COMT; EC 2.1.1.42; 0–160 h) are not altered by fungal infection. If the discs are restored to the tuber environment immediately after excision, by placing them inside a host tuber, the activity of PAL as well as those of CA4H and COMT remained at the constant low endogenous level for at least 60 h, irrespective of whether the discs had first been inoculated with P. infestans. The increase in PAL may not be an obligatory feature of the P. infestans/potato compatible interaction but dependent on an underlying wound response. The experiments provide further evidence that PAL is the rate limiting step of chlorogenic acid biosynthesis in potato tuber discs.Abbreviations PAL phenylalanine ammonia-lyase - CA4H cinnamic acid 4-hydroxylase - COMT caffeic acid o-methyltransferase - CGA chlrogenic acid (5-o-caffeoylquinic acid) - gfwt gram fresh weight