Manipulation and Engineering of Metabolic and ...

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Current Pllarmaceutica/ Design, 2013, 19, 6186-6206

Manipulation and Engineering of Metabolic and Biosynthetic Pathway of Plant Polyphenols Anthony Ananga 1·*, Vasil Georgiev 1' 2 and Violeta Tsolova 1 1

Centerfor Viticulture and Small Fniil Research, College of Agriculture and Food Sciences,Florida A & M University, 6505 Mahan Drive, Tallahassee, Fl 32317 USA; 1Laboratory of Applied Biotechnologies, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy ofSciences. 139 "Ruski" Blvd., 4000 Plovdiv, Bulgaria Abstract: Polyphenols are bioactivc natural molecules biogenerated through secondary metabolic pathways. They are involved in different functions in the ecology, physiology, and biochemistry of plants such as chemical defense against predators and in plant-plant interferences. These compounds are known to have important biological activities related to human health such as antioxidant action, antiinflammatory and antimicrobial effects. The immense health benefits as well as use of many polyphenolic compounds as anti-infective agents against human pathogens have heightened the need for continuous supply of rare and expensive secondary metabolites. It has been demonstrated that the chemical structure of dietary polyphenols, such as the number and position of hydroxyl groups, can change their biological properties and bioavailability. This review focuses on prospects fo r, and success in metabolic engineering, including manipulation of structural regulatory genes to develop plants with tailor-made, optimized levels and composition ofpolyphenols.

Keywords; Anthocyanins, fl avono ids, tannins, metabolic engineering, biosyn thesis. INTROD UCTION Polyphenolic compounds are natural products of secondary metabolism in plants. They have di fferent fu nctions in the plant ecology, physiology, and biochemistry [ I], includ ing form ation of p ig ments in fl owers and fru its, and contributing to b iotic and abioti c stress to lerance, and in pollen fertility [2, 3]. Other functions include structu ra l roles in different sup porting or protective tissues, being invo lved in defense strategies and signaling [4 , 5]. Common polyphenols include lignans, stilbenes, fl avono ids (anthocyanins, flavono ls, flavanols, tann ins, chalconcs), and pheno lic alcohols and acids. Polyphenolic compounds are among the most s tud ied phytochem icals in plants, owing to their properties in fru its, juices, and fenne nted beverages such as color, browning, bitterness and astringency [6]. Polyphenols are widely available in th e human diet and have been shown to p lay a major role in influencing human health [7, 8]. T hese compounds are among natural products in p lants that suppress the oxidatio n of low-density lipoprotein (LDL), thus making them potent antioxidants [9], hence the h igh correlation between their intake and reduced risk o f coronary heart disease [9]. Phenolic compound s have the ability to exhib it a number of ce ll protective actions such as modu lation and induction o f hum an cell receptors [ I OJ. On a daily basis, each p erson consum es substantial amounts o f tlavonoids and other polyphenols from fru its, vegetab les, food supplements, tea and wine. Studies have d emonstrated that dietary polyphenols help in figh ting maj or human d iseases [I 0 , 11]. Importantly flavonoids as an example of polyphenols, are excellent modu lators of major ABC d rug transporters [ 12- 15]. Overall , polyphenols can change the overall pharmacokinetics, including drug absorption, penetration and elimination by modulating the funct ions of ABC transporters [ 16, 17]. The bio logical properties and bioavailability of dietary polyphenols can change based on the influence o f the ir chem ical structure [18 19]. Some of the important var iables that can affect their biological properties include the number and position of hydroxyl •Address correspondence to this author at the Center for Viticulture and Small Fruit Research, College of Agriculture and food Sciences, Florida A & M University, 6505 Mahan Drive, Tallahassee, Fl 323 17 USA; Tel:+ 1-850-4 12-5 197; E-mail : [email protected] 1873-4286/13 $58.00+.00

groups and the structure of the heterocycle in ftavonoids. Further, acylation and glycosylatio n can influence ab sorption in the gut and bioavailability [20, 2 1]. In the last two decades, genomic approaches have been used extensively to broaden our understanding of the m echan ism underly ing the b iosynthesis of po lyphenolic compounds, specia lly on the regulation o f gene expression by external facto rs [22]. T his has included cloning and characterization of genes that code for enzym es fo und in the tlavonoid pathway [23]. As a result, m etabolic eng ineering of flavonoids, has been investigated extensive ly by d ifferent scienti fic groups for the modification o f fl owers in p lant species [22-31 ], m otivated by the w ide impact that polyphenols have on the quality of products in the agricultural sector. T his article reviews concepts of biosynlhesis and metabolic engineering of polyphenols as secondary metabolites. We review th e use of metabolic eng ineering in the biosynthesis of different polyphenols in plants, and d iscuss prospects for upscaling th e technology to direct tailor-mad e biosynthesis of desired secondary m etabolites. We recognize th e confusion surrounding the definition, exclusion and inclusion of compounds w ithin the meaning of polyphenols, however, this review examines these w ithin the context and relevance to application.

DEFINITION O F PLANT POLYPH ENOLS Polyphenols constitute one of the most abundant, complex and widely d istributed groups of plant secondary metabolites. Known as "vegetable tannins", these plant substances have been m ainly associated with th eir abilities to tan an imal hides into leather (the term "tannin" com es from the French word "tan", whi ch have been u sed to describe the powdered oak bark extracts u sed in leather industry) [32, 33]. With the advancement of ch emistry, during the second half of twentieth century, Wh ite [34] formulated th e fi rst d efin ition of tannins as po lyphenols: "All the vegetable tannins are phenolic,

but they constitute only a proportion of the polyphenols (i.e. polyhydric phenols) present in plants". According to White, polypheno ls sh ould have molecu lar masses between 500 and 300 0 Da and large number of phenolic groups, a llowing them to form crosslinked structures with collagen [34). Swain and Bate-Smith modified this definition , and according to them polyphenols are "water-

soluble phenolic compounds having molecular weights between 500

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Ma11ip11/atio11 a11d E11gi11eeri11g of Metabolic and Biasy11thetic Pathway

and 3000 Da and, besides giving the usual phenolic reactions, they have special properties such as the ability to precipitate alkaloids, gelatin and other proteins from solution" [35]. Decades later, Haslam expanded this definition by suggesting molecular weight range from 500 to 3000-4000 Da and focusing on the attention on their structure and polyphenolic character as: "Polyphenols, per I 000 relative molecular mass, possess some 12-16 phenolic groups and 5-7 aromatic rings" [36]. It is obvious that to be classified as polyphcnols the phenolic compounds should have more than one arene rings, substituted with at least one or more hydroxy groups, which significantly restrict the structures to be covered by the original definition. Following the expanded definition of Swain and Bate-Sm ith , Haslam recognized only three classes of natural polyphenols: I) condensed proan thocyanidins; 2) galloyl and hexahydroxyd iphenoyl esters and their derivatives; and 3) phlorotannins [37]. The last group, a phlorotannins, has been found only in redbrown algae [32, 33, 37]. According lo these understandings flavonoids, flavanoids-derived oligomers and polyhydroxystilbcnes cannot be classified as polyphcnols because of their low molecular weight and the lack of tanning action. However, from a chemical po int of view, those compounds have more than one phenolic moi ety and can undergo further polymerization to more complex structures with tannin-like properties. Definition also excludes some isolated proanlhocyanidins with large molecu lar masses up 20000 Da and clearly pronounced tanning properties. In addition, not all substances having tanning action can be classified as tannins and moreover, many compounds without possessing any tann ing properti es, but having similar phenolic structural characteristics w ith tann ins have been counted with the tann ins [38]. To distinguish tannin compounds from other plant derived phenolic compounds, Khanbabaee and Ree [38] introduced a modified definition: "Tannins are polyphenolic secondmy metabolites of higher plants, and are either galloyl esters and their derivatives, in which galloyl moieties or their derivatives are al/ached to a variety of polyol-, catechin- and triterpenoid cores (gallotannins, ellagilannins and complex tannins), or they are oligomeric and polymeric proanthocyanidins that can possess different inteiflavanyl coupling and substitution patlerns (condensed tannins)". According to this definition, tannins arc considered without a doubt as natural products of secondary metabolism of higher plants. Tannins are classified as a part of large group of plant polyphenols, but also th e definition permits some compounds with triterpenoid structure (complex tannins) to be classified as tannins [38]. Many classes of plant secondary metabolites, including tcrpenoids and alkaloids can contain one or more phenolic unit attached, but that does not mean that they can be classified as polyphenols. It is obviously that definition of polyphenols must be made separately from that of tannins. To distinguish the polyphenols from some tcrpenoids and tyrosine-derived alkaloids, Quideau et al. [33] proposed a new, revised definition: "The term ''polyphenol" should be used to define plant secondary metabolites derived exclusively from the shikimate-derived phenylpropanoid and/or the polyketide pathway (s), featuring more than one phenolic ring and being devoid of any nitrogen-based functional group in their most basic structural expression". Strictly, no monophenolic structures can be classified as polyphenols. Further, the biosynthelic origin of polyphenols has been clearly stated as products, derived from sh ikimate-dcrived phenylpropanoid and/or polyketide pathways. The large number and the wide diversity of chemical structures make the classification of polyphenols confusing even among scientists. This can be seen in single phenolic acids and many plant derived monophenolic compounds being loosely referred as polyphenols in formulations of cosmetic, pharmaceutical and food products, as well as by many scientists [32, 33, 39, 40]. These definitions are relevant in the context of what plant metabolites can be targeted fo r engineering via metabolic pathways. This review adopts the definition ofQuideau et al. [33] in relevant discussions.

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BIOSYNTHESIS OF POLYPHENOLS IN P LANTS The most important pathways, involved in plant polyphenol biosynthesis are shikimate and phenylpropanoid pathways, whereas polyketide (acetate-malonate) pathway is of less significance in higher plants. Shikimatc pathway is the common route, lead ing to production of chorismatc, which is a precursor for postchorismate pathways responsible for tryptophan and phenylalanine/tyrosine biosynthesis [41, 42]. These important biosynthesis pathways arc presented in bacteria, fungi and plants but are missing in animal s [4 1]. The shikimate pathway includes seven metabolic steps beginning w ith the condensation of erythrose-4 -phosphate with phosphoenolpyruvate and ending with the synthesis of chorismate (Fig. 1). This pathway has been studied in details in bacteria and yeasts, but the regulatory mechanisms in higher plants remains poorly understood [41, 43]. Detailed information on individual biosynthctie steps and the enzymes involved are available [4 1, 44]. The phenylpropanoid pathway is a major source of secondary metabol ites in plants [42]. It begins with three reactions leading lo the conversion of phenylalanine to 4-coumaroyl CoA [42, 45]. These steps, known as "general phenylpropanoid pathway" (Fig. 2) directs the carbon flow in plant cells from shikimate pathway (phenylalanine) to various branches of the phenypropanoid metabolism (flavonoid, volatile phenolics, hydrolyzable tannins, coumarins and lig nans) [42]. The pathway starts with the action of enzyme phenylalanine ammonia lyase (PAL), which catalyzes deamination of phenylalanine into trans-cinnamate [46]. Because of its key function, PAL is one of the best-characterized plant enzymes. It was fo und that several copies o f the PAL-encoding genes existed in genomes of different plant species [42]. However, it seems that only a single gene has been expressed in plant tissue, whereas the other members of the gene family remains effectively silenced [47]. The next two step s in phenylpropanoid biosynthesis are catalyzed by the enzymes cinnamate 4-hydroxylase (C4H) and 4-coumaroyl CoA-ligase (4CL) and this leads to the fo rmation of the intermediate product 4-coumaroyl CoA (Fig. 2). 4-coumaroyl CoA is the most important branch point in phenylpropanoid biosynthesis in plants, leading to the format ion of the most polyphenol structures. It is the direct precursor for flavonoid and sti lbcne biosynthesis (Fig. 3). Specific biosynthesis steps, leading to the formation of some major subclasses of polyphenols are discussed in later sections. Hyd rolyzable Tannins (Gallotannins, Ellagita nnins) and Ellagic Acid

llydro lyzable tann ins are a large group of water-soluble polyphenols, with a very restricted taxonomic distribution, found on ly in woody and herbaceous dicotyledonous plants. According to their chemical structure and the phenolic products released upon hydrolysis (gallic or ell agic acids), hydrolyzable tannins are generally subdivided into gallotannins and ellagitannins, respectively [48, 49]. The biosynthetic pathway of hydrolyzable tannins can be divided into three subsections (biosynthesis of simple galloylglucose esters, biosynthesis of complex gallotannins and biosynthesis of ellagitann ins), according to the typical chemical structures of the participating compounds [50]. Biosynthesis of simple galloylglueose esters starts with esterification of gallic acid and glucose catalyzed by UDP-glucose dependant glucosy ltransfcrase (UDP-GT) (Fig. 4). As a result, ft-glucogallin is formed [50]. The gallic acid, necessary for implementation of this reaction was derived from a branch of sh ikimate pathway by dehydrogenation of 5-dehydroshik imatc by the enzyme dehydroshikimate dehydrogenase (DSDG) [5 1] (Fig. 2). The biogenetic origin of gallic acid is unique, because this phenolic acid keep s the oxygen atoms of the alicyclic precursor, whereas in most of the other plant phenols, the hydroxyl groups are derived by direct oxygen insertion into the aromatic nucleus [5 I, 52]. At the next four steps, specifi c acy ltransferases converts the produced fi-glucogall in to 1,2,3,4,6-penta-0-galloyl-,B-Dglucopyranose (l ,2,3,4,6-pentagalloylglucose) (Fig. 4). In the first step, catalyzed by the enzyme ft-glucoga llin 0-galloyltransferase

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posphoenolpyruvate

erythrose-4-phosphate DAHP synthase

3-deoxy-D-arabino-heptulosonate-7-phosphate

1DHQS 3-dehydroquinate

j DHD-SDH

0

HO~OH

~ 3-dehydroshikimate. enol-form

HOY OH Gallic acid

=

3-dehydroshikimate

j DHD-SDH shikimate

l

SK

posphoenolpyruvate

shikimate-3-phosphate EPSP - -- - - - - - - l synthase

I

5-enolpyruvilshikimate-3-phosphate

l

cs

l

chorismate "-- - -

CM

ASa ASb

l

anthranilate PAT

5-phosphoribosylanthranilate

l

PAI

1-(o-carboxyphenylamino}-1-deoxy-ribulose-5-phosphate

!

IGPS

indole-3-glycerol phosphate TSa TSb

!

~ prephena t e l

j' ~· phenylpyruvate

PPA-AT

j PDH

4-hydroxyphenylpyruvate

PPY-AT

_u{, _____O_O_H ~ F'~'"'"l

Tryptophan Phenylalanine

ADH HPP-AT

B

Fig. (1). Schematic presentation of shikimate pathway in plants: DAHP synthase - 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase; DHQS - 3dehydroquinase; Dl-ID-SDH - 3-dehydroquinate dehydratasc- shikimate dehydrogenase; DSDG - dchydroshikimate dehydrogenase; SK - shikimate kinase; EPSP synthase - 5-enolpyruvylshikimate-3-phosphate synthase; CS - chorismate synthase; ASa and ASb - anthranilatc synthase a and b; PAT - phosphoribosyJanthranilate transferase; PAI - phosphoribosylanthranilate isomerasc; IGPS - indole-3 -glycerol phosphate synthase; TSa and TSb - tryptophan synthase; CM chorismate mutase; PDT - prephenate dehydratase; PP A-AT - prcphenate aminotransferase; PDH - prephcnate dehydrogenase; PPY-A T - phenylpyruvate aminotransferase; ADT - arogenate dehydratase; A DH - arogenate dehydrogenase; HPP-A T - 4-hydroxyphenylpyruvate aminotransferase.

(/JG-ATl) [53], /3-glucogallin exerted a dual functional ity as both acyl acceptor and as acyl donor [50]. Substitution of glucose hydroxyl groups is not randomly distributed but follows extremely speci fi c pattern, leading to formation of 1,6-digalloylglucose, 1,2,3trigalloylglucose, 1,2,3,6-tetragalloylglucose and 1,2,3,4,6-pentagalloylglucose (Fig. 4). In all steps j3-glucogallin is used as acyl donor. The reactions are catalyzed by the enzymesj3-glucogallin 0galloyltransferase (/JG-AT I), 13-glucogallin: 1,6-d i-0-galloyl-,8-Dglucose 2-0-galloyltransferase (/JG-AT2), ,8-glucogall in: 1,2,6-tri0-galloyl-,8-D -glucose 3-0-galloyltransferase (/JG-AT3) and ,8glucogallin: 1,2,3,6-tetra-O-galloylglucose 4-0-galloyltransferase (/JG-AT4), respective lly [53-55]. However, some unusual enzymes, cata lyzing direct transfer of galloyl residue between /3-glucogallin and free glucose or enzyme which used two molecu les 1,6digalloylglu cosc to produce 1,2,3-trigalloylglucose and 6-0galloylglucosc have been described [50]. The fina l galloylglucose

ester, 1,2,3,4,6-pentagalloylglucose is the key intermediate for which biosynthesis of complex gallotannins and biosynthesis of ellagitann ins begins. Complex gallotannins are produced by addition of further galloyl residue to 1,2,3,4,6-pentagalloylglucose by the enzymes known as galloyltransferases. The new galloyl residue usually substitute mhydroxyl group of linked galloyl unit and leads to formation of meta-digalloyl group (Fig. 5), which is the characteristic structure for meta-depsides (gallotannins) [38]. This process was catalyzed by the group of enzymes known as ,8-glucogallin-dependent galloyltransferases [56]. To date, five galloyltransferases, named A, B, C, D and E have been isolated and characterized from sumac (Rhus typhina L.) leaves [50, 56]. None of the isolated galloyltransferases has shown high substrate specificity, but each enzyme has shown preference for some substrate and trends to accumulate preferred

Manipulation and Engineering of Metabolic and Biosylllhetic Pathway

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0 v{,oH 2

phenylalanine

!

PAL

0

~OH cinnamate

!

0 JY{,oH HO

0

~OH

PAUTAL tyrosine

C4H

HO coumarate

!

4CL

0

~sJVCoA fl

4-coumaroyl-CoA

Fig. (2). Schematic presentation of general phenylpropanoid pathway: PAL - phenylalanine ammonia lyase; C4H - cinnamate 4-hydroxylase; 4CL - 4coumaroyl CoA-ligase; TAL- tyrosine ammonia lyase .

matonyl.CoA

m alonyl·COA

3x

------Stitbc ncs

!

3x

)

I

Chalconcs

6'-Oyc~O>___...

OH HO

F3'H

OH

trice tin

0 Apigenin

OH

0 luteolin

F3",5'H

Fig. (13). Biosynthesis of tlavanones, tlavones, and phlobaphenes: CHI - chalcone isomerase; DFR - dihydroflavonol reductase; F3'5 'H - tlavonoid-3',5' -

hydroxylase; F3 ' H - tlavonoid-3 '-hydroxy lase; FNS I and II - flavon synthses I and II, respectively. some other variant polymeric fo rms have also been reported [46, 82]. Flavan-4-o ls are produced by stereospecific conversion of corresponding fl avanones by the enzyme dihydroflavonol reductase (DFR) (Fig. 13). DFR belongs to the single-domain-reductase/ epimerasc/dehyd rogenase (RED) protein family and have been considered as key enzyme to both anthocyanidin and proanthocyanidin biosynthesis [46, 81, 82]. The polymerization of extension units (fl avan-4-ols) to condensed phlobaphenes is likely to be a nonenzymatic step (Fig. 13) [83, 84].

lsotlavones and lsotlavonoids lsofl avonoids have been principally found in legumes and in contrast to fl avonols o ccur only sporadically throughout the land plants. They have not been reported outside of the gy mnospenns and angiosperms [ l , 85]. Chemically iso fl avonoids are differenti-

ated from other flavonoids by the linking of the 8-ring to the C-3 rather than the C -2 position of the C-ring [46]. The entry point enzyme into the isoflavono id pathway, isoflavone synthase (IFS) catalyzes NADPH-dependent hydroxylation of the 2-position of the flavanone liquiritigenin with accompanying 8 -ring migration to yield 2.7.4 '-trihydro xyisoflavanone (Fig. 14). 2.7.4 '-trihydroxyisoflavanone is unstable intermed iates that can undergo furth er Cglycosylaton (catalyzed by g lycosyltransferase), 0 -methylation (catalyzed by isofl avone-0 -methyltransferase) or spontaneously dehydration to the corresponding isofl avones [86-89]. Al ternatively, corresponding 2-hyd roxyisofl avanone intermed iate cou ld be obtained by hydroxylation of fl avanone naringenin, which also can be used as substrate for IFS [82, 86]. The obtained isoflavones can undergo furth er mutilation by isofl avonc-0 -mcthyltransfcrase (IOMT), g lycosylation by g lycosyltransferases (GT) or h yd roxyla-

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Glu HOyYOH

r'i(oH

~OH

Hoy}yoH

~ --G_T_._~ 0 6'-deoxychalcone (isoliquiritigenin)

0 isoliquiritigenin-C-glucoside

!

!

CHI

OH

CHI

Glu HO

HO

OH

'-":

I ,,.::;

I 0

0

liquiritigenin-C-glucoside

liquiritigenin

!

IFS

H0% OH0

"" I

IOMT

,,.::;

Ho % o OH 1

""

I "' O

O

OCH3

2,7-dihydroxy-4'-methoxyisoflavanone

Glu

I

HO

!

IFS

0

GT

"'

,,.::; OH

OH

2, 7,4'-trihydroxyisonavanone

trihydroxyisonavanone-8-C-glucoside

!

!

HID

HID

Glu 0

HO

I

GT

"'

,,.::;

0

OH

puerarin

Glu-0%0

, ,__ I

I

O

l

isoformononetin

I "'

OH

,,.::;

daidzein-7-0-glucoside

IFR

~

""g:-__u

HOLJOl

HO

OCH3

vestitone

l

VR

HO % , , , _ _ I O

DMID

I "' HO HO

,,.::;

OCH3

7,2'-dihydroxy-4'-methoxyisoflavanol

HOL J O l

1~

"" ! J ; L OCH3

medicarpin

PTR

710 H o %

I "' HO

,,.::;

OCH3

vestitol

Fig. (14). Biosynthesis of isoflavones and isoflavonoids: GT - glycosyltransferase; C HI - chalcone isomerase; IFS - isoflavone synthase; IOMT - isoflavone0-methyltransferase; HID - 2-hydroxyisotlavanone dehydratase; 12'H - isotlavone-2'-hydroxylase; IFR - isotlavone reductase; VR - vestitione reductase; DMID - 7,2,-dihydroxy-4' -methoxyisoflavanol dehydratase; PTR - pterocarpan reductase. ti on b y isoflavone-2 '-hydroxy lase (12 ' H) (Fig. 14) [82, 86-88]. It was found in Lotus japonicas L. tha t 2'-hy droxyformononetin ca n be reduced to vestitone by the ac tion of isofl avone reductase (IFR), w hich are furth er reduced by vestitione reductase (YR) to 7,2' dihydroxy-4 ' -methoxyiso fl avano l [88). 7,2 '-dihydroxy-4 ' m ethoxyisoflavano l is th e p recu rsor for formation o f m edicarpin, catalyzed by 7,2,-d ihydroxy-4 ' -m e thoxyisofl avan ol d ehy d ratase

(DMID) (Fig. 14) [8 1, 82, 88). Medicarpin can also b e reduced by p terocarpan reductase (PTR), y ielding o f isoflavan vestitol [88].

Flavonols, Leucoanthocyanidins, Anthocyanidins, P roanthocyanidins (Condensed Tannins) F lavan oncs, an d specifically naringenin flavanone are key in termediates in flavonoid biosinthesys. Naringenine, a s well as eri-

Manipulation and Engineering of Metabolic and Biosynthetic Pathway

odictyol and 5,-hydroxyeriodictyol, which are products of its hydroxylation on C3' and C3 '/C5' positions catalyzed by F3 'H and F3 ' 5'H, respectively, can be modified by the further catalyzes by flavanone-3-/J-hydroxylase (F3H) to yields the corresponding dihydroflavonols (Fig. 15). In addition, dihydrokaempferol, produced by enzyme ox idation ofnatingenin can serve as substrate for F3'H and F3 '5' H to produce dihydroquercetin and dihydromyricetin, respectively (Fig. 15). Two genes encoding F3H, four encoding F3 ' H and only one encoding F3 '5 'H have been identified in grapes [72). It was found that the expression of F3'5 'II is higher than that of F3 'H. Moreover, the level and the type of expressed F3 ' H seems to be strongly specific in different varieties [72). The obtained dihydroflavonols could be further oxygenized by flavonol synthase (FLS) to corresponding flavonols (kaempferol, quercetin , myricetin) or reduced by the enzyme dihydroflavonol 4-reductase (DFR) to the corresponding leucoanthocyanidins. Despite the impo1tance of Oavonols as copigments in plants, little is known about the regulation of their biosynthesis. Existing data suggested that FLS is constitutively expressed in plants [84]. DFR has been considered to contribute to both anthocyanidin and proanthocyanidin biosynthesis, hence a good branch point to shift the metabolic flux in plants, either to anthocyanins, tlavonols or proanthocyanidin s fonnation [80, 81, 84]. Leucoanthocyanidins are the key intermediates between proanthocyanidins and anthocyanins biosynthesis. Anthocyanidin synthase (ANS) is the enzyme, responsible for transformation of leucoanthocyanidins to anthocyanins, whereas the leucoanthocyanidin reductase (LCR) yields to formation of flavan3-ols, which are the extension units for proanthocyanidin [81, 85] (Fig. 15). It was widely accepted that ANS was the first key enzyme that could redirect flavonoid flux into the anthocyanins formation, but recent research showed that its products, anthocyanidins, have significant role in proanthocyanidin production [72, 8 1] (Fig. 15). The enzyme anthocyanidin reductase (ANR), which reduced anthocyanidins to corresponding tlavan-3-ols have been found in Arabidopsis and Medicago [90, 9 1]. The enzyme showed a closely related sequence to dihydroflavonol reductase (DFR), cinnamoyl-CoA reductase, cinnamoyl alcohol dehydrogenase and vestitone reductase [8 1]. Curiously, ANR reduced anthocyanidin s to 2,3-cis-2R,3R-flavan-3-ols, whereas LRC reduce leucoanthocyanidins to 2.3-trans-2R,3S-tlavan-3-ols [81 ]. The action of both ANR and LRC could explain the different stereoisomers for flavonols and proanthocyanidin ex tension units, found in most plants [81 , 85]. Anthocyanins Anthocyanins are the major group of colored polyphenols, derived by the flavonoid biosynthesis pathway, with more than 635 structures isolated from natural sources [72, 92, 93]. They are glycosides of anthocyanidin s, since the last ones are inherently unstable under physiological conditions [72]. However, differen t anthocyanin structures are not equally distributed in nature. Thus, 90% of the naturally occurring anthocyanins (often referred as common anthocyanidins) are derived from on ly six anthocyanidins (cyanidin, delphinidin, pelargonidin, peonidin, malvidin and petunidin) [92). Anthocyanin biosynthesis has been studied in details in grapes, mainly because these compounds are essential for co lor formation in red wines [72]. UDP-glucose: Anthocianidin: Flavonoid glucosyltran sferases (UFGT) are the enzymes which catalyze the 0-glycosylation of anthocyanidin s or anthocyanins in plants (Fig. 16). The enzymes required UDP-activated sugars as the glycoside donors. Notably, both anthocyanidins and anthocyanins can be recognized as sugar acceptors [72). UFGT found in Vitis vinifera L. can catalyze only 0-glucosylation at the C3 position and often are referred 3-0-glu cosyltransferases (3-GT) [72] (Fig. 16). On the other hand, UFGT from other non-Vitis grapev ines as Muscadinia rotundifolia (Michx.) Small., Vitis labrusca L. and most of the hybrid varieties, 0 -glucosylation can be performed at both of the C3 and CS positions, yielding to fo rmation of anthocyanidin-3,5-0-

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diglucosides as well, which are characteristic compounds of those species [72, 80). However, the mechanism of glucosylation always involve first formation of anthocyanidin-3-0-glucosides and then if possible, their transformation to anthocyanidin-3,5-0-diglucosides [80, 92). However, research has demonstrated that this mechanism is completely different in roses (Rosa hybrida L.), where the rose glucosyltransferase (RhGT I) seems to catalyze first the fonnation of unstable anthocyanidin-5-0-dglucosides and then the fonnation of stable anthocyanidin-3,5-0-diglucosides (Fig. 16) [94]. Next step in anthocyanin biosynthesis in grape involve participation of enzymes 0 -methyltransferases (OMT), which are responsible for production of anthocyanins, containing aglycone of methylated anthocyanidins (peonidin, petunidin and malvidin) (Fig. 16). These compounds are obtain ed by methylation of hydroxyl groups, situated on B-rings of the cyanidin-3-0-glucoside, delphinidin-3-0glucoside and petunidin-3-0-glucoside (Fig. 16). Future modification of produced anthocyanins, yielding to more stable structures cou ld be performed by further acylation, catalyzed by different anthocyanin acyltransferases (ACT) [72, 80].

MOLECULAR METHOD FOR CONTROL AND MANIPULATION OF BIOSYNTHETIC PATHWAY IN SECONDARY METABOLITES: A MODEL FOR MODIFICATION OF POLYPHENOLS Metabolic Engineering Metabolic engineering refers to the science that integrates systematic analysis of metabolic and other pathways with molecular biological techniques to improve cellular properties by designing and implementing rational genetic modifications [95]. In that context, metabolic engineering deals with the measurement o f metabolic fluxes and eluc idation of their control as determinants of metabolic functi on and cell physiology [96]. According to Fridman and Pichersky, [97], specific secondary metabolites are otlen restri cted to a narrow set of species within a given group. However, metabolic engineering aims at modifying cellular metabolite composition, so that new compounds can be produced, existing compounds can be increased, and/or elimin ate undesirable compounds [97]. This can be achieved by either introducing novel genes or pathways, or enhancing or eliminating the expression of endogenous pathways [98]. The challenges and difficulties involved in genetic modification have been discussed [99- 103]. However, 'second generation ' GM crops and cell products that have traits beneficial for consumers, such as increased nutritional value, premium quality or low allergenicity are being developed [104]. Approaches have been taken to use metabolic engineering to improve amino acid and fatty acid content [I 05], as well as to synthesize therapeutic recombinant proteins [106- 108]. However, emphasis is on the use of plant cell cu ltures as potential matrices for the large-scale production of valuable compounds such as nutraceuticals, antioxidants, vitamins and flavours [ 109-112). In some cases, applications of plant metabolic engineering include biofuel production [11 3, 114] and phytoremediation [115 ). We di scuss these technologies with greater focus on metabolic engineering of secondary metabolites. Metabolic Engineering of Plant Secondary Metabolites The idea of increasing or adjusting concentrations of specific secondary metabolites as nutraceuticals to provide health benefits has gain ed widespread interest in recent years [98, 11 6). It is believed that plants that contain the correct amount of polyphenols as nutraceutical compounds may lead to the ingestion of sufficient healthful compounds, thus preventing humans from drastically changing their dietary habits. Notab ly, it has been established that the levels of polyphenolic compounds in plants vary signifi cantly among different varieties or cultivars. Even for the same variety in the same fi eld, different crop years might bring about more than a three-fold difference in poplyphenol levels [11 7]. Th e food, phar-

6200 Current Pharmaceutical Design, 2013, Vol. 19, No. 34

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Received: January 30, 2013

Accepted: February I 8, 2013

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