Biosynthetic Pathway and Metabolic Engineering of Plant

Subscriber access provided by READING UNIV

Review

Biosynthetic Pathway and Metabolic Engineering of Plant Dihydrochalcones Mwafaq Ibdah, Stefan Martens, and David R Gang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04445 • Publication Date (Web): 24 Nov 2017 Downloaded from http://pubs.acs.org on November 26, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

Journal of Agricultural and Food Chemistry

1

Biosynthetic Pathway and Metabolic Engineering of Plant Dihydrochalcones

2 3

Mwafaq Ibdah a*, Stefan Martens b, David R. Gang c

4 5

a

6

Yishay 30095, Israel

7

b

8

and Nutrition, Via E. Mach, 1 - 38010 San Michele all’Adige (TN), Italy

9

c

10

NeweYaar Research Center, Agriculture Research Organization, PO Box 1021, Ramat

Fondazione Edmund Mach, Centro Ricerca e Innovazione, Department of Food Quality

Institute of Biological Chemistry, Washington State University, PO Box 646340,

Pullman, WA 99164-6340, USA

11

*Corresponding Organization,

author: P.O.

Newe

Box

Yaar

1021,

Research

Ramat

Center,

Yishay,

Agricultural

30095,

Israel;

Research E-mail:

[email protected]; Telephone: +972-4-953-9509; Fax: +972-4-953-9509 12 13 14 15 16 17 18 19 20

1 ACS Paragon Plus Environment


21 22

Page 2 of 32

Abstract Dihydrochalcones are plant natural products containing the phenylpropanoid

23

backbone

and

derived

from

the

plant-specific

phenylpropanoid

pathway.

24

Dihydrochalcone compounds are important in plant growth and response to stresses and

25

thus can have large impacts on agricultural activity. In recent years, these compounds

26

have also received increased attention from the biomedical community for their potential

27

as anti-cancer treatments and other benefits for human health. However, they are

28

typically produced at relatively low levels in plants. Therefore, an attractive alternative is

29

to express the plant biosynthetic pathway genes in microbial hosts and to engineer the

30

metabolic pathway/host to improve the production of these metabolites. In the present

31

review, we discuss in detail the functions of genes and enzymes involved in the

32

biosynthetic pathway of the dihydrochalcones, and the recent strategies and achievements

33

used in the reconstruction of multi-enzyme pathways in microorganisms in efforts to be

34

able to attain higher amounts of desired dihydrochalcones.

35 36

Keywords: Dihydrochalcones, Biosynthesis, Biological activity, Metabolic engineering

37 38 39 40 41 42 43


Page 3 of 32


44

Introduction

45

Flavonoid compounds represent a highly different class of specialized plant

46

metabolites with more than 9000 structures, and dihydrochalcones (DHCs) define a major

47

sub-class of this group. Chemically, DHCs are open-chain flavonoids in which the two

48

aromatic rings are linked by a three-carbon α, β-saturated carbonyl system (Fig. 1). Like

49

other polyphenols, e.g. flavonoids, DHCs display a wide variation of hydroxyl and

50

glucosyl substitution patterns. For example, 3-hydroxyphloretin, phloridzin (phloretin-2'-

51

O-glucoside), trilobatin (phloretin-4'-O-glucoside), and sieboldin (3-hydroxyphloretin-4'-

52

O-glucoside) accumulate in diverse plant species, including Malus species (Fig. 2). 1-6

53

DHCs are perhaps best known as a class of compounds found in apple trees (Fig. 7

54

3) that have been intensively reviewed by Gosch et al.

but they are also occasionally

55

encountered in other edible plants. Importantly, they have been isolated from many

56

medicinal plants belonging to diverse plant families. DHC occurrence in general in the

57

plant kingdom has been recently intensively reviewed by Riviere.

58

diverse DHCs are presently known to be formed in over 46 plant families.

59

distributions are excessively heterogeneous in the plant kingdom, with members of this

60

compound class having been isolated and characterized from the Angiosperms and from

61

Pteridophytes. 8

8

Approximately 265 8, 9

DHC

62

DHCs, like many other classes of natural products, have been shown to play

63

different important roles in human health. High intake of apple fruits, which are rich in

64

DHCs, has been linked to lower risk of many degenerative diseases, particularly diabetes,

65

Alzheimer’s disease, and cardiovascular disease.

66

described the potential benefits of DHCs in human health, especially because of their

8, 10-13


Various investigations have


Page 4 of 32

67

antioxidant properties. Indeed, specific DHC compounds may be effective in preventing

68

different human physiological disorders, notably diabetes,

69

radical-involving disease, by inhibition of the formation of advanced glycation end-

70

products.

71

DHCs have also been reported to act as flavor enhancers and bitterness blockers with

72

various uses in the food, beverage and pharmaceutical industries. 17, 18

1

14

bone resorption,

15

and free

Also, DHCs have proven chemopreventative and antitumor activities.

16

73

DHCs are specialized metabolites (also called “secondary metabolites”) that

74

plants produce to protect themselves in their interactions with other organisms and the

75

environment. The physiological role for these compounds in planta appears to be largely

76

in defense mechanisms.

77

derivatives can also occurs when plants are faced with serious diseases, such as scab

78

and fire blight caused by the bacterium Erwinia amylovora. 20 Evidence for bioactivity of

79

DHCs as functional antioxidants was suggested by the lowering of oxidative stress of

80

apple leaves. 1

1, 2

The synthesis and accumulation of DHCs and their 19

81

As is the case for several natural products, the isolation of DHCs from different

82

plant species is limited by low production levels and the complexity of the mixtures

83

recovered from plants. Moreover, the total chemical syntheses of these structurally

84

complex metabolites are economically impractical in many cases. Therefore, an attractive

85

alternative is to express the plant biosynthetic pathway genes in microbial hosts such as

86

Escherichia coli, and Saccharomyces cerevisiae and to engineer the metabolic

87

pathway/host to improve the production of these metabolites.

88

offers many benefits over each field and plant cell cultivation because of the rapid growth

89

of microbes compared to plants, the convenience of genetic manipulation, and also the


21, 22

Microbial production

Page 5 of 32


90

well-established metabolic engineering tools developed to be used in microbes. In

91

addition, microbial biosynthesis is more environmentally friendly than chemical

92

synthesis, and can produce much more pure products from the culture than is obtainable

93

from plant tissues or plant cultures, thus allowing for simpler (and “greener”) purification

94

strategies. However, the functional reconstruction of plant biosynthetic pathways in

95

microbes and the application of microbial biosynthesis for the industrial production of

96

important compounds is still challenging. 21, 23, 24 It requires not only a full understanding

97

of the biochemical pathway of interest, but information about the interactions between

98

the pathway members (e.g. protein-protein interactions, steric hindrances, substrate

99

channeling, side reactions) as well as a strong foundational platform upon which to build

100

the production system (e.g. reduced/eliminated feedback inhibition of early pathway

101

steps, precursor availability within the production strains, high growth and production

102

capacity of the production strains, etc.). As an important foundational step for production

103

of DHCs in microbial culture systems, the biosynthesis of DHCs was recently

104

investigated, with genes for pathway members identified and recombinant enzymes

105

characterized. 25-31

106

The objective of this review article is to highlight the latest advances in DHC

107

biosynthetic research and the recent efforts in the microbial production of representative

108

compounds of the DHCs. The current challenges in and the potential of these approaches

109

are also briefly discussed.

110

Biosynthesis of DHCs

111

An Overview



112

The DHC pathway is a part of the large phenylpropanoid network, which

113

produces a range of other specialized metabolites, such as flavonoids, phenolic acids,

114

lignins, stilbenes, and lignans. The substitution pattern and the type of substituents

115

present on DHCs lead to the diversity within the chemical class.

116

From a biosynthetic point of view, and in contrast to the extensive literature on

117

flavonoid and chalcone biosynthesis, little information was available on the biosynthesis

118

of DHCs in plants until very recently (Table 1). Flavonoid compounds are

119

biosynthesized via the phenylpropanoid-acetate network. 32 Phenylalanine ammonia lyase

120

catalyzes the first step in this metabolic network: the conversion of L-phenylalanine to t-

121

cinnamic acid. Cinnamate 4-hydroxylase (C4H) then catalyzes the synthesis of p-

122

hydroxycinnamate (p-coumaric acid) from t-cinnamic acid. p-Coumaric acid is further

123

converted by p-coumarate:CoA ligase (4CL) to its coenzyme-A ester. From these central

124

intermediates, the pathway diverges into several side branches, each resulting in a

125

different class of phenolic compounds (Fig. 4). 33

126

The DHC pathway was originally thought to be a sub-pathway of the larger

127

flavonoid biosynthetic network, with the working hypothesis being that reduction of the

128

double bond of the propenyl linker occurred after the action of the type III polyketide

129

synthase known as chalcone synthase. However, work by us and by others clearly

130

demonstrated that DHCs belong to their own special network, being separated from

131

flavones, flavonols, flavanol, anthocyanins, proanthocyanidins, etc., by action of a double

132

bond reductase prior to the action of a CHS-like polyketide synthase (Fig. 4). 26, 29, 34

133

Significant recent advances in our understanding of DHC biosynthesis include

134

characterization of the formation of p-dihydrocoumaroyl-CoA from p-coumaroyl-CoA,


Page 6 of 32

Page 7 of 32


135

progress toward elucidating phloretin and other DHC derivative formation, the molecular

136

characterization of several genes encoding enzymes that modify the DHC core structures,

137

and analysis of enzyme function. Data are also starting emerge from knock-out studies of

138

DHC biosynthesis genes. However, there are still main areas where data are lacking. The

139

range of genes encoding secondary modification enzymes that have been characterized is

140

still limited compared to the great array of known DHC structures (Table 1). Moreover,

141

cDNAs or genes have not been described yet for some of the enzymes carrying out the

142

hydroxylation or the methylation of the core DHC structure.

143

Formation of p-dihydrocoumaroyl-CoA

144

The first committed enzyme in DHC biosynthesis is catalyzed by a double bond

145

reductase (DBR), which belongs to the medium-chain dehydrogenase/reductase (MDR)

146

superfamily and that catalyzes the reduction of the α, β unsaturated double bond of the

147

enoyl moiety in planta, such as p-coumaroyl-CoA. The resulting p-dihydrocoumaroyl-

148

CoA serves as the substrate for a CHS-like enzyme (perhaps canonical CHS in some

149

plant families) in subsequent substitution/modification reactions that produce DHCs (Fig.

150

4).

151

Several different enzymes from apple were suggested to catalyze this reduction of

152

the α, β unsaturated double bond of the enoyl moiety in planta. It had been assumed that

153

p-dihydrocoumaroyl-CoA is formed from p-coumaroyl-CoA by an NADPH-dependent

154

dehydrogenase (NADPH: p-coumaroyl-CoA oxidoreductase) or by a double bond

155

reductase. Gosch et al.

156

coumaric acid, radiolabeled malonyl-CoA and NADPH were incubated with protein

34

showed the formation of phloretin when the CoA ester of p-



Page 8 of 32

157

extracts from apple leaves. Enzyme assays with recombinant proteins from Psilotum

158

nudum showed that p-dihydrocoumaroyl-CoA can act as a precursor for phloretin. 35

159

To date, more than 1000 protein sequences have been identified as MDR

160

superfamily members, with a broad range of enzymatic activities. They are found in all

161

kingdoms of life and are involved in metabolism, regulatory processes, and protection

162

against cell damage. Despite their low sequence similarity, they have a similar size of 350

163

to 400 residues and a conserved overall structure formed by two domains, a cofactor

164

binding domain and a catalytic domain. While all MDRs use NAD(H) or NADP(H) as

165

cofactor, they can be divided into two classes with a different reaction mechanism: zinc-

166

containing and non-zinc-containing MDRs. 36, 37

167

Enone reductases are the best-characterized enzymes that can recognize and act

168

on the α,β unsaturated double bond of the enoyl moiety of specialized metabolites, such

169

as 3-methoxy-4-hydroxybenzalacetone, 4-hydroxybenzalacetone,

170

dehydro-diconiferyl aldehydes and p-coumarylaldehyde,

171

However, none of these enzymes have been shown to catalyze reduction of p-coumaroyl-

172

CoA or feruloyl-CoA. In Arabidopsis, a DBR specific for alkenals catalyzed the 7,8-

173

double

174

dihydrocompounds. 41 That enzyme was active with several of phenylpropanal substrates,

175

although phenylpropenoyl-CoA esters, including p-coumaroyl-CoA, were not tested as

176

substrates.

bond

reduction

of

phenylpropanal

substrates

39

to

38

coniferyl aldehydes,

and (+)-pulegone.

their

40

corresponding

177

An enoyl reductase-like (ENRL) enzyme that can generate p-dihydrocoumaroyl-

178

CoA from p-coumaroyl-CoA was cloned by Dare et al. 29 who showed in an RNAi-based


Page 9 of 32


179

study that reduction of the transcript levels of ENRL-3 in transgenic ‘Royal Gala’ led to a

180

66% decrease in the concentration of DHCs in the leaves in one silenced line.

181

The isolation of a cDNA for an NADPH-dependent hydroxycinnamoyl-CoA

182

double bond reductase gene was first reported from Malus domestica by us. 26 That gene

183

shared significant amino acid sequence homology to the Arabidopsis alkenal double bond

184

reductase.

185

hydroxycinnamoyl-CoA double bond reductase protein from M. domestica. 26 Similar Km

186

values toward p-coumaroyl-CoA and feruloyl-CoA, at 96 and 101 µM, respectively, were

187

determined for the plant-based and recombinant protein. p-Dihydrocoumaroyl-CoA and

188

dihydroferuloyl-CoA were found to be the in vivo products of this enzyme in apple

189

leaves, thus confirming its role as the first step in the DHC pathway and branch-point

190

enzyme off of the general phenylpropanoid network (Table 1).

191

excluded that other reductases play a similar role, e.g. the ENRL3/5 described by Dare et

192

al.

193

reduction can been performed by a apparently unrelated (yeast) enzyme, which is known

194

to have homologs in plants.

195

Formation of phloretin from p-dihydrocoumaroyl-CoA

29

41

Enzyme properties were also determined for the NADPH-dependent

or other as yet unidentified enzymes. Eichenberger et al.

26

21

Hence, it cannot be

demonstrate that the

196

The chemical structure similarity of p-coumaroyl-CoA and p-dihydrocoumaroyl-

197

CoA led to the assumption that CHS could utilize either molecule as substrate, with

198

condensation of three molecules of malonyl-CoA to form naringenin chalcone or

199

phloretin, respectively. Gosch et al.

200

catalyzed by a common CHS and not by a specific CHS-like enzyme. However, that is

201

yet to be determined across the plant kingdom.

34

suggested that the formation of phloretin is



Page 10 of 32

202

CHS catalyzes the first committed step in the flavonoid biosynthesis (via

203

naringenin chalcone) and potentially DHCs (via phloretin). The reaction catalyzed by

204

CHS includes one molecule of p-coumaryl-CoA for naringenin chalcone or one molecule

205

of p-dihydrocoumaroyl-CoA for phloretin as the starter substrate and three C2-units from

206

malonyl-CoA as the extender molecules (Fig. 4). 34, 42-44 CHS belongs to the superfamily

207

of enzymes called type III polyketide synthases (PKS) that also comprise stilbene

208

synthase, p-coumaroyltriacitic acid synthase, acridone synthase, pyrone synthase, and

209

bibenzyl synthase, among others.

210

best studied of the PKSs.

211

gene cloned for a flavonoid pathway enzyme. 47, 48 CHS sequences, and a series of CHS-

212

like sequence have been extensively studied in numerous plant species, e.g. M.

213

domestica,

214

Psilotum nudum,

215

identified about 650 CHS and CHS-like sequences in public sequence database.

216

number of sequences belonging to this class has ballooned since then, thanks to genome

217

sequencing efforts. GenBank now lists over 4250 genes as being annotated as CHS with

218

another 3450+ CHS-like genes being from the plant kingdom. It is likely (probable) that

219

most of these genes do not code for canonically functional CHSs, and that some may

220

play specific roles in biosynthesis of certain subfamilies of phenylpropanoids, such as

221

DHCs, in particular plant families. 44, 45

44

45, 46

42

CHS is structurally and mechanistically among the

The isolation of a cDNA for CHS represented the first

Solanum lycopersicum, 35

49

Medicaco sativa.

50

Petunia x hybrida,

and in strawberry (Fragaria x ananassa).

52

51

Austin and Noel 45

The

222

Recently, three genes of the CHS-superfamily (MdCHS1, MdCHS2, MdCHS3)

223

from M. domestica were functionally characterized. 44 All displayed kinetic parameters in

224

a range to be expected for CHS relative to its standard substrates. However, the three


Page 11 of 32


225

recombinant Malus CHSs converted cinnamoyl-CoA, p-coumaroyl-CoA, and p-

226

dihydrocoumaroyl-CoA substrates to their corresponding products with varying

227

conversion rates.

228

that had the ability to catalyze the formation of additional compounds beyond naringenin

229

chalcone, and thus could be viewed as perhaps being in the middle of new

230

functionalization.

44

These three enzymes appeared to be fully functional CHS enzymes

231 232

DHC Modification Enzymes

233

DHCs are substrates for a range of modification reactions, including

234

hydroxylation, glycosylation, prenylation, methylation, and polymerization. To date,

235

genes encoding proteins have been isolated that catalyze some of these conversions. For

236

example, two UDP-dependent glycosyltransferases (UGTs) from Oryza sativa (rice)

237

and Fagopyrum esculentum (buckwheat) were identified that are able to C-glycosylate

238

the 3'-position of a 2-hydroxyflavanone to form nothofagin (2',4,4',6'-tetrahydroxy-3-C-β-

239

D-glucopyranosyldihydrochalcone) (Fig. 5).

240

encoding an enzyme involved in the 3-hydroxylation of DHCs was accomplished. 55

54

53

Recently, the isolation of a cDNA

241

However, in vitro activities of recombinant proteins may not reflect their in vivo

242

activities. Factors such as the abundance of protein in relation to the potential substrate,

243

and involvement in the metabolic channeling affect in vivo activity. In several transgenic

244

experiments, endogenous flavonoid glucosyltransferase (GTs) have been shown to accept

245

substrates that are not naturally present in the recipient species, such as 6'-

246

deoxychalcones and isoflavonoids, suggesting that broad substrate acceptance for some

247

modification enzyme types may be common. 56-58



248

Page 12 of 32

DHCs O-Glycosyltransferase

249

cDNAs have been isolated for several UGTs with O-glycosylation activity on

250

phloretin. The recombinant proteins showed a wide substrate acceptance. 25, 59 In general,

251

UGTs characterized in flavonoid (and to date DHC) biosynthesis show high

252

regiospecificity but broad substrate acceptance, although there are some exceptions. 60-62

253

Several cDNAs encoding activities that can glycosylate the 2'-hydroxyl of

254

phloretin have been identified and characterized. MdPGT1 from M. domestica

255

glycosylates phloretin in the presence of UDP-glucose into phloridzin and accept only

256

phloretin as a substrate (Fig. 4).

257

UGT71K1 genes of M. domestica show activities against several types of flavonoids and

258

phenylpropanoids, including the DHC phloretin.

259

likely to be UDP-glucose: phloretin 2'-O-glycosyltransferase. 25, 27, 59, 63 The recombinant

260

Pyrus communis UGT71A16 and UGT71K2 proteins also showed wide regiospecificity,

261

adding glucose to the 2'-hydroxyl of phloretin and to position 5 of flavonoids, producing

262

monoglucosides. 25 The lack of phloretin in pear suggests that UGT71A16 and UGT71K2

263

accept other aglycones as substrates in vivo. Dianthus caryophyllus GT also showed

264

activity toward phloretin, although phloretin is not present in this plant species.

265

Recently, targeted downregulation of the apple phloretin-specific glycosyltransferase

266

UGT88F1 leads to changes in the concentration of a wide range of polyphenolic

267

compounds including the dihydrochalcone phloretin. 31

27

The recombinant proteins from the UGT71A15 and

25

However, their in vivo activities are

64

268

In contrast to these activities, a cDNA from M. domestica (Golden Delicious)

269

encoding a protein with UDP-glucose: phloretin 4'-O-glycosyltransferase (MdPh-4'-

270

OGT) activity was recently isolated and functionally characterized (Table 1, Fig. 4).


28

Page 13 of 32


271

The recombinant apple MdPh-4'-OGT was found to be position specific for its putative

272

substrate phloretin. It would accept trilobatin, phloridzin, quercetin, and naringenin as

273

substrates at lower efficiency. The Km value toward phloretin was 96 µM, and trilobatin

274

was found in the in vivo product. Unlike most of the GTs discussed previously, the

275

MdPh-4'-OGT showed strong catalytic efficiency with phloretin. 28

276 277 278

DHC C-Glycosyltransferase

279

C-Glycosides are characterized by their C-C bonds in which the anomeric carbon

280

of the sugar moieties is directly bound to a carbon atom of the aglycone. C-Glycosides

281

are unusually stable, as their C-C bonds are resistant to acid hydrolysis or glycosidase. A

282

diversity of plant species are known to accumulate C-glycosyl DHC and C-

283

glycosylflavonoids. 53, 54, 65, 66

284

Gutmann and Nidetzky 67 showed that OsCGT isolated from O. sativa formed the

285

3'-C-glycoside nothofagin exclusively. Two cDNAs were isolated from the dicot plant F.

286

esculentum, and the recombinant proteins [FeCGTa (UGT708C1) and FeCGTb

287

(UGT708C2)] were found to exhibit C-glucosylation activity towards phloretin.

288

recently, the isolation of two cDNAs for a CGT (CuCGT, FuCGT) genes were reported

289

from Citrus unshiu, and Frotunella crassifolia, respectively, that catalyzing the formation

290

of di-C-glucosyl phloretin. 68

291

DHCs Hydroxylase

54

More

292

Introduction of the hydroxyl groups in the B-ring of flavonoids and DHCs is

293

catalyzed by the well-known cytochrome P450 dependent monooxygenases flavonoid 3'-



Page 14 of 32

55, 69-71

294

hydroxylase (F3'H) and chalcone 3-hydroxylase (CH3H).

295

proteins of Perilla frutescens, Petunia hybrid, and A. thaliana accept flavones,

296

flavanones, and dihydroflavonols as substrates, as do enzyme preparations from plant

297

tissues. 72-74 Indeed, recombinant P. frutescens F3'H showed a similar Km (about 20 µM)

298

for naringenin, dihydrokaempferol, and apigenin

299

sequence has been used to generate a model of the enzyme and examine the active site

300

architecture and substrate recognition. 74

73

The F3'H recombinant

The A. thaliana F3'H amino acid

301

Hydroxylation of position 3 of dihydrochalcones shows high similarity to the

302

introduction of the second hydroxyl group in the B-ring of flavonoids and chalcone. This

303

was shown for the first time with microsomal enzyme preparations of Dahlia variabilis

304

petals, where conversion of the 6'-deoxychalcone, isoliquiritigenin, to the corresponding

305

3,4-hydroxylated product, butein, occurred. 75 Transgenic apple plants overexpressing the

306

Cosmos sulphureus CH3H gene show increased levels of 3-hydroxyphloridzin, but no 3-

307

hydroxyphloretin accumulation was observed. 55 This indicated that the highly reactive 3-

308

hydroxyphloretin is immediately converted to 3-hydroxyphloridzin to avoid undesired

309

cell damage.

310

Metabolic engineering of DHCs in a microbial cell factory

311

The increasing prevalence of several diseases, like Alzheimer’s disease, obesity,

312

cancer, and diabetes, in humans in recent decades worldwide, accompanied by rising

313

concern regarding the safety of many synthetic chemistry-based pharmaceuticals, has

314

raised public demand for phytochemical-based medicines.

315

increasing interest in metabolic engineering as an approach to produce such natural


6, 76

This in turn has led to

Page 15 of 32


316

products on an industrial scale, which has the potential to decrease production costs of,

317

for example, desired DHCs.

318

Eukaryotic and prokaryotic microbes such as S. cerevisiae and E. coli are widely 21, 77

319

used as a cell factories for the over-expression of targeted genes.

320

metabolically engineered microorganisms can be used to synthesize natural and non-

321

natural desired compounds via a precursor directed biosynthesis approach.

322

engineering involves enhancing or redirecting flux through metabolic pathways by

323

making genetic modifications e.g. deletion of genes, replacement of genes expression

324

signal, that alter the activity of specific enzymatic reactions. These strategies often

325

includes increasing activity at flux-controlling steps and introduction of irreversible

326

reactions of drive the flux in desired directions, and often comprises elimination of

327

unwanted activities. 23, 77

78

These

Metabolic

328

When it comes to industrial production and developing new platform strains, the

329

host strain matters. In addition, there are some central aims that the strains must achieve

330

to be ready for industrial production. For example, it should be considered whether the

331

chosen host can survive under the desired process conditions, e.g. temperature, ionic

332

strength, and pH, and if the host is genetically stable. In addition, the largest bottleneck

333

for industrial implementation of novel bioprocesses is often in the scale-up step that

334

usually appears late in the process, where the host strain has to be chosen absolutely, as it

335

will generally be too expensive to change at a late stage. 23, 77

336

Several compounds with high value such as pharmaceutical compounds (e.g.

337

artemisinin), fragrance (e.g. nootkatone), biofuels (e.g. ethanol and isobutanol), and food

338

chemicals (e.g. vanillin and resveratrol) were produced as end products in microbes.


23


339

Such biotechnological processes, compared to the extraction from plant material, are

340

environmentally friendly, less expensive, and allow efficient production of specific

341

desired compound. 79

342

Although, many genes responsible for DHC biosynthesis have been characterized,

343

there has been limited progress on the metabolic engineering of DHC production. To

344

date, there have been only four reports of the engineering of DHC production in E. coli

345

and S. cerevisiae.

346

thaliana and CHS from Hypericum androsaemum was achieved and the direct production

347

of phloretin by feeding phloretic acid was detected.

348

overexpression of 4CL, C4H, CHS, DBR and UGT from different sources greatly

349

increased the production of several DHCs, such as phloretin, phloridzin, 3-

350

hydroxyphloretin, nothofagin and the sweet tasting molecule naringin dihydrochalcone. 21

351

Thus, in order to increase the production of DHCs, several key steps must be followed:

352

(i) a host strain must be chosen that can be used under industrial conditions ( e.g. high

353

osmo-tolerance and tolerance to low pH), and (ii) several specific enzyme must be chosen

354

and over-expressed, e.g. DBR 26, CHS and UGT. 28, 44

21, 24, 80, 81

The co-expression of the 4CL gene from Arabidopsis

24

Recently, it was reported that

355

In conclusion, many DHCs with potential benefits for humans have been

356

identified in a wide diversity of plant species. Recent advances in our understanding of

357

the DHC biosynthetic pathway have revealed several key enzymes, but some of the

358

enzymes responsible for functional modifications on the phloretin (or other DHC)

359

backbone remain to be discovered. Furthermore, our ability to genetically engineer the

360

DHC biosynthetic pathway is still limited, and there is much work yet to be performed to


Page 16 of 32

Page 17 of 32


361

identify additional genes and enzymes involved in DHC formation and to develop a

362

better heterologous system for an economically feasible industrial production process.

363 364 365

Acknowledgments

366

This research was supported by the Ministry of Agriculture & Rural Development (Grant

367

No. 261-1043 to Mwafaq Ibdah), and partially funded by the European Region Tyrol-

368

South Tyrol-Trentino (EGTC) through the Euregio Science Fund, project ExpoApple2-

369

IPN 55, 2nd call 2016 and by the autonomous province of Trento (ADP 2010-2017; Italy)

370

(Stefan Martens).

371

Abbreviation Used

372

4CL: p-coumarate:CoA ligase; C4H: Cinnamate 4-hydroxylase; CHS: Chalcone

373

synthase; CH3H: Chalcone 3-hydroxylase; DBR: Double bond reductase; DHC:

374

Dihydrochalcones; ENRL: Enoyl reductase-like; F3'H: Flavonoid 3'-hydroxylase; GT:

375

Glucosyltransferase

376

dehydrogenase/reductase;

377

glycosyltransferase

378

glycosyltransferases.

Md:

PKS:

Malus

domestica;

MdPh-4'-OGT: Polyketide

MDR:

medium-chain

UDP-glucose:phloretin synthases;

379 380 381 382 383


UGT:

4'-O-

UDP-dependent


384 385 386 387 388 389 390 391 392 393 394 395

References

396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416

1. Dugé de Bernonville, T.; Guyot, S.; Paulin, J.-P.; Gaucher, M.; Loufrani, L.; Henrion, D.; Derbré, S.; Guilet, D.; Richomme, P.; Dat, J. F.; Brisset, M.-N., Dihydrochalcones: Implication in resistance to oxidative stress and bioactivities against advanced glycation end-products and vasoconstriction. Phytochemistry 2010, 71, 443452. 2. Dugé de Bernonville, T. D.; Gaucher, M.; Guyot, S.; Durel, C. E.; Dat, J. F.; Brisset, M. N., The constitutive phenolic composition of two Malus x domestica genotypes is not responsible for their contrasted susceptibilities to fire blight. Environ. Expe. Bot. 2011, 74, 65-73. 3. Ling, T.-J.; Lin, L.-D.; Wu, P.; Zhou, W.-H.; Ye, H.-G.; Liu, M.-F.; Wei, X.-Y., Dihydrochalcones from Symplocos vacciniifolia. Chinese Chem. Lett. 2004, 15, 11821184. 4. Qin, X. D.; Liu, J. K., A new sweet dihydrochalcone-glucoside from leaves of Lithocarpus pachyphyllus (Kurz) Rehd. (Fagaceae). Z. Naturforsch. C. 2003, 58, 759761. 5. Rui-Lin, N.; Tanaka, T.; Zhou, J.; Tanaka, O., Phlorizin and trilobatin, sweet dihydrochalcone-glucosides from leaves of Lithocarpus litseifolius (Hance) Rehd.(Fagaceae). Agr. Biol. Chem. Tokyo 1982, 46, 1933-1934. 6. Xiao, Z.; Zhang, Y.; Chen, X.; Wang, Y.; Chen, W.; Xu, Q.; Li, P.; Ma, F., Extraction, identification, and antioxidant and anticancer tests of seven dihydrochalcones from Malus ‘Red Splendor’ fruit. Food Chem. 2017, 231, 324-331.


Page 18 of 32

Page 19 of 32

417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461


7. Gosch, C.; Halbwirth, H.; Stich, K., Phloridzin: Biosynthesis, distribution and physiological relevance in plants. Phytochemistry 2010, 71, 838-843. 8. Rivière, C., Chapter 7 - Dihydrochalcones: Occurrence in the plant kingdom, chemistry and biological activities. In Studies in Natural Products Chemistry, Atta ur, Ed. Elsevier: 2016; Vol. Volume 51, pp 253-381. 9. Ninomiya, M.; Koketsu, M., Minor flavonoids (chalcones, flavanones, dihydrochalcones, and aurones). In Natural Products, Springer: 2013; pp 1867-1900. 10. Figtree, G. A.; Griffiths, H.; Liu, Y. Q.; Webb, C. M.; MacLeod, K.; Collins, P., Plant-derived estrogens relax coronary arteries in vitro by a calcium antagonistic mechanism. J. Am. Coll. Cardiol. 2000, 35, 1977-1985. 11. Stangl, V.; Lorenz, M.; Ludwig, A.; Grimbo, N.; Guether, C.; Sanad, W.; Ziemer, S.; Martus, P.; Baumann, G.; Stangl, K., The flavonoid phloretin suppresses stimulated expression of endothelial adhesion molecules and reduces activation of human platelets. J. Nutr. 2005, 135, 172-178. 12. Viet, M. H.; Chen, C.-Y.; Hu, C.-K.; Chen, Y.-R.; Li, M. S., Discovery of dihydrochalcone as potential lead for Alzheimer's disease: in silico and in vitro study. PLoS One 2013, 8, e79151. 13. Wojdyło, A.; Oszmiański, J.; Laskowski, P., Polyphenolic compounds and antioxidant activity of new and old apple varieties. J. Agr. Food Chem. 2008, 56, 65206530. 14. Ehrenkranz, J. R. L.; Lewis, N. G.; Ronald Kahn, C.; Roth, J., Phlorizin: a review. Diabetes Metab. Res. 2005, 21, 31-38. 15. Puel, C.; Quintin, A.; Mathey, J.; Obled, C.; Davicco, M.; Lebecque, P.; KatiCoulibaly, S.; Horcajada, M.; Coxam, V., Prevention of bone loss by phloridzin, an apple polyphenol, in ovariectomized rats under inflammation conditions. Calcif. Tissue Int. 2005, 77, 311-318. 16. Szliszka, E.; Czuba, Z. P.; Mazur, B.; Paradysz, A.; Krol, W., Chalcones and dihydrochalcones augment trail-mediated apoptosis in prostate cancer cells. Molecules 2010, 15, 5336. 17. Bar, A.; Borrego, F.; Benavente, O.; Castillo, J.; Delrio, J. A., Neohesperidin dihydrochalcone - properties and applications. Food Sci. Technol-Leb. 1990, 23, 371-376. 18. Tomas-Barberan, F.; Borrego, F.; Ferreres, F.; Lindley, M., Stability of the intense sweetener neohesperidine dihydrochalcone in blackcurrant jams. Food Chem. 1995, 52, 263-265. 19. Picinelli, A.; Dapena, E.; Mangas, J. J., Polyphenolic pattern in apple tree leaves in relation to scab resistance-a preliminary-study. J. Agric. Food Chem. 1995, 43, 22732278. 20. Roemmelt, S.; Fischer, T. C.; Halbwirth, H.; Peterek, S.; Schlangen, K.; Speakman, J.; Treutter, D.; Forkmann, G.; Stich, K., Effect of dioxygenase inhibitors on the resistance-relatedflavonoid metabolism of apple and pears: chemical, biochemical and molecular biological aspects. Eur. J. Hort. Sci. 2003, 68, 129-136. 21. Eichenberger, M.; Lehka, B. J.; Folly, C.; Fischer, D.; Martens, S.; Simón, E.; Naesby, M., Metabolic engineering of Saccharomyces cerevisiae for de novo production of dihydrochalcones with known antioxidant, antidiabetic, and sweet tasting properties. Metab. Eng. 2017, 39, 80-89.



462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505

22. Ververidis, F.; Trantas, E.; Douglas, C.; Vollmer, G.; Kretzschmar, G.; Panopoulos, N., Biotechnology of flavonoids and other phenylpropanoid-derived natural products. Part II: Reconstruction of multienzyme pathways in plants and microbes. Biotechnol. J. 2007, 2, 1235-1249. 23. Jullesson, D.; David, F.; Pfleger, B.; Nielsen, J., Impact of synthetic biology and metabolic engineering on industrial production of fine chemicals. Biotechnol. Adv. 2015, 33, 1395-1402. 24. Werner, S. R.; Chen, H.; Jiang, H.; Morgan, J. A., Synthesis of non-natural flavanones and dihydrochalcones in metabolically engineered yeast. J. Mol. Catal. B: Enzym. 2010, 66, 257-263. 25. Gosch, C.; Halbwirth, H.; Schneider, B.; Hölscher, D.; Stich, K., Cloning and heterologous expression of glycosyltransferases from Malus x domestica and Pyrus communis, which convert phloretin to phloretin 2′-O-glucoside (phloridzin). Plant Sci. 2010, 178, 299-306. 26. Ibdah, M.; Berim, A.; Martens, S.; Valderrama, A. L. H.; Palmieri, L.; Lewinsohn, E.; Gang, D. R., Identification and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase involved in dihydrochalcone formation in Malus x domestica Borkh. Phytochemistry 2014, 107, 24-31. 27. Jugde, H.; Nguy, D.; Moller, I.; Cooney, J. M.; Atkinson, R. G., Isolation and characterization of a novel glycosyltransferase that converts phloretin to phlorizin, a potent antioxidant in apple. FEBS J. 2008, 275, 3804-3814. 28. Yahyaa, M.; Davidovich-Rikanati, R.; Eyal, Y.; Sheachter, A.; Marzouk, S.; Lewinsohn, E.; Ibdah, M., Identification and characterization of UDP-glucose: Phloretin 4′-O-glycosyltransferase from Malus x domestica Borkh. Phytochemistry 2016, 130, 4755. 29. Dare, A. P.; Tomes, S.; Cooney, J. M.; Greenwood, D. R.; Hellens, R. P., The role of enoyl reductase genes in phloridzin biosynthesis in apple. Plant Physiol. Biochem. 2013. 30. Dare, A. P.; Tomes, S.; Jones, M.; McGhie, T. K.; Stevenson, D. E.; Johnson, R. A.; Greenwood, D. R.; Hellens, R. P., Phenotypic changes associated with RNA interference silencing of chalcone synthase in apple (Malus x domestica). Plant J. 2013, 74, 398-410. 31. Dare, A. P.; Yauk, Y. K.; Tomes, S.; McGhie, T. K.; Rebstock, R. S.; Cooney, J. M.; Atkinson, R. G., Silencing a phloretin-specific glycosyltransferase perturbs both general phenylpropanoid biosynthesis and plant development. Plant J. 2017. 32. Winkel-Shirley, B., Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol. 2001, 126, 485-493. 33. Andersen, O. M.; Markham, K. R., Flavonoids: chemistry, biochemistry and applications. CRC Press: 2005. 34. Gosch, C.; Halbwirth, H.; Kuhn, J.; Miosic, S.; Stich, K., Biosynthesis of phloridzin in apple (Malus domestica Borkh.). Plant Sci. 2009, 176, 223-231. 35. Yamazaki, Y.; Suh, D.-Y.; Sitthithaworn, W.; Ishiguro, K.; Kobayashi, Y.; Shibuya, M.; Ebizuka, Y.; Sankawa, U., Diverse chalcone synthase superfamily enzymes from the most primitive vascular plant, Psilotum nudum. Planta 2001, 214, 75-84.


Page 20 of 32

Page 21 of 32

506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550


36. Riveros-Rosas, H.; Julián-Sánchez, A.; Villalobos-Molina, R.; Pardo, J. P.; Piña, E., Diversity, taxonomy and evolution of medium-chain dehydrogenase/reductase superfamily. FEBS J. 2003, 270, 3309-3334. 37. Kavanagh, K.; Jörnvall, H.; Persson, B.; Oppermann, U., Medium-and short-chain dehydrogenase/reductase gene and protein families. Cell Mol. Life Sci. 2008, 65, 3895. 38. Koeduka, T.; Watanabe, B.; Suzuki, S.; Hiratake, J.; Mano, J.; Yazaki, K., Characterization of raspberry ketone/zingerone synthase, catalyzing the alpha, betahydrogenation of phenylbutenones in raspberry fruits. Biochem. Biophys. Res. Commun. 2011, 412, 104-108. 39. Kasahara, H.; Jiao, Y.; Bedgar, D. L.; Kim, S. J.; Patten, A. M.; Xia, Z. Q.; Davin, L. B.; Lewis, N. G., Pinus taeda phenylpropenal double-bond reductase: Purification, cDNA cloning, heterologous expression in Escherichia coli, and subcellular localization in P. taeda. Phytochemistry 2006, 67, 1765-1780. 40. Ringer, K. L.; McConkey, M. E.; Davis, E. M.; Rushing, G. W.; Croteau, R., Monoterpene double-bond reductases of the (−)-menthol biosynthetic pathway: isolation and characterization of cDNAs encoding (−)-isopiperitenone reductase and (+)-pulegone reductase of peppermint. Arch. Biochem. Biophys. 2003, 418, 80-92. 41. Youn, B.; Kim, S. J.; Moinuddin, S. G. A.; Lee, C.; Bedgar, D. L.; Harper, A. R.; Davin, L. B.; Lewis, N. G.; Kang, C., Mechanistic and structural studies of apoform, binary, and ternary complexes of the Arabidopsis alkenal double bond reductase At5g16970. J. Biol. Chem. 2006, 281, 40076-40088. 42. Dao, T.; Linthorst, H.; Verpoorte, R., Chalcone synthase and its functions in plant resistance. Phytochemistry Rev. 2011, 10, 397-412. 43. Ferrer, J.-L.; Jez, J. M.; Bowman, M. E.; Dixon, R. A.; Noel, J. P., Structure of chalcone synthase and the molecular basis of plant polyketide biosynthesis. Nat. Struct. Mol. Biol. 1999, 6, 775-784. 44. Yahyaa, M.; Ali, S.; Davidovich-Rikanati, R.; Ibdah, M.; Shachtier, A.; Eyal, Y.; Lewinsohn, E.; Ibdah, M., Characterization of three chalcone synthase-like genes from apple (Malus x domestica Borkh.). Phytochemistry 2017, 140, 125-133. 45. Austin, M. B.; Noel, J. P., The chalcone synthase superfamily of type III polyketide synthases. Nat. Prod. Rep. 2003, 20, 79-110. 46. Schröder, J., The chalcone/stilbene synthase-type family of condensing enzymes. Comprehensive natural products chemistry 1999, 1, 749-771. 47. Kreuzaler, F.; Ragg, H.; Fautz, E.; Kuhn, D. N.; Hahlbrock, K., UV-induction of chalcone synthase mRNA in cell suspension cultures of Petroselinum hortense. PNAS 1983, 80, 2591-2593. 48. Reimold, U.; Kröger, M.; Kreuzaler, F.; Hahlbrock, K., Coding and 3'non-coding nucleotide sequence of chalcone synthase mRNA and assignment of amino acid sequence of the enzyme. EMBO J. 1983, 2, 1801. 49. O'Neill, S. D.; Tong, Y.; Spörlein, B.; Forkmann, G.; Yoder, J. I., Molecular genetic analysis of chalcone synthase in Lycopersicon esculentum and an anthocyanindeficient mutant. Mol. Gen. Genet. 1990, 224, 279-288. 50. McKhann, H. I.; Hirsch, A. M., Isolation of chalcone synthase and chalcone isomerase cDNAs from alfalfa (Medicago sativa L.): highest transcript levels occur in young roots and root tips. Plant Mol. Biol. 1994, 24, 767-777.



551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594

51. Koes, R. E.; Spelt, C. E.; van den Elzen, P. J. M.; Mol, J. N. M., Cloning and molecular characterization of the chalcone synthase multigene family of Petunia hybrida. Gene 1989, 81, 245-257. 52. Lunkenbein, S.; Coiner, H.; de Vos, C. H. R.; Schaart, J. G.; Boone, M. J.; Krens, F. A.; Schwab, W.; Salentijn, E. M. J., Molecular characterization of a stable antisense chalcone synthase phenotype in strawberry (Fragaria x ananassa). J. Agr. Food Chem. 2006, 54, 2145-2153. 53. Brazier-Hicks, M.; Evans, K. M.; Gershater, M. C.; Puschmann, H.; Steel, P. G.; Edwards, R., The C-glycosylation of flavonoids in cereals. J. Biol. Chem. 2009, 284, 17926-17934. 54. Nagatomo, Y.; Usui, S.; Ito, T.; Kato, A.; Shimosaka, M.; Taguchi, G., Purification, molecular cloning and functional characterization of flavonoid Cglucosyltransferases from Fagopyrum esculentum M. (buckwheat) cotyledon. Plant J. 2014, 80, 437-448. 55. Hutabarat, O. S.; Flachowsky, H.; Regos, I.; Miosic, S.; Kaufmann, C.; Faramarzi, S.; Alam, M. Z.; Gosch, C.; Peil, A.; Richter, K., Transgenic apple plants overexpressing the chalcone 3-hydroxylase gene of Cosmos sulphureus show increased levels of 3-hydroxyphloridzin and reduced susceptibility to apple scab and fire blight. Planta 2016, 243, 1213-1224. 56. Davies, K. M.; Bloor, S. J.; Spiller, G. B.; Deroles, S. C., Production of yellow colour in flowers: redirection of flavonoid biosynthesis in Petunia. Plant J. 1998, 13, 259-266. 57. Cooper, J.; Qiu, F.; Paiva, N., Biotransformation of an exogenously supplied isoflavonoid by transgenic tobacco cells expressing alfalfa isoflavone reductase. Plant Cell. Rep. 2002, 20, 876-884. 58. Liu, C.-J.; Blount, J. W.; Steele, C. L.; Dixon, R. A., Bottlenecks for metabolic engineering of isoflavone glycoconjugates in Arabidopsis. PNAS 2002, 99, 14578-14583. 59. Gosch, C.; Flachowsky, H.; Halbwirth, H.; Thill, J.; Mjka-Wittmann, R.; Treutter, D.; Richter, K.; Hanke, M.-V.; Stich, K., Substrate specificity and contribution of the glycosyltransferase UGT71A15 to phloridzin biosynthesis. Trees 2012, 26, 259-271. 60. Griesser, M.; Vitzthum, F.; Fink, B.; Bellido, M. L.; Raasch, C.; Munoz-Blanco, J.; Schwab, W., Multi-substrate flavonol O-glucosyltransferases from strawberry (Fragaria x ananassa) achene and receptacle. J. Exp. Bot. 2008, 59, 2611-25. 61. Vogt, T.; Jones, P., Glycosyltransferases in plant natural product synthesis: characterization of a supergene family. Trends Plant Sci. 2000, 5, 380-386. 62. Witte, S.; Moco, S.; Vervoort, J.; Matern, U.; Martens, S., Recombinant expression and functional characterisation of regiospecific flavonoid glucosyltransferases from Hieracium pilosella L. Planta 2009, 229, 1135-1146. 63. Gutmann, A.; Bungaruang, L.; Weber, H.; Leypold, M.; Breinbauer, R.; Nidetzky, B., Towards the synthesis of glycosylated dihydrochalcone natural products using glycosyltransferase-catalysed cascade reactions. Green Chem. 2014, 16, 4417-4425. 64. Werner, S. R.; Morgan, J. A., Expression of a Dianthus flavonoid glucosyltransferase in Saccharomyces cerevisiae for whole-cell biocatalysis. J. Biotechnol. 2009, 142, 233-241.


Page 22 of 32

Page 23 of 32

595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640


65. Ferreyra, M. L. F.; Rodriguez, E.; Casas, M. I.; Labadie, G.; Grotewold, E.; Casati, P., Identification of a bifunctional maize C-and O-glucosyltransferase. J. Biol. Chem. 2013, 288, 31678-31688. 66. Hirade, Y.; Kotoku, N.; Terasaka, K.; Saijo-Hamano, Y.; Fukumoto, A.; Mizukami, H., Identification and functional analysis of 2-hydroxyflavanone Cglucosyltransferase in soybean (Glycine max). FEBS Lett. 2015, 589, 1778-1786. 67. Gutmann, A.; Nidetzky, B., Switching between O-and C-glycosyltransferase through exchange of active-site motifs. Angewandte Chemie International Edition 2012, 51, 12879-12883. 68. Ito, T.; Fujimoto, S.; Suito, F.; Shimosaka, M.; Taguchi, G., CGlycosyltransferases catalyzing the formation of di-C-glucosyl flavonoids in citrus plants. Plant J. 2017. 69. Schlangen, K.; Miosic, S.; Thill, J.; Halbwirth, H., Cloning, functional expression, and characterization of a chalcone 3-hydroxylase from Cosmos sulphureus. J. Exp. Bot. 2010, 61, 3451-3459. 70. Schlangen, K.; Miosic, S.; Topuz, F.; Muster, G.; Marosits, T.; Seitz, C.; Halbwirth, H., Chalcone 3-hydroxylation is not a general property of flavonoid 3′hydroxylase. Plant Sci. 2009, 177, 97-102. 71. Thill, J.; Miosic, S.; Gotame, T. P.; Mikulic-Petkovsek, M.; Gosch, C.; Veberic, R.; Preuss, A.; Schwab, W.; Stampar, F.; Stich, K., Differential expression of flavonoid 3′-hydroxylase during fruit development establishes the different B-ring hydroxylation patterns of flavonoids in Fragaria x ananassa and Fragaria vesca. Plant Physiol. Biochem. 2013, 72, 72-78. 72. Brugliera, F.; Barri-Rewell, G.; Holton, T. A.; Mason, J. G., Isolation and characterization of a flavonoid 3′-hydroxylase cDNA clone corresponding to the Ht1 locus of Petunia hybrida. Plant J. 1999, 19, 441-451. 73. Kitada, C.; Gong, Z.; Tanaka, Y.; Yamazaki, M.; Saito, K., Differential expression of two cytochrome P450s involved in the biosynthesis of flavones and anthocyanins in chemo-varietal forms of Perilla frutescens. Plant Cell Physiol. 2001, 42, 1338-1344. 74. Schoenbohm, C.; Martens, S.; Eder, C.; Forkmann, G.; Weisshaar, B., Identification of the Arabidopsis thaliana flavonoid 3'-hydroxylase gene and functional expression of the encoded P450 enzyme. Biol. Chem. 2000, 381, 749-753. 75. Wimmer, G.; Halbwirth, H.; Wurst, F.; Forkmann, G.; Stich, K., Enzymatic hydroxylation of 6′-deoxychalcones with protein preparations from petals of Dahlia variabilis. Phytochemistry 1998, 47, 1013-1016. 76. Janvier, S.; Goscinny, S.; Donne, C. L.; Loco, J. V., Low-calorie sweeteners in food and food supplements on the Italian market. Food Addit. Contam. B 2015, 8, 298308. 77. Keasling, J. D., Manufacturing molecules through metabolic engineering. Science 2010, 330, 1355-1358. 78. Kennedy, J., Mutasynthesis, chemobiosynthesis, and back to semi-synthesis: combining synthetic chemistry and biosynthetic engineering for diversifying natural products. Nat. Prod. Rep. 2008, 25, 25-34. 79. Krivoruchko, A.; Nielsen, J., Production of natural products through metabolic engineering of Saccharomyces cerevisiae. Curr. Opin. Biotech 2015, 35, 7-15. 23 ACS Paragon Plus Environment


641 642 643 644 645 646 647 648 649 650 651

80. Watts, K. T.; Lee, P. C.; Schmidt‐Dannert, C., Exploring recombinant flavonoid biosynthesis in metabolically engineered Escherichia coli. Chembiochem 2004, 5, 500507. 81. Pandey, R. P.; Li, T. F.; Kim, E.-H.; Yamaguchi, T.; Park, Y. I.; Kim, J. S.; Sohng, J. K., Enzymatic synthesis of novel phloretin glucosides. Appl. Environ. Microb. 2013, 79, 3516-3521. 82. Łata, B.; Trampczynska, A.; Paczesna, J., Cultivar variation in apple peel and whole fruit phenolic composition. Sci. Hort. 2009, 121, 176-181. 83. Duda-Chodak, A.; Tarko, T.; Tuszyński, T., Antioxidant activity of apples–an impact of maturity stage and fruit part. Acta. Sci. Pol. Technol. Aliment. 2011, 10, 443454.

652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668

Figure captions

669

Figure 1. Structure and numbering of phloretin as a representative backbone for all

670

dihydrochalcones.

671

Figure 2. Metabolic profiling of dihydrochalcones from four different Malus genotypes.

672

A: Malus sieversii; B: M. trilobata; C: M. hybrid “Evereste”; D: M. sieboldii syn.

673

toringo. 24 ACS Paragon Plus Environment

Page 24 of 32

Page 25 of 32


674

Figure 3. Phloretin and phloridzin distribution in leaves, seed, flesh, and peel of apples.

675

Data were re-elaborated from Lata et al, 82 Duda-Chodak et al, 83 and Yahyaa et al. 44

676

Figure 4. Proposed biosynthetic routes to several hydroxylated, glycosylated, and

677

methylated DHC derivatives. HCDBR, hydroxycinnamoyl-CoA double bond reductase;

678

CHS, chalcone synthase; Ph-2′-OGT, phloretin-2′-O-glycosyltransferase; Ph-4′-OGT,

679

phloretin-4′-O-glycosyltransferase;

680

cytochrome P450 reductase; CYP, cytochrome P450; OMT, O-methyltransferase. The

681

red labeled genes/enzymes have been biochemically characterized from Malus.

682

Figure 5. Structure and numbering of nothofagin (2',4,4',6'-tetrahydroxy-3-C-β-D-

683

glucopyranosyldihydrochalcone).

1,2

RhaT,

1,2-rhamnosyltransferase;

CPR,

684 685 686 687 688 689 690 691

Table 1. Biochemically characterized Malus enzymes involved in DHC biosynthesis

Enzyme name

Substrate

MdHCDBR

p-Coumaryl-CoA Feruloyl-CoA p-Dihydrocoumaryl-CoA p-Coumaryl-CoA Phloretin Phloretin

MdCHS3 MdPh-2′-OGT MdPh-2′-OGT

Km (µM) 96.6 92.9 5.07 5.09 0.62 82

Vmax (pkat µg-1 protein) 47.7 102.9 1.05 0.94 8


References 26

44

27 59


MdPh-4′-OGT

Phloretin

26.1

28

1.86

Figure 1 5 4

6 5'

HO

6' 4'

1

β

A

3'

B

OH

3 2

α 1'

2'

OH

O


Page 26 of 32

OH

Page 27 of 32


Figure 2



Figure 3


Page 28 of 32

Page 29 of 32




Page 30 of 32

Figure 4 O

O

OH

O

OH

OH

O

SCoA

O

SCoA OH

H2N

C4H

4CL

OH

p-Coumaric acid

HCDBR

OH

OH

p-Coumaroyl-CoA 3 x Malonyl-CoA

3 x Malonyl-CoA CHS

CHS

CoA-SH HO

O

Glc

O

Phloridzin

p-Dihydrocoumaroyl-CoA GT

Cinnamic acid

OH

Ph -2 `-O

PAL L-Phenylalanine

HO

NADP+

NADPH

OH

CoA-SH

OH

OH

HO

OH

OH

OH

HO

HO

OH

OH O

OH OH

O

CYP/CPR

OH O

Naringenin chalcone CHI HO

OH

O

DHC-OMT

3-OH-Hydroxyphloretin

Phloretin

OH O

4-Methoxy-phloretin

UDP-glc

OH

UDP

DHC-4'-OGT

DHC-4'-OGT

Ph-4'-OGT

O

OH

OH Glc

Glc

OH O

O

OH

Glc

O

O

OH OH O OH O

OH O

Glc

O

OH

Trilobatin

O

Glc

1,2 RhaT

O

OH

Glc

O

OH

OH Rha

OH O Rha

OH O

OH

Naringin dihydrochalcone O

Sieboldin 1,2 RhaT

1,2 RhaT

Prunin

Glc

4-Methoxy-phloretin-4'-O-glucoside

O

OH

O

OH

Naringenin GT

OH

OH

O

Rha

OH O

Naringin


Neohesperidin dihydrochalcone

OH O

Page 31 of 32


Figure 5.

5 6 5'

HO O

HO

4' 3'

A 2' OH

HO

6' OH

B 1

2

1' O

OH OH


OH

4 3


TOC Flavonoids O

O

OH

Dihydrochalcones

SCoA

O

SCoA

H2N NADPH

OH

L-Phenylalanine

NADP+

OH

HCDBR

p-Coumaroyl-CoA

p-Dihydrocoumaroyl-CoA 3 x Malonyl-CoA

CHS

CHS OH

CoA-SH OH HO

HO

OH

OH

OH O OH O

Phloretin

Naringenin chalcone

UDP-glc Ph-4'-OGT UDP Glc

O

OH OH

OH O

Trilobatin


Page 32 of 32

Biosynthetic Pathway and Metabolic Engineering of Plant

Recommend Documents