natural products - rensselaer polytechnic institute€¦ · natural products phytochemistry, botany...

Natural Products

1643

Kishan Gopal RamawatJean-Michel MerillonEditors

Natural Products

Phytochemistry, Botanyand Metabolism of Alkaloids,Phenolics and Terpenes

With 1569 Figures and 307 Tables

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Editors-in-Chief:Kishan Gopal RamawatBotany Department, M.L. Sukhadia UniversityUdaipur 313001India

Jean-Michel MerillonBiological-Active Plant Substances Study GroupUniversity of BordeauxInstitute of Vine and Wine SciencesVillenave d’OrnonFrance

ISBN 978-3-642-22143-9 ISBN 978-3-642-22144-6 (eBook)ISBN 978-3-642-22145-3 (Print and electronic bundle)DOI 10.1007/ 978-3-642-22144-6Springer Heidelberg New York Dordrecht London

Library of Congress Control Number: 2013934974

# Springer-Verlag Berlin Heidelberg 2013

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Comp. by: MohamedSameer Stage: Proof Chapter No.: 53 Title Name: HBNPDate:5/10/12 Time:21:30:02 Page Number: 1

1 Isoflavonoid Production by Genetically2 Engineered Micro-organisms 533 Brady F. Cress Au1, Robert J. Linhardt, and Mattheos A. G. Koffas

4 Contents

5 1 Metabolic Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

6 1.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

7 1.2 Metabolic Engineering Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

8 1.3 Microorganisms as a Production Platform for Plant Natural Products . . . . . . . . . . . . . . . 5

9 2 Plant Phenylpropanoid Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

10 2.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

11 2.2 Plant Phenylpropanoid Biosynthetic Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

12 2.3 Plant Flavonoid Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

13 3 Plant Isoflavonoid Production in Microbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

14 3.1 Construction of an Artificial Biosynthetic Pathway for Flavonoid Production in

Microbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

15 3.2 Engineering the Plant Isoflavonoid Pathway in Microbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

16 4 Mutasynthesis and Protein Engineering for Nonnatural Isoflavonoid Production in

Microbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

17 4.1 Mutasynthesis for Nonnatural Isoflavonoid Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

18 4.2 Protein Engineering for Nonnatural Isoflavonoid Production . . . . . . . . . . . . . . . . . . . . . . . . 21

19 4.3 Other Isoflavonoid Biotransformations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

20 5 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

21 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

B.F. Cress (*) • M.A.G. Koffas

Department of Chemical and Biological Engineering, Center for Biotechnology and

Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA

e-mail: [email protected]

R.J. Linhardt

Department of Chemical and Biological Engineering, Center for Biotechnology and

Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA

Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary

Studies, Rensselaer Polytechnic Institute, Troy, NY, USA

Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer

Polytechnic Institute, Troy, NY, USA

K.G. Ramawat, J.M. Merillon (eds.), Handbook of Natural Products,DOI 10.1007/978-3-642-22144-6_53, # Springer-Verlag Berlin Heidelberg 2013

1

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22 Abstract

23 Isoflavonoids are a class of plant natural products gaining attention due to their

24 pharmaceutical properties. These natural compounds constitute a subclass of flavo-

25 noids, which belong to a broader class of plant products known as phenylpropanoids.

26 Flavonoids have been associated with medicinal properties, while isoflavonoids have

27 shown anticancer, antioxidant, and cardioprotective properties due to their role as

28 inhibitors of estrogen receptors. Isoflavonoids are naturally produced by legumes and,

29 more specifically, organisms belonging to the pea family. Harvesting of these natural

30 products through traditional extraction processes is limited due to the low levels of

31 these phytochemicals in plants, so alternative production platforms are required to

32 reduce cost of production and increase availability. Over the last decade, researchers

33 have engineered artificial flavonoid biosynthesis pathways into Escherichia coli and34 Saccharomyces cerevisiae to convert simple, renewable sugars like glucose into

35 flavonoids at high production levels. This chapter outlines the metabolic engineering

36 research that has enabled microbial production of plant flavonoids and further details

37 the ongoing work aimed at producing both natural and nonnatural isoflavonoids in

38 microorganisms.

39 Keywords

40 Metabolic engineering • mutasynthesis • nonnatural isoflavonoids • protein

41 engineering • strain improvement

42 Abbreviations

43 3GT 3-O-glucosyltransferase44 4CL 4-Coumarate-CoA ligase

45 ACC Acetyl-CoA carboxylase

46 Ala Alanine

47 ANR Anthocyanidin reductase

48 ANS Anthocyanidin synthase

49 API Active pharmaceutical ingredient

50 Arg Arginine

51 BDO Biphenyl dioxygenase

52 BMC Bacterial microcompartment

53 C4H Cinnamate 4-hydroxylase

54 CHI Chalcone isomerase

55 CHS Chalcone synthase

56 CPR Cytochrome P450 reductase

57 CUS Curcuminoid synthase

58 DFR Dihydroflavonol reductase

59 DH Salmonella typhimurium LT2 TDP-glucose 4,6-dehydratase

60 EPI Streptomyces antibioticus Tu99 TDP-4-keto-6-deoxyglucose

3,5-epimerase

61 ER Endoplasmic reticulum

62 F7GAT Flavonoid 7-O-glucuronosyltransferase

2 B.F. Cress et al.

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63 FHT Flavanone 3b-hydroxylase64 FLS Flavonol synthase

65 FSI Soluble flavone synthase

66 FSII Membrane-bound flavone synthase

67 G1P Glucose-1-phosphate

68 G6P Glucose-6-phosphate

69 GALU Glucose-1-phosphate uridylyltransferase

70 GERF Streptomyces sp. KCTC 0041BP TDP-hexose 3-epimerase

71 GERK Streptomyces sp. KCTC 0041BP TDP-4-keto-6-deoxyglucose

reductase

72 Glu Glutamic acid

73 Gly Glycine

74 HEK Human embryonic kidney cells

75 hER Human estrogen receptor

76 HI40OMT 2,7,40-Trihydroxyisoflavanone 40-O-methyltransferase

77 HID 2-Hydroxyisoflavanone dehydratase

78 HIDH 2-Hydroxyisoflavanone dehydratase hydroxy type

79 HIDM 2-Hydroxyisoflavanone dehydratase methoxy type

80 IFR Isoflavone reductase

81 IFS Isoflavone synthase

82 Ile Isoleucine

83 kcat Turnover number

84 Km Michaelis constant

85 KR Streptomyces antibioticus Tu99 TDP-glucose 4-ketoreductase

86 LAR Leucoanthocyanidin reductase

87 LB Luria-Bertani medium

88 LDOX Leucoanthocyanidin dioxygenase

89 NADPH Nicotinamide adenine dinucleotide phosphate

90 NDK Nucleoside diphosphate kinase

91 NDO Naphthalene dioxygenase

92 PAL Phenylalanine ammonia-lyase

93 PGI Glucose-6-phosphate isomerase

94 PGM Phosphoglucomutase

95 Phe Phenylalanine

96 RCIFS Red clover isoflavone synthase

97 RCPR Rice cytochrome P450 reductase

98 SaOMT-2 Streptomyces avermitilis MA-4680 7-O-methyltransferase

99 ScCCL Streptomyces coelicolor A3 cinnamate/coumarate:CoA ligase

100 Ser Serine

101 SERM Selective estrogen receptor modulator

102 STS Stilbene synthase

103 TAL Tyrosine ammonia-lyase

104 TB Terrific broth

105 TDP Thymidyldiphosphate

53 Isoflavonoid Production by Genetically Engineered Micro-organisms 3

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106 TGS Thermus caldophilus GK24 thymidyldiphosphoglucose synthase

107 Thr Threonine

108 Trp Tryptophan

109 Tyr Tyrosine

110 UDG Uridine diphosphoglucose dehydrogenase

111 UDP Uridine diphosphate

112 UGT Uridine diphosphate glycosyltransferase

113 UTP Uridine triphosphate

114 UXS1 Uridine diphosphate glucuronic acid decarboxylase

115 Val Valine

116 Vmax Maximum reaction rate

117 1 Metabolic Engineering

118 1.1 Background

119 Metabolic engineering involves the genetic manipulation of metabolism for

120 a specific goal, often high-level production of a secondary metabolite. Second-

121 ary metabolites are those not critical to the survival of an organism in its normal

122 environment, and they are thus typically found in far lower quantities than

123 primary metabolites involved in energy maintenance and growth [1, 2]. As

124 secondary metabolites have evolved to serve in important ecological roles –

125 usually through interaction with other organisms – they possess unique proper-

126 ties and are thus the target of many metabolic engineering projects [3–5].

127 Although metabolic engineering has been a distinct discipline for over two

128 decades, advancing technologies in areas such as DNA sequencing and synthe-

129 sis, computational modeling and optimization, synthetic biology, and protein

130 engineering are enabling metabolic engineers to create economically viable

131 microbial production platforms for specialty chemicals like pharmaceuticals

132 and biofuels [6].

133 Throughout the past decade, much work has focused on both plant and

134 microbial metabolic engineering for production of pharmaceutically and

135 nutraceutically important plant isoflavonoids [7–11]. This class of phytochemi-

136 cals has been shown to possess a diverse array of pharmacological activities and

137 demonstrates potential for treatment of certain cancers, cardiovascular diseases,

138 and other conditions [12–17]. In particular, isoflavonoids have high affinity

139 toward human estrogen receptors (hERs) and are therefore being investigated

140 as estrogen receptor agonists and antagonists to modulate estrogen metabolism

141 [18–20]. The relatively low abundance of these valuable compounds in plants

142 makes microbial metabolic engineering an excellent alternative candidate for

143 large-scale isoflavonoid production.

4 B.F. Cress et al.

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144 1.2 Metabolic Engineering Products

145 The majority of work in the field of metabolic engineering has focused on the

146 production of commodity chemicals and biofuels from renewable, simple carbon

147 sources such as glucose and glycerol, or the production of pharmaceutical

148 chemicals and proteins [21–24]. In general, metabolic engineering can be viewed

149 as the process by which scientists combine genes from different sources to construct

150 a biosynthetic pathway in a host organism to convert an inexpensive feedstock into

151 a valuable product. Classic metabolic engineering projects range from the microbial

152 production of biofuels like ethanol and butanol to the production of commodity

153 chemicals like xylitol. Although these efforts are important for ensuring long-term

154 stability of commodity supply from renewable resources, microbial metabolic

155 engineering of valuable plant natural products and other active pharmaceutical

156 ingredients (APIs) with high overhead has the potential to make a much greater

157 impact on society by lowering cost and ensuring availability and widespread access

158 to medically important compounds [6].

159 1.3 Microorganisms as a Production Platform for Plant Natural160 Products

161 1.3.1162 Advantages of Microbial Hosts163 Microorganisms serve as excellent hosts for production of phytochemicals.

164 The relatively lower genetic complexity of microbes compared to multicellular

165 eukaryotes allows for more accurate prediction of the effects of genetic manip-

166 ulations in microbes than in plants. Modulation of gene copy or expression

167 level typically leads to an imbalance in reaction fluxes and, subsequently, the

168 accumulation of pathway intermediates. If a genetic pathway is not decoupled

169 from its native environment, accumulation of intermediates can become toxic

170 or elicit unintended regulatory effects like feedback inhibition. Such

171 uncharacterized genetic interactions in multicellular eukaryotic hosts are cur-

172 rently difficult to predict and can be largely avoided by transplanting

173 genes from evolutionarily distinct organisms into an artificial pathway in

174 a microbial host [25].

175 Perhaps the strongest argument for utilizing microorganisms for metabolic

176 engineering of plant natural products is the high degree of genetic tractability that

177 currently exists for microbial workhorses like Escherichia coli, Saccharomyces178 cerevisiae, and Bacillus subtilis. Thanks to decades of research, these hosts have

179 innumerable data sets and molecular biology tools available for facile genetic

180 manipulation, characterization, modeling, and scale-up. This genetic tractability

181 reduces experimental unknowns and allows for faster, more predictable experimen-

182 tation and data collection. Additionally, the high growth rates and simple media

183 requirements associated with microorganisms enable culturing with limited

184 resources [25, 26].


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185 1.3.2186 Alternative Production Platforms187 Plant natural products have traditionally been harvested through extraction

188 methods, as evidenced by the preparation of traditional medicines and the steeping

189 of tea leaves and coffee beans for millenia. Since plant natural products are

190 generally found at low levels in plant biomass, extraction is usually not

191 a sustainable mass production avenue. Although extraction is still utilized to harvest

192 APIs like the antimalarial drug artemisinin (from Artemisia annua, known as Sweet193 Wormwood) and the chemotherapeutic paclitaxel (from Taxus brevifolia, the

194 Pacific yew tree) when chemical synthesis is difficult or expensive, there is

195 a trend and growing necessity to shift toward alternative production platforms to

196 lower cost and increase availability [27, 28].

197 Alternative production platforms include organic synthesis, plant cell culture,

198 plant tissue culture, and even mammalian cell culture. The field of organic synthesis

199 of complex plant natural products has advanced significantly but is limited as an

200 industrial-scale flavonoid production platform by frequent use of toxic chemicals

201 and extreme reaction conditions, a high number of required steps, exorbitant costs,

202 relatively low overall yields, and nonspecific catalysts leading to by-products and

203 often difficult-to-separate racemic mixtures of target compounds [29–35].

204 Semisynthesis, which combines organic synthesis steps with biosynthetic steps, is

205 also limited by similar challenges. It is then reasonable to consider plant cell and

206 tissue culture as a closely related alternative production platform since the metab-

207 olites of interest are endogenously produced in undifferentiated plant cells [36].

208 A well-known example of industrial-scale production in plant cell lines is the

209 induction of paclitaxel production through methyl jasmonate elicitation, yielding

210 0.5 % of dry weight compared to 0.01 % of dry weight by extraction from the

211 Pacific yew [37, 38]. By contrast, chemical synthesis of paclitaxel requires 35–51

212 steps, with a yield of only 0.4 % [39]. Plant tissue culture is another option, as many

213 secondary metabolic biosynthetic pathways are only active in specific stages of

214 development or in certain tissues [40, 41]. Thus, elicitation of differentiated plant

215 cell tissues by small molecules or light can also be utilized to produce secondary

216 metabolites. Despite progress in plant cell and tissue culture, the elucidation and

217 characterization of all enzymes involved in plant secondary metabolite biosynthetic

218 pathways are still challenging tasks; moreover, the difficulty in unequivocally

219 discerning all sensitive, multilevel regulatory effects instigated by minimal varia-

220 tions in metabolite concentrations often makes the outcome of metabolic engineer-

221 ing in plant cell and tissue cultures unpredictable.

222 With advances in metabolic engineering of mammalian cells, it is foreseeable

223 that plant natural products might one day be produced and derivatized using

224 mammalian cell culture to take advantage of mammal-specific biotransforma-

225 tions and glycosylation patterns leading to improved pharmaceutical properties

226 and applications. Therapeutic phytochemical production pathways might even

227 be engineered into specific tissues to enable in situ biosynthesis for disease

228 treatment or prophylaxis. To date this alternative remains relatively unexplored;

229 however, engineering of a resveratrol artificial biosynthetic pathway into human

230 embryonic kidney cells (HEK293) circumvented purported difficulties

6 B.F. Cress et al.

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231 associated with yeast expression of tyrosine ammonia-lyase (TAL) and

232 highlighted the opportunity to move plant pathways into mammalian cells for

233 in situ production of phytochemical therapeutics in human tissue [42]. Just as

234 predictable metabolic engineering of plant cell and tissue cultures is currently

235 limited by cellular complexity, metabolic engineering of mammalian cells can

236 be encumbered with the same difficulties.

237 2 Plant Phenylpropanoid Biosynthesis

238 2.1 Background

239 Isoflavonoids belong to a broad class of compounds known as phenolics. Any

240 chemical containing one or more phenol group can be classified as a phenolic

241 compound, although the plant phenolics with the most biotechnological rele-

242 vance are flavonoids and other phenylpropanoids. Phenylpropanoids are sec-

243 ondary plant metabolites that are considered to be beneficial for human health

244 [43]. In particular, a subclass of phenylpropanoids known as flavonoids is

245 typified by bioactive compounds with antioxidant, antiviral and antibacterial,

246 anticancer, antiobesity, and estrogenic properties [9]. The microbial production

247 of flavonoids has attracted much attention due to the prospect of utilizing

248 flavonoids for personal health applications [44]. Flavonoids are currently used

249 as dietary supplements and are the subject of intense investigation as pharma-

250 ceutical precursors to treat chronic human pathological conditions like cancer

251 and diabetes [45–51]. Anthocyanins 17, another class of flavonoids, possess

252 brilliant natural colors and are potential replacements for artificial dyes that

253 have adverse health effects. The antioxidant properties of these glycosylated

254 flavonoids may have a positive health influence and make anthocyanins 17 well

255 suited as natural colorants for the food and beverage industry [52–54]. Antho-

256 cyanins 17 are good targets for metabolic engineering since glycosylations

257 remain a challenge from a chemical synthesis perspective. Furthermore, plant

258 extraction of phenolics seldom yields greater than 1 % of the dry weight.

259 Metabolic engineering of flavonoid biosynthesis has already gained traction

260 due to the long-standing interest in phenolic compounds and the corresponding

261 detailed characterization of related genetic pathways and enzymes [43].

262 As a general classification, phenolics do not contain nitrogen and may

263 contain multiple hydroxyl groups as well as heteroatom substituent groups.

264 Phenolics with greater than 12 phenolic groups are generally considered as

265 polyphenols, lignins, or tannins. Flavonoids are the most well characterized

266 and largest class of natural phenolics, and they are biosynthesized from the

267 aromatic amino acid phenylalanine 2 through the common precursor, chalcone

268 11. Further classification draws a distinction between five types of flavonoids

269 that are derived from the common flavanone 12 precursor: flavones 14, flavonols270 15, isoflavones 13, flavanols, and anthocyanins 17 [55]. Flavonoids are com-

271 posed of a C6-C3-C6 skeleton that serves as a 15-carbon phenylpropanoid core 1 for


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272 downstream decorations such as methylations, hydroxylations, reductions, oxidations,

273 glycosylations, acylations, methoxylations, alkylations, and various rearrangements

274 [44, 56–58]. The flavonoid core 1 consists of 3 rings, labeled A, B, and C.

4

7

6

5

8

A C

2

6�

5�

4�

BO

Flavonoid core1

3

2�

3�

275 Other phenylpropanoids, so named due to their common phenylalanine 2 pre-

276 cursor, include hydroxycinnamic acids, cinnamic aldehydes and monolignols,

277 coumarins, and stilbenoids 8.

278 2.2 Plant Phenylpropanoid Biosynthetic Pathway

279 Phenylpropanoid biosynthesis is initiated by the conversion of phenylalanine 2 to

280 cinnamic acid 5 as catalyzed by phenylalanine ammonia-lyase (PAL). Cinnamic acid

281 5 is then converted to flavanone 12 through a series of subsequent enzymatic reactions

282 involving the following steps: the hydroxylation of cinnamic acid 5 to p-coumaric acid

283 6 through cinnamate 4-hydroxylase (C4H); the ligation of p-coumaric acid 6 to a CoA284 group using 4-coumarate-CoA ligase (4CL); the sequential decarboxylative condensa-

285 tion of three acetate units from malonyl-CoA 10 to 4-coumaroyl-CoA 19 by chalcone

286 synthase (CHS), a type III polyketide synthase, to form chalcone 11 in a ring closing

287 step; and the stereospecific isomerization of chalcone 11 to flavanone 12 catalyzed by

288 chalcone isomerase (CHI). Downstream enzymes then catalyze the conversion of

289 flavanones 12 into compounds belonging to the various flavonoid subclasses.

290 Type III polyketide synthases are particularly relevant to this chapter because

291 they catalyze the formation of phenolic compounds. This group of polyketide

292 synthases consists of CHSs, stilbene synthase (STS), and curcuminoid

293 synthase (CUS), which perform decarboxylative condensations between a starter

294 unit, either p-coumaroyl-CoA 19 or cinnamoyl-CoA 18, and an extender unit,

295 malonyl-CoA 10. CHS, STS, and CUS convert the substrate molecules into flavo-

296 noids (C6-C3-C6), stilbenoids 8 (C6-C2-C6), and curcuminoids 9 (C6-C7-C6),

297 respectively [59]. Stilbenoids 8 and curcuminoids 9 are out of the scope of this

298 chapter but possess medicinal properties as well; resveratrol is a well-known

299 stilbenoid 8 associated with longevity, and curcumin is a common curcuminoid 9300 that is responsible for the yellow color in turmeric and can be utilized as a natural

301 pigment possessing antioxidant and anti-inflammatory properties [60–63]. For an

302 in-depth treatment of plant polyketide production in microbes, the reader is directed

303 to a recent comprehensive review by Boghigian et al. [64].

8 B.F. Cress et al.

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304 2.3 Plant Flavonoid Pathways

305 Plant flavanones 12 are enzymatically converted to five major subclasses of flavo-

306 noids. Flavanones 12 are oxidized to flavones 14 by the action of either a soluble

307 flavone synthase (FSI) or, as in most cases, a membrane-bound cytochrome P450

308 monooxygenase flavone synthase (FSII) [65]. Flavone synthases belong to the

309 oxidoreductase family of enzymes and effectively remove the stereocenter from

310 flavanones 12 by oxidation of C3 and introduction of a double bond between C2 and311 C3. Apigenin, luteolin, and chrysin are common flavones 14 that contribute to

312 human diet as glycosides and are found in large quantities in parsley and

313 celery [66–68].

314 Alternatively, isoflavone synthase (IFS) catalyzes the 1,2-aryl ring

315 B migration from C2 to C3 on ring C of the phenylpropanoid core 1 and the

316 hydroxylation of C2, converting flavanones 12 to 2-hydroxyisoflavanones

317 [69, 70]. Dehydration of 2-hydroxyisoflavanones into isoflavones 13 occurs

318 spontaneously through the 1,2-elimination of water, but accelerated dehydration

319 is catalyzed by one of two hydro-lyases known as 2-hydroxyisoflavanone

320 dehydratases (HID hydroxy type, HIDH; HID methoxy type, HIDM), depending

321 upon the occurrence of an intermediate 40-O-methylation catalyzed by 2,7,40-322 trihydroxyisoflavanone 40-O-methyltransferase (HI40OMT) [71]. Isoflavonoids

323 are characterized by a 3-phenylchroman skeleton, in contrast to the

324 2-phenylchroman core 1 possessed by flavonoids, and are incredibly diverse in

325 structure despite being limited to natural existence primarily in leguminous

326 plants [72]. Soy beans and soy bean food products contain high concentrations

327 of isoflavone 13 glycosides such as genistin 31 and daidzin 30 and relatively

328 lower quantities of their respective aglycones, daidzein 27 and genistein 26 [74].329 Isoflavones 13 are classified as phytoestrogens because of the structural simi-

330 larity shared with estrogens, and they are among the most highly studied poly-

331 phenols due to their affinities for steroid receptors and demonstrated

332 pharmacological properties [18–20, 74]. These characteristics make isoflavones

333 13 important metabolic engineering targets.

334 Flavanones 12 also serve as the substrate for flavanone 3b-hydroxylase (FHT),

335 which catalyzes the hydroxylation of C3 on the flavanone core 1 into

336 dihydroflavonol 16, the common precursor to both flavonols 15 and anthocyanins

337 17. Dihydroflavonols 16 are subsequently converted to flavonols 15 by reduction of338 C2 by the oxidoreductase enzyme flavonol synthase (FLS), again removing the

339 stereocenter and introducing a double bond between C2 and C3 [75]. Flavonols 15340 such as kaempferol and quercetin exist primarily as glycosides at appreciable levels

341 in onions and kale [67, 68].

342 Initiating another branch of the flavonoid pathway, C4 of dihydroflavonol 16343 can be reduced from a carbonyl group to a hydroxyl group by the oxidoreduc-

344 tase enzyme dihydroflavonol reductase (DFR), producing leucoanthocyanidins,

345 or the colorless precursors to anthocyanins 17. Leucoanthocyanidins are unsta-346 ble and are quickly converted to anthocyanidins by anthocyanidin synthase


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347 (ANS), synonymously leucoanthocyanidin dioxygenase (LDOX), working

348 jointly with DFR [76]. Anthocyanidins and leucoanthocyanidins can alterna-

349 tively be reduced to their corresponding flavan-3-ols (proanthocyanidins, or

350 condensed tannins) by anthocyanidin reductase (ANR) and leucoanthocyanidin

351 reductase (LAR), respectively. A flavonoid glycosyltransferase then adds

352 a sugar to the anthocyanidin, enabling pigment storage in the form of stable

353 anthocyanins 17 [77]. Many brilliant red, blue, and purple plant hues arise from

354 anthocyanin-mediated coloration. Figure 53.1 illustrates the alternative path-

355 ways for biosynthesis of various plant phenylpropanoid and flavonoid

356 subclasses.

Phenylalanine2

PAL

COOH COOH NH2

OH

6

Tyrosine3

TAL

OH

COOH4CL

CoASOCAcid-CoA complex

7R2

R1

R2

R1

R2

R1

R2

R1

R2

R1

R2

R1

R3

R1

R3

R1

DFRLAR3GT

HO

17

Flavones

14

HO O

O SCoA

COOH

Malonyl-CoA

10

x3

CUSO

O

4CL

4

Legend:

PAL – phenylalanine ammonia lyaseTAL – tyrosine ammonia lyaseC4H – cinnamate 4-hydroxylase4CL – 4-coumarate-CoA ligaseSTS – stilbene synthaseCUS – curcuminoid synthaseCHS – chalcone synthaseCHI – chalcone isomeraseIFS – isoflavone synthaseFSI – soluble flavone synthaseFSII – membrane-bound flavone synthaseFHT – flavanone 3b-hydroxylaseFLS – flavonol synthaseDFR – dihydroflavonol reductaseLAR – leucoanthocyanidin reductase3GT – 3-O-glucosyltransferase

Central flavanone biosynthetic pathway

COOH

OH

OHCaffeic acid

9Curcuminoids

OOH

FSI/FSII

OHO-Glc

O+

Anthocyanins

COOH

SCoAMalonyl-CoA

x3O10

CHS

OH

OH

HO

OChalcones

11

CHI

HO

OH

O

O

12

HO

OH OOH 16

O

Dihydroflavonols

Flavonoid subclass

Flavanones (12)Isoflavones (13)

Flavones (14)Flavonols (15)

Anthocyanin 3-O-glucosides (17)

Stilbenoids (8)Curcuminoids (9) Dicinnamoylmethane

Pinosylvin

Palargonidin 3-O-glucoside

Kaempferol

Apigenin

5,7-dihydroxyisoflavone

(2S)-pinocembrin (22)

Phenylalanine (2) precursor(R1 = H; R2= H)

Tyrosine (3) precursor(R1 = OH; R2= H)

Caffeic Acid (4) precursor(R1 = OH; R2= OH; R3 = OMe)

(2S)-naringenin (23)

Genistein (26)

Luteolin

Quercetin

Cyanidin 3-O-glucoside

Resveratrol

Bisdemthoxycurcumin Curcumin

Piceatannol

Delphinidin 3-O-glucoside

Myrecetin

Chrysin

Orobol

(2S)-eriodictyol

Flavanones

FHT

p-coumaricacid

NH2

Cinnamic acid5COOH

C4H

4CL

R1

R2

R1

R2

R1

R2

8

10SCoA

IFS

FLS

15OH OOH

OHO

Flavonols

Isoflavones

OHO

13 OH O

Malonyl-CoA

COOH

O

OH

HO

Stilbenoids

STS

x3

Fig. 53.1 Plant phenylpropanoid and flavonoid biosynthetic pathways; representative com-

pounds from each subclass are named

10 B.F. Cress et al.

1656


357 3 Plant Isoflavonoid Production in Microbes

358 3.1 Construction of an Artificial Biosynthetic Pathway for359 Flavonoid Production in Microbes

360 The first construction of an artificial plant flavonoid biosynthetic pathway in microbes

361 involved the transformation of E. coli with four heterologous genes. These genes are

362 required for the synthesis of flavanones 12 from phenylalanine 2 and tyrosine 3363 (through a promiscuous PAL having the ability to accept both phenylalanine 2 and

364 tyrosine 3 as substrates) [78–80]. This exercise provided a platform for the microbial

365 biosynthesis of a plethora of natural and nonnatural flavanone 12 derivatives. It should366 also be noted here that bacterial TAL catalyzes the conversion of tyrosine 3 to

367 p-coumaric acid 6 in one step and can replace the two-step conversion of phenylala-

368 nine 2 to p-coumaric acid 6 by PAL and C4H in an artificial biosynthetic pathway if so

369 desired [81]. Also, depending upon choice of aromatic amino acid precursor, two

370 parallel biosynthetic paths exist for phenylalanine-based flavonoids in contrast to

371 tyrosine-based flavonoids; other common natural and nonnatural aromatic acrylic

372 acids like caffeic acid 4 serve as substrates for 4CL in plants and microbes [82, 83].

373 The substrate flexibility of all enzymes involved allows for perpetuation of extra

374 hydroxyl side groups throughout the entire pathway, affording flavanones 12 or other

375 flavonoids with divergent hydroxylations. Another key distinction to note while read-

376 ing this section is whether the project being described utilizes an entirely fermentative

377 process to produce complex compounds from primary microbial metabolites or

378 whether the project takes advantage of intermediate chemical supplementation.

379 Although neither approach is absolutely superior to the other, distinctions can be

380 drawn between them.

381 For instance, a fermentative approach often suffers from low production due to

382 pathway complexity and increased number of steps, but it allows for production of

383 complex compounds such as phytochemicals from simple, renewable carbon com-

384 pounds like glucose. Conversely, intermediate supplementation is often utilized to

385 simplify pathway construction and is associated with higher product yields. Although

386 supplementing a microbial culture with an expensive precursor might be feasible for

387 a small-scale experiment, it severely hinders industrial applicability. However, if an

388 inexpensive, readily available intermediate can be utilized as a precursor, an entirely

389 fermentative process with lower titers might not be justifiable. A metabolic engineer

390 must then weigh the impact of generating a complex product entirely from primary

391 metabolites versus the value associated with significantly higher production levels.

392 As will be seen throughout this chapter, research efforts are often initiated with

393 intermediate supplementation in order to limit confounding variables, and full fer-

394 mentative pathways are constructed after significant breakthroughs are achieved and

395 once distinct metabolic pathways can be connected in vivo.

396 The experiment described in the beginning of this section involved the incorpo-

397 ration of four heterologous genes: S. cerevisiae PAL, Streptomyces coelicolor A3398 cinnamate/coumarate:CoA ligase (ScCCL) with substrate specificity toward both

399 cinnamic acid 5 and p-coumaric acid 6, licorice plant (Glycyrrhiza echinata) CHS,


1657


400 and Pueraria lobata CHI. Transformation of E. coli with a plasmid harboring these

401 four heterologous genes coupled with overexpression of the Corynebacterium402 glutamicum gene encoding two acetyl-CoA carboxylase subunits, accBC and

403 dtsR1, produced �60 mg L�1 of the flavanones 12 (2S)-naringenin 23 and (2S)-404 pinocembrin 22. The artificial biosynthetic pathway constructed for plant flavanone405 12 biosynthesis in microbes is shown in Fig. 53.2. Acetyl-CoA carboxylase (ACC)

406 was selected for overexpression to increase the intracellular pool of malonyl-CoA

407 10, which is required for synthesis of flavanones 12 from either 4-coumaroyl-CoA

408 19 or cinnamoyl-CoA 18. Further introduction of FSI from Petroselinum crispum,409 FHT from Citrus sinensis, and FLS from Citrus unshiu produced the flavones 14410 apigenin and chrysin, as well as the flavonols 15 kaempferol and galangin in low

411 concentrations [80, 84]. This seminal work has enabled the production in E. coli and412 S. cerevisiae of many valuable phenylpropanoid compounds, including natural and

413 nonnatural flavones 14, flavonols 15, anthocyanins 17, stilbenoids 8, and

414 curcuminoids 9 [42, 60, 65, 82, 84–107]. As this chapter focuses on isoflavonoids,

415 however, the reader is directed to a detailed review of microbial biosynthesis of

416 other valuable plant phenylpropanoids by Limem et al. [43].

417 3.2 Engineering the Plant Isoflavonoid Pathway in Microbes

418 3.2.1419 Production of Isoflavonoid Aglycones in Microbes420 The successful construction of an artificial plant flavonoid biosynthetic pathway in

421 microbes, combined with the first report of functional activity of IFS in yeast

422 microsomes by Akashi and coworkers in 1999, paved the way for high-level

423 isoflavonoid production [69]. However, a significant barrier to prokaryotic

Phenylalanine

COOH

32

Tyrosine

NH2 COOH

COOH

HO

OH

CHS

ScCCL

R

ScCCL

Acid-CoA complex

CoASOC

7

6 OH

CHI

Flavanones

12

R

OH

HO O

O

R = H,(2S )-PinocembrinR = OH,(2S)-Naringenin

R

OH O

11Chalcones

R = OH,

R = H,

x3

O SCoA

Malonyl-CoA Acetyl-CoA 24

ACCSCoAO

10

5

1819

R = H, Cinnamoyl-CoAR = OH, p-coumaroyl-CoA

COOH

COOH

NH2

PAL PAL

OH

p-coumaric acidCinnamic acid

Pinocembrinchalcone Naringeninchalcone

20

21

22

23

Fig. 53.2 Artificial construction of plant flavanone 12 biosynthetic pathway in microbes


1658


424 expression of IFS hampered progress and precluded taking advantage of the high

425 growth rate of E. coli and the abundance of molecular biology tools available for the

426 microbe. IFS is a membrane-bound cytochrome P450 that requires an electron

427 transfer system that is not present in bacterial cells; thus, coexpression of functional

428 IFS with the flavanone 12 pathway in recombinant E. coli required creative

429 engineering solutions. Eukaryotic microbes like S. cerevisiae and other unicellular

430 fungi possess the requisite machinery for cytochrome P450 enzyme expression;

431 specifically, they constitutively express an endogenous cytochrome P450 reductase

432 (CPR) that is an integral redox partner for IFS and other cytochrome P450s, and

433 they possess an endoplasmic reticulum (ER) on which the N-terminal signal-anchor

434 peptide sequences of cytochrome P450 enzymes can bind [108, 109].

435 Katsuyama et al. overcame this impediment by coculturing a flavanone-

436 producing E. coli strain with recombinant S. cerevisiae transformed with

437 a T7-inducible plasmid harboring IFS from G. echinata. To demonstrate the

438 production of the isoflavone 13 genistein 26 and the feasibility of coincubation,

439 the yeast strain was first transformed with a pESC vector containing the genes CHS

440 from G. echinata, CHI from P. lobata, and IFS from G. echinata, all under the441 control of galactose-inducible GAL promoters. Growth under supplementation with

442 the precursor, N-acetylcysteamine-attached p-coumarate (p-coumaroyl-NAC),

443 yielded �342 mg L�1 genistein 26. To examine the possibility of production

444 without precursor feeding, a naringenin-producing E. coli strain (57 mg L�1 of

445 (2S)-naringenin 23) was constructed as described in the previous section and

446 cocultured with a recombinant yeast strain transformed with a vector containing

447 G. echinata IFS under control of a galactose-inducible GAL promoter [80]. Simul-

448 taneous incubation of equal weights of engineered E. coli and S. cerevisiae, in449 addition to supplementation of the coculture media with 3 mM tyrosine 3 as

450 a substrate for E. coli, yielded �6 mg L�1 of genistein 26 [110]. This “one-pot

451 synthesis” scheme for production of genistein 26 from tyrosine 3 represented

452 the most valuable microbial isoflavonoid production platform at the time of its

453 publication. Optimization of coculture conditions subsequently improved genistein

454 26 production up to 100 mg L�1 [111].

455 In order to produce isoflavonoids in a model plant, a native flavonoid pathway must

456 be hijacked by diverting a common precursor away from its natural product and toward

457 the desired isoflavonoid product. Tian and colleagues accomplished production of

458 genistein 26 in the nonleguminous, model plant tobacco through protein engineering

459 of a fusion between IFS and CHI [112]. The spatial proximity between CHI and IFS

460 was engineered to increase the local concentration of the IFS substrate, naringenin 23,461 such that the production of nonnative genistein 26 was favored over the dominant,

462 endogenous pink anthocyanin 17 accumulation pathway. Localization of the protein

463 chimera at the ER was maintained by constructing the fusion with IFS at the

464 N-terminus so its innate, hydrophobic N-terminal membrane anchor, was free to target

465 the ER as usual [87, 113]. A flexible linker peptide composed of glycine-serine-glycine

466 (Gly-Ser-Gly) residues connected the C-terminus of IFS with the N-terminus of CHI to

467 ensure proper folding of the two independent catalytic domains. Expression of this

468 engineered protein fusion in transgenic tobacco successfully shifted flavonoid


1659


469 accumulation toward isoflavonoids and enabled production of isoflavonoids in

470 nonleguminous plants. Yeast expression of the protein fusion under precursor supple-

471 mentation conditions also produced isoflavonoids and highlighted the possibility to

472 utilize protein engineering to improve plant natural product titers in microbes [112].

473 Although E. coli and S. cerevisiae have both been utilized as model organisms

474 for plant flavonoid production, it is often beneficial to express entire biosynthetic

475 pathways in a single organism to avoid bidirectional metabolite transport limita-

476 tions through the cell walls of two organisms simultaneously and to obviate media

477 optimization for two different species at once. Functional expression of IFS in

478 E. coli would eliminate the necessity for coculture with yeast. As such, Leonard and

479 colleagues designed and expressed a set of artificial P450 enzymes that enabled

480 robust biosynthesis of the isoflavones 13 genistein 26 and daidzein 27 from the

481 flavanones 12 naringenin 23 and liquiritigenin in E. coli for the first time [114]. Two

482 challenges to functional prokaryotic expression of eukaryotic cytochrome P450

483 enzymes were overcome in this research: the translational fusion of Catharanthus484 roseus CPR to Glycine max IFS spatially organized the redox partners for efficient

485 electron shuttling from nicotinamide adenine dinucleotide phosphate (NADPH) to

486 substrate, and rational design of several IFS N-terminal membrane signal sequences

487 modulated activity of the protein fusion, enabling selection of a high-level isofla-

488 vone 13 producing chimera [114].

489 The protein engineering effort began with deletion of 71N-terminal amino acids

490 from CPR to minimize membrane association without hindering catalytic activity.

491 A glycine-serine-threonine (Gly-Ser-Thr) linker sequence was then designed to

492 connect the CPR N-terminus with the IFS C-terminus while thwarting any second-

493 ary structure formation that could lead to incorrect folding of the two domains.

494 The protein fusion was then truncated by a varying number of residues from the

495 N-terminus of IFS, and two peptide leader sequences (one mammalian and

496 one endogenous) were independently appended to these constructs in a semicombi-

497 natorial manner. Each chimera was separately expressed in E. coli and evaluated for498 production of isoflavone 13 from supplemented precursor. The most prominent

499 fusion produced 10 and 18 mg g�1 (dry cell weight) of genistein 26 and daidzein 27,500 respectively, and consisted of the deletion of 6 membrane-anchor residues and the

501 addition of an 8 residue mammalian leader sequence to the N-terminus of IFS.

502 To determine a baseline production level, plant IFS and CPR were coexpressed in

503 E. coli and found to yield negligible isoflavonoid concentrations compared to the

504 engineered strain. S. cerevisiae coexpressing plant IFS and CPR produced

505 isoflavones 13 at low concentrations approaching those of the poorly performing

506 protein fusion constructs expressed in E. coli. After accounting for the significantly507 higher biomass of yeast versus E. coli in minimal media, the specific production

508 level of isoflavones 13 in E. coli represented approximately 20-fold increase over

509 yeast [114]. The methodology implemented in this work provides an approach for

510 soluble expression of other eukaryotic membrane-bound cytochrome P450s in

511 prokaryotes. Although not performed in this set of experiments, this research

512 facilitated the impending construction of a complete artificial biosynthetic pathway

513 from aromatic amino acids to isoflavonoids in a single microorganism.


1660


514 A later report of functional expression of IFS in prokaryotes involved

515 construction of a protein fusion between red clover IFS (RCIFS) and rice CPR

516 (RCPR) in E. coli [115]. This work built upon previous results demonstrating that

517 coexpression in yeast of IFS with CPR from rice can convert 100 mM naringenin 23518 to 77 mM genistein 26, research predicated on the hypothesis that a plant CPR, as

519 opposed to a constitutively expressed yeast CPR, would interact more efficiently

520 with a plant IFS [103]. In this project RCIFS was truncated by deletion of the

521 codons for the first 21 amino acids on the N-terminus, a sequence predicted to code

522 for a helical region as indicated by computational secondary structure analysis.

523 Changing the first remaining codon to a start codon (encoded by the nucleotide

524 sequence ATG) enabled functional expression of RCIFS in E. coli, while removal

525 of the IFS stop codon and addition of a Gly-Ser-Thr linker sequence followed by the

526 RCPR coding sequence (also with the N-terminal membrane binding domain

527 deleted) enabled expression and proper folding of the two fused domains. It should

528 be noted here that this protein fusion design differs from that constructed previously

529 by Leonard primarily because, in this case, the hydrophobic N-terminal membrane-

530 associated domains were entirely removed from both enzyme constituents in the

531 fusion to enhance solubility of the final construct. The functional expression and

532 spatial proximity afforded by the soluble RCIFS-RCPR protein fusion enabled

533 conversion of 80 mM naringenin 23 into 56 mM genistein 26 in E. coli. Difference534 in conversion between yeast and E. coli was not investigated but could be due to

535 disparate expression and growth levels in the two distinct species. Again, it is likely

536 that higher-efficiency electron transfer from NADPH to substrate occurred in

537 E. coli due to the conjoined RCPR and RCIFS domains [115].

538 Coexpression in S. cerevisiae of all seven genes in the artificial isoflavone 13539 pathway (PAL and CPR from a hybrid poplar, Populus trichocarpa � Populus540 deltoides and C4H, 4CL, CHS, CHI, and IFS from soybean, G. max), with541 phenylalanine 2 supplementation, was ultimately achieved by Trantas et al.

542 and marked the first reported reconstitution of an entire isoflavonoid biosyn-

543 thetic pathway in microbes. Although yeast contains a chromosomal copy of

544 CPR, coexpression of a heterologous CPR from a the hybrid poplar increased

545 p-coumaric acid 6 production fourfold, once again demonstrating the advantage

546 of selecting a plant CPR to improve activity of the other enzymes in the

547 cytochrome P450 metabolon [101, 116]. Only 0.1 mg L�1 genistein 26 was

548 produced when the cultures were fed with phenylalanine 2 versus 7.7 mg L�1

549 when fed with naringenin 23, suggesting the presence of at least one limiting

550 enzyme or that cellular metabolism was burdened by the genes upstream of

551 naringenin 23. On average, the yeast strains in this work consumed 3.4 mmol L�1

552 phenylalanine 2, while the wild-type strain consumed 2.8 mmol L�1, a difference

553 in phenylalanine 2 uptake of 0.8 mmol L�1 that can be attributed to flux through

554 the heterologous flavonoid pathway. Stoichiometrically, this should lead to

555 0.8 mmol L�1 genistein 26, but production of only 0.4 mmol L�1 indicated

556 approximately 0.05 % efficiency of conversion of phenylalanine 2 to genistein

557 26 through the artificial biosynthetic pathway. Measurement of some upstream

558 intermediates showed 83 % flux efficiency through PAL and C4H, efficient


1661


559 conversion through 4CL as deduced from the rapid depletion of p-coumaric acid

560 6, and approximately 8 % efficiency to naringenin 23, which suggests that CHS

561 or CHI are rate limiting but could not be confirmed due to the inability to

562 quantify concentrations of the intermediate compounds 4-coumaroyl-CoA 19563 and naringenin chalcone 21 [101]. As described by Akashi, coexpression of an

564 HIDH in this engineered S. cerevisiae strain could potentially accelerate the

565 spontaneous conversion of naringenin 23 to genistein 26 but was not attempted

566 in this work [71].

567 The first attempt to coexpress HIDH with IFS and CPR confirmed this specu-

568 lation. Chemler and coworkers coexpressed IFS, CPR, and HIDH from five

569 various plant sources in yeast in a combinatorial fashion to determine the impact

570 of gene source on individual enzyme activity and coupled enzyme activities

571 [117]. IFS from G. max, Trifolium pratense, G. echinata, Pisum sativa, and572 Medicago truncatula was individually cloned into a pYES2.1 vector under con-

573 trol of the GAL1 promoter and transformed into S. cerevisiae strain INVSc1. After574 growth on minimal medium, the cultures were induced with galactose and

575 supplemented with naringenin 23. Genistein 26 production was monitored, and

576 T. pratense IFS was selected as the best enzyme because it showed significantly

577 higher in vivo activity than the IFS enzymes from other sources. Since it had

578 previously been shown that plant IFS activity in yeast is improved upon

579 coexpression of plant CPR, the researchers coexpressed CPR from C. roseus580 and G. max with IFS from either G. max or T. pratense to determine the enzyme

581 pair with highest coupled activity. Upon comparing genistein 26 production in

582 these engineered strains to yeast expressing IFS with endogenous CPR, the strain

583 coexpressing T. pratense IFS with G. max CPR was found to be the highest

584 producer at 15 mg L�1 day�1. This illustrates the value in combining different

585 gene sources to determine optimal protein pairing, particularly in the case of

586 enzyme-mediated redox reactions. To assess whether expression of plant HIDH

587 could increase genistein 26 production over its spontaneous generation from its

588 2-hydroxyisoflavanone precursor in yeast, coexpression of G. max or G. echinata589 HIDH was evaluated in the engineered strains. The best triple-enzyme combina-

590 tion was found to include T. pratense IFS, G. max CPR, and G. max HIDH,

591 followed closely by the cognate combination of G. max IFS, CPR, and HIDH.

592 Interestingly, T. pratense IFS holds some advantage over G. max IFS when

593 coexpressed with G. max CPR and HIDH, despite presumption that the G. max594 enzymes evolved to work optimally together. Ultimately the three-enzyme com-

595 bination showed greater than tenfold improvement in production rate over expres-

596 sion of IFS alone, but total production in all strains maximized at around

597 35 mg L�1 genistein 26. After further experimentation, it was shown that

598 isoflavones 13 like genistein 26 and biochanin A 29 strongly inhibit conversion

599 of naringenin 23 by IFS in yeast. It was speculated that isoflavone 13 glycosyl-

600 ations, methylations, and other enzymatic biotransformations might ameliorate

601 product inhibition and increase overall isoflavonoid production [117]. The basic

602 artificial biosynthetic pathway for plant isoflavone 13 production from flavanones

603 12 in microbes is illustrated in Fig. 53.3.


1662


604 3.2.2605 Production of Isoflavonoid Glycosides in Microbes606 Many flavonoids and other secondary metabolites exist as glycosides in plants, and

607 examples of engineered microbial glycosylation of various flavonoids like quercetin

608 and anthocyanidins have been reported over the last decade [85, 96, 118, 119].

609 Glycosylation of flavonoid aglycones is important because it often increases mamma-

610 lian bioavailability, solubility, and stability [68, 120–129]. In the first example of

611 microbial isoflavonoid glycosylation, expression of UGT71G1, a uridine diphosphate

612 glycosyltransferase (UGT) from the model legume M. truncatula, in heterologous E.613 coliwith supplementation of genistein 26 and biochanin A 29 yielded mg quantities at

614 greater than 70 % conversion to genistein 26 and biochanin A 29 7-O-glucosides615 (genistin 31 and sissotrin 33, respectively) after 24-h incubation (Fig. 53.4). Terrific

616 broth (TB) culture medium supported higher growth than Luria-Bertani (LB) culture

617 medium and thus provided 3.5-fold higher yield of 7-O-glucoside. Scale-up to 500 mL

618 culture achieved conversion rates of 30–60 %, about 80 % efficient compared to small

619 scale, yielding up to 20 mg L�1 of isoflavanone glycosides [98]. Of note, 90 % of the

620 glycosylated products were secreted from the cell, enabling facile collection and

621 suggesting that increased solubility or sugar moiety-related signaling affects efflux

622 from the cell. As such, this work suggests that coexpression of M. truncatula623 UGT71G1 in Chemler’s yeast strain (T. pratense IFS, G. max CPR, G. max HIDH)

624 could convert naringenin 23 to genistin 31, the genistein 26 7-O-glucoside, at much

625 higher rates than previously reported because feedback inhibition would be minimized

OHIFSCPR HO O OH

OH

HIDH

OH O

Genistein 26

OH

OHO

or

(2S)-Naringenin 23

IFS-CPRfusionOH O

OHO

OH O

2,4�,5,7-tetrahydroxyisoflavanone 25

Fig. 53.3 Aggregate artificial biosynthetic pathway for plant isoflavone 13 production from

flavanones 12 in microorganisms

UDP-glucose

UGT

UDP

Glu-O O

OR1

R1 = H, R2 = OH,DaidzinR1 = OH, R2 = OH,GenistinR1 = H, R2 = OCH3,OnoninR1 = OH, R2 = OCH3,Sissotrin

R1 = H, R2 = OH,DaidzeinR1 = OH, R2 = OH,GenisteinR1 = H, R2 = OCH3,FormononetinR1 = OH, R2 = OCH3,Biochanin A

27

O

OHO

R1R2

26

28

29 33

32

31

30

R2

Fig. 53.4 Microbial bioconversion of isoflavone 13 aglycones to isoflavone 13 glycosides


1663


626 by the glycosylation and subsequent export from the cell. Whereas extraction of plant

627 flavonoid glycosides is inefficient, and regioselective glycosylation of flavonoids

628 through chemical synthesis methods requires intermittent blocking and deblocking

629 of hydroxyl groups and yields only about 50 % conversion due to the occurrence of

630 nonspecific glycosylations, glycosylation through biotransformation offers a highly

631 efficient and cheap alternative [130–133]. A major barrier to high-level microbial

632 production of any flavonoid glycoside, however, is intracellular supply of uridine

633 diphosphate (UDP) glucose.

634 As seen in Fig. 53.4, nucleotide-activated sugars are required as donors for

635 glycosylation. In previous work, Yan and colleagues engineered a four-step metabolic

636 pathway for plant anthocyanin 17 biosynthesis in E. coli, which involved expression of637 four heterologous genes including Malus domestica FHT and ANS, Anthurium638 andraeanum DFR, and Petunia hybrida UDP-glucose:flavonoid 3-O-glucosyl-639 transferase (3GT). Anthocyanidins were converted by 3GT to the first stable glyco-

640 sidic anthocyanins 17 in the flavonoid biosynthetic pathway, pelargonidin 3-O-641 glucoside and cyanidin 3-O-glucoside [85]. The researchers identified UDP-glucose

642 as the rate-limiting step in anthocyanin 17 biosynthesis in E. coli and thereafter

643 optimized UDP-glucose production by supplementing with orotic acid, a cheap uri-

644 dine triphosphate (UTP) precursor, and performing a gene deletion and a set of gene

645 overexpressions. As synthesis of UDP-glucose interfaces nucleotide biosynthesis, the

646 pentose phosphate pathway, glycolysis, and energy production pathways, engineering

647 its overproduction is a nontrivial task. Episomal overexpression of endogenous phos-

648 phoglucomutase (PGM) and glucose-1-phosphate uridylyltransferase (GALU), which

649 convert glucose-6-phosphate (G6P) to glucose-1-phosphate (G1P) and produce UDP-

650 glucose from G1P and UTP, respectively, shunted carbon flux from the pentose

651 phosphate pathway toward UDP-glucose through the G6P branching point [57,

652 134]. These genetic modifications combined with the overexpression of endogenous

653 nucleoside diphosphate kinase (NDK), the limiting step in the linear UTP synthesis

654 pathway by orotic acid assimilation, and deletion of a gene encoding UDP-glucose

655 dehydrogenase (UDG), which consumes UDP-glucose to form UDP-glucuronic acid,

656 to yield increased UDP-glucose accumulation of 104 mg L�1 [96, 135, 136]. Due to

657 the natural production of UDP-glucose in E. coli for cell wall synthesis and the ability658 to achieve increased production of UDP-glucose, microbial glycosylation of

659 isoflavonoid aglycones with heterologous glycosyltransferases is an economically

660 viable option. To the best of our knowledge, Table 53.1 summarizes the most

661 representative studies of microbial production of plant natural isoflavonoids to date.

662 4 Mutasynthesis and Protein Engineering for Nonnatural663 Isoflavonoid Production in Microbes

664 Mutasynthesis is a common semisynthetic tool that hijacks natural product biosynthesis

665 through the feeding of nonnatural substrate analogs to produce nonnatural analogs to

666 natural products. This methodology takes advantage of the natural allowable range of

667 enzyme-substrate specificity and favors highly promiscuous enzymes that can convert


1664


t1:1 Table 53.1 Reports demonstrating microbial production of plant natural isoflavonoids

Isoflavonoid

target Precursor Host organism Genes: Donors Referencet1:2

Genistein 26 N-acetylcysteamine-

attached p-coumaric acid

E. coli CHS: G. echinata [110]t1:3

IFS: G. echinatat1:4

CHI: P. lobatat1:5

Genistein 26 Tyrosine 3 E. coli andS. cerevisiaecoculture

PAL: R. rubra [110]t1:6

4CL: S. coelicolort1:7

CHS: G. echinatat1:8

CHI: P. lobatat1:9

IFS: G. echinatat1:10

ACC: C. glutamicumt1:11

Genistein 26 Naringenin 23 S. cerevisiae IFS: T. pratense [103]t1:12

CPR: O. sativat1:13

Genistein 26 Phenylalanine 2 S. cerevisiae PAL: P. trichocarpa �P. deltoides

[101]t1:14

CPR: P. trichocarpa �P. deltoidest1:15

C4H: G. maxt1:16

4CL: G. maxt1:17

CHS: G. maxt1:18

CHI: G. maxt1:19

IFS: G. maxt1:20

Genistein 26 p-coumaric acid 6 S. cerevisiae PAL: P. trichocarpa �P. deltoides

[101]t1:21


C4H: G. maxt1:23

4CL: G. maxt1:24

CHS: G. maxt1:25

CHI: G. maxt1:26

IFS: G. maxt1:27

Genistein 26 Naringenin 23 S. cerevisiae PAL: P. trichocarpa �P. deltoides

[101]t1:28


C4H: G. maxt1:30

4CL: G. maxt1:31

CHS: G. maxt1:32

CHI: G. maxt1:33

IFS: G. maxt1:34

Genistein 26,Daidzein 27

Naringenin 23,Isoliquiritigenin

S. cerevisiae CHIa: M. sativa [112]t1:35

IFSa: G. maxt1:36

Genistein 26 Naringenin 23 S. cerevisiae IFS: G. max [137]t1:37


Naringenin 23,Liquiritigenin

S. cerevisiae IFS: G. echinata [69]t1:38

(continued)


1665


668 nonnatural analogs of the natural substrate to novel products. Since many plant natural

669 products possess valuable medicinal properties, it is of significant interest to explore the

670 space of nonnatural product analogs that has not yet been evolutionarily surveyed

671 because of the lack of nonnatural substrates in the environment. Presumably some of

672 these nonnatural analogs could have enhanced or even unique pharmaceutical proper-

673 ties. Production of flavonoids using mutasynthesis or substrate feeding has been

674 accomplished by several groups as reported elsewhere [60, 82, 97, 100].

675 Structural studies often utilize protein engineering tools such as site-directed muta-

676 genesis to evaluate the roles of various amino acid residues in catalytic mechanisms.

677 While this can furnish indispensable insight on enzyme-substrate interaction, it is of

678 significant interest to metabolic engineers because it also enables construction of tailor-

679 made enzyme mutants with improved kinetic properties, with the ability to accept

680 structurally related substrates, with reaction reversibility for substrate-product inter-

681 conversion, and with altered substrate and product regiospecificity. Protein engineering

682 tools such as site-directed mutagenesis and directed evolution have been applied to

683 improve production of both natural and nonnatural flavonoid, isoflavonoid, and other

684 plant natural product derivatives [87, 138–147]. Plant natural products can also be

685 microbially catalyzed by enzymes native to the microbe to form compounds not known

686 to exist in plants [82, 100, 119, 148–153]. These phytochemical derivatives have the

687 potential to be utilized as human therapeutics, as the microbes catalyzing these novel

688 reactions have been isolated from the human gut and are purported to have beneficial

689 health impacts on their human hosts [152–157].

690 4.1 Mutasynthesis for Nonnatural Isoflavonoid Production

691 Mutasynthesis involves the chemical synthesis of nonnatural substrates that are

692 similar in structure to natural substrates. After a library of nonnatural analogs are

t1:39 Table 53.1 (continued)

Isoflavonoid

target Precursor Host organism Genes: Donors Referencet1:40



E. coli CPRa: C. roseus [114]t1:41

IFSa: G. maxt1:42



S. cerevisiae CPR: C. roseus [114]t1:43

IFS: G. maxt1:44

Genistein 26 Naringenin 23 S. cerevisiae CPRa: O. sativa [116]t1:45

IFSa: T. pratenset1:46

Genistin 31,Sissotrin 33

Genistein 26, BiochaninA 29

E. coli UGT: M. truncatula [98]t1:47

See

Table 53.2

See Table 53.2 S. cerevisiae CPR: G. max [117]t1:48

IFS: T. pratenset1:49

HIDH: G. maxt1:50

t1:51aProtein fusion


1666


693 chemically synthesized, enzymatic conversion of the nonnatural analogs is

694 performed to isolate novel nonnatural compounds, and the results can then be

695 assessed to elucidate mechanisms of enzymatic catalysis and to determine sub-

696 strate specificity requirements. This so-called semisynthetic approach, or the

697 combination of chemical synthesis and biosynthesis, has also been utilized for

698 production of nonnatural isoflavonoids.

699 In a multiplex experiment, Chemler and colleagues evaluated the substrate

700 specificity of IFS enzymes from five different plant species (G. max, T. pratense,701 G. echinata, P. sativa, and M. truncatula) [117]. Each enzyme was cloned into

702 yeast and was supplemented with compounds from a library of natural and

703 nonnatural flavanones 12. Nonnatural flavanones 12 were synthesized to mimic

704 natural flavanones 12 and isoflavones 13; specifically, many library constituents

705 were 7-monohydroxylated or 5,7-dihydroxylated. The library also consisted of

706 flavanones 12 with B-ring substituents, such as single or multiple hydroxy,

707 methoxy, ethoxy, and halide side groups. Ultimately 19 nonnatural flavanones 12708 and 7 natural flavanones 12 were utilized to assess IFS substrate flexibility,

709 resulting in the biosynthesis of 4 natural isoflavones 13 and 14 nonnatural isofla-

710 vone 13 analogs which are tabulated in Table 53.2. IFS substrate requirements were

711 deduced from the rate of conversion of different flavanones 12, including the

712 necessity for hydroxylation at C7, the expendability of C5 hydroxylation, the

713 incompatibility of C20 or C60 substitutions, the toleration of small side-group sub-

714 stitutions at C30 or C50, and the absolute requirement of C40 hydroxylation for

715 production of 2-hydroxyisoflavones. Due to the high affinity of genistein 26 for

716 human estrogen receptors a (hERa) and b (hERb), isoflavones 13 are selective

717 estrogen receptor modulator (SERM) drug candidates [158–161]. SERMs can be

718 used to inhibit or stimulate estrogen receptors, thereby enabling their use as

719 hormone replacements and decreasing the risk of diseases such as

720 osteoporosis and breast cancer [17, 50, 53, 160]. In an effort to determine the

721 therapeutic potential of the semisynthetic isoflavones 13 in the previously described722 library, the interaction of each compound with hERa and hERb was assessed using

723 an in vitro competitive binding assay. As expected, the different isoflavones 13724 were found to show variable activity against the human estrogen receptors. Of

725 particular interest, both 30-bromo-40,5,7-trihydroxyflavone and the natural isofla-

726 vone 13 orobol displayed binding capabilities equal to genistein 26.727 Structure-activity relationships between isoflavones 13 and hERs were then

728 deduced to yield insight for future design of isoflavone 13 SERMs. Of note, the

729 authors suggest that novel isoflavones 13 with small substituents at

730 the C30 position should elicit improved interactions with estrogen receptors [117].

731 4.2 Protein Engineering for Nonnatural Isoflavonoid Production

732 Protein engineering has been utilized to study the mechanism by which

733 isoflavonoid aglycones are converted to isoflavonoid glycosides by uridine diphos-

734 phate glycosyltransferases, a large protein class catalyzing the transfer of activated


1667


t2:1 Table 53.2 Mutasynthesis for natural and nonnatural isoflavonoid production

O

OFlavanone

Isoflavoneor

Isoflavanol12

HO O

O

1334

IFSCPRHIDH

HO

R5

R5�

R5

R3�

R4�

R5�

R4�R3�

Side-group decorationt2:2

Flavanone precursor R5 R30 R40 R50 Primary biotransformation productt2:3

Naringenin 23 OH H OH H Genistein 26t2:4

Liquiritigenin H H OH H Daidzein 27t2:5

Eriodictyol OH OH OH H Orobolt2:6

Butin H OH OH H 30,40,7-Trihydroxyisoflavonet2:7

Homoeriodictyol OH OCH3 OH H 30-Methoxy-40,5,7-trihydroxyisoflavonet2:8

40,7-Dihydroxy-30-methoxyflavanone

H OCH3 OH H 40,7-Dihydroxy-30-methoxyisoflavonet2:9

30,50-Dimethoxy-40,5,7-trihydroxyflavanone

OH OCH3 OH OCH3 30,50-Dimethoxy-40,5,7-trihydroxyisoflavonet2:10

40,7-Dihydroxy-30,50-dimethoxyflavanone

H OCH3 OH OCH3 40,7-Dihydroxy-30,50-dimethoxyisoflavonet2:11

30-Ethoxy-40,5,7-trihydroxyflavanone

OH OCH2CH3 OH H 30-Ethoxy-40,5,7-trihydroxyflavanonet2:12

40,7-Dihydroxy-30-ethoxyflavanone

H OCH2CH3 OH H 40,7-Dihydroxy-30-ethoxyflavanonet2:13

30-Methyl-405,7-trihydroxyflavanone

OH CH3 OH H 30-Methyl-40,5,7-trihydroxyisoflavonet2:14

407-Dihydroxy-30-methylflavanone

H CH3 OH H 40,7-Dihydroxy-30-methylisoflavonet2:15

30,50-Dimethyl-40,5,7-trihydroxyflavanone

OH CH3 OH CH3 30,50-Dimethyl-40,5,7-trihydroxyisoflavonet2:16

40,7-Dihydroxy-30,50-dimethylflavanone

H CH3 OH CH3 40,7-Dihydroxy-30,50-dimethylisoflavonet2:17

30-Chloro-40,5,7-trihydroxyflavanone

OH Cl OH H 30-Chloro-40,5,7-trihydroxyisoflavonet2:18

30-Chloro-40,7-dihydroxyflavanone

H Cl OH H 30-Chloro-40,7-dihydroxyisoflavonet2:19

30-Bromo-40,5,7-trihydroxyflavanone

OH Br OH H 30-Bromo-40,5,7-trihydroxyisoflavonet2:20

30-Bromo-40,7-dihydroxyflavanone

OH Br OH H 30-Bromo-40,7-dihydroxyisoflavonet2:21


1668


735 sugars to various substrates. These studies yield insight into the interactions

736 between specific amino acid residues and substrate, enabling rational design of

737 enzyme mutants for specific purposes. Structure-guided enzyme engineering is

738 often directed at or around the active site or binding pocket region to alter substrate

739 specificity, enzymatic activity, and product regioselectivity. In the case of

740 UGT71G1 from M. truncatula, a point mutation in residue 202 from tyrosine

741 (Tyr) 3 to alanine (Ala), Y202A, enables the conversion of genistein 26 to both

742 7-O-glucoside and 5-O-glucoside, whereas the native enzyme only enables conver-

743 sion of genistein 26 to the 7-O-glucoside product. Residue 202 is located at one end744 of the acceptor (isoflavonoid aglycone) binding pocket, so this mutation from an

745 amino acid with a large aromatic side group to one with a small side group

746 presumably increases the volume of the pocket, providing the acceptor with an

747 increased number of possible docking configurations [140].

748 Another protein engineering effort for isoflavonoid production focused on M.749 truncatula UGT85H2. A point mutation in residue 305 from isoleucine (Ile) to

750 threonine (Thr), I305T, showed a 19-fold increase in enzyme activity with a 25-fold

751 decrease in the Michaelis constant (Km) for conversion of biochanin A 29 into

752 sissotrin 33. Additionally the mutation of residue 200 from valine (Val) to glutamic

753 acid (Glu), V200E, imparted deglycosylation activity in the presence of UDP in the

754 reaction mixture, enabling the removal of the glucose residue from sissotrin 33, the755 biochanin A 29 7-O-glucoside, to form biochanin A 29 aglycone. The mutation also

756 decreased Km by sevenfold, increased maximum velocity (Vmax) and turnover

757 number (kcat) by sevenfold, and increased catalytic efficiency by 54-fold. Amino

758 acid 200 resides on one end of the acceptor binding pocket, and docking studies

759 indicate that the negatively charged glutamic acid side group might interact with the

760 7-OH of biochanin A 29. This novel method utilizing mutagenesis to impart

761 reversibility could be applied to deglycosylation of other flavonoids [141].

762 The aforementioned UGT mutagenesis studies involved variations in activity

763 and regioselectivity. However, glycosylation of flavonoids with sugars other than

764 glucose occurs in nature and should be possible to engineer in microbes. In addition

765 to UDP-glucose, for instance, UDP-glucuronic acid, UDP-galactose, UDP-xylose,

766 and UDP-rhamnose are all known to act as nucleotide-activated sugar donors in

767 various plant species [162]. In Bellis perennis (red daisy) BpUGT94B1, the

768 positively charged guanidinium side group of a single arginine (Arg) residue at

769 position 25 is critical for UDP-glucuronic acid donor activity due to its interaction

770 with the negatively charged carboxylate group on glucuronic acid [139]. Similarly,

771 a family of UGTs known as flavonoid 7-O-glucuronosyltransferases (F7GATs)

772 found in plants from the Lamiales order share a conserved arginine residue in the

773 sugar donor binding pocket that is responsible for the specificity toward

774 UDP-glucuronic acid. Site-directed mutagenesis of Perilla frutescens UGT88D7

775 residue 350 containing arginine (which corresponds to tryptophan (Trp) 360 in

776 UGT71G1) to Trp abolished UDP-glucuronic acid specificity and instead invoked

777 UDP-glucose sugar donor specificity. Once again the cationic guanidinium moiety

778 on arginine is crucial for recognition and interaction with the anionic carboxylate

779 group on UDP-glucuronic acid.


1669


780 4.3 Other Isoflavonoid Biotransformations

781 In a series of recent reports, a G6P isomerase (PGI, catalyzing the isomerization of

782 G6P to fructose-6-phosphate) knockout strain of E. coli was engineered to produce

783 flavonoid glycosides from flavonoid aglycones. Specifically, the strain produced

784 7-O-xylosyl naringenin and 7-O-glucuronyl quercetin by overexpressing an

785 Arabidopsis thaliana UGT and an artificial UDP-sugar biosynthetic gene cluster

786 (containing E. coli K-12 GALU andMicromonospora echinospora spp. calichensis787 UDG and UDP-glucuronic acid decarboxylase, known as UXS1) in combination

788 with naringenin 23 and quercetin feeding [163, 164]. Continuing their efforts,

789 Simkhada and coworkers recently engineered E. coli for production of 3-O-790 rhamnosyl quercetin, 3-O-rhamnosyl kaempferol, and 3-O-allosyl quercetin by

791 assembling artificial thymidyldiphosphate (TDP)-sugar biosynthetic pathways for

792 TDP-L-rhamnose and TDP-6-deoxy-b-D-allose and feeding the strain with querce-

793 tin and kaempferol aglycones.

794 TDP-sugar production was enabled by the deletion of PGI to shunt flux toward

795 G1P and overexpression of TDP-glucose synthase (TGS) from Thermus796 caldophilus GK24 to form the activated nucleotide sugar [165]. TDP-L-rhamnose

797 was produced by overexpression of Salmonella typhimurium LT2 TDP-glucose

798 4,6-dehydratase (DH) and Streptomyces antibioticus Tu99 TDP-4-keto-6-

799 deoxyglucose 3,5-epimerase (EPI) and TDP-glucose 4-ketoreductase (KR); TDP-

800 6-deoxy-b-D-allose was produced by overexpression of T. caldophilus GK24 DH

801 and Streptomyces sp. KCTC 0041BP TDP-hexose 3-epimerase (GERF) and TDP-

802 4-keto-6-deoxyglucose reductase (GERK). Overexpression of a 3GT from A.803 thaliana completed the 3-O-glycosylation of the flavonoid aglycone precursors

804 with the TDP-sugars [166]. These engineering efforts demonstrate the potential

805 for regiospecific glycosylation of isoflavonoids with tailored sugar moieties that

806 could one day enable design of therapeutics with altered activities and varying

807 degrees of bioavailability; from a microbial production perspective, customizable

808 glycosylations might also mitigate cellular toxicity while improving isoflavonoid

809 solubility, stability, and transport from the cell, ultimately leading to higher product

810 yields [126, 167].

811 Other flavonoid biotransformations catalyzed by microbial enzymes will also

812 allow for production of novel, nonplant flavonoids from amino acid precursors.

813 Two bacterial nonheme dioxygenases, biphenyl dioxygenase (BDO) and naphtha-

814 lene dioxygenase (NDO), have recently been shown to regioselectively and

815 stereoselectively convert flavonoids, including isoflavones 13 and isoflavanols 34,816 to epoxides and dihydrodiols [151, 168–172]. BDO from Pseudomonas pseudoal-817 caligenes KF707 and NDO from Pseudomonas sp. strain NCIB9816-4 are able to

818 accept various flavonoids as substrates due to the presence of biphenyl and naph-

819 thalene moieties within the flavonoid core structure 1 [168]. Additionally, expres-

820 sion of Streptomyces avermitilis MA-4680 7-O-methyltransferase (SaOMT-2) in

821 E. coli shows substrate promiscuity and transfers a methyl group to flavones 14 and822 isoflavones 13 [173]. This is the first example of a methyltransferase known to act

823 upon both flavones 14 and isoflavones 13, opening up a route for biosynthesis of


1670


824 nonnatural methylated isoflavones 13 by feeding of nonnatural precursors. Another825 example of microbial isoflavonoid biotransformation is the reduction of daidzein 27826 to equol. Although several microorganisms isolated from mammalian digestive

827 tracts have been shown to catalyze the nonstereospecific transformation, a recently

828 isolated gram-negative anaerobic species, MRG-1, shares high homology with

829 Coprobacillus species and was shown to exhibit stereospecific reductase activity

830 for conversion of several isoflavones 13 to the corresponding isoflavanones.

831 Stereoselective reduction from the highly active MRG-1 isoflavone reductase

832 (IFR) opens new biotechnological routes for production of enantiopure flavanones

833 12 [153].

834 5 Concluding Remarks

835 Metabolic engineering of microbes for isoflavonoid biosynthesis showcases state-

836 of-the-art methodologies for high-level production of pharmaceutically and

837 nutraceutically relevant compounds. Decoupling production of plant secondary

838 metabolites from their native, convoluted regulatory backgrounds enables predict-

839 able control and design, while transplanting biosynthetic pathways into fast-grow-

840 ing, well-characterized microorganisms allows utilization of advanced genetic and

841 computational tools and an abundance of biological data. Genetically tractable

842 microbes such as E. coli and S. cerevisiae provide an unmatched platform for

843 combinatorial biosynthesis of complex plant natural products and their nonnatural

844 derivatives by transformation with heterologous genes from different organisms.

845 Though microbial production of plant natural products is a promising alternative

846 to traditional methods, further research will continue to improve titers and assist in

847 the discovery of novel isoflavonoid biotransformations. A significant challenge that

848 has not yet been accomplished is the expression of the entire isoflavonoid metabolic

849 pathway in E. coli, from aromatic amino acid precursors without supplementation

850 of intermediates. Given the propensity for feedback inhibition and host toxicity of

851 many flavonoid and isoflavonoid intermediates, protein engineering efforts will

852 likely be required to enable high-level isoflavonoid production [174–176]. Further-

853 more, in vivo characterization of all enzymes in the isoflavonoid pathway will help

854 determine rate-limiting steps that require higher relative promotion or expression

855 level. Stoichiometric-based modeling and computational algorithms can also be

856 utilized to predict genetic manipulations for maintaining high growth coupled with

857 high specific production. Several thorough reviews have addressed the relative

858 merits of various algorithms [177–182].

859 Feedback inhibition can be limited by optimizing both upstream and down-

860 stream enzyme expression such that the inhibitor does not significantly accumulate.

861 In instances where a metabolite inhibits an enzyme in the isoflavonoid pathway,

862 enzyme mutagenesis can alter the structural interaction between the enzyme and its

863 inhibitor to block the inhibition mechanism. Recently, allosteric feedback inhibi-

864 tion of a tomato peel 4CL by naringenin 23, a product several steps downstream,

865 was significantly reduced through directed evolution in E. coli [181].


1671


866 Cellular toxicity can also be ameliorated by various engineering strategies.

867 Toxicity caused by intracellular accumulation of an intermediate can be limited

868 by pathway optimization to ensure that the metabolite is utilized soon after it is

869 produced. Pathway optimization can be achieved by accurate in vivo characteriza-

870 tion of all enzymes in the pathway. Additionally, spatial localization of the enzymes

871 catalyzing subsequent steps in a pathway serves as a “pipeline” to channel inter-

872 mediate substrates to their respective catalyzing enzymes [184, 185]. This spatial

873 proximity effectively leads to increase local substrate concentration and can be

874 engineered by creating a protein fusion between adjacent enzymes, by docking

875 multiple enzymes to a protein scaffold at minimal distance from each other, or by

876 compartmentalizing all of the enzymes in a biosynthetic pathway in an isolated

877 enclosure, such as a bacterial microcompartment (BMC) or an artificial organelle

878 [186–188]. Such methodologies have enabled significant improvement in produc-

879 tion levels of other microbial products and are outlined in great detail in a recent

880 review by Agapakis and colleagues [185]. If the final product is toxic to the cell, one

881 method for reducing the toxicity is to engineer product transport. Overexpression of

882 a library of efflux pumps and extracellular transporters can pinpoint proteins

883 capable of selective export of a target product, while product glycosylation or

884 deglycosylation could also improve export from the cell [189–191]. It is also

885 important to consider if the product is natively transported into the cell from the

886 extracellular environment; blocking transport of the toxic compound back into the

887 cell can be accomplished by knocking out genes involved in product uptake.

888 Further work aimed at bioprospecting, culturing hard-to-culture microbes, searching

889 for “unknown” and “orphan” enzymes that have not yet been characterized, and

890 designing promiscuous enzymes capable of decorating and transforming flavonoids

891 and their unnatural analogs will increase the range of isoflavonoid derivatives produced

892 in microbes [192]. The search for enzymes capable of such manipulations should not

893 be limited to plants, however, as many microbes endemic to mammalian guts have

894 evolved to metabolize the plant phenylpropanoids ingested by their hosts. Current

895 research efforts in these areas will lead to economically viable microbial platforms for

896 production of isoflavonoids and products of high medicinal value.

897 References

898 1. Demain AL, Fang A (2000) The natural functions of secondary metabolites. Adv Biochem

899 Eng Biotechnol 69:1–39

900 2. Rhodes MJ (1994) Physiological roles for secondary metabolites in plants: some progress,

901 many outstanding problems. Plant Mol Biol 24:1–20

902 3. Demain AL, Sanchez S (2009) Microbial drug discovery: 80 years of progress. J Antibiot

903 62:5–16

904 4. Rodrıguez-Concepcion M, Boronat A (2002) Elucidation of the methylerythritol phosphate

905 pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved

906 through genomics. Plant Physiol 130:1079–1089

907 5. Demain AL (2000) Small bugs, big business: the economic power of the microbe. Biotechnol

908 Adv 18:499–514


1672


909 6. Keasling JD (2010) Manufacturing molecules through metabolic engineering. Sci (NY)

910 330:1355–1358

911 7. Dixon R, Steele C (1999) Flavonoids and isoflavonoids – a gold mine for metabolic

912 engineering. Trends Plant Sci 4:394–400

913 8. Dixon RA (2004) Phytoestrogens. Annu Rev Plant Biol 55:225–261

914 9. Forkmann G, Martens S (2001) Metabolic engineering and applications of flavonoids. Curr

915 Opin Biotechnol 12:155–160

916 10. Yu O, McGonigle B (2005) Metabolic engineering of isoflavone biosynthesis. Adv Agron

917 86:147–190

918 11. Yu O, Jez JM (2008) Nature’s assembly line: biosynthesis of simple phenylpropanoids and

919 polyketides. Plant J Cell MolBiol 54:750–762

920 12. Yamamoto S, Sobue T, Kobayashi M, Sasaki S, Tsugane S (2003) Soy, isoflavones, and

921 breast cancer risk in Japan. J Natl Cancer Inst 95:906–913

922 13. Siow RCM, Li FYL, Rowlands DJ, de Winter P, Mann GE (2007) Cardiovascular targets for

923 estrogens and phytoestrogens: transcriptional regulation of nitric oxide synthase and antiox-

924 idant defense genes. Free Radic Biol Med 42:909–925

925 14. Squadrito F, Altavilla D, Crisafulli A, Saitta A, Cucinotta D, Morabito N, D’Anna R, Corrado F,

926 Ruggeri P, Frisina N, Squadrito G (2003) Effect of genistein on endothelial function in

927 postmenopausal women: a randomized, double-blind, controlled study. Am J Med 114:470–476

928 15. Liu D, Zhen W, Yang Z, Carter JD, Si H, Reynolds KA (2006) Genistein acutely stimulates

929 insulin secretion in pancreatic beta-cells through a cAMP-dependent protein kinase pathway.

930 Diabetes 55:1043–1050

931 16. Rasbach KA, Schnellmann RG (2008) Isoflavones promote mitochondrial biogenesis.

932 J Pharmacol Exp Ther 325:536–543

933 17. Zhao L, Brinton RD (2007) WHI and WHIMS follow-up and human studies of soy

934 isoflavones on cognition. Expert Rev Neurother 7:1549–1564

935 18. Ji Z-N, Zhao WY, Liao GR, Choi RC, Lo CK, Dong TTX, Tsim KWK (2006) In vitro

936 estrogenic activity of formononetin by two bioassay systems. Gynecol EndocrinolOff J Int

937 Soc Gynecol Endocrinol 22:578–584

938 19. McCarty MF (2006) Isoflavones made simple – genistein’s agonist activity for the beta-type

939 estrogen receptor mediates their health benefits. Med hypotheses 66:1093–1114

940 20. Zhao L, Brinton RD (2005) Structure-based virtual screening for plant-based ERbeta-

941 selective ligands as potential preventative therapy against age-related neurodegenerative

942 diseases. J Med Chem 48:3463–3466

943 21. Lynd L, Wyman C, Gerngross T (1999) Biocommodity engineering. Biotechnol Prog

944 15:777–793

945 22. Causey TB, Zhou S, Shanmugam KT, Ingram LO (2003) Engineering the metabolism of

946 Escherichia coli W3110 for the conversion of sugar to redox-neutral and oxidized products:

947 homoacetate production. Proc Natl Acad Sci USA 100:825–832

948 23. Otero JM, Panagiotou G, Olsson L (2007) Fueling industrial biotechnology growth with

949 bioethanol. Adv Biochem Eng Biotechnol 108:1–40

950 24. Lee JW, Kim TY, Jang Y-S, Choi S, Lee SY (2011) Systems metabolic engineering for

951 chemicals and materials. Trends Biotechnol 29:370–378

952 25. Jiang M, Stephanopoulos G, Pfeifer BA (2012) Toward biosynthetic design and implemen-

953 tation towards E. coli-derived Taxol and other heterologous polyisoprene compounds. Appl

954 Environ Microbiol 78(8):2497–2504

955 26. Huang B, Guo J, Yi B, Yu X, Sun L, Chen W (2008) Heterologous production of secondary

956 metabolites as pharmaceuticals in Saccharomyces cerevisiae. Biotechnol Lett 30:1121–1137957 27. Chang MCY, Keasling JD (2006) Production of isoprenoid pharmaceuticals by engineered

958 microbes. Nat Chem Biol 2:674–681

959 28. Frense D (2007) Taxanes: perspectives for biotechnological production. Appl Microbiol

960 Biotechnol 73:1233–1240


1673


961 29. Kuboyama T, Yokoshima S, Tokuyama H, Fukuyama T (2004) Stereocontrolled total

962 synthesis of (+)-vincristine. Proc Natl Acad Sci USA 101:11966–11970

963 30. Miyazaki T, Yokoshima S, Simizu S, Osada H, Tokuyama H, Fukuyama T (2007) Synthesis

964 of (+)-vinblastine and its analogues. Org Lett 9:4737–4740

965 31. Uchida K, Yokoshima S, Kan T, Fukuyama T (2006) Total synthesis of (+/�)-morphine. Org

966 Lett 8:5311–5313

967 32. Yokoshima S, Ueda T, Kobayashi S, Sato A, Kuboyama T, Tokuyama H, Fukuyama

968 T (2002) Stereocontrolled total synthesis of (+)-vinblastine. J Am Chem Soc

969 124:2137–2139

970 33. Lee DYW, Zhang W-Y, Karnati VVR (2003) Total synthesis of puerarin, an isoflavone

971 C-glycoside. Tetrahedron Lett 44:6857–6859

972 34. Heemstra JM, Kerrigan SA, Doerge DR, Helferich WG, Boulanger WA (2006) Total

973 synthesis of (S)-equol. Org Lett 8:5441–5443

974 35. Granados-Covarrubias EH, Maldonado LA (2009) AWacker–Cook synthesis of isoflavones:

975 formononetin. Tetrahedron Lett 50:1542–1545

976 36. Stafford AM, Pazoles CJ, Siegel S, Yeh L-A (1998) Plant cell culture: a vehicle for drug

977 delivery. In: Harvey AL (ed) Advances in drug discovery techniques. CRC Press,

978 New York

979 37. Yukimune Y, Tabata H, Higashi Y, Hara Y (1996) Methyl jasmonate-induced overproduction of

980 paclitaxel and baccatin III in Taxus cell suspension cultures. Nat Biotechnol 14:1129–1132

981 38. Witherup KM, Look SA, Stasko MW, Ghiorzi TJ, Muschik GM, Cragg GM (1990) Taxus

982 spp. Needles contain amounts of taxol comparable to the bark of Taxus brevifolia: analysis983 and isolation. J Nat Prod 53:1249–1255

984 39. Ajikumar PK, Xiao W-H, Tyo KEJ, Wang Y, Simeon F, Leonard E, Mucha O, Phon TH,

985 Pfeifer B, Stephanopoulos G (2010) Isoprenoid pathway optimization for Taxol precursor

986 overproduction in Escherichia coli. Sci (NY) 330:70–74987 40. Filner P, Varner JE, Wray JL (1969) Environmental or developmental changes cause many

988 enzyme activities of higher plants to rise or fall. Sci (NY) 165:358–367

989 41. Shanks JV, Morgan J (1999) Plant “hairy root” culture. Curr Opin Biotechnol 10:151–155

990 42. Zhang Y, Li S-Z, Li J, Pan X, Cahoon RE, Jaworski JG, Wang X, Jez JM, Chen F, Yu O (2006)

991 Using unnatural protein fusions to engineer resveratrol biosynthesis in yeast and Mammalian

992 cells. J Am Chem Soc 128:13030–13031

993 43. Limem I, Guedon E, Hehn A, Bourgaud F, Chekir Ghedira L, Engasser J-M, Ghoul M (2008)

994 Production of phenylpropanoid compounds by recombinant microorganisms expressing

995 plant-specific biosynthesis genes. Process Biochem 43:463–479

996 44. Fowler ZL, Koffas MA (2010) Microbial biosynthesis of fine chemicals: an emerging technol-

997 ogy. In: Smolke CD (ed) The metabolic pathway engineering handbook. CRC Press, Boca Raton

998 45. Allister EM, Borradaile NM, Edwards JY, Huff MW (2005) Inhibition of microsomal

999 triglyceride transfer protein expression and apolipoprotein B100 secretion by the citrus

1000 flavonoid naringenin and by insulin involves activation of the mitogen-activated protein

1001 kinase pathway in hepatocytes. Diabetes 54:1676–1683

1002 46. Caltagirone S, Rossi C, Poggi A, Ranelletti FO, Natali PG, Brunetti M, Aiello FB, Piantelli

1003 M (2000) Flavonoids apigenin and quercetin inhibit melanoma growth and metastatic

1004 potential. Int J cancer 87:595–600, Journal international du cancer

1005 47. Hou D-X, Fujii M, Terahara N, Yoshimoto M (2004) Molecular mechanisms behind the

1006 chemopreventive effects of anthocyanidins. J Biomed Biotechnol 2004:321–325

1007 48. McDougall GJ, Stewart D (2005) The inhibitory effects of berry polyphenols on digestive

1008 enzymes. BioFactors (Oxf, Engl) 23:189–195

1009 49. Popiołkiewicz J, Polkowski K, Skierski JS, Mazurek AP (2005) In vitro toxicity evaluation in

1010 the development of new anticancer drugs-genistein glycosides. Cancer Lett 229:67–75

1011 50. Potter SM, Baum JA, Teng H, Stillman RJ, Shay NF, Erdman JW (1998) Soy protein and

1012 isoflavones: their effects on blood lipids and bone density in postmenopausal women. Am

1013 J Clin Nutr 68:1375S–1379S


1674


1014 51. Pouget C, Lauthier F, Simon A, Fagnere C, Basly JP, Delage C, Chulia AJ (2001) Flavonoids:

1015 structural requirements for antiproliferative activity on breast cancer cells. Bioorg Med

1016 Chem Lett 11:3095–3097

1017 52. Hannum SM (2004) Potential impact of strawberries on human health: a review of the

1018 science. Crit Rev Food Sci Nutr 44:1–17

1019 53. Greenwald P (2004) Clinical trials in cancer prevention: current results and perspectives for

1020 the future. J Nutr 134:3507S–3512S

1021 54. Nakajima J, Tanaka Y, Yamazaki M, Saito K (2001) Reaction mechanism from leucoantho-

1022 cyanidin to anthocyanidin 3-glucoside, a key reaction for coloring in anthocyanin biosyn-

1023 thesis. J Biol Chem 276:25797–25803

1024 55. Winkel-Shirley B (2001) Flavonoid biosynthesis. A colorful model for genetics, biochem-

1025 istry, cell biology, and biotechnology. Plant Physiol 126:485–493

1026 56. Du H, Huang Y, Tang Y (2010) Genetic and metabolic engineering of isoflavonoid biosyn-

1027 thesis. Appl Microbiol Biotechnol 86:1293–1312

1028 57. Leonard E, Yan Y, Fowler ZL, Li Z, Lim C-G, Lim K-H, Koffas MAG (2008) Strain

1029 improvement of recombinant Escherichia coli for efficient production of plant flavonoids.

1030 Mol Pharm 5:257–265

1031 58. Turnbull JJ, Nakajima J-I, Welford RWD, Yamazaki M, Saito K, Schofield CJ (2004) Mech-

1032 anistic studies on three 2-oxoglutarate-dependent oxygenases of flavonoid biosynthesis:

1033 anthocyanidin synthase, flavonol synthase, and flavanone 3b-hydroxylase. J Biol Chem

1034 279:1206–1216

1035 59. Gao X, Wang P, Tang Y (2010) Engineered polyketide biosynthesis and biocatalysis in

1036 Escherichia coli. Appl Microbiol Biotechnol 88:1233–1242

1037 60. Horinouchi S (2008) Combinatorial biosynthesis of non-bacterial and unnatural flavonoids,

1038 stilbenoids and curcuminoids by microorganisms. J Antibiot 61:709–728

1039 61. Maheshwari RK, Singh AK, Gaddipati J, Srimal RC (2006) Multiple biological activities of

1040 curcumin: a short review. Life Sci 78:2081–2087

1041 62. Anand P, Kunnumakkara AB, Newman RA, Aggarwal BB (2007) Bioavailability of

1042 curcumin: problems and promises. Mol Pharm 4:807–818

1043 63. Aggarwal BB, Sundaram C, Malani N, Ichikawa H (2007) Curcumin: the Indian solid gold.

1044 Adv Exp Med Biol 595:1–75

1045 64. Boghigian BA, Pfeifer BA (2008) Current status, strategies, and potential for the metabolic

1046 engineering of heterologous polyketides in Escherichia coli. Biotechnol Lett 30:1323–13301047 65. Leonard E, Yan Y, Lim KH, Koffas MAG (2005) Investigation of two distinct flavone

1048 synthases for plant-specific flavone biosynthesis in Saccharomyces cerevisiae. Appl Environ

1049 Microbiol 71:8241–8248

1050 66. Kyle JAM, Duthie GG (2005) Flavonoids in foods. In: Andersen Ø, Markham

1051 K (eds) Flavonoids: chemistry, biochemistry and applications. CRC Press, Boca Raton,

1052 pp 219–262

1053 67. Manach C, Scalbert A, Morand C, Remesy C, Jimenez L (2004) Polyphenols: food sources

1054 and bioavailability. Am J Clin Nutr 79:727–747

1055 68. Hollman PC, Arts IC (2000) Flavonols, flavones and flavanols – nature, occurrence and

1056 dietary burden. J Sci Food Agric 80:1081–1093

1057 69. Akashi T, Aoki T, Ayabe SI (1999) Cloning and functional expression of a cytochrome P450

1058 cDNA encoding 2-hydroxyisoflavanone synthase involved in biosynthesis of the

1059 isoflavonoid skeleton in licorice. Plant Physiol 121:821–828

1060 70. Steele CL, Gijzen M, Qutob D, Dixon RA (1999) Molecular characterization of the enzyme

1061 catalyzing the aryl migration reaction of isoflavonoid biosynthesis in soybean. Arch

1062 Biochem Biophys 367:146–150

1063 71. Akashi T, Aoki T, Ayabe S-I (2005) Molecular and biochemical characterization of 2-

1064 hydroxyisoflavanone dehydratase. Involvement of carboxylesterase-like proteins in legumi-

1065 nous isoflavone biosynthesis. Plant Physiol 137:882–891

1066 72. Grotewold E (ed) (2006) The science of flavonoids. Springer, New York


1675


1067 73. Fukutake M, Takahashi M, Ishida K, Kawamura H, Sugimura T, Wakabayashi K (1996)

1068 Quantification of genistein and genistin in soybeans and soybean products. Food Chem

1069 Toxicol 34:457–461

1070 74. Espın JC, Garcıa-Conesa MT, Tomas-Barberan FA (2007) Nutraceuticals: facts and fiction.

1071 Phytochemistry 68:2986–3008

1072 75. Au2Crozier A, Clifford MN, Ashihara H (eds) (2008) Plant secondary metabolites: occurrence,

1073 structure and role in the human diet. Wiley-Blackwell, Oxford

1074 76. Martens S, Preuß A, Matern U (2010) Multifunctional flavonoid dioxygenases: flavonol and

1075 anthocyanin biosynthesis in Arabidopsis thaliana L. Phytochemistry 71:1040–1049

1076 77. Davies KM, Schwinn KE (2005) Molecular biology and biotechnology of flavonoid biosyn-

1077 thesis. In: Andersen ØM, Markham KR (eds) Flavonoids: chemistry, biochemistry and

1078 applications. CRC Press, Boca Raton, pp 143–218

1079 78. Kaneko M, Hwang EI, Ohnishi Y, Horinouchi S (2003) Heterologous production of flava-

1080 nones in Escherichia coli: potential for combinatorial biosynthesis of flavonoids in bacteria.

1081 J Ind Microbiol Biotechnol 30:456–461

1082 79. Hwang EI, Kaneko M, Ohnishi Y, Horinouchi S (2003) Production of plant-specific flava-

1083 nones by Escherichia coli containing an artificial gene cluster. Appl Environ Microbiol

1084 69:2699–2706

1085 80. Miyahisa I, Kaneko M, Funa N, Kawasaki H, Kojima H, Ohnishi Y, Horinouchi S (2005)

1086 Efficient production of (2S)-flavanones by Escherichia coli containing an artificial biosyn-

1087 thetic gene cluster. Appl Microbiol Biotechnol 68:498–504

1088 81. Kyndt JA, Meyer TE, Cusanovich MA, Van Beeumen JJ (2002) Characterization of

1089 a bacterial tyrosine ammonia lyase, a biosynthetic enzyme for the photoactive yellow

1090 protein. FEBS Lett 512:240–244

1091 82. Chemler JA, Yan Y, Leonard E, Koffas MAG (2007) Combinatorial mutasynthesis of

1092 flavonoid analogues from acrylic acids in microorganisms. Org Lett 9:1855–1858

1093 83. Chemler JA (2009) Metabolic engineering of Escherichia coli and Saccharomyces1094 cerevisiae to mutasynthesize natural phenylpropanoids and novel analogs

1095 84. Miyahisa I, Funa N, Ohnishi Y, Martens S, Moriguchi T, Horinouchi S (2006) Combinatorial

1096 biosynthesis of flavones and flavonols in Escherichia coli. Appl Microbiol Biotechnol

1097 71:53–58

1098 85. Yan Y, Chemler J, Huang L, Martens S, Koffas MAG (2005) Metabolic engineering of

1099 anthocyanin biosynthesis in Escherichia coli. Appl Environ Microbiol 71:3617–3623

1100 86. Leonard E, Chemler J, Lim KH, Koffas MAG (2006) Expression of a soluble flavone

1101 synthase allows the biosynthesis of phytoestrogen derivatives in Escherichia coli. Appl1102 Microbiol Biotechnol 70:85–91

1103 87. Leonard E, Yan Y, Koffas MAG (2006) Functional expression of a P450 flavonoid hydrox-

1104 ylase for the biosynthesis of plant-specific hydroxylated flavonols in Escherichia coli. Metab

1105 Eng 8:172–181

1106 88. Yan Y, Huang L, Koffas MAG (2007) Biosynthesis of 5-deoxyflavanones in microorgan-

1107 isms. Biotechnol J 2:1250–1262

1108 89. Leonard E, Lim K-H, Saw P-N, Koffas MAG (2007) Engineering central metabolic path-

1109 ways for high-level flavonoid production in Escherichia coli. Appl Environ Microbiol

1110 73:3877–3886

1111 90. Chemler JA, Lock LT, Koffas MAG, Tzanakakis ES (2007) Standardized biosynthesis of

1112 flavan-3-ols with effects on pancreatic beta-cell insulin secretion. Appl Microbiol Biotechnol

1113 77:797–807

1114 91. Jiang H, Wood KV, Morgan JA (2005) Metabolic engineering of the phenylpropanoid

1115 pathway in Saccharomyces cerevisiae. Appl Environ Microbiol 71:2962

1116 92. Yan Y, Kohli A, Koffas MAG (2005) Biosynthesis of natural flavanones in Saccharomyces1117 cerevisiae. Appl Environ Microbiol 71:5610–5613

1118 93. Lim CG, Fowler ZL, Hueller T, Schaffer S, Koffas MAG (2011) High-yield resveratrol

1119 production in engineered Escherichia coli. Appl Environ Microbiol 77:3451–3460


1676


1120 94. Beekwilder J, Wolswinkel R, Jonker H, Hall R, de Vos CHR, Bovy A (2006) Production of

1121 resveratrol in recombinant microorganisms. Appl Environ Microbiol 72:5670–5672

1122 95. Watts KT, Lee PC, Schmidt-Dannert C (2006) Biosynthesis of plant-specific stilbene

1123 polyketides in metabolically engineered Escherichia coli. BMC Biotechnol 6:22

1124 96. Yan Y, Li Z, Koffas MAG (2008) High-yield anthocyanin biosynthesis in engineered

1125 Escherichia coli. Biotechnol Bioeng 100:126–140

1126 97. Watts KT, Lee PC, Schmidt-Dannert C (2004) Exploring recombinant flavonoid biosynthesis

1127 in metabolically engineered Escherichia coli. Chembiochem: Eur J Chem Biol 5:500–507

1128 98. He X-Z, Li W-S, Blount JW, Dixon RA (2008) Regioselective synthesis of plant (iso)flavone

1129 glycosides in Escherichia coli. Appl Microbiol Biotechnol 80:253–260

1130 99. Katsuyama Y, Hirose Y, Funa N, Ohnishi Y, Horinouchi S (2010) Precursor-directed

1131 biosynthesis of curcumin analogs in Escherichia coli. Biosci Biotechnol Biochem

1132 74:641–645

1133 100. Katsuyama Y, Funa N, Miyahisa I, Horinouchi S (2007) Synthesis of unnatural flavonoids

1134 and stilbenes by exploiting the plant biosynthetic pathway in Escherichia coli. Chem Biol

1135 14:613–621

1136 101. Trantas E, Panopoulos N, Ververidis F (2009) Metabolic engineering of the complete

1137 pathway leading to heterologous biosynthesis of various flavonoids and stilbenoids in

1138 Saccharomyces cerevisiae. Metab Eng 11:355–366

1139 102. Jiang H, Morgan JA (2004) Optimization of an in vivo plant P450 monooxygenase system in

1140 Saccharomyces cerevisiae. Biotechnol Bioeng 85:130–137

1141 103. Kim DH, Kim BG, Lee HJ, Lim Y, Hur HG, Ahn J-H (2005) Enhancement of isoflavone

1142 synthase activity by co-expression of P450 reductase from rice. Biotechnol Lett 27:1291–

1143 1294

1144 104. Ralston L, Subramanian S, Matsuno M, Yu O (2005) Partial reconstruction of flavonoid and

1145 isoflavonoid biosynthesis in yeast using soybean type I and type II chalcone isomerases.

1146 Plant Physiol 137:1375–1388

1147 105. Becker JVW, Armstrong GO, van der Merwe MJ, Lambrechts MG, Vivier MA, Pretorius IS

1148 (2003) Metabolic engineering of Saccharomyces cerevisiae for the synthesis of the wine-

1149 related antioxidant resveratrol. FEMS Yeast Res 4:79–85

1150 106. Ro D-K, Douglas CJ (2004) Reconstitution of the entry point of plant phenylpropanoid

1151 metabolism in yeast (Saccharomyces cerevisiae): implications for control of metabolic flux

1152 into the phenylpropanoid pathway. J Biol Chem 279:2600–2607

1153 107. Vannelli T, Wei Qi W, Sweigard J, Gatenby AA, Sariaslani FS (2007) Production of

1154 p-hydroxycinnamic acid from glucose in Saccharomyces cerevisiae and Escherichia coli1155 by expression of heterologous genes from plants and fungi. Metab Eng 9:142–151

1156 108. Barnes HJ, Arlotto MP, Waterman MR (1991) Expression and enzymatic activity of recom-

1157 binant cytochrome P450 17 alpha-hydroxylase in Escherichia coli. Proc Natl Acad Sci USA1158 88:5597–5601

1159 109. Williams PA, Cosme J, Sridhar V, Johnson EF, McRee DE (2000) Microsomal cytochrome

1160 P450 2C5: comparison to microbial P450s and unique features. J Inorg Biochem 81:183–190

1161 110. Katsuyama Y, Miyahisa I, Funa N, Horinouchi S (2007) One-pot synthesis of genistein from

1162 tyrosine by coincubation of genetically engineered Escherichia coli and Saccharomyces1163 cerevisiae cells. Appl Microbiol Biotechnol 73:1143–1149

1164 111. Horinouchi S (2009) Combinatorial biosynthesis of plant medicinal polyketides by micro-

1165 organisms. Curr Opin Chem Biol 13:197–204

1166 112. Tian L, Dixon RA (2006) Engineering isoflavone metabolism with an artificial bifunctional

1167 enzyme. Planta 224:496–507

1168 113. Porter TD, Wilson TE, Kasper CB (1987) Expression of a functional 78,000 dalton mam-

1169 malian flavoprotein, NADPH-cytochrome P-450 oxidoreductase, in Escherichia coli. Arch1170 Biochem Biophys 254:353–367

1171 114. Leonard E, Koffas MAG (2007) Engineering of artificial plant cytochrome P450 enzymes for

1172 synthesis of isoflavones by Escherichia coli. Appl Environ Microbiol 73:7246–7251


1677


1173 115. Kim DH, Kim B-G, Jung NR, Ahn J-H (2009) Production of genistein from naringenin using

1174 Escherichia coli containing isoflavone synthase-cytochrome P450 reductase fusion protein.

1175 J Microbiol Biotechnol 19:1612–1616

1176 116. Ro D-K, Ehlting J, Douglas CJ (2002) Cloning, functional expression, and subcellular

1177 localization of multiple NADPH-cytochrome P450 reductases from hybrid poplar. Plant

1178 Physiol 130:1837–1851

1179 117. Chemler JA, Lim CG, Daiss JL, Koffas MAG (2010) A versatile microbial system for

1180 biosynthesis of novel polyphenols with altered estrogen receptor binding activity. Chem

1181 Biol 17:392–401

1182 118. Lim E-K, Ashford DA, Hou B, Jackson RG, Bowles DJ (2004) Arabidopsis glycosyl-

1183 transferases as biocatalysts in fermentation for regioselective synthesis of diverse quercetin

1184 glucosides. Biotechnol Bioeng 87:623–631

1185 119. Willits MG, Giovanni M, Prata RT, Kramer CM, De Luca V, Steffens JC, Graser G (2004)

1186 Bio-fermentation of modified flavonoids: an example of in vivo diversification of secondary

1187 metabolites. Phytochemistry 65:31–41

1188 120. Deavours BE, Dixon RA, Division PB, Roberts S, Foundation N (2005) Metabolic engi-

1189 neering of isoflavonoid biosynthesis in alfalfa. Plant Physiol 138:2245–2259

1190 121. Liu C-J, Blount JW, Steele CL, Dixon RA (2002) Bottlenecks for metabolic engineering of

1191 isoflavone glycoconjugates in Arabidopsis. Proc Natl Acad Sci USA 99:14578–14583

1192 122. Yu O, Jung W, Shi J, Croes RA, Fader GM, McGonigle B, Odell JT (2000) Production of the

1193 isoflavones genistein and daidzein in non-legume dicot and monocot tissues. Plant Physiol

1194 124:781–794

1195 123. Yu O, Shi J, Hession AO, Maxwell CA, McGonigle B, Odell JT (2003) Metabolic engineer-

1196 ing to increase isoflavone biosynthesis in soybean seed. Phytochemistry 63:753–763

1197 124. Hollman PC, Katan MB (1999) Health effects and bioavailability of dietary flavonols. Free

1198 Radic Res 31(Suppl):S75–S80

1199 125. Hollman PC, Katan MB (1998) Bioavailability and health effects of dietary flavonols in man.

1200 Archives of toxicology. Supplement. ¼ Archiv f€ur Toxikologie. Supplement. 20:237–248

1201 126. Hollman PC, Bijsman MN, van Gameren Y, Cnossen EP, de Vries JH, Katan MB (1999) The

1202 sugar moiety is a major determinant of the absorption of dietary flavonoid glycosides in man.

1203 Free Radic Res 31:569–573

1204 127. Smith GJ, Thomsen SJ, Markham KR, Andary C, Cardon D (2000) The photostabilities of

1205 naturally occurring 5-hydroxyflavones, flavonols, their glycosides and their aluminium

1206 complexes. J Photochem Photobiol: Chem 136:87–91

1207 128. Crespy V, Morand C, Besson C, Manach C, Demigne C, Remesy C (2001) Comparison of the

1208 intestinal absorption of quercetin, phloretin and their glucosides in rats. J Nutr 131:2109–2114

1209 129. Graefe EU, Wittig J, Mueller S, Riethling AK, Uehleke B, Drewelow B, Pforte H, Jacobasch

1210 G, Derendorf H, Veit M (2001) Pharmacokinetics and bioavailability of quercetin glycosides

1211 in humans. J Clin Pharmacol 41:492–499

1212 130. Bouktaib M, Atmani A, Rolando C (2002) Regio- and stereoselective synthesis of the major

1213 metabolite of quercetin, quercetin-3-O-b-d-glucuronide. Tetrahedron Lett 43:6263–6266

1214 131. Harborne JB, Baxter H, Harborne JB (1999) The handbook of natural flavonoids. Wiley,

1215 Chichester

1216 132. Lim SS, Jung SH, Ji J, Shin KH, Keum SR (2001) Synthesis of flavonoids and their effects on

1217 aldose reductase and sorbitol accumulation in streptozotocin-induced diabetic rat tissues.

1218 J Pharm Pharmacol 53:653–668

1219 133. Mavel S, Dikic B, Palakas S, Emond P, Greguric I, de Gracia AG, Mattner F, Garrigos M,

1220 Guilloteau D, Katsifis A (2006) Synthesis and biological evaluation of a series of flavone

1221 derivatives as potential radioligands for imaging the multidrug resistance-associated protein

1222 1 (ABCC1/MRP1). Bioorg Med Chem 14:1599–1607

1223 134. Mao Z, Shin H-D, Chen RR (2006) Engineering the E. coli UDP-glucose synthesis pathway1224 for oligosaccharide synthesis. Biotechnol Prog 22:369–374


1678


1225 135. Li Z (2008) High yield anthocyanin biosynthesis in metabolic engineering Escherichia coli.1226 Master of Science, Department of Chemical and Biological Engineering

1227 136. Yan Y (2008) Constructing microbial production platform for the biosynthesis of natural

1228 drug candidates-flavonoids

1229 137. Jung W, Yu O, Lau SM, O’Keefe DP, Odell J, Fader G, McGonigle B (2000) Identification

1230 and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in

1231 legumes. Nat Biotechnol 18:208–212

1232 138. Noguchi A, Horikawa M, Fukui Y, Fukuchi-Mizutani M, Iuchi-Okada A, Ishiguro M, Kiso

1233 Y, Nakayama T, Ono E (2009) Local differentiation of sugar donor specificity of flavonoid

1234 glycosyltransferase in Lamiales. Plant Cell 21:1556–1572

1235 139. Osmani SA, Bak S, Imberty A, Olsen CE, Møller BL (2008) Catalytic key amino acids and

1236 UDP-sugar donor specificity of a plant glucuronosyltransferase, UGT94B1: molecular

1237 modeling substantiated by site-specific mutagenesis and biochemical analyses. Plant Physiol

1238 148:1295–1308

1239 140. He X-Z, Wang X, Dixon RA (2006) Mutational analysis of the Medicago glycosyltransferase

1240 UGT71G1 reveals residues that control regioselectivity for (iso)flavonoid glycosylation.

1241 J Biol Chem 281:34441–34447

1242 141. Modolo LV, Escamilla-Trevino LL, Dixon RA, Wang X (2009) Single amino acid mutations

1243 of Medicago glycosyltransferase UGT85H2 enhance activity and impart reversibility. FEBS

1244 Lett 583:2131–2135

1245 142. Funa N, Ohnishi Y, Ebizuka Y, Horinouchi S (2002) Alteration of reaction and substrate

1246 specificity of a bacterial type III polyketide synthase by site-directed mutagenesis. Biochem

1247 J 367:781–789

1248 143. Morita H, Yamashita M, Shi S-P, Wakimoto T, Kondo S, Kato R, Sugio S, Kohno T,

1249 Abe I (2011) Synthesis of unnatural alkaloid scaffolds by exploiting plant polyketide

1250 synthase. Proc Natl Acad Sci USA 108:13504–13509

1251 144. Wang Y, Halls C, Zhang J, Matsuno M, Zhang Y, Yu O (2011) Stepwise increase of

1252 resveratrol biosynthesis in yeast Saccharomyces cerevisiae by metabolic engineering.

1253 Metab Eng 13:455–463

1254 145. Chen H, Jiang H, Morgan JA (2007) Non-natural cinnamic acid derivatives as substrates of

1255 cinnamate 4-hydroxylase. Phytochemistry 68:306–311

1256 146. Williams GJ, Zhang C, Thorson JS (2007) Expanding the promiscuity of a natural-product

1257 glycosyltransferase by directed evolution. Nat Chem Biol 3:657–662

1258 147. Felnagle EA, Chaubey A, Noey EL, Houk KN, Liao JC (2012) Engineering synthetic

1259 recursive pathways to generate non-natural small molecules. Nat Chem Biol 8:518–526

1260 148. Minami H, Kim J-S, Ikezawa N, Takemura T, Katayama T, Kumagai H, Sato F (2008)

1261 Microbial production of plant benzylisoquinoline alkaloids. Proc Natl Acad Sci USA

1262 105:7393–7398

1263 149. Challis GL, Hopwood DA (2007) Chemical biotechnology: bioactive small molecules –

1264 targets and discovery technologies. Curr Opin Biotechnol 18:475–477

1265 150. Straathof AJJ, Panke S, Schmid A (2002) The production of fine chemicals by biotransfor-

1266 mations. Curr Opin Biotechnol 13:548–556

1267 151. Kagami O, Shindo K, Kyojima A, Takeda K, Ikenaga H, Furukawa K, Misawa N (2008)

1268 Protein engineering on biphenyl dioxygenase for conferring activity to convert 7-

1269 hydroxyflavone and 5,7-dihydroxyflavone (chrysin). J Biosci Bioeng 106:121–127

1270 152. Laparra JM, Sanz Y (2010) Interactions of gut microbiota with functional food components

1271 and nutraceuticals. Pharmacol Res: Off J Italian pharmacol soc 61:219–225

1272 153. Park H-Y, Kim M, Han J (2011) Stereospecific microbial production of isoflavanones from

1273 isoflavones and isoflavone glucosides. Appl Microbiol Biotechnol 91:1173–1181

1274 154. van Duynhoven J, Vaughan EE, Jacobs DM, Kemperman RA, van Velzen EJJ, Gross G, Roger

1275 LC, Possemiers S, Smilde AK, Dore J, Westerhuis JA, Van de Wiele T (2011) Metabolic fate of

1276 polyphenols in the human superorganism. Proc Natl Acad Sci USA 108(Suppl):4531–4538


1679


1277 155. Jin J-S, Zhao Y-F, Nakamura N, Akao T, Kakiuchi N, Min B-S, Hattori M (2007)

1278 Enantioselective dehydroxylation of enterodiol and enterolactone precursors by human

1279 intestinal bacteria. Biol Pharm Bull 30:2113–2119

1280 156. Larrosa M, Gonzalez-Sarrıas A, Garcıa-Conesa MT, Tomas-Barberan FA, Espın JC

1281 (2006) Urolithins, ellagic acid-derived metabolites produced by human colonic microflora,

1282 exhibit estrogenic and antiestrogenic activities. J Agric Food Chem 54:1611–1620

1283 157. Kim M, Kim S-I, Han J, Wang X-L, Song D-G, Kim S-U (2009) Stereospecific biotransfor-

1284 mation of dihydrodaidzein into (3S)-equol by the human intestinal bacterium Eggerthella

1285 strain Julong 732. Appl Environ Microbiol 75:3062–3068

1286 158. Setchell KD (2001) Soy isoflavones – benefits and risks from nature’s selective estrogen

1287 receptor modulators (SERMs). J Am Coll Nutr 20:354S–362S; discussion 381S–383S (2001)

1288 159. Basly J-P, Lavier M-CC (2005) Dietary phytoestrogens: potential selective estrogen enzyme

1289 modulators? Planta Med 71:287–294

1290 160. Zhao X, Li L, Wang Z (2006) Chemoprevention of breast cancer: current status and future

1291 prospects. Front biosci 11:2249–2256

1292 161. Ho S-M (2004) Estrogens and anti-estrogens: key mediators of prostate carcinogenesis and

1293 new therapeutic candidates. J Cell Biochem 91:491–503

1294 162. Bowles D, Lim E-K, Poppenberger B, Vaistij FE (2006) Glycosyltransferases of lipophilic

1295 small molecules. Annu Rev Plant Biol 57:567–597

1296 163. Simkhada D, Kim E, Lee HC, Sohng JK (2009) Metabolic engineering of Escherichia coli1297 for the biological synthesis of 7-O-xylosyl naringenin. Mol Cells 28:397–401

1298 164. Simkhada D, Kurumbang NP, Lee HC, Sohng JK (2010) Exploration of glycosylated

1299 flavonoids from metabolically engineered E. coli. Biotechnol Bioprocess Eng 15:754–760

1300 165. Kurumbang NP, Liou K, Sohng JK (2010) Biosynthesis of paromamine derivatives in

1301 engineered Escherichia coli by heterologous expression. J Appl Microbiol 108:1780–1788

1302 166. Simkhada D, Lee HC, Sohng JK (2010) Genetic engineering approach for the production of

1303 rhamnosyl and allosyl flavonoids from Escherichia coli. Biotechnol Bioeng 107:154–162

1304 167. Day AJ, Canada FJ, Dıaz JC, Kroon PA, Mclauchlan R, Faulds CB, Plumb GW,MorganMR,

1305 Williamson G (2000) Dietary flavonoid and isoflavone glycosides are hydrolysed by the

1306 lactase site of lactase phlorizin hydrolase. FEBS Lett 468:166–170

1307 168. Seo J, Kang S-I, Kim M, Han J, Hur H-G (2011) Flavonoids biotransformation by bacterial

1308 non-heme dioxygenases, biphenyl and naphthalene dioxygenase. Appl Microbiol Biotechnol

1309 91:219–228

1310 169. Wang A, Zhang F, Huang L, Yin X, Li H, Wang Q (2010) New progress in biocatalysis and

1311 biotransformation of flavonoids. J Med Plant Res 4:847–856

1312 170. Chun H-K, Ohnishi Y, Shindo K, Misawa N, Furukawa K, Horinouchi S (2003) Biotrans-

1313 formation of flavone and flavanone by Streptomyces lividans cells carrying shuffled biphenyl

1314 dioxygenase genes. J Mol Catal B: Enzym 21:113–121

1315 171. Seeger M, Gonzalez M, Camara B, Munoz L, Ponce E, Mejıas L, Mascayano C, Vasquez Y,

1316 Sepulveda-Boza S (2003) Biotransformation of natural and synthetic isoflavonoids by two

1317 recombinant microbial enzymes. Appl Environ Microbiol 69:5045–5050

1318 172. Seo J, Kang S-I, Ryu J-Y, Lee Y-J, Park KD, Kim M, Won D, Park H-Y, Ahn J-H, Chong Y,

1319 Kanaly RA, Han J, Hur H-G (2010) Location of flavone B-ring controls regioselectivity and

1320 stereoselectivity of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

1321 Appl Microbiol Biotechnol 86:1451–1462

1322 173. Kim B-G, Jung B-R, Lee Y, Hur H-G, Lim Y, Ahn J-H (2006) Regiospecific flavonoid 7-O-

1323 methylation with Streptomyces avermitilis O-methyltransferase expressed in Escherichia1324 coli. J Agric Food Chem 54:823–828

1325 174. Santos CNS, Koffas M, Stephanopoulos G (2011) Optimization of a heterologous pathway

1326 for the production of flavonoids from glucose. Metab Eng 13:392–400

1327 175. Wang Y, Chen S, Yu O (2011) Metabolic engineering of flavonoids in plants and microor-

1328 ganisms. Appl Microbiol Biotechnol 91:949–956


1680


1329 176. Xu P, Koffas MA (2010) Metabolic engineering of Escherichia coli for biofuel production.1330 Biofuels 1:493–504

1331 177. Lee SY, Park JM, Kim TY (2011) Application of metabolic flux analysis in metabolic

1332 engineering. Methods Enzymol 498:67–93. Elsevier Inc

1333 178. Haggart CR, Bartell JA, Saucerman JJ, Papin JA (2011) Whole-genome metabolic network

1334 reconstruction and constraint-based modeling. Methods Enzymol 500:411–433. Elsevier Inc

1335 179. Kim HU, Kim TY, Lee SY (2008) Metabolic flux analysis and metabolic engineering of

1336 microorganisms. Mol Biosyst 4:113–120

1337 180. Maertens J, Vanrolleghem PA (2010) Modeling with a view to target identification in

1338 metabolic engineering: a critical evaluation of the available tools. Biotechnol Prog

1339 26:313–331

1340 181. Medema MH, van Raaphorst R, Takano E, Breitling R (2012) Computational tools for the

1341 synthetic design of biochemical pathways. Nat Rev Microbiol 10:1–12

1342 182. Santos F, Boele J, Teusink B (2011) A practical guide to genome-scale metabolic models and

1343 their analysis. Methods Sys Biol 500:509–532. Elsevier Inc

1344 183. Alberstein M, Eisenstein M, Abeliovich H (2012) Removing allosteric feedback inhibition of

1345 tomato 4-coumarate:CoA ligase by directed evolution. Plant J 69:57–69

1346 184. Siddiqui MS, Thodey K, Trenchard I, Smolke CD (2012) Advancing secondary metabolite

1347 biosynthesis in yeast with synthetic biology tools. FEMS Yeast Res 12:144–170

1348 185. Agapakis CM, Boyle PM, Silver PA (2012) Natural strategies for the spatial optimization of

1349 metabolism in synthetic biology. Nat Chem Biol 8:527–535

1350 186. Dueber JE, Wu GC, Malmirchegini GR, Moon TS, Petzold CJ, Ullal AV, Prather KLJ,

1351 Keasling JD (2009) synthetic protein scaffolds provide modular control over metabolic flux.

1352 Online 27:5–8

1353 187. Weeks A, Lund L, Raushel FM (2006) Tunneling of intermediates in enzyme-catalyzed

1354 reactions. Curr Opin Chem Biol 10:465–472

1355 188. Bonacci W, Teng PK, Afonso B, Niederholtmeyer H, Grob P, Silver PA (2011) Modularity

1356 of a carbon-fixing protein organelle. Proc Natl Acad Sci USA 109:478

1357 189. Dunlop MJ, Dossani ZY, Szmidt HL, Chu HC, Lee TS, Keasling JD, Hadi MZ,

1358 Mukhopadhyay A (2011) Engineering microbial biofuel tolerance and export using efflux

1359 pumps. Mol Syst Biol 7:487

1360 190. Dunlop MJ (2011) Engineering microbes for tolerance to next-generation biofuels.

1361 Biotechnol biofuels 4:32

1362 191. Wang M, Si T, Zhao H (2012) Biocatalyst development by directed evolution. Bioresour

1363 Technol 115:117–125

1364 192. Hanson AD, Pribat A, Waller JC, de Crecy-Lagard V (2010) “Unknown” proteins and “orphan”

1365 enzymes: the missing half of the engineering parts list–and how to find it. Biochem J 425:1–11


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