Natural Products
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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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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
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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.
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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
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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].
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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.
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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,
53 Isoflavonoid Production by Genetically Engineered Micro-organisms 11
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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
12 B.F. Cress et al.
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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
53 Isoflavonoid Production by Genetically Engineered Micro-organisms 13
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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.
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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
53 Isoflavonoid Production by Genetically Engineered Micro-organisms 15
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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.
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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
53 Isoflavonoid Production by Genetically Engineered Micro-organisms 17
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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
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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
CPR: P. trichocarpa �P. deltoidest1:22
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
CPR: P. trichocarpa �P. deltoidest1:29
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
Genistein 26,Daidzein 27
Naringenin 23,Liquiritigenin
S. cerevisiae IFS: G. echinata [69]t1:38
(continued)
53 Isoflavonoid Production by Genetically Engineered Micro-organisms 19
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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
Genistein 26,Daidzein 27
Naringenin 23,Liquiritigenin
E. coli CPRa: C. roseus [114]t1:41
IFSa: G. maxt1:42
Genistein 26,Daidzein 27
Naringenin 23,Liquiritigenin
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
20 B.F. Cress et al.
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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
53 Isoflavonoid Production by Genetically Engineered Micro-organisms 21
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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
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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.
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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
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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].
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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.
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