Engineering Plant Secondary Metabolism in …...108 alkaloids, giving rise to more than 3,000...
Transcript of Engineering Plant Secondary Metabolism in …...108 alkaloids, giving rise to more than 3,000...
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Engineering Plant Secondary Metabolism in Microbial Systems 2
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Michael E. Pyne*, Lauren Narcross
*, Vincent J.J. Martin
*,1 4
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*Department of Biology, Centre of Applied Synthetic Biology, Centre for Structural and 7
Functional Genomics, Concordia University, Montréal, Québec, Canada 8
1 Corresponding author: E-mail address: [email protected] 9
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Short title: Producing plant secondary metabolites in microbes 11
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One-sentence summary: An overview of common challenges and 13
strategies underlying efforts to reconstruct plant isoprenoid, alkaloid, 14
phenylpropanoid, and polyketide biosynthetic pathways in microbial 15
systems. 16
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Author Contributions: M.E.P. and L.N. prepared the manuscript. 18
V.J.J.M supervised and edited the manuscript. All authors participated 19
in discussion and revision of the manuscript. 20
Plant Physiology Preview. Published on January 14, 2019, as DOI:10.1104/pp.18.01291
Copyright 2019 by the American Society of Plant Biologists
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Introduction 21
Secondary metabolites are broadly defined as natural products synthesized by an 22
organism that are not essential to support growth and life. The plant kingdom 23
manufactures over 200,000 distinct chemical compounds, most of which arise from 24
specialized metabolism. While these compounds play important roles in interspecies 25
competition and defense, many plant natural products have been exploited for use as 26
medicines, fragrances, flavors, nutrients, repellants, and colorants. 27
Despite this vast chemical diversity, many secondary metabolites are present at 28
very low concentrations in planta, eliminating crop-based manufacturing as a means of 29
attaining these important products. The structural and stereochemical complexity of 30
specialized metabolites hinders most attempts to access these compounds using chemical 31
synthesis. Although native plants can be engineered to accumulate target pathway 32
metabolites (Zhou et al., 2009; Glenn et al., 2013; Lange and Ahkami, 2013; Wilson and 33
Roberts, 2014; Tatsis and O’Connor, 2016), metabolic engineering is technically more 34
challenging in plants than in microbes. 35
Advancements in synthetic biology have stimulated the synthesis of valuable 36
natural products in tractable laboratory microbes by interfacing plant secondary pathways 37
with core host metabolism. Microbial synthesis overcomes many of the obstacles 38
hindering traditional chemical synthesis and plant metabolic engineering, thus providing 39
an alternative avenue for exploring plant specialized pathways. 40
This Update provides a brief overview of engineering plant secondary metabolism 41
in microbial systems. We briefly outline biosynthetic pathways mediating formation of 42
the major classes of natural products with an emphasis on high-value terpenoids, 43
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alkaloids, phenylpropanoids, and polyketides. We also highlight common themes, 44
strategies, and challenges underlying efforts to reconstruct and engineer these pathways 45
in microbial hosts. We focus chiefly on de novo biosynthetic approaches in which plant 46
specialized metabolites are synthesized directly from sugar feedstocks rather than 47
supplemented precursors or intermediates. Readers are directed to a selection of 48
pioneering supplementation studies within the context of microbially-sourced plant 49
natural products (Becker et al., 2003; Kaneko et al., 2003; Yan et al., 2005; Watts et al., 50
2006; Leonard et al., 2007; Hawkins and Smolke, 2008; Leonard et al., 2008; Fossati et 51
al., 2014; Fossati et al., 2015). 52
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OVERVIEW OF KEY PLANT SECONDARY METABOLIC PATHWAYS 54
Terpenoids 55
Terpenoids (also called isoprenoids) are the largest class of plant secondary metabolites, 56
comprising more than 50,000 natural products (Connolly and Hill, 1991). Whereas the 57
terpene classification refers strictly to hydrocarbons, terpenoids possess a range of 58
chemical functionalities. The central precursors geranyl pyrophosphate (GPP; C10), 59
farnesyl pyrophosphate (FPP; C15), and geranylgeranylpyrophosphate (GGPP; C20) form 60
the structural basis of most higher-order terpenoids (Figure 1). Plant terpene synthases 61
convert these pyrophosphate intermediates to terpenes, which are then functionalized in 62
downstream reactions. 63
GPP forms the backbone of most monoterpenoids (C10), including linear (geraniol 64
and linalool) and cyclic (camphor and eucalyptol) terpenoids, as well as monoterpenes 65
(limonene and pinene). Geraniol can be modified to the pest repellant citronellol, while 66
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limonene gives rise to menthol, a flavoring agent and decongestant. GPP also supplies the 67
prenyl group in the biosynthesis of cannabinoids (Ahmed et al., 2015; Vickery et al., 68
2016), a class of natural products with promising pharmaceutical properties (Aizpurua-69
Olaizola et al., 2016). Sesquiterpenes (C15) are derived from FPP, itself generated through 70
condensation of GPP and isopentenyl pyrophosphate (IPP) in yeast or directly from IPP 71
and dimethylallyl pyrophosphate (DMAPP) units in plants. The sesquiterpene 72
amorphadiene gives rise to the antimalarial drug precursor artemisinic acid. The addition 73
of another IPP unit to FPP in yeast gives rise to GGPP, which forms the scaffold of the 74
diterpenes and diterpenoids (C20), such as taxadiene, a precursor to the taxane family of 75
chemotherapeutics. Condensation of two FPP units yields the linear triterpene squalene 76
(C30), which serves as the universal building block of all sterols, including ergosterol in 77
yeast. In plants, squalene gives rise to a number of pharmacologically active 78
triterpenoids, such as β-amyrin and lupeol. Tetraterpenes (C40) are produced through 79
condensation of two molecules of GGPP, yielding phytoene, the precursor to the 80
carotenoids, such as lycopene and β-carotene. Polyterpenes such as natural rubber (cis-81
polyisoprene) comprise thousands of isoprene units. 82
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Alkaloids 84
In the broadest sense, alkaloids are defined as low-molecular-weight metabolites 85
containing heterocyclic (true alkaloids) or exocyclic (protoalkaloids, amines, and 86
polyamines) nitrogen atoms. Approximately 20,000 natural alkaloids are known, many of 87
which exhibit analgesic (morphine), stimulant (caffeine and ephedrine), psychotropic 88
(mescaline and cocaine), antibacterial (sanguinarine), anticancer (vinblastine and 89
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vincristine), antitussive (codeine), anti-inflammatory (berberine), antispasmodic 90
(papaverine), or antimalarial (quinine) activities. 91
Phenylalanine, tyrosine, and their derivatives (e.g., phenethylamine, tyramine, and 92
dopamine) are the source of a tremendous number of alkaloids, including the 93
benzylisoquinoline alkaloids (BIAs) and the phenethylisoquinoline alkaloids (Figure 2). 94
BIAs are a large class of roughly 2,500 metabolites that includes berberine, noscapine, 95
sanguinarine, and morphine. These important medicines are derived from the 96
condensation of dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA), both of which 97
are synthesized from tyrosine. Dopamine can also condense with derivatives of 98
phenylalanine (4-hydroxydihydrocinnamaldehyde), yielding the phenethylisoquinoline 99
class of alkaloids. Phenylalanine and tyrosine form the basis of a number of simpler 100
protoalkaloids and catecholamines, including modified amphetamines (ephedrine and 101
cathinone), dopamine, mescaline, and adrenaline. Owing to the ubiquity of the indole 102
group in nature, tryptophan also forms the basis of many important alkaloids, such as 103
simple indoles, β-carbolines (serotonin and harmine), and the monoterpenoid indole 104
alkaloids (MIAs). 105
MIAs, derived from the condensation of the tryptophan analog tryptamine and a 106
monoterpenoid (secologanin), are one of the largest and most complex classes of 107
alkaloids, giving rise to more than 3,000 structures. The tryptophan precursor anthranilate 108
also serves as a precursor to several alkaloid sub-classes, including the quinazolines. 109
Beyond the aromatic amino acids, arginine and ornithine form the basis of the tropane, 110
pyrrolidine, pyrrolizidine, and pyridine alkaloids, whereas lysine is the precursor to the 111
piperidines and quinolizidines. Nucleosides also form the basis of some alkaloids, such as 112
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caffeine and theobromine. 113
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Phenylpropanoids and polyketides 115
The phenylpropanoids represent a class of more than 8,000 plant phenolics derived from 116
tyrosine and phenylalanine via the phenylpropanoid pathway (Wu and Chappell, 2008). 117
The name phenylpropanoid refers to the distinctive C6-C3 structure of metabolites within 118
this pathway. The key phenylpropanoid branch point intermediate is p-coumaroyl-CoA 119
(Figure 3), which forms the basis of the flavonoids, stilbenoids, coumarins, lignans, 120
catechins, and aurones. In addition to the C6-C3 skeleton, the phenylpropanoid pathway 121
also diverts at the level of cinnamic or ferulic acid to yield an array of C6-C1 benzoates, 122
such as vanillin, benzaldehyde, and gallic acid (Vogt, 2010; Kallscheuer et al., 2019). 123
Although the stilbenoids and flavonoids originate from the phenylpropanoid pathway, 124
they are elongated by type III plant polyketide synthases (PKSs), underscoring the mixed 125
biosynthetic nature of these specialized metabolites (Box 1). PKSs accept CoA-bound 126
substrates (Yu et al., 2012), most often p-coumaroyl-CoA, although cinnamoyl- and 127
feruloyl-CoA form the basis of some phenylpropanoid polyketides, such as pinosylvin 128
and curcumin (Preisig-Müller et al., 1999; Kita et al., 2008). Once loaded with a starter 129
molecule, malonyl-CoA moieties are incorporated into the growing polyketide chain. 130
A number of different polyketide backbones can be produced, though the most 131
heavily targeted for metabolic engineering are the naringenin chalcone and resveratrol 132
scaffolds, which give rise to the respective flavonoid and stilbenoid subclasses (Lussier et 133
al., 2012). These compounds are functionalized in downstream reactions including 134
aromatic hydroxylation, nicotinamide adenine dinucleotide phosphate (NADPH)-135
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dependent reduction, O-methylation, and glycosylation. The flavonoids alone encompass 136
more than 6,000 natural products, including chalcones, aurones, catechins, flavanones, 137
flavanols, isoflavonoids, and anthocyanins (Yu et al., 2012). In contrast to the flavonoids 138
and stilbenoids, coumarins are not polyketides, as their lactone ring structure is derived 139
from hydroxylation, isomerization, and lactonization of phenolic acids (cinnamic, p-140
coumaric, caffeic, or ferulic acid) or the corresponding phenolic acyl-CoA esters (Kai et 141
al., 2008; Yao et al., 2017). 142
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PIONEERING STUDIES IN THE MICROBIAL SYNTHESIS OF PLANT 144
METABOLITES 145
The most famous application of synthetic biology for the synthesis of valuable plant 146
products is semisynthetic artemisinin, an antimalarial terpenoid natively produced by 147
sweet wormwood (Artemisia annua). Over a period of more than a decade, artemisinic 148
acid production has reached titers of 25 g/L in yeast (Paddon et al., 2013), whereas 149
Escherichia coli has been engineered to produce 27 g/L of the amorphadiene precursor 150
(Tsuruta et al., 2009). The yeast artemisinic acid production strain has been repurposed as 151
a sesquiterpenoid platform, facilitating industrial-scale production of more than 130 g/L 152
farnesene (Meadows et al., 2016). Paclitaxel is another terpenoid medicine and microbes 153
have been engineered to produce up to 1 g/L of the taxadiene scaffold (Ajikumar et al., 154
2010; Ding et al., 2014; Zhou et al., 2015). Other notable terpenoid pathway 155
reconstructions include limonene, pinene, geraniol, eucalyptol, humulene, β-156
carophyllene, patchoulol, santalene, and bisabolene (Table 1). 157
Because geraniol forms half of the MIA scaffold, strategies successfully used to 158
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produce monoterpenoids were exploited to synthesize MIA intermediates in yeast, such 159
as nepetalactol (Campbell et al., 2016; Billingsley et al., 2017) and strictosidine (Brown 160
et al., 2015). These efforts have yet to be extended to the de novo synthesis of 161
functionalized MIAs in a microbial system, although impressive partial pathway 162
reconstructions have been reported (Qu et al., 2015). The BIA class has also recently 163
experienced several breakthrough microbial pathway reconstitutions. As a result of 164
successes in resolving upstream pathways involving conversion of tyrosine to dopamine, 165
de novo formation of (S)-reticuline was demonstrated for the first time (Nakagawa et al., 166
2011; DeLoache et al., 2015). This pathway was later extended for de novo biosynthesis 167
of morphinan alkaloids in E. coli and yeast (Galanie et al., 2015; Nakagawa et al., 2016), 168
as well as noscapine in yeast (Li et al., 2018). Titers of (S)-reticuline have recently been 169
increased to 160 mg/L in E. coli (Matsumura et al., 2018). Stilbenoids have been the 170
target of several microbial synthesis efforts and the de novo titer of resveratrol has 171
reached 5 g/L in yeast (Li et al., 2016; Katz et al., 2017). Other stilbenoids produced in 172
microbes include pinostilbene, pterostilbene, pinosylvin, and piceatannol (Table 1). 173
Similarly, the branch point flavonoid naringenin has been produced from glucose at titers 174
above 200 mg/L (Lehka et al., 2016). The flavonoid pathway has been diversified in E. 175
coli and yeast to yield the dihydrochalcone phloretin and its derivatives, the flavanone 176
liquiritigenin, and the flavonols kaempferol, quercetin, and fisetin, among others (Table 177
1). This pathway was recently extended to produce anthocyanin pigments using E. coli 178
polycultures (Jones et al., 2017) and a single engineered yeast strain (Eichenberger et al., 179
2018; Levisson et al., 2018). 180
Not all natural product syntheses involve straightforward maximization of product 181
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titer, as some applications require careful tuning of product concentrations. One such 182
study involved combinatorial engineering of industrial brewing yeasts (S. cerevisiae) to 183
generate strains with a diverse range of geraniol and linalool production profiles, 184
culminating in the formulation of beers with a more desirable hops flavor compared to 185
commercial varieties (Denby et al., 2018). Studies such as these highlight the wide range 186
of applications afforded through the manipulation of plant metabolic pathways in 187
microbial systems. 188
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ELUCIDATION OF PLANT METABOLIC PATHWAYS 190
In planta 191
Traditionally, the elucidation of metabolic pathways in source plants has been the driving 192
force and often limiting factor of microbial biosynthesis. Owing to the complexity of 193
plant metabolism, gaps in pathways are common, particularly within downstream 194
reactions mediating the formation of functionalized products. Pathway gaps in the context 195
of microbial synthesis refer to a lack of enzyme identification, as the biochemical nature 196
of metabolic transformations is typically known. 197
To bypass gaps in enzyme elucidation, pathway intermediates are often supplied 198
to cells exogenously, granted they are available and an uptake system exists. Although 199
enzyme gaps have plagued many natural product pathways, a flurry of activity has 200
recently led to the completion of several important pathways. While it was known that 201
production of the nepetalactol backbone within the MIA pathway involved reductive 202
cyclization of 10-oxogeranial, the enzyme mediating this transformation was finally 203
discovered in 2012 (Geu-Flores et al., 2012). Critical to this success was the search of 204
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source plant transcriptomes for redox enzymes co-expressed with MIA pathway 205
transcripts. Other key enzymes within the early MIA pathway were discovered in 2014 206
(Miettinen et al., 2014), prompting the synthesis of the key branch point MIA 207
strictosidine in yeast soon thereafter (Brown et al., 2015). 208
The MIA pathway leading to vinblastine was fully elucidated in 2018 (Caputi et 209
al., 2018); thus, it is only a matter of time before the de novo synthesis of this potent 210
anticancer medicine is achieved in a microbial system. Similarly, the final enzymes 211
involved in the formation of substituted amphetamines, such as ephedrine and 212
pseudoephedrine (Hagel et al., 2012), were also identified as of 2018 (Morris et al., 213
2018), providing rationale for their synthesis in a microbial system. The opium poppy 214
(Papaver somniferum) enzyme responsible for the stereochemical inversion of (S)- to (R)-215
reticuline eluded identification for many years, limiting morphine pathway 216
reconstructions to upstream [glucose to (S)-reticuline] and downstream [(R)-reticuline to 217
morphine] modules. Eventual identification of the fusion enzyme by three research 218
groups led to the reconstruction of the de novo morphinan pathway within a microbial 219
host (Galanie et al., 2015). Candidate epimerase genes were identified using 220
transcriptome database mining and candidates were silenced in opium poppy plants, 221
sequenced from mutants that accumulate (S)-reticuline, or cloned in S. cerevisiae for 222
functional analysis (Farrow et al., 2015; Galanie et al., 2015; Winzer et al., 2015). While 223
this sequence of events permeates the field of microbial synthesis, in which pathway 224
elucidation in planta precedes pathway reconstruction in microbes, a recent paradigm has 225
arisen in response to this laborious and time-consuming process. 226
227
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In microbes 228
Due to the declining cost of DNA synthesis, rapidly advancing bioinformatics tools, and 229
expanding omics databases, it has become possible to resolve single and multi-enzyme 230
gaps within a heterologous microbial host. Using this synthetic biology approach, 231
libraries of candidate gap-filling enzymes are first compiled by querying plant 232
transcriptome databases, such as 1000 Plants (Matasci et al., 2014) or PhytoMetaSyn 233
(Xiao et al., 2013) Projects. The corresponding genes are then codon-optimized, 234
synthesized, and expressed in a laboratory microbe. This workflow facilitates the 235
resolution of enzyme gaps in plant metabolic pathways without having to obtain physical 236
plant material or cDNAs (Narcross et al., 2016). In this regard, synthetic biologists do not 237
need to wait for gap-filling enzymes to be isolated and characterized from natural sources 238
to reconstitute target pathways in a microbial species. One of the groups involved in 239
identifying the aforementioned reticuline epimerase gene employed this strategy by 240
querying plant transcriptome databases and integrating synthetic genes into a yeast strain 241
harboring a downstream module for thebaine synthesis (Galanie et al., 2015). An 242
ambitious combinatorial variation of this technique was attempted to fill a seven-enzyme 243
gap within the vinblastine MIA pathway (Casini et al., 2018). While the authors were 244
unable to resolve this intricate pathway, they succeeded in building 74 strains, each 245
possessing a distinct combination of the seven gene candidates. This study illustrates the 246
immense potential of synthetic biology for elucidating and reconstructing complex 247
biochemical pathways in microbial systems. 248
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HOST SELECTION 250
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Host selection is a fundamental facet of microbial synthesis and a key determinant of 251
pathway performance. Since it is virtually impossible to forecast the ideal host for 252
synthesizing a target metabolite, a number of factors must be weighed, including access 253
to a complete genome sequence and the availability of genetic tools. Precursor 254
availability is perhaps the most important criterion of host selection, as an abundant 255
precursor pool, such as acetyl-CoA or shikimate, sidesteps the need to rewire core 256
metabolism. Host selection is further complicated by the existence of phenotypically 257
distinct strains within a given species, for instance the E. coli K and B lineages or the 258
S288C and CEN.PK strains of S. cerevisiae. In a dramatic instance, utilization of E. coli 259
DH1 improved oxidation of amorphadiene by a factor of 1,000 relative to related strains 260
(Chang et al., 2007). Screening of E. coli lineages for polyketide synthesis has revealed 261
not only strain-specific differences in baseline polyketide production but also differences 262
in response to the same genetic manipulations (Yang et al., 2018). Due to differences in 263
amino acid biosynthesis between S. cerevisiae (Canelas et al., 2010), production of 264
vanillin-β-glucoside was significantly higher in S288C than in CEN.PK (Strucko et al., 265
2015). Similar yeast-specific differences were observed for the synthesis of the 266
polyketide triacetic acid lactone (Saunders et al., 2015). These studies highlight the often 267
overlooked task of screening strains to identify optimal producers prior to 268
comprehensively engineering a production strain. 269
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Workhorses and specialized hosts 271
To date, the overwhelming majority of microbial syntheses have employed E. coli or S. 272
cerevisiae owing to their ease of genetic and metabolic manipulation, a comprehensive 273
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understanding of their genetics, metabolism, and physiology, and their highly active 274
central metabolic pathways. Several critical differences between these industry-proven 275
hosts inform many microbial synthesis campaigns (Box 2). 276
Recently, the diversity of microbial hosts exploited for the synthesis of plant 277
natural products has expanded dramatically. Some microorganisms naturally synthesize 278
aromatic amino acids at levels well above those of E. coli and S. cerevisiae, indicating a 279
major potential of these hosts for the production of alkaloids and phenylpropanoids. Of 280
particular importance is Corynebacterium glutamicum, an industrial bacterium with a 281
tremendous capacity to synthesize glutamate and lysine, as well as aromatic amino acids 282
(Azuma et al., 1993; Ikeda et al., 1993). Lactococcus lactis is another industry-proven 283
bacterium with a demonstrated capacity to synthesize plant terpenoids and 284
phenylpropanoids (Gaspar et al., 2013; Dudnik et al., 2018). The yeast Scheffersomyces 285
stipitis has gained interest for its ability to utilize C5 sugars, suggesting that this organism 286
has a more active pentose phosphate pathway than does S. cerevisiae. Indeed, the highest 287
shikimate titer reported to date in yeast was achieved using S. stipites (Gao et al., 2016). 288
Within the polyketide class of natural products, bacteria from the genus 289
Streptomyces are major natural producers, providing rationale for diverting these native 290
pathways to plant-derived polyketides (Baltz, 2010). The oleaginous yeast Yarrowia 291
lipolytica has also been demonstrated to be a promising host for terpenoids, polyketides, 292
and other compounds derived from acetyl-CoA (Abdel-Mawgoud et al., 2018). Other 293
hosts are desirable for superior growth characteristics, such as Pichia pastoris, a yeast 294
that attains higher cell densities than S. cerevisiae (Wriessnegger et al., 2014). 295
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RECONSTITUTION AND OPTIMIZATION OF PLANT PATHWAYS IN 297
MICROBIAL SYSTEMS 298
Pathway assembly and delivery 299
Pathway assembly refers to the design and organization of genetic parts required to 300
reconstruct heterologous pathways in microbes. This endeavor begins by selecting and 301
synthesizing gene variants responsible for mediating the synthesis of the target product. 302
The selected candidates are placed within transcriptional units containing a promoter and 303
terminator. Traditionally, heterologous genes and metabolic pathways have been 304
delivered and expressed from plasmids. While this technique has certain merits, such as 305
facilitating the rapid screening of prospective hosts (Casini et al., 2018) and easily 306
assessing the performance of pathway modules (Fossati et al., 2014), access to efficient 307
genome editing tools has led to the adoption of chromosomal expression systems 308
(Horwitz et al., 2015; Jakočiūnas et al., 2015). Furthermore, plasmid-borne expression 309
leads to significant variability in gene expression and copy number between cells (Ryan 310
et al., 2014). 311
With respect to transcriptional control, most pathways are assembled by placing 312
all genes under control of constitutive promoters. For instance, amorphadiene and 313
artemisinic acid production strains were first constructed using galactose induction, 314
which was later converted to constitutive expression without affecting production or 315
fitness (Westfall et al., 2012; Paddon et al., 2013). Because even “constitutive” promoters 316
exhibit distinct transcriptional profiles, which vary based on carbon source or medium 317
formulation (Reider Apel et al., 2016), major efforts have focused on comprehensively 318
characterizing both promoter and terminator elements (Yamanishi et al., 2013; Lee et al., 319
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2015). These toolkits facilitate the exploration of vast pathway design spaces comprised 320
of a staggering number of possible gene and expression combinations. 321
Product titer, rate, and yield following initial pathway reconstitution are often 322
very poor, necessitating many design-build-test engineering cycles to reach 323
commercially-viable levels. Historically, this process lasts 6-8 years and costs more than 324
$50 million (Nielsen and Keasling, 2016). These metrics are declining at a dramatic rate 325
as a result of the standardization of synthetic biology and the advent of high-throughput 326
strain engineering facilities (‘biofoundries’), which aim to miniaturize and automate 327
strain construction workflows (Chao et al., 2017; Casini et al., 2018). 328
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Expression of plant genes in microbial species 330
The functional expression of plant genes in microbial species poses a number of technical 331
challenges. Many plant enzymes possess localization signals for targeting to specific 332
organelles, such as the chloroplast. Implementation of these plant genes in microbial 333
hosts typically leads to poor gene expression or insolubility of the corresponding protein 334
(Williams et al., 1998). Most terpene synthases are plastid-localized and thus N-terminal 335
truncation is an effective strategy for improving activity (Alonso-Gutierrez et al., 2013; 336
Zhao et al., 2016). Production of BIAs is also improved through N-terminal truncation of 337
norcoclaurine synthase (Li et al., 2016). While software tools exist for predicting optimal 338
signal peptide cleavage sites (Petersen et al., 2011), assessing various-sized truncations 339
affords a range of improvements (Nishihachijo et al., 2014; Jiang et al., 2017). 340
Perhaps the most challenging facet of synthesizing plant-derived metabolites in 341
microbial hosts is the functional expression of cytochrome P450 (CYP) enzymes. This 342
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obstacle provides much of the rationale for using eukaryotic expression systems over 343
bacteria (Box 2). CYPs are membrane-bound enzymes that carry out many of the 344
complex functionalizations in secondary metabolic pathways, including hydroxylations, 345
double bond epoxidations, and dealkylations (Meunier et al., 2004). Because CYPs 346
mediate oxidation reactions, catalysis is coupled to reduction of NADPH, which in turn 347
requires a cytochrome P450 reductase (CPR) partner to shuttle electrons between 348
NADPH and CYP. Additional components can also be involved, such as cytochrome b5 349
and a cytochrome b5 reductase (Porter, 2002; Paddon et al., 2013; Li et al., 2016). 350
Some species of plant contain hundreds of CYPs and multiple CPRs, leading to 351
immense challenges in optimizing heterologous CYP-CPR pairings. Although yeast 352
contains a native CPR, most plant CYPs exhibit greater activity when paired with CPRs 353
from the same plant species (Fossati et al., 2014). Production of highly functionalized 354
secondary metabolites, such as paclitaxel and hydrocodone, involves numerous CYP-355
catalyzed reactions, requiring the coordinated expression of multiple CPR genes. To 356
overcome this hurdle, most microbial synthesis studies employ a “one-size-fits-all” 357
strategy by pairing multiple CYPs with a single CPR. CYP and CPR expression must be 358
carefully balanced to ensure efficient shuttling of electrons and avoid the production of 359
toxic reactive oxygen species. Optimization of CYP-CPR pairing was paramount to the 360
success of the semi-synthetic artemisinin process (Paddon et al., 2013) and enabled 361
production of 500 mg/L oxygenated taxadiene in E. coli (Biggs et al., 2016). 362
The most efficient natural CYP is a primitive bacterial CYP-CPR fusion from 363
Bacillus megaterium (Munro et al., 2002). This architecture has been exploited for the 364
expression of plant CYPs in E. coli, in which the N-terminal membrane-binding domain 365
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is cleaved or modified for more efficient insertion within bacterial membranes (Hausjell 366
et al., 2018). Conversely, it has been shown that a direct CYP-CPR fusion in E. coli was 367
less active than carefully balanced expression of both components for oxygenated 368
taxadiene synthesis (Biggs et al., 2016). The authors suggest that since plant CYPs and 369
CPRs do not interact in a 1:1 ratio, a direct fusion approach is not appropriate. 370
371
Precursor supply 372
Engineering microbial systems for the production of phenylpropanoids and most 373
alkaloids demands an abundant supply of aromatic amino acid precursors (Figure 2 and 374
Figure 3). Tremendous strides have been made in engineering platform strains producing 375
high levels of tyrosine, phenylalanine, tryptophan, and their derivatives (Table 1). 376
Deregulating host precursor pathways is regarded as the chief hurdle to the 377
production of heterologous metabolites (Nielsen and Keasling, 2016). The yeast 378
shikimate and aromatic amino acid pathways are subject to multiple layers of 379
transcriptional and post-transcriptional control. The entry point to the shikimate pathway 380
involving condensation of E4P and PEP is catalyzed by 3-deoxy-D-arabino-381
heptulosonate-7-phosphate (DAHP) synthase, for which two isoenzymes exist in yeast 382
(Aro3 and Aro4). Allosteric inhibition by tyrosine can be overcome in both enzymes 383
using analogous mutations (Aro3K222L
and Aro4K229L
) (Luttik et al., 2008; Brückner et al., 384
2018). Similarly, chorismate mutase (Aro7) is feedback-inhibited by tyrosine, prompting 385
discovery of the Aro7G141S
allele for deregulating tyrosine biosynthesis (Luttik et al., 386
2008). Implementation of Aro4K229L
and Aro7G141S
in the same host leads to a 200-fold 387
increase in extracellular levels of aromatic compounds. 388
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The terpenoid branch of secondary metabolism has also been the focal point of 389
engineering precursor supply. The rate-limiting step of the mevalonate pathway catalyzed 390
by HMG-CoA reductase (HMGR) can be improved by implementing a truncated mutant 391
(tHMGR). Owing to the importance of this enzymatic step, studies achieving high-level 392
terpenoid or MIA production have incorporated up to four gene copies of tHMGR 393
(Paddon et al., 2013; Brown et al., 2015). 394
The production of GPP-derived monoterpenoids poses a unique engineering 395
challenge pertaining to the dual nature of the yeast Erg20 protein. This enzyme catalyzes 396
the synthesis of GPP from IPP and DMAPP, as well as the subsequent condensation of 397
GPP and an additional unit of IPP to give FPP (Figure 1). Owing to the central role of 398
FPP in ergosterol biosynthesis, Erg20 is an essential enzyme, signifying that its FPP 399
synthase activity can not be abolished. Instead, Erg20 variants with reduced GPP-binding 400
properties have been described (Chambon et al., 1990) and exploited for the production 401
of various monoterpenoids and MIAs (Brown et al., 2015; Campbell et al., 2016). 402
Relatedly, the essential squalene synthase enzyme (Erg9) generates squalene from two 403
FPP units, thus placing constraints on the production of all terpenoids deriving from FPP 404
(Figure 1). 405
The most fundamental means of circumventing host regulation is replacing native 406
precursor pathways with a heterologous counterpart. This approach was utilized in early 407
efforts to produce amorphadiene in E. coli, wherein the native MEP pathway was 408
replaced by the yeast mevalonate pathway (Martin et al., 2003). Reconstructing the yeast 409
pathway within a bacterial operon eliminates both E. coli and S. cerevisiae modes of 410
regulation, facilitating constitutive production of terpenoids. 411
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18
Acetyl-CoA plays a pivotal role in the biosynthesis of terpenoids, polyketides, 412
phenylpropanoids, and cannabinoids (Figure 1 and Figure 3), and its overproduction has 413
also been a target for improvement. In yeast, multiple acetyl-CoA synthesis pathways 414
exist in different subcellular compartments, which acetyl-CoA can not directly cross 415
(Strijbis and Distel, 2010). This provides options for strain engineering based on the 416
pathway that is most suited for a particular heterologous product (van Rossum et al., 417
2016). Alternatively, heterologous cytosolic acetyl-CoA pathways are of great interest 418
(Kozak et al., 2014; Kozak et al., 2014; Zhang et al., 2015; Cardenas and Da Silva, 2016; 419
Kozak et al., 2016; Meadows et al., 2016; Rodriguez et al., 2016). The highest reported 420
titer of a heterologous product derived from cytosolic acetyl-CoA is the synthesis of 130 421
g/L of the sesquiterpene farnesene under fermentative conditions (Meadows et al., 2016). 422
This approach utilized multiple alternative cytosolic acetyl-CoA pathways to overproduce 423
farnesene. 424
In prokaryotes, all routes to acetyl-CoA are equally accessible, reducing the 425
necessity for alternative pathways, although they have been demonstrated (Wang et al., 426
2018). However, buildup of acetyl-CoA results in its conversion to acetate, which is an 427
unproductive carbon sink. In E. coli, native acetate assimilation enzymes can be 428
overexpressed to convert acetate back to acetyl-CoA, resulting in improved flavanone 429
biosynthesis (Leonard et al., 2007; Zha et al., 2009). Gene knockouts have been identified 430
that prevent acetate synthesis and improve heterologous production without reducing 431
cellular fitness (Zha et al., 2009; Liu et al., 2017). Eliminating pathway bottlenecks to 432
improve flux through acetyl-CoA can also reduce acetate overflow (Li et al., 2015; King 433
et al., 2017; Wang et al., 2018). Relatively little attention has been placed on the 434
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19
availability of free Coenzyme A to accept increased carbon flux. Supplementation of the 435
Coenzyme A precursor pantothenate can improve the yield of heterologous products 436
requiring acetyl-CoA. Several studies have demonstrated that overexpression of the rate-437
limiting step of Coenzyme A synthesis, pantothenate kinase, improves product synthesis 438
two-fold (Fowler et al., 2009; Schadeweg and Boles, 2016; Liu et al., 2017). 439
Phenylpropanoid, polyketide and cannabinoid synthesis require malonyl-CoA, 440
which is used for fatty acid biosynthesis in E. coli and S. cerevisiae. In these organisms, 441
fatty acid synthesis is tightly-regulated (Davis et al., 2000). Improving synthesis and 442
reducing off-target consumption of malonyl-CoA are the major areas for overproduction. 443
Acetyl-CoA is converted to malonyl-CoA by acetyl-CoA carboxylase (Acc) and 444
improvements in this activity have been achieved by overexpression (Zha et al., 2009; Xu 445
et al., 2011; Yang et al., 2018), ablating post-translational modification sites (Choi and 446
Da Silva, 2014; Shi et al., 2014; Chen et al., 2018), and improving the availability of its 447
biotin cofactor (Leonard et al., 2007). Acc-independent malonyl-CoA synthesis has also 448
been demonstrated (Leonard et al., 2008), though the pathway requires supplemented 449
malonate. Reducing the diversion of malonyl-CoA to essential pathways is a challenge, 450
because fatty acids are required for biomass production. In yeast, knockout of genes 451
regulating fatty acid biosynthesis improved titers of malonyl-CoA-derived products (Zha 452
et al., 2009; Chen et al., 2017), yet the strain was auxotrophic for inositol. More 453
promisingly, biomass accumulation prior to conditional gene knockdown has been 454
demonstrated to improve polyketide titers in E. coli (Yang et al., 2015; Liang et al., 2016; 455
Wu et al., 2016; Yang et al., 2018). 456
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20
As a ubiquitous cofactor and the source of reductant for plant CYPs, NADPH is 457
an important engineering target for the synthesis of natural products. A proven strategy 458
for enhancing NADPH availability is the inactivation of native NADPH-consuming 459
reactions. A caveat of this approach pertains to the targeting of enzymes involved in 460
central carbon metabolism, such as glucose-6-phosphate isomerase (Pgi) and 461
phosphoenolpyruvate carboxylase (Ppc) in E. coli (Chemler et al., 2010) or glucose-6-462
phosphate dehydrogenase (Zwf1) in S. cerevisiae (Gold et al., 2015). Glutamate 463
dehydrogenase (GldH or Gdh1) is a promising candidate in both species (Asadollahi et 464
al., 2009; Chemler et al., 2010). Inactivation of these central targets often has deleterious 465
effects on strain fitness, including the generation of amino acid auxotrophy (Gold et al., 466
2015), demanding a delicate balancing of core pathways and sophisticated metabolic 467
models for predicting combinatorial modifications (Chemler et al., 2010). 468
The counter approach of upregulating NADPH-generating enzymes is a more 469
viable avenue for engineering NADPH supply. In yeast, these enzymes include 470
prephenate dehydrogenase (Tyr1) and cytosolic aldehyde dehydrogenase (Ald6) (Li et al., 471
2018). Curiously, both overexpression and deletion of ZWF1 have been shown to 472
enhance formation of tyrosine products (Gold et al., 2015; Li et al., 2018), underscoring 473
the complexity of yeast central metabolism and redox homeostasis. 474
475
Improving flux to heterologous pathways 476
Flux improvement involves iterative rounds of strain engineering with the aim of 477
elevating product titer, rate, and yield. Efforts to improve flux through a target pathway 478
begin with the identification of rate-limiting enzymatic conversions, which is diagnosed 479
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21
by monitoring levels of pathway intermediates. The most fundamental means of relieving 480
metabolic bottlenecks is to increase levels of rate-limiting enzymes by increasing gene 481
expression or copy number. In addition to high-level expression of the heterologous 482
pathway, the entire eight-gene yeast pathway from acetyl-CoA to FPP was overexpressed 483
in the industrial artemisinic acid strain (Paddon et al., 2013). 484
Poor pathway flux can also arise from the diversion of pathway intermediates to 485
off-target routes, either by host activities or promiscuous plant enzymes. Because 486
phenylpropanoid biosynthesis derives from phenylalanine or tyrosine, the yeast pathway 487
for aromatic amino acid degradation (Ehrlich pathway) is typically inactivated through 488
deletion of amino acid decarboxylase genes (ARO10 and PDC5) (Rodriguez et al., 2015). 489
Yeast BIA biosynthesis involves a more intricate balancing of tyrosine pathways, as the 490
dopamine and 4-HPAA precursors are derived from its biosynthesis and degradation, 491
respectively (Narcross et al., 2016). The physiological fate of 4-HPAA in yeast involves 492
conversion to tyrosol or 4-hydroxyphenylacetate, which can be mediated by more than 20 493
candidate enzymes (Hazelwood et al., 2008; DeLoache et al., 2015). These enzymes have 494
also been implicated in the irreversible reduction of aldehyde intermediates within the 495
MIA pathway (Billingsley et al., 2017), suggesting that any pathway possessing carbonyl 496
intermediates is subject to diversion by host activities. In a similar scenario, a yeast 497
reductase was shown to divert p-coumaroyl-CoA within the phenylpropanoid pathway to 498
an unwanted side product (Lehka et al., 2016). 499
Pathway flux can also be diverted to off-target routes by heterologous pathway 500
enzymes. O- and N-methyltransferases within the core BIA pathway have been shown to 501
exhibit a wide substrate range (Frick et al., 2001; Ounaroon et al., 2003; Fossati et al., 502
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22
2014), which was circumvented by screening of plant homologues for variants exhibiting 503
a more stringent substrate specificity (Narcross et al., 2016). In addition to catalyzing the 504
reductive cyclization of 10-oxogeranial, iridoid synthase within the MIA pathway was 505
found to exhibit activity on other intermediates (Campbell et al., 2016). In this case, 506
enzyme activity was higher on the unwanted substrates than the physiological substrate. 507
Enzyme promiscuity may be avoided in co-cultures, in which a pathway is split 508
between strains such that promiscuous enzymes are sequestered from pathway 509
intermediates (Nakagawa et al., 2014). Loss of intermediates is also mitigated through 510
enzyme fusion, as in the cases of geraniol synthase or patchoulol synthase through fusion 511
to yeast farnesyl diphosphate synthase (Erg20) (Albertsen et al., 2011; Jiang et al., 2017). 512
Fusions have the potential to facilitate more efficient substrate channeling by maintaining 513
close physical proximity of sequential pathway enzymes, thus limiting competition from 514
host enzymes. In addition to terpenoid systems, fusion of resveratrol biosynthetic 515
enzymes facilitated a 15-fold improvement in titer in engineered S. cerevisiae (Zhang et 516
al., 2006). Enzyme fusions have the added potential to improve protein stability or 517
solubility, as demonstrated through fusions of taxadiene synthase with the soluble E. coli 518
maltose binding protein, yielding a 25-fold improvement in taxadiene titer (Reider Apel 519
et al., 2016). Fusions with maltose-binding protein also improved production of α-ionone 520
up to 50-fold (Zhang et al., 2018). 521
522
EXPANDING THE PLANT SECONDARY METABOLIC SPACE IN 523
MICROBIAL SYSTEMS 524
An emerging application of synthetic biology aims to exploit microbial systems for drug 525
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23
discovery. In this manner, plant enzyme promiscuity and combinatorial biochemistry are 526
exploited to synthesize new structural scaffolds and functionalizations not found in 527
nature. 528
A common chemical modification within the pharmaceutical industry is 529
halogenation, in which fluoro or chloro moieties are introduced to drug candidates to 530
modify the physicochemical properties of the compound. Because many natural products 531
derive from amino acids, halogenated analogs can be supplemented to engineered 532
microbes leading to the formation of halogenated products. This strategy was used to feed 533
yeast fluoro- or chloro-tyrosine, resulting in the synthesis of halogenated BIA pathway 534
intermediates (Li et al., 2018). Halogenated end products were not detected, suggesting a 535
more stringent substrate specificity of downstream BIA enzymes. Moreover, this method 536
relies on the supplementation of costly amino acid analogs and could be overcome by 537
engineering de novo production of halogenated BIAs. 538
O-sulphated reticuline has been produced de novo by implementing a human 539
sulphotransferase into E. coli engineered for reticuline biosynthesis (Matsumura et al., 540
2018). Promiscuity of BIA pathway enzymes has also been exploited for the production 541
of novel scaffolds, such as disubstituted- and spiro-tetrahydroisoquinolines, by harnessing 542
the capacity of norcoclaurine synthase to accept an exceptional range of carbonyl 543
substrates (Lichman et al., 2017). Again, this strategy relies on the supplementation of 544
costly substrates, in this case carbonyl species that are not synthesized by 545
microorganisms. Moving these in vitro assays to robust de novo biosynthetic processes 546
presents a formidable challenge. 547
548
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CONCLUDING REMARKS 549
Microbial synthesis provides a new avenue for the production of natural plant 550
pharmaceuticals, fragrances, nutrients, and colorants. Although this field is in its infancy 551
and faces many challenges (see Outstanding Questions), the wealth of successes in recent 552
years has solidified microbial biosynthetic production as a viable alternative to natural 553
product extraction and total chemical synthesis. The elucidation of increasingly complex 554
plant secondary pathways continues to drive the reconstruction of longer and more 555
challenging routes in microbial systems. Indeed, the discovery of missing enzymes in 556
several highly sought-after plant specialized pathways has primed a number of untapped 557
natural products for de novo biosynthesis in a microbial species. Several microbially-558
derived natural products have reached commercialization and these successes will 559
continue to pave the way for new opportunities. The exploitation of microbial systems for 560
drug discovery promises to deliver important new structural scaffolds and chemical 561
functionalizations that have hitherto not been observed in nature. 562
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563
ACKNOWLEDGMENTS 564
This work was supported financially by a NSERC-Industrial Biocatalysis Network (IBN) grant, 565
and a NSERC Discovery grant. M.E.P. is supported by a NSERC postdoctoral fellowship, and 566
L.N. is supported by a FQRNT DE Doctoral Research Scholarship for Foreign Students. V.J.J.M. 567
is supported by a Concordia University Research Chair. 568
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Table 1 – Selection of de novo plant secondary pathway reconstructions in microbial systems 569
Titer (mg/L)
Metabolite class Metabolite S. cerevisiae E. coli Reference(s)
PRECURSORS tyrosine 1,930a 55,000 (Patnaik et al., 2008)
p-coumaric acid 1,930 2,510 (Rodriguez et al., 2015; Jones et al., 2017)
dopamine 24 2,150 (Nakagawa et al., 2014; DeLoache et al., 2015)
TERPENOIDS Monoterpenoids
geraniol 1,680 2,000 (Liu et al., 2016; Jiang et al., 2017)
pinene ND 970 (Yang et al., 2013)
limonene 0.49 2,700 (Willrodt et al., 2014; Jongedijk et al., 2015)
eucalyptol 400 653 (Ignea et al., 2011; Mendez‐Perez et al., 2017)
linalool 0.75 505 (Mendez‐Perez et al., 2017; Camesasca et al.,
2018)
Sesquiterpenoids
β-farnesene 130,000 8,700 (Meadows et al., 2016; You et al., 2017)
bisabolene 5,200 912 (Peralta-Yahya et al., 2011; Özaydın et al., 2013)
α-humulene 1,300 ND (Zhang et al., 2018)
β-caryophyllene ND 1,520 (Yang et al., 2016)
amorphadiene 37,000 27,400 (Tsuruta et al., 2009; Westfall et al., 2012)
artemisinic acid 25,000 ND (Paddon et al., 2013)
santalene 163 ND (Tippmann et al., 2016)
patchoulol 40.9 ND (Albertsen et al., 2011)
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Diterpenoids
jolkinol C 800 ND (Wong et al., 2018)
taxadiene 72.8 1,020 (Ajikumar et al., 2010; Ding et al., 2014)
Triterpenoids
β-amyrin 108.1 NR (Takemura et al., 2017; Zhu et al., 2018)
Tetraterpenoids
β-carotene 162.1 3,200 (Yang and Guo, 2014; Wang et al., 2016)
lycopene 1,650 3,520 (Sun et al., 2014; Chen et al., 2016)
ALKALOIDS Benzylisoquinoline
alkaloids
(S)-reticuline 0.082 160 (DeLoache et al., 2015; Matsumura et al., 2018)
thebaine 0.0064 2.1 (Galanie et al., 2015; Nakagawa et al., 2016)
hydrocodone 0.0003 0.36 (Galanie et al., 2015; Nakagawa et al., 2016)
noscapine 2.2 ND (Li et al., 2018)
Monoterpene indole
alkaloids
strictosidine 0.5 ND (Brown et al., 2015)
PHENYLPROPANOIDS Stilbenoids
resveratrol > 5,000 51.8 (Katz et al., 2017; Yang et al., 2018)
pinostilbene 5.52 ~2.5 (Kang et al., 2014; Li et al., 2016)
pterostilbene 34.93 33.6 (Li et al., 2016; Heo et al., 2017)
pinosylvin 130.02 281 (Katz et al., 2017; Wu et al., 2017)
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Flavonoids
naringenin >200 103.8 (Lehka et al., 2016; Yang et al., 2018)
scutellarin 108 ND (Liu et al., 2018)
genistein 7.7 ND (Trantas et al., 2009)
pinocembrin 2.6 432.4 (Wu et al., 2016; Eichenberger et al., 2017)
quercetin 20.38 ND (Rodriguez et al., 2017)
kaempferol 66.29 57b (Yang et al., 2014; Duan et al., 2017)
dihydrokaempferol 44 ND (Levisson et al., 2018)
pelargonidin-3-O-
glucoside 0.02 9.5 (Jones et al., 2017; Levisson et al., 2018)
phloretin 42.7 ND (Eichenberger et al., 2017)
Coumarins
umbelliferone ND 66.1 (Yang et al., 2015)
esculetin ND 61.4 (Yang et al., 2015)
4-hydroxycoumarin ND 483.1 (Lin et al., 2013) atiter reported for the downstream metabolite p-coumaric acid 570
btiter reported for kaempferol 3-O-rhamnoside 571
ND: no data available 572 NR: titer not reported 573
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Figure legends 574
Figure 1. Interfacing plant terpenoid secondary metabolic pathways with microbial 575
metabolism. Plant secondary metabolic reactions and pathways (green) are shown linked 576
to core microbial metabolism (beige shading; black font). S. cerevisiae is shown as the 577
prospective host species. Key yeast enzymes discussed in the main text are shown. 578
Abbreviations: Bts1, geranylgeranyl diphosphate synthase; DMAPP, dimethylallyl 579
pyrophosphate; Erg9, squalene synthase; Erg20, farnesyl pyrophosphate synthetase; FPP, 580
farnesyl pyrophosphate; GPP, geranyl pyrophosphate; GGPP, geranylgeranyl 581
pyrophosphate; IPP, isopentenyl pyrophosphate; MEV, mevalonate pathway. 582
583
Figure 2. Interfacing plant alkaloid secondary metabolic pathways with microbial 584
metabolism. S. cerevisiae is shown as the prospective host species. Refer to Figure 1 for 585
abbreviations and color coding. Additional abbreviations: AAA path, aromatic amino 586
acid pathway; Aro1, pentafunctional arom protein; Aro2, chorismate synthase and Flavin 587
reductase; Aro3+Aro4, DAHP synthase isoenzymes; Aro7, chorismate mutase; E4P, 588
erythrose 4-phosphate; 4-HPAA, 4-hydroxyphenylacetaldehyde; R5P, ribose 5-589
phosphate; PPP, pentose phosphate pathway; PEP, phosphoenolpyruvate; TCA cycle, 590
tricarboxylic acid cycle. 591
592
Figure 3. Interfacing plant phenylpropanoid and polyketide secondary metabolic 593
pathways with microbial metabolism. A selection of natural plant pathways in a 594
prospective S. cerevisiae host is shown. More extensive networks of natural and synthetic 595
phenylpropanoid routes have been shown previously (Wang et al., 2015; Zhao et al., 596
2015). Refer to Figure 1 and Figure 2 for abbreviations and color coding. Additional 597
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abbreviations: Acc1, acetyl-CoA carboxylase; PAL, phenylalanine ammonia-lyase; TAL, 598
tyrosine ammonia-lyase; PKS, polyketide synthase. 599
600
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1152 1153
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ADVANCES
• Microbial synthesis of plant secondary metabolites is in the midst of a renaissance.
• The elucidation of major plant biosynthetic routes has stimulated the production of high-value natural products in microbial systems.
• Efforts are expanding beyond precursors and branch point intermediates to functionalized end products.
• Although baker’s yeast (Saccharomyces cerevisiae) remains the host of choice for reconstructing plant secondary pathways, the range of alternative hosts has diversified.
• Synthetic biology has enabled the expansion of the plant secondary metabolic space in microbial species.
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OUTSTANDING QUESTIONS
• Can microbial production of plant metabolites compete with traditional methods of chemical synthesis and crop-based manufacturing?
• Pathways for several untapped sub-classes of plant specialized metabolites have been elucidated recently (e.g., modified amphetamines and vinblastine). Can these pathways be reconstituted in a microbial species?
• Other functionalized metabolites of immense pharmaceutical value (e.g., paclitaxel) have proven inaccessible to date. What are the limitations and how can they be overcome to realize the microbial synthesis of these important drugs?
• To what extent will emerging synthetic biology workflows reduce the time and cost of advancing bioprocesses from proof-of-concept to large-scale production?
• Will microbial systems provide a viable avenue for drug discovery through the exploitation of enzyme promiscuity and combinatorial biochemistry?
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BOX 1. Metabolites of mixed biosynthetic
origin
A number of important plant natural products are derived from mixed biosynthetic origins, obscuring the boundaries between secondary metabolite classes (Box 1 Figure). Within the alkaloid class, MIAs arise through condensation of the tryptophan analog tryptamine and a secoiridoid monoterpene (secologanin). Secologanin can also condense with dopamine, giving rise to emetine and cephaeline, the active alkaloids of the emetic drug ipecac. Nicotine is formed through coupling of two heterocyclic rings of distinct biosynthetic origin. Whereas the pyrrolidine ring derives from ornithine via the tropane alkaloid pathway (Figure 1), the pyridine ring is formed from niacin, itself synthesized from aspartate. Several other alkaloids are formed from products of the phenylpropanoid pathway, such as substituted amphetamines through condensation of pyruvate and benzoic acid, a C6-C1 analog of cinnamic acid (Hagel et al., 2012). Modified amphetamines are also called phenylpropylamino alkaloids to reflect their dual biosynthetic origin. Similarly, the autumnaline scaffold of the phenethylisoquinoline alkaloids is derived through condensation of dopamine and 4-hydroxydihydrocinnamaldehyde, another cinnamic acid derivative. The capsaicinoids produced by chili peppers have a unique tripartite classification, as they are exocyclic alkaloids that arise in part from the phenylpropanoid pathway and polyketide synthesis.
Specifically, coupling of ferulic-acid-derived vanillylamine and a fatty acyl-CoA (8-methyl-6-nonenoyl CoA) derived from valine or leucine gives rise to the capsaicin scaffold. Many other functionalized phenylpropanoids, notably the flavonoids and stilbenoids, are also polyketides involving chain elongation by malonyl-CoA (Figure 3). The curcuminoids and gingerols, responsible for the color and pungency of turmeric and ginger, respectively, are two additional subclasses comprising phenylpropanoid CoA esters linked by a polyketide synthase. Aromatic plant secondary metabolites are also frequently prenylated, which requires a terpenoid donor (Vickery et al., 2016). One such class of natural products is the cannabinoids, of which more than 100 distinct compounds are produced by the cannabis plant (Ahmed et al., 2015). A polyketide synthase catalyzes the decarboxylative condensation of hexanoyl-CoA with three molar equivalents of malonyl-CoA to generate olivetolic acid, which is then prenylated by GPP, generating cannabigerolic acid (CBGA). CBGA is the precursor to tetrahydrocannabinol (THC), cannabidiol, and their derivatives. These bioactive natural products have the potential to treat various medical conditions, including cancer, anorexia, epilepsy, and Alzheimer’s and Huntington’s diseases (Aizpurua-Olaizola et al., 2016).
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BOX 2. E. coli versus S. cerevisiae
Most microbial synthesis projects start with the question: E. coli or S. cerevisiae? Yeast alcohol production exemplifies the most ancient industrial bioprocess and both hosts have been exploited extensively in industrial biotechnology. Although many plant CYPs and CPRs can be functionally expressed in bacteria, often following extensive engineering of N-terminal membrane domains, CYPs are typically functional in yeast without modification. Because yeast produces only three native CYPs, interference from heterologous CYP and CPR proteins is generally not problematic. Therefore, we surmise that yeast is the superior host to support the biosynthesis of highly functionalized plant secondary metabolites, particularly those arising from multi-CYP pathways. The occurrence of eight CYP-catalyzed reactions in the downstream taxane pathway precludes the use of bacterial systems for producing these important products. E. coli has been engineered to synthesize the highly functionalized morphinan BIA hydrocodone (Nakagawa et al., 2016), yet the full de novo pathway was split between four engineered strains. The same pathway has been reconstructed within a single S. cerevisiae strain
(Galanie et al., 2015), albeit at lower titers, again highlighting the potential advantages of eukaryotic hosts for synthesizing complex plant secondary metabolites. Analogously, de novo anthocyanin synthesis in E. coli employed four engineered strains (Jones et al., 2017), which again contrasts strategies using S. cerevisiae in which a single strain hosts the full anthocyanin pathway (Eichenberger et al., 2018; Levisson et al., 2018). Although yeast is clearly favored for harboring downstream pathways, precursor titers are often higher in E. coli (e.g., for production of tyrosine, p-coumaric acid, dopamine, reticuline, and taxadiene; Table 1). For instance, E. coli has been engineered to produce 1 g/L of taxadiene (Ajikumar et al., 2010), which contrasts yeast titers of only 72.8 mg/L (Ding et al., 2014). This disparity in precursor production and downstream functionalization points to co-culture strategies in which natural product pathways are divided between E. coli and S. cerevisiae hosts. This approach proved highly effective for the synthesis of functionalized taxanes, in which the taxadiene precursor was secreted by E. coli and imported and derivatized by S. cerevisiae harboring multiple CYP enzymes (Zhou et al., 2015).
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Figure 1. Interfacing plant terpenoid secondary metabolic pathways with microbial
metabolism. Plant secondary metabolic reactions and pathways (green) are shown linked to core
microbial metabolism (beige shading; black font). S. cerevisiae is shown as prospective host
species. Key yeast enzymes discussed in the main text are shown. Abbreviations: Bts1,
geranylgeranyl diphosphate synthase; DMAPP, dimethylallyl pyrophosphate; Erg9, squalene
synthase; Erg20, farnesyl pyrophosphate synthetase; FPP, farnesyl pyrophosphate; GPP, geranyl
pyrophosphate; GGPP, geranylgeranyl pyrophosphate; IPP, isopentenyl pyrophosphate; MEV,
mevalonate pathway.
Sugars
Pyruvate
Acetyl-CoA
IPP
MONO-
TERPENOIDS
SESQUI-
TERPENOIDS
DI-
TERPENOIDS
GPPGeraniol
Taxadiene
Squalene
TETRA-TERPENOIDS
FPP
GGPP
Phytoene
Citronellol
OH
OH
β-carotene
TRI-TERPENOIDS
α-Pinene
HH
OH
Menthol
Isoprene Prenol
HEMI-
TERPENOIDS
Lycopene
Bisabolene
POLY-
TERPENOIDS
IPP
Farnesol
Erg20
Amorphadiene Artemisinic acid
Limonene
FerruginolGGPP
Natural rubber
(polyisoprene)
n IPP
n IPP
β-Amyrin Lupeol
Erg20
Erg9
Bts1
Ergosterol
Farnesene
OH
OH
OH
O
Camphor
OH
Thymol
OH
Linalool
OH
n>5,000
DMAPP
FPP
OH
MEV
IPP
OH
O
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Figure 2. Interfacing plant alkaloid secondary metabolic pathways with microbial metabolism. S. cerevisiae is shown as
prospective host species. Refer to Figure 1 for abbreviations and color coding. Additional abbreviations: AAA path, aromatic amino
acid pathway; Aro1, pentafunctional arom protein; Aro2, chorismate synthase and Flavin reductase; Aro3+Aro4, DAHP synthase
isoenzymes; Aro7, chorismate mutase; E4P, erythrose 4-phosphate; 4-HPAA, 4-hydroxyphenylacetaldehyde; R5P, ribose 5-
phosphate; PPP, pentose phosphate pathway; PEP, phosphoenolpyruvate; TCA cycle, tricarboxylic acid cycle.
Sugars
PEP
Pyruvate
Acetyl-CoA
G6P
R5P
PPP Glycolysis
NucleosidesPURINE ALKALOIDS
Caffeine
BENZYLISOQUINOLINE
ALKALOIDS
MONOTERPENOID INDOLE
ALKALOIDS
Noscapine Morphine Berberine Sanguinarine
TROPANE, PYRROLIDINE, PYRROLIZIDINE,
PYRIDINE ALKALOIDS
PIPERIDINE, QUINOLIZIDINE ALKALOIDS
E4P
Chorismate
Vasicine
QUINAZOLINE
ALKALOIDS
Anthranilate
Prephenate
Nicotine Cocaine Atropine
GPPTryptophan
AMPHETAMINE ANALOGS
Phenylalanine
Tyrosine
NH2
OH
O
NH2
OH
O
OH
Shikimate
path
MEV
Piperine
AAA path
Ephedrine Cathinone
Secologanin
NH2
O
OH
NH
Tryptamine
Dopamine 4-HPAA
H
H
OH
N
O
OH
H
H
OO
O
OO
NO
O
O
O
N
N
N
N
PHENETHYL-
ISOQUINOLINE
ALKALOIDS
Colchicine
L-DOPA
OH
N
N
O
NH
O
O
O
O
O
O
NH2
Vinblastine Quinine Camptothecin
OO
N+
O
O
OH
O
O
OO
OO
O
OH
N
NNH
N
H
H
N
NOH
O
OOH
O
N
N
OOO
N+
O
O
Sparteine
Pyruvate
N
N
O
O O
O
N
H
H
OO
OHN
H
H
O
O
O
N
SIMPLE INDOLES +
β-CARBOLINES
Serotonin
α-Ketoglutarate Glutamate
Arginine
Lysine
Cadaverine
Putrescine
Ornithine
TCA cycle
Harmine
OH
NH2
NH
O NH
N
N
NH
H
Aro3+Aro4
Aro1
Aro2
Aro7
OH
NH
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Figure 3. Interfacing plant phenylpropanoid and polyketide secondary metabolic pathways with
microbial metabolism. A selection of natural plant pathways in a prospective S. cerevisiae host is
shown. More extensive networks of natural and synthetic phenylpropanoid routes have been shown
previously (Wang et al., 2015; Zhao et al., 2015). Refer to Figure 1 and Figure 2 for abbreviations and
color coding. Additional abbreviations: Acc1, acetyl-CoA carboxylase; PAL, phenylalanine ammonia-
lyase; TAL, tyrosine ammonia-lyase; PKS, polyketide synthase.
Sugars
PEP E4P
Prephenate
Shikimate
path
Malonyl-CoA
Phenylalanine Tyrosine
NH2
OH
O
NH2
OH
O
OH
OH
O
Cinnamic acidOH
OH
O
p-Coumaric acid
PAL TAL
p-Coumaroyl-CoA
Acc1
COUMARINS
FLAVONOIDS
STILBENOIDS
Naringenin
(flavanone)
OH O
OH O
OH
3 Malonyl-CoA
3 Malonyl-
CoA
OH
OH
OH
Resveratrol
Umbelliferone
Pelargonidin 3-glucoside
(anthocyanin)
Quercetin
(flavonol)
Genistein
(isoflavonoid)
Pinosylvin
Pterostilbene
Pinostilbene
OOH O
O
O
OH
OH
OH
OH
O
OH
PPPGlycolysis
AAA
path
PKS
PKS
Caffeoyl-CoA Feruloyl-CoA
OH
S-CoA
O
Scopoletin
OH
OH
S-CoA
O
Acetyl-CoACoumarin
OO
Esculetin
OH
OOH O
O
OOH O
4-Hydroxycoumarin
OH
OO
Cinnamoyl-CoA
S-CoA
O
PKS3 Malonyl-CoA
OH
OH
OH O
OH O
OH
OH
OH O
OH O GlcO
OH O+
OH
OH
Caffeic acid Ferulic acid
OH
OH
OH
O
OH
O
S-CoA
O
OHOH
O
O
OTHER
PHENOLS
Vanillin
Angelicin
OOO
O
OH
O
Dhydrokaempferol
(dihydroflavonol)
OH
OH O
OH O
OH
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