Embed Size (px)
Citation preview
1
Biosynthetic design and implementation towards E. coli-derived Taxol and other 1
heterologous polyisoprene compounds 2
3
Running title: E. coli-derived Taxol Biosynthetic Efforts 4
5
Ming Jiang1, Gregory Stephanopoulos2, Blaine A. Pfeifer1* 6
7
1Department of Chemical and Biological Engineering, State University of New York at 8
Buffalo, Buffalo, NY 14260 9
2Department of Chemical Engineering; Massachusetts Institute of Technology, 10
Cambridge, MA 02139 11
12
*Corresponding author: 13
Tel: 716-645-1198 14
Fax: 716-645-3822 15
Email: [email protected] 16
17
18
Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.07391-11 AEM Accepts, published online ahead of print on 27 January 2012
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
2
Abstract 19
Escherichia coli offers unparalleled engineering capacity in the context of 20
heterologous natural product biosynthesis. However, like other heterologous hosts, 21
cellular metabolism must be designed or re-designed to support final compound 22
formation. This task is at once complicated and aided by the fact that the cell does not 23
natively produce an abundance of natural products. As such, the metabolic engineer 24
will avoid complicated interactions with native pathways closely associated with the 25
outcome of interest, but this convenience is tempered by the need to implement the 26
required metabolism to allow functional biosynthesis. This review will focus on 27
engineering E. coli for the purpose of polyisoprene formation as related to those 28
isoprenoid compounds currently being pursued through a heterologous approach. In 29
particular, the review will feature the Taxol compound and early efforts to design and 30
overproduce intermediates through E. coli. 31
Introduction 32
Isoprenoids are an important class of natural products that have been converted 33
to several therapeutic medicines (2, 41, 42). However, this process can be complicated 34
by the need to harness or re-construct native biosynthesis. As an example, the well-35
known isoprenoid natural product Taxol is natively produced by the Pacific yew tree 36
(Taxus brevifolia); yet, utilizing production from the native host proved both 37
uneconomical and environmentally destructive (5, 17, 25). An economical total 38
chemical synthetic route to the compound is challenging due to the molecule’s complex 39
final architecture. This common scenario has driven alternative approaches towards the 40
viable production of this and other therapeutically-relevant natural products. One such 41
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
3
approach is the use of heterologous biosynthesis in which the genetic content 42
responsible for a given natural compound is transplanted from the native organism to a 43
surrogate host for the purpose of leveraging the new host’s innate biological and 44
engineering capabilities. This review will feature the approach of heterologous 45
biosynthesis, using the Taxol compound as a primary example. More specifically, 46
emphasis will be placed on the logic and initial steps guiding heterologous biosynthesis 47
including the choice of host organism, the provision of required intracellular substrates, 48
and the initiation of polyisoprene formation. For more comprehensive or alternative 49
discussions of isoprenoid biosynthesis, readers are directed to several additional review 50
articles (1, 11, 21, 30, 31, 33, 40, 46, 67). 51
Isoprenoids are often associated with plant natural products, but they are also 52
produced by microorganisms (19, 58). The compounds serve several roles in basic 53
cellular function including light absorption, electron transport, and protein modification. 54
In addition, they are perhaps better known for roles in less clearly defined chemical 55
communication scenarios. For example, cellular damage or potential foreign organism 56
invasion both spur isoprenoid biosynthesis in an attempt to protect plant viability. Such 57
capacity for isoprenoids to serve as chemical cues in response to environmental stimuli, 58
including as repellents to foreign organism invasion, provides one supporting theory as 59
to their medicinal properties, which have been widely recognized (42). In addition to 60
Taxol, other isoprenoids with therapeutic value include: artemisinin (anti-malarial), 61
lycopene (antioxidant), astaxanthin (antioxidant). 62
From the standpoint of harnessing this medicinal potential, production processes 63
may then take advantage of the native hosts. From one perspective, this approach 64
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
4
simplifies production efforts by utilizing a host that has been evolutionarily optimized for 65
compound formation. Attempts to produce Taxol have included semi-synthesis in which 66
a key intermediate harvested from the needles of the Yew tree is chemically converted 67
to the final active compound (24). More recently, plant cell culture has been used to 68
generate Taxol, and there is active research dedicated to characterizing and optimizing 69
this process (53). As mentioned, harvesting the full compound from the bark of the Yew 70
tree proved impractical, spurring the more environmentally-friendly and scale-able 71
semisynthetic and plant cell culture approaches. However, plant-derived production 72
processes, while taking advantage of native Taxol biosynthetic capabilities, are also 73
dictated by the intrinsic biological traits associated with the plant host (17). The 74
resulting cell cycle kinetics associated with plants will clearly fall behind the rapid growth 75
rates associated with microbial hosts like Escherichia coli or Saccharomyces cerevisiae. 76
As such, the classical heterologous production situation is presented: can the 77
biosynthesis of a complex therapeutic natural product be established in a technically 78
superior, yet evolutionarily unoptimized, alternative host? 79
Within the last 15 years, this question has been asked and pursued with 80
increasing frequency. Driven by the possibility of expediting and simplifying the 81
production of therapeutic isoprenoid compounds, heterologous biosynthesis has also 82
taken advantage of rapid technology development for candidate surrogate hosts. As a 83
result, a series of successful examples have emerged, prompting continued efforts to 84
apply and extend the technology. Each success story, however, must carefully address 85
the biosynthetic needs that are in-built within the original producers. In essence, 86
heterologous biosynthesis attempts to couple the medicinal value of therapeutic natural 87
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
5
products, like isoprenoids, with the engineering possibilities associated with the chosen 88
alternative hosts. This review will explain this process, featuring the recent application 89
of the approach towards early stage Taxol intermediates, and the emergence of 90
technical advances that have aided previous and will continue to aid future heterologous 91
biosynthetic attempts. 92
Heterologous Host Choice 93
Generally, the traits that define a promising heterologous host relate to technical 94
convenience and engineering potential. Growth speed and advanced molecular biology 95
protocols are an understated reason why E. coli and S. cerevisiae are commonly 96
selected as heterologous host options. In particular, the abundance of experimental 97
protocols associated with these hosts derived from the rapid growth kinetics associated 98
with each. Prior to heterologous biosynthetic applications, many experimental protocols 99
and tools were developed for recombinant protein production. As such, these same 100
tools have been adopted or modified for heterologous biosynthetic efforts, providing a 101
formidable arsenal of options to aid heterologous reconstitution of desired isoprenoid 102
pathways. 103
Another criterion for the selection of a heterologous host is the intrinsic ability to 104
support heterologous biosynthesis. This is the main advantage of basing production 105
upon a native host system; the original host will have the innate, evolutionarily optimized 106
ability to support biosynthesis. In choosing a heterologous host, the selection may be 107
biased by the potential of the alternative host to contribute to the biosynthetic effort, and 108
this potential must be weighed against growth rate advantages and engineering 109
opportunities. 110
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
6
In this regard, it is fortuitous that the most convenient heterologous host options 111
have the native metabolism required to support isoprenoid biosynthesis. E. coli, S. 112
cerevisiae, and Bacillus subtilis, arguably the most commonly used recombinant 113
microbial systems, each have the ability to generate isoprenoid compounds, although 114
none display the health-related properties associated with those isoprenoids from plant 115
or more fastidious microbial sources. Nonetheless, this in-built capability can be then 116
be utilized in the context of heterologous biosynthesis. 117
Table 1 summarizes the use of E. coli and S. cerevisiae in the heterologous 118
production of isoprenoid compounds. The literature featured in the table summarizes 119
efforts in establishing heterologous biosynthesis according to compound and host 120
organism. There have been several highly visible examples of successful compound 121
formation through both E. coli and S. cerevisiae, implying that both have the potential to 122
be viable production options. The choice may then be decided by compound-specific 123
requirements, host-specific potential to meet these requirements, and experimental 124
preference by the individual researcher. Interestingly, although the Bacillus genus has 125
an apparently robust ability to produce isoprene (34), only a few efforts have been made 126
to use the strain for isoprene or polyisoprene formation (68). 127
Substrate Provision, Pathway Selection, and Impact on Heterologous 128
Biosynthesis 129
There are two distinct metabolic pathways capable of providing the required 130
isopentenyl diphosphate (IPP) starting substrate for isoprenoid biosynthesis. The first 131
route, the mevalonate (MVA) pathway, was identified in the 1950s and completely 132
elucidated shortly thereafter (7, 44). The MVA pathway is primarily found in eukaryotes 133
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
7
and starts with the common intracellular metabolite acetyl-CoA which is converted 134
through a series of six reactions to IPP. As Figure 1 indicates, multiple routes have 135
been used to provide the acetoacetyl-CoA intermediate, including an acetoacetyl-CoA 136
synthase and an acetoacetyl-CoA ligase (22, 47). With regards to the heterologous 137
hosts previously referenced, the MVA pathway is natively found in S. cerevisiae. 138
E. coli possesses the alternative pathway for IPP formation which begins with a 139
molecule each of pyruvate and glyceraldehyde-3-phosphate. This route is referred to 140
as the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway and produces both IPP and 141
dimethylallyl diphosphate (DMAPP) (20, 35, 55). The MVA pathway includes an 142
essential isomerase to convert between IPP and DMAPP; whereas, the MEP pathway 143
isomerase is not required but such activity is used to alter the ratio between IPP and 144
DMAPP for the purpose of subsequent product formation. The MEP pathway features 145
seven steps, and both the MVA and MEP pathways require energy units (ATP and 146
CTP) and reducing equivalents (NADPH) during substrate generation (Figure 1). 147
Table 1 also summarizes the substrate provision pathways used in heterologous 148
production attempts. It is interesting to note that the MVA pathway has been 149
successfully implemented into E. coli despite the existence of the organism’s 150
endogenous MEP pathway. A primary reason given for this approach is the unknown 151
regulatory features associated with the native MEP pathway and how these might limit 152
subsequent engineering. As such, a completely heterologous pathway would then be 153
decoupled from native control and would be easier to alter for the purpose of supporting 154
eventual isoprenoid biosynthesis (39). Alternatively, the MEP pathway has been used 155
extensively and successfully during E. coli-based heterologous efforts. More recently, 156
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
8
studies have emerged that compare both substrate provision pathways during efforts to 157
generate a single isoprenoid compound using E. coli (45, 54). In both reports, the MVA 158
pathway supported higher isoprenoid titers under the specific conditions of the 159
experimental systems employed. As will be further discussed in the case of E. coli-160
derived Taxol intermediate biosynthesis, such head-to-head comparisons of the MVA 161
and MEP pathways can be difficult to interpret without an exhaustive effort to balance 162
and optimize precursor supply towards final isoprenoid biosynthesis. 163
Use of a heterologous substrate provision pathway has been successful in 164
supporting accompanying biosynthetic efforts and also helps to emphasize general 165
considerations when attempting heterologous biosynthesis. The reliance on a non-166
native pathway carries an initial requirement for eventual success: functional gene 167
expression. This would appear to be of little concern with a system native to E. coli, like 168
the MEP pathway. But a heterologous MVA pathway may pose the common problems 169
that have long plagued the use of E. coli for the production of recombinant proteins. 170
Namely, foreign gene expression elements may not transition to the new host. 171
However, thanks to the range of expression and control elements (promoter, operator, 172
ribosomal binding site, and termination sequences) identified and utilized for E. coli, 173
challenges in foreign gene expression initiation are becoming more manageable with a 174
key result being a greater frequency of successful final enzyme activity. Another 175
advance that has aided heterologous gene expression is the use of gene synthesis to 176
address codon bias between organisms. In this way, native codons associated with the 177
heterologous MVA pathway genes can be replaced with those optimal for E. coli. Such 178
technical advances have developed in parallel with heterologous biosynthesis and it is 179
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
9
reasonable to conclude that the latter has benefited from the former. Besides gene 180
expression, additional steps have also been useful in assuring functional enzyme 181
activity including the adjustment of culture temperature, the use of chaperonin proteins, 182
and the further modification of gene sequences to remove any foreign signaling or 183
localization amino acids (or in some cases, to add such sequences) (2, 39, 73). 184
The considerations of the previous paragraph accompany any heterologous 185
biosynthetic effort. As such, there is a lessened degree of alert when trying to provide 186
substrate provision through the endogenous MEP pathway. There have also been 187
stoichiometric and energetic arguments that the MEP pathway is capable of more 188
efficient substrate conversion (2, 74). However, there is the question of native utility 189
and how the evolutionary control elements associated with endogenous activity will 190
respond to genetic and metabolic engineering. For the purpose of isoprenoid 191
heterologous biosynthesis, either the native or heterologous substrate provision 192
pathways will potentially overlap with native regulatory or metabolic networks. This 193
awareness heightens the need for metabolic engineering, which will be further 194
discussed later in this review. 195
In the case of E. coli Taxol heterologous biosynthetic attempts, the MEP pathway 196
was selected for substrate provision (2). This choice was made, in part, due to the 197
previously mentioned potential for improved theoretical yield and as a result of a 198
growing number of examples in which the pathway had supported polyisoprene 199
formation (Table 1). However, the native chromosomal version of the MEP pathway 200
was incapable of supporting Taxol overproduction. Instead, based upon information 201
regarding MEP pathway bottleneck points (72), additional copies of the dxs, ispD, ispF, 202
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
10
and idi genes were introduced at a range of gene dosage levels. This particular 203
strategy was adopted because of wide experimental variation in resulting taxadiene 204
titers (the first dedicated intermediate in the Taxol pathway) without a systematic 205
approach to expressing either those genes associated with substrate provision or those 206
needed for eventual taxadiene formation. The results suggested the potential for high 207
titer biosynthesis, but a careful experimental design was required to assess and 208
establish the correct metabolic environment for consistent over-production. This was 209
accomplished by balancing the flux of carbon through both substrate provision (referred 210
to as the upstream pathway) and subsequent taxadiene formation (the downstream 211
pathway). There may very well be remaining inefficiencies with this system and the 212
MEP pathway, but this result demonstrated the potential of the MEP pathway to serve 213
high level isoprenoid biosynthesis in the context of heterologous taxadiene production. 214
The Formation of Polyisoprene Units 215
Prior to taxadiene biosynthesis, which is the result of a dedicated terpene 216
synthase, the polyisoprene unit geranylgeranyl diphosphate (GGPP) must be formed. 217
From IPP and DMAPP, polyisoprene units are generated through the consecutive 218
condensation of IPP to allylic diphosphate compounds (typically beginning with a 219
DMAPP unit). The resulting C10 (geranyl diphosphate [GPP]), C15 (farnesyl diphosphate 220
[FPP]), and C20 (geranylgeranyl diphosphate) chains are the basis for conversion by 221
additional enzymes into isoprenoid classes of monoterpenes, sesquiterpenes, and 222
diterpenes, respectively (57). E. coli has the native capability to produce FPP as a 223
result of the FPP synthase IspA (18). However, the cell accumulates only limited 224
amounts of GGPP (6, 28). 225
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
11
As a result, a GGPP synthase from Taxus canadensis was used to allow the 226
production of Taxol intermediates from E. coli (2, 23). Subject to the same design 227
parameters introduced above for heterologously expressed genes, the GGPP synthase 228
gene was synthesized with care to optimize codon usage for E. coli and to remove any 229
unneeded native plastidial insertion regions. This gene, in conjunction with a taxadiene 230
synthase and bottleneck MEP genes, was then tested over a wide expression range. 231
Chromosomal, low, medium, and higher gene dosages were implemented through 232
chromosomal integration or the use of plasmids with well-established copy number 233
values. Gene dosage was coupled to a range of promoters also varying in strength. As 234
a result, a comprehensive landscape of metabolic control was mapped to taxadiene 235
production titers and a maximum was identified for this particular system. The resulting 236
balance of upstream substrate provision and downstream biosynthetic pathways 237
resulted in a taxadiene titer of ~300 mg/L. Figure 2 depicts the steps involved with 238
heterologous isoprenoid biosynthesis featuring the Taxol case. 239
Post Biosynthesis: Protein, Metabolic, and Process Engineering 240
A heterologous biosynthetic attempt can be classified a success if any level of 241
production is accomplished. However, such success will not serve the larger objective 242
of viable mass production. Here, the choice of heterologous biosynthesis offers a 243
second level of engineering to improve upon initial titers, yields, and productivities. 244
Similar to the advantages afforded by the heterologous host during attempts at pathway 245
reconstitution, the engineering tools available with hosts like E. coli and S. cerevisiae 246
offer a number of engineering opportunities across scales. 247
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
12
At the protein level, specific enzymes within the precursor or polyisoprene 248
formation stages of biosynthesis have been targeted for improvements in final titer 249
values. The GGPPS enzyme responsible for initiating the downstream isoprenoid 250
formation process has been a frequent target for protein engineering strategies. Both 251
direct and random mutations have been used to alter or improve the capabilities of this 252
enzyme (37, 50, 65). In these studies (and numerous others), the engineering schemes 253
were aided or enabled by the colorimetric properties of carotenoid compounds. Other 254
groups have used dedicated foreign GGPPS enzymes to aid in a particular isoprenoid 255
biosynthetic effort (similar to the Taxol case). This is done to channel substrate flow to 256
the correct chain-length polyisoprene unit prior to subsequent isoprenoid formation 257
reactions (16, 66). 258
Metabolic engineering in the context of isoprenoid heterologous biosynthesis has 259
primarily been dedicated to the MEP or MVA pathways. Numerous studies have 260
focused on individual components of these pathways to identify and address potential 261
bottlenecks (and a comprehensive assessment of these studies is presented in the 262
reviews cited at the beginning of this article). With regards to the Taxol case, several 263
groups have optimized production of related compounds through chromosomal 264
modification of the MEP pathway (12, 72). This approach appears particularly well-265
suited for the native E. coli MEP pathway and has the potential to both minimize 266
metabolic burden and eliminate the need for plasmids as well as to maintain the 267
dynamic range of gene expression through variation in promoter strength. The well-268
curated metabolic backgrounds of hosts like E. coli and S. cerevisiae also offer the 269
application of computational modeling to predict and implement beneficial metabolic 270
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
13
changes. This has been demonstrated in a number of cases using stoichiometric 271
modeling of background metabolism to identify genes to be either deleted or over-272
expressed for the purpose of improving final titer values (3, 13, 61, 62). The advantage 273
of such models is the ability to identify alternative native metabolism contributing to or 274
detracting from heterologous production. In a recent effort focused on identifying gene 275
over-expression targets capable of improving E. coli-derived taxadiene, the ppk, sthA, 276
purN, and folD targets were computationally and experimentally over-expressed with 277
three out of four targets resulting in improved taxadiene levels (8). Finally, given the 278
native and heterologous components associated with or influencing total biosynthesis, 279
studies have begun to use various -omics technologies as another route to characterize 280
and potentially engineer the biosynthetic process (9, 32, 49, 51). 281
Hosts like E. coli also have well-developed bioreactor protocols available to boost 282
cell density and volumetric productivity (36, 60, 69). These strategies were applied in 283
the Taxol case to build upon the metabolic engineering of upstream and downstream 284
pathway balances to push final taxadiene titers to >1 g/L. Other efforts in isoprenoid 285
heterologous biosynthesis have shown similarly impressive final titers (52, 63). In 286
summary, E. coli and other well-characterized heterologous host systems offer more 287
than excellent molecular biology tools available to facilitate pathway transfer and 288
reconstitution. The new hosts have been the primary platforms for bio-product protein, 289
metabolic, and process engineering for the last 3 decades and this knowledge base is 290
supportive of the product development strategies that are now being applied to 291
isoprenoid heterologous biosynthesis. 292
Complete Isoprenoid Biosynthesis and Heterologous Host Bias 293
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
14
This review has primarily focused on the logic and steps needed to establish 294
polyisoprene formation (i.e., GGPP) without providing the detailed reactions that support 295
full isoprenoid compound formation (i.e., Taxol). These reactions will be unique for 296
each individual isoprenoid compound, but biosynthetic similarities include multiple 297
reaction steps that modify a polyisoprene frame. Oftentimes, these reactions involve 298
dedicated cytochrome P450 hydroxylase enzymes. The Taxol compound is a good 299
example in which 17 steps are predicted to convert taxadiene to the final compound 300
with the majority of these transformations catalyzed by P450 enzymes (14, 27). 301
Establishing full heterologous Taxol biosynthesis will require the same dedication 302
that has led to the success of high-level E. coli taxadiene production. Namely, a 303
combination of heterologous and pathway metabolic engineering will be required to first 304
establish Taxol intermediate and full compound formation followed by efforts to 305
maximize specific and volumetric productivities. As before, the transfer of genes from 306
plant sources will require special attention to establish active gene expression, both 307
from a mechanistic standpoint (through gene synthesis and expression technology) and 308
post-expression functionality (as a by-product of protein localization, folding, and 309
activity). As a key example, in the same study to balance substrate and biosynthetic 310
requirements for taxadiene overproduction, the resulting E. coli strain was engineered to 311
generate the subsequent Taxol pathway intermediate taxadien-5α-ol, catalyzed by a 312
cytochrome P450 enzyme (2). As for the case of the foreign GGPPS and taxadiene 313
synthase, the P450 gene and that for an accompanying NADPH reductase was 314
synthesized and altered to maximize gene expression, cellular protein localization 315
(through removing unwanted and introducing beneficial localization and fusion 316
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
15
sequences), and final enzyme activity. Even so, the 58 mg/L titer for the taxadien-5α-ol 317
product was significantly below the optimized value obtained for taxadiene. Such a 318
result then points to renewed efforts in pathway metabolic engineering to boost yields. 319
Furthermore, this process must repeatedly take place to reach the Taxol endpoint or a 320
pathway intermediate capable of being converted semisynthetically to the final product. 321
An additional complication is that not all of the pathway enzymes have been identified; 322
thus, additional research into pathway elucidation or substitution must occur prior to 323
next-step pathway heterologous production. The positive viewpoint is that there is now 324
precedence to support future attempts at heterologously generating and overproducing 325
downstream Taxol pathway intermediates. However, this venture will no doubt test the 326
current limits of metabolic engineering and, most likely, will require advances in current 327
technology to obtain ultimate success. Still, considering the advances that have 328
occurred to this point and the implications in overall success, there is reason for 329
optimism in pursuing such a challenging objective. 330
Finally, Table 2 presents a literature analysis of the heterologous production of 331
recent and common isoprenoid compounds. Several points can be made about the 332
results. First, there has clearly been significantly more research dedicated to the 333
production of carotenoid compounds. This is due in part to the earlier sequence 334
availability of such pathways which then spurred the first heterologous attempts (29, 43, 335
48, 56, 59). Since the first heterologous publications, the majority of the remaining 336
studies have been dedicated to metabolic engineering and production optimization. As 337
mentioned previously, for the carotenoid compounds, engineering studies are aided 338
greatly by the colorimetric nature of the final compounds, most-likely bolstering the 339
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
16
associated number of research efforts. There is a drop in research citations with 340
increasing complexity of the carotenoid compound (from lycopene to zeaxanthin to 341
astaxanthin). This again may reflect the order or availability associated with pathway 342
knowledge, but it may also reflect the difficulty associated with introducing downstream 343
steps dedicated to polyisoprene adornment. It should also be stated that the above 344
carotenoid trend holds for E. coli but not for S. cerevisiae. It is interesting to observe 345
such a disparity between these two technically convenient hosts within the carotenoid 346
family of isoprenoids. Our speculation is that the biosynthetic requirements did not 347
favor the use of either E. coli or S. cerevisiae, and as such, the research community 348
then relied on E. coli which would generally be considered more technically convenient 349
than S. cerevisiae. 350
Second, the more recent trends associated with Taxol and artemisinin 351
intermediates show a more even distribution of the E. coli and S. cerevisiae 352
heterologous hosts. Unlike the carotenoid compounds, these studies have only begun 353
in earnest over the last 5-10 years. But the more prevalent use of S. cerevisiae may 354
point to the additional complexity associated with both final compounds. The yeast 355
cellular background will obviously have more commonalities with the plant cell structure 356
when compared to E. coli. As such, this may greatly facilitate the introduction of the 357
downstream pathways required for final compound formation. Essentially, the nature 358
and complexity of the pathway then biases the use of a yeast system and this may 359
reflect the early trends in heterologous host choice. Time will tell if the final artemisinin 360
or Taxol (or other complex isoprenoid) compounds can be completely synthesized 361
heterologously and which host will serve this purpose. 362
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
17
363
Acknowledgements 364
The authors wish to recognize support from the Milheim Foundation (2006-17) and the 365
NIH (GM085323) for projects related to heterologous Taxol biosynthesis. 366
367
References 368
1. Ajikumar, P. K., K. Tyo, S. Carlsen, O. Mucha, T. H. Phon, and G. Stephanopoulos. 369 2008. Terpenoids: opportunities for biosynthesis of natural product drugs using engineered 370 microorganisms. Mol Pharm 5:167-190. 371
2. Ajikumar, P. K., W. H. Xiao, K. E. Tyo, Y. Wang, F. Simeon, E. Leonard, O. Mucha, T. H. 372 Phon, B. Pfeifer, and G. Stephanopoulos. 2010. Isoprenoid pathway optimization for Taxol 373 precursor overproduction in Escherichia coli. Science 330:70-74. 374
3. Alper, H., Y. S. Jin, J. F. Moxley, and G. Stephanopoulos. 2005. Identifying gene targets 375 for the metabolic engineering of lycopene biosynthesis in Escherichia coli. Metab Eng 7:155-376 164. 377
4. Anthony, J. R., L. C. Anthony, F. Nowroozi, G. Kwon, J. D. Newman, and J. D. Keasling. 378 2009. Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia 379 coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metab Eng 11:13-380 19. 381
5. Arbuck, S. G. and B.A. Blaylock. 1995. Taxol: clinical results and current issues in 382 development. CRC Press, Boca Raton. 383
6. Asai, K., S. Fujisaki, Y. Nishimura, T. Nishino, K. Okada, T. Nakagawa, M. Kawamukai, 384 and H. Matsuda. 1994. The identification of Escherichia coli ispB (cel) gene encoding the 385 octaprenyl diphosphate synthase. Biochemical and Biophysical Research Communications 386 202:340-345. 387
7. Bloch, K. 1992. Sterol molecule: structure, biosynthesis, and function. Steroids 57:378-383. 388 8. Boghigian, B. A., J. Armando, D. Salas, and B. A. Pfeifer. 2011. Computational 389
identification of gene over-expression targets for metabolic engineering of taxadiene 390 production. Appl Microbiol Biotechnol. In press. 391
9. Boghigian, B. A., D. Salas, P. K. Ajikumar, G. Stephanopoulos, and B. A. Pfeifer. 2011. 392 Analysis of heterologous taxadiene production in K- and B-derived Escherichia coli. Appl 393 Microbiol Biotechnol. In press. 394
10. Chang, M. C., R. A. Eachus, W. Trieu, D. K. Ro, and J. D. Keasling. 2007. Engineering 395 Escherichia coli for production of functionalized terpenoids using plant P450s. Nat Chem Biol 396 3:274-277. 397
11. Chang, M. C., and J. D. Keasling. 2006. Production of isoprenoid pharmaceuticals by 398 engineered microbes. Nat Chem Biol 2:674-681. 399
12. Chiang, C. J., P. T. Chen, and Y. P. Chao. 2008. Replicon-free and markerless methods for 400 genomic insertion of DNAs in phage attachment sites and controlled expression of 401 chromosomal genes in Escherichia coli. Biotechnol Bioeng 101:985-995. 402
13. Choi, H. S., S. Y. Lee, T. Y. Kim, and H. M. Woo. 2010. In silico identification of gene 403 amplification targets for improvement of lycopene production. Appl Environ Microbiol 404 76:3097-3105. 405
14. Croteau, R., R. E. Ketchum, R. M. Long, R. Kaspera, and M. R. Wildung. 2006. Taxol 406 biosynthesis and molecular genetics. Phytochemistry Reviews 5:75-97. 407
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
18
15. Dejong, J. M., Y. Liu, A. P. Bollon, R. M. Long, S. Jennewein, D. Williams, and R. B. 408 Croteau. 2006. Genetic engineering of taxol biosynthetic genes in Saccharomyces 409 cerevisiae. Biotechnol Bioeng 93:212-224. 410
16. Engels, B., P. Dahm, and S. Jennewein. 2008. Metabolic engineering of taxadiene 411 biosynthesis in yeast as a first step towards Taxol (Paclitaxel) production. Metab Eng 412 10:201-206. 413
17. Frense, D. 2007. Taxanes: perspectives for biotechnological production. Appl Microbiol 414 Biotechnol 73:1233-1240. 415
18. Fujisaki, S., H. Hara, Y. Nishimura, K. Horiuchi, and T. Nishino. 1990. Cloning and 416 nucleotide sequence of the ispA gene responsible for farnesyl diphosphate synthase activity 417 in Escherichia coli. J Biochem 108:995-1000. 418
19. Gershenzon, J., and N. Dudareva. 2007. The function of terpene natural products in the 419 natural world. Nat Chem Biol 3:408-414. 420
20. Grawert, T., M. Groll, F. Rohdich, A. Bacher, and W. Eisenreich. 2011. Biochemistry of 421 the non-mevalonate isoprenoid pathway. Cellular and Molecular Life Sciences 68:3797-422 3814. 423
21. Harada, H., and N. Misawa. 2009. Novel approaches and achievements in biosynthesis of 424 functional isoprenoids in Escherichia coli. Appl Microbiol Biotechnol 84:1021-1031. 425
22. Harada, H., F. Yu, S. Okamoto, T. Kuzuyama, R. Utsumi, and N. Misawa. 2009. Efficient 426 synthesis of functional isoprenoids from acetoacetate through metabolic pathway-engineered 427 Escherichia coli. Appl Microbiol Biotechnol 81:915-925. 428
23. Hefner, J., R. E. Ketchum, and R. Croteau. 1998. Cloning and functional expression of a 429 cDNA encoding geranylgeranyl diphosphate synthase from Taxus canadensis and 430 assessment of the role of this prenyltransferase in cells induced for taxol production. Arch 431 Biochem Biophys 360:62-74. 432
24. Holton, R. A., Biediger, R.J., Boatman, P.D. 1995. Semisynthesis of taxol and taxotere. 433 CRC Press, Boca Raton. 434
25. Horwitz, S. B. 1994. How to make taxol from scratch. Nature 367:593-594. 435 26. Huang, Q., C. A. Roessner, R. Croteau, and A. I. Scott. 2001. Engineering Escherichia coli 436
for the synthesis of taxadiene, a key intermediate in the biosynthesis of taxol. Bioorg Med 437 Chem 9:2237-2242. 438
27. Jennewein, S., M. R. Wildung, M. Chau, K. Walker, and R. Croteau. 2004. Random 439 sequencing of an induced Taxus cell cDNA library for identification of clones involved in 440 Taxol biosynthesis. Proc Natl Acad Sci U S A 101:9149-9154. 441
28. Kainou, T., K. Okada, K. Suzuki, T. Nakagawa, H. Matsuda, and M. Kawamukai. 2001. 442 Dimer formation of octaprenyl-diphosphate synthase (IspB) is essential for chain length 443 determination of ubiquinone. J Biol Chem 276:7876-7883. 444
29. Kajiwara, S., T. Kakizono, T. Saito, K. Kondo, T. Ohtani, N. Nishio, S. Nagai, and N. 445 Misawa. 1995. Isolation and functional identification of a novel cDNA for astaxanthin 446 biosynthesis from Haematococcus pluvialis, and astaxanthin synthesis in Escherichia coli. 447 Plant Molecular Biology 29:343-352. 448
30. Kirby, J., and J. D. Keasling. 2009. Biosynthesis of plant isoprenoids: perspectives for 449 microbial engineering. Annu Rev Plant Biol 60:335-355. 450
31. Kirby, J., and J. D. Keasling. 2008. Metabolic engineering of microorganisms for isoprenoid 451 production. Nat Prod Rep 25:656-661. 452
32. Kizer, L., D. J. Pitera, B. F. Pfleger, and J. D. Keasling. 2008. Application of functional 453 genomics to pathway optimization for increased isoprenoid production. Appl Environ 454 Microbiol 74:3229-3241. 455
33. Klein-Marcuschamer, D., P. K. Ajikumar, and G. Stephanopoulos. 2007. Engineering 456 microbial cell factories for biosynthesis of isoprenoid molecules: beyond lycopene. Trends 457 Biotechnol 25:417-424. 458
34. Kuzma, J., M. Nemecek-Marshall, W. H. Pollock, and R. Fall. 1995. Bacteria produce the 459 volatile hydrocarbon isoprene. Curr Microbiol 30:97-103. 460
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
19
35. Kuzuyama, T., and H. Seto. 2003. Diversity of the biosynthesis of the isoprene units. Nat 461 Prod Rep 20:171-183. 462
36. Lee, S. Y. 1996. High cell-density culture of Escherichia coli. Trends Biotechnol 14:98-105. 463 37. Leonard, E., P. K. Ajikumar, K. Thayer, W. H. Xiao, J. D. Mo, B. Tidor, G. 464
Stephanopoulos, and K. L. Prather. 2010. Combining metabolic and protein engineering of 465 a terpenoid biosynthetic pathway for overproduction and selectivity control. Proc Natl Acad 466 Sci U S A 107:13654-13659. 467
38. Lindahl, A. L., M. E. Olsson, P. Mercke, O. Tollbom, J. Schelin, M. Brodelius, and P. E. 468 Brodelius. 2006. Production of the artemisinin precursor amorpha-4,11-diene by engineered 469 Saccharomyces cerevisiae. Biotechnol Lett 28:571-580. 470
39. Martin, V. J., D. J. Pitera, S. T. Withers, J. D. Newman, and J. D. Keasling. 2003. 471 Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat 472 Biotechnol 21:796-802. 473
40. Maury, J., M. A. Asadollahi, K. Moller, A. Clark, and J. Nielsen. 2005. Microbial 474 isoprenoid production: an example of green chemistry through metabolic engineering. Adv 475 Biochem Eng Biotechnol 100:19-51. 476
41. McCaskill, D., and R. Croteau. 1997. Prospects for the bioengineering of isoprenoid 477 biosynthesis. Adv Biochem Eng Biotechnol 55:107-146. 478
42. McGarvey, D. J., and R. Croteau. 1995. Terpenoid metabolism. Plant Cell 7:1015-1026. 479 43. Misawa, N., M. Nakagawa, K. Kobayashi, S. Yamano, Y. Izawa, K. Nakamura, and K. 480
Harashima. 1990. Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by 481 functional analysis of gene products expressed in Escherichia coli. J Bacteriol 172:6704-482 6712. 483
44. Miziorko, H. M. 2011. Enzymes of the mevalonate pathway of isoprenoid biosynthesis. Arch 484 Biochem Biophys 505:131-143. 485
45. Morrone, D., L. Lowry, M. K. Determan, D. M. Hershey, M. Xu, and R. J. Peters. 2010. 486 Increasing diterpene yield with a modular metabolic engineering system in E. coli: 487 comparison of MEV and MEP isoprenoid precursor pathway engineering. Appl Microbiol 488 Biotechnol 85:1893-1906. 489
46. Muntendam, R., E. Melillo, A. Ryden, and O. Kayser. 2009. Perspectives and limits of 490 engineering the isoprenoid metabolism in heterologous hosts. Appl Microbiol Biotechnol 491 84:1003-1019. 492
47. Okamura, E., T. Tomita, R. Sawa, M. Nishiyama, and T. Kuzuyama. 2010. Unprecedented 493 acetoacetyl-coenzyme A synthesizing enzyme of the thiolase superfamily involved in the 494 mevalonate pathway. Proc Natl Acad Sci U S A 107:11265-11270. 495
48. Perry, K. L., T. A. Simonitch, K. J. Harrison-Lavoie, and S. T. Liu. 1986. Cloning and 496 regulation of Erwinia herbicola pigment genes. J Bacteriol 168:607-612. 497
49. Redding-Johanson, A. M., T. S. Batth, R. Chan, R. Krupa, H. L. Szmidt, P. D. Adams, J. 498 D. Keasling, T. S. Lee, A. Mukhopadhyay, and C. J. Petzold. 2011. Targeted proteomics 499 for metabolic pathway optimization: application to terpene production. Metab Eng 13:194-500 203. 501
50. Reiling, K. K., Y. Yoshikuni, V. J. Martin, J. Newman, J. Bohlmann, and J. D. Keasling. 502 2004. Mono and diterpene production in Escherichia coli. Biotechnol Bioeng 87:200-212. 503
51. Ro, D. K., M. Ouellet, E. M. Paradise, H. Burd, D. Eng, C. J. Paddon, J. D. Newman, and 504 J. D. Keasling. 2008. Induction of multiple pleiotropic drug resistance genes in yeast 505 engineered to produce an increased level of anti-malarial drug precursor, artemisinic acid. 506 BMC Biotechnology 8:83. 507
52. Ro, D. K., E. M. Paradise, M. Ouellet, K. J. Fisher, K. L. Newman, J. M. Ndungu, K. A. 508 Ho, R. A. Eachus, T. S. Ham, J. Kirby, M. C. Chang, S. T. Withers, Y. Shiba, R. Sarpong, 509 and J. D. Keasling. 2006. Production of the antimalarial drug precursor artemisinic acid in 510 engineered yeast. Nature 440:940-943. 511
53. Roberts, S. C. 2007. Production and engineering of terpenoids in plant cell culture. Nat 512 Chem Biol 3:387-395. 513
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
20
54. Rodriguez-Villalon, A., J. Perez-Gil, and M. Rodriguez-Concepcion. 2008. Carotenoid 514 accumulation in bacteria with enhanced supply of isoprenoid precursors by upregulation of 515 exogenous or endogenous pathways. J Biotechnol 135:78-84. 516
55. Rohmer, M. 1999. The discovery of a mevalonate-independent pathway for isoprenoid 517 biosynthesis in bacteria, algae and higher plants. Nat Prod Rep 16:565-574. 518
56. Ruther, A., N. Misawa, P. Boger, and G. Sandmann. 1997. Production of zeaxanthin in 519 Escherichia coli transformed with different carotenogenic plasmids. Appl Microbiol Biotechnol 520 48:162-167. 521
57. Ruzicka, L. 1953. The isoprene rule and the biogenesis of terpenic compounds. Experientia 522 9:357-367. 523
58. Sacchettini, J. C., and C. D. Poulter. 1997. Creating isoprenoid diversity. Science 524 277:1788-1789. 525
59. Sandmann, G., W. S. Woods, and R. W. Tuveson. 1990. Identification of carotenoids in 526 Erwinia herbicola and in a transformed Escherichia coli strain. FEMS Microbiol Lett 59:77-82. 527
60. Shiloach, J., and R. Fass. 2005. Growing E. coli to high cell density--a historical perspective 528 on method development. Biotechnol Adv 23:345-357. 529
61. Suthers, P. F., A. P. Burgard, M. S. Dasika, F. Nowroozi, S. Van Dien, J. D. Keasling, 530 and C. D. Maranas. 2007. Metabolic flux elucidation for large-scale models using 13C 531 labeled isotopes. Metab Eng 9:387-405. 532
62. Suthers, P. F., Y. J. Chang, and C. D. Maranas. 2010. Improved computational 533 performance of MFA using elementary metabolite units and flux coupling. Metab Eng 534 12:123-128. 535
63. Tsuruta, H., C. J. Paddon, D. Eng, J. R. Lenihan, T. Horning, L. C. Anthony, R. 536 Regentin, J. D. Keasling, N. S. Renninger, and J. D. Newman. 2009. High-level 537 production of amorpha-4,11-diene, a precursor of the antimalarial agent artemisinin, in 538 Escherichia coli. PLoS One 4:e4489. 539
64. Verwaal, R., J. Wang, J. P. Meijnen, H. Visser, G. Sandmann, J. A. van den Berg, and A. 540 J. van Ooyen. 2007. High-level production of beta-carotene in Saccharomyces cerevisiae by 541 successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous. 542 Appl Environ Microbiol 73:4342-4350. 543
65. Wang, C., M. K. Oh, and J. C. Liao. 2000. Directed evolution of metabolically engineered 544 Escherichia coli for carotenoid production. Biotechnol Prog 16:922-926. 545
66. Wang, C. W., M. K. Oh, and J. C. Liao. 1999. Engineered isoprenoid pathway enhances 546 astaxanthin production in Escherichia coli. Biotechnol Bioeng 62:235-241. 547
67. Withers, S. T., and J. D. Keasling. 2007. Biosynthesis and engineering of isoprenoid small 548 molecules. Appl Microbiol Biotechnol 73:980-990. 549
68. Xue, J., and B. K. Ahring. 2011. Enhancing isoprene production by genetic modification of 550 the 1-deoxy-d-xylulose-5-phosphate pathway in Bacillus subtilis. Appl Environ Microbiol 551 77:2399-2405. 552
69. Yamane, T., Shimizu, S. 1984. Fed-batch techniques in microbial processes. Adv. Biochem. 553 Eng. 30:147-194. 554
70. Yamano, S., T. Ishii, M. Nakagawa, H. Ikenaga, and N. Misawa. 1994. Metabolic 555 engineering for production of beta-carotene and lycopene in Saccharomyces cerevisiae. 556 Biosci Biotechnol Biochem 58:1112-1114. 557
71. Yoon, S. H., Y. M. Lee, J. E. Kim, S. H. Lee, J. H. Lee, J. Y. Kim, K. H. Jung, Y. C. Shin, 558 J. D. Keasling, and S. W. Kim. 2006. Enhanced lycopene production in Escherichia coli 559 engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from 560 mevalonate. Biotechnol Bioeng 94:1025-1032. 561
72. Yuan, L. Z., P. E. Rouviere, R. A. Larossa, and W. Suh. 2006. Chromosomal promoter 562 replacement of the isoprenoid pathway for enhancing carotenoid production in E. coli. Metab 563 Eng 8:79-90. 564
73. Zhang, H., B. A. Boghigian, J. Armando, and B. A. Pfeifer. 2011. Methods and options for 565 the heterologous production of complex natural products. Nat Prod Rep 28:125-151. 566
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
21
74. Zhao, Y., J. Yang, B. Qin, Y. Li, Y. Sun, S. Su, and M. Xian. 2011. Biosynthesis of 567 isoprene in Escherichia coli via methylerythritol phosphate (MEP) pathway. Appl Microbiol 568 Biotechnol 90:1915-1922. 569
570
571
Figure Legends 572
Figure 1. The mevalonate (MVA) and 2C-methyl-D-erythritol-4-phosphate (MEP) 573
polyisoprene substrate provision pathways. Those enzymes that have been targeted 574
during the course of metabolic engineering studies for improved Taxol intermediate 575
titers have been circled. Specific E. coli enzyme designations have been included in 576
parentheses. AACS, acetoacetyl-CoA synthase; AACT, acetoacetyl-CoA thiolase; 577
AACL, acetoacetyl-CoA ligase; HMGS, hydroxymethylglutaryl (HMG)-CoA synthase; 578
HMGR, HMG-CoA reductase; MVK, mevalonate kinase; PMK, phosphomevalonate 579
kinase; PMD, mevalonate pyrophosphate decarboxylase; IDI, isopentenyl diphosphate 580
isomerase; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; G3P, 581
glyceraldehyde-3-phosphate; DXS, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase; 582
DXR, DXP reductase; MEP, 2-C-methyl-D-erythritol 4-phosphate; CMS, 4-583
diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) synthase; CMK, CDP-ME kinase; 584
CDP-MEP, CDP-ME 2-phosphate; MCS, 2C-methyl-D-erythritol 2,4-cyclodiphosphate 585
(MEcPP) synthase; HDS, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP) 586
synthase; HDR, HMBPP reductase. 587
588
Figure 2. Heterologous taxadiene design, implementation, and optimization. Strain 589
MG1655 was engineered for plasmid stabilization (ΔrecAΔendA) and to support the T7 590
promoter (DE3). Having been previously identified and sequenced, the heterologous 591
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
22
geranylgeranyl diphosphate synthase (ggpps) and taxadiene synthase (txs) genes were 592
synthesized, codon optimized, and truncated to remove native plastidial integration 593
regions before being introduced via plasmid. Both native bottleneck MEP genes and 594
the heterologous genes were designed for a range of expression levels through 595
modifications to promoter (black circle preceding operon) strength (trc, T5, and T7) and 596
gene dosage. Upstream and downstream pathway balancing allowed optimized 597
taxadiene titer levels. 598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
23
Tables 615
Table 1. Representative (not comprehensive) examples of heterologously produced 616
isoprenoids, the host cell chosen, and the supporting substrate provision pathway 617
utilized. Also included are related product titers and engineering steps that influenced 618
subsequent studies or titer improvements. 619
COMPOUND HETEROLOGOUS HOST
SUBSTRATE PROVISION PATHWAY
FINAL TITER (INTERMEDIATE
OR FINAL COMPOUND)
PATHWAY ENGINEERING
STEP TO ENABLE OR
OVERPRODUCE PRODUCT
REFERENCES
Taxol E. coli MEP 1.3 mg/L (taxadiene)
Overexpression of dxs and idi
(26)
300 mg/L (taxadiene)
58 mg/L (taxadiene-5α-ol)
Overexpression and variation of
copy number and promoter of dxs,
idi, ispD, and ispF
(2)
S. cerevisiae MVA 1.0 mg/L (taxadiene) 0.025 mg/L
(taxadiene-5α-ol)
None (15)
8.7 mg/L (taxadiene)
Overexpression of a truncated HMG-CoA reductase;
reduction of competing steroid
pathway
(16)
Artemisinin E. coli MVA 155 mg/L (amorphadiene;
initial titer) 1,084 mg/L*
(amorphadiene; optimized titer)
Introduction and overexpression of
MVA pathway; foreign gene
codon optimization;
promoter variation; plasmid copy
number variation; specific
overexpression of mevalonate kinase
(39) (4)
105 mg/L (artemisinic acid)
Overexpression of MVA pathway; foreign gene
codon
(10)
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
24
optimization; promoter variation
S. cerevisiae MVA 0.6 mg/L None (38)
153 mg/L (amorphadiene)
32 mg/L (artemisinic acid)
Overexpression of a truncated HMG-CoA reductase;
reduction of competing steroid
pathway
(52)
Carotenoids E. coli MEP 6 mg/g DCW (β-carotene)
Overexpression of dxs, ispD, ispF, ispB, and idi.
(72)
MVA 22 mg/g DCW (lycopene)
Overexpression of mvaK1 (mvk), mvaK2 (pmk),
mvaD (pmd), and idi
(71)
12.5 mg/g DCW (lycopene)
Overexpression of MVA pathway with acetoacetyl-CoA ligase introduced
(21)
S. cerevisiae MVA 0.113 mg/g DCW (lycopene)
None (70)
5.9 mg/g DCW (β-carotene)
Overexpression of a truncated HMG-
CoA reductase and endogenous geranylgeranyl
diphosphate synthase
(64)
*Estimated based upon an OD600 value of 3.7 (39); actual value reported is 293 mg/L/OD600. 620
DCW-dry cell weight 621
622
623
624
625
626
627
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
25
Table 2. Literature analysis of isoprenoid compounds generated heterologously. 628
Search conducted (10/26/2011) using the PubMed database with either “coli” or 629
“cerevisiae” in conjunction with the molecules below in parentheses. The numbers 630
presented include both primary research and review articles and may include articles 631
not directly related to heterologous biosynthesis. However, the results illustrate the 632
differences in compound-specific host choice and the associated research volume since 633
heterologous biosynthesis initiated. 634
COMPOUND HETEROLOGOUS HOST
ARTICLE NUMBER
FIRST REPORT
Taxol (taxadiene) E. coli 11 2001 S. cerevisiae 9 2006
Artemisinin (amorphadiene)
E. coli 24 2000
S. cerevisiae 18 2006
Carotenoid (lycopene) E. coli 158 1986 S. cerevisiae 9 1994
Carotenoid (zeaxanthin) E. coli 64 1990 S. cerevisiae 2 2009
Carotenoid (astaxanthin) E. coli 35 1995 S. cerevisiae 4 2009
635
636
637
638
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
yl CoA malonyl CoA acetyl CoA
AACTAACS
AACL
ATP/CoA
HMGS
acetoacetyl-CoA
acetyl-CoA
toacetate
HMGR
HMG-CoA
2 NADPH
mevalonatemevalonate
MVKATP
5-phosphomevalonate
PMKATP
5-pyrophosphomevalonate
OPPIPPAPP
PMD
IDI
A pyruvate G3P
DXS
DXP DXRNADPH
MEPCMS (IspD)CTP
CDP-ME
CMKATP
CDP-MEP
MCS (IspF)
MEcPP
HMBPP
HDS
IPPDMAPP
IDI
HDRNADPH
on May 21, 2020 by guest
http://aem.asm
.org/D
ownloaded from
E. coli K12MG1655ΔrecAΔendADE3
dxs idi ispD F ggpps txs
Glucose DXS IspD IspF
Upstream Pathway
IPP
ggpps txs
IDI GGPPS
GGPPTaxadiene
DMAPP
TXS
y DownstreamPathwayy on M
ay 21, 2020 by guesthttp://aem
.asm.org/
Dow
nloaded from
