Using chemobiosynthesis and synthetic mini-polyketide synthases to produce pharmaceutical intermediates in Escherichia coli.
ABSTRACT Recombinant microbial whole-cell biocatalysis is a valuable approach for producing enantiomerically pure intermediates for the synthesis of complex molecules. Here, we describe a method to produce polyketide intermediates possessing multiple stereogenic centers by combining chemobiosynthesis and engineered mini-polyketide synthases (PKSs). Chemobiosynthesis allows the introduction of unnatural moieties, while a library of synthetic bimodular PKSs expressed from codon-optimized genes permits the introduction of a variety of ketide units. To validate the approach, intermediates for the synthesis of trans-9,10-dehydroepothilone D were generated. The designer molecules obtained have the potential to greatly reduce the manufacturing cost of epothilone analogues, thus facilitating their commercial development as therapeutic agents.
- [Show abstract] [Hide abstract]
ABSTRACT: Microbes from two of the three domains of life, the Prokarya, and Eukarya, continue to serve as rich sources of structurally complex chemical scaffolds that have proven to be essential for the development of anticancer therapeutics. This review describes only a handful of exemplary natural products and their derivatives as well as those that have served as elegant blueprints for the development of novel synthetic structures that are either currently in use or in clinical or preclinical trials together with some of their earlier analogs in some cases whose failure to proceed aided in the derivation of later compounds. In every case, a microbe has been either identified as the producer of secondary metabolites or speculated to be involved in the production via symbiotic associations. Finally, rapidly evolving next-generation sequencing technologies have led to the increasing availability of microbial genomes. Relevant examples of genome mining and genetic manipulation are discussed, demonstrating that we have only barely scratched the surface with regards to harnessing the potential of microbes as sources of new pharmaceutical leads/agents or biological probes.Journal of Industrial Microbiology 09/2013; · 1.80 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The efficient production of functional proteins in heterologous hosts is one of the major bases of modern biotechnology. Unfortunately, many genes are difficult to express outside their original context. Due to their apparent "silent" nature, synonymous codon substitutions have long been thought to be trivial. In recent years, this dogma has been refuted by evidence that codon replacement can have a significant impact on gene expression levels and protein folding. In the past decade, considerable advances in the speed and cost of gene synthesis have facilitated the complete redesign of entire gene sequences, dramatically improving the likelihood of high protein expression. This technology significantly impacts the economic feasibility of microbial-based biotechnological processes by, for example, increasing the volumetric productivities of recombinant proteins or facilitating the redesign of novel biosynthetic routes for the production of metabolites. This review discusses the current applications of this technology, particularly those regarding the production of small molecules and industrially relevant recombinant enzymes. Suggestions for future research and potential uses are provided as well.Frontiers in Microbiology 01/2014; 5:21. · 3.90 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Engineering modular polyketide synthases (PKSs) has the potential to be an effective methodology to produce existing and novel chemicals. However, this potential has only just begun to be realized. We propose the adoption of an iterative design-build-test-learn paradigm to improve PKS engineering. We suggest methods to improve engineered PKS design by learning from laboratory-based selection; adoption of DNA design software and automation to build constructs and libraries more easily; tools for the expression of engineered proteins in a variety of heterologous hosts; and mass spectrometry-based high-throughput screening methods. Finally, lessons learned during iterations of the design-build-test-learn cycle can serve as a knowledge base for the development of a single retrosynthesis algorithm usable by both PKS experts and non-experts alike.Current opinion in biotechnology 05/2014; 30C:32-39. · 7.82 Impact Factor
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2010, p. 5221–5227
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 15
Using Chemobiosynthesis and Synthetic Mini-Polyketide Synthases To
Produce Pharmaceutical Intermediates in Escherichia coli?†
Hugo G. Menzella,* John R. Carney,‡ Yong Li,§ and Daniel V. Santi¶
Kosan Biosciences Inc., Hayward, California 94545
Received 8 December 2009/Accepted 29 May 2010
Recombinant microbial whole-cell biocatalysis is a valuable approach for producing enantiomerically pure
intermediates for the synthesis of complex molecules. Here, we describe a method to produce polyketide
intermediates possessing multiple stereogenic centers by combining chemobiosynthesis and engineered mini-
polyketide synthases (PKSs). Chemobiosynthesis allows the introduction of unnatural moieties, while a library
of synthetic bimodular PKSs expressed from codon-optimized genes permits the introduction of a variety of
ketide units. To validate the approach, intermediates for the synthesis of trans-9,10-dehydroepothilone D were
generated. The designer molecules obtained have the potential to greatly reduce the manufacturing cost of
epothilone analogues, thus facilitating their commercial development as therapeutic agents.
Whole-cell biocatalysis is a rapidly developing technology
used to assist in developing synthetic routes to complex mole-
cules in the pharmaceutical industry. The exquisite regio- and
stereoselectivity of enzymes allows the facile introduction of
stereogenic centers with complete enantiomeric control, which
may result in a significant reduction in the number of synthesis
steps and therefore the final production cost (2, 14, 18). In
addition, biocatalysis is one of the greenest technologies cur-
rently available; since the protection and deprotection of func-
tional groups are not required, high- and low-temperature
reactions can be circumvented, and organic solvents are not
Type I modular polyketide synthase (PKS) genes determine
the biosynthesis of valuable polyketide natural products, such
as erythromycin, epothilone, and many others. These genes
encode enzymes consisting of modules of active sites (do-
mains) that build the carbon chain of the final product in a
stepwise fashion using acyl-coenzyme A (CoA) starter and
extender units (15).
Most of the known PKSs are microbial enzymes and possess
a variable number of modules (Mod) preceded by a loading
didomain (LM). The LM is composed of an acyl transferase
(AT) domain that selects the starter acyl-CoA unit and an acyl
carrier protein (ACP) domain that receives the acyl group
from the loading AT. The acyl group then is transferred to the
first extender module and successively to downstream mod-
ules. All extender modules contain an essential set of three
domains: ketosynthase (KS), AT, and ACP. The KS receives
the acyl unit from the preceding module, while the AT trans-
fers an appropriate acyl extender unit from its CoA ester to the
ACP. The KS then catalyzes a condensation between the
acyl-KS and the ?-carbon of the extender acyl-ACP to give an
acyl-ACP. Additional domains may be present in some mod-
ules and are responsible for the reduction of the keto groups of
the growing polyketide chain. For example, modules may con-
tain a ketoreductase (KR) that reduces the ?-keto group ste-
reospecifically to an alcohol. At the end of the assembly line, a
thioesterase (TE) domain on the C terminus of the last ex-
tender module cleaves the polyketide chain from the PKS and
converts it to a lactone.
Thus, the structure of the two-carbon unit dictated by a
module is determined by the specificity of its AT domain, its
complement of reductive domains, and carbon branch stereo-
chemistry; the order of modules determines the sequence of
two-carbon units in the polyketide product, and the number of
modules determines carbon chain length.
Since the early 1990s, many research groups have been in-
terested in understanding the rules of module-module interac-
tions so as to genetically engineer microorganisms to create
novel polyketides (7). An ultimate goal is to produce complex
molecules by creating synthetic PKSs to be used directly as
drugs or as lead compounds for chemical optimization. Mean-
while, even the combination of a few PKS modules can pro-
duce molecules with multiple chiral centers (up to two per
module) that are difficult to obtain by chemical synthesis (5,
21), thus assisting in the production of complex molecules
currently made by total chemical synthesis.
Although the biosynthesis of polyketides found in nature is
confined to those that can be assembled with natural acyl-CoA
precursors, this limitation often can be overcome using che-
mobiosynthesis (3, 9, 12, 20). Here, unusual chemical moieties
may be introduced as the first unit of a polyketide chain by
feeding a PKS that has been disabled or deleted in an early
extension module with a chemically synthesized carboxylic acid
N-acetyl-cysteamine thioester (SNAC). In successful cases, the
synthetic thioester acylates the KS of the module immediately
* Corresponding author. Present address: Facultad de Ciencias Bio-
quimicas y Farmace ´uticas, Universidad Nacional de Rosario, Suipacha
531, Rosario 2000, Argentina. Phone and fax: 54-341-4350661. E-mail:
‡ Present address: Solazyme, Inc., 561 Eccles Avenue, South San
Francisco, CA 94080.
§ Present address: TerraBay Pharmaceuticals Inc., 2 HuaTian Rd.,
Suite 7012, Tianjin, P. R. China, 300384.
¶ Present address: Department of Pharmaceutical Chemistry, Box
2240, 600 16th Street N457B, University of California, San Fran-
† Supplemental material for this article may be found at http://aem
?Published ahead of print on 11 June 2010.
downstream of the disabled/deleted one and is faithfully
lengthened by subsequent extender modules.
There are several challenges in using chemobiosynthesis as a
general approach to making a desired polyketide. First, it is not
possible to rationally predict whether a particular SNAC will
be accepted and processed by a given PKS module and, if it is,
whether the extension of the unnatural starter unit will provide
an acceptable yield of product. Second, if the SNAC is ex-
tended by the first module, it cannot be predicted whether the
foreign polyketide chain will be extended by subsequent mod-
ules. Finally, it is improbable that a naturally occurring PKS
will possess the appropriate sequence of modules necessary
to extend the SNAC and produce the desired unnatural
polyketide, hence the need for imaginative genetic engineering
approaches to overcome these challenges.
The epothilones, a family of polyketide compounds naturally
produced by the myxobacterium Sorangium cellulosum, have
emerged as promising anti-cancer agents (10, 13, 19). The
potent synthetic analogue trans-9,10-dehydroepothilone D
(Fig. 1A), which is in human clinical trials, currently is pre-
pared by complete chemical synthesis requiring 23 operations
to provide an overall yield of only about 1% (4). The analogue
26-trifluoro trans-9,10-dehydroepothilone D, another promis-
ing clinical candidate (16), likewise is difficult to prepare. Bio-
synthetic approaches to making such analogues are especially
challenging, because the C-4-gem-dimethyl group and the dou-
ble bond at C-9–C-10 are rarely found in natural polyketides,
and the routes for their incorporation into engineered biosyn-
thetic pathways are largely unknown.
In this work, we describe an approach to create novel
polyketides with an unusual starter unit and an engineered
pattern of extension. Our strategy was validated by the biosyn-
thesis of intermediates that facilitate the synthesis of epothi-
MATERIALS AND METHODS
Host and vectors. The Escherichia coli polyketide producer strain K207-3
(BL21?prpBCD::T7prom prpE, T7prom accA1-pccB, T7prom sfp) and the pAng
and pBru series of vectors for the expression of PKS constructs have been
described previously (8).
E. coli strain DH5? was used for plasmid preparation. The pAngII series of
vectors for the expression of PKS modules flanked by the N-terminal linker of
ery5 (LNery5) and the C-terminal linker of ery2 (LCery2), designated LNery5-
Mod-LCery2, were created by removing the NdeI-MfeI fragment of the pAng
plasmids and inserting a DNA fragment containing a codon-optimized version of
LNery5flanked by identical restriction sites. All of the vectors used in this work
are listed in Table S1 in the supplemental material.
SNAC feeding to bimodules. K207-3 bacteria harboring pAngII donor plas-
mids and pBru acceptor plasmids were grown in 2.5 ml LB with carbenicillin (50
?g/ml) at 37°C to an optical density at 600 nm of 0.5. Cultures were induced with
isopropyl-?-D-thiogalactopyranoside (IPTG; 0.5 mM) and arabinose (2 mg/ml),
and 0.5 ml of a mixture of sodium glutamate (50 mM), sodium succinate (50
mM), sodium propionate (5 mM), and SNAC 1 (1 mM) was added. After
incubation at 22°C for 24 h with agitation, bacteria were removed by centrifu-
gation, and supernatants were acidified with phosphoric acid to pH 2.5 and
analyzed after at least 30 min for polyketide production by liquid chromatogra-
phy-mass spectrometry-mass spectrometry (LC-MS-MS).
Protein expression analysis. Samples (1 ml) of each culture were centrifuged
at 14,000 ? g for 3 min, resuspended in 1 ml 20 mM Tris, 150 mM NaCl, pH 7.5,
and lysed by sonication. After 10 min of centrifugation at 14,000 ? g, soluble
fractions equivalent to 10-?l cell suspensions were separated on NuPAGE Novex
3 to 8% Tris-acetate gels (Invitrogen), stained by Sypro-red staining (Molecular
Probes), and quantified with a Typhoon scanner using bovine serum albumin
Polyketide detection. Samples were analyzed by using a system consisting of a
Leap Technologies HTC PAL sample handler, an Agilent 1100 high-perfor-
mance liquid chromatography (HPLC) pump, and an Applied Biosystems API-
3000 triple quadrupole mass spectrometer equipped with a Turbo ion spray
source. For the identification/characterization of triketides, samples (10 ?l) were
chromatographed on an Agilent Zorbax Eclipse XDB-C8 column (3.5-mm di-
ameter, 2.1 by 150 mm) at 250 ?l/min by holding a mobile phase of 10%
acetonitrile (MeCN) (0.1% acetic acid [HOAc]) in H2O (0.1% HOAc) for 3 min,
followed by a linear gradient to MeCN (0.1% HOAc) over 9 min. Additional
conditions were the following: source temperature, 375°C; declustering and fo-
cusing potentials, 51 and 180 V, respectively; spray tip potential, 5,000 V; and
collision energy, 15 eV. Triketide 2 was identified by comparing characteristic
mass spectra to that of an authentic synthetic standard.
The same LC-MS system was used for the detection of the tetraketides with
different HPLC and mass spectrometry conditions. Samples (10 ?l) were injected
into the same column, and its temperature was maintained at 45°C; the mobile
phase used was a linear gradient from 10% MeCN (0.1% HOAc) in H2O (0.1%
HOAc) to MeCN (0.1% HOAc) over 10 min. Multiple reaction monitoring
(MRM) in positive-ion mode was used for detection.
The parent/daughter pairs of m/z 229/211, 229/193, 229/165, and 229/127 each
were acquired with a dwell time of 200 ms and at unit resolution in the first and
Additional conditions were the following: source temperature, 375°C; declus-
tering and focusing potentials, 26 and 200 V, respectively; spray tip potential,
4,600 V; and collision energy, 19 eV. The tetraketides were identified by com-
paring their characteristic mass spectra to that of an authentic synthetic standard.
Concentrations were estimated from the MRM data by comparing the area
response of samples to that of the standard at a known concentration.
RESULTS AND DISCUSSION
As an alternative to the complete biosynthesis of epothilone
analogues, we considered using genetically engineered mi-
crobes to prepare polyketide fragments that would serve as
advanced intermediates for chemical synthesis. A key interme-
diate in the synthesis of compounds in this series is the C-3–
C-9 aldehyde (Fig. 1A), which is made from a chiral material
and requires 12 chemical operations to produce. The diastereo-
selectivity of the synthetic steps to create the two new stereo-
genic centers is low (?4:1), and the purification of the desired
diastereomers is problematic, making the large-scale manufac-
turing of these epothilone analogues extremely costly and,
therefore, precluding their clinical development. We sought to
develop a biosynthetic method to produce tetraketide 3 or 4,
which can be converted to the C-3–C-9 aldehyde by only five
straightforward steps (4). Clearly, the economics of the semi-
synthetic approach are attractive, but since the key intermedi-
ates, tetraketides 3 and 4, have a double-bond moiety at a
position rarely found in polyketides, their biosynthesis was a
The tetraketide target molecules 3 and 4 (Fig. 1B) were
shown previously to serve as starting material for the complete
synthesis of trans-9,10-dehydroepothilone D (4). While there
are two possible stereomers of the ?-methyl group of keto-
lactone 4, in practice the rapid keto-enol equilibration of the
final ?-keto-ester results in the loss of stereochemical infor-
mation at this position. Fortunately, either of the diaste-
reomers 4a or 4b is a suitable biosynthetic target that would
provide equivalent access to the C-3–C-9 aldehyde (4).
We previously reported an approach to rapidly discover
pairs of heterologous PKS modules that can interact efficiently
to produce a polyketide (8). In this approach, members of a
library of modules encoded by redesigned genes in a donor
plasmid (pAng in Fig. 2) are coexpressed with members of a
library of modules in an acceptor plasmid (pBru), and triketide
5222 MENZELLA ET AL.APPL. ENVIRON. MICROBIOL.
FIG. 1. (A) Scheme for the synthesis of trans-9,10-dehydroepothilone D. (B) Production of tetraketide intermediate 3 by the double extension
of SNAC 1 with two D-type modules, 4a by extension with a D-G bimodular PKS and 4b by extension with a D-H bimodular PKS. (C) Structures
of two-carbon units added by the extension modules used in this work. D-type modules: eryM2, eryM5, eryM6, gldM3, sorM6, lepM10, rifM5, and
epoM7; G-type modules: eryM3, rapM3, lepM4, and rapM6; H-type module: pikM6. ery, erythromycin; sor, soraphen; rap, rapamycin; gdm,
geldanamycin; rif, rifamycin; lep, leptomycin.
production is analyzed by LC-MS in culture supernatants.
More than 150 module-module interactions tested using this
assay yielded a library of ?70 novel active bimodular PKSs;
subsequently, the library has been expanded to ?200 mini-
PKSs that provide a library of some 100 active bimodular
combinations (unpublished results).
We decided to explore a systematic, stepwise method toward
creating novel polyketides with unusual starter units using our
preexisting bimodular library. First, the SNAC containing the
starter unit is fed to an engineered E. coli strain expressing
members of the Mod-TE (pBru) library that catalyze the first
desired ketide extension, and the production of the expected
molecule in the cultures is determined. Second, modules that
best catalyze this first extension are cloned into donor vectors
and coexpressed with Mod-TEs (represented by further pBru
plasmids) in the library that are known to extend the ketide
product offered by the first module, and cultures are analyzed
for the presence of the final product (Fig. 3). To validate our
approach, we sought to create designer PKSs to produce in-
termediates that would facilitate the chemical synthesis of
We have reported previously the abbreviated codes D, H,
and G (8) for the two-carbon ketide units and PKS modules
that encode them (Fig. 1C). The successful extension of SNAC
1 by a bimodular PKS comprising two D-type modules is ex-
pected to yield intermediate 3, while D-G and D-H combina-
tions are expected to produce 4a and 4b, respectively. Of the
eight D-type extension Mod-TEs in our library, only sorM6
naturally receives and processes a substrate that resembles
SNAC 1. Nevertheless, LC-MS analysis revealed that six of the
eight D-type extension Mod-TEs available in our library pro-
duced detectable levels of triketide 2 when expressed in the E.
coli polyketide producer strain K207-3 (11) in the presence of
SNAC 1 to provide the expected triketide 2 in yields of 1 to 20
mg/liter, significantly more than those produced by sorM6, which
In keeping with earlier observations (1), all of the synthetic con-
structs gave similar levels of soluble protein in the range of 70 to
100 mg/liter. The high levels of protein expression likely are due
to the codon-optimized PKS genes, which typically produce 5- to
10-fold more PKS than wild-type sequences.
FIG. 2. Two classes of expression plasmid used to test bimodular interactions in E. coli. pAng vectors contain a CloDF13 replication origin, a
streptomycin resistance selection marker, and a PBADpromoter to drive the expression of LM-Module-LCeryM2ORFs. pBru vectors contain a
ColE1 replication origin, a carbenicillin resistance selection marker, and a PBADpromoter to drive the expression of LNeryM3-Module-TE ORFs.
Productive combinations of modules are revealed by the formation of triketide lactones (TKL).
5224MENZELLA ET AL.APPL. ENVIRON. MICROBIOL.
The individual Mod-TEs containing a His6tag also were
purified and tested for the production of triketide 2 in vitro;
they showed a similar ranking of production, as was the case in
experiments with whole bacteria (data not shown).
The four modules that most successfully catalyzed the first
extension of SNAC 1 were reformatted into pAngII donor
vectors in which the loading module was replaced by LNeryM5
(Fig. 3). Although the modules initially were evaluated in the
context of the N-terminal linker of ery3 (LNery3), we have
shown in our previous work that the yields of Mod-TEs pre-
ceded by LNery3or LNery5are essentially identical (6). In the
resultant open reading frames (ORFs) encoding LNery5-Mod-
LCery2, the LM-to-LNery5alteration precludes the priming of
the module by intracellular acyl-CoAs and facilitates PKS pro-
tein expression (6). As acceptors, we selected from 11 LNery3-
Mod-TEs fulfilling two criteria: (i) they were, in theory, capa-
ble of adding the desired second extensions (D type) to provide
the 3-hydroxy-tetraketide 3 or the equilibrated mixture of keto-
lactones 4a and 4b (G or H type); and (ii) each was shown
previously to accept a substrate from one or more of the four
donor modules that could catalyze the first extension of SNAC
1 (8). Therefore, 6 D-type, 4 G-type, and 1 H-type modules
were used as acceptors, each of which was shown previously to
accept substrate from one or more of the four selected donor
modules. From the 44 possible combinations, 30 were created
and evaluated, 15 to provide tetraketide 3, 12 to give 4a, and 3
to yield 4b. Bacteria were grown and PKS expression induced
in the presence of SNAC 1, and culture supernatants were
analyzed for the expected tetraketide products. As shown in
Table 2 and Fig. S1 in the supplemental material, all of the
PKSs made products that showed LC retention times and
MS-MS spectra identical to those of the synthetic standards.
As observed with a chemically synthesized standard, the proton
nuclear magnetic resonance (NMR) of purified tetraketide 4 ob-
tained from fermentation showed about 15% of the enol form,
confirming chemical the equilibration of 4a and 4b, and that the
same product(s) could be obtained from either type of bimodule.
FIG. 3. Method used to produce polyketide intermediates. PKS modules capable of catalyzing the extension of the unnatural starter unit are
identified by feeding the SNAC to bacteria expressing appropriate LNery3-Mod-TEs and analyzing for product. Modules active in the first SNAC
extension then are reformatted as LNery5-Mod-LCery2donor modules in pAngII vectors, and these are coexpressed with appropriate LNery3-Mod-
TEs from pBru vectors to determine which bimodular combinations can perform two extensions of the SNAC.
TABLE 1. In vivo extension of SNAC 1 by modules expressed as
Mod-TEs in E. coli strain K207-3
Triketide 2 production
VOL. 76, 2010POLYKETIDE PHARMACEUTICAL INTERMEDIATES IN E. COLI5225
The bimodule eryM6 ? eryM5-TE made the largest amount
of tetraketide 3. For tetraketide 4, similar yields were obtained
from the combinations eryM2 ? eryM3-TE and eryM2 ?
pikM6-TE. Such results could not have been predicted. For
example, one natural sequence of two modules (eryM2 ?
eryM3), presumably adapted by evolution, produced the highest
level of anticipated product, whereas another (eryM5 ? eryM6)
did not. The latter observation is in agreement with earlier results
showing that eryM5 is considerably less efficient at processing
SNACs than eryM6 (Table 1) (22). In all cases, the expected
compound was the major product, and no other tetraketide prod-
2 were found. This compound presumably originated from the
early release of the polyketide chain after the extension of the
SNAC by the first module. We conclude that the guided combi-
natorial approach presented here is currently the most effective
route to success.
Although our strategy was successful in producing either the
hydroxy- or keto-tetraketide, 3 or 4, respectively, the latter has
the clear advantage of requiring one fewer chemical conver-
sions to provide the C-3–C-9 aldehyde. Thus, future efforts
directed toward optimizing the biosynthetic production of tet-
raketide 4 by further protein engineering and fermentation
process development should yield titer improvements to pro-
duce this starting material in a cost-effective manner.
In summary, a procedure has been established to utilize a
preexisting library of bimodular PKSs to create polyketides
with unnatural starter units. First, PKS modules capable of
catalyzing the extension of the unnatural starter unit are iden-
tified by feeding the SNAC to bacteria expressing appropriate
Mod-TEs and analyzing for product. Next, modules active in
the first SNAC extension are reformatted as donor modules,
and these are coexpressed with appropriate Mod-TEs to de-
termine which bimodular combinations can perform two ex-
tensions of the SNAC. The process can, in principle, be con-
tinued to provide polyketides of increasing size. The feasibility
of this approach has been validated by developing bimodular
systems producing tetraketide lactones with multiple chiral
centers and an unusual starter, which serve as valuable inter-
mediates in the chemical synthesis of 9,10-dehydro-12,13-des-
oxyepothilone and its analogues. We anticipate that the use of
the semisynthetic C-3–C-9 aldehyde intermediate described
here will eliminate seven steps in the synthesis, including those
involved in the creation of stereogenic centers and the prob-
lematic purification operations associated with them.
We thank Janice Lau for helping with fermentation and the isolation of
polyketides, Gary Ashley and Yue Chen for assisting with the preparation
of SNACs, and David Hopwood for the critical review of the manuscript.
1. Chandran, S. S., H. G. Menzella, J. R. Carney, and D. V. Santi. 2006.
Activating hybrid modular interfaces in synthetic polyketide synthases by
cassette replacement of ketosynthase domains. Chem. Biol. 13:469–474.
2. Chotani, G., T. Dodge, A. Hsu, M. Kumar, R. LaDuca, D. Trimbur, W.
Weyler, and K. Sanford. 2000. The commercial production of chemicals
using pathway engineering. Biochim. Biophys. Acta 1543:434–455.
3. Jacobsen, J. R., C. R. Hutchinson, D. E. Cane, and C. Khosla. 1997. Pre-
cursor-directed biosynthesis of erythromycin analogues by an engineered
polyketide synthase. Science 277:367–369.
4. Li, Y., and Y. Chen. 2008. Process for the preparation of epothilones. Patent
5. McDaniel, R., M. Welch, and C. R. Hutchinson. 2005. Genetic approaches to
polyketide antibiotics. 1. Chem. Rev. 105:543–558.
6. Menzella, H. G., J. R. Carney, and D. V. Santi. 2007. Rational design and
assembly of synthetic trimodular polyketide synthases. Chem. Biol. 14:143–
7. Menzella, H. G., and C. D. Reeves. 2007. Combinatorial biosynthesis for drug
development. Curr. Opin. Microbiol. 10:238–245.
8. Menzella, H. G., R. Reid, J. R. Carney, S. S. Chandran, S. J. Reisinger, K. G.
Patel, D. A. Hopwood, and D. V. Santi. 2005. Combinatorial polyketide
biosynthesis by de novo design and rearrangement of modular polyketide
synthase genes. Nat. Biotechnol. 23:1171–1176.
9. Menzella, H. G., T. T. Tran, J. R. Carney, J. Lau-Wee, J. Galazzo, C. D.
Reeves, C. Carreras, S. Mukadam, S. Eng., Z. Zhong, P. B. Timmermans, S.
Murli, and G. W. Ashley. 2009. Potent non-benzoquinone ansamycin heat
shock protein 90 inhibitors from genetic engineering of Streptomyces hygro-
scopicus. J. Med. Chem. 52:1518–1521.
10. Michaud, L. B. 2009. The epothilones: how pharmacology relates to clinical
utility. Ann. Pharmacother. 43:1294–1309.
11. Murli, S., J. Kennedy, L. C. Dayem, J. R. Carney, and J. T. Kealey. 2003.
Metabolic engineering of Escherichia coli for improved 6-deoxyerythronolide
B production. J. Ind. Microbiol. Biotechnol. 30:500–509.
12. Murli, S., K. S. MacMillan, Z. Hu, G. W. Ashley, S. D. Dong, J. T. Kealey,
C. D. Reeves, and J. Kennedy. 2005. Chemobiosynthesis of novel 6-deoxy-
erythronolide B analogues by mutation of the loading module of 6-deoxy-
erythronolide B synthase 1. Appl. Environ. Microbiol. 71:4503–4509.
13. Perez, E. A. 2009. Microtubule inhibitors: differentiating tubulin-inhibiting
agents based on mechanisms of action, clinical activity, and resistance. Mol.
Cancer Ther. 8:2086–2095.
14. Pollard, D. J., and J. M. Woodley. 2007. Biocatalysis for pharmaceutical
intermediates: the future is now. Trends Biotechnol. 25:66–73.
15. Reeves, C. D. 2003. The enzymology of combinatorial biosynthesis. Crit. Rev.
16. Rivkin, A., F. Yoshimura, A. E. Gabarda, Y. S. Cho, T. C. Chou, H. Dong,
and S. J. Danishefsky. 2004. Discovery of (E)-9,10-dehydroepothilones
through chemical synthesis: on the emergence of 26-trifluoro-(E)-9,10-dehy-
dro-12,13-desoxyepothilone B as a promising anticancer drug candidate.
J. Am. Chem. Soc. 126:10913–10922.
17. Tao, J., and J. H. Xu. 2009. Biocatalysis in development of green pharma-
ceutical processes. Curr. Opin. Chem. Biol. 13:43–50.
TABLE 2. In vivo production of tetraketide 3 or 4a, 4b, and 4c in
E. coli strain K207-3 by extension of SNAC 1 by bimodular PKSs
Production (mg/liter) of tetraketide:
34a, 4b, and 4c
eryM6 ? eryM5-TE
eryM6 ? eryM2-TE
eryM6 ? eryM6-TE
eryM6 ? sorM6-TE
eryM6 ? gldM3-TE
eryM2 ? eryM2-TE
eryM2 ? eryM5-TE
eryM2 ? eryM6-TE
eryM2 ? sorM6-TE
eryM5 ? eryM6-TE
eryM5 ? eryM2-TE
eryM5 ? eryM5-TE
eryM5 ? rifM5-TE
gldM3 ? eryM6-TE
gldM3 ? eryM2-TE
eryM2 ? eryM3-TE
eryM2 ? pikM6-TE
eryM2 ? rapM3-TE
eryM2 ? lepM4-TE
eryM2 ? rapM6-TE
eryM6 ? eryM3-TE
eryM6 ? pikM6-TE
eryM6 ? rapM3-TE
eryM6 ? lepM4-TE
eryM6 ? rapM6-TE
eryM5 ? eryM3-TE
eryM5 ? pikM6-TE
eryM5 ? rapM3-TE
eryM5 ? lepM4-TE
eryM5 ? rapM6-TE
5226MENZELLA ET AL.APPL. ENVIRON. MICROBIOL.
18. Walsh, C. 2001. Enabling the chemistry of life. Nature 409:226–231.
19. Wang, J., H. Zhang, L. Ying, C. Wang, N. Jiang, Y. Zhou, H. Wang, and H.
Bai. 2009. Five new epothilone metabolites from Sorangium cellulosum strain
So0157-2. J. Antibiot. (Tokyo) 62:483–487.
20. Weissman, K. J. 2007. Mutasynthesis–uniting chemistry and genetics for
drug discovery. Trends Biotechnol. 25:139–142.
21. Weissman, K. J., and P. F. Leadlay. 2005. Combinatorial biosynthesis of
reduced polyketides. Nat. Rev. Microbiol. 3:925–936.
22. Wu, N., S. Y. Tsuji, D. E. Cane, and C. Khosla. 2001. Assessing the balance
between protein-protein interactions and enzyme-substrate interactions in
the channeling of intermediates between polyketide synthase modules.
J. Am. Chem. Soc. 123:6465–6474.
VOL. 76, 2010 POLYKETIDE PHARMACEUTICAL INTERMEDIATES IN E. COLI5227