Interfacing Microbial Styrene Production with a Biocompatible Cyclopropanation Reaction

Article abstract of DOI:10.1002/anie.201502185

Abstract The introduction of new reactivity into living organisms is a major challenge in synthetic biology. Despite an increasing interest in both the development of small-molecule catalysts that are compatible with aqueous media and the engineering of enzymes to perform new chemistry in vitro, the integration of non-native reactivity into metabolic pathways for small-molecule production has been underexplored. Herein we report a biocompatible iron(III) phthalocyanine catalyst capable of efficient olefin cyclopropanation in the presence of a living microorganism. By interfacing this catalyst with E.coli engineered to produce styrene, we synthesized non-natural phenyl cyclopropanes directly from D-glucose in single-vessel fermentations. This process is the first example of the combination of nonbiological carbene-transfer reactivity with cellular metabolism for small-molecule production.

Full text of DOI:10.1002/anie.201502185

Angewandte
Chemie
International Edition: DOI: 10.1002/anie.201502185
German Edition: DOI: 10.1002/ange.201502185
Synthetic Biology
Interfacing Microbial Styrene Production with a Biocompatible
Cyclopropanation Reaction**
Stephen Wallace and Emily P. Balskus*
Abstract: The introduction of new reactivity into living
organisms is a major challenge in synthetic biology. Despite
an increasing interest in both the development of small-
molecule catalysts that are compatible with aqueous media and
the engineering of enzymes to perform new chemistry in vitro,
the integration of non-native reactivity into metabolic pathways
for small-molecule production has been underexplored.
Herein we report a biocompatible iron(III) phthalocyanine
catalyst capable of efficient olefin cyclopropa-
nation in the presence of a living microorgan-
ism. By interfacing this catalyst with E. coli
engineered to produce styrene, we synthesized
non-natural phenyl cyclopropanes directly from
d-glucose in single-vessel fermentations. This
process is the first example of the combination
of nonbiological carbene-transfer reactivity with
cellular metabolism for small-molecule produc-
tion.
Exploration of the reactivity of carbenes has been
particularly fruitful for enzyme engineering efforts. Carbene
intermediates are accessed and utilized in cells by thiamine
diphosphate dependent enzymes (Figure 1A).[4] In previous
studies, ruthenium carbene complexes have been introduced
within artificial metalloenzymes for olefin ring-closing meta-
thesis in water and phosphate buffer.[5] More recent studies by
the Arnold and Fasan research groups have extended the
T
he field of synthetic biology is changing how
important small molecules are manufactured.[1]
By using renewable starting materials (e.g.
sugar, plant biomass, CO2) metabolic engineers
overproduce small molecules in single-vessel
fermentations by optimizing both known and
de novo biosynthetic pathways in host micro-
organisms. Despite significant progress in this
discipline, a major remaining challenge is the
engineering of organisms to access compounds
of non-natural origin. This objective is impor-
tant because many small molecules of commer-
cial interest are not currently accessible by the
use of known enzymatic chemistry. Two strat-
Figure 1. Design of a biocompatible cyclopropanation reaction. A) Carbene intermedi-
ates in biological catalysis. B) Current approaches for accessing ethyl 2-phenylcyclopro-
pane-1-carboxylate (1) from styrene by transition-metal-mediated carbene chemistry.
C) Phenyl cyclopropane production from d-glucose by combining in vivo styrene
production with biocompatible chemistry.
egies toward this goal have emerged: the engineering of
nonbiological reactivity into enzymes and the development of
nonenzymatic catalysts that can be interfaced with cellular
metabolism.[2,3]
types of carbenes involved in enzymatic catalysis to include
iron carbenes by the engineering of hemin-binding proteins to
catalyze enantioselective olefin cyclopropanation with ethyl
diazoacetate (EDA) both in vitro and in a whole-cell format
(Figure 1B).[2b,f,g,6] These studies were inspired by the mech-
anistic similarities between cytochrome P450 oxene-transfer
catalysis and transition-metal-mediated carbene-transfer
reactions. This research provides a biocatalytic route to
cyclopropanes, which are found in many bioactive synthetic
molecules.[7] Despite the success of these engineering efforts,
enzymes that utilize metallocarbene intermediates have not
yet been integrated into engineered metabolic pathways.
We envisioned approaching this challenge by using
biocompatible chemistry: nonenzymatic reactions capable
of modifying metabolites as they are made by living
organisms (Figure 1C).[3] Herein we describe the identifica-
tion of an iron phthalocyanine catalyst that cyclopropanates
[*] Dr. S. Wallace, Prof. E. P. Balskus
Department of Chemistry and Chemical Biology, Harvard University
12 Oxford Street, Cambridge, MA 02138 (USA)
E-mail: balskus@chemistry.harvard.edu
Homepage: http://scholar.harvard.edu/balskus
[**] This research was supported by the National Institutes of Health
(DP2 GM105434), the Searle Scholars Program, and the European
Commission (Marie Curie IOF fellowship to S.W.). We acknowledge
Prof. David Nielsen (Arizona State University) for generously
providing plasmids, and Prof. Kristala Jones Prather (MIT) for
helpful discussions during the preparation of this manuscript.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201502185.
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Table 2: Catalyst screen in the presence of E. coli.[a]
styrene generated by engineered microbial metabolism. This
reaction is both a new biocompatible iron-mediated trans-
formation and, to the best of our knowledge, the first example
of metallocarbene chemistry interfaced with cellular metab-
olism for small-molecule production.
We began our studies by investigating the efficiency of
olefin cyclopropanation under conditions compatible with the
growth of Escherichia coli. We initially examined the tetra-
phenylporphyrin iron(III) chloride (FeTPPCl) catalyzed
cyclopropanation of 4-vinylanisole and EDA. This reaction
has been previously reported to occur under aqueous alkaline
conditions with diazomethane, and also in aqueous phosphate
buffer (pH 7.0) under anaerobic conditions with the hemin
cofactor as a catalyst (29% conversion).[2g,8] We performed
the FeTPPCl-mediated reaction in water, phosphate buffer
(pH 7.0), and growth media of increasing complexity. We
found that although the reaction was moderately efficient in
water, phosphate buffer and growth media provided signifi-
cantly higher conversions and diastereoselectivities (Table 1).
Unlike other biocompatible transformations, cyclopropana-
tion proceeded efficiently in the complex medium Luria–
Bertani broth (LB).[3a] The reasons for the increased con-
version in phosphate buffer and growth media relative to
water are unclear; however, similar effects observed for other
reactions carried out in highly ionic solvents are hypothesized
to arise from rate acceleration due to an increased hydro-
phobic effect.[9] Finally, the addition of bacterial cells (E. coli
BL21(DE3), OD600 = 0.5) had no detrimental effect on the
reaction yield or selectivity (Table 1, entries 5 and 7).
Entry
Catalyst (mol%)
Yield [%]
trans/cis
1
2
3
4
5
6
7
8
FeTPPCl (10)
Fe(F20TPP)Cl (10)
Fe(OMe)4TPPCl (10)
hemin (10)
FePcCl (10)
FePcCl (5)
75
55
7
3.9:1
5.1:1
2.9:1
–
3.4:1
3.4:1
3.7:1
4.0:1
0
95
93[b]
90[c]
80[c]
FePcCl (2.5)
FePcCl (1)
[a] Reactions were performed as described in Table 1. All data are shown
as an average of three experiments. [b] The reaction was carried out for
24 h. [c] The reaction was carried out for 48 h.
Replacement of the four aromatic rings of the TPP ring
system with aliphatic substituents abolished catalytic activity
(see Table S1). Hemin was unreactive under the reaction
conditions (Table 2, entry 4), despite having previously been
reported to catalyze this transformation in vitro.[2g] Ferric
phthalocyanine (FePcCl) proved to be an exceptional cyclo-
propanation catalyst and afforded the product in 95% yield
(Table 2, entry 5). The catalyst loading of FePcCl could be
reduced to as low as 1 mol% with only a moderate loss in
conversion. No product was detected when FeCl3 was used or
in the absence of an added catalyst, thus ruling out the
possibility that either endogenously biosynthesized metal-ion
complexes or ferric ligands produced by E. coli contribute to
catalysis in vivo.
We next conducted a catalyst screen in M9CA–glucose
media containing E. coli (Table 2; see also Table S1 in the
Supporting Information). The presence of electron-withdraw-
ing and electron-donating substituents on the porphyrin ring
system influenced product diastereoselectivity but diminished
conversion relative to FeTPPCl (Table 2, entries 2 and 3).
Table 1: FeTPPCl-catalyzed cyclopropanation reactions in aqueous
media and in the presence of E. coli.[a]
The major by-product of the reaction at a catalyst loading
of 10 mol% was diethyl maleate, which arises from EDA
dimerization. Unexpectedly, at lower catalyst loadings we
observed a new by-product, diethyl succinate (Figure 2; see
Entry Growth medium E. coli cells added? Yield [%] trans/cis
1
2
3
4
5
6
7
H2O
no
no
no
no
yes
no
yes
46
77
71
72
75
71
70
3:1
3.7:1
3.7:1
4:1
3.9:1
4:1
0.1m K2HPO4
M9–glucose
M9CA–glucose
M9CA–glucose
LB
LB
4:1
[a] Reactions were performed with 4-vinylanisole (5 mm), EDA (10 mm),
and FeTPPCl (0.5 mm) in sealed Hungate tubes under an atmosphere of
air. All cultures were grown in the presence of kanamycin (50 mgLÀ1) and
isopropyl b-d-1-thiogalactopyranoside (IPTG; 0.2 mm). E. coli BL21 cells
transformed with an empty pET-29b(+) expression plasmid
(OD600 =0.5) were used. Product concentrations in crude culture
extracts were determined by 1H NMR spectroscopy relative to an internal
standard of 1,3,5-trimethoxybenzene. All data are shown as an average of
three experiments to one standard deviation.
Figure 2. A reaction by-product reports on E. coli survival under the
cyclopropanation reaction conditions. Reactions were performed as
described in Table 1.
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also Table S2). We hypothesized that this product arose from
the use of diethyl maleate as a terminal electron acceptor by
E. coli for anaerobic respiration under the progressively
oxygen limiting conditions of the reaction setup. We also
verified that living cells were required for diethyl succinate
production. The formation of this by-product at a catalyst
loading of 2.5 mol% therefore confirms that the E. coli
bacteria are alive under our optimal reaction conditions.
Having shown that the FePcCl-catalyzed cyclopropana-
tion was compatible with E. coli, we next attempted the
reaction with styrene generated by bacterial metabolism.
Styrene production from d-glucose was achieved in the l-
phenylalanine-overproducing strain E. coli NST74 by intro-
ducing two enzymes: l-phenylalanine ammonia lyase from
Arabidopsis thaliana (PAL2), which converts l-phenylalanine
into trans-cinnamic acid, and a decarboxylase from Saccha-
romyces cerevisiae (FDC1) that generates styrene from trans-
cinnamic acid (Figure 3A).[10]
without reaction components showed no difference in sur-
vival (see Figure S1 in the Supporting Information). Together,
these results suggested that our reaction would be compatible
with in vivo styrene production. Accordingly, the PAL2 and
FDC1 genes were introduced into E. coli NST74 on a single
expression plasmid (pTrc99A_PAL2-FDC1). Under opti-
mized conditions, this strain accumulated 1.65 mm styrene in
the culture medium over 48 h (see Figure S5). We next
attempted the cyclopropanation reaction by adding FePcCl
(2.5 mol%) and EDA (2 equiv) to cultures at the point of
induction of the styrene-producing pathway (OD600 = 0.5–0.6,
t = 0 h). After 48 h we observed cyclopropane 1 by GC (44%
conversion, d.r. 3.0:1). 1H NMR spectroscopic analysis of the
reaction extract showed no unreacted EDA and significant
levels of EDA by-products, thus indicating that competing
carbene dimerization was probably limiting reaction conver-
sion. This issue was circumvented by adding EDA portionwise
over the course of the fermentation; in this way, the yield of
1 was increased to 81%. By adding an additional equivalent
of EDA and extending the reaction time to 60 h we obtained
1 in 95% yield (as determined by GC; Figure 3B).
We performed a series of control experiments to confirm
that cyclopropanation required the presence of the catalyst,
EDA, and living E. coli (Figure 3C). To obtain information
about the timing and rate of cyclopropanation
relative to in vivo styrene production, we analyzed
product distributions in fermentations with and
without the reaction components (Figure 3D; see
also Figure S4). In the presence of FePcCl and
EDA, styrene reached a maximum concentration
of 0.6 mm after 18 h and then steadily depleted as
1 accumulated. Ultimately, the concentration of
1 equaled the concentration of styrene produced in
the absence of the reaction components. This
observation confirms that the biocompatible cyclo-
propanation is interfaced with styrene output from
E. coli and that after 18 h the rate of styrene
consumption by cyclopropanation probably
exceeds that of styrene generation by metabolism.
This analysis also demonstrates that the reaction
components have a minimal effect on the overall
levels of styrene production. Interestingly, in the
absence of FePcCl, accumulating EDA signifi-
cantly inhibited styrene production after 12 h (P <
0.05), thus indicating that this reagent is toxic to
E. coli and that the activity of the catalyst prevents
this adverse effect in the full reaction (see Fig-
ure S5). A preliminary investigation of the cyclo-
propanation by using a three-phase test suggested
that catalysis by FePcCl occurs at a solid–liquid
We confirmed that FePcCl was effective under the
conditions required for styrene production by performing
the cyclopropanation reaction with 1.5 mm styrene in phos-
phate-limited minimal media (MM1) containing E. coli
BL21(DE3) and d-glucose (82% yield, d.r. 1.7:1). Serial
dilutions and plate counts directly from cultures with and
interface, as no product was detected when a poly-
mer-supported styrene was used (see Figure S6).
This result indicates that FePcCl is probably
functioning as a heterogeneous catalyst in this
transformation.
We evaluated the scope of the in vivo cyclo-
propanation by examining other diazoacetate
derivatives. Using additional electron-poor
(acceptor) carbenes afforded cyclopropanes 2–4,
Figure 3. The biocompatible cyclopropanation reaction can be interfaced with micro-
bial styrene production. A) Engineered pathway for styrene production in the l-
phenylalanine overproducer E. coli NST74. B) Cyclopropanation of metabolically
generated styrene. C) Graph showing that cyclopropane production requires all
reaction components and living E. coli. D) Metabolite production during fermenta-
tions. E) Additional cyclopropanes accessed by this approach. Metabolite concen-
trations were determined by GC relative to an internal standard of 1,3,5-trimethoxy-
benzene. Yields in (E) are for isolated material from 800 mL cultures. All data are
shown as an average of three independent experiments to one standard deviation.
[a] The product was isolated in 93% yield. [b] The reaction was carried out for 72 h.
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which were isolated in good yields (Figure 3E). We also
reexamined hemin as a catalyst to test the extent to which the
results of our initial screening procedure predicted catalyst
performance with metabolically generated styrene. Hemin
remained a less efficient cyclopropanation catalyst (27%
conversion after 60 h; see Table S4), thus confirming the
utility of our catalyst-identification strategy. The low reac-
tivity of hemin also resulted in EDA accumulation, which
dramatically reduced overall styrene production levels to
0.23 mm. This finding shows that the identity of the non-
enzymatic catalyst can influence multiple variables of in vivo
reactions. Overall, these studies not only provide a new route
to cyclopropanes but also a potential roadmap for the
discovery of other biocompatible reactions.
The combined use of enzymatic and nonenzymatic
catalysis for chemical synthesis can provide unique benefits
over the use of chemo- or biocatalysis alone.[3c,11] In the case
of this biocompatible cyclopropanation, the generation of
styrene from d-glucose is attractive, as it avoids the use of
nonrenewable petroleum streams. By intercepting biological-
ly produced styrene in the fermentation vessel we also
circumvent the challenges associated with its isolation,
including volatility and reactivity (polymerization during gas
stripping).[12] Additionally, the use of bench- and air-stable
phthalocyanines circumvents the need for strictly anaerobic
conditions during cyclopropanation (as are required by many
engineered biocatalysts). This feature is critical for overall
cyclopropane production, as an aerobic growth environment
is needed for maximum styrene production by engineered
E. coli.[10] Notably, this requirement has most likely made the
incorporation of cyclopropanating enzymes into engineered
metabolic pathways challenging.
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Keywords: iron · metabolism · phthalocyanine ·
Received: March 8, 2015
synthetic biology · synthetic methods
Published online: && &&, &&&&
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Synthetic Biology
S. Wallace, E. P. Balskus*
&&&& — &&&&
Interfacing Microbial Styrene Production
with a Biocompatible Cyclopropanation
Reaction
The beauty of bugs: Iron(III) phthalocya-
nine was identified as a highly active and
biocompatible carbene-transfer catalyst
capable of cyclopropanating styrene gen-
erated by the metabolism of engineered
Escherichia coli. The biocompatible reac-
tion is nontoxic to the producing organ-
ism, thus enabling the near-quantitative
production of various phenyl cyclopro-
panes directly from d-glucose in single-
vessel fermentations.
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