An enzymatic toolbox for cascade reactions: A showcase for an in vivo redox sequence in asymmetric synthesis

Article abstract of DOI:10.1002/cctc.201300604

Joined forces: Alcohol dehydrogenase, enoate reductase, and Baeyer-Villiger monooxygenase are combined in a cascade reaction by coexpression in E. coli to have a recombinant whole-cell biocatalyst. Such an artificial metabolic mini -pathway provides access to functionalized chiral compounds in high yields and optical purities as exemplified for kinetic resolutions, desymmetrizations, and regiodivergent biotransformations. Copyright

Full text of DOI:10.1002/cctc.201300604

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DOI: 10.1002/cctc.201300604
An Enzymatic Toolbox for Cascade Reactions: A Showcase
for an In Vivo Redox Sequence in Asymmetric Synthesis
Nikolin Oberleitner,[b] Christin Peters,[a] Jan Muschiol,[a] Maria Kadow,[a] Stefan Saß,[a]
Thomas Bayer,[b] Patricia Schaaf,[b] Naseem Iqbal,[b] Florian Rudroff,*[b]
Marko D. Mihovilovic,*[b] and Uwe T. Bornscheuer*[a]
Single-step transformations of non-natural substrates by en-
zymes have been successfully established in the last decades
as highly valuable techniques for the synthesis of chiral build-
ing blocks.[1] Owing to their properties, biocatalysts can be em-
ployed as catalytic tools for the facile transformation of func-
tional groups in organic synthesis. By exploiting the manifold-
ness of enzymes and their different catalytic activities, it is pos-
sible to design new artificial biosynthetic pathways on the
basis of the “retrosynthetic approach”[2] that is commonly ap-
plied in chemical synthesis and that was only very recently
proposed as a novel concept for biocatalysis.[3] This design
principle is used in the strategic planning of organic syntheses
by transforming a target molecule into simpler precursors in
which molecular complexity is reduced by manipulation of
functional groups.
ways.[4a,5] However, this approach is largely limited to already-
existing metabolic reaction sequences (e.g., 1,3-propane diol[6]
and 1,4-butane diol[7]) and does not necessarily deliver the
high structural diversity of compounds utilized by the chemical
industry.
The major difference between biocatalysis and classical fer-
mentation is the extension of the enzymatic transformations to
non-natural substrates. Consequently, the extension of single-
step biotransformations, which are particularly powerful in
asymmetric synthesis, is a logical development.[4a,8] In this con-
text, we recently reported the successful conversion of unsatu-
rated fatty acids into medium-chain alcohols, w-hydroxy acids,
and dicarboxylic acids through the combination of appropriate
enzymes in a cascade reaction by employing a combined
in vivo/in vitro strategy towards nonchiral products.[9]
Very recently, Schrewe et al.[10] presented an interesting
three-step synthetic approach including two enzymes for the
production of terminal alkylamino functionalization in a re-
combinant E. coli strain. Notably, however, only a single sub-
strate was investigated, but even so, the versatility of the pre-
sented enzyme was highlighted. Another approach was pub-
lished very recently by the group of Li[11] who applied a two-
strain-mixed-culture strategy for the synthesis of d-lactones.
Both studies showed the potential of redox cascades in living
organisms and at the same time pointed out their limitations.
A simple transfer from a single-step biotransformation (includ-
ing broad substrate acceptance and high selectivity) to
enzyme cascades still remains a challenge. The prime novelty
and major aim of the present study was to combine the effi-
ciency of biosynthetic redox pathways, the modularity of syn-
thesis by simple functional group transformations, and the
substrate promiscuity of enzymes. Designing and evaluating
the feasibility of a multienzyme-catalyzed cascade process in
living microbial cells enabled the creation of an artificial
“mini”-metabolic pathway connected to primary metabolism
through redox-cofactor regeneration. This approach was ap-
plied in an in vivo environment and concomitantly provided
access to diverse chemical entities through divergent reaction
pathways.
The concept of multistep one-pot reactions has caught the
attention of synthetic chemists in recent years.[4] To address
the increasing demands by society to further improve the sus-
tainability of chemical processes, one-pot cascades of reaction
sequences can substantially decrease the amount of chemicals
used for each reaction and subsequent down-stream process-
ing by concomitantly optimizing the energy requirements and
operation expenses. As pointed out in a recent review,[4a] such
cascades not only improve processes by saving time and re-
ducing waste, but they also offer advantages if unstable or
toxic intermediates are involved, as these do not accumulate.
Nature uses the design principle in a highly successfully
manner, as all metabolic pathways are interconnected and con-
ducted within the “single-vessel” environment of a cell. The
concerted interaction of numerous enzymes within the cell
allows exceptionally high yields in multistep biosynthetic path-
[a] C. Peters,+ J. Muschiol, Dr. M. Kadow, S. Saß, Prof. Dr. U. T. Bornscheuer
Institute of Biochemistry, Dept. of Biotechnology & Enzyme Catalysis
Greifswald University
Felix-Hausdorff-Str. 4, 17487 Greifswald (Germany)
Fax: (+49)3834-86-794367
E-mail: uwe.bornscheuer@uni-greifswald.de
[b] N. Oberleitner,+ T. Bayer, P. Schaaf, Dr. N. Iqbal, Dr. F. Rudroff,
Prof. Dr. M. D. Mihovilovic
Figure 1 illustrates the straightforward concept of this work.
In lieu of looking at different specific target compounds, for
which one synthetic step is performed biocatalytically, we re-
duced the complexity of the molecule to the functional groups
of the compound regardless of the residual structure. From
a synthetic point of view, oxidation of a simple allylic alcohol
starting material to the corresponding a,b-unsaturated ketone
Institute of Applied Chemistry
Technical University Vienna
Getreidemarkt 9/163-OC, 1060 Vienna (Austria)
E-mail: marko.mihovilovic@tuwien.ac.at
florian.rudroff@tuwien.ac.at
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201300604.
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tion of the corresponding lac-
tone (as exemplified for sub-
strate 1a in Scheme 1). This par-
ticular sequence was realized as
a
proof-of-concept study to
demonstrate the principal feasi-
bility of the construction of en-
zymatic cell factories for the syn-
thesis of fine chemicals and to
determine and resolve limiting
aspects.
Starting with the simple race-
mic substrate 2-cyclohexenol
(1a), the first aim of this study
was dedicated to the identifica-
tion of suitable enzymes for all
Figure 1. Basic concept of the developed biocatalytic redox toolbox.
functional group transformations
that are conducted in this reac-
tion sequence. In an initial study
needed to be achieved. In the second step, (stereo)selective re-
duction of the double bond in the presence of a ketone func-
tionality was required. Finally, the obtained saturated ketone
needed to be further oxidized to the corresponding target
molecule, a chiral lactone.
(see the Supporting Information), two suitable and preferen-
tially nonselective ADHs were investigated, that is, LK-ADH
from Lactobacillus kefir[13] and RR-ADH from Rhodococcus ruber.
According to the literature and our preliminary results, LK-ADH
was the most suitable for the first reaction step. Alcohol 1a
was fully oxidized to corresponding cyclohexenone 1b with
concomitant consumption of nicotinamide adenine dinucleo-
tide phosphate (NADP+). Subsequent reduction of the a,b-un-
saturated ketone was performed by an ERED from Pseudomo-
nas sp., that is, XenB.[14] The artificial “mini”-pathway was com-
pleted by the well-known cyclohexanone monooxygenase
(CHMOAcineto) from Acinetobacter sp.[15]
Each transformation is quite well known in classical synthetic
chemistry, but the combination of these transformations in
a one-pot fashion is rather difficult to achieve. A simple reason
for that is the possible need for different solvents, reaction
temperatures, reaction atmosphere, and, predominantly, suita-
ble catalysts with desired substrate scope, specific activity, and
chemo-, regio-, and stereoselectivity. Judicious choice of the
enzymes also offers a major advantage over chemical process-
es, which often lack the required chemoselectivity if more than
one functional group is present in the target molecule. This
can only be circumvented by either the introduction of pro-
tecting groups or the design of “domino reactions”. The latter
type of reaction has a major disadvantage in that the subse-
quent reaction can only be started once the preceding trans-
formation is complete to avoid byproduct formation.
Having all catalysts identified, a first one-pot in vivo experi-
ment—by expressing all enzymes at once in E. coli—gave
promising results, and reasonable amounts of final lactone 1d
(Figure 2, Table 1) were formed. The possible rate-limiting step
of the cascade, the ADH-catalyzed oxidation to unsaturated
ketone 1b, was successfully shifted by subsequent reduction
of the C=C bond by the ERED to yield 1c. This further under-
We developed a three-step artificial metabolic “mini”-path-
way comprising three types of biocatalysts with broad sub-
strate scopes that are naturally not connected in a single or-
ganism in a metabolic context:[12] An alcohol dehydrogenase
(ADH) for the oxidation of an allylic secondary cyclic alcohol to
the corresponding a,b-unsaturated ketone, an enoate reduc-
tase (ERED) for the subsequent reduction of the double bond,
and a Baeyer–Villiger monooxygenase (BVMO) for the forma-
Scheme 1. Artificial metabolic pathway composed of an alcohol dehydro-
genase (LK-ADH), an enoate reductase (XenB), and a Baeyer–Villiger-mono-
oxygenase (CHMOAcineto) starting from 2-cyclohexenol. All enzymes were ex-
pressed recombinantly in E. coli. See the Supporting Information for details.
Figure 2. In vivo cascade starting from 2-cyclohexenol (1a) to yield lactone
1d.
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cofactor recycling, but the final oxygenation step requires an
additional equivalent of NADPH (Scheme 1). Implementation of
the multistep cascade in the viable microorganism E. coli pro-
vides all redox cofactors and thus can close the gap in the
redox cycle, and this leads to improved performance of this
multistep biotransformation and reduces the metabolic burden
of the cell significantly.
Table 1. Results of the enzymatic cascade reactions.
Substrate[a]
Product
Conv.
[%]
ee/de[b]
[%]
t
[h]
H
>99
>99
n.a.[c]
21
20
Given that the first aim, a proof of concept, was pervaded
successfully, we next addressed the questions of flexibility and
modularity of our approach and increased the complexity of
the process by also considering stereoselective transforma-
tions. Selection of various substrates enabled us to address all
important reaction types: an achiral transformation, desymmet-
rization, and kinetic resolution (Scheme 2). For this, we synthe-
sized a set of cyclohexenol derivatives for sequential biocata-
lytic reactions with stepwise-increased complexity of these
transformations (Table 1).
4-Me
>99
2-Me
3-Me
64
>99
20
n.a.[c,d]
n.a.[c]
n.a.[c]
86
63
>99
>99
20
20
carveol
(1R,5R)
carveol
carveol
93
>99
20
20
Scheme 2. Substrate scope of the proposed enzyme cascade including all
important biocatalytic reactions. Classifications of transformations are based
on the BVMO-mediated step. All substrates and intermediates were synthe-
sized according to literature or adapted protocols (see the Supporting
Information).
(1S,5R)
(1S,5S)
>99
>99
Introduction of chirality by desymmetrization of prochiral
substrates is of common interest in chiral synthesis, as a 100%
theoretical yield can be achieved directly with high optical
purity. (cis/trans)-4-Methylcyclohexenol (2a) was chosen as
a model substrate for this type of transformation and oxidation
by LK-ADH gave racemic 2b in very good yield (see the Sup-
porting Information). Subsequent reduction of the double
bond by the ERED XenB to prochiral 4-methylcyclohexanone
(2c) and final desymmetrization by the BVMO led to lactone
2d in excellent GC yield (100%) and perfect optical purity
[>99% enantiomeric excess (ee)].
[a] In all cases the substrate concentration was 4 mm, with the exception
of substrate 5a (2.5 mm). [b] de=diastereomeric excess. [c] n.a.=not ap-
plicable. [d] No formation of proximal lactone 4d was observed. [e] 5d=
6d, owing to the stereopreference of XenB.
scored the advantages enabled in a cascade reaction setting.
Moreover, as the involved enzymes are not evolutionary inter-
connected, there was no regulation process and the synthetic
pathway towards the final product was not restricted. This is
a particularly important feature for exploitation in chemical
synthesis.
LK-ADH converts NADP+ into NADPH, which serves as a co-
factor for XenB and BVMO. Consequently, the first and the
second steps of the cascade are self-sustaining with respect to
On the basis of these promising results, attention was shift-
ed to 2-substituted cyclohexenol derivatives by employing ki-
netic resolution in the BVMO-mediated step (Table 1). Oxida-
tion of 3a by LK-ADH gave 3b, and reduction of 3b with the
ERED delivered optically pure 3c, which was then converted
stereoselectively into chiral lactone 3d catalyzed by
CHMOAcineto (64% GC yield, >99% ee). By applying this cascade
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sequence we were able to circumvent classical kinetic resolu-
tion, which resulted in very low E values for several tested
BVMOs (data not shown), to obtain enantiomerically pure lac-
tone 3d. Again, we demonstrated the particular advantages of
this atom-efficient multistep redox cascade, which yielded
64% (GC) of the desired product after three steps.
Regiodivergent biotransformations are a particularly valuable
feature of BVMOs, as this conversion enables access to regio-
isomeric products from antipodal precursors. Substrate 4a was
easily oxidized by LK-ADH to 3-methylcyclohexenone (4b), but
XenB did not accept 4b, and this stopped the cascade at this
point. To solve this issue, we investigated an alternative ERED
from Saccharomyces carlsbergensis—also named old yellow
enzyme (OYE1)[16]—previously reported to accept 4b. Indeed,
optically pure (S)-3-methylcyclohexanone (4c) was obtained by
applying OYE1, and this cyclohexanone served as a substrate
for the final Baeyer–Villiger oxidation with the use of
CHMOAcineto to ultimately obtain distal lactone 4e (86% GC
yield, >99% ee).[17]
Having now established a toolbox of recombinantly ex-
pressed enzymes in one cell in proper combination (two ADHs,
two EREDs, and one BVMO; see the Supporting Information),
we approached the cascade conversion of three different iso-
mers of carveol (i.e., 5a–7a) representing the most complex
substrates for this study. Applying our designed pathway
offers the advantage that different diastereomers of carveol
can be directly converted into the desired product in a stereo-
selective manner by choosing the appropriate enzymes. Fur-
thermore, the availability of BVMOs with opposite regioselec-
tivity[18] can be exploited to afford different regioisomeric lac-
tones. Notably, both ADHs showed excellent stereopreference
with respect to the alcohol group and were completely unse-
lective towards the stereogenic center in the 5-position. RR-
ADH converted exclusively (1R,5R)-carveol (5a), whereas LK-
ADH accepted only the (1S,5R) and (1S,5S) stereoisomers, that
is, 6a and 7a, respectively. Experiments with resting E. coli
cells containing the appropriate enzymes revealed the forma-
tion of the desired product after 20 h for all three carveol iso-
mers, as exemplified in Figure 3 for substrate 7a.
The expected intermediates from the cascade reaction were
detected only in low amounts as a result of fast biotransforma-
tion. A closer look at each individual reaction step revealed
that an E. coli background transformation competes with the
enoate reductase step in our cascade. A literature survey iden-
tified N-ethylmaleimide reductase (NemR) as a disturbing re-
ductase.[19] After a first glance, we found that both EREDs
showed the same stereopreference for both carvone substrates
and that NemR did not accept the other a,b-unsaturated sub-
strates of the cascades (i.e., 1–4b). Nevertheless further exten-
sion of the cascade, including the accessibility of different iso-
mers, would require a DnemA E. coli strain. First, in vivo studies
with BL21(DE3)DnemA showed the complete absence of the
background reaction (Table S2, Supporting Information).
Figure 3. In vivo cascade for the conversion of (1S,5S)-carveol (7a) into
corresponding lactone 7d.
100 mg of either 3-methylcyclohexanone (4a) or carveol (7a)
were used to produce desired lactones 4e and 7d (Table 2) at
a conversion of 94 and 93%, respectively. Classical extractive
workup was performed followed by column chromatography
to yield 4e and 7d in 55 and 60% (isolated material) yield, re-
spectively. In summary, we conducted a three-step enzymatic
redox cascade and achieved an average of 82% (for 4a!4e)
and 84% (for 7a!7d) yield in each individual step.
Table 2. Preparative-scale cascade biotransformations.
Substrate
(4 mm)
Product
Yield[a]
[%]
ee/de[b]
[%]
t
[h]
3-Me
4a
4d[c]
4e
n.a.[d]
55
n.a.[d]
>99
>99
n.a.[d]
24
18
carveol
7a (1S,5S)
7d
60
[a] Yield of isolated product after column chromatography. [b] de=dia-
stereomeric excess. [c] No formation of proximal lactone 4d was ob-
served. [d] n.a.=not applicable.
By this proof-of-concept study, we were able to design and
create a modular enzymatic multistep redox toolbox in the mi-
crobial host E. coli. The design principle of this artificial “mini”-
metabolic pathway was predicated on the basis of the retro-
synthetic approach derived from strategic planning of organic
syntheses. By increasing the complexity, we managed to cover
the full range of important reaction types known in chiral syn-
thesis. Hence, by selection of proper enzymes—not present in
a single natural microorganism—we enabled the efficient
three-step synthesis of various chiral building blocks in a one-
pot fashion simply by using resting E. coli cells. We met the
challenge of combining relatively unselective enzymes in the
Ultimately, to demonstrate the applicability of the presented
concept for the synthesis of high-value compounds for the
chemical industry, we performed two preparative-scale experi-
ments. Thus, freshly prepared resting cells supplemented with
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We thank the Deutsche Forschungsgemeinschaft (DFG) (grant no.
Bo1862/6-1), the Fonds zur Fçrderung der wissenschaftlichen For-
schung (FWF) (grant no. I723-N17 & P24483-B20), and Vienna
University of Technology (grant no. GEV-TOP163) for financial
support. We are also grateful to Prof. W. Hummel (University of
Dꢀsseldorf) for supplying the genes encoding LK-ADH and RR-
ADH and to Prof. J. D. Stewart (University of Florida) for the gene
encoding OYE1.
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Keywords: artificial pathway · biocatalysis · domino reactions ·
redox chemistry · retrosynthesis
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Received: July 24, 2013
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N. Oberleitner, C. Peters, J. Muschiol,
M. Kadow, S. Saß, T. Bayer, P. Schaaf,
N. Iqbal, F. Rudroff,* M. D. Mihovilovic,*
U. T. Bornscheuer*
Joined forces: Alcohol dehydrogenase,
enoate reductase, and Baeyer–Villiger
monooxygenase are combined in a cas-
cade reaction by coexpression in E. coli
to have a recombinant whole-cell bio-
catalyst. Such an artificial metabolic
“mini”-pathway provides access to func-
tionalized chiral compounds in high
yields and optical purities as exempli-
fied for kinetic resolutions, desym-
metrizations, and regiodivergent bio-
transformations.
&& – &&
An Enzymatic Toolbox for Cascade
Reactions: A Showcase for an In Vivo
Redox Sequence in Asymmetric
Synthesis
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemCatChem 0000, 00, 1 – 5
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6
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These are not the final page numbers!