University of Groningen
Multi-Enzymatic Cascades In Vitro
Schmidt, Sandy; Schallmey, Anett; Kourist, Robert
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Enzyme Cascade Design and Modelling
DOI:
10.1007/978-3-030-65718-5_3
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Schmidt, S., Schallmey, A., & Kourist, R. (2021). Multi-Enzymatic Cascades In Vitro. In S. Kara, & F.
Rudroff (Eds.),
Enzyme Cascade Design and Modelling
(pp. 31-48). Springer International Publishing.
https://doi.org/10.1007/978-3-030-65718-5_3
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Multi-Enzymatic Cascades In Vitro
3
Sandy Schmidt, Anett Schallmey, and Robert Kourist
Abstract
The combination of enzymatic reactions in a
simultaneous or sequential fashion by design-
ing artificial synthetic cascades allows for the
synthesis of complex compounds from simple
precursors. Such multi-catalytic cascade
reactions not only bear a great potential to
minimize downstream processing steps but
can also lead to a drastic reduction of the
produced waste. With the growing toolbox of
biocatalysts, alternative routes employing
enzymatic transformations towards manifold
and diverse target molecules become accessi-
ble. In vitro cascade reactions open up new
possibilities for efficient regeneration of the
required cofactors such as nicotinamide
cofactors or nucleoside triphosphates. They
are represented by a vast array of
two-enzyme cascades that have been designed
by coupling the activity of a cofactor
regenerating enzyme to the product generating
enzyme. However, the implementation of cas-
cade reactions requires careful consideration,
particularly with respect to whether the path-
way is constructed concurrently or sequen-
tially. In this regard, this chapter describes
how biocatalytic cascades are classified, and
how such cascade react ions can be employed
in order to solve synthetic problems. Recent
developments in the area of dynamic kinetic
resolution or cofactor regeneration and
showcases are presented. We also highlight
the factors that influence the design and imple-
mentation of purely enzymatic cascades in
one-pot or multi-step pathways in an industrial
setting.
Keywords
Biocatalysis · Enzymatic cascades · In vitro
biotransformations · Enzymes · Cofactor
regeneration
3.1 Introduction
Reaction systems combining two or more chemi-
cal steps in one pot without isolation of reaction
intermediates are commonly referred to as
cascades [1]. In such systems, individual chemi-
cal steps can be enzyme catalyzed or involve
chemical (metal or organo-) catalysts. Accord-
ingly, multi-enzymatic cascades include several
S. Schmidt
Groningen Research Institute of Pharmacy, Chemical and
Pharmaceutical Biology, Groningen, The Netherlands
e-mail: s.schmidt@rug.nl
A. Schallmey
Technische Universität Braunschweig, Institute for
Biochemistry, Biotechnology and Bioinformatics,
Braunschweig, Germany
e-mail: a.schallmey@tu-braunschweig.de
R. Kourist (*)
Graz University of Technology, Institute of Molecular
Biotechnology, Graz, Austria
e-mail: kourist@tugraz.at
#
Springer Nature Switzerland AG 2021
S. Kara, F. Rudroff (eds.), Enzyme Cascade Design and Modelling,
https://doi.org/10.1007/978-3-030-65718-5_3
31
biocatalytic steps, which can be either performed
simultaneously or sequentially. In the first case,
also called concurrent cascade or tandem reac-
tion, all enzymes and reagents are present from
the beginning of the reaction, meaning that all
reaction steps take place at the same time. In
contrast, in a sequential multi-enzymatic cascade,
certain enzymes and/or reagents are added at a
later point in time after a certain sequence is
completed. Hence, all reaction steps of a sequen-
tial cascade are still performed in one pot but must
be separated in time. The latter might be neces-
sary if, e.g., two or more enzymes of the same
cascade require different reaction conditions, one
enzyme is inhibited by a compound appearing
prior or later in the sequence, or to prevent unde-
sired side reactions if cross-reactivities of the
involved enzymes occur. Moreover, in vitro and
in vivo multi-enzymatic cascades are distin-
guished depending on the biocatalyst preparation
(compare also Chap. 4). Whereas in in vivo
cascades all enzymes of the cascade are included
in whole living cells, in vitro cascades make use
of isolated enzymes in purified form, as cell-free
extracts, freeze-dried preparations, immobilized
versions, etc. [2].
In addition, cascade reactions in general can
exhibit different topologies (Fig. 3.1)[1, 3]. In a
linear c ascade (Fig. 3.1a), the product of one
chemical step serves as substrate of the
subsequent chemical step. This is probably the
most straightforward type of cascade reaction as
it avoids the isolation of (unstable) reaction
intermediates with the final goal to increase the
overall product yield while saving time and
resources.
Additionally, a linear cascade can be used to
shift an unfavorable reaction equilibrium of one
step by combination with a subsequent irrevers-
ible reaction step that pulls the product out of the
reaction. A recent example is the combination of
the hydroxynitrile lyase from Manihot esculenta
for the synth esis of optically pure (S)-4-
methoxymandelonitrile with the Candida
antarctica lipase A-catalyzed acylation of the
formed α-cyanohydrin yielding a stable ester
product (Scheme 3.1)[4]. This way, the equilib-
rium of the hydrocyanation reaction could be
shifted towards product formation and isolation
of the unstable cyanohydrin intermediate was
avoided.
Next to linear cascades, also orthogonal
(Fig. 3.1b), cyclic (Fig. 3.1c), and parallel,
interconnected (Fig. 3.1d) cascades have been
described. In an orthogonal enzyme cascade, the
conversion of a substrate into the desired product
is coupled with a second reaction to remove one
or more by-products. An example is the combina-
tion of a transaminase with lactate dehydroge-
nase, where the by-product pyruvate (when
using alanine as amine donor) of the
transaminase-catalyzed reaction is further
converted to lactic acid in order to shift the equi-
librium of the transaminase reaction (Scheme 3.2)
[5]. In a cyclic cascade, one enantiomer out of a
racemic substrate mixture is converted to an inter-
mediate product, which is then transformed back
to the racemic starting material yielding the
unreacted substrate enantiomer as final product.
The same applies if the unreacted substrate enan-
tiomer is racemized to yield enantiomerically
pure product (dynamic kinetic resolution).
Hence, cyclic cascades are commonly applied in
deracemization processes, e.g. of amino acids,
hydroxy acids, or amines [6, 7]. Finally, in a
parallel, interconnected cascade, two separate
biocatalytic reactions are connected by comple-
mentary cofactor requirements of the two
enzymes. Therefore, parallel, interconnected
cascades are commonly associated with cofactor
recycling systems. While in one biocatalytic step
a substrate is transform ed into the desired prod-
uct, a cheap co-substrate is converted to a
co-product in the parallel enzyme-catalyzed step
to recycle the required cofactor for the first
enzyme.
As briefly mentioned, cascade reactions offer
several advantages compared to conventional
reaction schemes [1]. This includes the avoidance
of operational work up steps, which saves time,
resources, and reagents, can reduce waste forma-
tion and, at the same time, allows for higher final
product yields. Additionally, different reaction
steps can be smartly combined in a cascade to
solve synthetic problems of a reaction sequence
such as cofactor regeneration, shift of reaction
32 S. Schmidt et al.
equilibria, in situ generation of toxic or instable
reagents, etc. On the other hand, the setup of
efficient cascade reactions is usually a complex
and challenging task [1]. Compatible reaction
conditions have to be identified and, in case of a
simultaneous cascade, the reaction rates of indi-
vidual steps have to be balanced. Moreover, pos-
sible problems, such as the formati on of
undesired side products due to cross-reactivities
of catalysts or the inhibition of an enzyme by a
compound appearing earlier or later in the reac-
tion sequence, can occur that have to be
addressed. Multi-enzymatic cascades are usually
easier to establish than chemoenzymatic or
chemo-catalytic cascade reactions as enzymes
commonly work in aqueous reaction media and
often display similar temperature and pH
requirements for optimal performance. Neverthe-
less, also several examples for the successful
combination of enzymatic and chemical reaction
steps in a cascade have been described in litera-
ture (compare also Cha p. 5) [8].
In an impressive way, nature successfully
evolved a multitude of highly complex conc urrent
cascade reactions. Living organisms built every
minute thousands of highly complex molecules
from simple precursors in an astonishing variety
and efficiency. To achieve such high efficiencies,
individual enzymatic transformations are
arranged in cascading sequences (biosynthetic
S
P
1
P
2
Cat. 1
Cat. 2
S
P
1
Cat. 1
P
2
P
2
Cat. 2
A
)
B)
C)
S
1
S
2
P
1
S
2
Cat. 1
Cat. 2
S
P
1
Cat. 1
S
2
P
2
Cat. 2
cofactor
red/ox
cofactor
ox/red
D)
Fig. 3.1 Possible
topologies of cascade
reactions. (a) Linear. (b)
Orthogonal. (c) Cyclic. (d)
Parallel, interconnected.
The scheme was adapted
from [1]
Scheme 3.1 Combination of Manihot esculenta hydroxynitrile lyase (MeHNL) and Candida antarctica lipase A (CAL
A) in a linear cascade to shift the reaction equilibrium of the first hydrocyanation reaction [4]
3 Multi-Enzymatic Cascades In Vitro 33
pathways) in living cells [9]. This strategy can be
mimicked in vitro by the design of artificial meta-
bolic pathways [10] through combination of mul-
tiple isolated enzymes in a homogeneous phase or
by combination of two or more catalytic activities
in a single protein, e.g., by fusion of genes
encoding different enzymes or by crosslinking
several enzymes [11–14]. Recently, a novel
approach has been mentioned in literature
named systems biocata lysis, which aims for the
in vitro setup of synthetic metabolic cycles for the
production of valuable compounds [15].
To illustrate the synthetic potential of multi-
enzyme cascades, but also potent ial challenges in
the development of cascade reactions, different
cascade examples have been selected and are
described in more detail in Sect. 3.2. Perhaps the
most frequent motivation for currently used
enzyme cascades is the concurrent regeneration
of (redox-) cofactors or expensive reagents. A
second application is the combination of
isomerizing enzymes or catalysts with highly
enantioselective enzymes for dynamic kinetic
resolutions, which allow to overcome the yield
limitation of 50% of kinetic resolutions. Cofactor
regeneration and dynamic kinetic resolutions
require concurrent cascades, which often makes
it very challenging to provide optimal reaction
conditions for both catalysts used. To overcome
compatibility issues, compartmentalization is a
possibility to enable different operating
conditions for all reaction steps of a concurrent
or step-wise cascade [8].
3.2 Cascades to Solve Synthetic
Problems
3.2.1 Combination of Selective
Enzymatic Steps
with Isomerizing Reactions
Due to their availability and ease to use,
hydrolases mainly constituted the first wave of
biocatalysts in the first biocatalytic processes
[16]. Kinetic resolution of racemic mixtures was
often the preferred reaction form (Fig. 3.2)
[17]. While this reaction is simple and robust, it
suffers from an intrinsic limitation of 50% yield,
which in turn requires the physical separation of
substrate and product. The addition of a second
catalyst, which racemizes the unreacted substrate,
but not the product (Fig. 3.2) in a cyclic cascade
allows for the complete conversion of starting
material to the desired product enantiomer. The
depletion of the faster-reacting substrate enantio-
mer leads to an increased conversion of the
slower-reacting substrate enantiomer as its rela-
tive concentration increases, which reduces the
optical purity of the product. To prevent this, the
racemization should be one order of magnitude
faster than the enantioselective reaction. This
makes the racemization steps often the bottleneck
of dynamic kinetic resolution reactions.
In 1997, Bäckvall et al. combined Ruthenium-
catalyzed hydrogen transfer reactions for the race-
mization of aryl aliphatic secondary alcohols with
their lipase-catalyzed kinetic resolution in a
R
1
R
2
O
R
1
R
2
NH
2
transaminase
+
H
3
N
COO
-
O
COO
-
lactate
dehydrogenase
HO
COO
-
NAD
+
NADH
+ H
+
cofactor
regeneration
co-substrate
co-product
Scheme 3.2 Combination
of transaminase and lactate
dehydrogenase in an
orthogonal cascade to shift
the reaction equilibrium of
the transaminase-catalyzed
reaction
34 S. Schmidt et al.
![Fig. 3.1 Possible topologies of cascade reactions. (a) Linear. (b) Orthogonal. (c) Cyclic. (d) Parallel, interconnected. The scheme was adapted from [1]](/figures/fig-3-1-possible-topologies-of-cascade-reactions-a-linear-b-29mtkyej.webp)