Abstract

In natural metabolic networks, more than 2000 different biochemical reactions are operated and spatially and temporally co-ordinated in a reaction volume of <1 µm3. A similar level of control and precision has not been achieved in chemical synthesis, so far. Recently, synthetic biology succeeded in reconstructing complex synthetic in vitro metabolic networks (SIVMNs) from individual proteins in a defined fashion bottom-up. In this review, we will highlight some examples of SIVMNs and discuss how the further advancement of SIVMNs will require the structural organization of these networks and their reactions to (i) minimize deleterious side reactions, (ii) efficiently energize these networks from renewable energies, and (iii) achieve high productivity. The structural organization of synthetic metabolic networks will be a key step to create novel catalytic systems of the future and advance ongoing efforts of creating cell-like systems and artificial cells.

The power of natural metabolic networks

Nature is an incredible chemist. Photosynthesis uses the energy of light to convert inorganic carbon into the central building blocks of life. These are further diversified and give rise to more than 200 000 different chemical compounds that have been purified from various organisms to date [1]. All this is achieved in an incredibly dynamic, self-regulated, self-optimized and synchronized fashion. For instance, there are more than 2000 different reactions that can take place at the same time in one Escherichia coli cell [2,3] with a volume of ∼1 µm3, corresponding to 10−15 l [4].

The synthetic capabilities of Nature are unmatched by humankind. Even though chemists have achieved exquisite control over individual chemical reactions [5], synthetic organic chemistry is to a large extent still pursued in individual, sequential steps, which makes the build-up of a chemical structure a tedious and multi-step process (Figure 1). This modus operandi requires several follow-up steps between individual reactions, such as separation, purification, and sometimes even crystallization. This, in turn, needs additional resources, such as solvents, and energy and most importantly also time, which makes stepwise chemical synthesis very often a cost-intense, wasteful undertaking [6,7]. As an example, the total synthesis of vitamin B12 (cobalamin) requires more than 70 steps at a total yield that ‘never should be asked for' (A. Eschenmoser) [8], probably below 0.01‰ [9]. In contrast the same compound can be produced by the bacterium Pseudomonas denitrificans at 200 mg l−1 over a 180 h fermentation [10,11].

Comparison of synthetic chemistry, biotechnology and SIVMNs.

Figure 1.
Comparison of synthetic chemistry, biotechnology and SIVMNs.

In synthetic chemistry, the synthesis of compound D from substrate A is carried out in separate steps, which requires the separation and purification of intermediates between the different steps. This is a time- and resource-intense process. The overall yield is lowered due to losses during synthesis and purification. Biotechnology is an integrated production process, however, some of the substrate is used by the cell to provide the energy for the synthesis and some of the substrate is drained into biomass to allow the cells to grow and divide, which leads to an overall decrease in yield. Interactions of the metabolites with the native metabolic network of the cell make engineering of the biotechnological process a complicated task. SIVMNs ideally combine the advantages of both systems. SIVMNs are defined multi-reaction networks that are assembled in a bottom-up fashion, which do not drain metabolites into other processes. External energy from renewable sources or even electricity might be used to power the catalytic network.

Figure 1.
Comparison of synthetic chemistry, biotechnology and SIVMNs.

In synthetic chemistry, the synthesis of compound D from substrate A is carried out in separate steps, which requires the separation and purification of intermediates between the different steps. This is a time- and resource-intense process. The overall yield is lowered due to losses during synthesis and purification. Biotechnology is an integrated production process, however, some of the substrate is used by the cell to provide the energy for the synthesis and some of the substrate is drained into biomass to allow the cells to grow and divide, which leads to an overall decrease in yield. Interactions of the metabolites with the native metabolic network of the cell make engineering of the biotechnological process a complicated task. SIVMNs ideally combine the advantages of both systems. SIVMNs are defined multi-reaction networks that are assembled in a bottom-up fashion, which do not drain metabolites into other processes. External energy from renewable sources or even electricity might be used to power the catalytic network.

Limitations of natural metabolic networks

So, can we not simply harness the power of natural metabolism to create a more efficient, sustainable and integrated chemistry? For more than three decades, biotechnology and in particular metabolic engineering has striven to program cells into chemical factories that produce a wanted compound from a given starting material, very often a renewable carbon source. Impressive progress was made; there are yeast strains that are able to produce complex chemical structures at 25 g l−1 [12] and there is an ever-growing number of complex molecules that can be derived through metabolically engineered cells [13,14]. Vitamin B12, for instance, can now be produced at 300 µg g−1 cell dry weight within 24 h in a recombinant E. coli strain after introduction of 28 additional genes [15].

However, the biosynthetic reaction space of cells is limited by the number of naturally existing enzymes, as well as the narrow reaction conditions that can be operated inside of the cell (pH, salts, ionic strengths) [16]. Also, the engineering of novel biosynthetic processes very often requires complex manipulations of the regulatory and metabolic networks of a cell. As a simple exercise, imagine the introduction of five new non-native reactions and metabolites into an E. coli strain. There are potentially 5 × 2000 interactions with the native metabolic network of the cell possible (e.g. in form cross-reactivities, degradation, side reactions, inhibitory and allosteric interactions). This number even increases, when considering the potential interactions of these new metabolites with the regulatory network of the cell (i.e. several hundred different transcription factors, regulators, etc.). Lastly, the fraction of substrate carbon that can be channeled into a desired biosynthetic product is very often constrained, because of the many parallel operating reaction networks inside of a living cell, which overall reduces the possible yield and atom efficiency of a given process (Figure 1). Overall, these facts limit the possibilities to replace synthetic routes by biotechnological processes.

The vision of synthetic in vitro metabolic networks

From one — arguably provocative — point of view, the use of biotechnology can be understood as an effort to overcome our current technical incapability to achieve a similar level of precision, coordination, as well as temporal and spatial control of catalysis in technical systems. Ideally, instead of the laborious re-wiring of metabolism and dealing with the disadvantages of cellular systems, it would be more desirable if we were able to build catalytic networks that allow the multi-step synthesis of the desired compound at high yields and atom efficiency (Figure 1). An important step towards achieving this goal is the successful assembly of (bio)catalytic reaction networks in a bottom-up fashion in vitro with purified proteins [17].

Several recent examples demonstrated that it is possible to construct and operate complex multi-enzyme reaction cascades outside of a cellular context. These synthetic in vitro metabolic networks (SIVMNs) are drafted based on physical and chemical principles, assembled from individual enzymes and further optimized in several rounds [17,18]. Examples are the formation of polyhydroxybutyrate from glucose through a 18-enzyme network [19], a synthetic pathway that converts glucose in more than 20 steps into geranyl pyrophosphate, which in turn is used to prenylate different compounds [20], as well as a synthetic pathway (‘CETCH cycle’) for the continuous capture and conversion of CO2 that was constructed from 17 different enzymes, including three re-engineered enzymes [18].

Although these systems are fascinating examples of what can be achieved today, they are only the first steps on the way of building more efficient catalytic systems in the future. Automation will allow fast prototyping to determine optimal concentrations of individual components of a given SIVMN. Enzyme engineering will extend the range pf possible transformations that can be used in SIVMNs. Lastly, the prospects to operate SIVMNs at non-physiological reaction conditions, include new-to-nature materials and even synthetic-chemical catalysts will help to further expand the solution space of SIVMNs beyond the realm of natural metabolism. In other words, the real potential of SIVMNs lies not in the efforts to establish naturally existing metabolic routes outside of a living cell, but in the possibility to create completely new bio-inspired catalytic networks that outcompete naturally evolved metabolism in efficiency and productivity.

The challenges of building SIVMNs

What are the challenges in building and operating SIVMNs? One particular challenge is that many enzymes show inherent promiscuity [21], which becomes a problem for the assembly of complex networks and scales with the number of reaction steps as well as the diversity of the biological origin of the individual components used. Deleterious side reactions and unfavorable interactions between different metabolites and enzymes of a network can cause the accumulation of unwanted metabolites and ultimately the breakdown of SIVMNs [22]. To prevent this from happening, the construction of synthetic in vitro metabolic networks very often requires the engineering and/or replacement of problematic enzymes, or the introduction of metabolite proof-reading strategies that recycle dead-end metabolites [18,23].

An alternative mitigation strategy is to spatially separate problematic reactions, as happens in natural systems, which show a high degree of structural organization. A yeast cell, for instance, features several organelles that serve as separate reaction compartments in which complicated and incompatible chemistries can be operated in parallel (Figure 2). The discovery of phase separation as an active principle to create distinct non-membrane bound compartments is another example for the spatial organization of biological components inside living cells. Within microbial cells, organelle-like structures can be found, too. Examples are bacterial microcompartments, which serve as carbon concentrating and/or detoxification chambers or encapsulins (Figure 2). Lastly, spatial confinement can also be achieved on the single enzyme level (Figure 2). The recent report of a dynamically regulated multi-catalytic reaction chamber that sequesters a toxic metabolite illustrates the elaborate design of such naturally evolved protein nanoreactors [24,25].

Spatial organization of metabolism in natural systems and potential realization in SIVMNs.

Figure 2.
Spatial organization of metabolism in natural systems and potential realization in SIVMNs.

Natural organization spans several orders of magnitude. From organelles, such as the chloroplast and its thylakoids that convert light into chemical energy, bacterial microcompartments and encapsulins that encapsulate various proteins, metabolic enzymes and cofactors, to single protein nanoreactors, such as propionyl-CoA synthase, that sequester toxic metabolites. Data and pictures from [24,51,52]. Thylakoid scheme obtained with permission from Miller T. Building an artificial chloroplast drop-by-drop. Lecture presented at Engineering Life; 2019; Dresden.

Figure 2.
Spatial organization of metabolism in natural systems and potential realization in SIVMNs.

Natural organization spans several orders of magnitude. From organelles, such as the chloroplast and its thylakoids that convert light into chemical energy, bacterial microcompartments and encapsulins that encapsulate various proteins, metabolic enzymes and cofactors, to single protein nanoreactors, such as propionyl-CoA synthase, that sequester toxic metabolites. Data and pictures from [24,51,52]. Thylakoid scheme obtained with permission from Miller T. Building an artificial chloroplast drop-by-drop. Lecture presented at Engineering Life; 2019; Dresden.

Altogether, these examples emphasize the importance of spatially structuring individual reaction sequences in natural metabolic networks and show the great potential of this organizational principle to achieve improved and robust operation of SIVMNs (Figure 2). While the community has started to re-purpose and re-design spatially organized enzymes [26], protein complexes and reactors, application of these principles remains still to a large extent underexplored in the design of SIVMNs. Most promising are current efforts to engineer naturally existing microcompartments for new cargo [2729] or to build and load protein cages de novo [30,31]. Further progress in protein scaffold design will expand our possibilities of spatially structuring catalysis in SIVMNs the future [3234].

Energizing SIVMNs

Another challenge is how to power SIVMNs. In some cases, the supply of reducing power and chemical energy is deliberately designed into the network to create a stochiometrically balanced process [19]. An example is the production of bioplastics from glucose. However, in many cases, this is not possible, so that the required reducing power and chemical energy either needs to be balanced with purge valves [35] and molecular rheostats [36] or provided through dedicated external energy modules [18]. Well-known are polyphosphate- and creatine phosphate-based ATP regeneration systems [3740] and regeneration systems for NADH and NADPH based on formate-, glucose- or alcohol-dehydrogenase. So far, all these regeneration systems require the supply in the form of chemical energy, which is very often provided from petrochemical or biological sources, representing an indirect way to energize SIVMNs.

In the long run, hydrogen-, light- or even electricity-based systems could be used to provide an alternative, renewable ways to energize in vitro metabolic networks directly. Again, the three-dimensional organization of these systems will become important for these efforts, especially if membrane-bound regeneration systems featuring photosynthetic or ATP-synthesizing complexes are to be used. Recent work has shown that naturally isolated and synthetically created vesicle systems are indeed able to provide energy and reducing equivalents [4143]. The further development of efficient microfluidics-based tools for the creation and manipulation of droplets and vesicles [44], as well as the use of coacervates and polymers [45] will open the way to assemble higher organized systems in which SIVMNs are powered by compartmentalized energy modules, resembling the architecture of a natural cells with its organelles [46].

Structural confinement to achieve high yields

Lastly, the productivity of SIVMNs is still an issue that needs to be solved, for any long-term operation, upscale or ultimately commercial application of these systems. Strategies to enhance productivity will include increasing protein stability, introducing effective metabolite detoxification and proof-reading systems (see above), but also protection against oxidative stress and damage. The latter task can be achieved by introducing enzymes to the SIVMN that maintain a reducing reaction environment or lower the concentration of oxygen and reactive oxygen species. Structural elements can augment these efforts and provide alternative solutions, e.g. by creating protein- or membrane-bound reaction compartments for the SIVMN that would allow operating the network in a protected environment.

Note that encapsulation provides another advantage. Introducing a physical barrier that separates a reaction compartment from the environment automatically creates a source-sink situation. This will, in turn, allow the establishment of a vectorial gradient, if the barrier (capsule or membrane) is specifically permeable for a given metabolite. In other words, if the product of a given SIVMN can be specifically exported to the outside of the reaction compartment, this would allow longer operation of the SIVMN by keeping the system in an out-of-the-equilibrium state and removing any product feedback inhibition loops, plus, it would enable an easier separation of the product from the SIVMN. While the introduction of such selective membranes is a technical challenge that needs to be solved, it would allow the creation of a similar situation that is found in naturally existing cells, which features specific exporters and importers. Also, it should be mentioned that the possibility to use non-biological materials, such as polymers or coacervates [47] could open new options for building selectively permissive compartments.

Controlling SIVMNs

Power is nothing without control. While this review has explored the chances of structurally organizing SIVMNs, it should not go unmentioned that temporal organization and control of metabolic networks are as important, especially when we aim at creating robust catalytic reaction networks that are resilient against perturbations and stochastic fluctuations [48,49]. This challenge, however, will be discussed by another article in this mini-review series [50].

Conclusions

SIVMNs have the potential to provide several advantages compared with chemical and biotechnological synthesis processes. However, to overcome the current limitations of SIVMNs it will be essential to spatially organize SIVMNs, which will allow improvement of their performance by suppressing deleterious interactions between enzymes and metabolites, coupling them to renewable energy modules and increasing product yield. The implementation of the spatial design into SIVMNs will be a necessary prerequisite to leverage their full potential and move catalytic networks into the next dimension.

Summary

  • Natural metabolic networks, provide synthetic routes to many different chemical compounds, but are limited in malleability and productivity.

  • Synthetic chemistry is currently unable to build, operate and control complex catalytic networks.

  • Synthetic in vitro metabolic networks (SIVMN) provide a new approach to build novel biosynthetic routes in a rational fashion, thus bridging the natural and the synthetic world.

  • To increase the performance of SIVMN, structural organization of the enzymes and components will be required.

  • The structural organization of SIVMN will be a key step to create catalytic systems of the future and advance ongoing efforts of creating cell-like systems and artificial cells.

Abbreviations

     
  • SIVMNs

    synthetic in vitro metabolic networks

Acknowledgements

The author acknowledges support through the MaxSynBio network of the Max Planck Society and the European Research Council.

Competing Interests

The Author declares that there are no competing interests associated with this manuscript.

References

References
1
Wink
,
M.
(
2003
)
Evolution of secondary metabolites from an ecological and molecular phylogenetic perspective
.
Phytochemistry
64
,
3
19
2
Weaver
,
D.S.
,
Keseler
,
I.M.
,
Mackie
,
A.
,
Paulsen
,
I.T.
and
Karp
,
P.D.
(
2014
)
A genome-scale metabolic flux model of Escherichia coli K-12 derived from the EcoCyc database
.
BMC Syst. Biol.
8
,
79
3
Orth
,
J.D.
,
Conrad
,
T.M.
,
Na
,
J.
,
Lerman
,
J.A.
,
Nam
,
H.
,
Feist
,
A.M.
et al.  (
2011
)
A comprehensive genome-scale reconstruction of Escherichia coli metabolism — 2011
.
Mol. Syst. Biol.
7
,
535
4
Kubitschek
,
H.E.
(
1969
)
Growth during the bacterial cell cycle: analysis of cell size distribution
.
Biophys. J.
9
,
792
809
5
Nicolaou
,
K.C.
,
Vourloumis
,
D.
,
Winssinger
,
N.
and
Baran
,
P.S.
(
2000
)
The art and science of total synthesis at the Dawn of the twenty-first century
.
Angew. Chem. Int. Ed. Engl.
39
,
44
122
6
Horváth
,
I.T.
and
Anastas
,
P.T.
(
2007
)
Innovations and green chemistry
.
Chem. Rev.
107
,
2169
2173
7
Li
,
C.-J.
and
Trost
,
B.M.
(
2008
)
Green chemistry for chemical synthesis
.
Proc. Natl Acad. Sci. U.S.A.
105
,
13197
13202
8
Seeman
,
J.I.
(
2013
)
The Nozoe autograph books: stories behind the stories
.
Chem. Rec.
13
,
483
514
9
Eschenmoser
,
A.
and
Wintner
,
C.E.
(
1977
)
Natural product synthesis and vitamin B12
.
Science
196
,
1410
1420
10
Li
,
K.-T.
,
Liu
,
D.-H.
,
Chu
,
J.
,
Wang
,
Y.-H.
,
Zhuang
,
Y.-P.
and
Zhang
,
S.-L.
(
2008
)
An effective and simplified pH-stat control strategy for the industrial fermentation of vitamin B(12) by Pseudomonas denitrificans
.
Bioprocess Biosyst. Eng.
31
,
605
610
11
Xia
,
W.
,
Chen
,
W.
,
Peng
,
W.-F.
and
Li
,
K.-T.
(
2015
)
Industrial vitamin B12 production by Pseudomonas denitrificans using maltose syrup and corn steep liquor as the cost-effective fermentation substrates
.
Bioprocess Biosyst. Eng.
38
,
1065
1073
12
Paddon
,
C.J.
,
Westfall
,
P.J.
,
Pitera
,
D.J.
,
Benjamin
,
K.
,
Fisher
,
K.
,
McPhee
,
D.
et al.  (
2013
)
High-level semi-synthetic production of the potent antimalarial artemisinin
.
Nature
496
,
528
532
13
Galanie
,
S.
,
Thodey
,
K.
,
Trenchard
,
I.J.
,
Filsinger Interrante
,
M.
and
Smolke
,
C.D.
(
2015
)
Complete biosynthesis of opioids in yeast
.
Science
349
,
1095
1100
14
Li
,
Y.
and
Smolke
,
C.D.
(
2016
)
Engineering biosynthesis of the anticancer alkaloid noscapine in yeast
.
Nat. Commun.
7
,
12137
15
Fang
,
H.
,
Li
,
D.
,
Kang
,
J.
,
Jiang
,
P.
,
Sun
,
J.
and
Zhang
,
D.
(
2018
)
Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12
.
Nat. Commun.
9
,
4917
16
Claassens
,
N.J.
,
Burgener
,
S.
,
Vögeli
,
B.
,
Erb
,
T.J.
and
Bar-Even
,
A.
(
2019
)
A critical comparison of cellular and cell-free bioproduction systems
.
Curr. Opin. Biotechnol.
60
,
221
229
17
Erb
,
T.J.
,
Jones
,
P.R.
and
Bar-Even
,
A.
(
2017
)
Synthetic metabolism: metabolic engineering meets enzyme design
.
Curr. Opin. Chem. Biol.
37
,
56
62
18
Schwander
,
T.
,
Schada von Borzyskowski
,
L.
,
Burgener
,
S.
,
Cortina
,
N.S.
and
Erb
,
T.J.
(
2016
)
A synthetic pathway for the fixation of carbon dioxide in vitro
.
Science
354
,
900
904
19
Opgenorth
,
P.H.
,
Korman
,
T.P.
and
Bowie
,
J.U.
(
2016
)
A synthetic biochemistry module for production of bio-based chemicals from glucose
.
Nat. Chem. Biol.
12
,
393
395
20
Valliere
,
M.A.
,
Korman
,
T.P.
,
Woodall
,
N.B.
,
Khitrov
,
G.A.
,
Taylor
,
R.E.
,
Baker
,
D.
et al.  (
2019
)
A cell-free platform for the prenylation of natural products and application to cannabinoid production
.
Nat. Commun.
10
,
565
21
Copley
,
S.D.
(
2017
)
Shining a light on enzyme promiscuity
.
Curr. Opin. Struct. Biol.
47
,
167
175
22
Lerma-Ortiz
,
C.
,
Jeffryes
,
J.G.
,
Cooper
,
A.J.
,
Niehaus
,
T.D.
,
Thamm
,
A.M.
,
Frelin
,
O.
et al.  (
2016
)
‘Nothing of chemistry disappears in biology’: the Top 30 damage-prone endogenous metabolites
.
Biochem. Soc. Trans.
44
,
961
971
23
Sun
,
J.
,
Jeffryes
,
J.G.
,
Henry
,
C.S.
,
Bruner
,
S.D.
and
Hanson
,
A.D.
(
2017
)
Metabolite damage and repair in metabolic engineering design
.
Metab. Eng.
44
,
150
159
24
Bernhardsgrütter
,
I.
,
Vögeli
,
B.
,
Wagner
,
T.
,
Peter
,
D.M.
,
Cortina
,
N.S.
,
Kahnt
,
J.
et al.  (
2018
)
The multicatalytic compartment of propionyl-CoA synthase sequesters a toxic metabolite
.
Nat. Chem. Biol.
14
,
1127
1132
25
Alber
,
B.E.
and
Fuchs
,
G.
(
2002
)
Propionyl-coenzyme A synthase from Chloroflexus aurantiacus, a key enzyme of the 3-hydroxypropionate cycle for autotrophic CO2 fixation
.
J. Biol. Chem.
277
,
12137
12143
26
Bernhardsgrütter
,
I.
,
Schell
,
K.
,
Peter
,
D.M.
,
Borjian
,
F.
,
Saez
,
D.A.
,
Vöhringer-Martinez
,
E.
et al.  (
2019
)
Awakening the sleeping carboxylase function of enzymes: engineering the natural CO2-binding potential of reductases
.
J. Am. Chem. Soc.
141
,
9778
9782
27
Ferlez
,
B.
,
Sutter
,
M.
and
Kerfeld
,
C.A.
(
2019
)
A designed bacterial microcompartment shell with tunable composition and precision cargo loading
.
Metab. Eng.
54
,
286
291
28
Hagen
,
A.R.
,
Plegaria
,
J.S.
,
Sloan
,
N.
,
Ferlez
,
B.
,
Aussignargues
,
C.
,
Burton
,
R.
et al.  (
2018
)
In vitro assembly of diverse bacterial microcompartment shell architectures
.
Nano Lett.
18
,
7030
7037
29
Tetter
,
S.
and
Hilvert
,
D.
(
2017
)
Enzyme encapsulation by a ferritin cage
.
Angew. Chem. Int. Ed. Engl.
56
,
14933
6
30
Azuma
,
Y.
,
Bader
,
D.L.V.
and
Hilvert
,
D.
(
2018
)
Substrate sorting by a supercharged nanoreactor
.
J. Am. Chem. Soc.
140
,
860
863
31
Sasaki
,
E.
,
Böhringer
,
D.
,
van de Waterbeemd
,
M.
,
Leibundgut
,
M.
,
Zschoche
,
R.
,
Heck
,
A.J.
et al.  (
2017
)
Structure and assembly of scalable porous protein cages
.
Nat. Commun.
8
,
14663
32
Lee
,
M.J.
,
Mantell
,
J.
,
Hodgson
,
L.
,
Alibhai
,
D.
,
Fletcher
,
J.M.
,
Brown
,
I.R.
et al.  (
2018
)
Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm
.
Nat. Chem. Biol.
14
,
142
147
33
Ross
,
J.F.
,
Bridges
,
A.
,
Fletcher
,
J.M.
,
Shoemark
,
D.
,
Alibhai
,
D.
,
Bray
,
H.E.V.
et al.  (
2017
)
Decorating self-assembled peptide cages with proteins
.
ACS Nano
11
,
7901
7914
34
Burton
,
A.J.
,
Thomson
,
A.R.
,
Dawson
,
W.M.
,
Brady
,
R.L.
and
Woolfson
,
D.N.
(
2016
)
Installing hydrolytic activity into a completely de novo protein framework
.
Nat. Chem.
8
,
837
844
35
Opgenorth
,
P.H.
,
Korman
,
T.P.
and
Bowie
,
J.U.
(
2014
)
A synthetic biochemistry molecular purge valve module that maintains redox balance
.
Nat. Commun.
5
,
4113
36
Opgenorth
,
P.H.
,
Korman
,
T.P.
,
Iancu
,
L.
and
Bowie
,
J.U.
(
2017
)
A molecular rheostat maintains ATP levels to drive a synthetic biochemistry system
.
Nat. Chem. Biol.
13
,
938
942
37
Nocek
,
B.
,
Kochinyan
,
S.
,
Proudfoot
,
M.
,
Brown
,
G.
,
Evdokimova
,
E.
,
Osipiuk
,
J.
et al.  (
2008
)
Polyphosphate-dependent synthesis of ATP and ADP by the family-2 polyphosphate kinases in bacteria
.
Proc. Natl Acad. Sci. U.S.A.
105
,
17730
17735
38
Andexer
,
J.N.
and
Richter
,
M.
(
2015
)
Emerging enzymes for ATP regeneration in biocatalytic processes
.
Chembiochem
16
,
380
386
39
Mordhorst
,
S.
,
Singh
,
J.
,
Mohr
,
M.K.F.
,
Hinkelmann
,
R.
,
Keppler
,
M.
,
Jessen
,
H.J.
et al.  (
2019
)
Several polyphosphate kinase 2 enzymes catalyse the production of adenosine 5′-polyphosphates
.
Chembiochem
20
,
1019
1022
40
Shih
,
Y.H.
and
Whitesides
,
G.M.
(
1977
)
Large-scale ATP-requiring enzymatic phosphorylation of creatine can be driven by enzymatic ATP regeneration
.
J. Org. Chem.
42
,
4165
4166
41
Berhanu
,
S.
,
Ueda
,
T.
and
Kuruma
,
Y.
(
2019
)
Artificial photosynthetic cell producing energy for protein synthesis
.
Nat. Commun.
10
,
1325
42
Beneyton
,
T.
,
Krafft
,
D.
,
Bednarz
,
C.
,
Kleineberg
,
C.
,
Woelfer
,
C.
,
Ivanov
,
I.
et al.  (
2018
)
Out-of-equilibrium microcompartments for the bottom-up integration of metabolic functions
.
Nat. Commun.
9
,
2391
43
Lee
,
K.Y.
,
Park
,
S.-J.
,
Lee
,
K.A.
,
Kim
,
S.-H.
,
Kim
,
H.
,
Meroz
,
Y.
et al.  (
2018
)
Photosynthetic artificial organelles sustain and control ATP-dependent reactions in a protocellular system
.
Nat. Biotechnol.
36
,
530
535
44
Weiss
,
M.
,
Frohnmayer
,
J.P.
,
Benk
,
L.T.
,
Haller
,
B.
,
Janiesch
,
J.-W.
,
Heitkamp
,
T.
et al.  (
2018
)
Sequential bottom-up assembly of mechanically stabilized synthetic cells by microfluidics
.
Nat. Mater.
17
,
89
96
45
Ma
,
B.C.
,
da Silva L
,
C.
,
Jo
,
S.M.
,
Wurm
,
F.R.
,
Bannwarth
,
M.B.
,
Zhang
,
K.A.I.
et al.  (
2019
)
Polymer-based module for NAD(+) regeneration with visible light
.
Chembiochem
46
Schwille
,
P.
,
Spatz
,
J.
,
Landfester
,
K.
,
Bodenschatz
,
E.
,
Herminghaus
,
S.
,
Sourjik
,
V.
et al.  (
2018
)
Maxsynbio: avenues towards creating cells from the bottom up
.
Angew. Chem. Int. Ed. Engl.
57
,
13382
13392
47
Li
,
M.
,
Huang
,
X.
,
Tang
,
T.-Y.
and
Mann
,
S.
(
2014
)
Synthetic cellularity based on non-lipid micro-compartments and protocell models
.
Curr. Opin. Chem. Biol.
22
,
1
11
48
Helwig
,
B.
,
van Sluijs
,
B.
,
Pogodaev
,
A.A.
,
Postma
,
S.G.J.
and
Huck
,
W.T.S.
(
2018
)
Bottom-up construction of an adaptive enzymatic reaction network
.
Angew. Chem. Int. Ed. Engl.
57
,
14065
14069
49
Pogodaev
,
A.A.
,
Wong
,
A.S.Y.
and
Huck
,
W.T.S.
(
2017
)
Photochemical control over oscillations in chemical reaction networks
.
J. Am. Chem. Soc.
139
,
15296
15299
50
Maguire
,
O.
and
Huck
,
W.
On the importance of reaction networks for synthetic living systems
.
Emerg. Top. Life Sci.
3
,
51
Engel
,
B.D.
,
Schaffer
,
M.
,
Kuhn Cuellar
,
L.
,
Villa
,
E.
,
Plitzko
,
J.M.
and
Baumeister
,
W.
(
2015
)
Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography
.
eLife
4
,
e04889
52
Sutter
,
M.
,
Boehringer
,
D.
,
Gutmann
,
S.
,
Günther
,
S.
,
Prangishvili
,
D.
,
Loessner
,
M.J.
et al.  (
2008
)
Structural basis of enzyme encapsulation into a bacterial nanocompartment
.
Nat. Struct. Mol. Biol.
15
,
939
947
This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology and distributed under the Creative Commons Attribution License 4.0 (CC BY-NC-ND).