Team:Aachen/Implementation

What M.A.R.S. can achieve in the real world

Good scientific project is even better with practical applications

Adenosine triphosphate is one of the most abundant molecules in the metabolism of all organisms [1]. As the universal energy currency in every organism, most metabolic pathways are somehow dependent on ATP as an energy donor, or directly connected to some form of energy conversion inside the cell, which ultimately leads to the production of ATP. With its central role in the complex reaction networks of biochemical systems, due to its role as a crucial product of catabolism as well as a ubiquitous cofactor in various anabolic reactions, ATP is closely connected to nearly every reaction catalysed by enzymes. Even if ATP itself is not directly used to overcome the activation energy of enzymatic reactions, its role as a regulatory molecule reaches from the direct posttranslational modification of enzymes to partaking in signalling cascades down to the control of the expression of genomic material [2].

ATP can really be considered “the energy of life”.

In synthetic biology, scientists aim to unravel the mysteries of biological systems and to transform them into applications in the development of new fundamental biotechnological techniques or the industrial production of various products like pharmaceuticals, fine and bulk chemicals. An important trend in industrial biotechnology is the use of cell-free systems [3]. The natural environments of biochemical systems, the complex metabolic networks of cells, pose challenges for scientists.

How can the biochemical background noise of cells be minimized?

How can we achieve the most efficient state of a given pathway or reaction?

How can we provide the best results with the fewest need of resources to ensure a sustainable industrial application?

How can we guarantee a responsible and safe technology when using genetically modified systems in the industry?

One possible solution to provide the most efficient, sustainable, and safest way of harnessing the power of synthetic biology is the use of cell-free systems. It gives us an incredible head start to answer all the questions above, and many more. But there is also a new question, scientists must think about: How can our systems work, when we take away the organisms that produced them? The organisms that embedded these reactions in an intricate dance of equilibria. By trying to ensure efficiency, sustainability, and safety at the same time, we usually have to sacrifice the ideas of scientist completely, before any beyond conceptual work can be done, because we strip the processes of their fine tuned surroundings and of the energy sources that power them. This should not be the case! A crucial part of this complex problem is the lack of cofactor regeneration in cell free systems [4]. Here, M.A.R.S. enters the stage as a first of its kind cofactor regeneration system for the most abundant energy carrier in all life. Find out more about some of the possible applications in the real world. Learn about some of the various processes that heavily rely on ATP Recycling. Concerning the actual first application trials of our system in the synthetic biology industry, the examples demonstrated the focus on the use cases of ATP-dependent Enzymes, especially Carboxylic Acid Reductases (CARs).

Click on the light bulb to turn it on and discover the various applications of our biorecactor M.A.R.S.

M.A.R.S.

ATP-Regeneration

↓

Enzyme systems

free

immobilized

←

Pharmaceuticals ➤ One of the many ATP-dependent enzyme families is Carboxylic Acid Reductases (CARs). Their broad spectrum of catalysed reactions as well as their high stereoselectivity makes them an ideal tool for the production of complicated pharmaceuticals or precursors that could not be synthesized easily before [5]. CARs can be used to catalyse reactions by themselves, yet they are often part of more sophisticated enzyme cascades. An example for an important precursor for the production of pharmaceuticals is cinnamyl alcohol, which can be produced from glucose by the combination of CARs with other reductases [10].

A more recent proposal is the usage of CARs to synthesize (S)-2-aminobutanol, an important chiral precursor for the pharmaceutical compound Ethambutol, an antibiotic to treat tuberculosis [11] [12].

Another example for the relevance of ATP recycling systems in the industry is the use of ATP itself as a treatment for mitochondriopathy [13]. This shows that ATP’s potential as a vital compound in the production of pharmaceuticals is not purely secondary, as a cofactor of reaction. There is much more to it.

↓

Fine chemicals ➤ An example of the heavy dependence on fine chemicals apart from pharmaceutical production is the agrochemical sector. CARs find application in simple two-step reaction cascades to synthesize arbitrary secondary amines [7][8].

↓

Fundamental research
➤ ATP is crucial in every part of the body, from muscle to brain. Because it means to discover the secrets of the body itself, we need to understand the role of ATP in every part of it. In the brain, it plays an important role as messenger and activator for pain receptors like rP2X2 [9]. In electrophysiological experiments, these receptors get expressed in Xenopus laevis oocytes and their behavior is studied. Supplying new ATP for every experiment is very expensive and cannot be regenerated after experiments yet.

↓

Food production ➤ The promising CARs enzyme family has broad application in the synthesis of aldehyde components. [5]. In the food industry, many aldehydes are relevant as flavor and fragrance compounds like vanillin. The annual production of synthetic vanillin was estimated to be as high as 16,000 metric tons in 2019 [6].

→

Bulk chemicals ➤ CARs can be applied in various ways to produce classic bulk chemicals like alkanes or aliphatic alcohols. Many application pathways were proposed, especially for a chain length from six to 16 carbons for alcohols and seven to 15 carbons for alkanes [14]. These include the most popular and important aliphatic compounds in the whole chemical industry.

References

[1] Alberts, B., Bray, D., Hopkin, K., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P. (2012). Lehrbuch der Molekularen Zellbiologie, Kapitel 14 – Energiegewinnung in Mitochondrien und Chloroplasten
[2] Berg, J. M., Tymoczko, J. L., Stryer, L. (2014). Biochemie, Kapitel 15 – Der Stoffwechsel: Konzepte und Grundmuster
[3] Swartz, J. Developing cell-free biology for industrial applications. J IND MICROBIOL BIOTECHNOL 33, 476–485 (2006). https://doi.org/10.1007/s10295-006-0127-y
[4] G. A. Strohmeier, I. C. Eiteljörg, A. Schwarz, M. Winkler, Chem. Eur. J. 2019, 25, 6119
[5] France, S.P., Hepworth, L.J., Turner, N.J., Flitsch, S.L., 2017. Constructing biocatalyticcascades: in vitro and in vivo approaches to de novo multi-enzyme pathways. ACSCatal. 7, 710–724.https://doi.org/10.1021/acscatal.6b02979.
[6] Banerjee, G., Chattopadhyay, P., 2019. Vanillin biotechnology: the perspectives and fu-ture. J. Sci. Food Agric. 99, 499–506.https://doi.org/10.1002/jsfa.9303.
[7] Ramsden, J.I., Heath, R.S., Derrington, S., Montgomery, S.L., Mangas-Sanchez, J.,Mulholland, K.R., Turner, N.J., 2019. Biocatalytic N-alkylation of amines using eitherprimary alcohols or carboxylic acids via reductive aminase cascades. J. Am. Chem.Soc. 141, 1201–1206.https://doi.org/10.1021/jacs.8b11561.
[8] Schaffer, S., Corthals, J., Wessel, M., Hennemann, H.-G., Haeger, H., Volland, M., Roos,M., 2017. Producing amines and diamines from a carboxylic acid or dicarboxylic acidor a monoester thereof. US Patent USO09725746 B2
[9] Eva-Verena Bongartz, Jürgen Rettinger & Ralf Hausmann: Aminoglycoside block of P2X2 receptors heterologously expressed in Xenopus laevis oocytes; Purinergic Signalling (2010) 6:393–403 DOI 10.1007/s11302-010-9204-9
[10] Stereoselective synthesis of (S)-dapoxetine starting from trans-cinnamyl alcohol (08-3482EP), K. Venkatesan and K.V. Srinivasan, DOI: https://doi.org/10.3998/ark.5550190.0009.g28
[11] Dobrikov, G.M., Valcheva, V., Stoilova-Disheva, M., Momekov, G., Tzvetkova, P., Chimov,A., Dimitrov, V., 2012. Synthesis and in vitro antimycobacterial activity of com-pounds derived from (R)- and (S)-2-amino-1-butanol – the crucial role of the con-figuration. Eur. J. Med. Chem. 48, 45–56.https://doi.org/10.1016/j.ejmech.2011.11.035.
[12] Schwendenwein, D., Fiume, G., Weber, H., Rudroff, F., Winkler, M., 2016. Selective en-zymatic transformation to aldehydes in vivo by fungal carboxylate reductase from Neurospora crassa. Adv. Synth. Catal. 358, 3414–3421.https://doi.org/10.1002/adsc.201600914
[13] Sperl, W., Prokisch, H., Karall, D. et al. Mitochondriopathien. Monatsschr Kinderheilkd 159, 848 (2011). https://doi.org/10.1007/s00112-011-2447-x
[14] Akhtar, M.K., Dandapani, H., Thiel, K., Jones, P.R., 2015. Microbial production of 1-octanol: a naturally excreted biofuel with diesel-like properties. Metab. Eng.Commun. 2, 1–5.https://doi.org/10.1016/j.meteno.2014.11.001