Periodic Reporting for period 3 - CLASSY (Cell-Like ‘Molecular Assembly Lines’ of Programmable Reaction Sequences as Game-Changers in Chemical Synthesis)
Reporting period: 2022-05-01 to 2024-04-30
CLASSY’s overarching objective is to create a microfluidic platform of microreactors, to emulate living cells in their capacity to self-regulate and catalyse programmable multistep synthetic processes. This microfluidic platform of microreactors will be able to synthesise complex molecules through programmable reaction sequences in molecular assembly lines. The main scientific challenges in creating cell-like molecular assembly lines include achieving an efficient compartmentalisation of the different steps for each reaction sequence and regulating such reaction sequences in a way that enables the same chemical reactor to produce a wide range of different molecules. Overcoming these challenges, however, will revolutionise chemical synthesis and provide a solution for close-to-zero waste streams and a sustainable ‘green chemical factory of the future’. Bringing together the expertise of leading scientists in systems chemistry, biocatalysis and microfluidics, the consortium has set three specific objectives that will be addressed: (1) the development of a microfluidic platform for the immobilisation of multiple enzymes or peptide catalysts in microfluidic compartments, so to produce a versatile set of flow reactors that can catalyse a variety of single-step reactions; (2) the delivery of a new type of hybrid molecules capable to selectively control the catalysis of specific single-step reactions through programmable activation/deactivation of self-synthesising catalysts; (3) the study of microfluidic programming of cascade reactions by selective activation/deactivation of catalysts that operate sequentially.
During the third reporting period of the CLASSY project, significant results have been achieved according to the general project goals (Figure 1). The main results include:
In the last project stage, the consortium has kept advancing various concepts that relate catalytic synthetic approaches to the way in which catalysis occurs in biology. That implies, for example, the efforts carried out to tune catalytic activity through self-assembly processes, making use of nucleic acid analogues and peptide (NA-pep) chimeras where the peptide is responsible for the catalytic activity and the NA sequence to control the self-assembled structure. Extending such an approach to NA-pep replicating systems, allows us to control two complementary orthogonal functions such as replication and catalysis (Figure 1B). Currently, we are optimising the dynamics of both processes so that they reinforce each oher. In all these studies it is very important to understand the systems behaviour of the involved assemblies.
Another concept of biology that affects catalysis and has been investigated within the CLASSY project is that of compartmentalisation. The compartmentalisation of catalytic reactions can be performed in different ways, and CLASSY has explored various of them, including the use of microfluidic flow reactors, different types of NA-pep assemblies (e.g. liquid droplets as a consequence of liquid-liquid phase separation), and photoswitchable NA-based hydrogels. Importantly, in studies with peptide replication networks, it has been demonstrated that compartmentalisation in microfluidic flow reactors can lead to chemical oscillations. For studies in flow (Figure 1A), two different microfluidic setups have been developed. The first involves a microfluidic platform of multiple reactors that are connected and can be filled with enzyme-loaded beads. As a second strategy, an alternative way to deal with reaction networks (e.g. enzymatic ones) has been explored intensively in the third reporting period, and is based on a prototype of a microfluidic cell-like molecular assembly line where the syringe pumps, the microfluidic reactor, an ultraviolet–visible (UV-vis) spectrophotometer and a trapped ion mobility spectrometry – time of flight (timsTOF) mass spectrometer are all connected.
Finally, various teams of the CLASSY consortium have concentrated on the demonstration of life-like enzymatic reaction sequences and networks, employing microfluidic flow setups to facilitate the experiments (Figure 1C). On one hand, aiming for cascades with separated sites of reaction and also the possibility to control the activity with light, a light-dependent decarboxylase has been successfully combined with the biocatalytic hydrolysis of triolein in flow. On the other hand, for developing a prototype of a cell-like molecular assembly line, a suitable microfluidic setup (see above) has allowed ten distinct species to be mixed directly in the reaction chamber, where a cascade reaction takes place. Advanced trapped ion mobility spectrometry – time of flight (timsTOF) instrumentation was then used to quantitively monitor species of interest online. As a proof of concept of the first cell-like molecular assembly line, the pentose phosphate pathway has been investigated using free enzymes in flow conditions.
In the last project stage, the consortium has kept advancing various concepts that relate catalytic synthetic approaches to the way in which catalysis occurs in biology. That implies, for example, the efforts carried out to tune catalytic activity through self-assembly processes, making use of nucleic acid analogues and peptide (NA-pep) chimeras where the peptide is responsible for the catalytic activity and the NA sequence to control the self-assembled structure. Extending such an approach to NA-pep replicating systems, allows us to control two complementary orthogonal functions such as replication and catalysis (Figure 1B). Currently, we are optimising the dynamics of both processes so that they reinforce each oher. In all these studies it is very important to understand the systems behaviour of the involved assemblies.
Another concept of biology that affects catalysis and has been investigated within the CLASSY project is that of compartmentalisation. The compartmentalisation of catalytic reactions can be performed in different ways, and CLASSY has explored various of them, including the use of microfluidic flow reactors, different types of NA-pep assemblies (e.g. liquid droplets as a consequence of liquid-liquid phase separation), and photoswitchable NA-based hydrogels. Importantly, in studies with peptide replication networks, it has been demonstrated that compartmentalisation in microfluidic flow reactors can lead to chemical oscillations. For studies in flow (Figure 1A), two different microfluidic setups have been developed. The first involves a microfluidic platform of multiple reactors that are connected and can be filled with enzyme-loaded beads. As a second strategy, an alternative way to deal with reaction networks (e.g. enzymatic ones) has been explored intensively in the third reporting period, and is based on a prototype of a microfluidic cell-like molecular assembly line where the syringe pumps, the microfluidic reactor, an ultraviolet–visible (UV-vis) spectrophotometer and a trapped ion mobility spectrometry – time of flight (timsTOF) mass spectrometer are all connected.
Finally, various teams of the CLASSY consortium have concentrated on the demonstration of life-like enzymatic reaction sequences and networks, employing microfluidic flow setups to facilitate the experiments (Figure 1C). On one hand, aiming for cascades with separated sites of reaction and also the possibility to control the activity with light, a light-dependent decarboxylase has been successfully combined with the biocatalytic hydrolysis of triolein in flow. On the other hand, for developing a prototype of a cell-like molecular assembly line, a suitable microfluidic setup (see above) has allowed ten distinct species to be mixed directly in the reaction chamber, where a cascade reaction takes place. Advanced trapped ion mobility spectrometry – time of flight (timsTOF) instrumentation was then used to quantitively monitor species of interest online. As a proof of concept of the first cell-like molecular assembly line, the pentose phosphate pathway has been investigated using free enzymes in flow conditions.
Key steps towards the achievement of the CLASSY objectives have been completed in the last project stage. Results at the project endpoint include having set up two different (prototypical) microfluidic reactor strategies to study compartmentalised catalysts. The second one, where the syringe pumps, the microfluidic reactor, an ultraviolet–visible (UV-vis) spectrometer and a timsTOF mass spectrometer are all connected, is really promising to implement cell-like molecular assembly lines and to study complex enzymatic reaction networks. The different strategies to compartmentalise and control the activity of peptides, NA-peptide hybrids and enzymes in a number of interesting reactions will also open new concepts and avenues of research in the field of catalysis. Now, from these outstanding results, the next steps beyond the project lifetime will be to bring this generated scientific knowledge into new technology that helps to achieve a high degree of programmability, selectivity, efficiency, sustainability and multi-functionality in cell-like catalytic systems. This will affect science and technology at two different levels. On the one hand, it will lead to an improved understanding of the collective processes of living systems, which poses a formidable challenge that needs to be faced for a complete understanding of life. On the other hand, applying this new understanding to the synthesis of functional systems and materials will revolutionise current approaches to chemical engineering, emphasising energy balances, recycling of building blocks and multi-functionality. From an applied perspective, there is a growing industrial need to better understand and integrate complex physicochemical and biological phenomena relevant to the mastering of eco-efficient processes, which are currently subject to global threats, such as climate change and the crisis of raw material resources. The development of the new CLASSY technology is expected to trigger new business opportunities revolving around the commercialisation of catalytic technology and to create fertile ground for future research endeavours. The advances that CLASSY will bring in the scientific and technological realm are expected to result in a key enabling technology for a cleaner and sustainable future, with the potential to bring long-term impacts on societal challenges, such as the development of less expensive drugs or the eradication of infectious diseases, by improving production methods and thus lowering costs.