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Redesigning biocatalysis: Thermal-tuning of one-pot multienzymatic cascades by nanoactuation

Periodic Reporting for period 3 - HOTZYMES (Redesigning biocatalysis: Thermal-tuning of one-pot multienzymatic cascades by nanoactuation)

Reporting period: 2021-04-01 to 2022-09-30

- What is the problem being addressed in HOTZYMES?

The use of multi-enzyme processes for in vitro synthetic biology is currently considered a promising biomanufacturing platform. Enzymatic cascades open a path to the efficient implementation of complex biotransformations for the production of high-cost pharmaceuticals to low-cost biocommodities. They are valuable alternative routes to traditional step-wise chemical synthetic processes, since substrate specificity, stereo- and regioselectivity of enzymes are outstanding. This allows to circumvent the isolation of by-products and reaction intermediates and, thus, to increase the eco-efficiency of the overall process. Besides, one-pot multi-enzyme biocatalysis has numerous manufacturing advantages, such as higher product yield, faster reaction rates, less energy input needed, and less waste generation. Unfortunately, the development of efficient cell-free metabolic systems perfectly orchestrated and regulated is still an unmet need.

- Why this is important for society?

In addition to a clear potential economic impact, the development of well-coordinated one-pot enzymatic cascades of industrial interest is a fundamental pillar in the roadmap for the transition from a fossil fuel-based economy to a bio-economy based on climate change.

- What are HOTZYMES overall objectives?

HOTZYMES overall objective consists of the establishment of a new groundbreaking concept to exert functional control over different enzymes using magnetic heating to be able to achieve well-coordinated one-pot-systems. Its achievement will pave the road for changing industrial biotransformations from an unsatisfactory current paradigm (uncoordinated enzyme function, sequential reactions, disposable bioprocesses) into a game-changing breakthrough (coordinated enzyme function, concurrent reactions, recyclable bioprocesses).
HOTZYMES´s consortium has worked hard during this first year of the project to obtain promising results on the development of a Toolbox of Nanoparticles (NPs) with adequate and tunable magnetic heating efficiencies. This allows us to think that they could enable the development of the proposed concept to exert spatio-temporal remote control over enzymatic cascades. Besides, the partners had also been working on other aspects that are also key-enabling to achieve this aim. In this sense, they not only have been working on the development of the Toolbox of recombinant enzymes needed but also in the development of tailor-designed functionalization strategies for the obtained NPs and in the study of their safety.

During the second year of the project, HOTZMES´s consortium has shown the feasibility of triggering multiple local hotspots using a mixture of MNPs with different magnetic heating efficiencies. The results obtained are compatible with our aim of gaining spatio-temporal remote control over enzymatic cascades by the application of alternating magnetic fields (AMF). Besides, the partners have been working on the development of hybrids integrating MNPs and enzymes to avoid aggregation issues observed when using enzymes attached to MNPs directly on the biotransformations that were selected for exploring the feasibility of their AMF-tuning. In addition, the partners have been working on the development of different tools (recombinant protein toolkits, thermodynamic studies, nanothermometry measurements, etc) in order to help to understand the parameter governing and influencing the heat transferences processes triggered at the nanoscale by AMF.

During the final period of the project, partners have been working on obtaining a third generation of MNPs changing their anisotropy but maintaining their core size, also focusing on the development of more sustainable methods for their synthesis. Progress has also been made in modeling studies and the use of molecular dynamics to understand the heating at the nanoscale. A recombinant toolkit of enzymes has been developed that would allow studying the effect of enzyme orientation/distance to the MNP surface on heat transference processes triggered by AMF. The feasibility of using the selected enzymes to perform the selected bioprocesses was optimized. Integration of enzymes and MNPs within hybrid materials resulted in a suitable strategy to avoid MNP aggregation issues. Indeed, different AMF-tunable enzyme micro and nanohybrids were obtained. In addition, a pilot-scale AMF reactor was designed, prototyped, and manufactured. Lixiviation of metal ions upon AMF application and an original “omic” epigenetic study have been conducted to go beyond traditional nanotoxicity approaches. All this work has resulted in the identification of exploitable results including: i) self-standing scientific and technical knowledge providing results for further research activities, ii) development of products, and iii) development of results for their use in standardization activities. Within these exploitable results, a European patent has been granted (EP21382585.4). This patent could form part of the portfolio of an innovative SME company, for whose creation several actions have already been initiated such as: i) contacting investment funds, and ii) receiving coaching from Business Development Consultancies. Besides, another patent is being prepared. Although many results obtained could not be disseminated until this patent is filed, the consortium has produced so far 21 open-access publications that could also be found in HOTZYME´s Zenodo community.
Through an interdisciplinary work plan based on synergies between all the partners, HOTZYMES pursued developing the proposed magnetic-driven heating technology with spatiotemporal control for applied biocatalysis. In this sense, the partners were able to show the feasibility of achieving multiplex local heating simultaneously or sequentially in a single-pot vessel by the combined use of MNPs with different anisotropies. Besides, the integration of these MNPs within hybrids materials had shown to be an effective strategy to overcome aggregation issues observed during MNPs functionalization, under reaction conditions and/or AMF application. This allows achieving the local control of enzyme activity via the application of contactless magnetic heating while the temperature of the reaction media or of the microenvironment surrounding those enzymes not attached to the MNPs is not affected. Thus, the achieved results could clearly help to boost the use of multi-enzymatic cascades which are in their infancy in terms of their industrial application. Indeed, they pave the way to overcome drawbacks they currently face including (a) incompatibility between the thermal conditions underlying bioprocesses requiring high temperatures and the thermal lability of reactants (substrates, reaction intermediates, and/or products), (b) optimal temperature incompatibilities among enzymes involved in the targeted bioprocess,(c) cross-reactivity encountered in enzyme cascades involving two or more enzymes competing for the same substrate. Thus, the developed technology would enable in the long term to accomplish a quantum leap in the production of pharmaceuticals or biobased chemicals.