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

Periodic Reporting for period 2 - HOTZYMES (Redesigning biocatalysis: Thermal-tuning of one-pot multienzymatic cascades by nanoactuation)

Reporting period: 2020-04-01 to 2021-03-31

- 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 in the establishment of a new ground breaking concept to exert functional control over different enzymes using magnetic heating in 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). This would enable in the long term to accomplish a quantum leap in the production of pharmaceuticals or biobased chemicals.
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 of 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.
Through an inter-disciplinary work plan based on scientific synergies between all the partners, HOTZYMES pursues developing the proposed magnetic-driven heating technology with spatio-temporal control for applied biocatalysis. Magnetic heating has been successfully applied in biomedicine to kill tumoral cells and control drug delivery. However, the use of this property to improve biocatalytic processes of industrial interest remains almost unexplored.

In this sense, HOTZYMES partners expect to develop a new ground breaking concept to provide optimal reaction conditions for different enzymes, to ultimately implement efficient multienzyme “one-pot” processes of industrial interest. In particular, it is expected to achieve the magnetic-driven remote control of three different enzymatic bioprocesses for obtaining from high-value intermediates in the synthesis of drugs (eg. antitumoral, antiparasitic, anticholinergic, etc) to a zero-calorie sweetener with probiotic activity. In all cases, their chemical synthesis is highly inefficient and thus, this is a great opportunity to tackle their production in a more efficient way.

Thus, the accomplishment of the goals herein proposed has clear potential economic impact. Specifically, it will engender modernity among the industrial biotechnology community, solving current challenges that are hampering the development of new, sustainable, eco-efficient and competitive biocatalytic ex-vivo cascades to access high added-value products. Thus, HOTZYMES´s vision is clearly aligned with that of Paris protocol of transforming industrial biocatalysis into the blueprint for tackling global climate change beyond 2020.