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Light-controlled synthetic enzyme cascades

Periodic Reporting for period 3 - LightCas (Light-controlled synthetic enzyme cascades)

Reporting period: 2021-01-01 to 2022-06-30

There is an urgent need for the development of greener syntheses procedures if mankind wants to maintain an environment worth living in but is at the same time unwilling to accept a reduction in material comfort. The establishment of more biocatalytic steps in chemical syntheses is one possible solution, as enzymes and whole cells offer sustainable advantages, such as biodegradability, intoxicity, high selectivity, and many more. As a myriad of enzymatic reactions exist for almost any product, their potential is immense. Great scientific achievements and new techniques recently developed have enabled the design of economically and ecologically feasible multi-step enzyme cascades. However, with these new opportunities, also new challenges arise. The more enzyme steps are combined in one pot, the higher the risk of undesired cross-reactivity is. There is thus need for a tight control of each biocatalytic step in a cascade in order to obtain the desired product in a high purity and to make use of all advantages that enzyme cascades intrinsically offer. LightCas aims to break new grounds in the area of multi-step (bio)catalysis by enabling an orthogonal, selective and thus flexible on/off tuning of enzymes in a cascade. By entrapping enzymes into stimuli-switchable microgels (WP A), using photo-switchable active site lids (WP B) and light-induced enzyme deactivation (WP C), three methods providing the opportunity to control enzyme activity in vitro and in vivo on demand are (further) developed. The ultimate goal is to set up a one-pot multistep light-controlled enzyme reactor yielding the desired product in high selectivity and concentration in a technically self regulated manner (WP D). Beyond the ground-breaking direct impact in the field of enzyme catalysis, huge gains in knowledge are expected from LightCas with respect to the application of intelligent stimuli-responsive materials as well as new, advanced methods for applications in the clinical and research environment.
Enzyme entrapment in stimuli-switchable microgels: The aim of this LightCas work package was to enable a reversible and selective on/off switching of enzymes involved in multi-step cascade reactions using stimuli switchable micrgogels. To achieve this, enzymes were immobilised in temperature-responsive hydrogels. These hydrogels swell and shrink with temperature shifts. As the mesh size of the material changes in the process, it is expected that the activity of the enzyme is influenced by less flexible movement with smaller mesh size. Here, enzymes were encapsulated in a temperature-sensitive hydrogel, which was produced by our cooperation partner Prof. Seema Agarwal (University of Bayreuth), an expert in novel temperature-sensitive materials. In fact, the encapsulated enzyme was effectively immobilised and still active when trapped in the hydrogel. However, as a bottleneck, the gels showed less swelling capacity when salts and/or substrate were added. This reduced the stimulation efficiency and thus the controllability of enzyme activity in the hydrogels.

Sensitive online monitoring of enzyme activity: Recently, the company Magritek brought a novel benchtop nuclear-magnet-resonance (NMR) device to the market. We could show, that this technique is not only applicable for chemical transformations, but also to online monitor enzymatic conversions with very high sensitivity, selectivity and reliability. Even low concentrations of product could be detected in a buffered reaction environment without removing the enzyme from the vessel. In future, online analytic systems might not only enable direct monitoring of (enzymatic) reactions, but may also form the basis for self-regulation in biocatalytic reactions, as we test in the second half of the LightCas project.

Light-induced enzyme deactivation using genetically encoded photosensitizers: In this LightCAS work package, enzymes prone to side-reactivities are fused to genetically encoded photosensitisers. These photosensitisers are light-active proteins, which produce highly reactive oxygen species (ROS) after illumination with a specific wavelength. These ROS inactivate all kinds of biological structures adjacent to the photosensitiser, including proteins. The inactivation is not reversible, but this technique can be used for e.g. targeted enzyme inactivation in enzyme cascades (also in whole cells), a novel approach in biocatalysis. Different photosensitizer enzyme fusions were produced and characterised. With the best fusion construct, the activity of the attached enzyme was even enhanced (before light exposure), probably due to increased stability. Upon blue light exposure, the tagged enzyme was inactivation within a few minutes. We have hoped for exactly this result. Currently, the superimposition to other catalysts and the regulation of the enzyme in cascades is tested with very promising first results. Compared to a non-regulated process, the blue light exposure and selective inactivation of the enzymes led to less side-product formation in a three step enzymatic process.

Design of light-regulated enzyme reactors: With the promising results achieved with genetically encoded photosensitizers, we aim to scale up the reaction to an order of magnitude that is interesting for application. The challenge here is that the entire reaction mixture in the photodeactivation process must be uniformly illuminated. The first prototypes with LED strips wrapped around the vessels meet our expectations.
In summary, the following novel techniques and scientific results could be achieved:

1. Online bench-top NMR can be used to track enzymatic transformations over time. The bench-top analytical device allows sensitive, selective and reliable detection of substrate depletion and product formation, which was not possible before.

2. Genetically encoded photosensitisers can be used to selectively stop enzymatic reactions. This allows enzymatic transformations to be regulated on demand and avoids the production of undesired by-products.

3. First prototypes for light-controlled reactors on a preparative scale show promising results in terms of uniform illumination. In combination with the novel online analysis tool, the basis for self-regulating process control is being laid.

The feedback from the scientific community on the results and ideas of the LightCas project is great. Our review on stimulus-responsive enzyme regulation was selected as a 'very important article' by the journal Advanced Synthesis and Catalysis. Several proposals for oral presentations were accepted and Dörte Rother was invited to present at renowned national and international conferences in the field of biocatalysis. Two prestigious prizes were awarded to the PI: Biotrans Junior Prize 2019 (international prize of the biocatalytic community) and DECHEMA Prize 2018 (prize of the German Society for Chemical Engineering and Biotechnology).
Blue light reaction set-up