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TIMEnzyme Report Summary

Project ID: 701512
Funded under: H2020-EU.1.3.2.

Periodic Reporting for period 1 - TIMEnzyme (Implementation of Enzymatic Activity in a Naïve, de novo Designed Protein Scaffold by Rational Design and Laboratory Evolution)

Reporting period: 2016-03-01 to 2018-02-28

Summary of the context and overall objectives of the project

The chemical and pharmaceutical industry is under increasing pressure to replace traditional chemical catalysis with environmentally benign approaches for the synthesis of high-value compounds. Biocatalytic transformations, that is using enzymes as catalysts, generally offer sustainability in combination with high selectivity and catalytic activity. However, most natural enzymes do not meet the requirements of large-scale industrial processes and/or do not catalyze the relevant non-natural reactions with sufficient efficiency.

A powerful strategy to tackle this problem is the development of de novo enzymes using a combined approach of computational design and directed evolution. The TIMEnzyme project is centered around recently produced de novo protein scaffolds, which have no sequence homology with natural proteins, and may thus provide an unbiased starting point for the design of novel enzymes and their subsequent optimization by laboratory evolution. The overall goal was to functionalize these protein scaffolds and test their evolvability towards a synthetically valuable activity. We chose to design de novo aldolases catalyzing the stereoselective cleavage or formation of carbon-carbon bonds (Fig. 1).

While the work on this project is still in progress at the time this report has been filed, important milestones were achieved and published during the time period of the fellowship, as described in further detail below.

This study has been performed in close collaboration with the laboratory of Prof. David Baker (University of Washington, Seattle, USA).

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

As computationally designed enzymes usually possess low initial catalytic efficiency, directed evolution is used for optimization, which is done in iterative cycles of gene diversification by mutagenesis and screening for improved catalysts. Laboratory evolution thus relies on high-throughput screening techniques that must be applicable for enzymes with low starting activity. A first work package of this project was hence to establish that microfluidic-based fluorescence-activated droplet sorting (FADS), a ultrahigh-throughput method used to screen for (retro-)aldolase activity, is suitable for low initial catalytic activity. We could successfully demonstrate this by evolving a previously designed de novo (retro-)aldolase (RA95), which is based on a natural protein scaffold. The results showed that alternative evolutionary trajectories leading to enzymes with opposite enantioselectivity can be explored efficiently when starting straight from the computational design. This part of the project has been published (Ref. 1) and provides the basis for the evolution of aldolases implemented in de novo protein scaffolds, which is in progress.

Another important aspect of the project was to gain a mechanistic understanding of changes that occur during the directed evolution of artificial aldolases. Here, the evolutionary history of a previously optimized (retro-)aldolase (RA95) was analyzed using single turnover enzyme kinetics, isotope exchange and kinetic isotope effect measurements in combination with structural analysis. The results have been published (Ref. 2) and provide important feedback for enzyme design. Specifically, a shift in the rate-determining step was identified and the importance of a catalytic tetrad in the enzyme’s active site that emerged during evolution was emphasized. As a consequence, this catalytic motif will be introduced in a new generation of de novo aldolases.

The first generation of (retro-)aldolase designs in the actual de novo scaffolds showed severe problems with solubility and stability and were generally not sufficiently active to start the optimization by directed evolution. A second generation of designs with modifications in both the active sites and the scaffolds themselves shows promising initial results and is currently being optimized.

In addition to the aldolase approach, de novo protein scaffolds were equipped with binding sites for metal cofactors, which provide a broad range of non-natural activities. A crystal structure of a metal-binding de novo protein has been determined and provides the basis for our future work. This part of the project is still at a preliminary stage, but will be continued.

In summary, the overall goal of this study, generating an efficient enzyme from a naïve de novo protein scaffold, has not yet been fully achieved, but work in progress. The results obtained in the period of the fellowship represent important milestones and were disseminated in two peer-reviewed publications and contributions two several international conferences.

[1] *Obexer, R., *Pott, M., *Zeymer, C., Griffiths, A.D., and Hilvert, D. (2016) „Efficient Laboratory Evolution of Computationally Designed Enzymes with Low Starting Activities Using Fluorescence-Activated Droplet Sorting”
Protein Engineering, Design & Selection 29, 355-366
[2] Zeymer, C., Zschoche, R., and Hilvert, D. (2017) „Optimization of Enzyme Mechanism along the Evolutionary Trajectory of a Computationally Designed (Retro-)Aldolase”
Journal of the American Chemical Society 139, 12541-12549

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The results of this work have impact for the fields of mechanistic enzymology as well as enzyme design and evolution. The detailed reconstruction of mechanistic changes that occurred along the evolutionary trajectory of a computationally designed enzyme is progress beyond state of the art and will influence future enzyme design efforts. Proof-of-concept that de novo protein scaffolds are suitable starting points for the development of novel enzymes may have a broad impact on the field of biocatalysis, as more and more de novo enzymes may find applications in industrial processes in the future.

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