Periodic Reporting for period 1 - E-VOLUTION (Electrifying Peptide Synthesis for Directed Evolution of Artificial Enzymes)
Periodo di rendicontazione: 2022-09-01 al 2025-02-28
Global climate and energy challenges require efficient, robust and scalable catalysts for the conversion of renewable energies. Nature has evolved extremely active catalysts (enzymes) for the conversion of small molecules relevant to energy (H2, CO2, N2). The scalability of these enzymes offers distinct advantages over the rare, precious metals that are currently used in energy conversion.
Unfortunately, the enzymes are unable to tolerate the extreme conditions of operating fuel cells or electrolyzers. Directed evolution is a powerful approach for improving enzymes, but is mostly restricted to natural amino acids and biological conditions, with limited compatibility for evolving enzymes toward enhanced resistance in abiotic systems. The project E-VOLUTION aims to establish directed evolution in fully abiotic systems, using artificial amino acids to make artificial enzymes that are stable even in extreme conditions. Towards this, I will establish new electrochemical peptide synthesis platforms to enable the generation of enzyme-length peptides using both natural and artificial amino acids. Extended libraries of artificial enzyme variants will be produced and screened directly on electrode microarrays. Top enzyme candidates for the conversion of H2 will be selected using fuel cell/electrolyzer conditions as the evolutionary criteria. A new procedure for synthesizing libraries of full-length artificial proteins will enable the creation of thousands of enzyme variants using artificial building blocks. The generation of high-quality datasets will be transformative to drive future machine learning-based evolution steps for both full size enzymes and small-molecule catalysts with applications beyond H2 evolution. This will support the discovery of highly active catalysts able to sustain conditions of large-scale energy conversion devices, accelerating breakthroughs toward the economically competitive use of renewable energies for fuel and chemical production.
Unfortunately, the enzymes are unable to tolerate the extreme conditions of operating fuel cells or electrolyzers. Directed evolution is a powerful approach for improving enzymes, but is mostly restricted to natural amino acids and biological conditions, with limited compatibility for evolving enzymes toward enhanced resistance in abiotic systems. The project E-VOLUTION aims to establish directed evolution in fully abiotic systems, using artificial amino acids to make artificial enzymes that are stable even in extreme conditions. Towards this, I will establish new electrochemical peptide synthesis platforms to enable the generation of enzyme-length peptides using both natural and artificial amino acids. Extended libraries of artificial enzyme variants will be produced and screened directly on electrode microarrays. Top enzyme candidates for the conversion of H2 will be selected using fuel cell/electrolyzer conditions as the evolutionary criteria. A new procedure for synthesizing libraries of full-length artificial proteins will enable the creation of thousands of enzyme variants using artificial building blocks. The generation of high-quality datasets will be transformative to drive future machine learning-based evolution steps for both full size enzymes and small-molecule catalysts with applications beyond H2 evolution. This will support the discovery of highly active catalysts able to sustain conditions of large-scale energy conversion devices, accelerating breakthroughs toward the economically competitive use of renewable energies for fuel and chemical production.
A new electrochemical system toward the synthesis of high purity, long-chain peptides has been established (WP1). It is based on the coupling of amino acids modified with electroactive functionalities. Out of multiple N-terminal protecting and C-terminal activating group that were tested so far, two are advantageous because they allow a minimum side product formation compared to the other electroactive building blocks. These redox-active functionalities enabled the proof-of-concept electrochemical synthesis of dipeptides and tripeptide. The key and most challenging part so far was the optimization of the synthesis of the amino acid building blocks.
We also designed electrochemical microarrays with individually addressable droplets for the synthesis and characterization of peptides (WP2) based on a three-electrode configuration. The first prototype based on a 16x16 electrochemical microarray was produced by screen printing. We identified a specific configuration of the three-electrode system that significantly decrease cross talks between droplets, which is typically occurring in high density arrays of individual electrochemical cell. This configuration will be further optimized and supported by a specific potentiostat design to enable peptide synthesis and read-out without interference from neighboring droplets.
We additionally developed and optimized protocols for the production, assembly, and reconstitution of semi-synthetic [FeFe]-hydrogenase (WP3), a key step toward enhancing its catalytic performance and expanding its applicability due to the integration of non-canonical amino acids. The semi-synthetic approach combines chemical synthesis and recombinant techniques to assemble these fragments to the full-length polypeptide of the enzyme.
We also designed electrochemical microarrays with individually addressable droplets for the synthesis and characterization of peptides (WP2) based on a three-electrode configuration. The first prototype based on a 16x16 electrochemical microarray was produced by screen printing. We identified a specific configuration of the three-electrode system that significantly decrease cross talks between droplets, which is typically occurring in high density arrays of individual electrochemical cell. This configuration will be further optimized and supported by a specific potentiostat design to enable peptide synthesis and read-out without interference from neighboring droplets.
We additionally developed and optimized protocols for the production, assembly, and reconstitution of semi-synthetic [FeFe]-hydrogenase (WP3), a key step toward enhancing its catalytic performance and expanding its applicability due to the integration of non-canonical amino acids. The semi-synthetic approach combines chemical synthesis and recombinant techniques to assemble these fragments to the full-length polypeptide of the enzyme.
The novel procedure for generating and evolving new artificial enzymes will be the highest impact of the project with high potential for commercialization of either the method itself or of enzymes discovered through the method. This is expected by the end of the project.
The novel design of electrode miroarray and bespoke potentiostat is an advance over the state-of-the-art that we are currently assessing for IP protection and future commercialization
The novel design of electrode miroarray and bespoke potentiostat is an advance over the state-of-the-art that we are currently assessing for IP protection and future commercialization