Bio-Click: A cell-free high-throughput platform for the engineering of enzymatic group transfer reactions

Project Abstract – Invizyne Technologies, Inc Biocatalytic group transfer reactions are essential in all biological systems and have emerged as powerful tools in synthetic biology and cell-free biomanufacturing. Their characteristics and mechanisms bear striking similarity to click chemistry reactions (‘BioClick Reactions’) and their ability to regio- and stereo-specifically modulate natural products and synthetic pharmaceuticals can dramatically improve physiochemical, biological, and pharmacological properties of molecules. We propose a novel cell-free, high-throughput platform to expedite the engineering of enzymes for BioClick reactions. Our platform integrates cell-free protein synthesis (CFPS) with multiplexed mass spectrometry for rapid screening of vast enzyme variant libraries against diverse substrate pairings. Invizyne has developed the cell-free biochemistry pipeline SimplePath™ which can produce cofactors for a variety of BioClick reactions at low cost. Phase I of this project focuses on our most mature technology – the biocatalytic prenylation of aromatic pharmaceutical and nutraceutical scaffolds using our workhorse enzyme NphBM34S. Our project pursues two primary aims: i) establishing an in-house CFPS system for efficient and rapid expression of site-saturation libraries of NphBM34S variants and ii) utilizing low-cost prenyl-donors to screen variant libraries against a collection of prenyl-acceptor substrates using robotics and high-throughput experimentation (HTE), and subsequently analyzing results with multiplexed mass spectrometry and cloud-based computational tools for data deconvolution. Successful completion will establish a rapid and efficient platform for BioClick reaction engineering, identify promising NphBM34S variants for diverse prenylation reactions, and validate the utility of CFPS and HTE for BioClick reaction discovery. This not only paves the way for AI-based protein engineering and natural product development during Phase II of this project but may also offer a transformative strategy for ‘Generative Biocatalysis’ with potential applications across drug discovery, combinatorial medicinal (bio)chemistry and natural product synthesis.