LyGo: A platform for rapid screening of lytic polysaccharide monooxygenase production

Abstract

AbstractEnvironmentally friendly sources of energy and chemicals are essential constituents of a sustainable society. An important step towards this goal is the utilization of non-edible biomass as supply of building blocks for future biorefineries. Lytic polysaccharide monooxygenases (LPMOs) are enzymes that play a critical role in breaking the chemical bonds in the most abundant polymers found in recalcitrant biomass, such as cellulose and chitin. Predicting optimal strategies for producing LPMOs is often non-trivial, and methods allowing for screening several strategies simultaneously are therefore needed. Here, we present a standardized platform for cloning LPMOs. The platform allows users to combine gene fragments with different expression vectors in a simple 15-minute reaction, thus enabling rapid exploration of several gene contexts, hosts and expression strategies in parallel. The open-source LyGo platform is accompanied by easy-to-follow online protocols for both cloning and expression. As a demonstration, we utilize the LyGo platform to explore different strategies for expressing several different LPMOs in Escherichia coli, Bacillus subtilis, and Komagataella phaffii.