The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, 2800, Denmark.
Mycropt ApS, Kongens Lyngby, 2800, Denmark.
ACS Synth Biol. 2021 Apr 16;10(4):897-906. doi: 10.1021/acssynbio.1c00034. Epub 2021 Apr 2.
Environmentally friendly sources of energy and chemicals are essential constituents of a sustainable society. An important step toward this goal is the utilization of biomass to supply 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. To use them in industrial processes they need to be produced in high titers in cell factories. Predicting optimal strategies for producing LPMOs is often nontrivial, 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 14 different expression vectors in a simple 15 min 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 of its utility, we explore different strategies for expressing several different LPMOs in , , and .
环境友好型能源和化学品是可持续社会的重要组成部分。实现这一目标的重要一步是利用生物质为未来的生物精炼厂提供构建模块。溶细胞多糖单加氧酶（LPMO）是一种在分解最丰富的聚合物中发挥关键作用的酶，这些聚合物存在于木质纤维素和几丁质等难处理的生物质中。为了在工业过程中使用它们，需要在细胞工厂中以高滴度生产。预测生产 LPMO 的最佳策略通常并不简单，因此需要能够同时筛选几种策略的方法。在这里，我们提出了一个用于克隆 LPMO 的标准化平台。该平台允许用户在简单的 15 分钟反应中，将基因片段与 14 种不同的表达载体组合在一起，从而能够并行快速探索几种基因背景、宿主和表达策略。开源 LyGo 平台还提供了克隆和表达的简单在线协议。作为其实用性的证明，我们探索了在 、 、 和 中表达几种不同 LPMO 的不同策略。
