A fast and easy strategy for lytic polysaccharide monooxygenase-cleavable His6-Tag cloning, expression, and purification

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are industrially important enzymes able to enhance the enzymatic lignocellulose saccharification in synergism with classical glycoside hydrolases. Fungal LPMOs have been classified as AA9, AA11, and AA13-16 families showing a diverse specificity for substrates such as soluble and insoluble beta-glucans, chitin, starch, and xylan, besides cellulose. These enzymes are still not fully characterized, and for example this is testify by their mechanism of oxidation regularly reviewed multiple times in the last decade. Noteworthy is that despite the extremely large abundance in the entire Tree of Life, our structural and functional knowledge is based on a restricted pool of LPMO, and probably one of the main reason reside in the challenging posed by their heterologous expression. Notably, the lack of a simple cloning protocol that could be universally applied to LPMO, hinders the conversion of the ever-increasing available genomic information to actual new enzymes. Here, we provide an easy and fast protocol for cloning, expression, and purification of active LPMOs in the following architecture: natural signal peptide, LPMO enzyme, TEV protease site, and His6-Tag. For this purpose, a commercial methanol inducible expression vector was initially modified to allow the LPMO expression containing the above characteristics. Gibson assembly, a one-step isothermal DNA assembly, was adopted for the direct assembly of intron-less or intron-containing genes and the modified expression vector. Moreover, His6-tagged LPMO constructs can be submitted to TEV proteolysis for removal of the questionable C-terminal His6-Tag, obtaining a close-to-native form of LPMO. We successfully applied this method to clone, express, and purify six LPMOs from AA9 family with different regioselectivities. The proposed protocol, provided as step-by-step, could be virtually applied in many laboratories thanks to the choice of popular and commons materials.