Cloning of an AA13 Polysaccharide Monooxygenase Gene from Neurospora crassa into the plasmid pEX2B

Abstract

Polysaccharide monooxygenases (PMOs) cleave glycoside linkages in polysaccharides, which has great potential in manufacturing biofuel from polysaccharide biomasses. Therefore, heterologous production of PMOs is required for the development of their application. We aim to clone PMO genes into the plasmid pEX2B to express PMOs in Aspergillus oryzae, a filamentous fungus widely used for industrial enzyme production.Here we cloned the NCU08746 gene, an AA13 PMO from Neurospora crassa, into pEX2B. We successfully designed primers and amplified the NCU08746 gene from the N. crassa genome. NCU08746 was then cloned into the pEX2B vector. The recombinant plasmid pEX2B-NCU08746 was subsequently transferred into A. tumefaciens AGL1 prior to being transferred into A. oryzae. This work provides the foundation for further study on PMOs expression in A. oryzae.