Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes that
help break down lignocellulose, making them highly attractive for improving
biomass utilization in industrial biotechnology. The catalytically essential
N-terminal histidine (His1) of LPMOs is post-translationally modified by
methylation in filamentous fungi to protect them from auto-oxidative inactivation,
however, the responsiblemethyltransferase enzyme is unknown. Using
mass-spectrometry-based quantitative proteomics in combination with systematic
CRISPR/Cas9 knockout screening in Aspergillus nidulans, we identify
the N-terminal histidine methyltransferase (NHMT) encoded by the gene
AN4663. Targeted proteomics confirm that NHMT was solely responsible for
His1 methylation of LPMOs. NHMT is predicted to encode a unique seventransmembrane
segment anchoring a soluble methyltransferase domain. Colocalization
studies show endoplasmic reticulum residence of NHMT and coexpression
in the industrial production yeast Komagataella phaffii with LPMOs
results in His1 methylation of the LPMOs. This demonstrates the biotechnological
potential of recombinant production of proteins and peptides harbouring
this specific post-translational modification.