Characterisation of a monooxygenase in Shiraia bambusicola

A monooxygenase-encoding gene (Mono) is located in the hypocrellin gene cluster of Shiraia sp. SUPER-H168 and was targeted by a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. The ΔMono mutant abolished hypocrellin production, whereas the ΔMono complement mutant restored hypocrellin production. Relative expression levels of the Mono and its adjacent genes were abolished in the ΔMono mutant compared with the wild-type strain. These results indicate the essential role of Mono in hypocrellin biosynthesis. The Mono gene of Shiraia bambusicola was further expressed in Pichia pastoris and salicylate monooxygenase activity was detected, which suggested that this monooxygenase has the ability to catalyse decarboxylative hydroxylation. The relative growth ratio of the ΔMono mutant was significantly improved compared with the wild-type strain. In contrast to the wild-type strain, the ΔMono mutant also represented excellent oxidative stress tolerance after exposure to high concentrations of H2O2 (16 mM) based on the increasing activities of superoxide dismutase, catalase, and glutathione peroxidase. These results suggest that ΔMono mutants could be used as microbial cell factories to produce metabolites that will cause oxidative stress. This study also enhances our understanding of hypocrellin biosynthesis and opens an avenue for decoding the hypocrellin pathway.