Characterisation of a monooxygenase in Shiraia bambusicola

Abstract

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.

Advances and perspectives on perylenequinone biosynthesis

“…The complex of Cas9 and sgRNA triggers specific double-strand breaks, induces cellular repair pathways, and modifies the target genes (Sander and Joung, 2014; Figure 1C). Our group has recently constructed a CRISPR the presence of their corresponding intermediates (Deng et al, 2018). These findings remain to be confirmed by heterologous expression of these enzymes and substrate selectivity/ product characterization.…”

“…The complex of Cas9 and sgRNA triggers specific double-strand breaks, induces cellular repair pathways, and modifies the target genes ( Sander and Joung, 2014 ; Figure 1C ). Our group has recently constructed a CRISPR system in S. bambusicola and generated several mutants ( Deng et al, 2017a , c , 2018 ), including mutants of TF, polyketide synthase, monooxygenase, and a major facilitator superfamily transporter, all of which decrease hypocrellin production. Compared with the red wild-type S. bambusicola , transcription factor and polyketide synthase mutants are colorless, whereas mutants of monooxygenase and major facilitator superfamily transporter are brown.…”

Fungal perylenequinones

“…The CTB , elc , and HYP gene clusters contain multiple shared homologs (Fig. 3 ) (Chen et al 2007 ; Yang et al 2014 ; Newman and Townsend 2016 ; Chooi et al 2017 ; Deng et al 2018 ; Hu et al 2019 ). The phylogenetic analysis of elcA and allied fungal non-reducing PKSs revealed that elcA is more closely related to Shiraia sp.…”

“…Elsinochrome A ( 45 ) is synthesized via a radical process triggered by a single electron transfer from an enolate at the side chain to the FAD in elcH via the putative perylenequinone intermediate V . In the absence of elcH , hypocrellins ( 49 – 50 ) are formed, suggesting that they are most likely derived from the putative intermediate V via a transannular aldol reaction (Newman and Townsend 2016 ; Deng et al 2018 ; Hu et al 2019 ). CTB5 , 6 , 7 , 9 , and 10 afford cercosporin ( 30 ) through different reactions including reduction and homodimerization of the intermediates.…”

“…Slf14 (Yang et al 2014 ), and subsequently verified by targeted gene deletion, the monooxygenase-encoding gene (Mono) which is located in the hypocrellin gene cluster of Shiraia sp. (Deng et al 2018 ).…”