Deng Huaxiang, Gao Ruijie, Liao Xiangru, Cai Yujie
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, PR China.
Microbiology (Reading). 2018 Sep;164(9):1180-1188. doi: 10.1099/mic.0.000694. Epub 2018 Jul 20.
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.
一个单加氧酶编码基因（Mono）位于竹黄菌Shiraia sp. SUPER-H168的竹红菌素基因簇中，并被成簇规律间隔短回文重复序列（CRISPR）/Cas9系统靶向。ΔMono突变体消除了竹红菌素的产生，而ΔMono互补突变体恢复了竹红菌素的产生。与野生型菌株相比，ΔMono突变体中Mono及其相邻基因的相对表达水平被消除。这些结果表明Mono在竹红菌素生物合成中起关键作用。竹黄菌的Mono基因在毕赤酵母中进一步表达，并检测到水杨酸单加氧酶活性，这表明这种单加氧酶具有催化脱羧羟基化的能力。与野生型菌株相比，ΔMono突变体的相对生长率显著提高。与野生型菌株不同，基于超氧化物歧化酶、过氧化氢酶和谷胱甘肽过氧化物酶活性的增加，ΔMono突变体在暴露于高浓度H2O2（16 mM）后也表现出优异的氧化应激耐受性。这些结果表明，ΔMono突变体可作为微生物细胞工厂来生产会引起氧化应激的代谢物。本研究还增强了我们对竹红菌素生物合成的理解，并为解码竹红菌素途径开辟了一条道路。
