Construction of CRISPR-Cas9 genome editing platform for white-rot fungus Cerrena unicolor BBP6 and its effects on extracellular ligninolytic enzyme biosynthesis

Abstract

White-rot fungi (WRF) produce extracellular ligninolytic enzymes such as laccase and manganese-dependent peroxidase (MnP). They have a remarkable potential in degrading a wide range of pollutants including polycyclic aromatic hydrocarbons and synthetic dyes. However, the lack of WRF genetic manipulation methods limits their further applications. Cerrena unicolor BBP6 is a wild-type WRF for laccase and MnP hyper production. This study is the first attempt to develop a genome editing platform for the genus Cerrena sp. based on CRISPR-Cas9 technology. Firstly, the pyrG gene in C. unicolor BBP6 was successfully disrupted and used as an effective screening marker. Subsequently, two genes, LacA and mnp3, which respectively encode a laccase and a MnP protein in strain BBP6, were disrupted. The laccase activity of the LacA mutant strain was reduced by 53.5% compared to the wild-type strain while the MnP activity of the mnp3 mutant strain was reduced by 64.7%. Our study demonstrated that the CRISPR-Cas9 genome-editing platform for C. unicolor BBP6 was successfully established, and it was effective on perturbation of ligninolytic enzyme biosynthesis by this fungus.