(85b) Metabolic Engineering of Filamentous Fungi Using the CRISPR/Cas9 Systems

Filamentous fungi, which are a kind of important industrial microorganism, play important roles in the food, agricultural, and pharmaceutical industries. However, it is relatively difficult to carry out genetic manipulation on the fungi, which hampered the metabolic engineering modification to obtain the target phenotype. In recent years, CRISPR/Cas9 genome editing technology, which has emerged as a potential candidate to solve the problem of low gene editing frequency, will greatly facilitates the genetic improvement of filamentous fungi. In the present study, we focused on a filamentous fungus Fusarium fujikuroi, which is well-known for its production of natural plant growth hormones: a series of gibberellic acids (GAs). Some GAs, including GA1, GA3, GA4, and GA7, are biologically active and have been widely applied in agriculture. However, the low efficiency of traditional genetic tools limits the further research towards making this fungus more efficient and able to produce tailor-made GAs. We established an efficient CRISPR/Cas9-based genome editing tool for F. fujikuroi. First, we compared three different nuclear localization signals (NLS) and selected an efficient NLS from histone H2B (HTB_NLS) to enable the import of the Cas9 protein into the fungal nucleus. Then, different sgRNA expression strategies, both in vitro and different promoter-based in vivo strategies, were explored. The promoters of the U6 small nuclear RNA and 5S rRNA, which were identified in F. fujikuroi, had the highest editing efficiency. The 5S rRNA-promoter-driven genome editing efficiency reached up to 79.2%. What’s more, multi-gene editing was also explored and showed good results. Finally, we used the developed genome editing tool to engineer the metabolic pathways responsible for the accumulation of a series GAs in the filamentous fungus F. fujikuroi, and successfully changed its GA product profile, from GA3 to tailor-made GA4 and GA7 mixtures. Since these mixtures are more efficient for agricultural use, especially for fruit growth, the developed strains will greatly improve industrial GA production.