**4. Application of Gene Knockout Strategy in Biosynthesis of SMs of Fungi**

Gene knockout is one of the important methods for studying the biosynthesis pathway of fungal SMs; it mainly focuses on the in vivo verification of the gene function of the studied strains (Figure 2). The studied genes (clusters) are usually predicted to be expressed in the original strain, rather than silent genes. The research idea is usually to determine the existence of interesting compounds in the fermentation broth of the original strain first, then to sequence the gene of the strains, predict the biosynthesis pathway (gene clusters) of the interesting compounds after homologous gene mining and bioinformatics analysis of the genomes, and then verify the correlation between the gene cluster and the biosynthesis of the natural products by the gene knockout. The function of the studied genes in the natural product biosynthesis can be roughly judged by the construction of single or multiple gene mutants and the accumulation of intermediate metabolites, which preliminarily analyzed the biosynthetic pathway of the products.

**Figure 2.** Application of gene knockout strategy in the study of fungal secondary metabolites. It mainly includes the determination of target BGCs, construction of mutants, and identification of intermediate products, which verifies the biosynthetic pathway.
