*4.1. Application of Different Gene Knockout Methods*

Different strains have formed different physiological and biochemical characteristics in the long process of evolution. Even if the evolutionary tree shows that the strains are in the same genus, their morphological, physiological, and biochemical characteristics are also quite different. Therefore, the establishment of the genetic transformation system needs to be investigated and selected according to the actual situation of the different strains. Currently, PEG-mediated homologous recombination, *Agrobacterium*-mediated transformation, and PEG-mediated CRISPR/Cas technology are the main methods used in the research on the gene knockout related to the biosynthesis of fungal secondary metabolites.

### 4.1.1. PEG-Mediated Homologous Recombination

PEG-mediated homologous recombination is a classic method for fungal gene research, based on the preparation of high-quality protoplasts, which induces foreign DNA into cells by PEG/CaCl2 and other methods. PEG is a cell fusion agent which can interfere

with the recognition between cells by causing the disorder of the surface charge of the cell membrane, thus facilitating the intercellular fusion and the entry of foreign DNA molecules into the protoplast. The homologous recombination reaction strictly depends on the homology between the DNA molecules. The homologous recombination reaction is usually based on the formation and resolution of cross molecules or the Holliday junction structure, that is, the precursor stage, the formation of the synaptonemial complex, and the resolution of the Holliday structure.

It has been widely used in the biosynthesis of SMs derived from *Aspergillus*. In knocking out the key genes *GedF* and *GedK* of the anthraquinones biosynthesis from *A. fumigatus*, a revised questin ring-opening mechanism was elucidated; this caused a classic Baeyer–Villiger oxidation hypothesis, which has been challenged [32]. By comparing the metabolites of wildtype *A. fumigatus* and *crmA*, a deleted strain grown under Cu2+, it was found that at the level of trace Cu2+ CrmA participated in two different biosynthetic pathways to improve the adaptability under environmental pressure [33]. After knocking out the oxepinamides biosynthesis gene derived from *A. ustus*, the necessary intermediates were obtained, and the biosynthesis pathway was analyzed for the first time [34]. Analyzing the azaphilones biosynthesis gene derived from *A. terreus*, synthesized by two independent gene clusters, provides a new idea for the biological mechanism of complex compounds synthesized by filamentous fungi [35]. At the same time, it has also been applied in the study of the biosynthesis pathways of natural products, such as the oxygenated phenethyl derivative from *A. ustus* [36] and the hopane-type triterpenoid glycoside from *A. fumigatus* [37], indicating that it is generally applicable to *Aspergillus* fungi. In addition, for xylomyrocins from *Paramyrothecium* sp., their biosynthesis pathway was identified through gene knockout and stable isotope feeding, which clarified the fusion coordination between carbohydrate metabolism and NRPS skeleton synthesis and enriched the biosynthesis sources of the special assembly units of non-ribosomal peptides [38]. Liu et al. confirmed the biosynthetic gene cluster of sordarin in the *Sordaria araneosa*, proving that four P450 oxidases play an important role in the rearrangement process [39]. The PEG-mediated homologous recombination method has also been used in the study of biosynthetic genes in epidithiodiketopiperazines derived from *Trichoderma hypoxylon* [40] and indolizidine alkaloids derived from *Curvularia* sp. [41], showing that this method has been widely used in gene knockout.

### 4.1.2. PEG-Mediated CRISPR/Cas Technique

The CRISPR/Cas technique uses RNA to guide the Cas protein to modify the targeted sequences; this has been widely used in various fields as a hot spot. The CRISPR-Cas9 gene editing technology is to identify the target genome sequence through the artificially designed sgRNA (guide RNA) and to guide the Cas9 protease to effectively cut the double strands of DNA to form double strand breaks. The damage repair will cause gene knockout or knock-in and finally achieve the goal of modifying the genome DNA.

In the fungi gene study, CRISPR/Cas technology often requires PEG-mediated protoplast transformation. Scientists knocked out the meroterpenoids biosynthesis gene from marine fungus *Talaromyces purpureogenus* and evaluated two NHI proteins from a heterodimer for catalysis, analyzing the biosynthesis of heteroterpene [42]. Several biosynthetic genes of aculenes derived from *A. aculeatus* were inactivated, which provided reference for the synthesis and derivation of daucane sesquiterpenes [43]. In addition, CRISPR/Cas technology has played an important role in the functional research on phomoxanthone A, derived from marine fungus *Diaporthe* sp. [44], and flavoprotein monooxygenase, derived from *A. terreus* [45]. This technique is becoming mature, with more and more applications in the future study of fungi genes.

#### 4.1.3. *Agrobacterium*-Mediated Transformation

There are relatively few studies on fungal gene knockout mediated by *Agrobacterium* because of the difficulty of the genetic operation and the low transformation efficiency of the fungi. Mycotoxin patulin isolated from *Penicillium expansum* can cause fruit and product pollution. Li et al. used *Agrobacterium*-mediated transformation to study the patulin biosynthesis gene cluster and confirmed the function of all the genes involved in its biosynthesis, providing the support for the prevention and treatment of pathogenic microorganisms [46]. Zhang et al. knocked out a pyrone meroterpenoid oxalicine B gene cluster from *P. oxalicum* and further elucidated its biosynthesis pathway and oxidase catalytic mechanism through in vitro biochemical verification. Oxalicine B possessed good anti-influenza virus activity [47]. Research on a "super" gene cluster of *Metarhizium robertsii* shows that this cluster contains three secondary metabolic gene clusters. It is confirmed that different gene deletions do not affect the insecticidal virulence of *M. robertsii* but do significantly affect the ability of *M. robertsii* to resist different bacteria because of the different gene deletions leading to the production of different structural compounds [48].
