*4.2. Other Applications of Gene Knockout Strategy*

Gene knockout could also be applied to the diverse study of SMs in fungi, except for the verification of the function of genes, such as for the activating of silent gene clusters and increasing product diversity. Wei et al. knocked out the key genes in the biosynthesis of rubratoxins, the main product of *P. dangeardi*, which makes it easier to inhibit the production of the main compounds and competitively obtain common precursors of polyketide synthesis and isolate novel skeleton compounds [49]. Qi et al. realized the abundant accumulation of emodin, the precursor of physcion, by knocking out the key emodin-1-OH-O-methyltransferase gene in *A. terreus* [50]. This method could also apply in the agricultural pathogenic bacteria, such as the biosynthesis of toxin ustilaginoidins derived from *Ustilaginoidea virens* [51], the infection of fusaoctaxin B derived from *Fusarium graminearum* on the plant virulence factor [52], and the verification of the multiple gene function in the biosynthesis pathway of penifulvin, an anti-insect compound derived from *P. griseofulvum*, which provides an important reference for the prevention and control of agricultural pathogenic microorganisms and the development of new green biological pesticides [53].

#### *4.3. Limitations of Gene Knockout Strategy*

There are also many defects and limitations in the study of fungal SM biosynthesis by gene knockout technology. First of all, because of the high level of evolution and incomplete genetic system of fungi, it is difficult for most strains to establish a genetic transformation system by conventional methods. Regardless of the PEG-mediated homologous recombination or CRISPR/Cas technology, they both require the high-quality protoplasts, while the low transformation efficiency is common in the protoplasts [54]. In addition, there were usually a lot of verification works needed in the screening of mutant strains. Secondly, it is difficult to analyze and identify all the metabolites in the fermentation by the current separation and identification techniques because of the unknown metabolic pathways of most of the studied strains and the complex metabolite compositions. Based on this, the gene deletion is easy to ignore in the regulation of biological metabolites. In the process of studying the biosynthesis pathway of mycotoxin flavipucine derived from *A. nidulans*, gene knockout could only determine the protein involved in the synthesis of the toxin. When the single gene in the BGC was knocked out, there were no intermediates observed; this needs to use the heterologous expression strategy to clarify its specific biosynthesis pathway [55]. Gene knockout strategies play the role of "verifier" in most studies focusing on whether genes (clusters) are involved in the synthesis of certain SMs, but they cannot explain how genes participate in biosynthesis exactly. This rough verification can be used as a guiding tool to study the primary stage of fungal SMs.

### **5. Application of Heterologous Expression Strategy in Biosynthesis of SMs of Fungi**

Because of the disadvantages and limitations of the gene knockout strategy, the heterologous expression strategy has become the other important method in the study of the biosynthesis of the SMs of fungi. Gene data mining reveals that many fungi possess cryptic BGCs that appear to be silent when cultivated in the conventional fermentation conditions [8]. It is difficult to study the function of these genes by gene knockout when carrying out a detailed study of the biosynthetic pathway of complex SMs, while it is necessary to use heterologous hosts with a clear genetic background and mature genetic transformation system to express these silent or complex biosynthetic genes (clusters) (Figure 3). The general research idea of heterologous expression mainly includes the whole genome sequencing of the original strain, blasting and searching for the homologous genomic data to obtain the gene clusters encoding the biosynthesis of the SMs of interest. After the bioinformatics analysis of these genes (clusters), the meaningful genes (clusters) are cloned. Finally, the genes were heterologously expressed by genetic transformation, and the corresponding biosynthetic pathway was clarified by the identification and analysis of the products. There could be the heterologous expression not only of a single gene, but also of the whole gene cluster. When a single gene was heterologously expressed, it mainly combined with the in vitro enzyme experiments to characterize the expressed proteins in detail; when the gene cluster was heterologously expressed, it mainly combined with precursor feeding to verify the hypothetical biosynthesis pathway.

**Figure 3.** Application of heterologous expression strategy in the study of fungal secondary metabolites. It mainly includes the screening of target BGCs, heterologous expression of recombinant strain, feeding of precursor, in vitro enzymatic verification, and identification of intermediate products, in order to predict the biosynthetic pathway.
