**1. Introduction**

Microorganisms can produce a wide variety of SMs (e.g., polyketides, terpenoids, saponins, and non-ribosomal peptides), most of which show good biological activity, such as antibacterial, anti-tumor, and immunoregulation properties [1–3]. The research on SMs from prokaryotes (such as *Actinomyces*, *Streptomyces*, and other bacteria) started earlier and more thoroughly, while the research on SMs from eukaryotes is relatively scarce. However, with the in-depth study of fungi in recent years, it has been shown that fungi possess more potential to produce SMs than bacteria, and the products have better biological activity, which has attracted more extensive attention and research [4,5]. At the same time, with the ongoing advancement of gene sequencing technology and bioinformatics, a large amount of genomic information on bacteria and fungi has been analyzed and annotated [6,7]. After "mining" this gene information, numerous "silent" biosynthetic gene clusters (BGCs) of SMs from bacteria and fungi still have not been characterized, while fungi show more powerful production potential for SMs than bacteria because of their larger and more complex genomes [8]. The continuous progress of molecular biology technology, such as gene knockout and heterologous expression, as well as the application of combinatorial biosynthesis strategies, makes the manipulation of genes increasingly convenient, which greatly expands the research on microbial SMs [9,10]. All of these promote the biosynthetic pathway study for microbial SMs and show great advantages in the field of biosynthesis.

**Citation:** Ning, Y.; Xu, Y.; Jiao, B.; Lu, X. Application of Gene Knockout and Heterologous Expression Strategy in Fungal Secondary Metabolites Biosynthesis. *Mar. Drugs* **2022**, *20*, 705. https://doi.org/10.3390/ md20110705

Academic Editor: Dehai Li

Received: 27 September 2022 Accepted: 9 November 2022 Published: 10 November 2022

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Gene knockout and heterologous expression are common and mature strategies for the study of the biosynthesis of microbial SMs under the genome mining. The common gene knockout methods mainly include PEG/CaCl2-mediated homologous recombination, *Agrobacterium*-mediated transformation, and the CRISPR/Cas technology [11]. In the heterologous expression, the model strains mainly include *Escherichia coli* and *Bacillus subtilis* in the prokaryotes and *Saccharomyces cerevisiae* and filamentous fungi such as *Aspergillus nidulans* and *Aspergillus oryzae* in the eukaryotes [12]. For the prokaryotes, gene knockout and heterologous expression techniques have been well experienced and established because the BGCs of the SMs are usually distributed in clusters and exist in the form of operons, which are convenient for the investigation and operation of target genes (clusters) [13]. For eukaryotes, because of the intact nucleus and genetic system and the larger and more complex genome of fungi, the BGCs of the SMs are usually scattered, which makes it very challenging to dig out and analyze the fungal target genes [14]. Additionally, due to the high level of fungal evolution, it is difficult to establish genetic operating systems, which consequently started late and are relatively scarce in the study of the biosynthesis of fungal SMs [15]. Nowadays, more and more scholars are dedicated to the genome mining of fungal SMs and have obtained a series of achievements. Therefore, it is important and necessary to summarize the research on the molecular biology technology in the secondary metabolites of fungi, especially the gene knockout and the heterologous expression techniques.

In this paper, we primarily concentrated on the representative studies of the biosynthesis of fungal SMs by gene knockout and heterologous expression under the fungal genome mining in the last three years, of which the techniques and methods were briefly introduced. The purpose of this paper is to classify and summarize the strains which have been studied, elucidating the fact that some gene knockout and heterologous expression methods are indeed applicable to the gene manipulation of certain species of fungi, which can provide some ideas and references for future research. In addition, we look forward to the prospect and direction of biosynthesis in the future and to providing new ideas for the biosynthesis.

#### **2. Traditional Strategies of Diversity of SMs from Fungi**

Microorganisms are important sources of natural products, most of which are isolated from bacteria and fungi. The SMs from fungi have attracted extensive attention and research because of their novel structure (e.g., terpenoids, polyketides, anthraquinones, steroids, and non-ribosomal polypeptides), diverse biological activity (e.g., antibacterial, anti-inflammatory, anti-tumor, and immunoregulation), and rich yield [1–3].

Studies on the diversity of SMs from fungi primarily followed the traditional natural product discovery strategy before information on fungal genomes became available (Figure 1). To obtain more SMs with diverse structures and biological activity from the same fungus, the most representative method is the OSMAC strategy. Generally, the same strain may produce various amounts or even distinct kinds of natural products from the different culture conditions (such as the composition of the culture medium, the fermentation conditions, the added precursors, etc.). The scientists have discovered numerous novel and bioactive natural products by the OSMAC method, showing that different culture conditions may activate the silent genes to produce new secondary metabolites [16–18]. The OSMAC strategy will continue to be used as a valuable method to exploit the biosynthetic potential of strains. Co-culture, the other traditional natural product mining method, can activate silent genes or clusters through interspecific interactions [19]. In the two-way chemical communications between the co-cultured strains, the signal molecules are transmitted back and forth to interfere with the compound library of co-cultured strains to enrich the quantity of the compounds. Epigenetic regulation is to activate the silent gene clusters through DNA methylation and histone modification without changing the DNA sequence to regulate the secondary metabolic pathway of the strain to obtain new products [18,20]. However, no matter what the traditional method is, the essence is a random selection of the de-silencing of the secondary metabolic pathway, and any conditions affecting the

response of a strain to the external conditions may be used to change the transcriptome and then to change the proteome; finally, it can be read out in the variable SMs. This "blind" selection and these changes make it neither possible to accurately understand the law of the biosynthesis of the strains from the perspective of genes, nor to directionally discover the SMs that interested us under the guidance of the genomic information, which still has great limitations in the process of mining natural products.

**Figure 1.** Traditional strategies for fungal secondary metabolites discovery. These mainly include OSMAC strategy, strains co-culture, and epigenetic regulation, aiming to stimulate the expression of biosynthetic gene clusters of SMs through changes of external conditions or exchange of signal molecules between strains in order to obtain more SMs.

## **3. Gene Mining and Bioinformatics Broaden the Discovery of SMs of Fungi**

With the rapid development of genome sequencing technology, many fungal genome data have been identified and reported, which makes it possible to predict which kinds of compounds may be produced. More and more bioinformatics tools have been developed with the continuous improvement of bioinformatics analysis. BLAST and FASTA are currently the most commonly used database search programs based on local similarity and are tools for the sequence similarity search, which can be used for homologous gene retrieval in public databases [21]. TOUCAN [22] and ARTS [23] can be used for gene mining; antiSMASH [24–26] and cluster finder [27,28] were used for analyzing and predicting BGCs; these have greatly promoted the research on fungal SM BGCs. A new stage in the study of the SMs of fungi has been entered. The fungi have large and complex genomes, restricting the analysis of gene clusters. After mining the BGCs of the SMs of fungi, it has been shown that most of the gene clusters (>90%) were unknown [29–31]. The genomes of the marine fungi *Calcarisporium* sp. and *Pestalotiopsis* sp. possessed 60 and 67 BGCs, respectively, by bioinformatics analysis, of which the new clusters accounted for 98% and 97%, respectively, and only a small number of BGCs were expressed after RNA-seq verification [30]. Gao et al. reported the high-quality genome sketch sequence of the endophytic fungus *Neonectria* sp. DH2, of which 14,163 genes are predicted to encode proteins, and 557 of the genes are unique. According to the neighborhood-linked phylogenetic tree of the ITS region, there were 47 BGCs in the DH2 genome, of which only 5 BGCs were previously reported, showing the huge production potential of the SMs of fungi [31].

Unlike the traditional research strategy, the study of the fungal SMs based on the gene mining is to associate the SMs with the BGCs by bioinformatics analysis, predict the potential biosynthesis pathway, and then verify the prediction by molecular biology techniques and to analyze the biosynthesis pathway of the SMs. In this research strategy, it is more definite to discover what we want, master the law of biosynthesis, and to develop it more accurately. Gene knockout and heterologous expression have been widely used in the study of fungal secondary metabolic biosynthesis and have played an important role.
