*5.1. Application of Different Heterologous Hosts*

The selection of the heterologous host is the key to the successful application of heterologous expression. Firstly, it is important to determine the clear genetic background of the heterologous expression strain. Secondly, the heterologous expression strain should have the advantages of simple culture and fast growth. Finally, the simple and easy manipulation and high transformation efficiency of the genetic transformation were chosen. At present, the mature and commonly used expression systems in the biosynthesis of SMs of fungi include *Escherichia coli* and *Bacillus subtilis* in prokaryotes and *Saccharomyces cerevisiae* and filamentous fungi in eukaryotes, such as *Aspergillus nidulans* and *Aspergillus oryzae*. The selection of model strains should consider the sources of different genes (clusters) and the physical and chemical properties of the expressed products.

### 5.1.1. Application of Filamentous Fungi as Heterologous Hosts

With the in-depth study of filamentous fungi, there are more outstanding advantages than the model strains with, for example, prokaryotes and yeast as the heterologous hosts. The filamentous fungi can correctly splice introns from heterogenous fungi and express multiple biosynthetic genes in fungi concurrently, which plays a role in protein translation and post-modification to obtain the target product. At present, the filamentous fungi expression systems commonly used in fungal heterologous expression were mainly *A. nidulans* and *A. oryzae*.

As a filamentous fungus, *A. nidulans* has a relatively clear genetic background after years of research, and a relatively mature genetic transformation system has been established. It has been widely used as a model strain in the study of the heterologous expression of the SMs of fungi. With the combined heterologous expression of the biosynthesis gene cluster of sordarinane, derived from *Sordaria araneosa* with enzyme experiments in vitro, a group of P450 multi-enzyme systems was confirmed. The new catalytic function of the P450 family oxidases was demonstrated, which laid a foundation for the development of P450 multi-enzyme synergistic catalysis for the synthesis of new chemical entities [56]. Wei et al. identified the functions of some P450 enzymes and sesquiterpene synthetases by the heterologous expression of the BGC of asperaculin A, isolated from *A. aculeatus* [57]. Due to the failure of the gene knockout strategy, Zhong et al. heterogeneously expressed the BGC of rumbrin, isolated from *Auxarthron umbrinum*, and found an autoantibody gene, verifying that the product had anti-HIV activity [58]. In addition, the BGCs of flavunoidine-1 derived from *A. flavus* were recombinantly arranged and heterologously expressed in *A. nidulans* in different combinations, and the BGCs containing both TC and NRPS core enzymes were identified [59]. In addition, the BGCs of harzianic acid [60] and trichoxide [61] from *Trichoderma*, citridone [62] and ilicicolin H [63] from *Penicillium*, and decarestricitine from *Beauveria bassiana* were heterologously expressed in *A. nidulans*, combined with precursor feeding, which clarified the biosynthesis pathway to some extent, respectively [64].

*A. oryzae*, a heterologous host, has similar characteristics and advantages to *A. nidulans*, which is also widely used as a model strain of biosynthetic genes in the SMs of fungi. In the reconstruction of the biosynthetic pathway of phlegmacins derived from *Talaromyces* sp., an unprecedented laccase-involved unsymmetrically regioselective oxidative coupling reaction was shown, which provides a new reference for the synergistic catalytic mechanism of laccases and other proteins [65]. The BGC of funiculolides derived from *A. funiculosus* were heterogeneously expressed to elucidate the fact that α-ketoglutarate-dependent dioxygenase FncG catalyzed spirocyclopentanone [66]. By the heterologous expression of CJ-20557 biosynthetic gene clusters from *A. duricaulis*, in combination with in vitro enzyme experiments, it was clarified that the SAT domain of polyketide synthase DrcA was responsible for the formation of the depside bond, which enriched the understanding of the mechanism of fungal NR-PKS biosynthesis [67]. Chen et al. confirmed that the enzyme IlIS, a sesquiterpene synthase from *Irpex lactous*, was responsible for the synthesis of the tremulane skeleton through heterologous expression and in vitro enzymatic reaction, and four new tremulane sesquiterpene products were isolated [68]. The specific PKS synthase GrgF in the BGC of gregatin A derived from *Penicillium* sp. [69] and the trichobrasilenol terpene cyclase derived from *Trichoderma atroviride* [70] were verified by the heterologous expression of *A. oryzae*. In addition, the NRPS-PKS gene cluster from *A. candidus* was heterologously expressed to obtain a pyrrolobenzazepine alkaloid [71]. Additionally, conidiogenone [72], brevianamide A [73], and brevione E [74] derived from *Penicillium* were heterologously expressed in *A. oryzae*, combined with the precursor feeding, which is helpful in analyzing the biosynthesis pathway.

#### 5.1.2. Application of *Saccharomyces cerevisiae* as Heterologous Host

Because of the early study of the genetic background and the mature genetic manipulation system, *Saccharomyces cerevisiae* was widely used as the heterologous expression model strain in the study of the SMs of fungi in the past. Eukaryotic *S. cerevisiae* could translate proteins correctly and post-modify. Zhang et al. heterologously expressed *P. funiculosum* source chrodrimanin-type meroterpenoids BGCs and verified them in in vitro catalytic experiments to clarify the function of CdnC protein, obtaining a series of new

chrodrimanins compounds [75]. In the biosynthetic path of cyclohexanoid terpenoids derived from *Aspergillus* sp.*,* two key enzymatic functions were characterized by heterologous expression and in vitro enzymology experiments [76]. In addition, *S. cerevisiae* has also been applied for the expression of entire BGCs. *S. cerevisiae* was used as an expression vector in the heterologous expression of the BGC of the formation central C ring in the tetracyclic ergoline derived from *A. fumigatus* [77]. In the study of the biosynthesis pathway of shimalactones derived from *Emericella variecolor* [78], and flavunoidine 1 [59] and diorcinol [79] derived from *Aspergillus, S. cerevisiae* was also the heterologous expression vector. However, compared with filamentous fungi as expression vectors, *S. cerevisiae* still has some shortcomings, such as the lack of an advanced mRNA splicing system and the difficult in expressing complex BGCs; it is only suitable for expression of single or simple BGCs, which restricts its further application in the study of fungal SMs.
