*5.2. Application of Heterologous Expression Strategy in Mining Silent BGCs*

The rapid development of gene mining and bioinformatics has brought new breakthroughs to the discovery of fungal SMs. A large number of terpenoid, PKS, and NRPS gene clusters have been predicted from the genome sequences of many fungi, most of which are unknown. The actual kinds of obtained secondary metabolites of the strains were far less than the predicted kinds based on the BGCs' functions because of the gene silence under conventional experimental conditions. How to activate these silent genes to obtain lots of SMs is the current research focus. At present, there are generally two ways to activate the silent gene clusters. One is to overexpress the silent gene clusters by adding strong promoters to or regulating the transcription factors of the original strains. Seven new compounds were obtained by overexpressing the specific transcription factor tenS of *Beauveria bassiana*-derived silent gene clusters [82]. However, it has not been widely used in fungi because of the difficulty in establishing genetic system in the original strain. The other is to clone these silent gene clusters and transform them into model strains for heterologous expression, which is a common research method at present. Nine new sesquiterpenes were discovered after the heterologous expression of silent gene clusters derived from *A. ustus* in *A. oryzae*, which strongly supports the application of heterologous expression in the gene mining of silent BGCs [83].

#### *5.3. Limitations of Heterologous Expression Strategy*

Although heterologous expression technology has significant advantages in the study of fungal secondary metabolite biosynthesis, it still has some limitations, especially in terms of the compatibility of heterologous strains with foreign genes. The proteins could be expressed and modified well by heterologous host filamentous fungi, but filamentous fungi still have some defects. When the silent hancockinone A BGC, derived from *A. hancockii*, was heterologously expressed in *A. nidulans*, the target product was not obtained due to intron cleavage and inactive expressed protein [84]. Moreover, the genetic stability of the recombinant strain is very important. How to maintain stable genetic characteristics is a problem to be solved. In short, the heterologous expression system still needs continuous technical optimization in order to better serve the biosynthesis of SMs of fungi.
