*Review* **Application of Gene Knockout and Heterologous Expression Strategy in Fungal Secondary Metabolites Biosynthesis**

**Yaodong Ning †, Yao Xu †, Binghua Jiao and Xiaoling Lu \***

Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Naval Medical University, Shanghai 200433, China

**\*** Correspondence: luxiaoling80@126.com

† These authors contributed equally to this work.

**Abstract:** The in-depth study of fungal secondary metabolites (SMs) over the past few years has led to the discovery of a vast number of novel fungal SMs, some of which possess good biological activity. However, because of the limitations of the traditional natural product mining methods, the discovery of new SMs has become increasingly difficult. In recent years, with the rapid development of gene sequencing technology and bioinformatics, new breakthroughs have been made in the study of fungal SMs, and more fungal biosynthetic gene clusters of SMs have been discovered, which shows that the fungi still have a considerable potential to produce SMs. How to study these gene clusters to obtain a large number of unknown SMs has been a research hotspot. With the continuous breakthrough of molecular biology technology, gene manipulation has reached a mature stage. Methods such as gene knockout and heterologous expression techniques have been widely used in the study of fungal SM biosynthesis and have achieved good effects. In this review, the representative studies on the biosynthesis of fungal SMs by gene knockout and heterologous expression under the fungal genome mining in the last three years were summarized. The techniques and methods used in these studies were also briefly discussed. In addition, the prospect of synthetic biology in the future under this research background was proposed.

**Keywords:** fungi; secondary metabolites; gene knockout; heterologous expression
