Monascus, a genus of fungi known for its fermentation capability and production of bioactive compounds, such as Monascus azaphilone pigments and Monacolin K, have received considerable attention because of their potential in biotechnological applications. Understanding the genetic basis of these metabolic pathways is crucial for optimizing the fermentation and enhancing the yield and quality of these products. However, Monascus spp. are not model fungi, and knowledge of their genetics is limited, which is a great challenge in understanding physiological and biochemical phenomena at the genetic level. Since the first application of particle bombardment to explore gene function, it has become feasible to link the phenotypic variation and genomic information on Monascus strains. In recent decades, accurate gene editing assisted by genomic information has provided a solution to analyze the functions of genes involved in the metabolism and development of Monascus spp. at the molecular level. This review summarizes most of the genetic manipulation tools used in Monascus spp. and emphasizes Agrobacterium tumefaciens-mediated transformation and nuclease-guided gene editing, providing comprehensive references for scholars to select suitable genetic manipulation tools to investigate the functions of genes of interest in Monascus spp.

Monascus spp. are renowned for producing valuable Monascus azaphilone pigments (MonAzPs), yet their biosynthesis is intrinsically linked to the co-production of the mycotoxin citrinin, posing a significant safety challenge and limiting industrial application. Conventional approaches to disrupt citrinin synthesis often inadvertently reduce MonAzPs yield. To circumvent this limitation, we employed a dual-targeting strategy in Monascus ruber. In this study, we selected the mresa1-overexpressed strain—which can produce more MonAzPs and citrinin—as wild strain to construct a pksCT-deleted strain and explore whether pksCT deletion can affect the enhancement of MonAzPs caused by MrEsa1 overexpression. The results showed that the growth, development, and production of MonAzPs in △pksCT-M7::PtrpC-mresa1 were comparable to those in M7::PtrpC-mresa1, showing accelerated growth and higher MonAzPs yields than in M7. In addition, the relative expression levels of genes involved in MonAzPs synthesis in △pksCT-M7::PtrpC-mresa1 and M7::PtrpC-mresa1 showed the same trend compared with M7, indicating that MrEsa1 overexpression can resist the reduction in MonAzPs caused by pksCT deletion. This study establishes a novel and effective paradigm for decoupling desirable metabolite production from toxin synthesis in fungi, providing a strategic framework for the safe and enhanced production of MonAzPs.