Activation of Secondary Metabolism in Aspergillus and Related Filamentous Fungi Through Regulatory Engineering

Filamentous fungi are major contributors to diverse secondary metabolites with broad applications to medicine, agriculture, and biotechnology. Advances in genome sequencing and bioinformatic tools have revealed that fungal genomes encode far more biosynthetic gene clusters (BGCs) than are expressed under normal laboratory conditions, leaving much biosynthetic potential transcriptionally silent. Overcoming this gap between predicted and observed secondary metabolism has become a major challenge in fungal natural product discovery. In this review, we summarize current strategies for activating silent or weakly expressed fungal BGCs through regulatory engineering, with an emphasis on approaches validated in Aspergillus, Penicillium, Monascus, and related filamentous fungi. We focus on genetic and chemical manipulations that enable coordinated activation of multiple biosynthetic pathways through chromatin-level modifiers, global transcriptional regulators, and developmental regulators. By framing these regulators as practical tools rather than solely biological components, we demonstrate their strengths, limitations, and applications in Aspergillus and related filamentous fungi. We further discuss emerging combinatorial and integrative approaches that use regulatory engineering alongside omics technologies and predictive tools, outlining alternatives and future directions for improving the interpretability of silent pathway activation.