Activation of a silent gene cluster in Aspergillus nidulans through the development of a hybrid transcription factor

Michelle Grau 1 Ruth Entwistle 2 Christine Elizabeth Oakley 2 Yi-Ming Chiang 1 Berl R. Oakley 2 Clay C.C. Wang 1
1Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, California, USA
2Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA

Many efforts have focused on activating silent gene clusters in fungal species since they are a rich source of structurally diverse compounds with medicinal and agricultural value. Fungal genome analyses have revealed that many species contain a surprising large number of secondary metabolite (SM) genes, the products of which are mostly unknown. In this investigation, we use a genetically engineered host strain of Aspergillus nidulans, developed in earlier work, which allows genes to be deleted or have their promoters replaced with relative ease. This genetic system has allowed us to express cryptic SM pathways in A. nidulans through promoter replacement, and through deletion analysis, define the genes responsible for the synthesis of each SM. While most studies have focused on nonreducing polyketide synthase (NR-PKS) and non-ribosomal peptide synthetase pathways, this investigation highlights the discovery of a novel metabolite produced by one of the less-studied, highly reducing (HR)-PKS pathways. Preliminary work focused on generating an overexpression strain of the HR-PKS, AN11191, and subsequent experiments determined the product released by AN11191 to be octatrienoic acid (OTA). Efforts to overexpress the entire AN11191 pathway through overexpression of the cluster-specific transcription factor (TF), AN9221, proved to be unsuccessful, and prompted the design of a highly activated hybrid TF. This chimeric TF is characterized by the native DNA binding domain of AN9221 fused to the TF activation domain from the highly expressed pathway for asperfuranone. Heterologous expression of this hybrid TF in place of AN9221 resulted in the successful production of the SM derived from the AN11191 cluster. Gene deletion studies have allowed us to elucidate the biosynthetic pathway for this compound.