Extension of the ergot alkaloid gene cluster

Abstract

Specialized metabolites produced by fungi impact human health. A large portion of the pharmaceuticals currently on the market are derived from metabolites biosynthesized by microbes. Ergot alkaloids are a class of fungal metabolites that are important in the interactions of environmental fungi with insects and mammals and also are used in the production of pharmaceuticals. In animals, ergot alkaloids can act as partial agonists or antagonists at receptors for 5-hydroxytryptamine (serotonin), dopamine, and noradrenaline as ergot alkaloids have chemical structures similar to those neurotransmitters. Therefore, they affect insects and mammals that consume them and can be used to produce drugs that can aid in treatment of migraines, dementia, Parkinson’s and gynecological issues. The conserved ergot alkaloid synthesis pathway is responsible for the biosynthesis of these tryptophan-derived toxins in ergot alkaloid-producing fungi. Clusters of ergot alkaloid synthesis (eas) genes present in genomes of ergot alkaloid-producing fungi encode enzymes that catalyze synthesis of these chemicals. The fungus Metarhizium brunneum produces lysergic acid α-hydroxyethylamide (LAH), an ecologically and pharmaceutically important compound. The final steps of LAH synthesis involve two monomodular nonribosomal peptide synthetases: lysergyl peptide synthetase 2 (Lps2); encoded by lpsB and Lps3,encoded by lpsC. A novel two-module Lps gene, lpsD, was discovered via genome mining in three species of lysergic acid amide-producing Aspergillus. Heterologous expression of lpsD from Aspergillus leporis in a lysergic acid-accumulating strain of Aspergillus fumigatus yielded ergonovine and lysergyl-alanine, end products typically derived from Lps2 and Lps3 in Clavicipitaceous fungi. Addition of the A. leporis easO gene to this strain resulted in accumulation of LAH, the pathway end product observed in both A. leporis and M. brunneum. This study established that the product of the lpsD gene is the functional equivalent of the two monomodular peptide synthetases of the Clavicipitaceae. While most biosynthetic steps in the ergot alkaloid pathway are well characterized, the regulation of the genes is unknown. Understanding the regulatory mechanisms may aid in insect biocontrol strategies and may be of value for the pharmaceutical industry, as upregulation of the pathway could result in increased biosynthetic yields. In this study, the role of two novel genes with regulatory potential, easR and easS were studied to determine their role in ergot alkaloid biosynthesis. CRISPR-mediated knockout of easR in M. brunneum resulted in a strain with no detectable ergot alkaloids. Infection of Galleria mellonella larvae with the easR knockout of M. brunneum compared to a wild-type control showed a significantly lower virulence rate for the knockout strain, supporting previous evidence that ergot alkaloids contribute to fungal virulence to this insect. Knockout of easR greatly reduced or eliminated accumulation of mRNA from the eas genes. This study provided evidence that easR plays a role in regulating ergot alkaloid biosynthesis. The functional copy of the easS gene identified in M. majus was transformed into M. brunneum. The introduction of this potential repressor gene did not yield any significant difference in ergot alkaloid accumulation. Thus, despite its presence in the eas cluster, the easS gene may not play a role in ergot alkaloid accumulation. Collectively, the components of this study expanded the ergot alkaloid synthesis cluster and provided information on novel biosynthetic and regulatory genes