Activation of hamiformamide production in the thermotolerant fungus Hamigera avellanea triggered by co-culture with animal immune cells.

Understanding the mechanisms by which silent biosynthetic genes of secondary metabolites in microorganisms are activated is critical to developing ways to enhance natural product production. Our laboratory previously showed that co-culturing fungi with animal immune cells induces the production of secondary metabolites. In this study, we cultured cells at 37 °C (the optimal temperature for human cells) with thermotolerant fungi that can grow at this temperature. This optimized condition was expected to enhance production of otherwise silent metabolite genes. Eight thermotolerant fungi were co-cultured with J774.1 mouse macrophage-like cells at 28 or 37 °C. By comparing extracts, we identified metabolites specifically induced-or markedly enhanced-by co-culture at each temperature. Co-culture of Hamigera avellanea IFM 52957 with J774.1 cells at 37 °C enhanced the expression of two compounds, 1 and 2. Structural analyses identified 1 as 4-hydroxybenzaldehyde and 2 as (Z,Z)-N,N'-[1-[(4-hydroxyphenyl)methylene]-2-[(4-methoxyphenyl)methylene]-1,2-ethanediyl]bis-formamide, designated hamiformamide. Compound 2 inhibited nitric oxide production (IC50 = 49.1 µM), indicating its potential for modulating host immune signaling. In addition, the production of 2 increased under iron-depleted conditions, suggesting it is induced by iron competition with host immune cells.