Activation of Secondary Metabolism and Protease Activity Mechanisms in the Black Koji Mold <i>Aspergillus luchuensis</i> through Coculture with Animal Cells

The activation of secondary metabolism plays a pivotal role in the discovery of novel natural products. We recently developed a coculture method involving actinomycetes and mouse macrophage-like cells to stimulate the production of bioactive compounds. A black koji mold, <i>Aspergillus luchuensis</i> IFM 61405, markedly enhanced the production of (3<i>S</i>,8<i>R</i>)-8-hydroxy-3-carboxy-2-methylenenonanoic acid (<b>1a</b>), (3<i>S</i>,8<i>S</i>)-8-hydroxy-3-carboxy-2-methylenenonanoic acid (<b>1b</b>), and (3<i>S</i>)-9-hydroxy-3-carboxy-2-methylenenonanoic acid (<b>2</b>) when coincubated with J774.1 mouse macrophage cells. The production of <b>1</b> and <b>2</b> increased by at least 3.5-fold and 2.7-fold, respectively, compared to monoculture after 7 days. A mechanistic investigation revealed that a protease from strain IFM 61405 plays a key role in enhancing the production of <b>1</b> and <b>2</b>. This enhancement was not replicated in <i>A. niger</i> IFM 59706, a nonkoji mold, despite the presence of biosynthetic genes for <b>1</b> and <b>2</b> in <i>A. niger</i> IFM 59706. Furthermore, the addition of protease inhibitors suppressed the production of <b>1</b> and <b>2</b>, suggesting that proteins secreted from animal cells, likely degraded by proteases secreted by strain IFM 61405, serve as precursors for <b>1</b> and <b>2</b>. The results show that the strategy of coculturing koji mold with animal cells has the potential to enhance the production of natural products.