Activation of Secondary Metabolism and Protease Activity Mechanisms in the Black Koji Mold 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, IFM 61405, markedly enhanced the production of (3,8)-8-hydroxy-3-carboxy-2-methylenenonanoic acid (), (3,8)-8-hydroxy-3-carboxy-2-methylenenonanoic acid (), and (3)-9-hydroxy-3-carboxy-2-methylenenonanoic acid () when coincubated with J774.1 mouse macrophage cells. The production of and 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 and . This enhancement was not replicated in IFM 59706, a nonkoji mold, despite the presence of biosynthetic genes for and in IFM 59706. Furthermore, the addition of protease inhibitors suppressed the production of and , suggesting that proteins secreted from animal cells, likely degraded by proteases secreted by strain IFM 61405, serve as precursors for and . The results show that the strategy of coculturing koji mold with animal cells has the potential to enhance the production of natural products.