Unconventional semi-solid cultivation enhances cytochalasins production by the Colombian fungus Xylaria sp. CM-UDEA-H199.

Fungal species of the order Xylariales, particularly those from tropical and untapped areas like the Amazon region, denote an intriguing reservoir of biodiversity and chemically varied metabolites. Based on this potential and by implementing the One Strain Many Compounds (OSMAC) approach, herein we have cultivated a Colombian Xylaria strain in several liquid, solid or semi-solid media, under different nutrient compositions and culture conditions. Metabolomic studies of Xylaria sp. CM-UDEA-H199 across these conditions led to the isolation of diverse metabolites. Six compounds were purified from rice (BRFT) cultures, identified as griseofulvin (1), xylaropyrones B/C (2/3), akolitserin (4), hypoxylin A (5), and (-)-(R)-5-(methoxycarbonyl)mellein (6). Three compounds were isolated from liquid YM cultivation: 2-hexylidene-3-methylsuccinic acid (7), its 4-methyl ester (8), and akoenic acid (9). Notably, cultivation in the newly designed semi-solid (S-BRFT) medium significantly altered the metabolome, leading to the predominant production of cytochalasins, with five derivatives (10-14) purified and structurally characterized.Among the isolated cytochalasins, compound 12 was identified as a previously undescribed natural diepoxy derivative of cytochalasin D. Structure elucidation of all isolated compounds was achieved based on their MS and comprehensive 1D/2D NMR analyses in addition to comparisons with the reported literature. Compounds 4-6, 10 and 11 revealed mild antifungal activity, while compounds (1, 5, 6, 8, 10, 11, 13 and 14) exhibited cytotoxic activity, with hypoxylin A (5) being the most potent, displaying IC(50) values in the nanomolar range. In cellulo studies revealed that the epimerization at C-5 of cytochalasin D (10) backbone, as in hypoxylin A (5), neither affected its activity nor reversibility on actin dynamics. However, the epoxylated variant of 10, cytochalasin R (14), enhanced actin activity accompanied by reduced cytotoxicity compared to 5 and 10. The occurrence of diverse epoxy-substituted cytochalasins suggests that specific biosynthetic enzymes were activated in response to the applied fermentation conditions. These findings provide a basis for further bioprocess optimization strategies aimed at enhancing cytochalasan production, a chemical class recognized for its promising bioactivities in recent decades.