The synthetic cytokinin forchlorfenuron (FCF), while seemingly presenting relatively low toxicity for mammalian organisms, has been the subject of renewed scrutiny in the past few years due to its increasing use in fruit crops and potential for bioaccumulation. Despite many toxicological properties of FCF being known, little research has been conducted on the toxicological effects of its secondary metabolites. Given this critical gap in the existing literature, understanding the formation of relevant FCF secondary metabolites and their association with mammalian metabolism is essential. To investigate the formation of FCF metabolites in sufficient quantities for toxicological studies, a panel of four fungi were screened for their ability to catalyze the biotransformation of FCF. Of the organisms screened, Cunninghamella elegans (ATCC 9245), a filamentous fungus, was found to convert FCF to 4-hydroxyphenyl-forchlorfenuron, the major FCF secondary metabolite identified in mammals, after 26 days. Following the optimization of biotransformation conditions using a solid support system, media screening, and inoculation with a solid pre-formed fungal mass of C. elegans, this conversion time was significantly reduced to 7 days-representing a 73% reduction in total reaction time as deduced from the biotransformation products and confirmed by LC-MS, NMR spectroscopic data, as well as a comparison with synthetically prepared metabolites. Our study provides the first report of the metabolism of FCF by C. elegans. These findings suggest that C. elegans can produce FCF secondary metabolites consistent with those produced via mammalian metabolism and could be used as a more efficient, cost-effective, and ethical alternative for producing those metabolites in useful quantities for toxicological studies.
Keywords: Cunninghamella elegans; cytokinins; forchlorfenuron; hydroxylation; liquid chromatography–mass spectrometry; metabolite identification and synthesis; microbial transformation.