Rhizopus stolonifer as a biocatalyst for cyclic dipeptide production and artemisinin/artemether biotransformation

Abstract Fungi are prolific producers of chemically diverse natural products and biocatalytically active enzymes, yet the biosynthetic and biotransformation potential of many fungal species remains underexplored. This study establishes Rhizopus stolonifer as a promising platform for both natural-product discovery and selective biotransformation of bioactive molecules. Ten cyclic dipeptides (CDPs) and over seventy additional metabolites were tentatively identified using an integrated metabolomics approach combining gas chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, and one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Three major CDPs, cyclo(L-Leu-L-Pro), cyclo(L-Ile-L-Pro), and cyclo(L-Phe-L-Pro), were subsequently isolated and characterized, with their stereochemical configurations established. In parallel, R. stolonifer was utilized as a microbial platform for the biotransformation of antimalarial drugs artemisinin and artemether. The resulting derivatives were generated through deoxygenation, site-selective hydroxylation, and structural rearrangement. Time-course NMR analyses provided evidence for the proposed biotransformation pathways. Genome mining revealed six putative non-ribosomal peptide synthetase biosynthetic gene clusters and thirteen cytochrome P450 monooxygenases, suggesting a genetic basis for CDP biosynthesis and artemisinin biotransformation, respectively. These results underscore the dual utility of R. stolonifer as a rich source of CDPs and a versatile biocatalyst for the transformation of artemisinin derivatives. Key points Rhizopus stolonifer is a rich source of cyclic dipeptides (CDPs). R. stolonifer biotransforms artemisinin and artemether into multiple derivatives. Non-ribosomal peptide synthetase pathways may underpin CDP production.