Phosphatidylcholine biosynthesis via ChoC is crucial for cellular integrity and virulence in Rhizopus microsporus.

Rhizopus microsporus is an opportunistic fungal pathogen and a prominent etiological agent of mucormycosis, a life-threatening invasive fungal infection. In this study, we investigated the role of choC, a gene encoding a putative phospholipid methyltransferase involved in phosphatidylcholine (PC) biosynthesis through the Bremer-Greenberg pathway. Deletion of choC using CRISPR-Cas9 led to severe defects in growth and asexual development, which persisted even at 37°C, a temperature relevant to host infection. Supplementation with choline or its precursor PDEA restored growth, confirming the importance of choC in maintaining PC levels. Lipidomic and transcriptomic analyses revealed broad disruptions in PC and phosphatidylethanolamine (PE) levels, glycerol-3-phosphate and fatty acid metabolism, endoplasmic reticulum (ER) stress responses, and vesicle trafficking. Notably, ΔchoC mutants exhibited reduced melanin and chitin content, while ergosterol levels remained unaffected, indicating specific impairment in cell envelope assembly. In a murine infection model, the ΔchoC mutant showed attenuated virulence, linking lipid imbalance to impaired host adaptation. Together, these findings highlight choC as a key regulator of membrane homeostasis and fungal pathogenicity. As the methylation-dependent PC synthesis pathway is barely present in human tissues, choC represents a promising target for selective antifungal therapies.