Overcoming Barriers to Heterologous Gene Expression Uncovers Regulatory Constraints in the Mucoralean Pathogen Rhizopus delemar

Rhizopus species are a leading cause of mucormycosis, an aggressive and life-threatening invasive fungal disease that affects immunocompromised individuals. Despite their clinical importance, mucoralean pathogens, especially R. delemar, are difficult to manipulate genetically because of coenocytic growth, multinucleate hyphae, difficulty with transformation, and limited selectable markers. These obstacles hinder the study of innate stress resistance and disease mechanisms. Previous efforts to express heterologous genes or implement gene-editing systems with vectors designed for model fungi have been largely unsuccessful or inefficient in R. delemar, suggesting unique requirements for gene expression in this organism. In this study, we tested nuclear-targeted fluorescent protein expression and identified differences in proline and arginine codon usage between heterologous and endogenous coding sequences. We then evaluated gene expression efficiency using various combinations of heterologous and endogenous regulatory elements and coding sequences, including six promoters, two terminators, three histone fusion proteins, and two fluorescent reporters, with or without an exogenous nuclear localization signal (NLS). Endogenous promoters orthologous to those with high transcriptional activity produced robust fluorescence, in contrast to weak fluorescence from constructs with heterologous sequences. Notably, fluorescence was also observed in a construct lacking an experimentally provided terminator, and the H2B-GFP fusion protein did not require an exogenous NLS for nuclear localization. These results show that efficient heterologous gene expression in R. delemar requires optimization at several levels, including promoter selection, coding sequence compatibility, and expression-cassette design. Although the direct impact of codon usage on expression efficiency remains to be tested, the expression systems developed here provide a platform for molecular analysis of R. delemar biology and pathogenesis.