Comprehensive Genome-Wide Analyses and Functional Validation of C22 Desaturases and C24 Methyltransferases for the Regulatory Dynamics between Steroidal Saponins and Brassinosteroids in Three Asparagus L. Species

Abstract

Asparagus L. species are recognized for producing steroids, including brassinosteroids (BRs) and steroidal saponins (SSs), crucial for development and environmental adaptation. C24 methyltransferases (C24SMTs) and C22 desaturases (C22SDs) are key enzymes in steroid biosynthesis, and their synergistic actions may optimize substrate availability, therefore rebalancing the metabolic flux between SS and BR pathways. Despite their significance, C22SDs and C24SMTs have yet to be functionally characterized in Asparagus L. Thus a comprehensive genome-wide analysis of C22SDs and C24SMTs across three genome available species: Asparagus officinalis, Asparagus taliensis and Asparagus setaceus was conducted, identifying 6 C22SD candidates each and 4, 7, and 5 C24SMT candidates respectively in these species. Due to the difficulty in transformation of A. officinalis and the lack of established transformation system in the other two species, Neurospora crassa was employed to characterize these representative enzymatic activities. Targeted disruptions of the homologous erg5 (NcC22SDs) and erg6 (NcC24SMT) led to; significant declines in ergosterol (ERG) levels, reduced growth rates and altered membrane characteristics. Complementation with selected representatives of C22SDs and C24SMTs from the three species resulted in; partial restoration of ERG biosynthesis, improved growth rates and maintained membrane fluidities. 3D structural predictions revealed strong homology of representative C22SDs and C24SMTs candidates to fungal erg5 and erg6 enzymes while molecular docking demonstrated high affinity substrate interactions supporting our hypothesis of a cooperative interaction between these enzymes, underscores the dynamic interplay between SSs and BRs biosynthesis suggesting metabolic channeling that optimizes substrate availability and has implications for understanding steroid metabolism in Asparagus species.

Keywords: C22 desaturases, C24 methyltransferases, steroids biosynthesis, Asparagus L., heterologous expression.