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Cross-organelle Coordination to Advance Plant Cytochrome P450 Functionality in Yeast

ABSTRACT: Sustainable production of plant secondary metabolites remains a significant challenge. Engineered yeast platforms offer a promising alternative, yet reconstituting plant cytochrome P450s (CYPs), key enzymes in these pathways, remains a major burden. Plant Membrane-Steroid-Binding Proteins (MSBPs) have been suggested to enhance CYP functionality, possibly by facilitating CYP-CYP reductase (CPR) interactions. In this study, Arabidopsis thaliana MSBP1 (AtMSBP1) was engineered with diverse plant CYPs to assess its broad CYP activity-enhancing effects. Surprisingly, CYP-CPR interactions are not essential for AtMSBP1’s function. Instead, AtMSBP1 orchestrates inter-organelle communication, involving not only the endoplasmic reticulum (ER), but mitochondria and vacuoles. Co-expression of AtMSBP1 with CYPs triggered significant subcellular reorganization, including tubular ER expansion, increased mitochondrial volume, and vacuole fission. This study highlights the critical role of organelle coordination in supporting CYP functionality, emphasizing a shift from traditional ER-based approaches to engineering cross-organelle interactions and tailoring the cellular microenvironment to enhance plant CYP activity in yeast.

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ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Songbo Wei

LAB HEAD: Yanran Li

PROVIDER: PXD060429 | Pride | 2025-09-22

REPOSITORIES: Pride

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Project description:Cytochrome P450s (CYPs) are a family of enzymes responsible for metabolizing nearly 80% of small molecule drugs. Variants in CYPs can substantially alter drug metabolism, which may result in improper dosing and severe adverse drug reactions. CYPs have low sequence conservation, making it difficult to anticipate whether variant effects measured in one CYP may extend to others based on sequence alone. Even closely related CYPs, like CYP2C9 and its closest homolog CYP2C19, have distinct phenotypic properties despite sharing 92% of their sequences. Thus, we used Variant Abundance by Massively Parallel sequencing (VAMP-seq) to measure the protein abundance of 7,660 missense variants in CYP2C19 expressed in cultured human cells. Our results confirmed positions and structural features critical for CYP function and revealed how variants at positions conserved across all eukaryotic CYPs influence abundance. We jointly analyzed 4,670 variants whose abundance was measured in both CYP2C19 and CYP2C9, finding that the homologs have different variant abundances in substrate recognition sites within the hydrophobic core, and that substitutions in some regions reduced abundance in CYP2C19 but not CYP2C9. We also measured the abundance of all single and some multiple WT amino acid exchanges between CYP2C19 and CYP2C9. While most exchanges had no effect, substitutions in substrate recognition site 4 (SRS4) reduced abundance in CYP2C19. When nearby amino acids were exchanged in double and triple mutants, we found distinct interactions between the sites in CYP2C19 and CYP2C9, revealing a region that is partially responsible for the difference in thermodynamic stability between the two homologs. Since these positions are also important for determining substrate specificity, there may be an evolutionary tradeoff between stability and altered enzymatic function. Finally, we used our data to analyze 368 previously unannotated human variants, finding that 43% had decreased abundance. Thus, by comparing variant effects between two closely related, important human genes, we have uncovered regions underlying their functional differences and paved the way for a more complete understanding of one of the most versatile families of enzymes.

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	SX_Customized_search-Proteingroups_ProteomeXchange.txt	Txt
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Cross-organelle Coordination to Advance Plant Cytochrome P450 Functionality in Yeast

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