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Targeted proteomics (parallel reaction monitoring) to quantify starch-biosynthetic proteins in yeast

ABSTRACT: Heterologous protein expression in Saccharomyces cerevisiae is a powerful testbed to dissect the functions of plant starch biosynthetic enzymes and create diverse starch-like polymers. Here we modulated the expression each starch synthase together with a branching enzyme (derived from Arabidopsis thaliana) in a suite of yeast strains and quantified protein abundances using targeted proteomics. Specifically, we developed parallel reaction monitoring assays to quantify the Arabidopsis proteins SS1, SS2, SS3, SS4, BE2, BE3, ISA1, ISA2, among others (see Supplementary File 1, sheet 3) when expressed in yeast. We selected multiple surrogate peptides for each protein and characterized the assays with respect to their dynamic range (Supplementary Files 1 and 2). Data calibration was supported using crude stable isotope labeled (SIL) peptides.

ORGANISM(S): Arabidopsis Thaliana Saccharomyces Cerevisiae

SUBMITTER: Barbara Pfister

PROVIDER: PXD033364 | panorama | Mon Sep 05 00:00:00 BST 2022

REPOSITORIES: PanoramaPublic

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Project description:Steroidal alkaloids are FDA-approved drugs (e.g., Zytiga) and promising drug candidates/leads (e.g., cyclopamine); yet many of the ≥ 697 known steroidal alkaloid natural products remain underutilized as drugs because it can be challenging to scale their biosynthesis in their producing organisms. Cyclopamine is a steroidal alkaloid produced by corn lily (Veratrum spp.) plants, and it is an inhibitor of the Hedgehog (Hh) signaling pathway. Therefore, cyclopamine is an important drug candidate/lead to treat human diseases that are associated with dysregulated Hh signaling, such as basal cell carcinoma and acute myeloid leukemia. Cyclopamine and its semi-synthetic derivatives have been studied in (pre)clinical trials as Hh inhibitor-based drugs. However, challenges in scaling the production of cyclopamine have slowed efforts to improve its 1 efficacy and safety profile through (bio)synthetic derivatization, often limiting drug development to synthetic analogs of cyclopamine such as the FDA-approved drugs Odomzo, Daurismo, and Erivedge. If a platform for the scalable and sustainable production of cyclopamine were established, then its (bio)synthetic derivatization, clinical development, and, ultimately, widespread distribution could be accelerated. Ongoing efforts to achieve this goal include the biosynthesis of cyclopamine in Veratrum plant cell culture and the semi-/total chemical synthesis of cyclopamine. Herein, this work advances efforts towards a promising future approach: the biosynthesis of cyclopamine in engineered microorganisms. We completed the heterologous microbial production of verazine (biosynthetic precursor to cyclopamine) from simple sugars (i.e., glucose and galactose) in engineered Saccharomyces cerevisiae (S. cerevisiae) through the inducible upregulation of the native yeast mevalonate and lanosterol biosynthetic pathways, diversion of biosynthetic flux from ergosterol (i.e., native sterol in S. cerevisiae) to cholesterol (i.e., biosynthetic precursor to verazine), and expression of a refactored five-step verazine biosynthetic pathway containing eight heterologous enzymes sourced from seven different species. Importantly, S. cerevisiae-produced verazine was indistinguishable via liquid chromatography-mass spectrometry from both a commercial standard (Veratrum spp. plant-produced) and Nicotiana benthamiana-produced verazine. To the best of our knowledge, this is the first report describing the heterologous production of a steroidal alkaloid in an engineered yeast. Verazine production was increased through design-build-test-learn cycles to a final titer of 27 ± 2 μg/L (1.7 ± 0.1 μg/g DCW). Together, this research lays the groundwork for future microbial biosynthesis of cyclopamine, (bio)synthetic derivatives of cyclopamine, and other steroidal alkaloid natural products.

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Targeted proteomics (parallel reaction monitoring) to quantify starch-biosynthetic proteins in yeast

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