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ARS Home » Midwest Area » Columbia, Missouri » Plant Genetics Research » Research » Publications at this Location » Publication #367775

Research Project: Soybean Seed Improvement Through Translational Genomics, Assessments of Elemental Carbon Metabolism, and Lipid Profiles

Location: Plant Genetics Research

Title: Application of stable isotope tracing to elucidate metabolic dynamics during Yarrowia lipolytica a-ionone fermentation

item CZAJKA, JEFFREY - Washington University
item KAMBHAMPATI, SHRIKAAR - Danforth Plant Science Center
item TANG, YINJIE - Washington University
item WANG, YECHUN - Arch Innotek, Llc
item Allen, Douglas - Doug

Submitted to: iScience
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/15/2020
Publication Date: 2/21/2020
Citation: Czajka, J.J., Kambhampati, S., Tang, Y.J., Wang, Y., Allen, D.K. 2020. Application of stable isotope tracing to elucidate metabolic dynamics during Yarrowia lipolytica a-ionone fermentation. iScience. 23(2):100854.

Interpretive Summary: Secondary metabolites are important for human nutrition. They are used as health supplements, medicines, and can have application to renewable feed stock demands for polymers, fuels and other societal needs that currently rely on non-renewable petroleum reserves. Though a number of organisms are capable of making secondary metabolites along with lipids and other precious commodities, controlling increased production is often not economically feasible and therefore a better understanding of the underlying metabolism is necessary to engineer efficient biosynthesis of valuable compounds. Here, we developed methods that can separate these types of molecules for analysis and comparison through mass spectrometry that can accurately detect and quantify them. We then used these methods to explore production in a type of yeast of an important compound for fragrance and aroma as a proof of concept. We describe the changes in yeast metabolism based on our developed methods. Our methods can provide insight to production of valuable compounds from a variety of organisms.

Technical Abstract: Targeted metabolite analysis in combination with 13C-tracing is a convenient strategy to determine pathway activity in biological systems; however, metabolite analysis is limited by challenges in separating and detecting pathway intermediates with current chromatographic methods. Here, a hydrophilic interaction chromatography (HILIC) tandem mass spectrometry approach was developed for improved metabolite separation, isotopologue analysis and quantification. The physiological responses of a Yarrowia lipolytica strain engineered to produce ~400 mg/L a-ionone and temporal changes in metabolism were quantified (e.g., mevalonate secretion, then uptake) indicating bottleneck shifts in the engineered pathway over the course of fermentation. Dynamic labeling results indicated limited TCA cycle label incorporation and, combined with a measurable ATP shortage during the high ionone production phase, suggested that electron transport and oxidative-phosphorylation may limit energy supply and strain performance. The results provide insights into terpenoid pathway metabolic dynamics of non-model yeasts and offer guidelines for sensor development and modular engineering.