(532f) Understanding Yarrowia Lipolytica Triacylglycerol Catabolism Using 13c-Metabolic Flux Analysis, Genome Scale Modeling, and Transcriptomic Analysis

Yarrowia lipolytica is a notable yeast species for its role in industrial biotechnology, particularly in the conversion of waste oil to high-value products. Despite its capabilities, the specifics of how Y. lipolytica metabolizes lipid substrates including triacylglycerol (TAG) remain not fully elucidated. This study uses ¹³C-metabolic flux analysis (¹³C-MFA), a genome-scale model (GSM), and transcriptomics to analyze the metabolic mechanisms Y. lipolytica uses to catabolize TAG substrates by focusing on the utilization of oleic acid, glycerol, and glucose as carbon sources. This approach offers insights into the regulation of metabolic pathways during the catabolism of different components of TAG substrates and how these pathways are regulated compared to glucose metabolism.

Methods.

We integrated ¹³C-MFA with GSM and transcriptomics to study the metabolic behavior of Y. lipolytica. The genome-scale model was informed by transcriptomic data to validate carbon source specific metabolic networks, which were then used to make and refined using ¹³C-MFA results. This integration allowed for a detailed analysis of the flux distributions of Y. lipolytica's metabolic network when it’s grown on different carbon sources. The model results help us to understand the metabolic adjustments made by the yeast in response to changes in carbon substrate.

Results.

Our findings reveal significant metabolic flexibility in Y. lipolytica, with distinct pathways preferred based on the carbon source. Notably, a large portion of oleic acid is directed through the glyoxylate shunt in a route that minimizes CO₂ loss. This pathway preference contrasts with the metabolism of glucose and glycerol, where the EMP pathway is the perfered route for carbon and energy utilization. Moreover, the integration of transcriptomic data with ¹³C-MFA and GSM analyses further confirmed the crucial role of the oxidative pentose phosphate pathway as the primary source of NADPH.

Implications.

This study demonstrates the importance of using a multidisciplinary approach to study the metabolic pathways and regulatory networks in industrially relevant microorganisms. The insights gained from the metabolic modeling and analysis of Y. lipolytica advance our understanding of its metabolic capabilities and flexibility and highlight potential metabolic engineering targets for enhancing the production of value-added products from lipid-based substrates. The findings offer a foundation for future efforts to optimize Y. lipolytica as a cell factory in the bioeconomy, capable of converting waste lipids into a range of bio-based products.