Date of Award
12-20-2024
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Sustainability efforts have driven significant advancements in synthetic biology, particularly through the development of microbial cell factories designed for waste reduction and resource conservation. This dissertation seeks to characterize a microbial biorefinery platform for the biosynthesis of terpene compounds from yeast, with an emphasis on the industrial bioconversion of waste lipids. Terpene compounds play crucial roles in various industrial applications; however, traditional methods of terpene production have sustainability and productivity challenges that could be overcome by using microbial biosynthesis. The yeast Yarrowia lipolytica is a microbial host well suited for converting waste oils into value-added terpenes due to its native mevalonate pathway and ability to metabolize hydrophobic substrates. However, challenges exist in transferring the production to industrial scale processes. This work aims to evaluate Y. lipolytica for its use as a lipid-to-terpenoid platform host. First, 13C-isotopic tracing, cell composition, and cell morphology analyses were utilized to investigate feedstock utilization in Y. lipolytica. Comparative analyses revealed that oil-based feedstocks enhance biomass yield and cell size but reduce protein content and enzyme abundance for product synthesis. Importantly, the co-catabolism of sugar and fatty acids revealed a segregated metabolic network that allows for optimized resource allocation without catabolite repression. Metabolic flux analysis (MFA) and RNA-sequencing were conducted to further elucidate changes in metabolic regulation during lipid-based substrate utilization. Transcriptomics data were used to guide 13C-MFA model construction and to validate the 13C-MFA results. A genome-scale model was constrained by the resulting 13C-MFA data to predict Y. lipolytica fluxes and theoretical yields of terpene products. This work provided new insights into cellular regulation during catabolism of oil-based feedstocks. Specifically, the carnitine shuttle was found to be a key regulator of acetyl-CoA pools in cells cultivated in oil-based media. Further, in addition to the oxidative pentose phosphate pathway, the mannitol cycle provides another source of NADPH generation as well as a route for balancing excess NADH generated from the β-oxidation of long-chain fatty acids. Lastly, the performance of the engineered β-carotene producing Y. lipolytica strain was assessed to evaluate titer stability for improving the understanding of culture heterogeneity during process scale-up. Continuous bioreactor fermentations were conducted in 2-liter reactors and semi-continuous fermentations were conducted in the Sartorius Ambr® 250 high-throughput system. Successive passaging in shaking flasks was conducted alongside these fermentation methods to assess the similarities in titer stability over more cell generations. Ultimately, the results showed that cultivation with oil as the carbon source allowed for extractive fermentation and demonstrated significantly prolonged β-carotene production compared to glucose-grown cells. Further, successive passaging in shaking flasks demonstrated production over more generations than observed in bioreactor fermentations, demonstrating the challenge of strains performing differently at laboratory and industrial scales. Together, the outcomes of the metabolic and production stability mechanisms investigated in this dissertation provide new insights into the non-model yeast Y. lipolytica and can be extended to different microbial systems for biorefinery applications.
Language
English (en)
Chair
Yinjie Tang
Committee Members
Douglas Allen; Fuzhong Zhang; Joshua Yuan; Marcus Foston; Wei Wen Su