Assessing Gene Expression of NAD+-Dependent Pathways in Non-Model Yeast for Biofuel Production

open_access
Open Access
Author:
Williams, Lily
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
Melanie Mc Reynolds, Thesis Supervisor
Wendy Hanna-Rose, Thesis Honors Advisor
Keywords:
NAD+
Rhodotorula Toruloides
Gene Expression
Biofuel Production
Metabolism
Abstract:
The burning of fossil fuels continues to accelerate climate change, underscoring the urgent need for renewable alternatives such as biofuels. While most biofuels are currently derived from crops that require substantial resources to cultivate, microbes like yeast provide a cost-effective and efficient platform for biofuel production. Oleaginous yeasts, such as Rhodotorula toruloides, provide an excellent alternative due to their ability to produce high levels of lipids and fatty alcohols, which serve as additives in biofuels and other bioproducts. However, as a non-model organism, it remains understudied. Lipid and fatty alcohol biosynthesis depend on NADP(H) and its precursor NAD⁺, which is known to decline over time in most species. However, the intricacies contributing to this decline remain poorly understood, particularly in R. toruloides. This raises the central question: How do NAD⁺-dependent pathways change during the growth of this non-model yeast? Understanding these changes is critical to optimizing bioproduct yields. We hypothesize that there is decreased expression of NAD⁺ biosynthetic genes over time, coupled with increased expression of degradation pathway genes. Using quantitative reverse transcription PCR, we profiled expression of genes involved in NAD⁺ recycling over time in three nutrient conditions: rich media, minimal media, and nitrogen limitation. Utilizing these different conditions highlights how nutrient availability can alter the pathways of the NAD metabolome. This experiment highlights vulnerabilities in NAD⁺ biosynthesis that may alter lipid and fatty alcohol production. Changes in gene expression were shown between the different growth conditions tested. Identifying and addressing critical bottlenecks in a variety of environmental conditions with different nutrient sources helps to inform strategies for engineering R. toruloides into a more robust and sustainable platform for biofuel and bioproduct generation.