PhD Oral Exam - Mackenzie Thornbury, Biology

When studying for a doctoral degree (PhD), candidates submit a thesis that provides a critical review of the current state of knowledge of the thesis subject as well as the student’s own contributions to the subject. The distinguishing criterion of doctoral graduate research is a significant and original contribution to knowledge.

Once accepted, the candidate presents the thesis orally. This oral exam is open to the public.

Abstract

The yeast Kluyveromyces marxianus is an attractive candidate for industrial bioproduction due to its rapid growth rate, thermotolerance, and ability to utilize a wide range of substrates, including lactose from dairy byproducts. However, its use in metabolic engineering has been limited by underdeveloped genetic tools. This thesis addresses this gap by developing these tools with a focus on enabling fumaric acid biosynthesis as a model pathway for organic acid biomanufacturing.

To build a foundation for engineering, we developed and characterized a standardized promoter library compatible with the Yeast Toolkit system and optimized CRISPR-based editing methods, allowing precise and efficient control of gene expression and genome modification. These tools enabled the development of CRISPR activation, interference and deletion system in K. marxianus which facilitated a genome-wide study of fumaric acid tolerance, where a pooled CRISPR screen identified new contributors to acid stress resistance. In particular, the DHA1-family transporters QDR2 and QDR3 were found to improve growth in fumaric acid, underscoring the role of transport in tolerance mechanisms.

Metabolic engineering strategies were then applied to increase fumaric acid production. Deletion of FUM1 enabled fumarate accumulation via the TCA cycle, while a modified reductive TCA pathway from Rhizopus oryzae further improved yields. When combined with QDR2 and QDR3 co-overexpression, fumaric acid titers doubled relative to the base strain. Additional interventions, including heterologous glutamate dehydrogenase expression, provided modest improvements in redox balance and by-product reduction, though acetate accumulation persisted as a major challenge.

Overall, this work establishes enabling genetic engineering tools for K. marxianus, expands knowledge of its acid stress responses, and demonstrates rational design strategies for fumaric acid biosynthesis. While acetic acid overflow remains a barrier to high titers, these advances strengthen the case for K. marxianus as a versatile platform for bioproduction and open new opportunities for valorizing dairy byproducts.