Strategies to design bioprocesses for sustainable yeast-based production of recombinant proteins and bioproducts
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Date
2025-12-30
Authors
Belloch-Molina, Carlos
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Publisher
University College Cork
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Abstract
Kluyveromyces marxianus is a fast-growing, thermotolerant yeast with the ability to metabolise diverse sugars and naturally secrete proteins, making it a promising candidate for sustainable biorefineries. However, its industrial adoption has been limited by a lack of standardised genetic tools and incomplete understanding of its complex regulatory networks. This thesis addresses these bottlenecks by integrating transcriptomics, synthetic biology, and Adaptive Laboratory Evolution to characterise, engineer, and evolve K. marxianus into a robust and versatile industrial chassis.
First, transcriptome analysis was used to elucidate the nitrogen requirements of K. marxianus during growth on whey permeate, revealing activation of nitrogen scavenging pathways and unique adaptations. These insights provide a foundation for optimising growth and productivity in dairy-based waste streams. Next, a modular, Golden Gate compatible toolkit was developed to expand protein secretion capabilities. Native secretion signals were functionally characterised using a Yeast Surface Display platform, enabling standardised study of heterologous protein secretion in K. marxianus. Finally, Adaptive Laboratory Evolution was applied to generate a non-Genetically Modified strain with enhanced ethanol tolerance and operational robustness. This evolved strain was validated both in laboratory-scale fermentations and, most importantly, in full-scale trials at the Carbery Food Ingredients plant, demonstrating its industrial applicability for converting whey permeate into bioethanol.
Together, this work delivers functional insights, practical synthetic biology tools, and industrial validation, advancing K. marxianus as a resilient microbial cell factory for circular dairy biorefineries. By bridging these fields, this thesis establishes a foundation for future deployment of K. marxianus as a cornerstone organism in the bioeconomy.
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Keywords
Synthetic biology , Sustainability , Whey permeate , Secretion , Yeast , Adaptive Laboratory Evolution , Heterologous protein production , Transcriptome
Citation
Belloch-Molina, C. 2025. Strategies to design bioprocesses for sustainable yeast-based production of recombinant proteins and bioproducts. PhD Thesis, University College Cork.