Engineering diverse yeast species for optimal sugar consumption and enhanced product formation

Yeasts are employed ubiquitously within industry and academia for basic studies in biology and production of value-added compounds. Although the term yeast generally reminds people of the common baker’s yeast, Saccharomyces cerevisiae, yeasts are a diverse group of organisms with an extensive evolutionary history. This thesis focuses on two vastly differing yeasts: Yarrowia lipolytica and S. cerevisiae. Y. lipolytica is an obligate aerobic oleaginous yeast best known for its capabilities at digesting alkanes and strong capacity for producing lipids. The species is commonly used as a model yeast for studying lipogenesis and has an extensive history in the academic literature. In industry, this yeast has been highlighted as having strong potential for production of a wide range of molecules. Recently, a genetically engineered strain of this yeast has been employed by DuPont in production of omega-3 fatty acids. Additionally, the species has been proposed for usage in environmental cleanup applications as well as bioconversion of industrial wastes, specifically glycerol from biodiesel production. While recent work has identified that this species possesses genes within the cellobiose and xylose metabolic pathways, most strains of species do not naturally possess the ability to digest lignocellulosic sugars. This thesis describes the creation of a cellobiose-consuming strain of Y. lipolytica. We investigate the necessary modifications to enable cellobiose utilization and physiologically characterizes a cellobiose-consuming strain. The strain is then used in a simultaneous saccharification and fermentation process to produce citric acid from cellulose. The second half of this thesis focuses on S. cerevisiae. While this yeast is a hallmark of the phenomenon known as glucose repression, wherein glucose is consumed before all other sugars, bypassing this effect and enabling simultaneous consumption of multiple sugars is desirable for robust fermentations and implementing continuous fermentation processes. This thesis demonstrates that slowing the rate of glucose consumption leads to simultaneous cofermentation of glucose and other sugars. Experiments are shown with glucose/xylose and glucose/galactose mixtures, however the mechanism described to explain this design indicates that other fermentable carbon sources may also be co-utilized in this fashion. The final research chapter of this thesis investigates the consequences of glucose sensing through the membrane sensors Snf3 and Rgt2 on non-glucose sugar consumption and non-ethanol product formation. Fermentations are performed with strains devoid of these glucose sensors but capable of consuming the lignocellulosic sugars xylose and cellobiose. Later, the dual role of glucose sensing and import on product formation is investigated with sensor-deleted and inducible-hexokinase strains expressing a lactic acid dehydrogenase LdhA. Finally, deletion of sensors is found to increase yield of the amino acid intermediate 2-isopropylmalate.

Name thesis:degree_name

M.S.

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Food Science & Human Nutrition

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2016

Author dc:creator

• Lane, Stephan Thomas

Contributors dc:contributor

• Jin, Yong-Su
• Miller, Michael J.
• Rao, Christopher V.

• Saccharomyces cerevisiae
• Glucose repression
• Sugar signalling
• glucose signalling
• Yarrowia lipolytica
• cellobiose
• xylose
• galactose
• glucose
• 2-isopropylmalate
• lactic acid
• lipids
• fermentation
• cofermentation

Statement dc:rights

• Copyright 2016 Stephan Lane

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/90973

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F90973