Precision fermentation for enhanced production of food ingredients, fermented foods, and chemicals

Kim, Chanwoo

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https://hdl.handle.net/2142/121273 Copy

Description

Title

Precision fermentation for enhanced production of food ingredients, fermented foods, and chemicals

Author(s)

Kim, Chanwoo

Issue Date

2023-07-13

Director of Research (if dissertation) or Advisor (if thesis)

Jin, Yong-Su

Doctoral Committee Chair(s)

Miller, Michael J

Committee Member(s)

• Cadwallader, Keith R
• Rao, Christopher V

Department of Study

Food Science & Human Nutrition

Discipline

Food Science & Human Nutrition

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• precision fermentation
• yeast
• metabolic engineering

Abstract

Precision fermentation is a promising biotechnology that uses microbial hosts as cell factories to produce value-added products, such as biofuels and biochemicals. This approach has the potential to transform the food industry by providing an efficient and cost-effective method for producing food ingredients and fermented foods while addressing the challenges of food security and sustainability. The study begins with the development of industrial genome-edited yeast strains for improving the quality and flavor of fermented foods through precision fermentation. The first objective is based on four approaches. First, a transcriptional regulator (URE2) of nitrogen catabolite repression was deleted to increase the accumulation of savory amino acids. Second, acetate esters producing and degrading genes (ATF1, alcohol acetyltransferase 1 and IAH1, isoamyl alcohol dehydrogenase 1) were perturbated in yeast to intensify the production of ester-based compounds in fermented foods. Third, the control of expression levels of ATF1 and mixed culture fermentation was conducted to provide various types and levels of flavor molecules. Finally, the resulting genome-edited strains were applied to produce Makgeolli (Korean rice wine). The Makgeolli made by the genome-edited strains exhibited increased levels of savory amino acids and flavor-active esters compared to the Makgeolli made by their parental strain. The second goal of this study was to efficiently produce galactitol and tagatose from lactose-rich permeates by changing expression levels of genes (XYL1, xylose reductase and GDH, galactitol dehydrogenase). The dairy industry generates lactose-rich permeates as a by-product, which can be fermented to produce value-added compounds like tagatose, a low-calorie sweetener. Engineered yeast strains capable of producing tagatose from lactose-rich dairy byproducts were constructed by varying the copy numbers of the expression cassettes with XYL1 and GDH to change the flux of lactose metabolites and increase tagatose production. The resulting strains do not rely on plasmid-based expression which requires antibiotics, making them a feasible approach for large-scale industrial and food fermentation processes without using antibiotics. The use of lactose-rich permeates as a carbon source for fermentation resulted in increased tagatose productivity, cell growth, and lactose consumption rates as compared with the use of pure lactose. The third goal of this study was to establish a CRISPR-Cas9 ribonucleoprotein (RNP)-mediated genome engineering technique in Saccharomyces cerevisiae. While the CRISPR-Cas9 system allows for precise genome modifications, safety concerns have arisen in the food industry due to the use of antibiotic markers and the introduction of genetic constructs. In this study, we introduced purified Cas9 and in vitro transcribed single guide RNA (sgRNA) to delete the ADE2 gene in yeast. By optimizing the concentration of the RNP complex, pink colonies derived from ADE2 deletion were observed, and the deletion of ADE2 in the pink colonies was confirmed through PCR amplification. The RNP-based method does not require antibiotic selection and heterologous genetic construct, making it a promising approach for generating genome-edited food microorganisms without concerns for genetic modification (GM) regulation. Finally, Issatchenkia orientalis, a food yeast which is highly tolerant under low pH and high temperature, was engineered to produce 3-hydroxypropinoic acid (3-HP) from xylose via the β-alanine pathway. 3-HP serves as a platform chemical with the capability to produce diverse value-added products including absorbents and bioplastics. In this study, we introduced a xylose oxidoreductase pathway (XYL1, xylose reductase, XYL2, xylitol dehydrogenase, and XYL3, xylulokinase) into a 3-HP producing strain, resulting in efficient 3-HP production from xylose with a yield of 0.34 g/g xylose. Efficient xylose utilization and production of 3-HP using the engineered I. orientalis can enable economical and sustainable bioconversion to produce value-added products from abundant lignocellulosic feedstocks.

Graduation Semester

2023-08

Type of Resource

Thesis

Copyright and License Information

Copyright 2023 Chanwoo Kim

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