3,6-Anhydro-L-galactose Production from Red Algal Agarobiose through Evolution of Saccharomyces boulardii and Introduction of a Heterologous β-Galactosidase

다국어 초록 (Multilingual Abstract)

Red macroalgae (Rhodophyta) are promising renewable biomass resources owing to their high carbohydrate and low lignin content. Agarose, the principal polysaccharide of red algae, consists of repeating agarobiose (AB) units that yield a rare sugar, 3,6-anhydro-L-galactose (AHG) with diverse biological activities, including anti-inflammatory, skin-whitening, anticariogenic, and anti-colon cancer effects. Despite its potential as a functional ingredient in pharmaceuticals, health supplements, and cosmetics, large-scale AHG production remains costly and unstable owing to inefficient catalytic systems and the lack of cell-based production platforms. Therefore, establishing an efficient catalytic system and a cell-based platform for continuous AHG production is essential. In this study, the goal was to construct a strain capable of hydrolyzing AB to AHG. As a first step, AB production conditions were optimized to secure a sufficient substrate supply. Subsequently, several recombinant β-galactosidases (Bga42A, Bga42B, Bga42C, and Bga2A) from Bifidobacterium longum subsp. infantis ATCC 15697 were evaluated by using E. coli DE3 for their AB-hydrolyzing performance, and Bga42A was identified as the most effective enzyme, exhibiting the highest specific activity (6,870 U/μmol) and catalytic efficiency (24.70 mM-1·s-1). Saccharomyces boulardii was selected as the host to develop a production platform with combined AB-degrading capacity and probiotic/prebiotic properties. Prior to enzyme introduction, the yeast was adaptively evolved under galactose conditions to enhance utilization of galactose, a major AB hydrolysis product. The evolved strain expressing Bga42A was then subsequently evaluated in batch and fed-batch fermentations, representing AHG productivity of 0.12 g/L/h (0.41 g AHG / g AB) and 0.12 g/L/h (0.38 g AHG per g AB), respectively. These results highlight the synergistic potential of genetic engineering and adaptive evolution in optimizing probiotic yeast for rare sugar production.

목차 (Table of Contents)

• List of Tables vi
• List of Scheme and Figures viii
• ABSTRACT xii
• 1. Introduction 1
• 2. Materials and methods 7
• 2.1 Plasmids and strains 7
• 2.2 Media and culture conditions 12
• 2.3 AB production from agarose 14
• 2.4 Overexpression and purification of recombinant proteins 15
• 2.5 Specific activity assay of β-galactosidase 16
• 2.6 Kinetic parameters of β-galactosidase 17
• 2.7 Serial transfer-based evolutionary cultivation 17
• 2.8 Whole genome sequencing 19
• 2.9 Protein structure prediction and interface analysis 21
• 2.10 Intracellular metabolite extraction 21
• 2.11 AB tolerance test 22
• 2.12 Batch fermentation 23
• 2.13 Fed-batch fermentation 24
• 2.14 Thin-layer chromatography and high-performance liquid
• chromatography analysis 25
• 2.15 Gas chromatography-mass spectrometry analysis 26
• 2.16 Data processing and statistical analysis 27
• 3. Results and Discussion 29
• 3.1 Hydrolysis of agarose to AB 29
• 3.2 Specific activity of β-galactosidase for the hydrolysis of AB 36
• 3.3 Kinetic parameters of β-galactosidases for the hydrolysis of AB 40
• 3.4 Serial transfer of SB-HTU and isolation of the evolved YR10-2 strain 45
• 3.5 Genetic mutations of the evolved strain YR10-2 53
• 3.6 Predicted structure of Gal1-ATP and Gal80 58
• 3.7 Comparative intracellular metabolite analysis between SB-HTU and YR10-2 strain under
• glucose and galactose condition 62
• 3.8 Assessment of AB toxicity in SB-HTU 70
• 3.9 Batch fermentation of AB for AHG production 73
• 3.10 Fed-batch fermentation of AB for AHG production 82
• 4. Conclusion 85
• 5. References 87
• 국문 논문제출서 97
• 국문 인준서 98
• 국문 초록 99