Characterization of mannan-overproducing yeast through genomic analysis = 유전체 분석을 통한 Mannan 고생산 효모의 특성 규명

다국어 초록 (Multilingual Abstract)

Mannan, a polysaccharide derived from yeast cell walls, is known as an immune enhancing and physiologically active material, and is in the limelight as a functional food material. This is because the monosaccharides constituting the yeast cell wall-derived mannan are all mannose. Mannan composed only of pure mannose is known to perform much better as a probiotics than mannan composed of other sugars and mannose. In particular, yeast cell wall-derived beta-glucan has been registered as a health functional food ingredient that helps improve immune function and is currently being sold as a health functional food, but mannan has not yet been registered as a health functional food ingredient, so research on this is necessary. In this study, using two yeasts, Saccharomyces cerevisiae and Saccharomyces boulardii, which are generally reconized as safe (GRAS) microorganisms approved by the US Food and Drug Administration (FDA), high-yielding Mannan strains were selected and the Mannose quantification method was optimized. Genes involved in the cell wall-derived mannan biosynthetic pathway of S. cerevisiae and S. bouladii were identified, and gene recombination technology using CRISPR-Cas9 was introduced to establish high and low mannan production strains for each yeast. Through this, it was confirmed that the identified gene was involved in the production of mannan in the yeast cell wall. The mannan content was compared with the mannose content after mannan was lysed. The recombinant Mannan low-producing strain in S. cerevisiae had about 1.2 times lower mannose content than the wild type.
The recombinant Mannan high-producing strain had about 1.2 times higher mannose content than the wild type. Also, in the structure of the yeast cell wall through Field Emission Transmission Electron Microscopy (FE-TEM), both S. cerevisiae and S. boulardii yeasts were different from the recombinant strains and wild strains. Significant differences were is shown in the strains. Therefore, after generating a mutant library through UV irradiation in S. cerevisae, strains with high mannose content were finally selected compared to recombinant Mannan high-producing strains, and strain characteristics were identified through NSG-based whole genome sequence analysis. As a result, if the mass production of Mannan is possible through optimization of culture using the finally selected high-production strain of Mannan, and it is recognized as a raw material for health functional food, it is considered to be highly likely to be a high value-added material in the food industry.

국문 초록 (Abstract)

효모 세포벽 유래 다당류인 만난은 면역증강 및 생리활성 소재로 알려져 있어 기능성 식품소재로 각광받고 있다. 이는 효모 세포벽 유래 만난을 구성하고 있는 단당류들이 전부 만노스이기 때문이다.순수 만노스로만 구성된 만난은 다른 당과 만노스로 구성된 만난에 비해 훨씬 프로바이오틱스 기능을 잘 수행한다고 알려져있다. 특히 효모 세포벽 유래 베타글루칸은 면역기능 증진에 도움을 주는 건강기능식품 원료로 등록되어 현재 건강기능식품으로 판매가 되고있지만 아직까지 만난은 건강기능식품 원료로 등록되지 않아 이에 대한 연구가 필요하다. 본 연구에서는 미국 식품의약안전처(FDA)에서 승인한 generally reconized as safe (GRAS) 미생물인 Saccharomyces cerevisiae, Saccharomyces boulardii 효모 2종을 이용, Mannan 고생산 균주 선별하고 Mannose 정량법을 최적화했다. S. cerevisiea, S. bouladii의 세포벽 유래 mannan 생합성 경로에 관여하는 유전자를 식별하고 이를 CRISPR-Cas9을 이용한 유전자 재조합 기술을 도입하여 각 효모별 Mannan 고생산 및 저생산 균주를 구축했다. 이를 통해 식별한 유전자가 효모 세포벽 내 Mannan 생성에 관여하는지 확인할 수 있었다. Mannan 함량은 Mannan을 lysis하여 mannose 함량으로 비교했다. S. cerevisiae에서 재조합 Mannan 저생산 균주는 야생계 대비 약 1.0배 Mannose 함량이 낮았고, 재조합 Mannan 고생산 균주는 야생계 대비 약 1.1배 Mannose 함량이 높았다.또한 Field Emission Transmission Electron Microscopy (FE-TEM)촬영을 통한 효모 세포벽 구조에서도 S. cerevisiae, S. boulardii 두 효모 모두 재조합 균주들과 야생계 균주에서 유의미한 차이를 나타냈다. 이에 S. cerevisae에 UV 조사를 통한 Mutant library 생성후 재조합 Mannan 고생산 균주 대비 Mannose 함량이 높은 균주를 최종선별하고 NSG 기반Whole genome sequence 분석을 통해 균주 특성까지 규명하였다. 결과적으로 최종 선별된 Mannan 고생산 균주를 이용하여 배양최적화를 통해 Mannan의 대량생산을 가능케하고, 건강기능식품 원료로써 인정이 된다면 이는 식품산업에서의 고부가가치 소재로서 가능성이 높다고 사료된다.

목차 (Table of Contents)

• CONTENTS
• List of figures Ⅲ
• List of tables Ⅴ
• Abstract 1
• Ⅰ. Introduction 3
• Ⅱ. Materials and Methods 8
• 2.1. Strains 8
• 2.2. Plasmid construction 8
• 2.3. Gene editing process using CRISPR-Cas9 8
• 2.4. Cell wall mutant library by UV irradiation (1st screening) 10
• 2.5. Cell wall mutant screening by laminarinase resistance (2nd screening) 10
• 2.6. Mannan acid-hydrolysis method andAnylsis of mannose through HPLC-RID 11
• 2.7. Capturing cell wall images using Bio-TEM 12
• Ⅲ. Results and Discussion
• 3.1. Cell wall observation of 5 yeast types using Bio-TEM 28
• 3.2. Comparison of mannose content by strain 30
• 3.3. Cell wall mutant screening by Laminarinase resistance 32
• 3.4. Analysis of mannose content through HPLC-RID 34
• 3.5. Mannan high production yeast mutant screening 36