KR-102962039-B1 - Engineered Saccharomyces boulardii for metabolizing L-fucose

Abstract

The present invention relates to a recombinant Saccharomyces boulardii capable of metabolizing L-fucose, a sugar derived from intestinal mucin. By providing a genetically modified Saccharomyces boulardii that metabolizes L-fucose and exhibits improved intestinal survival and metabolic activity, the present invention enables the greater utilization of various functionalities possessed by the strain, as well as probiotics.

Inventors

• 김경헌
• 김정연
• 정유은

Assignees

• 주식회사 바이오크래프트

Dates

Publication Date

20260508

Application Date

20230905

Priority Date

20220906

Claims (4)

1. Recombinant Saccharomyces boulardii metabolizing L-fuse, comprising a gene encoding fucose mutarotase; a gene encoding fucose isomerase; a gene encoding fuculose kinase; a gene encoding fuculose-1-phosphate aldolase; and a gene encoding a hexose transporter.
2. In Article 1, A recombinant Saccharomyces boulardii metabolizing L-fuse, in which the gene encoding fucose mutarotase; the gene encoding fucose isomerase; the gene encoding fuculose kinase; and the gene encoding fuculose-1-phosphate aldolase are derived from Escherichia coli, and the gene encoding a hexose transporter is derived from Saccharomyces cerevisiae.
3. In Article 1, A recombinant Saccharomyces boulardii metabolizing L-fuse, wherein the gene encoding fucose mutarotase is denoted by SEQ No. 1; the gene encoding fucose isomerase is denoted by SEQ No. 2; the gene encoding fuculose kinase is denoted by SEQ No. 3; the gene encoding fuculose-1-phosphate aldolase is denoted by SEQ No. 4; and the gene encoding a hexose transporter is denoted by SEQ No. 5.
4. In Article 1, The above strain is a recombinant Saccharomyces boulardii that metabolizes L-fusose, which is transfected with a recombinant vector comprising a gene encoding fucose mutarotase; a gene encoding fucose isomerase; a gene encoding fuculose kinase; a gene encoding fuculose-1-phosphate aldolase; and a gene encoding a hexose transporter.

Description

Engineered Saccharomyces boulardii for metabolizing L-fucose The present invention relates to a recombinant Saccharomyces boulardii capable of metabolizing L-fucose, a sugar derived from intestinal mucin. Saccharomyces boulardii is a well-known probiotic strain that exhibits antimicrobial activity, antitoxin effects, and immunomodulatory effects in the human gut. Although it has a genome similar to that of the same species, Saccharomyces cerevisiae, it has a distinct phenotype. A representative example is that due to a point mutation in Pgm2 , Saccharomyces cerevisiae rapidly metabolizes galactose, whereas Saccharomyces boulardii has a slow metabolic rate. Furthermore, Saccharomyces boulardii can easily grow at 37°C, the normal human body temperature, and has the characteristic of attaching to human or mouse intestinal epithelial cells. It also has higher resistance to environmental stresses, such as high temperatures and acidity, than Saccharomyces cerevisiae. Due to these phenotypic characteristics, Saccharomyces boulardii is reported to have higher intestinal metabolic activity than Saccharomyces cerevisiae and provide various health benefits to humans. In particular, its preventive and therapeutic effects against many gastrointestinal diseases caused by pathogens such as pathogenic Escherichia coli , Salmonella enterica serotype Typhimurium, Campylobacter jejuni , and Clostridium difficile have been reported through numerous clinical trials. These treatments have been used for decades as general medicines to treat or prevent diarrhea in Europe, Africa, and the United States. Generally, Saccharomyces boulardii can remain in the human and mouse intestines only after high-dose oral administration and is completely excreted within 3 to 7 days after administration. Furthermore, Saccharomyces boulardii has been reported to colonize intestinal epithelial cells in humans or notobiotic mice treated with antibiotics. This suggests that while Saccharomyces boulardii can grow in the intestine and exhibit various metabolic activities, it can be easily inhibited by competition with other intestinal microorganisms. Gut microorganisms colonizing the intestines metabolize molecules secreted by some intestinal epithelial cells. Dietary nutrients provided by the host are consumed competitively by the host and other gut microorganisms, resulting in an insufficient nutrient supply. To overcome this, some microorganisms metabolize mucin secreted by mammalian hosts. Mucin is primarily composed of L-fucose, D-galactose, N-acetylgalactosamine, N-acetylglucosamine, D-glucuronic acid, and salicylic acid. Among these, gut microorganisms utilize L-fucose most extensively as a carbon source. Microorganisms such as Bacteroides thetaiotaomicron , Bacteroides fragilis , and Bifidobacterium bifidum are known to secrete fucosidase to cleave L-fuse at the glycoprotein ends of mucin mucus molecules. The isolated L-fuse is metabolized through a process of phosphorylation by other microorganisms. Accordingly, the inventors aimed to improve the colonization and viability of Saccharomyces boulardii, which exhibits various functionalities, and completed a Saccharomyces boulardii strain transformed with a recombinant vector containing genes that express enzymes required for L-fucose metabolism so that it can metabolize L-fucose, which is most frequently used by intestinal microorganisms as a carbon source. Figure 1 shows the results of an in silico genome-scale metabolic model analysis of the growth of Saccharomyces cerevisiae during the metabolism of L-fucose. (A) Detailed schematic diagram of fucose metabolism for cell growth and each reaction (B) In silico calculations of fucose uptake and growth rates (C) Relationship between oxygen supply and D-glucose or L-fucose growth. Figure 2 shows the results of comparing the growth profile and fucose transport efficiency of Saccharomyces cerevisiae during L-fucose metabolism. (A) Growth profile of wild-type Saccharomyces cerevisiae (B) Growth profile of Saccharomyces cerevisiae FCT (C) Intracellular L-fucose concentrations of HXT-Null strain and HXT1-7 overexpressing strain (D) Growth profile of Saccharomyces cerevisiae FCT Figure 3 shows the growth profiles of Saccharomyces boulardii during the metabolism of L-fucose under various aerobic conditions. (A) Growth profile of wild-type Saccharomyces boulardii (B) Growth profile of Saccharomyces boulardii FCT under aerobic conditions (C) Growth profile of Saccharomyces boulardii FCT under microaerobic conditions (D) Growth profile of Saccharomyces boulardii FCT under anaerobic conditions. Figure 4 shows the results of the basic flux mode analysis to explain fucose metabolism under aerobic and anaerobic conditions. (A) Schematic diagram of estimated fucose metabolism under aerobic conditions and (B) anaerobic conditions. (C) Amounts of 1,2-PDO and biomass produced by metabolizing fucose under aerobic conditions. (D) Stoichiometry for maximum production of