Background: Saccharomyces cerevisiae var. boulardii is a representative probiotic yeast that has been widely used in the food and pharmaceutical industries. However, S. boulardii has not been studied as a microbial cell factory for producing useful substances. Agarose, a major component of red macroalgae, can be depolymerized into neoagarooligosaccharides (NAOSs) by an endo-type β-agarase. NAOSs, including neoagarotetraose (NeoDP4), are known to be health-benefiting substances owing to their prebiotic effect. Thus, NAOS production in the gut is required. In this study, the probiotic yeast S. boulardii was engineered to produce NAOSs by expressing an endo-type β-agarase, BpGH16A, derived from a human gut bacterium Bacteroides plebeius. Results: In total, four different signal peptides were compared in S. boulardii for protein (BpGH16A) secretion for the first time. The SED1 signal peptide derived from Saccharomyces cerevisiae was selected as optimal for extracellular production of NeoDP4 from agarose. Expression of BpGH16A was performed in two ways using the plasmid vector system and the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system. The production of NeoDP4 by engineered S. boulardii was verified and quantified. NeoDP4 was produced by S. boulardii engineered using the plasmid vector system and CRISPR-Cas9 at 1.86 and 0.80 g/L in a 72-h fermentation, respectively. Conclusions: This is the first report on NAOS production using the probiotic yeast S. boulardii. Our results suggest that S. boulardii can be considered a microbial cell factory to produce health-beneficial substances in the human gut. Graphical abstract: [Figure not available: see fulltext.]
Bibliographical noteFunding Information:
This work was supported by the Mid-career Researcher Program (2020R1A2B5B02002631) through the National Research Foundation of Korea (NRF), the Ministry of Oceans and Fisheries, Korea (20200367), and the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry, and Fisheries, funded by the Ministry of Agriculture, Food, and Rural Affairs (321036051SB010). This research was also funded by National Institute of Food and Agriculture, U.S. Department of Agriculture: ILLU-698-914 awarded to YSJ and DNS Vision 20/20 awarded to EJY and YSJ.
Facility support from the Institute of Biomedical Science and Food Safety at the Korea University Food Safety Hall is acknowledged.
© 2021, The Author(s).
- Saccharomyces boulardii
ASJC Scopus subject areas
- Applied Microbiology and Biotechnology