Metabolic Engineering of Probiotic Saccharomyces boulardii

Liu, Jingjing; Kong, In Iok; Zhang, Guo-Chang; Jayakody, Lahiru N.; Kim, Heejin; Xia, Peng‐Fei; Kwak, Suryang; Sung, Bong Hyun; Sohn, Jung‐Hoon; Walukiewicz, Hanna E.; Rao, Christopher V.; Jin, Yong‐Su

doi:10.1128/aem.00057-16

Cited by 83 publications

(77 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…We reasoned that the truncated Pgm2 protein might cause the growth defect of S. boulardii on galactose. In order to examine the relationship between the truncated Pgm2 and the growth defect on galactose, we introduced an S. cerevisiae PGM2-overexpressing plasmid (37) into an auxotrophic S. boulardii strain (38). The resulting S. boulardii transformant with the S. cerevisiae PGM2-overexpressing plasmid showed improved growth on galactose ( Fig.…”

Section: Resultsmentioning

confidence: 99%

A Mutation in PGM2 Causing Inefficient Galactose Metabolism in the Probiotic Yeast Saccharomyces boulardii

Liu

Zhang

Kong

et al. 2018

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

The probiotic yeast has been extensively studied for the prevention and treatment of diarrheal diseases, and it is now commercially available in some countries. displays notable phenotypic characteristics, such as a high optimal growth temperature, high tolerance against acidic conditions, and the inability to form ascospores, which differentiate from The majority of prior studies stated that exhibits sluggish or halted galactose utilization. Nonetheless, the molecular mechanisms underlying inefficient galactose uptake have yet to be elucidated. When the galactose utilization of a widely used strain, ATCC MYA-796, was examined under various culture conditions, the strain could consume galactose, but at a much lower rate than that of While all genes were present in the genome, according to analysis of genomic sequencing data in a previous study, a point mutation (G1278A) in , which codes for phosphoglucomutase, was identified in the genome of the strain. As the point mutation resulted in the truncation of the Pgm2 protein, which is known to play a pivotal role in galactose utilization, we hypothesized that the truncated Pgm2 might be associated with inefficient galactose metabolism. Indeed, complementation of in restored galactose utilization. After reverting the point mutation to a full-length in by Cas9-based genome editing, the growth rates of wild-type (with a truncated gene) and mutant (with a full-length) strains with glucose or galactose as the carbon source were examined. As expected, the mutant (with a full-length ) was able to ferment galactose faster than the wild-type strain. Interestingly, the mutant showed a lower growth rate than that of the wild-type strain on glucose at 37°C. Also, the wild-type strain was enriched in the mixed culture of wild-type and mutant strains on glucose at 37°C, suggesting that the truncated might offer better growth on glucose at a higher temperature in return for inefficient galactose utilization. Our results suggest that the point mutation in might be involved in multiple phenotypes with different effects. is a probiotic yeast strain capable of preventing and treating diarrheal diseases. However, the genetics and metabolism of this yeast are largely unexplored. In particular, molecular mechanisms underlying the inefficient galactose metabolism of remain unknown. Our study reports that a point mutation in, which codes for phosphoglucomutase, is responsible for inferior galactose utilization by After correction of the mutated via genome editing, the resulting strain was able to use galactose faster than a parental strain. While the mutation made the yeast use galactose slowly, investigation of the genomic sequencing data of other strains revealed that the mutation is evolutionarily conserved. Interestingly, the mutation was beneficial for growth at a higher temperature on glucose. We speculate that the mutation was enriched due to selection of in the natural habitat (sugar-rich fruits in tropical areas).

show abstract

Section: Resultsmentioning

confidence: 99%

A Mutation in PGM2 Causing Inefficient Galactose Metabolism in the Probiotic Yeast Saccharomyces boulardii

Liu

Zhang

Kong

et al. 2018

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

show abstract

“…Therefore, a search of the NCBI database for sequences related to each of the 17 proteins, using BLASTP without keyword limits identified high scoring alignments with related proteins from many molds and yeasts. In all cases, these included Saccharomyces cerevisiae and Saccharomyces bayanus , which are commonly used in making wine, bread, and beer and Saccharomyces boulardii which is widely used as a probiotic . The long history of consumption of organisms producing these close homologs of the 17 Pichia sp .…”

Section: Resultsmentioning

confidence: 99%

Evaluating Potential Risks of Food Allergy and Toxicity of Soy Leghemoglobin Expressed in Pichia pastoris

Yuan

Andoh-Kumi

et al. 2017

Molecular Nutrition Food Res

View full text Add to dashboard Cite

ScopeThe Soybean (Glycine max) leghemoglobin c2 (LegHb) gene was introduced into Pichia pastoris yeast for sustainable production of a heme‐carrying protein, for organoleptic use in plant‐based meat. The potential allergenicity and toxicity of LegHb and 17 Pichia host‐proteins each representing ≥1% of total protein in production batches are evaluated by literature review, bioinformatics sequence comparisons to known allergens or toxins, and in vitro pepsin digestion.Methods and resultsLiterature searches found no evidence of allergenicity or toxicity for these proteins. There are no significant sequence matches of LegHb to known allergens or toxins. Eleven Pichia proteins have modest identity matches to minor environmental allergens and 13 Pichia proteins have significant matches to proteins from toxic sources. Yet the matched allergens and toxins have similar matches to proteins from the commonly consumed yeast Saccharomyces cerevisiae, without evidence of food allergy or toxicity. The demonstrated history of safe use indicates additional tests for allergenicity and toxicity are not needed. The LegHb and Pichia sp. proteins were rapidly digested by pepsin at pH 2.ConclusionThese results demonstrate that foods containing recombinant soy LegHb produced in Pichia sp. are unlikely to present an unacceptable risk of allergenicity or toxicity to consumers.

show abstract

“…S. boulardii is closely related to the famous budding yeast Saccharomyces cerevisiae , indicating that it may be similarly amenable to engineering for the production of biomolecules, including biologics requiring post-translational modifications 2, 32 . Supporting this, S. cerevisiae expression vectors can be transformed and propagated in S. boulardii , and CRISPR/Cas9-mediated genome editing is functional in both strains 33 . However, compared to S. cerevisiae, S. boulardii better tolerates low pH environments and grows more rapidly at human body temperature 32, 34, 35 .…”

Section: Introductionmentioning

confidence: 99%

“…Given its regulatory status, genetically tractability, and well-studied yeast relative, S. boulardii is uniquely poised as a potential model chassis for the delivery of therapeutics to the human gut. Indeed, S. boulardii has been engineered to secrete human synthetic lysozyme 33 and HIV-1 Gag 36 in cell culture, as well as IL-10 in an IBD mouse model 37 . However, while the rules governing high-level biomolecule production in S. cerevisiae have been extensively studied, these rules remain to be defined in culture- and gut-resident S. boulardii .…”

Section: Introductionmentioning

confidence: 99%

Establishing ProbioticSaccharomyces boulardiias a Model Organism for Synthesis and Delivery of Biomolecules

Durmusoglu

Al'Abri

Collins

et al. 2020

Preprint

View full text Add to dashboard Cite

12Saccharomyces boulardii is a widely used yeast probiotic which can counteract various 13 gastrointestinal disorders 1 . As a relative of Saccharomyces cerevisiae, S. boulardii exhibits rapid 14 growth and is easy to transform 2 and thus represents a promising chassis for the engineered 15 secretion of biomolecules. To establish S. boulardii as a platform for delivery of biomolecules to 16 the mammalian gut, we measured the amount and variance in protein expression enabled by 17 promoters, terminators, selective markers, and copy number control elements in this organism. 18These genetic elements were characterized in plasmidic and genomic contexts, revealing 19 strategies for tunable control of gene expression and CRISPR-mediated genome editing in this 20 strain. We then leveraged this set of genetic parts to combinatorially assemble pathways 21 enabling a wide range of drug and vitamin titers. Finally, we measured S. boulardii's residence 22 time in the gastrointestinal tracts of germ-free and antibiotic-treated mice, revealing the 23 relationships between dosing strategy and colonization level. This work establishes S. boulardii 24 as a genetically tractable commensal fungus and provides a set of strategies for engineering S. 25 boulardii to synthesize and deliver biomolecules during gut colonization. 26 27 abundant in the human gut, 21 and difficulty in producing high levels of post-translationally-42 modified proteins 22 . 43 44In addition to bacteria, a diverse fungal population also exists in the human gastrointestinal tract 45 (GIT) [23][24][25][26] . While the numerical abundance of fungal cells in the GIT is much lower than bacterial 46 cells, fungal cells are on average 100-fold larger, indicating that their biomass and role in the gut 47 may be larger than metagenomic surveys indicate 25,27 . Recent studies have shown that while 48 3 commensal fungi play important roles in the development of inflammatory bowel diseases, they 49 also provide protective heterologous immunity to pathogenic organisms by training the immune 50 system 28,29 . Furthermore, fungi are not susceptible to bacteriophage predation and are easily 51 engineered to secrete high titers of proteins that are post-translationally modified. Thus, in this 52 study, we established a pipeline to engineer the only eukaryotic probiotic that is approved by the 53 FDA: Saccharomyces boulardii 30 . 54 55 S. boulardii is a non-pathogenic yeast that was originally isolated from lychee and mangosteen in 56 1923 and has since been used to treat ulcerative colitis, diarrhea, and recurrent Clostridium 57 difficile infection 1,31 . S. boulardii is closely related to the famous budding yeast Saccharomyces 58 cerevisiae, indicating that it may be similarly amenable to engineering for the production of 59 biomolecules, including biologics requiring post-translational modifications 2,32 . Supporting this, 60 S. cerevisiae expression vectors can be transformed and propagated in S. boulardii, and 61 CRISPR/Cas9-mediated genome editing is functional in both ...

show abstract

Metabolic Engineering of Probiotic Saccharomyces boulardii

Cited by 83 publications

References 46 publications

A Mutation in PGM2 Causing Inefficient Galactose Metabolism in the Probiotic Yeast Saccharomyces boulardii

A Mutation in PGM2 Causing Inefficient Galactose Metabolism in the Probiotic Yeast Saccharomyces boulardii

Evaluating Potential Risks of Food Allergy and Toxicity of Soy Leghemoglobin Expressed in Pichia pastoris

Establishing ProbioticSaccharomyces boulardiias a Model Organism for Synthesis and Delivery of Biomolecules

Contact Info

Product

Resources

About