Dataset Information

Strain engineering and metabolic flux analysis of a probiotic yeast Saccharomyces boulardii for metabolizing L-fucose, a mammalian mucin component.

ABSTRACT:

Background

Saccharomyces boulardii is a probiotic yeast that exhibits antimicrobial and anti-toxin activities. Although S. boulardii has been clinically used for decades to treat gastrointestinal disorders, several studies have reported weak or no beneficial effects of S. boulardii administration in some cases. These conflicting results of S. boulardii efficacity may be due to nutrient deficiencies in the intestine that make it difficult for S. boulardii to maintain its metabolic activity.

Results

To enable S. boulardii to overcome any nutritional deficiencies in the intestine, we constructed a S. boulardii strain that could metabolize L-fucose, a major component of mucin in the gut epithelium. The fucU, fucI, fucK, and fucA from Escherichia coli and HXT4 from S. cerevisiae were overexpressed in S. boulardii. The engineered S. boulardii metabolized L-fucose and produced 1,2-propanediol under aerobic and anaerobic conditions. It also produced large amounts of 1,2-propanediol under strict anaerobic conditions. An in silico genome-scale metabolic model analysis was performed to simulate the growth of S. boulardii on L-fucose, and elementary flux modes were calculated to identify critical metabolic reactions for assimilating L-fucose. As a result, we found that the engineered S. boulardii consumes L-fucose via (S)-lactaldehyde-(S)-lactate-pyruvate pathway, which is highly oxygen dependent.

Conclusion

To the best of our knowledge, this is the first study in which S. cerevisiae and S. boulardii strains capable of metabolizing L-fucose have been constructed. This strategy could be used to enhance the metabolic activity of S. boulardii and other probiotic microorganisms in the gut.

SUBMITTER: Kim J

PROVIDER: S-EPMC9541068 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Publications

Strain engineering and metabolic flux analysis of a probiotic yeast Saccharomyces boulardii for metabolizing L-fucose, a mammalian mucin component.

Kim Jungyeon J Cheong Yu Eun YE Yu Sora S Jin Yong-Su YS Kim Kyoung Heon KH

Microbial cell factories 20221007 1

<h4>Background</h4>Saccharomyces boulardii is a probiotic yeast that exhibits antimicrobial and anti-toxin activities. Although S. boulardii has been clinically used for decades to treat gastrointestinal disorders, several studies have reported weak or no beneficial effects of S. boulardii administration in some cases. These conflicting results of S. boulardii efficacity may be due to nutrient deficiencies in the intestine that make it difficult for S. boulardii to maintain its metabolic activit ...[more]

PMID: 36207743

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Project description:The probiotic yeast Saccharomyces boulardii (Sb) is a promising chassis to deliver therapeutic proteins to the gut due to Sb's innate therapeutic properties, resistance to phage and antibiotics, and high protein secretion capacity. To maintain therapeutic efficacy in the context of challenges such as washout, low rates of diffusion, weak target binding, and/or high rates of proteolysis, it is desirable to engineer Sb strains with enhanced levels of protein secretion. In this work, we explored genetic modifications in both cis- (i.e. to the expression cassette of the secreted protein) and trans- (i.e. to the Sb genome) that enhance Sb's ability to secrete proteins, taking a Clostridioides difficile Toxin A neutralizing peptide (NPA) as our model therapeutic. First, by modulating the copy number of the NPA expression cassette, we found NPA concentrations in the supernatant could be varied by sixfold (76-458 mg/L) in microbioreactor fermentations. In the context of high NPA copy number, we found a previously-developed collection of native and synthetic secretion signals could further tune NPA secretion between 121 and 463 mg/L. Then, guided by prior knowledge of S. cerevisiae's secretion mechanisms, we generated a library of homozygous single gene deletion strains, the most productive of which achieved 2297 mg/L secretory production of NPA. We then expanded on this library by performing combinatorial gene deletions, supplemented by proteomics experiments. We ultimately constructed a quadruple protease-deficient Sb strain that produces 5045 mg/L secretory NPA, an improvement of > tenfold over wild-type Sb. Overall, this work systematically explores a broad collection of engineering strategies to improve protein secretion in Sb and highlights the ability of proteomics to highlight under-explored mediators of this process. In doing so, we created a set of probiotic strains that are capable of delivering a wide range of protein titers and therefore furthers the ability of Sb to deliver therapeutics to the gut and other settings to which it is adapted.

Project description:Saccharomyces boulardii has been a subject of growing interest due to its potential as a probiotic microorganism with applications in gastrointestinal health, but the molecular cause for its probiotic potency has remained elusive. The recent discovery that S. boulardii contains unique mutations causing high acetic acid accumulation and inhibition of bacterial growth provides a possible clue. The natural S. boulardii isolates Sb.P and Sb.A are homozygous for the recessive mutation whi2S270* and accumulate unusually high amounts of acetic acid, which strongly inhibit bacterial growth. However, the homozygous whi2S270* mutation also leads to acetic acid sensitivity and acid sensitivity in general. In the present study, we have constructed a new S. boulardii strain, derived from the widely therapeutically used CMCN I-745 strain (isolated from the pharmaceutical product Enterol), producing even higher levels of acetic acid while keeping the same tolerance toward low pH as the parent Enterol (ENT) strain. This newly engineered strain, named ENT3, has a homozygous deletion of ACH1 and strong overexpression of ALD4. It is also able to accumulate much higher acetic acid concentrations when growing on low glucose levels, in contrast to the ENT wild-type and Sb.P strains. Moreover, we show the antimicrobial capacity of ENT3 against gut pathogens in vitro and observed that higher acetic acid production might correlate with better persistence in the gut in healthy mice. These findings underscore the possible role of the unique acetic acid production and its potential for improvement of the probiotic action of S. boulardii.IMPORTANCESuperior variants of the probiotic yeast Saccharomyces boulardii produce high levels of acetic acid, which inhibit the growth of bacterial pathogens. However, these strains also show increased acid sensitivity, which can compromise the viability of the cells during their passage through the stomach. In this work, we have developed by genetic engineering a variant of Saccharomyces boulardii that produces even higher levels of acetic acid and does not show enhanced acid sensitivity. We also show that the S. boulardii yeasts with higher acetic acid production persist longer in the gut, in agreement with a previous work indicating competition between probiotic yeast and bacteria for residence in the gut.

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Dataset Information

Strain engineering and metabolic flux analysis of a probiotic yeast Saccharomyces boulardii for metabolizing L-fucose, a mammalian mucin component.

Background

Results

Conclusion

Publications

Strain engineering and metabolic flux analysis of a probiotic yeast Saccharomyces boulardii for metabolizing L-fucose, a mammalian mucin component.

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