Novel insights in genetic transformation of the probiotic yeast<i>Saccharomyces boulardii</i>

Douradinha, Bruno; Reis, Viviane Castelo Branco; Rogers, Matthew B.; Torres, Fernando Araripe Gonçalves; Evans, Jared D.; Marques, Ernesto T. A.

doi:10.4161/bioe.26271

Cited by 33 publications

(30 citation statements)

References 40 publications

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“…There have been several reports showing that S. boulardii is different from S. cerevisiae in terms of transformation procedures and limited genetic perturbation tools (2). In this study, we showed that existing plasmids and genetic tools currently used for S. cerevisiae could be readily applied to S. boulardii.…”

Section: Discussionmentioning

confidence: 68%

“…Also, constitutive promoters, including pTDH3, pPGK1, and pTEF, have been employed for the overexpression of a target gene in conjunction with the use of the pRS series plasmids (27). While previous studies examined whether genetic tools of S. cerevisiae can be utilized for introducing genetic perturbations into S. boulardii (1,2), the examinations were limited because four common auxotrophic strains (ura3, his3, trp1, and leu2) of S. boulardii did not exist. As we obtained isogenic single or double mutants, which can be complemented by four auxotrophic markers, we were able to perform comprehensive evaluations of S. cerevisiae genetic tools in S. boulardii.…”

Section: Discussionmentioning

confidence: 99%

“…This yeast has been widely used in food and nutraceutical industries, because it is known to be effective for limiting diarrheal diseases. S. boulardii is acknowledged as generally regarded as safe (GRAS) by the Food and Drug Administration (FDA) (2). S. boulardii is the only yeast probiotic that has been proven effective in double-blind studies (3,4), and it outperformed other known probiotics, such as Bifidobacterium and Lactobacillus, regarding immunomodulation (5).…”

mentioning

confidence: 99%

“…S. boulardii is the only yeast probiotic that has been proven effective in double-blind studies (3,4), and it outperformed other known probiotics, such as Bifidobacterium and Lactobacillus, regarding immunomodulation (5). Additionally, S. boulardii can survive in the human gastrointestinal tract due to its resistance to high temperature and low pH (2,4). S. boulardii can compete with diarrhea-causing pathogens for growth in the gut, making it effective for treating and preventing diarrhea (6).…”

mentioning

confidence: 99%

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Metabolic Engineering of Probiotic Saccharomyces boulardii

Liu

Kong

Zhang

et al. 2016

Appl Environ Microbiol

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Saccharomyces boulardii is a probiotic yeast that has been used for promoting gut health as well as preventing diarrheal diseases. This yeast not only exhibits beneficial phenotypes for gut health but also can stay longer in the gut than Saccharomyces cerevisiae. Therefore, S. boulardii is an attractive host for metabolic engineering to produce biomolecules of interest in the gut. However, the lack of auxotrophic strains with defined genetic backgrounds has hampered the use of this strain for metabolic engineering. Here, we report the development of well-defined auxotrophic mutants (leu2, ura3, his3, and trp1) through clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-based genome editing. The resulting auxotrophic mutants can be used as a host for introducing various genetic perturbations, such as overexpression or deletion of a target gene, using existing genetic tools for S. cerevisiae. We demonstrated the overexpression of a heterologous gene (lacZ), the correct localization of a target protein (red fluorescent protein) into mitochondria by using a protein localization signal, and the introduction of a heterologous metabolic pathway (xylose-assimilating pathway) in the genome of S. boulardii. We further demonstrated that human lysozyme, which is beneficial for human gut health, could be secreted by S. boulardii. Our results suggest that more sophisticated genetic perturbations to improve S. boulardii can be performed without using a drug resistance marker, which is a prerequisite for in vivo applications using engineered S. boulardii.

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Section: Discussionmentioning

confidence: 68%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Metabolic Engineering of Probiotic Saccharomyces boulardii

Liu

Kong

Zhang

et al. 2016

Appl Environ Microbiol

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“…Thanks to molecular genetic techniques, S. boulardii considered originally as a yeast species by itself [7] has been proven in recent years to be a variant of S. cerevisiae [8]. In this chapter, a diploid yeast strain similar to those strains used in most research laboratories, amenable to genetic manipulation when using conventional yeast protocols is shown.…”

Section: Introductionmentioning

confidence: 99%

The Benefits of Saccharomyces boulardii

Altmann¹

2018

The Yeast Role in Medical Applications

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A Saccharomyces boulardii strain, which does not carry any auxotrophic markers, was transformed with knockout constructs for the genes HIS3 and ADE2 using the dominant antibiotic marker genes encoding for kanamycin/G418-and nourseothricin/NAT R resistance. Thereby, homozygous derivatives that were histidine or adenine deicient were obtained. Histidine prototrophy was easily reconstituted by transforming hisdefective diploid derivatives with yeast plasmids carrying the HIS3 gene. Despite different atempts, for example, by creating a rme1::KANX rme1::NATR double-deleted S.boulardii yeast strain (RME1 encodes for Regulator of Meiosis), no visible sporulation to obtain haploid derivatives could be obtained. Besides, no ilamentation properties of S. boulardii were observed. As previously mentioned, this yeast strain was conirmed to thrive at 37°C, a temperature disliked by some but not all S. cerevisiae strains used in the laboratory. S. boulardii is a diploid derivative of S. cerevisiae that does not sporulates and survives at temperatures as those found in the human gut. It can be easily manipulated by using conventional yeast methods to introduce auxotrophic markers and obtain heterozygous diploid knockout derivatives that can be transformed with yeast plasmids following conventional yeast protocols, thereby it could be even suited for biochemical and genetic research purposes.

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4D Printing of Engineered Living Materials

Rivera‐Tarazona

Shukla

Singh

et al. 2021

Adv Funct Materials

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Herein, a method that uses direct-ink-write printing to fabricate engineering living materials (ELMs) that respond by undergoing a programmed shape change in response to specific molecules is reported. Stimuli-responsiveness is imparted to ELMs by integrating genetically engineered yeast that only proliferate in the presence of specific biomolecules. This proliferation, in turn, leads to a shape change in the ELM in response to that biomolecule. These ELMs are fabricated by coprinting bioinks that contain multiple yeast strains. Locally, cellular proliferation leads to controllable shape change of the material resulting in up to a 370% increase in volume. Globally, the printed 3D structures contain regions of material that increase in volume and regions that do not under a given set of conditions, leading to programmable changes in form in response to target amino acids and nucleotides. Finally, this printing method is applied to design a reservoir-based drug delivery system for the on-demand delivery of a model drug in response to a specific biomolecule.

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Novel insights in genetic transformation of the probiotic yeastSaccharomyces boulardii

Cited by 33 publications

References 40 publications

Metabolic Engineering of Probiotic Saccharomyces boulardii

Metabolic Engineering of Probiotic Saccharomyces boulardii

The Benefits of Saccharomyces boulardii

4D Printing of Engineered Living Materials

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