Fungal Genetics and Biology 137 (2020) 103333

Contents lists available at ScienceDirect

Fungal Genetics and Biology

journal homepage: www.elsevier.com/locate/yfgbi

Yeasts as probiotics: Mechanisms, outcomes, and future potential T

a a,b,⁎
Swastik Sen , Thomas J. Mansell
a
Interdepartmental Graduate Microbiology Program, Iowa State University, 4122A, BRL, 617 Bissel Rd, Ames, IA 50011, USA
b
Department of Chemical and Biological Engineering, Iowa State University, 2112 Sweeney Hall, 618 Bissel Rd, Ames, IA 50011, USA

A R T I C LE I N FO A B S T R A C T

Keywords: The presence of commensal fungal species in the human gut indicates that organisms from this kingdom have the
Saccharomyces boulardii potential to benefit the host as well. Saccharomyces boulardii, a yeast strain isolated about a hundred years ago, is
Saccharomyces cerevisiae the most well-characterized probiotic yeast. Though for the most part it genetically resembles Saccharomyces
Mycobiome cerevisiae, specific phenotypic differences make it better suited for the gut microenvironment such as better acid
Probiotics
and heat tolerance. Several studies using animal hosts suggest that S. boulardii can be used as a biotherapeutic in
Genome engineering
humans. Clinical trials indicate that it can alleviate symptoms from gastrointestinal (GI) tract infections to some
extent, but further trials are needed to understand the full therapeutic potential of S. boulardii. Improvement on
probiotic function using engineered yeast is an attractive future direction, though genome modification tools for
use in S. boulardii have been limited until recently. However, some tools available for S. cerevisiae should be
applicable for S. boulardii as well. In this review, we summarize the observed probiotic effect of this yeast and the
state of the art for genome engineering tools that could help enhance its probiotic properties.

1. Introduction
The human microbiome consists not only of bacterial species but
also an abundance of fungal and archaeal species (Hoffmann et al.,
2013). Studies on the interactions between resident the mycobiome and
the rest of the microbiota and the host have revealed complex and
multifaceted relationships. One of the key aspects of this discovery is
that there are multiple fungal species involved in gastrointestinal
homeostasis in human beings and other mammals. The Human Micro-
biome Project found that the most abundant genera of fungi in the
human gut are Saccharomyces, Malassezia, and Candida in descending
order of abundance (Nash et al., 2017), with eight out of 15 genera
comprising ascomycetes and Saccharomyces comprising approximately
5–65% of observed fungi. Since fungi are harbored in the gut en-
vironment, it follows that some resident species might provide a sym-
biotic benefit to the human host. These benefits may exist in the form of
modulating immunological homeostasis, adaptive immunity, or
through the general maintenance of microbial homeostasis in the gut
through specific interactions (Lai et al., 2019).
Probiotics are living microorganisms that have the potential to be
beneficial to host organisms when administered at the correct dosage
(Hill et al., 2014). Humans have benefitted from microorganisms in
food in various forms throughout history. The benefits of including
certain live microbes in food were first indirectly observed in the health
effects of fermented foods, though the cause would have been almost
certainly unknown at the time (Gogineni et al., 2013). While most well-
characterized probiotic microbes are bacteria such as Bifidobacteria and
Lactobacillus (Bermudez-Brito et al., 2012), certain yeasts have been
shown to have health benefits across various studies (Czerucka et al.,
2007).
The most common yeast with proposed probiotic effects is
Saccharomyces boulardii. Also known as Saccharomyces cerevisiae var.
boulardii or Saccharomyces cerevisiae Hansen CBS 5926, over-the-
counter preparations of this yeast are typically recommended for the
treatment of acute gastrointestinal diseases such as rotoviral and bac-
terial diarrhea (Kelesidis and Pothoulakis, 2012) and chronic conditions
such as inflammatory bowel disease (Madsen, 2001). In this review, we
describe several proposed probiotic mechanisms of this yeast and up-
date readers on recent clinical and preclinical studies. Finally, we ad-
dress a new frontier in the use of probiotics: the potential for the use of
engineered yeast as live biotherapeutics.
2. Saccharomyces boulardii: A probiotic yeast
Saccharomyces strains have been observed in up to 96.8% of samples
in recent mycobiome studies (Dollive et al., 2012; Nash et al., 2017).
The prevalence of Saccharomyces strains in the human GI tract is not
surprising since live S. cerevisiae and related species have been

⁎
Corresponding author at: Department of Chemical and Biological Engineering, Iowa State University, 2112 Sweeney Hall, 618 Bissel Rd, Ames, IA 50011, USA.
E-mail addresses: ssen@iastate.edu (S. Sen), mansell@iastate.edu (T.J. Mansell).

https://doi.org/10.1016/j.fgb.2020.103333
Received 25 May 2019; Received in revised form 18 October 2019; Accepted 4 January 2020
Available online 07 January 2020
1087-1845/ © 2020 Elsevier Inc. All rights reserved.
S. Sen and T.J. Mansell Fungal Genetics and Biology 137 (2020) 103333

purposely consumed by humans for thousands of years in bread, beer,

(Soyturk et al., 2012)

(Martins et al., 2013)

(Everard et al., 2014)

(Justino et al., 2014)

(Duman et al., 2013)

(Sakarya and Gunay,

(Pontier-Bres et al.,

(Buccigrossi et al.,
and other fermented foods and beverages. However, there are very few

(Lee et al., 2016)

(de Avila et al.,

S. cerevisiae strains with demonstrated benefits to the host (Fernandez-
Pacheco et al., 2018). In 1920, Henri Boulard, a French scientist, ob-

Reference
served that certain people in French Indochina were not affected by a

2014)

2012)

2012)

2014)
cholera outbreak. These people were drinking a specific kind of tea
made from the extract of lychee and mangosteen (McFarland, 2010). He
isolated a Saccharomyces strain from the peels of these fruits, naming it

CNCM I-

CNCM I-

CNCM I-

CNCM I-

CNCM I-

CNCM I-

CNCM I-

CNCM I-
Saccharomyces boulardii. The strain is currently available around the

Strain

745

745

745

745

745

745

745

745
n/s

n/s
world as an over-the-counter probiotic supplement.
That S. boulardii preparations have been manufactured and sold

Lower Disease Activity Index(DAI) scores for colitis in rats treated with

increased survival rate, inhibits bacterial translocation, inflammatory

Reduced adherence of H. pylori to gastric and mucin epithelial cells.

Reduced Chloride secretion and diarrhea symptoms upon treatment

since the 1950 s and used in clinical studies since 1977 is indicative of

Reduced Diarrhea and weight loss upon treatment with S. boulardii

its relative safety for use and non-pathogenic nature (McFarland, 2010).

Prophylactic Administration of S. boulardii reduced weight loss,

Intestinal and hepatic damage reversed upon treatment with S.

Reduced cecal tissue damage, TNFα protein expression, NF-κB
An extensive review of the role of S. boulardii as probiotic was published

cytokines, and protects against intestinal and liver damage

No in vitro antimicrobial effect of S. boulardii on H. pylori

in 2012 (Kelesidis and Pothoulakis, 2012). This work seeks to update

Reduced Salmonella motility in presence of S. boulardii

Reduced fat mass, hepatic steatosis and inflammation
readers on clinical progress and new developments since then, as well
as to explore the role of S. boulardii in treating non-GI tract disorders.

2.1. Animal studies

phosphorylation and actin disruption

Reduced parasite infection intensity
Studies of S. boulardii in pre-clinical trials have predominantly used
rodent hosts and have had varying degrees of success. The range of
disorders that have been targeted is mostly limited to the disorders of
the gastrointestinal tract, but some studies have explored the impact of
S. boulardii on infections leading to hepatic disorders (Duman et al.,

with S. boulardii
2013; Everard et al., 2014). Most studies use a proprietary strain known
Study outcome
as CNCM I-745, which is commercially available around the globe

S. boulardii
under the brand names Florastor, Reflor, Bioflor, Codex, Ultra-leveur,

boulardii
Floratil and several others. Table 1 summarizes recent studies that have
been conducted in various hosts. While several studies demonstrate the
potential of S. boulardii in treating GI tract disorders caused due to the

5-fluorouracil-induced intestinal mucositis

C. difficile -associated cecal inflammation
infection by various pathogens such as Helicobacter pylori and Salmo-

Hepatic Steatosis, Inflammation, obesity

Toxocariasis(chronic tissue parasitosis)

nella, other studies show that the prophylactic administration of this

Liver and intestine injury caused by

antibiotic and immunosuppressant
organism does not necessarily prevent such infections altogether
H. pylori induced peptic ulcers

(Hudson et al., 2016). This apparent discrepancy may suggest that the
Systemic Salmonella infection

potential benefits of administering this probiotic may vary largely

based upon the nature of the infection or the host itself. Corthier et al.

Salmonella infection
studied the benefit of using S. boulardi in gnotobiotic mice in 1986 in

Rotavirus Diarrhea
Target disorder/

what was one of the first studies to demonstrate the efficacy of S.

boulardii administration in treating Clostridium difficile infections

treatment
(Corthier et al., 1986). Duman et al. studied the effect of S. boulardii on
Colitis

Antibiotic Associated Diarrhea (AAD) in rats (Jolivet et al., 1983;

McKenna et al., 2001). In this study, administration of antibiotics in-
linked Sialic acid, which is the substrate for H. pylori

Improved gut barrier function and intestinal integrity

creased intestinal transit and reduced levels of the antioxidant glu-

Inhibition of Pro-inflammatory cytokine production

Inhibition of Pro-inflammatory cytokine production

Prevention of Inflammatory response (Inhibition of

S.boulardii neuraminidase removes surface α(2,3)-

Enhanced retention of intestinal mucosa integrity

Selected recent pre-clinical trial outcomes (n/s = strain not specified).

tathione in the hepatic and ileal tissues. The administration of S. bou-

(Reduced Nitrite, Glutathione and inflammatory
Partial inhibition of host inflammatory response
Proposed Mechanism of action for S. boulardii

TNFα expression and NF-κB phosphorylation)

lardii completely reversed these effects to control levels.

In 2016 it was demonstrated that S. boulardii can also be effective in
treating C. difficile infections (Lee et al., 2016). In this study hamsters
Inhibition of Neutrophil recruitment

were infected with C. difficile strains associated with common out-

Inhibition of Chloride Secretion

breaks. Upon infection, the hamsters developed various symptoms such

as cecal inflammation, damage to the mucus membrane, increase in the
cytokine concentrations)

expression of TNFα and the phosphorylation of NF-κB, all of which are

implicated in various inflammatory pathways (Hing et al., 2013; Kim
Steric Hindrance

et al., 2006). S. boulardii administration in these hamsters not only

helped significantly alleviate these symptoms, but also played an im-
portant role in neutralizing the damage done to the epithelial actin
adhesin

cytoskeleton by C. difficile toxins A and B. The success of S. boulardii in

fighting a range of infections in various hosts demonstrates that it can
be a promising candidate for therapeutic use in humans as well.
Wistar albino female rats

Human colonic T84 cells

Human Caco-2 Cell Line

Germ free swiss outbred

Duodenal cell line

Type 2 Diabetic Mice

2.2. Recent human clinical trials with S. boulardii

In vitro, HuTo 80
Host organism

The efficacy as well as safety of using S. boulardii in a variety of

Hamsters
mice

different organisms has prompted many different clinical trials on hu-

Table 1

Mice

Mice

Rats

mans with varying success. Previous work with humans has established
some of the systemic as well as GI tract specific effects of S. boulardii.

2
S. Sen and T.J. Mansell Fungal Genetics and Biology 137 (2020) 103333

For example, S. boulardii can enhance humoral and innate immunity as

(Consoli et al., 2016)

(Abbas et al., 2014)

(Villar-García et al.,

(Serce et al., 2013)

well as improve the composition of the gut microflora in healthy in-

(Costanza et al.,
(Demirel et al.,

(Namkin et al.,
dividuals (Caetano et al., 1986; Vanhoutte et al., 2006), laying the
groundwork for using the probiotic for the treatment of various dis-
Reference

orders. Some older studies that targeted common GI tract disorders such
2013)

2015)

2015)

2016)
as C. difficile infections and Crohn’s Disease using S. boulardii as a part
of the treatment regimen provided crucial preliminary data on how the
probiotic can curtail the recurrence of these diseases (Guslandi et al.,
CNCM I-745

CNCM I-745

CNCM I-745

CNCM I-745

Hansen CBS
2000; Surawicz et al., 1989). Table 2 lists some of the recent and pro-
Strain

minent trials for S. boulardii mediated therapy in human clinical studies.

5926
n/s

n/s

Since 2013, several human trials have focused on treating infection-

associated inflammation in patients. These studies have reported var-
TNFα) and increased Anti-inflammatory cytokine concentration (IL-

Down regulation of both pro and anti inflammatory cytokines in the

No significant decrease was observed in H. pylori numbers between

Reduced cholesterol and uric acid levels in group treated with S.

ious degrees of success, often case-specific. Abbas et al. demonstrated

Reduced feeding intolerance and clinical sepsis in the probiotic

No reduction in number of Necrotizing Enterocolitis or sepsis

Reduced Pro-inflammatory cytokine concentrations (IL-8 and

Drastic reduction in microbial translocation across intestinal

that S. boulardii administration in inflammatory bowel disease patients

mucosa achieved through lowered intestinal permeability

led to a drastic reduction in serum levels of pro-inflammatory cytokines

such as interleukin-8 and tumor necrosis factor-α (TNF-α) and in-
the control and the S. boulardii administered group

creased concentrations of anti-inflammatory cytokines such as inter-

leukin-10 (Abbas et al., 2014). In another double-blind study it was
shown that S. boulardii restored the integrity of the GI tract intestinal
gut, no effect on postoperative infections

epithelium in patients undergoing treatment for HIV-1 infections and as

group compared to the control group

a result reduced microbial translocation across the epithelium as well as

inflammation in the patients (Villar-García et al., 2015). However, in a
different trial where researchers tried to determine the effect of S.
boulardii on necrotizing enterocolitis, an acquired inflammatory disease
almost exclusively limited to newborns (Sylvester et al., 2012), they
found that S. boulardii administration did not lead to any significant
Study outcome

decrease in sepsis (Serce et al., 2013). In a study to determine the in-

10) in blood

occurrences

fluence of S. boulardii on the H. pylori population in human GI tracts

boulardii

(Namkin et al., 2016), the authors again found the probiotic to have no
significant effect over a study of 28 children. Prophylaxis against C.
difficile infection was noted in an elderly population in a recent small
study (Carstensen et al., 2018). Differential outcomes between human
Post HIV-1 infection inflammation

Post colon resection infection and

Post heart failure inflammation

and animal trials may be attributed to differences in the makeup of the

due to microbial translocation

gut microbiome as compared to animal models such as mice which

Irritable Bowel Syndrome
Invasive fungal infections

Necrotizing Enterocolitis

usually have a more controlled diet during studies. For example,

abundance of S. boulardii can depend on the host microbiome (Edwards-
H. pylori infection

Ingram et al., 2007). Furthermore, differing expression of mucins and

Target disorder

inflammation

O-glycosylation variation might also contribute. These results suggest

that further study of the putative mechanisms of probiotic effect of this
organism is needed to resolve the differential outcomes between animal
and human trials.
Reducing inflammation by limiting Th1 cell mobility

Antimicrobial production, enhancement of intestinal

maturation through cell growth and differentiation

2.3. Risks: Fungal probiotics and fungemia

Polyamine production, which leads to intestinal
Inhibition of C. albicans adhesion, filamentous
Proposed Mechanism of action for S. boulardii

Downregulation of Inflammatory cytokines

Although S. boulardii is generally regarded as safe (Czerucka et al.,

Reduced intracellular cytokine production

2007) and has been extensively used as a probiotic without many ad-
verse effects, fungemia and sepsis are possible concerns, particularly in
immunocompromised patients. Fungemia is a systemic infection char-
growth and biofilm formation
Alteration of cytokine profile

acterized by the presence of fungal species in blood. Although an

overwhelming majority of reported fungemia cases are caused by
Candida spp. (Bodey et al., 2002; Huttova et al., 1998; Nguyen et al.,
1998), some instances of Saccharomyces fungemia have also been
documented. Though they are rare, occurrences of fungemia have been
Recent human clinical trials of S. boulardii.

reported in people receiving therapeutic doses of S. boulardii (Appel-da-

barriers

Silva et al., 2017; Cassone et al., 2003; Lherm et al., 2002; Thygesen
et al., 2012). Hennequin et al. and Cassone et al. reviewed some of
these cases where patients across an age range from 30 months to
78 years developed fungemia as a result of S. boulardii administration
Low birth weight infants

Asymptomatic H. pylori

either post-surgery or as part of a treatment regimen of other chronic

HIV-treated patients

disorders (Cassone et al., 2003; Hennequin et al., 2000). These cases

Target population

make it evident that Saccharomyces fungemia is a distinct but rare

carrier children
Preterm infants

possibility in patients severely compromised health conditions, espe-

cially those involving the GI tract or the circulatory system. Approaches
Adults

Adults

Adults

to minimize the possibility of invasion leading to fungemia will be an

Table 2

important consideration for addressing regulatory and safety concerns

in this organism. Gut colonization of S. cerevisiae has been reported in

3
S. Sen and T.J. Mansell
patients, leading to a rare condition known as auto-brewery syndrome,
in which yeast convert fermentable sugars to ethanol in the anaerobic
gut environment (Hafez et al., 2017; Welch et al., 2016), leading to
toxicity. Safety and standardization of dosage will likely benefit from
reduction of the probability of colonization, perhaps by the adminis-
tration of consortia rather than monocultures of S. boulardii.
3. Genetic and biochemical mechanisms of probiotic action
3.1. Genetic differences between S. boulardii and S. cerevisiae
S. boulardii and S. cerevisiae are genetically very similar, each con-
taining 16 chromosomes with greater than 99% relatedness by average
nucleotide identity (Khatri et al., 2017). Some of the important differ-
ences include those in the genes expressing some flocculation proteins,
which contribute to a different adhesion profile of S. boulardii when
compared to S. cerevisiae (Edwards-Ingram et al., 2007). A major ge-
netic difference between S. boulardii and other S. cerevisiae is chromo-
some IX trisomy in S. boulardii, though its impact on the probiotic at-
tributes of S. boulardii has not been definitively demonstrated
(Edwards-Ingram et al., 2007).
Complex phenotypes are often difficult to map to specific genomic
loci. Thus, research on physiological and biochemical aspects relevant
to the use of S. cerevisiae and S. boulardii show a much greater dis-
tinction than genetic comparisons. Fig. 1 summarizes known char-
acteristics that may make S. boulardii a desirable biotherapeutic for
human hosts.
In a study comparing molecular and physiological characteristics in
S. cerevisiae and S. boulardii, S. boulardii strains were more resistant to
high temperatures and grew faster than S. cerevisiae W303, which is a
well characterized yeast lab strain, at both 30 °C and 37 °C (Fietto et al.,
2004). The higher viability at 37 °C and higher temperatures (up to
55 °C) of S. boulardii as compared to S. cerevisiae is advantageous for its
use in mammalian hosts as well as for viability during industrial
Fig. 1. Properties of S. boulardii that may improve probiotic function.
4
Fungal Genetics and Biology 137 (2020) 103333
processing (Fietto et al., 2004). In this study the authors also demon-
strated that S. boulardii maintained roughly 75% viability at simulated
gastric environments at pH 2, which was far greater than S. cerevisiae
which had a viability of 30%. However, in a different study by Graff
et al., it was demonstrated that S. boulardii viability drops significantly
after 5 min when subjected to pH 1.1 (Graff et al., 2008a). Scanning
electron microscopy confirmed damage to the yeast cell walls in these
conditions explaining the drop in viability. There was no significant
decrease in viability at intestinal pH. This study also assessed the via-
bility of the probiotic in its aqueous and freeze-dried forms, and for-
mulated microspheres and tablets of these forms respectively. It was
demonstrated that encapsulated S. boulardii performed better both in
terms of viability at the low pH of 1.1, as well as efficiency of release at
pH 6.8. Indeed, most probiotic preparations are sold lyophilized and
encapsulated. Fietto et al. also evaluated the susceptibility of S. bou-
lardii to various concentrations of bile salts, which are secreted by the
liver and important for fat breakdown (Fietto et al., 2004). They found
that S. boulardii is sensitive to bile salts, as the maximum concentration
at which cells were viable was 0.1%. These results suggest that though
S. boulardii can survive in the mammalian gut, the success of oral ad-
ministration could be improved with greater protection against the
harsh gut environment. Various groups have attempted to protect cells
by, for example, encapsulation in polymeric microspheres (Arslan et al.,
2015; Graff et al., 2008b).
Phenotypic characteristics of S. boulardii that help ease safety con-
cerns for use in humans include lack of sporulation (McCullough et al.,
1998) and the lack of stable colonization of the gut (Edwards-Ingram
et al., 2007) in the presence of normal enteric flora. These attributes are
particularly important since fungal spores can serve as infectious pro-
pagules (Giles et al., 2009) and can survive various harsh conditions
(Coluccio et al., 2008). Although Saccharomyces spores are not in-
fectious, their presence can be counter-productive to efforts of limiting
their presence in the gut when not required in a therapeutic context. An
innate lack of colonization is helpful since colonization increases the
S. Sen and T.J. Mansell
risk of subsequent infections (Tacconelli et al., 2009). In addition, lack
of colonization allows for controlled dosing of live biotherapeutics, as
seen in previous studies with non-colonizing bacteria (Isabella et al.,
2018; Kurtz et al., 2019; Martín et al., 2014).
Besides exhibiting a difference in growth in gastrointestinal condi-
tions, S. boulardii has also been shown to have different sugar con-
sumption profiles than S. cerevisiae (Mitterdorfer et al., 2001). Ga-
lactose metabolism in particular is less efficient in S. boulardii than in S.
cerevisiae due to a mutation in the gene coding for phosphoglucomu-
tase, PGM2 (Liu et al., 2018). This may be relevant in the consumption
of other carbon sources as well since some of the downstream genes of
the GAL regulon which are required for growth are highly conserved
irrespective of the carbon source and Gal4p acts as a global regulator
(Endalur Gopinarayanan and Nair, 2018). The differential utilization of
carbon sources between S. cerevisiae and S. boulardii may be implicated
in prebiotic requirements of S. boulardii as well.
3.2. Proposed mechanisms of probiotic action
Although not all mechanisms by which probiotics benefit their hosts
are well understood, there are some established mechanistic connec-
tions. These include either enhancement of physical barriers of the gut
epithelia and enhancement of innate immunity, or regulating cytokine
secretion and helping modulate the immune system (O’Toole and
Cooney, 2008). Enhancement of the epithelial barrier can emerge from
improving tight junctions (Anderson et al., 2010) and competitively
excluding certain pathogenic microbes by adhering to the intestinal
mucosa (Servin, 2004). Recently, S. boulardii supernatant was shown to
help restore tight junctions via E-cadherin and p120 catenin expression
in colonic explants taken from inflammatory bowel disease patients
(Terciolo et al., 2017).
Modulation of the immune system by probiotics involves two dis-
tinct mechanisms. Some probiotic bacteria can have anti-inflammatory
effects by creating a tolerant state through the action of toll-like re-
ceptors (TLRs) on the effector components of the inflammation
pathway, such as dendritic cells (Wells, 2011). Other probiotics have
been shown to directly down-regulate host inflammatory responses by
stimulating the secretion of anti-inflammatory cytokines such as IL-10
(Azad et al., 2018). There have also been studies in which probiotic
lactic acid bacteria have been used as a vector to deliver IL-10 directly
into the human gut (Martín et al., 2014; Steidler et al., 2000). S. bou-
lardii administration has proven to have a positive effect in the con-
centrations of short-chain fatty acids (SCFAs, e.g., acetate, propionate,
and butyrate), which are known to have immunomodulatory proper-
ties. Schneider et al. showed that administration of S. boulardii in pa-
tients requiring total enteral nutrition (TEN) induced long lasting in-
crease in butyrate concentrations in the gut (Schneider et al., 2005).
This intervention also plays an important role in inhibiting the pro-
liferation of harmful pathogens such as C. difficile and reducing the
incidence of TEN-associated diarrhea. Competition for ecological niches
enhances the impact of probiotics on infections (Vidjeadevan et al.,
2018); a recent study has shown that S. boulardii can secrete anti-
microbial peptides as observed by inhibition of Bacillus cereus (Naimah
et al., 2018).
4. Future prospects: Engineering fungal probiotics for targeted
therapy
4.1. Heterologous gene expression
While some the qualities of S. boulardii make it an attractive pro-
biotic, its efficacy could theoretically be improved by genetic en-
gineering to amplify inherent benefits or add new probiotic character-
istics. Previous work in the well characterized probiotic Escherichia coli
Nissle 1917 has demonstrated the value that can be added to a probiotic
through genetic engineering (Kurtz et al., 2018). In this study, the
5
Fungal Genetics and Biology 137 (2020) 103333
essential gene thymidylate synthase was deleted from the genome, re-
sulting in a lack of replication in vivo. This resulted in a faster clearance
rate compared to replicating strain, in cynomolgous monkeys as well as
humans rendering a greater dosage-mediated control. The same or-
ganism was more recently engineered to treat hyperammonemia, where
the E. coli Nissle genome was edited to overproduce arginine in order to
sequester excess ammonia present in the blood (Kurtz et al., 2019).
Palma et al. described in great detail the various instances wherein the
researchers have attempted to express heterologous proteins in S.
boulardii (Palma et al., 2015). Engineered S. boulardii administered to
mice and subsequently recovered from their feces were hygromycin
resistant indicating heterologous expression of the hygromycin B
phosphotransferase enzyme (Edwards-Ingram et al., 2007). Researchers
were also able to express heterologous IL-10 in mice as a potential
treatment for colitis, though no significant difference in colitis was
found in that study (Michael et al., 2013). In another study, researchers
were able to build a single vector-mediated surface display system for
multiple antigens in S. boulardii for immunotherapy against eukaryotic
pathogens. (Wang et al., 2014). In addition, green fluorescent protein
(GFP) (Hudson et al., 2014) and LacZ (Liu et al., 2016) were expressed
in S. boulardii which is an attractive prospect since these proteins can be
useful tools in genome engineering as well (Dymecki, 1996; March
et al., 2003).
One possible advantage that S. boulardii has over most prokaryotic
probiotics is that as opposed to eukaryotic systems, prokaryotes often
lack post-translational modification machinery which would limit the
range of proteins that can be expressed in their native form (Tokmakov
et al., 2012). In a recent study, a uracil auxotroph of S. boulardii was
engineered to heterologously express OVA-CPE (Bagherpour et al.,
2018), a fusion of ovalbumin, a highly immunogenic protein (Koch
et al., 1996) and Clostridium perfringens enterotoxin, an intestinal epi-
thelium-targeting ligand that helps in reacting with immune cells un-
derlying the epithelium (Suzuki et al., 2012). Oral administration of
this recombinant S. boulardii resulted in elevated serum IgG and fecal
IgA levels, confirming the expression of the OVA-CPE recombinant
protein. All of the above are examples of heterologous protein expres-
sion from extrachromosomal plasmids, which are easier to manipulate
in vitro than the genomic DNA. However, further engineering of this
organism can be accomplished by making insertions, mutations, or
deletions at the genome level.
4.2. Genome engineering in S. boulardii
Many previous studies have reported heterologous protein expres-
sion in S. boulardii, but few of them involve engineering the genome to
enhance any intrinsic probiotic properties. One of the bottlenecks for
genome engineering of S. boulardii in the past was a lack of available
auxotrophic strains (Hashimoto et al., 2005). The use of antibiotic re-
sistance markers leads to lower efficiency, a higher number of false
positives and the potential spread of antibiotic resistance (Jonas et al.,
2001; Schilter and Constable, 2002), in addition to potential disruption
of the myco/microbiome. One of the first studies to engineer auxo-
trophy in S. boulardii was performed in 2013, in which it was demon-
strated that S. boulardii uracil auxotrophs can be generated using clas-
sical UV mutagenesis (Hamedi et al., 2013). This was achieved by
selecting for URA3- mutants via resistance to the toxic uracil precursor
analog 5-fluoroorotic acid. More recently, researchers generated aux-
otrophs for histidine, leucine, tryptophan and uracil for using a CRISPR-
Cas9 genome engineering approach originally designed for S. cerevisiae
(Liu et al., 2016). Using this method, editing efficiency is enhanced by
using the Streptococcus pyogenes CRISPR-Cas9 complex to create double
stranded breaks in the genome, stimulating homologous recombination
and/or non-homologous end joining. The group was also able to de-
monstrate that these auxotrophs can be used for the heterologous ex-
pression of various proteins such as β-galactosidase, lysozyme and red
fluorescent protein. Another significant aspect of this work was the
S. Sen and T.J. Mansell
introduction of the xylose utilization pathway into the S. boulardii
genome. This is especially important from a probiotic standpoint since
differential substrate utilization can confer a selective advantage in the
gut microenvironment (Watson et al., 2013).
The above studies demonstrate that even though genome en-
gineering tools for S. boulardii have been limited in the past, existing
genetic tools, especially those designed for the model organism S. cer-
evisiae can be leveraged for S. boulardii as well.
4.2.1. Genome engineering tools for S. cerevisiae with potential applications
in S. boulardii
Because of the rapidly growing significance of various yeast strains
in industrial manufacturing of value-added products as well as biome-
dical research, there have been significant advances in the tools and
strategies available for genome engineering in yeast. Fraczek et al.
described in great detail the history of various genome engineering
tools and techniques available for engineering the yeast genome
(Fraczek et al., 2018). Among them are techniques that leverage native
homologous recombination in yeast, which is a high efficiency double-
strand break DNA repair system that requires a homology of just 38 to
50 base pairs on either side of a DNA cassette (Lorenz et al., 1995).
Their work also covers the Cre-LoxP (Sauer, 1987) and delitto perfetto
(Storici et al., 2001) systems for gene editing in yeast.
More recent advances leverage the CRISPR-Cas9 system due to its
significantly higher efficiency, versatility and robustness in eukaryotic
systems than previously used tools (Ran et al., 2013). The use of
CRISPR-Cas9 based gene editing in yeast was reviewed in 2017 by
Stovicek et al. (Stovicek et al., 2017). Since then, Si et al. developed a
single step automated pathway for multiplex genome engineering in
yeast where they successfully built a strain library containing over-
expression or knockdown mutants in over 90% of yeast genes (Si et al.,
2017). Their work was motivated by the lack of readily available au-
tomation strategies needed to build and screen diverse genome scale
libraries. In this work, the authors were able to build a full length cDNA
library to be used as donor DNA for δ integration in yeast. They were
able to automate the entire multiplex genome engineering workflow
using the biological foundry iBioFAB, which is an integrated robotic
platform for automated biomanufacturing (Chao et al., 2017).
A tool to integrate exogenous pathways into the yeast genome with
several genes having multiple copy numbers was recently developed by
Hou et al. (Hou et al., 2018) using a DNA cassette called WICKET.
WICKET consists of universal homology arms flanking a Cas9 target
site. Once multiple copies of WICKET are integrated into the genome
using CRISPR-Cas9, it can accept entire exogenous gene pathways fol-
lowing a double-strand break created by Cas9. Using this tool, the au-
thors were able to integrate a β-carotene pathway into the S. cerevisiae
genome, paving the way for genomic integrations of more such path-
ways.
These genome engineering tools aided by CRISPR-Cas9 can greatly
enhance our ability to engineer the S. boulardii genome, since the tools
used for S. cerevisiae may be applicable to S. boulardii largely due to the
genetic similarities between the two species. This can also be sub-
stantiated by the fact that tools have been successfully implemented in
various other yeast and non yeast systems such as Schizosaccharomyces
pombe and Kluyveromyces lactis (Fraczek et al., 2018). The tools used in
these cases were Cre/loxP and CRISPR-Cas9 respectively. However, it is
worth bearing in mind that these systems can often be strain dependent,
with gene editing efficacies sometimes varying widely among related
strains of the same species (Leenay et al., 2019) and may require further
characterization for use in S. boulardii.
4.3. Other candidate probiotic Fungi:
While S. boulardii is the most well-studied probiotic fungus and es-
sentially a model yeast, some studies indicate that other fungi may have
potential as probiotics. Aspergillus awamori, a fungus regularly used in
6
Fungal Genetics and Biology 137 (2020) 103333
food processing, was administered to broiler chickens in a study where
the authors attempted to determine the effect of the fungus on body
weight and feeding habits (Saleh et al., 2014). The study revealed a
greater increase in body weight in the group that was administered the
fungus as compared to the control group that was fed a basal diet. In
another study, the fungus Chrysonilia crassa was administered to broiler
chickens in order to study its probiotic effects in comparison to con-
ventional feed additives, including Bacillus subtilis (Sugiharto et al.,
2018). Some of the parameters tested were body weight, number of
leukocytes and lymphocytes, total protein and globulin. The group
administered with C. crassa showed better growth and immune re-
sponses, but did not outperform the B. subtilis group. A different study
in broiler chickens demonstrated potential for the well-characterized
workhorse yeast Pichia pastoris along with S. boulardii in improving feed
efficiency (Gil de Los Santos et al., 2018). These preliminary studies are
a good starting point for exploring other fungi for probiotic use and may
have the potential to circumvent some of the shortcomings of bacterial
probiotics.
5. Conclusion
S. boulardii is a probiotic yeast that has been shown to be useful in
fighting various GI tract infections in rat models as well as human
beings. This organism’s probiotic attributes largely stem from being
able to modulate host immunity as well as the ability to competitively
exclude pathogenic bacteria. S. boulardii is also suited for survival in
mammalian hosts as compared to other yeasts. Preclinical and clinical
studies have demonstrated the safety of using this yeast, with systemic
infections being very rare. Human trials for the most part have yielded
various degrees of success in alleviating most gastrointestinal disorders,
suggesting that the strains of S. boulardii being used currently could be
modified for maximum efficacy. Unlike S. cerevisiae, genome en-
gineering in S. boulardii has been limited so far. However, since
Saccharomyces strains are so genetically similar, most of the tools for S.
cerevisiae might be applicable for S. boulardii as well. Application of
these techniques can help open up exciting avenues in strain im-
provement for S. boulardii with the potential to drastically improve its
probiotic attributes.
Acknowledgements
This work was supported by Iowa State University startup funds.
TJM is partially supported by the Karen and Denny Vaughn Faculty
Fellowship. The authors declare no conflict of interest. We thank Dr.
Zengyi Shao for helpful discussions regarding this manuscript.
References
Abbas, Z., Yakoob, J., Jafri, W., Ahmad, Z., Azam, Z., Usman, M.W., Shamim, S., Islam,
M., 2014. Cytokine and clinical response to Saccharomyces boulardii therapy in
diarrhea-dominant irritable bowel syndrome: a randomized trial. Eur. J.
Gastroenterol. Hepatol. 26, 630–639.
Anderson, R.C., Cookson, A.L., McNabb, W.C., Park, Z., McCann, M.J., Kelly, W.J., Roy,
N.C., 2010. Lactobacillus plantarum MB452 enhances the function of the intestinal
barrier by increasing the expression levels of genes involved in tight junction for-
mation. BMC Microbiol. 10, 316.
Appel-da-Silva, M.C., Narvaez, G.A., Perez, L.R.R., Drehmer, L., Lewgoy, J., 2017.
Saccharomyces cerevisiae var. boulardii fungemia following probiotic treatment.
Med. Mycol. Case Rep. 18, 15–17.
Arslan, S., Erbas, M., Tontul, I., Topuz, A., 2015. Microencapsulation of probiotic
Saccharomyces cerevisiae var. boulardii with different wall materials by spray
drying. LWT - Food Sci. Technol. 63, 685–690.
Azad, M.A.K., Sarker, M., Wan, D., 2018. Immunomodulatory effects of probiotics on
cytokine profiles. Biomed Res. Int. 2018, 8063647.
Bagherpour, G., Ghasemi, H., Zand, B., Zarei, N., Roohvand, F., Ardakani, E.M., Azizi, M.,
Khalaj, V., 2018. Oral administration of recombinant saccharomyces boulardii ex-
pressing ovalbumin-CPE fusion protein induces antibody response in mice. Front.
Microbiol. 9, 723.
Bermudez-Brito, M., Plaza-Díaz, J., Muñoz-Quezada, S., Gómez-Llorente, C., Gil, A., 2012.
Probiotic mechanisms of action. Ann. Nutr. Metab. 61, 160–174.
Bodey, G.P., Mardani, M., Hanna, H.A., Boktour, M., Abbas, J., Girgawy, E., Hachem,
S. Sen and T.J. Mansell
R.Y., Kontoyiannis, D.P., Raad, I.I., 2002. The epidemiology of Candida glabrata and
Candida albicans fungemia in immunocompromised patients with cancer. Am. J.
Med. 112, 380–385.
Buccigrossi, V., Laudiero, G., Russo, C., Miele, E., Sofia, M., Monini, M., Ruggeri, F.M.,
Guarino, A., 2014. Chloride secretion induced by rotavirus is oxidative stress-de-
pendent and inhibited by Saccharomyces boulardii in human enterocytes. PLoS One
9, e99830.
Caetano, J.A., Paramés, M.T., Babo, M.J., Santos, A., Ferreira, A.B., Freitas, A.A., Coelho,
M.R., Mateus, A.M., 1986. Immunopharmacological effects of Saccharomyces bou-
lardii in healthy human volunteers. Int. J. Immunopharmacol. 8, 245–259.
Carstensen, J.W., Chehri, M., Schønning, K., Rasmussen, S.C., Anhøj, J., Godtfredsen,
N.S., Andersen, C.Ø., Petersen, A.M., 2018. Use of prophylactic Saccharomyces
boulardii to prevent Clostridium difficile infection in hospitalized patients: a con-
trolled prospective intervention study. Eur. J. Clin. Microbiol. Infect. Dis. 37,
1431–1439.
Cassone, M., Serra, P., Mondello, F., Girolamo, A., Scafetti, S., Pistella, E., Venditti, M.,
2003. Outbreak of Saccharomyces cerevisiae subtype boulardii fungemia in patients
neighboring those treated with a probiotic preparation of the organism. J. Clin.
Microbiol. 41, 5340–5343.
Chao, R., Liang, J., Tasan, I., Si, T., Ju, L., Zhao, H., 2017. Fully automated one-step
synthesis of single-transcript TALEN pairs using a biological foundry. ACS Synth.
Biol. 6, 678–685.
Coluccio, A.E., Rodriguez, R.K., Kernan, M.J., Neiman, A.M., 2008. The yeast spore wall
enables spores to survive passage through the digestive tract of Drosophila. PLoS One
3, e2873.
Consoli, M.L.D., da Silva, R.S., Nicoli, J.R., Bruña-Romero, O., da Silva, R.G., de
Vasconcelos Generoso, S., Correia, M.I.T.D., 2016. Randomized clinical trial: impact
of oral administration of saccharomyces boulardii on gene expression of intestinal
cytokines in patients undergoing colon resection. JPEN J. Parenter. Enteral Nutr. 40,
1114–1121.
Corthier, G., Dubos, F., Ducluzeau, R., 1986. Prevention of Clostridium difficile induced
mortality in gnotobiotic mice by Saccharomyces boulardii. Can. J. Microbiol. 32,
894–896.
Costanza, A.C., Moscavitch, S.D., Faria Neto, H.C.C., Mesquita, E.T., 2015. Probiotic
therapy with Saccharomyces boulardii for heart failure patients: a randomized,
double-blind, placebo-controlled pilot trial. Int. J. Cardiol. 179, 348–350.
Czerucka, D., Piche, T., Rampal, P., 2007. Review article: yeast as probiotics –
Saccharomyces boulardii. Aliment. Pharmacol. Ther. 26, 767–778.
de Avila, L.F., Conceição, F.R., de Lima, Telmo P., Dutra, G.F., de los Santos, D.G.,
Martins, L.H., Berne, M.E., da Silva, P.E., Scaini, C.J., 2012. Saccharomyces boulardii
reduces infection intensity of mice with toxocariasis. Vet. Parasitol. 187, 337–340.
Demirel, G., Celik, I.H., Erdeve, O., Saygan, S., Dilmen, U., Canpolat, F.E., 2013.
Prophylactic Saccharomyces boulardii versus nystatin for the prevention of fungal
colonization and invasive fungal infection in premature infants. Eur. J. Pediatr. 172,
1321–1326.
Dollive, S., Peterfreund, G.L., Sherrill-Mix, S., Bittinger, K., Sinha, R., Hoffmann, C.,
Nabel, C.S., Hill, D.A., Artis, D., Bachman, M.A., Custers-Allen, R., Grunberg, S., Wu,
G.D., Lewis, J.D., Bushman, F.D., 2012. A tool kit for quantifying eukaryotic rRNA
gene sequences from human microbiome samples. Genome Biol. 13, R60.
Duman, D.G., Kumral, Z.N.Ö., Ercan, F., Deniz, M., Can, G., Cağlayan Yeğen, B., 2013.
Saccharomyces boulardii ameliorates clarithromycin- and methotrexate-induced in-
testinal and hepatic injury in rats. Br. J. Nutr. 110, 493–499.
Dymecki, S.M., 1996. A modular set of Flp, FRT and lacZ fusion vectors for manipulating
genes by site-specific recombination. Gene 171, 197–201.
Edwards-Ingram, L., Gitsham, P., Burton, N., Warhurst, G., Clarke, I., Hoyle, D., Oliver,
S.G., Stateva, L., 2007. Genotypic and physiological characterization of
Saccharomyces boulardii, the probiotic strain of Saccharomyces cerevisiae. Appl.
Environ. Microbiol. 73, 2458–2467.
Endalur Gopinarayanan, V., Nair, N.U., 2018. A semi-synthetic regulon enables rapid
growth of yeast on xylose. Nat. Commun. 9, 1233.
Everard, A., Matamoros, S., Geurts, L., Delzenne, N.M., Cani, P.D., 2014. Saccharomyces
boulardii administration changes gut microbiota and reduces hepatic steatosis, low-
grade inflammation, and fat mass in obese and type 2 diabetic db/db mice. MBio 5,
e01011–e1014.
Fernandez-Pacheco, P., Arévalo-Villena, M., Bevilacqua, A., Corbo, M.R., Briones Pérez,
A., 2018. Probiotic characteristics in Saccharomyces cerevisiae strains: properties for
application in food industries. LWT 97, 332–340.
Fietto, J.L.R., Araújo, R.S., Valadão, F.N., Fietto, L.G., Brandão, R.L., Neves, M.J., Gomes,
F.C.O., Nicoli, J.R., Castro, I.M., 2004. Molecular and physiological comparisons
between Saccharomyces cerevisiae and Saccharomyces boulardii. Can. J. Microbiol.
50, 615–621.
Fraczek, M.G., Naseeb, S., Delneri, D., 2018. History of genome editing in yeast. Yeast 35,
361–368.
de Los, Gil, Santos, D., de Los, Gil, Santos, J.R., Gil-Turnes, C., Gaboardi, G., Fernandes
Silva, L., França, R., Gevehr Fernandes, C., Rochedo Conceição, F., 2018. Probiotic
effect of Pichia pastoris X-33 produced in parboiled rice effluent and YPD medium on
broiler chickens. PLoS One 13, e0192904.
Giles, S.S., Dagenais, T.R.T., Botts, M.R., Keller, N.P., Hull, C.M., 2009. Elucidating the
pathogenesis of spores from the human fungal pathogen Cryptococcus neoformans.
Infect. Immun. 77, 3491–3500.
Gogineni, V.K., Morrow, L.E., Gregory, P.J., Malesker, M.A., 2013. Probiotics: history and
evolution. J. Infect. Dis. Prevent. Med. 1, 1–7.
Graff, S., Chaumeil, J.-C., Boy, P., Lai-Kuen, R., Charrueau, C., 2008a. Influence of pH
conditions on the viability of Saccharomyces boulardii yeast. J. Gen. Appl. Microbiol.
54, 221–227.
Graff, S., Hussain, S., Chaumeil, J.-C., Charrueau, C., 2008b. Increased intestinal delivery
7
Fungal Genetics and Biology 137 (2020) 103333
of viable Saccharomyces boulardii by encapsulation in microspheres. Pharm. Res. 25,
1290–1296.
Guslandi, M., Mezzi, G., Sorghi, M., Testoni, P.A., 2000. Saccharomyces boulardii in
maintenance treatment of Crohn’s disease. Dig. Dis. Sci. 45, 1462–1464.
Hafez, E.M., Hamad, M.A., Fouad, M., Abdel-Lateff, A., 2017. Auto-brewery syndrome:
ethanol pseudo-toxicity in diabetic and hepatic patients. Hum. Exp. Toxicol. 36,
445–450.
Hamedi, H., Misaghi, A., Modarressi, M.H., Salehi, T.Z., Khorasanizadeh, D., Khalaj, V.,
2013. Generation of a uracil auxotroph strain of the probiotic yeast saccharomyces
boulardii as a host for the recombinant protein production. Avicenna J. Med.
Biotechnol. 5, 29–34.
Hashimoto, S., Ogura, M., Aritomi, K., Hoshida, H., Nishizawa, Y., Akada, R., 2005.
Isolation of auxotrophic mutants of diploid industrial yeast strains after UV muta-
genesis. Appl. Environ. Microbiol. 71, 312–319.
Hennequin, C., Kauffmann-Lacroix, C., Jobert, A., Viard, J.P., Ricour, C., Jacquemin, J.L.,
Berche, P., 2000. Possible role of catheters in Saccharomyces boulardii fungemia.
Eur. J. Clin. Microbiol. Infect. Dis. 19, 16–20.
Hill, C., Guarner, F., Reid, G., Gibson, G.R., Merenstein, D.J., Pot, B., Morelli, L., Canani,
R.B., Flint, H.J., Salminen, S., et al., 2014. Expert consensus document: The
International Scientific Association for Probiotics and Prebiotics consensus statement
on the scope and appropriate use of the term probiotic. Nat. Rev. Gastroenterol.
Hepatol. 11, 506.
Hing, T.C., Ho, S., Shih, D.Q., Ichikawa, R., Cheng, M., Chen, J., Chen, X., Law, I.,
Najarian, R., Kelly, C.P., Gallo, R.L., Targan, S.R., Pothoulakis, C., Koon, H.W., 2013.
The antimicrobial peptide cathelicidin modulates Clostridium difficile-associated
colitis and toxin A-mediated enteritis in mice. Gut 62, 1295–1305.
Hoffmann, C., Dollive, S., Grunberg, S., Chen, J., Li, H., Wu, G.D., Lewis, J.D., Bushman,
F.D., 2013. Archaea and fungi of the human gut microbiome: correlations with diet
and bacterial residents. PLoS One 8, e66019.
Hou, S., Qin, Q., Dai, J., 2018. Wicket: a versatile tool for the integration and optimi-
zation of exogenous pathways in saccharomyces cerevisiae. ACS Synth. Biol. 7,
782–788.
Hudson, L.E., Fasken, M.B., McDermott, C.D., McBride, S.M., Kuiper, E.G., Guiliano, D.B.,
Corbett, A.H., Lamb, T.J., 2014. Functional heterologous protein expression by ge-
netically engineered probiotic yeast Saccharomyces boulardii. PLoS One 9, e112660.
Hudson, L.E., McDermott, C.D., Stewart, T.P., Hudson, W.H., Rios, D., Fasken, M.B.,
Corbett, A.H., Lamb, T.J., 2016. Characterization of the probiotic yeast sacchar-
omyces boulardii in the healthy mucosal immune system. PLoS One 11, e0153351.
Huttova, M., Hartmanova, I., Kralinsky, K., Filka, J., Uher, J., Kurak, J., Krizan, S.,
Krcmery Jr, V., 1998. Candida fungemia in neonates treated with fluconazole: report
of forty cases, including eight with meningitis. Pediatr. Infect. Dis. J. 17, 1012–1015.
Isabella, V.M., Ha, B.N., Castillo, M.J., Lubkowicz, D.J., Rowe, S.E., Millet, Y.A.,
Anderson, C.L., Li, N., Fisher, A.B., West, K.A., Reeder, P.J., Momin, M.M., Bergeron,
C.G., Guilmain, S.E., Miller, P.F., Kurtz, C.B., Falb, D., 2018. Development of a syn-
thetic live bacterial therapeutic for the human metabolic disease phenylketonuria.
Nat. Biotechnol. 36, 857–864.
Jolivet, J., Cowan, K.H., Curt, G.A., Clendeninn, N.J., Chabner, B.A., 1983. The phar-
macology and clinical use of methotrexate. N. Engl. J. Med. 309, 1094–1104.
Jonas, D.A., Elmadfa, I., Engel, K.H., Heller, K.J., Kozianowski, G., König, A., Müller, D.,
Narbonne, J.F., Wackernagel, W., Kleiner, J., 2001. Safety considerations of DNA in
food. Ann. Nutr. Metab. 45, 235–254.
Justino, P.F.C., Melo, L.F.M., Nogueira, A.F., Costa, J.V.G., Silva, L.M.N., Santos, C.M.,
Mendes, W.O., Costa, M.R., Franco, A.X., Lima, A.A., Ribeiro, R.A., Souza, M.H.L.P.,
Soares, P.M.G., 2014. Treatment with Saccharomyces boulardii reduces the in-
flammation and dysfunction of the gastrointestinal tract in 5-fluorouracil-induced
intestinal mucositis in mice. Br. J. Nutr. 111, 1611–1621.
Kelesidis, T., Pothoulakis, C., 2012. Efficacy and safety of the probiotic Saccharomyces
boulardii for the prevention and therapy of gastrointestinal disorders. Therap. Adv.
Gastroenterol. 5, 111–125.
Khatri, I., Tomar, R., Ganesan, K., Prasad, G.S., Subramanian, S., 2017. Complete genome
sequence and comparative genomics of the probiotic yeast Saccharomyces boulardii.
Sci. Rep. 7, 371.
Kim, J.M., Lee, J.Y., Yoon, Y.M., Oh, Y.-K., Youn, J., Kim, Y.-J., 2006. NF-kappa B acti-
vation pathway is essential for the chemokine expression in intestinal epithelial cells
stimulated with Clostridium difficile toxin A. Scand. J. Immunol. 63, 453–460.
Koch, C., Jensen, S.S., Øster, A., Houen, G., 1996. A comparison of the immunogenicity of
the native and denatured forms of a protein. APMIS 104, 115–125.
Kurtz, C.B., Millet, Y.A., Puurunen, M.K., Perreault, M., Charbonneau, M.R., Isabella,
V.M., Kotula, J.W., Antipov, E., Dagon, Y., Denney, W.S., Wagner, D.A., West, K.A.,
Degar, A.J., Brennan, A.M., Miller, P.F., 2019. An engineered E. coli Nissle improves
hyperammonemia and survival in mice and shows dose-dependent exposure in
healthy humans. Sci. Transl. Med. 11. https://doi.org/10.1126/scitranslmed.
aau7975.
Kurtz, C., Denney, W.S., Blankstein, L., Guilmain, S.E., Machinani, S., Kotula, J., Saha, S.,
Miller, P., Brennan, A.M., 2018. Translational development of microbiome-based
therapeutics: kinetics of E. coli nissle and engineered strains in humans and non-
human primates. Clin. Transl. Sci. 11, 200–207.
Lai, G.C., Tan, T.G., Pavelka, N., 2019. The mammalian mycobiome: A complex system in
a dynamic relationship with the host. Wiley Interdiscip. Rev. Syst. Biol. Med. 11,
e1438.
Lee, E.C., Ortiz, C., Wang, J., Lee, J.E., Ho, S., 2016. Probiotic Saccharomyces boulardii
CNCM I-745 prevents outbreak-associated Clostridium difficile-associated cecal in-
flammation in hamsters. American J.
Leenay, R.T., Vento, J.M., Shah, M., Martino, M.E., Leulier, F., Beisel, C.L., 2019. Genome
editing with CRISPR-Cas9 in lactobacillus plantarum revealed that editing outcomes
can vary across strains and between methods. Biotechnol. J. 14, 1700583.
S. Sen and T.J. Mansell
Lherm, T., Monet, C., Nougière, B., Soulier, M., Larbi, D., Le Gall, C., Caen, D., Malbrunot,
C., 2002. Seven cases of fungemia with Saccharomyces boulardii in critically ill pa-
tients. Intensive Care Med. 28, 797–801.
Liu, J.-J., Kong, I.I., Zhang, G.-C., Jayakody, L.N., Kim, H., Xia, P.-F., Kwak, S., Sung, B.H.,
Sohn, J.-H., Walukiewicz, H.E., Rao, C.V., Jin, Y.-S., 2016. Metabolic engineering of
probiotic saccharomyces boulardii. Appl. Environ. Microbiol. 82, 2280–2287.
Liu, J.-J., Zhang, G.-C., Kong, I.I., Yun, E.J., Zheng, J.-Q., Kweon, D.-H., Jin, Y.-S., 2018. A
mutation in PGM2 causing inefficient galactose metabolism in the probiotic yeast
Saccharomyces boulardii. Appl. Environ. Microbiol. 84. https://doi.org/10.1128/
AEM.02858-17.
Lorenz, M.C., Muir, R.S., Lim, E., McElver, J., Weber, S.C., Heitman, J., 1995. Gene dis-
ruption with PCR products in Saccharomyces cerevisiae. Gene 158, 113–117.
Madsen, K.L., 2001. The use of probiotics in gastrointestinal disease. Can. J.
Gastroenterol. 15, 817–822.
March, J.C., Rao, G., Bentley, W.E., 2003. Biotechnological applications of green fluor-
escent protein. Appl. Microbiol. Biotechnol. 62, 303–315.
Martín, R., Martín, R., Chain, F., Chain, F., Miquel, S., Miquel, S., Natividad, J.M.,
Natividad, J.M., Sokol, H., Sokol, H., Verdu, E.F., Verdu, E.F., Langella, P., Langella,
P., Bermúdez-Humarán, L.G., Bermúdez-Humarán, L.G., 2014. Effects in the use of a
genetically engineered strain of Lactococcus lactis delivering in situ IL-10 as a
therapy to treat low-grade colon inflammation. Hum. Vaccin. Immunother. 10,
1611–1621.
Martins, F.S., Vieira, A.T., Elian, S.D.A., Arantes, R.M.E., Tiago, F.C.P., Sousa, L.P.,
Araújo, H.R.C., Pimenta, P.F., Bonjardim, C.A., Nicoli, J.R., Teixeira, M.M., 2013.
Inhibition of tissue inflammation and bacterial translocation as one of the protective
mechanisms of Saccharomyces boulardii against Salmonella infection in mice.
Microbes Infect. 15, 270–279.
McCullough, M.J., Clemons, K.V., McCusker, J.H., Stevens, D.A., 1998. Species identifi-
cation and virulence attributes of Saccharomyces boulardii (nom. inval.). J. Clin.
Microbiol. 36, 2613–2617.
McFarland, L.V., 2010. Systematic review and meta-analysis of Saccharomyces boulardii
in adult patients. World J. Gastroenterol. 16, 2202–2222.
McKenna, S., Evans, G., 2001. Canadian infectious disease society antimicrobial agents
committee, macrolides: a canadian infectious disease society position paper. Can. J.
Infect. Dis. 12, 218–231.
Michael, S., Keubler, L.M., Smoczek, A., Meier, M., Gunzer, F., Pöhlmann, C., Krause-
Buchholz, U., Hedrich, H.-J., Bleich, A., 2013. Quantitative phenotyping of in-
flammatory bowel disease in the IL-10-deficient mouse by use of noninvasive mag-
netic resonance imaging. Inflamm. Bowel Dis. 19, 185–193.
Mitterdorfer, G., Kneifel, W., Viernstein, H., 2001. Utilization of prebiotic carbohydrates
by yeasts of therapeutic relevance. Lett. Appl. Microbiol. 33, 251–255.
Naimah, A.K., Al-Manhel, A.J.A., Al-Shawi, M.J., 2018. Isolation, purification and char-
acterization of antimicrobial peptides produced from saccharomyces boulardii. Int. J.
Pept. Res. Ther. 24, 455–461.
Namkin, K., Zardast, M., Basirinejad, F., 2016. Saccharomyces Boulardii in helicobacter
pylori eradication in children: a randomized trial from Iran. Iran. J. Pediatr. 26,
e3768.
Nash, A.K., Auchtung, T.A., Wong, M.C., Smith, D.P., Gesell, J.R., Ross, M.C., Stewart,
C.J., Metcalf, G.A., Muzny, D.M., Gibbs, R.A., Ajami, N.J., Petrosino, J.F., 2017. The
gut mycobiome of the Human Microbiome Project healthy cohort. Microbiome 5,
153.
Nguyen, M.H., Clancy, C.J., Yu, V.L., Yu, Y.C., Morris, A.J., Snydman, D.R., Sutton, D.A.,
Rinaldi, M.G., 1998. Do in vitro susceptibility data predict the microbiologic response
to amphotericin B? Results of a prospective study of patients with Candida fungemia.
J. Infect. Dis. 177, 425–430.
O’Toole, P.W., Cooney, J.C., 2008. Probiotic bacteria influence the composition and
function of the intestinal microbiota. Interdiscip. Perspect. Infect. Dis. 2008, 175285.
Palma, M.L., Zamith-Miranda, D., Martins, F.S., Bozza, F.A., Nimrichter, L., Montero-
Lomeli, M., Marques Jr, E.T., Douradinha, B., 2015. Probiotic Saccharomyces cere-
visiae strains as biotherapeutic tools: is there room for improvement? Appl.
Microbiol. Biotechnol. 99, 6563–6570.
Pontier-Bres, R., Prodon, F., Munro, P., Rampal, P., Lemichez, E., Peyron, J.F., Czerucka,
D., 2012. Modification of Salmonella Typhimurium motility by the probiotic yeast
strain Saccharomyces boulardii. PLoS One 7, e33796.
Ran, F.A., Hsu, P.D., Wright, J., Agarwala, V., Scott, D.A., Zhang, F., 2013. Genome en-
gineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308.
Sakarya, S., Gunay, N., 2014. Saccharomyces boulardii expresses neuraminidase activity
selective for α2,3-linked sialic acid that decreases Helicobacter pylori adhesion to
host cells. APMIS 122, 941–950.
Saleh, A.A., Hayashi, K., Ijiri, D., Ohtsuka, A., 2014. Beneficial effects of Aspergillus
awamori in broiler nutrition. Worlds. Poult. Sci. J. 70, 857–864.
Sauer, B., 1987. Functional expression of the cre-lox site-specific recombination system in
the yeast Saccharomyces cerevisiae. Mol. Cell. Biol. 7, 2087–2096.
Schilter, B., Constable, A., 2002. Regulatory control of genetically modified (GM) foods:
Fungal Genetics and Biology 137 (2020) 103333
likely developments. Toxicol. Lett. 127, 341–349.
Schneider, S.-M., Girard-Pipau, F., Filippi, J., Hebuterne, X., Moyse, D., Hinojosa, G.-C.,
Pompei, A., Rampal, P., 2005. Effects of Saccharomyces boulardii on fecal short-chain
fatty acids and microflora in patients on long-term total enteral nutrition. World J.
Gastroenterol. 11, 6165–6169.
Serce, O., Benzer, D., Gursoy, T., Karatekin, G., Ovali, F., 2013. Efficacy of Saccharomyces
boulardii on necrotizing enterocolitis or sepsis in very low birth weight infants: a
randomised controlled trial. Early Hum. Dev. 89, 1033–1036.
Servin, A.L., 2004. Antagonistic activities of lactobacilli and bifidobacteria against mi-
crobial pathogens. FEMS Microbiol. Rev. 28, 405–440.
Si, T., Chao, R., Min, Y., Wu, Y., Ren, W., Zhao, H., 2017. Automated multiplex genome-
scale engineering in yeast. Nat. Commun. 8, 15187.
Soyturk, M., Saygili, S.M., Baskin, H., Sagol, O., Yilmaz, O., Saygili, F., Akpinar, H., 2012.
Effectiveness of Saccharomyces boulardii in a rat model of colitis. World J.
Gastroenterol. 18, 6452–6460 discussion p. 6459.
Steidler, L., Hans, W., Schotte, L., Neirynck, S., Obermeier, F., Falk, W., Fiers, W., Remaut,
E., 2000. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10.
Science 289, 1352–1355.
Storici, F., Lewis, L.K., Resnick, M.A., 2001. In vivo site-directed mutagenesis using oli-
gonucleotides. Nat. Biotechnol. 19, 773–776.
Stovicek, V., Holkenbrink, C., Borodina, I., 2017. CRISPR/Cas system for yeast genome
engineering: advances and applications. FEMS Yeast Res. 17. https://doi.org/10.
1093/femsyr/fox030.
Sugiharto, S., Yudiarti, T., Isroli, I., Widiastuti, E., Wahyuni, H., Sartono, T., 2018. The
effect of fungi-origin probiotic Chrysonilia crassa in comparison to selected com-
mercially used feed additives on broiler chicken performance, intestinal micro-
biology, and blood indices. J Adv Vet Anim Res 5, 332.
Surawicz, C.M., McFarland, L.V., Elmer, G., Chinn, J., 1989. Treatment of recurrent
Clostridium difficile colitis with vancomycin and Saccharomyces boulardii. Am. J.
Gastroenterol. 84, 1285–1287.
Suzuki, H., Kondoh, M., Takahashi, A., Yagi, K., 2012. Proof of concept for claudin-tar-
geted drug development. Ann. N. Y. Acad. Sci. 1258, 65–70.
Sylvester, K.G., Liu, G.Y., Albanese, C.T., 2012. Chapter 94 - Necrotizing Enterocolitis. In:
Coran, A.G. (Ed.), Pediatric Surgery, Seventh Ed. Mosby, Philadelphia, pp.
1187–1207.
Tacconelli, E., De Angelis, G., Cataldo, M.A., Mantengoli, E., Spanu, T., Pan, A., Corti, G.,
Radice, A., Stolzuoli, L., Antinori, S., Paradisi, F., Carosi, G., Bernabei, R., Antonelli,
M., Fadda, G., Rossolini, G.M., Cauda, R., 2009. Antibiotic usage and risk of colo-
nization and infection with antibiotic-resistant bacteria: a hospital population-based
study. Antimicrob. Agents Chemother. 53, 4264–4269.
Terciolo, C., Dobric, A., Ouaissi, M., Siret, C., Breuzard, G., Silvy, F., Marchiori, B.,
Germain, S., Bonier, R., Hama, A., Owens, R., Lombardo, D., Rigot, V., André, F.,
2017. Saccharomyces boulardii CNCM I-745 Restores intestinal barrier integrity by
regulation of E-cadherin recycling. J. Crohns. Colitis 11, 999–1010.
Thygesen, J.B., Glerup, H., Tarp, B., 2012. Saccharomyces boulardii fungemia caused by
treatment with a probioticum. BMJ Case Rep. 2012. https://doi.org/10.1136/bcr.06.
2011.4412.
Tokmakov, A.A., Kurotani, A., Takagi, T., Toyama, M., Shirouzu, M., Fukami, Y.,
Yokoyama, S., 2012. Multiple post-translational modifications affect heterologous
protein synthesis. J. Biol. Chem. 287, 27106–27116.
Vanhoutte, T., De Preter, V., De Brandt, E., Verbeke, K., Swings, J., Huys, G., 2006.
Molecular monitoring of the fecal microbiota of healthy human subjects during ad-
ministration of lactulose and Saccharomyces boulardii. Appl. Environ. Microbiol. 72,
5990–5997.
Vidjeadevan, D., Vinoth, S., Ramesh, S., 2018. Role of Saccharomyces boulardii and
Bacillus clausii in reducing the duration of diarrhea: a three-armed randomised
controlled trial. Int. J. Contemporary Pediatrics 5, 1811–1814.
Villar-García, J., Hernández, J.J., Güerri-Fernández, R., González, A., Lerma, E., Guelar,
A., Saenz, D., Sorlí, L., Montero, M., Horcajada, J.P., Knobel Freud, H., 2015. Effect of
probiotics (Saccharomyces boulardii) on microbial translocation and inflammation in
HIV-treated patients: a double-blind, randomized, placebo-controlled trial. J. Acquir.
Immune Defic. Syndr. 68, 256–263.
Wang, T., Sun, H., Zhang, J., Liu, Q., Wang, L., Chen, P., Wang, F., Li, H., Xiao, Y., Zhao,
X., 2014. The establishment of Saccharomyces boulardii surface display system using
a single expression vector. Fungal Genet. Biol. 64, 1–10.
Watson, D., O’Connell Motherway, M., Schoterman, M.H.C., van Neerven, R.J.J., Nauta,
A., van Sinderen, D., 2013. Selective carbohydrate utilization by lactobacilli and
bifidobacteria. J. Appl. Microbiol. 114, 1132–1146.
Welch, B.T., Coelho Prabhu, N., Walkoff, L., Trenkner, S.W., 2016. Auto-brewery
Syndrome in the Setting of Long-standing Crohn’s Disease: A Case Report and Review
of the Literature. J. Crohns. Colitis 10, 1448–1450.
Wells, J.M., 2011. Immunomodulatory mechanisms of lactobacilli. Microb. Cell Fact. 10
(Suppl 1), S17.