Appl Microbiol Biotechnol (2013) 97:503–517
DOI 10.1007/s00253-012-4497-y
MINI-REVIEW
Biotechnology of non-Saccharomyces
yeasts—the ascomycetes
Eric A. Johnson
Received: 15 February 2012 / Revised: 3 October 2012 / Accepted: 6 October 2012 / Published online: 27 November 2012
# Springer-Verlag Berlin Heidelberg 2012
Abstract Saccharomyces cerevisiae and several other yeast
species are among the most important groups of biotechnolog-
ical organisms. S. cerevisiae and closely related ascomycetous
yeasts are the major producer of biotechnology products
worldwide, exceeding other groups of industrial microorgan-
isms in productivity and economic revenues. Traditional in-
dustrial attributes of the S. cerevisiae group include their
primary roles in food fermentations such as beers, cider, wines,
sake, distilled spirits, bakery products, cheese, sausages, and
other fermented foods. Other long-standing industrial process-
es involving S. cerevisae yeasts are production of fuel ethanol,
single-cell protein (SCP), feeds and fodder, industrial enzymes,
and small molecular weight metabolites. More recently, non-
Saccharomyces yeasts (non-conventional yeasts) have been
utilized as industrial organisms for a variety of biotechnolog-
ical roles. Non-Saccharomyces yeasts are increasingly being
used as hosts for expression of proteins, biocatalysts and multi-
enzyme pathways for the synthesis of fine chemicals and small
molecular weight compounds of medicinal and nutritional
importance. Non-Saccharomyces yeasts also have important
roles in agriculture as agents of biocontrol, bioremediation, and
as indicators of environmental quality. Several of these prod-
ucts and processes have reached commercial utility, while
others are in advanced development. The objective of this
mini-review is to describe processes currently used by industry
and those in developmental stages and close to commerciali-
zation primarily from non-Saccharomyces yeasts with an em-
phasis on new opportunities. The utility of S. cerevisiae in
heterologous production of selected products is also described.
E. A. Johnson (*)
Department of Bacteriology, Food Research Institute,
University of Wisconsin,
Madison, WI 53706, USA
e-mail: eajohnso@wisc.edu
Keywords Yeast . Biotechnology . Ascomycetes .
Non-Saccharomyces
Prominent non-Saccharomyces ascomycetous yeast
species of biotechnological importance
A landmark three-volume treatise “Yeasts: A Taxonomic
Study” was recently published that authoritatively describes
the taxonomy, ecology, physiology, and other aspects of the
large and diverse world of yeasts (Kurtzman et al. 2011).
This treatise has provided current updates on the taxonomic
designation of yeasts according to the International Code of
Taxonomy as well as chapters on ecological and applied
aspects, including developments in biotechnology (Johnson
and Echavarri-Erasun 2011). The primary species of asco-
mycetous non-Saccharomyces yeasts of current and poten-
tial importance in biotechnology are described in this Mini-
Review. Yeasts of basidiomycetous affinity and biotechno-
logical importance are described in the second Mini-Review
(Johnson 2013). An overview of various areas of yeast
biotechnology are provided in Fig. 1.
Debaryomyces hansenii
The primary non-Saccharomyces ascomycetous yeasts of
importance in biotechnology are listed in Table 1 and de-
scribed in the following sections.
Debaryomyces spp., particularly D. hansenii, have bio-
technological potential and utility in a diversity of areas
(Breuer and Harms 2006; Fleet 2007). Debaryomyces spp.
belong to the lipid-accumulating or “oleaginous” yeasts, and
some strains can accumulate 20–70 % of their biomass as
lipids (Cohen and Ratledge 2005; Papanilolaou and George
2011a, b; Beopolous et al. 2011; Ratledge 2002). In addition
to D. hansenii, other oleaginous yeasts include species of
504 Appl Microbiol Biotechnol (2013) 97:503–517

Fig. 1 An overview of various TRADITIONAL YEAST

areas of yeast biotechnology FERMENTATIONS
FOOD AND
ENVIRONMENTAL FEED INGREDIENTS
Beer, Wine, Sake, Soy Sauce,
BIOTECHNOLOGY Other Food Fermentations
Enzymes, Flavors,
Bioremediation, Pollutant Pigments, Amino acids,
Degradation Organic acids

BIOCONTROL BIOCATALYSIS

Crop protection, YEAST Pharmaceuticals, Chiral

Food and Feed Safety, BIOTECHNOLOGY Chemical Intermediates,
Probiotics Biotransformations

BIOMEDICAL HETEROLOGOUS
RESEARCH PROTEIN
PRODUCTION
Drug discovery, Drug FUNDAMENTAL
Resistance and Metabolism, BIOLOGICAL RESEARCH Protein Pharmaceuticals,
Elucidation of Disease Enzymes, Hormones,
Mechanisms Vaccines, Toxins
Molecular and Cellular Biology, Genomics,
Functional Genomics, Pathway Engineering,
Systems Biology Mechanisms

Candida, Cryptococcus, Pichia (Hansenula), Lipomyces, offers the commercial potential for production of lipids or
Pseudozyma, Rhodosporidium, Rhodotorula, Trichosporon, “single-cell oils” (Beopolous et al. 2011; Fickers et al. 2005;
Trigonopsis, Yarrowia, Saccharomycopsis, and potentially Papanikolaou et al. 2002; Papanilolaou and George 2011a, b),
Phaffia rhodozyma, and Xanthophyllomyces dendrorhous. biodiesel generation (Dai et al. 2007), and accumulation of
Several of these yeast species are of basidiomycetous affinity lipid-soluble fine chemicals of high value, such as carote-
and are covered in the accompanying Mini-Review. The abil- noids, surfactants, and flavorants.
ity of oleaginous yeasts to accumulate high quantities of lipids Debaryomyces spp. are remarkable (Gunde-Cinerman et
al. 2009), being able to grow in culture media and complex
Table 1 Principal ascomycetous yeast species of biotechnology substrates such as foods containing up to 4 M NaCl (∼10 % w/
importance w) and glucose (∼5 % w/w). In contrast, the most osmotolerant
strains of S. cerevisiae are restricted in growth to media con-
Yeast species Updated nomenclaturea
taining less than 1.7 M NaCl (Onishi 1963). Consequently,
Candida spp. Variousa Debaryomyces spp. are frequently found in salted, sugared,
Debaryomyces hansenii Unchanged
and fermented foods of high osmolarity as well as in marine
Hansenula spp. Ogataea spp.
environments (Kutty and Philp 2008). Osmotolerance of
Kluyveromyces lactis Unchanged
Debaryomyces is desirable from a biotechnological perspec-
tive as the yeast can be grown in media that are resistant to
Kluyveromyces marxianus Unchanged
contamination, thus reducing costs of the fermentation
Lipomyces spp. Unchanged
(Breuer and Harms 2006). It is intriguing that D. hansenii also
Pichia stipitis Schefferomyces stipitis
has high resistance to chlorine dioxide (ClO2) (Ramirez-
Pichia spp. Komagataella spp.
Orozco et al. 2001), a property that could be exploited to
Saccharomyces cerevisiae Unchanged
maintain asepsis in fermentations. D. hansenii produces killer
Schizosaccharomyces pombe Unchanged
toxins active against various yeast species, which may be of
Saccharomycopsis spp. Unchanged
value in maintaining aseptic conditions in industrial fermen-
Schwanniomyces occidentalis Unchanged
tations and for potential control of yeast infections (Buzzini
Yarrowia lipolytica Unchanged
and Martini 2001). The ability of D. hansenii to tolerate
These yeasts were chosen on the basis of the potential and realized extreme stresses could be highly advantageous in the imple-
importance in industrial fermentations and biotechnological processes. mentation of low-cost fermentation processes (Breuer and
The list does not encompass the large number of yeast species involved Harms 2006).
in food spoilage and defects (Fleet 2007), or in human and animal
D. hansenii produces xylitol from D-xylose in wood
disease
hydrolysates, yielding relatively high xylitol:ethanol ratios
Candida is an extremely heterogenous genus encompassing ascomy-
cetous and basidiomycetous yeasts for which the teleomorphic state (Breuer and Harms 2006). This capability is of potential
has not been demonstrated (Lachance 2011) importance in biomass conversion since a major bottleneck
a
For updated nomenclature, see Kurtzman et al. (2011) in this process is the bioconversion of pentoses to ethanol
Appl Microbiol Biotechnol (2013) 97:503–517
(Hahn-Hägerdahl et al. 2007; Jeffries 2006; Saha 2003). D.
hansenii also produces other sugar alcohols from pentoses,
including arabinose (Breuer and Harms 2006). D. hansenii
synthesizes several exo-enzymes including β-glucosidases,
esterases, and inulinases, enzyme systems of current and
growing industrial importance (Ricca et al. 2007; Sandhya
and Pandey 2006). The draft genome sequence of
Debayomyces hansenii has become available (Kumar et al.
2012) which should enable increased understanding of salt
tolerance and exploitation of this ascomycetous yeast in
biotechnology.
Kluyeromyces lactis and Kluyveromyces marxianus
The systematics of Kluyveromyces spp. has been extensively
studied, particuarly by two prominent yeast biologists
(Lachance 2003, 2007; van der Walt 1970). Kluyveromyces
is an ascomycetous yeast that readily forms asci. Several
genetic techniques have been developed for manipulation of
K. lactis for basic studies and industrial development (Spencer
et al. 2002; van Ooyen et al. 2006). The availability of genetic
and molecular tools and genomic information for for K.lactis
and K. marxianus (Dujon 2006; Dujon et al. 2004) has facil-
itated the development of the yeast for heterologous protein
production and potentially other industrial applications (van
Ooyen et al. 2006).
Species of Kluyveromyces produce β-galactosidase
(lactase) that enables the utilization of lactose as a primary
carbon source (Ansari and Satar 2012; Oliveira et al. 2011;
Rubio-Texeira 2006), an attribute of particular importance
for the dairy industry. Kluyveromyces spp. can be grown on
inexpensive substrates and waste streams containing lactose
such as cheese whey (Siso 1996). This yeast genus also has
industrial potential for production of single-celled protein,
exopolysaccharides, and fine chemicals (Antoni et al. 2003;
van Ooyen et al. 2006).
Kluyveromyces lactis has been investigated for produc-
tion of heterologous proteins, and has the capability for
efficient expression and secretion of a variety of peptides
and high molecular weight proteins in attractive yields
(Fleer 1992; Romanos et al. 1992; van Ooyen et al. 2006).
High cell densities have been obtained in fermentations,
reaching approximately 100 g dry cell weight (DCW) per
liter concomitant with secretion of recombinant human se-
rum albumin in several grams per liter (Fleer 1992;
Romanos et al. 1992). A drawback to the use of K. lactis
for the production of heterologous human proteins is that the
glycosylation does not conform to the human pattern. It may
be possible to adapt a “humanized” glycosylation system to
K. lactis, such as that developed for Komagataella (Pichia)
pastoris and Saccharomyces cerevisiae (Hamilton and
Gerngross 2007; Li et al. 2007; Wildt and Gerngross
2005). K. lactis also forms the killer toxin zymocin that is
505
active against certain ascomycetous yeasts (Jablonowski
and Schaffrath 2007). Since K. lactis is present in many
dairy products that are consumed by humans, it is consid-
ered to be generally recognized as safe (GRAS), and the
yeast can be utilized for production of food ingredients,
flavors and fragrances, and as a dietary supplement
(Gounaris 2010; van Ooyen et al. 2006). The potential for
production of lactic acid by Kluyveromyces and other yeasts
has been assessed (Sauer et al. 2010).
Methanol-utilizing yeasts—K. (P.) pastoris, other Komagataella
spp., Ogataea (Hansenula) polymorpha, Ogataea minuta,
and Candida spp.
The methylotrophic yeasts were initially isolated in the late
1960s and early 1970s (Kato et al. 1974; Ogata et al. 1969),
and methanol-assimilating yeasts have been continued to be
isolated from various habitats (Ji and Bai 2008; Peter et al.
2011). These yeasts have the intriguing property of being
able to very efficiently use methanol as a sole source of
carbon and energy. Methanol utilization is associated with
the strong expression of the enzyme methanol oxidase
(Mox) (also referred to as alcohol oxidase), and the mox
promoter governing the expression of the gene has been
exploited for high-level expression of native and heterolo-
gous genes (Boettner et al. 2007; Cregg 2007; Gellissen et
al. 2005; Houard et al. 2002; Porro et al. 2005). The heter-
ologous proteins accumulate in peroxisomes, and at high
levels of expression, peroxisomes can occupy as much as
80 % of the cell volume, imparting a striking cellular mor-
phology (Gellisen 2002; Tanaka and Ueda 1993; Veenhuis
et al. 1983).
Initially, the methylotrophic yeasts were considered for
the production of biomass and single-cell protein from an
inexpensive source of carbon (methanol). Industrial devel-
opment of the yeast was performed at Phillips Petroleum
Company for the production of single-cell protein for feeds
(Sreekrishna and Kropp 1996; Wegner 1983). High levels of
biomass (ca. 130 g DCW per liter) and high yeast produc-
tivity (>10 g DCW/l-h) was achieved with a specialized
fermentation system including injection of pure oxygen
and high mass transfer conditions. A similar process was
developed by Imperial Chemical Industries using the bacte-
rium Methylophilus methylotrophus for the production of
Pruteen (Macauley-Patrick et al. 2005). However, the eco-
nomics of protein production became unfavorable compared
to proteins from soybean and other sources, and efforts were
turned to the development of K. pastoris and O. polymorpha
for heterologous protein production (Macauley-Patrick et al.
2005; Romanos et al. 1992; Wegner 1990).
Of the methylotrophic yeasts, K. pastoris and O. poly-
morpha have been intensely investigated for heterologous
protein production (Cregg 2007; Jahic et al. 2006; http://
506
faculty.kgi.edu/cregg). In O. polymorpha, several heterolo-
gous proteins including membrane-bound and labile
enzymes have been efficiently produced (Cregg 2007;
Gellisen 2002, 2005). The yield of heterologous protein
production in K. pastoris and O. polymorpha is often higher
than other yeasts including S. cerevisiae, and grams per liter
can be obtained for certain proteins. Secretion is generally
more efficient and protein degradation diminished in these
yeasts compared to S. cerevisiae. Expression systems, fer-
mentation conditions, downstream processing, and other
aspects of heterologous protein production in K. pastoris
and O. polymorpha have been extensively reviewed
(Boettner et al. 2007; Cregg 2007; Daly and Hearn 2005;
Gellisen 2002; Gellissen et al. 2005; Macauley et al. 2005).
Heterologous protein production in K. pastoris, particularly
optimization of glycosylation, is discussed more thoroughly
below. The genome sequence of P. pastoris has become
available (Kuberi et al. 2011) which will enable more de-
finitive studies of factors controlling heterologous protein
synthesis.
Yarrowia (Candida) lipolytica
Y. lipolytica is an ascomycetous yeast that is the teleomorph
of C. lipolytica. Industrial interest in Y. lipolytica derived
initially from its unique physiological capabilities to utilize
polyalcohols, organic acids, and long-chain hydrocarbons as
substrates (Fickers et al. 2005; Gellissen et al. 2005; Khanna
et al. 2012; Papanikolaou and Aggelis 2011a, b). Y. lip-
olytica also produces high levels of lipolytic enzymes, sev-
eral of which have industrial utility (Beopolous et al. 2011;
Fickers et al. 2011). The yeast has been evaluated as a
source of single cell protein (SCP) from lipid substrates,
such as crude oils and hydrocarbons (Cohen and Ratledge
2005; Khanna et al. 2012; Ratledge 2002). The yeast pro-
duces relatively large quantities of organic acids (e.g., py-
ruvate, citric acid, and isocitrate) from various carbon
sources (Zhou et al. 2012). Y. lipolytica also produces higher
value compounds, such as lactones as flavorants (Garcia et
al. 2007; Waché et al. 2006; Ye et al. 2012) and enzymes
including cytochrome P450s and lipases with commercial
uses in biotransformations of steroids, synthesis of pharma-
ceutical intermediates, and production of fine chemicals
(Fickers et al. 2005). The availability of the genome se-
quence (Dujon 2006) should lead to advances in genetic
manipulation of this yeast.
Candida spp.
Candida is an extremely heterogenous genus that comprises
a wide diversity of yeasts of ascomycetous affinity that lack
known sexual states (Lachance 2011). The commercial util-
ity and potential of Candida spp. includes synthesis of
Appl Microbiol Biotechnol (2013) 97:503–517
cytochrome P450 monooxygenases for biocatalysis, produc-
tion of SCP from a variety of substrates including hydro-
carbons, formation of D-amino acids from racemic mixtures,
production of hydrophobic intermediates of the alkane oxi-
dation pathway such as fatty alcohols, fatty acids, and
especially dicarboxylic acids of commercial interest, reduc-
tion of xylose to xylitol, and reclamation of chemicals,
paricularly solvents (Guo et al. 2006; Klein and Favreau
1995; Spencer et al. 2002; Wolf 1996; Yadav et al. 2012).
Several Candida spp. grow on a diversity of substrates
including n-alkanes, fatty acids, xylose, and several other
carbohydrates (Kapoor and Gupta 2012; Tanaka and Ueda
1993; Klein and Favreau 1995; Wolf 1996).
Candida species developed for industrial processes in-
clude alkane and phenol-utilizing species, particularly
Candida maltosa and Candida tropicalis (Klein and
Favreau 1995). C. maltosa is able to utilize aromatic and
certain other amino acids as the sole nitrogen source and has
been utilized for the resolution of D- and L-racemic mixtures
of amino acids by metabolism of the undesired isomer. The
utilization of n-alkanes and certain other lipid compounds
requires cytochrome P450 (P450, CYP) enzymes, which are
increasingly being utilized as biocatalysts (Becher and
Wirsel 2012; Faber 2004; Kristan and Rizner 2012; Li et
al. 2012; Panke et al. 2004; Patel 2007; Straathof and
Aldercreutz 2000; Straathof et al. 2002). Two species that
have yielded valuable industrial biocatalysts and are exten-
sively described in the chemical literature are Pseudozyma
antarctica and Candida cylindracea. Other species investi-
gated for their P450 systems include C. tropicalis, C. mal-
tosa, and Candida rugosa. C. tropicalis and Candida utilis
have also been used industrially for the synthesis of SCP
(Hagel et al. 2012; Idris and Bukhari 2012; Klein and
Favreau 1995). These organisms grow and produce biomass
on a variety of substrates and waste streams including oils,
plant hydrolysates, apple pomace, sulfite waste liquor, and
many others (Halász and Lásztity 1991; Klein and Faveau
1995; Bekatorou 2006).
Scheffersomyces (Pichia) stipitis
S. (P.) stipitis is an ascomycetous yeast that has been exten-
sively investigated for the fermentation of xylose in hemi-
cellulose to ethanol, L-lactic acid, and other products (Hahn-
Hägerdal et al.2007; Ilmén et al. 2007; Jeffries 2006; Jordan
et al. 2012). Hemicellulose is the second most abundant
component of cellulosic biomass. Laboratory strains of S.
stipitis, which are amenable to genetic and physiological
manipulation, have been developed by metabolic engineer-
ing for xylose utilization (Jeffries 2006; Jeffries et al. 2007;
Hahn-Hägerdahl et al. 2007). The genome sequence recent-
ly obtained for S. stipitis will provide a valuable resource for
enhancement of xylose utilization (Jeffries et al. 2007).
Appl Microbiol Biotechnol (2013) 97:503–517
Genes from S. stipitis have been introduced into S. cerevi-
siae to enable fermentation of pentoses but with variable
yields (Cai and Zhang 2012; Hahn-Hägerdahl et al. 2007;
Jeffries 2006; Jeffries et al. 2007).
Selected industrial processes of ascomycetous yeasts
Bioethanol
Rapid global population growth is occurring concurrently
with increased demands for food, fuel, and energy
(Goldemberg et al. 2007; Nass et al. 2007; Ragauskas et
al. 2006). World oil production will not keep pace with
increase in demand for petrochemical fuels (Kendon et al.
1986). Limited oil reserves, pollution concerns, global
warming, and political instability have renewed interest
and led to financial support for the generation of sustainable
sources of energy (Agrawal et al. 2007; Antoni et al. 2007;
Cai and Zhang 2012; Farell et al. 2006; Hill et al. 2006;
Kuhad et al. 2011; Ragauskas et al. 2006). Due to the
outstanding capacity of S. cerevisiae and certain other yeasts
to produce ethanol and other organics products, yeasts will
continue to be developed for manufacture of ethanol and
other alcohols.
Biofuels including bioethanol and biodiesel rely on the
conversion of renewable feedstocks to fuels, with current
emphasis mainly on digestion of biomass and lignocellulose
sources such as crop residues, perennial grasses, and other
abundant materials. These materials are plentiful in many
terrestrial environments, and it has been estimated that more
than 1.3 billion tons of biomass could be produced annually in
the United States on a sustainable basis (Himmel et al. 2007).
Recently, the U.S. government set forth the Biofuel Initiative,
which has the goal of replacing 75 % of the current fossil fuel
resources with biofuel by 2025 (Luque et al. 2008).
A primary limitation in biomass fermentations is that
yeasts cannot directly convert cellulose, hemicellulose, lig-
nin, and associated components inherent in biomass to eth-
anol (Hahn-Hägerdahl 2006; Jeffries 2006; Jeffries et al.
2007; Cai and Zhang 2012; Kuhad et al. 2011; Lynd et al.
2002; Weber et al. 2010). The lignin component in terrestrial
biomass is exceptionally recalcitrant to digestion and con-
version to bioproducts by most microorganisms, except for
white rot fungi, but degradation by these fungi is slow and
inefficient. Cellulose and hemicellulose utilization requires
digestion to fermentable sugars before they can be fer-
mented by yeasts (Hahn-Hägerdahl 2006; Jeffries 2006;
Jeffries et al. 2007; Kuhad et al. 2011; Lynd et al. 2002;
Walker 2011). The current rate of enzymatic cellulosic bio-
mass conversion to sugars is about two orders of magnitude
slower than the average fermentation rate of simple sugars
with yeast (Antoni et al. 2007). In addition, xylose and most
507
other pentoses, which constitute about 25 % of biomass, are
not efficiently fermented to ethanol by S. cerevisiae and
other yeasts, such as S. stipitis, which only slowly use
xylose and other pentoses resulting in low cell and ethanol
productivity (Gray et al. 2006; Hahn-Hägerdahl et al. 2007;
Jeffries 2006; Lynd et al. 2002; Saha 2003). Two primary
strategies are being pursued for fermentation of cellulosic
biomass by yeast: (a) enhance and optimize xylose and other
pentose utilization by S. stipitis or other pentose-fermenting
yeasts or (b) introduction of the pathways for fermentation
of xylose and other pentoses into S. cerevisiae to be
exploited by its well-known strong fermentation capacity
and high ethanol tolerance (Chu and Lee 2007; Jeffries
2006; Lynd et al. 2002; Ostergaard et al. 2000; Saha 2003;
van Maris et al. 2006). Metabolic engineering and modeling
of the biochemical pathways involved in the fermentation of
pentoses and other carbon sources are being exploited to
enhance fermentation productivity, as well as other limiting
factors such as ethanol resistance (Alper et al. 2006; Flores
et al. 2000; Ostergaard et al. 2000; Stephanopolous 2007).
The availability of the genome sequence from S. stipitis
(Jeffries et al. 2007) and other yeasts has provided important
resources for improvement of biomass utilization. Marked
improvements have been made in fermentation of cellulosic
hydrolyzates by yeasts, yet the processes have not reached
the stage of economic viability and industrialization.
Selected yeast enzymes for industrial processes
The term “enzyme” (literally “in yeast”) was coined by Kühne
in 1876 (Aehle 2004). Enzyme technology utilizing cell-free
enzymes and whole-cell biocatalysts is integral to several
large-scale chemical, pharmaceutical, and agricultural biopro-
cesses (Aehle 2004; Pandey et al. 2006; Panke et al. 2004).
The economic value of the industrial enzyme market in 2005
was approximately $3 billion and was expected to reach more
than $8 billion by 2009 (Porro et al. 2005).
In recent years, growth of the bulk enzyme industry has
been particularly robust in the baking and animal feed
sectors, in organic syntheses for fine chemicals and pharma-
ceuticals, and to a lesser degree for paper and pulp process-
ing, production of biofuels, and for personal care (Aehle
2004; Kirk et al. 2002). The bulk enzyme market is domi-
nated by enzymes produced by bacteria and filamentous
fungi cultivated in submerged culture, particularly Bacillus
and Aspergillus species (Aehle 2004; Pandey et al. 2006).
Only a limited number of yeast enzymes are currently used
as bulk enzymes in commodity processes, the majority
derived from ascomycetous yeasts (Tables 2 and 4).
However, several yeast enzymes have found application in
the production of high-value specialized fine chemicals and
for biotransformation of pharmaceutical intermediates (see
below). Enzymes and whole cells used as biocatalysts for
508 Appl Microbiol Biotechnol (2013) 97:503–517

Table 2 Industrial enzymes from yeasts Table 3 Examples of industrial recombinant enzymes produced in
yeasts
Enzyme Yeast Industry
Enzyme Recombinant yeast host
Chymosin Kluyeromyces spp. Food processing
Saccharomyces cerevisae Chymosin Kluyveromyces marxianus var. lactis
α-Galactosidase Saccharomyces spp. Feed applications Glycolate oxidase Komagataella pastoris
L-Glutaminase Zygosaccharomyces rouxii Therapeutic Phytase Komagataella pastoris
Analytical
Sources: Cowan 1996; Cregg 2007; see text for additional references
Inulinases Candida spp. Food applications
Kluyveromyces marxianus
Invertase Saccharomyces cerevisiae Food applications Biocatalysts for pharmaceutical and fine chemical
Lactase Candida pseudotropicalis Food processing production
Kluyveromyces spp.
Lipase Candida rugosa Food processing
Catalysis in the chemical and pharmaceutical industries has
Pseudozyma antactica A, B Flavors
traditionally been dominated by chemical syntheses using
Candida lipolytica Detergent
non-biological catalysts (Bernhardt 2006; Blaser 2003;
Geotrichum candidum Wastewater
Blaser et al. 2005; Bonrath and Netscher 2005; Kuoda and
Trichosporon fermentum Degreasing
Ueda 2011). However, several disadvantages are inherent to
Bioremediation certain industrial chemical synthetic processes, including rel-
Yarrowia lipolytica Therapeutic atively low catalytic efficiency for many reactions, lack of
L-Phenylalanine Rhodotorula spp. Pharmaceutical enantiomeric specificity for chiral syntheses, the need for
ammonia lyase Rhodosporidium spp.
reaction conditions of high temperature, low pH and high
Phenylalanine Candida boidinii Pharmaceutical
dehydrogenase pressure requirements, as well as the extensive use of organic
Phytase Ogataea polymorpha Feed solvents with consequent formation of organic waste and
Nutrition pollutants. These concerns and drawbacks have stimulated
keen interest in biocatalytic processes using enzymes
Sources: Aehle 2004; Hasan et al. 2006; Hilterhaus and Liese 2007; (Bonrath and Netscher 2005; Rozzell 1999). Advantages of
Pandey et al. 2006; Vakhlu and Klur 2006. Also see text for additonial
references
enzymes for chemical processes include mild reaction con-
ditions, i.e., operation at temperatures from 20 °C to 80 °C, at
neutral or slightly acidic pHs, and under atmospheric condi-
tions (Koeller and Wong 2001; Rozell 1999). In contrast to
high-value pharmaceuticals and fine chemicals is a rapidly organic catalysts, enzymes typically catalyze reactions with-
growing sector of the enzyme industry. out substrate functional-group protection, and many enzymes
Currently, about 90 % of industrial enzymes are produced have a long half-life, typically several days to months. Other
as heterologous proteins by recombinant methods (Cherry beneficial properties are the stereoselectivity of enzymes for
and Fidanstef 2003). Certain yeasts, especially K. pastoris their substrates that yields stereo- and regiochemically defined
and S. cerevisiae, are increasingly being utilized for heter- reaction products at the enormous rate acceleration of 105 to
ologous production of enzymes and a variety of other pro- 108 (Aehle 2004; Koeller and Wong 2001; Rozell 1999). They
teins (Aehle 2004; Cregg 2007; van Beilen and Li 2002). also catalyze reactions on unnatural substrates, leading to
Blastobotrys adeninivorans, O. polymorpha, K. lactis, enormous compound diversity.
Schizosaccharomyces pombe, Y. lipolytica, Pseudozyma Another advantage of enzymes as industrial catalysts is
spp., and other yeast species have also been developed for that they can be genetically selected and chemically modi-
the production of heterologous proteins (Avis et al. 2005; fied to enhance stability, substrate specificity, and specific
Buckholz and Gleeson 1991; Gellissen 2005; Gellissen et al. activity (Aehle 2004; Koeller and Wong 2001; Rozell
2005; Madzak et al. 2004; van Ooyen et al. 2006). Industrial 1999). Several enzymes are active in organic solvents, su-
recombinant enzymes and those close to commercialization percritical fluids, high pressures, and ionic solvents (“Green
produced by yeasts are presented in Tables 3 and 4. Yeasts are solvents”) (Klibanov 2001; Rezaei et al. 2007; Yang and
desirable hosts of enzymes for food uses because of their lack Pan 2005). Hydrophobic environments and ionic solvents
of production of toxic secondary metabolites (Olempska-Beer are optimal for some hydrophobic reactions using biocata-
et al. 2006). K. lactis is currently approved by the FDA for lysts, such as those catalyzed by lipases (Park et al. 2009;
production of recombinant enyzmes (chymosin) for uses in Yang and Pan 2005). Immobilized enzyme processes can
foods. A much more detailed description of industrial enzymes have advantages over enzymes in solution including stability,
from yeasts is described in Johnson and Echavarri (2011). process duration, and catalytic efficiency (Sheldon 2007). A
Appl Microbiol Biotechnol (2013) 97:503–517
Table 4 Yeast enzymes/whole cells used in biocatalytic processes (adapted from Hilterhaus and Liese 2007; see text for additional references)
Enzyme class Enzyme
I. Oxidoreductases EC 1 Dehydrogenase
Dehydrogenase
Dehdrogenase
Reductase
Reductase
Reductase
D-amino acid oxidase
β-Galactosidase
Formate dehydrogenase
Transferases EC2 Lipase
Lipase
Hydrolases EC 3 β-Galactosidase
Lactamase/racemase
Lyases EC 4 Pyruvate carboxylase
Enoyl-CoA hydratase
L-Phenylalanine ammonia lyase
Isomerases EC 5 No significant commercial sources from yeasts
Ligases EC6 No significant commercial sources from yeasts
drawback to certain enzyme syntheses is that they require
cofactors, but this problem can be minimized by various
approaches, including cofactor recycling (Straathof and
Aldercreutz 2000) and the use of whole cells, such as engi-
neered S. cerevisiae (Faber 2004).
Although a large number of studies and a voluminous
literature have been devoted to the production of fine chem-
icals using enantioselective catalysis by enzymes, relatively
few enzymatic processes are currently used on an industrial
scale (Blaser 2003; Bonrath and Netscher 2005; De Mot and
Verachtert 1982). It has been estimated that about 150
industrial syntheses currently utilize enzymes or microbial
whole cell catalysts (Straathof et al. 2002; Woodley 2006;
Yazbeck et al. 2004), but the use of enzymes in syntheses is
increasing particularly for complex and chiral molecules.
Several yeasts produce enzymes and bioacatalysts of
realized and potential value in the traditional enzyme indus-
try and as chiral-specific biocatalysts for the fine chemical
and pharmaceutical industries (Table 5) (Aehle 2004; Liese
et al. 2000; Matsuda et al. 2009; Pandey et al. 2006; Patel
2004, 2007; Straathof and Adlercreutz 2000). Useful case
studies of commercial enzymatic and microbial biotransfor-
mations, including several involving yeasts, have been de-
scribed (Table 5) (Cheetham 2004). Lipases and esterases,
commonly produced by yeasts, are the most frequently used
biocatalysts in industrial organic syntheses (Chenevert et al.
2006; Li et al. 2012; Pscheldt and Glieder 2008).
Despite the advantages of biocatalysts, enzymes and whole
yeast cells are still an underutilized technology (Bernhardt
2006; Rozzell 1999). Recently, the discovery of enzymes and
509
Yeast source
Zygosaccharomyes rouxii
Geotrichum candidum
Candida sorbophila
Ogataea methanolica
Aureobasidium pullulans
Saccharomyces cerevisiae
Trigonopsis variabilis
Kluyveromyces lactis
Candida boidinii
Candida cylindracea
Candida antarctica A, B0Pseudozyma antarctica
Kluyveromyces lactis
Cryptococcus laurentii
Saccharomyces cerevisiae
Candida rugosa
Rhodotorula rubra
metabolites for use in industrial biotechnology has been vital-
ized through metagenomics (Ferrer et al. 2007a, b; Lorenz and
Eck 2005). This approach has led to the discovery of enzymes,
antimicrobials, and other compounds of industrial potential
(Ferrer et al. 2007a, b; Lorenz and Eck 2005; Podar and
Reysenbach 2006). However, most metagenomic studies have
been directed towards understanding bacterial diversity, and
not yeasts, filamentous fungi, and other eukaryotes (Lorenz
and Eck 2005). The paucity of metagenomic studies conducted
in yeasts, filamentous fungi, and other eukaryotes, such as
protozoa, is primarily due to the comparatively large size of
genomes in fungi compared to bacteria and viruses and the
relatively large nucleotide sequencing efforts required to obtain
a representation of species diversity (Hall 2007). It is antici-
pated that more metagenomic studies will be devoted to fungi
and yeasts due to advances in technology in sequencing and
will be devoted to fungi because of their enormous biodiversity
and ability to produce a multitude of secondary metabolites.
Heterologous protein production in yeasts
Since the early 1980s, yeasts have been utilized for heterol-
ogous production of a variety of proteins (Cregg 2007;
Gellissen 2005; Gellissen et al. 2005; Hitzeman et al.
1981; Hou et al. 2012; Smith et al. 1985; Romanos et al.
1992). The production of heterologous proteins in yeasts
holds enormous potential for biotechnological processes. A
breakthrough in heterologous protein expression in yeast
was the cloning, expression, processing, and secretion of
human proinsulin in S. cerevisiae in the 1980s (Cousens et
510 Appl Microbiol Biotechnol (2013) 97:503–517

Table 5 Examples of yeast enzymes used in biotransformation/biocatalytic processes in the pharmaceutical and fine chemicals industries

Yeast Enzyme or Whole Cell Product Reference

Lipasea Taxol Koeller and Wong 2001

Monooxygenase (S. cerevisiae) Cyclic lactones Stewart et al. 1998
Nuceloside phosphorylasea Antiviral Ribavarin Koeller and Wong 2001
Zygosaccharomyces rouxii (dehydrogenase) Benzodiazepines Rozell 1999
Hilterhaus and Liese 2007
Dehydrogenasesa Optically active Sturmer and Breuer 2006
Alcohols
Dehdrogenasesa Ephedrine Sturmer and Breuer 2006
Esterasea Racemic alcohols Koul et al. 2005
Reductasea Ataxanavir (HIV protease inhibitor) Patel 2004
Lipase (Candida cylindraceae) Lobucavir (antiviral) Patel 2004, 2007
Lipase (C. cylindraceae) Hepatitis B antiviral Patel 2004, 2007
Lipase (Candida antarctica) Ribavarin (antiviral) Patel 2004
Reductase (Pichia spp.) Pacliataxel (anticancer drug) Patel 2004, 2007
Reductase (Aureobasidium pullulans, Candida spp.) Retinoid receptor agonists Patel 2004
Lipase (Candida cylindracea) Pharmaceutical intermediate Patel 2004
Reductase (Candida spp.) Pichia spp., Rhodotorula spp., Pharmaceutical intermediate Patel 2004
Saccharomyces spp.)
Epoxide hydrolase (Rhodotorula glutinis) Melantonin receptor agonist Patel 2004
Whole cell racemate resolution (Candida boidinii, Pharmaceutical intermediate Patel 2004, 2007
Ogataea methanolica, Ogataea polymorpha)
Reductase (Geotrichum candidum) Hydroxy methyl glutaryl Patel 2004
CoA inhibitors (cholesterol lowering agents)
Lipase (Pseudozyma antarctica), Hyphozyma Tachykinin receptor agonists) Patel 2004
sp., Cryptococcus tsukubaensis
(S)-2-pentanol, (S)-2-heptanol Anti-Alzheimer drugs Patel 2007
(R)–(S) oxidase Mixed racemate resolution Patel 2007
(Ogataea polymorpha)
(Candida boidinii)
(Ogataea methanolica)
Lipase, reductase (Pseudozyma antarctica Beta-blockers Zelaszczyk and Kiec-Kononowicz 2007
S. cerevisiae)
a
Yeast species not referenced. See text for additional references

al. 1987; Hadfield et al. 1993; Ladisch and Kohlmann 1992;
Thim et al. 1986). Insulin from S. cerevisiae had increased
pharmacological efficacy and reduced side effects compared
to porcine insulin. Subsequent studies showed that S. cer-
evisiae effectively synthesized and secreted many mamma-
lian and human proteins including α-interferon, epidermal
growth factor, human serum albumin, hepatitis B surface
antigen, epidermal growth factor, β-endorphin, and prochy-
mosin. Several of of these proteins have been developed and
commercialized as high-value human pharmaceuticals or
enzymes (Hadfield et al. 1993; Romanos et al. 1992;
Smith et al. 1985; Gellissen 2002; Gellissen et al. 2005;
Hou et al. 2012). Yeasts have several advantages as hosts for
native and heterologous production, including ease and
rapidity of growth, excellent methods of genetic manipula-
tion, high expression with optimized systems, the ability to
perform eukaryotic post-translational modifications includ-
ing intron splicing, proteolytic processing, folding, forma-
tion of disulfide linkages, glycosylation, and mainentance of
quality suitable for biochemical and structural analyses
(Gellissen 2005; Gellissen et al. 2005).
Yeast species investigated for heterologous protein pro-
duction included the non-methylotrophs S. cerevisiae,
Schizosaccharomyces pombe, K. lactis, Y. lipolytica,
Zygosaccharomyces rouxii, Zygosaccharomyces bailii, and
B. adeninivorans (Böer et al. 2007; Gellissen 2005;
Gellissen et al. 2005; Madzak et al. 2004; Porro et al. 2005;
van Ooyen et al. 2006), and the methylotrophs K. pastoris, O.
polymorpha, Candida boidinii, Ogataea methanolica, and
Schwanniomyces occidentalis (Table 4)(Böer et al. 2007;
Gellissen 2002; Houard et al. 2002; Jahic et al. 2006;
Labuschagne and Albertyn 2007). Of these yeasts, S.
Appl Microbiol Biotechnol (2013) 97:503–517
cerevisiae and K. pastoris have been most extensively devel-
oped for heterologous protein production. More than 500
heterologous proteins have been producted in K. pastoris,
including plant, animal and human proteins, as well as
membrane-associated proteins which are often difficult to
functionally express in many other systems (Cregg 2007;
Labarre et al. 2007; Macauley-Patrick et al. 2005; Porro et
al. 2005; http://faculty.kgi.edu/cregg). K. pastoris has the
exceptional ability to produce high quantities of proteins,
glycosylate them in patterns similar to that of humans,
efficiently secrete them into the medium, and avoid ag-
gregation and proteolysis during recovery for many pro-
teins. Proteins can be expressed in sufficient quantity and
quality for applications in protein engineering and stuc-
tural analyses (Daly and Hearn 2005).
Protein pharmaceuticals from yeasts
Proteins currently constitute about 25 % of new approved
drugs in the USA, with the majority being monoclonal
antibodies (Gellissen 2005; Sethuraman and Stadheim
2006; Walsh 2005, 2006). As of 2006, more than 165
protein biopharmaceuticals have been approved by
European and US regulatory agencies, and more than 500
product candidates are in preclinical and clinical develop-
ment (Walsh 2006). In 2004, the global market for recom-
binant protein therapeutics and nucleic acid-based products
was more than than $33 billion and was expected to reach
$60–70 billion by 2010 (Evans and Das 2005; Pavlou and
Reichert 2005; Schmidt 2004; Walsh 2006).
Yeasts are intensively being developed as protein expres-
sion systems, which in comparison to mammalian cell lines
have higher productivity, higher cell yields, shorter fermen-
tation cycles, and can be cultured in defined media under
relatively inexpensive conditions. In addition, yeasts can
efficiently secrete proteins, possess posttranslational modi-
fication pathways, and are nonpathogenic and non-
pyrogenic (Schmidt 2004). The primary yeast species being
developed for protein pharmaceutical and enzyme produc-
tion are S. cerevisiae, K. pastoris, O. polymorpha, B. adeni-
nivorans, Y. lipolytica, and K. lactis (Schmidt 2004). Certain
peptide and protein therapeutics produced in S. cerevisiae
and more recently O. polymorpha and K. pastoris have been
commercialized (Table 5) (Gerngross 2004; Walsh 2003).
One disadvantage to the synthesis of protein biopharma-
ceuticals in yeasts is that the resulting products often have a
non-human glycosylation pattern (Gemmill and Trimble
1999; Gerngross 2004; Schmidt 2004; van Ooyen et al.
2006). Non-human glycosylation of therapeutic proteins can
affect various biological properties including half-life, tissue
distribution, pharmacokinetics, and immunogenicity
(Hamilton and Gerngross 2007). Most yeast species have the
propensity to glycosylate proteins with high mannose-
511
containing N-glycans (Gemmill and Trimble 1999). To re-
solve the problem of glycosylation, yeasts are being devel-
oped for “humanization” of post-translational modifications
(Li et al. 2007; Wildt and Gerngross 2005). Emphasis in this
area has been with K. pastoris, which is being optimized by
“glycoengineering” through gene alterations to eliminate un-
desirable protein modifications (Gerngross 2004; Hamilton
and Gerngross 2007).
Yeasts are currently commercially used or are close to
commercialization for the production of a variety of heter-
ologous protein pharmaceuticals. High-level expression of
human insulin (Cousens et al. 1987), interferons (Gasmi et
al. 2011), tetanus fragment C (Clare et al. 1998), HIV-1
envelope glycoprotein gp120 (Jordan and Gibbins 2006),
and others have been achieved in S. cerevisiae, K. pastoris,
Y. lipolytica, and other yeast species (Hadfield et al. 1993).
Monoclonal antibodies (mAbs) are the most prevalent
and high-value classes of protein drugs (Gasser and
Mattanovich 2007; Maggon 2007; Pavlou and Reichert
2005), which presently have a global market of approx.
$30 billion (Evans and Das 2005; Gasser and Mattanovich
2007). mAbs have traditionally been produced in mamma-
lian cell lines, mostly recombinant CHO cells and other
lines, but this process is slow and expensive (Farrid 2006).
The production of antibodies in yeasts and fungi is also
being actively pursued, but it has been very challenging to
develop antibody-producing systems in yeasts (Gasser and
Mattanovich 2007).
Probiotics and prebiotics
Certain yeast species have been used as prebiotic and pro-
biotic agents for preventing or treating various intestinal,
nutritional, and toxicological disorders (Mosiehi-Jenabian et
al. 2010; Williams 2010). In particular, Saccharomyces
boulardii, originally isolated from fruit in Indochina, has
been used for treatment of intestinal diseases in children and
adults since 1950 (Buts and Bernasconi 2005; Edwards-
Ingram et al. 2007). Recent studies have shown that it is
conspecific with S. cerevisiae, but S. boulardii appears to
have certain genotypic and phenotypic properties that may
contribute to its probiotic properties (Edwards-Ingram et al.
2007). Controlled clinical trials have shown efficacy of S.
boulardii for prevention or elimination of several intestinal
disorders, including infection by Clostridium difficile asso-
ciated with antibiotic-induced diarrhea (Fric 2007). Yeasts
have also been hypothesized to prevent other intestinal
infections, including inflammatory bowel disease and
Crohn's disease (Besirbellioglu et al. 2006; Buts 2009;
Dalmasso et al. 2006; Edwards-Ingram et al. 2007;
Sougioultzis 2006; Villarruel et al. 2007). The mechanisms
of probiotic activity have not been elucidated but may
involve alteration of inflammatory and immune responses
512
or destruction of toxic factors (Ozkan et al. 2007). Although
yeasts may be promising therapeutic agents, they need to
undergo controlled clinical trials to critically evaluate their
efficacy.
Preliminary evidence indicates that certain yeasts can
produce prebiotics, which are compounds (generally oligo-
saccharides) that stimulate the growth of bifidobacteria and
other beneficial bacteria in the gut of humans and animals or
oligopeptides that have beneficial health benefits by stimu-
lation of immune response. Prebiotics are often sugar deriv-
atives such as fructooligosaccharides (Maugeri and
Hernalsteens 2007), of which certain yeasts can catabolize
due to their formation of inulinases. K. lactis was found to
synthesize prebiotic oligosaccharides and immunostimula-
tory peptides from whey (Belem and Lee 1998).
Yeast glucans and cell wall polysaccharides
Yeast cell wall polysaccharides have been used as adjuncts
for animal and fish feeds (Sauerwein et al. 2007; Singh et al.
2008). These polysaccharides have been shown to promote
animal growth and health by various mechanisms including
immunomodulation, oxidative status, binding of toxins and
pathogens, and interactions with gut constituents. Glucans
and mannans have also been found to have a myriad of
biological functions in animal models and potentially in
humans, including modulation of histamine release (Holck
et al. 2007) and antitumor activities (Ghoneum et al. 2007).
Further studies are needed in this area to evaluate health
promotion by yeast cell wall polysaccharides.
Genetically modified and recombinant yeast for use in food
Because of its long history of safe use and consumption, S.
cerevisiae was among the first organisms to be designated
“generally recognized as safe” (GRAS) and was the first
genetically modified (micro)organism (GMO) used for re-
combinant production of food and feed additives. In 1990, a
genetically modified strain of S. cerevisiae became one of the
first GMOs to be approved for food use in the UK (Aldhous
2000). It has been used in bakery products for enhanced
production of carbon dioxide. Many genes encoding desirable
traits mentioned above have been cloned in S. cerevisiae,
Saccharommyces pastorianus, and Saccharomyces bayunus
that have primary roles in food fermentations (Bisson 2004;
Pretorius 2003; Schuller and Casal 2005). However, most of
these strains have not been used commercially because of
regulatory and public hurdles.
Concerns have been raised that certain yeast species and
strains may be opportunistically pathogenic or toxigenic to
immunocompromised humans (Klein and Favreau 1995;
Fenn 2007; Lin and Heitman 2006). In addition to the
well-known pathogens, Candida albicans, Cryptococcus
Appl Microbiol Biotechnol (2013) 97:503–517
neoformans, and certain other yeasts (Cooper 2011), select-
ed strains of S. cerevisiae, Candida, Trichosporon, and
Cryptococcus have shown pathogenic potential (Murphy
and Kavanagh 1999; Wei et al. 2007), and careful assess-
ment of safety is needed for applications in food and bio-
technology uses (Flamm 1991; Olemska-Beer et al. 2006;
Pariza and Johnson 2001; Spok 2006). Allergenicity has
been a major concern of uses of yeasts in human diets
(Airola et al. 2006; Lehrer 1996; Smith et al. 1996).
The advent of functional genomics has created opportu-
nities for in depth understanding, analysis, and improvement
of yeasts in foods and other industrial applications.
Scientific, public, and regulatory concerns with GMOs, such
as spread of antibiotic resistance genes, insecticide or other
resistance genes can be obviated with modern genetic
manipulations (Lehrer et al. 1996; Pariza and Johnson
2001; Plahuta and Raspor 2007; Pretorius and Bauer 2002;
Stewart 2006). S. cerevisiae is also used for the production
of insulin, hepatitis B vaccine, and other products for human
treatment, and it is of utmost importance that these strains
and their by products do not pose a risk for human health.
Summary and perspectives
Yeasts have a rich history and a bright future in biotechnol-
ogy. Their involvement and importance in traditional food
fermentations is unparalled by other organisms of biotech-
nological relevance. The utility of yeasts in biotechnological
processes is accelerating due to a number of properties and
developments. Most yeast species are nonpathogenic to
humans and animals, and thus are anticipated to see more
utility in a variety of disciplines of importance to human
activities. It is likely that yeasts will be increasingly used as
GMOs in traditional processes as their safety is more exten-
sively established. S. cerevisiae and certain other species are
being developed for the production of biofuels from cellu-
losic materials and potentially other substrates and will have
importance in the generation of new sources of energy.
During the past decade, yeast systems have been developed
for the production of heterologous proteins in high yields
and with post-translational modifications equivalent or sim-
ilar to modifications performed in humans. Yeasts have
increasing importance as sources of biocatalysts for the
production of high-value fine chemicals and protein phar-
maceuticals. Due to their metabolic capabilities, they have
important roles in environmental bioremediation. S. cerevi-
siae has become the premier model eukaryotic organism for
the understanding of human physiology, including disease
processes, and will increase in importance for the under-
standing and treatment of disease. Tremendous advances
have been made in the development of S. cerevisiae as the
premier eukaryotic organism for the development of
Appl Microbiol Biotechnol (2013) 97:503–517
functional genomics and systems biology. Other species of
ascomycetous yeast also have tremendous utility and poten-
tial for biotechnologial processes.
Acknowledgments I would like to acknowledge Professor Herman
Jan Phaff (deceased) and Professor Michael J. Lewis and their post-
odoctoral fellows for encouragment and guidance in working on Phaf-
fia/Xanthopyllomyces over the years. I also thank the students and
scientists who have studied this special yeast.
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