fermentation

Review
The Xylose Metabolizing Yeast Spathaspora
passalidarum is a Promising Genetic Treasure for
Improving Bioethanol Production
Khaled A. Selim 1, *, Saadia M. Easa 2 and Ahmed I. El-Diwany 1
1 Pharmaceutical and Drug Industries Research Division, National Research Centre, 33-El-Bohouth St. (former
El Tahrir St.), Dokki, P.O. Box, Giza 12622, Egypt; ahmed_eldiwany@yahoo.com
2 Microbiology Department, Faculty of Science, Ain Shams University, Cairo 11566, Egypt;
elkas_1111@yahoo.com
* Correspondence: Khaled.a.selim@gmail.com




Received: 15 February 2020; Accepted: 16 March 2020; Published: 18 March 2020


Abstract: Currently, the fermentation technology for recycling agriculture waste for generation of
alternative renewable biofuels is getting more and more attention because of the environmental
merits of biofuels for decreasing the rapid rise of greenhouse gas effects compared to petrochemical,
keeping in mind the increase of petrol cost and the exhaustion of limited petroleum resources. One of
widely used biofuels is bioethanol, and the use of yeasts for commercial fermentation of cellulosic and
hemicellulosic agricultural biomasses is one of the growing biotechnological trends for bioethanol
production. Effective fermentation and assimilation of xylose, the major pentose sugar element of
plant cell walls and the second most abundant carbohydrate, is a bottleneck step towards a robust
biofuel production from agricultural waste materials. Hence, several attempts were implemented
to engineer the conventional Saccharomyces cerevisiae yeast to transport and ferment xylose because
naturally it does not use xylose, using genetic materials of Pichia stipitis, the pioneer native xylose
fermenting yeast. Recently, the nonconventional yeast Spathaspora passalidarum appeared as a founder
member of a new small group of yeasts that, like Pichia stipitis, can utilize and ferment xylose.
Therefore, the understanding of the molecular mechanisms regulating the xylose assimilation in such
pentose fermenting yeasts will enable us to eliminate the obstacles in the biofuels pipeline, and to
develop industrial strains by means of genetic engineering to increase the availability of renewable
biofuel products from agricultural biomass. In this review, we will highlight the recent advances in
the field of native xylose metabolizing yeasts, with special emphasis on S. passalidarum for improving
bioethanol production.

Keywords: fermentation; xylose metabolism; genetic engineering; biofuel; Spathaspora passalidarum;

Pichia stipitis

1. Fermentation Technology and Challenges

The modern biotechnological applications for generation of alternative and renewable sources
of biofuels are receiving more attention due to global worries over the climate change, rapid global
warming, and the rising of fossil fuel costs. One of such growing biotechnological trends is the
fermentation technology to convert the sugar-rich agriculture waste into bioethanol by conventional
or non-conventional yeasts [1–4]. In general, yeasts have advantages over bacteria for commercial
fermentation due to the thickness of their cell walls, less stringent nutritional requirements, large sizes,
utmost resistance to contamination, and better growth at acidic pH of bioreactor fermenters.
In nature, the second most abundant hemicellulosic sugar in fast-growing hardwoods and
agricultural biomass is xylose. Xylose sugar forms up to 15–25% of all angiosperm biomass,

Fermentation 2020, 6, 33; doi:10.3390/fermentation6010033 www.mdpi.com/journal/fermentation

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and it could supply an alternative fuel source for its ability to be commercially fermented into
Several
ethanol. approaches have been
Several approaches haveemployed
been employed to engineer
to engineerxylose assimilation
xylose assimilationmetabolism
metabolisminto into
conventional fermentingyeasts,
conventional fermenting yeasts,
suchsuch as Saccharomyces
as Saccharomyces cerevisiae
cerevisiae [4–6]. [4–6]. efficient
Therefore, Therefore, efficient
hemicellulosic
hemicellulosic sugar is
sugar fermentation fermentation
crucial for is crucial
the for the
economic economic of
conversion conversion of lignocellulose
lignocellulose biomass tobiomass
renewable to
renewable biofuels
biofuels [4,6–8]. The[4,6–8].
discovery Theofdiscovery of xylose‐fermenting
xylose-fermenting yeasts in newyeasts
nichesinand
new nichesengineering
genetic and genetic of
engineering of yeastsoftorapid
yeasts to be capable be capable of rapid
fermentation of fermentation of xylose
xylose and other sugarsand other sugars
to recoverable to recoverable
concentrations of
concentrations
bioethanol couldof bioethanol could provide
provide alternative biofuelalternative
sources forbiofuel sources
the future for the
(Figure future (Figure 1) [4,9].
1) [4,9].

Figure 1. Model of yeast fermentation machinery for bioethanol production using the agriculture waste
Figure 1. Model of yeast fermentation machinery for bioethanol production using the agriculture
to feed yeasts [4]. The metabolic pathways for xylose and glucose assimilation and fermentation are
waste to feed yeasts [4]. The metabolic pathways for xylose and glucose assimilation and fermentation
indicated including the pentose phosphate pathway and glycolysis. The agriculture waste is treated
are indicated including the pentose phosphate pathway and glycolysis. The agriculture waste is
through the enzymatic and chemical simultaneous saccharification and fermentation (SSF) processes to
treated through the enzymatic and chemical simultaneous saccharification and fermentation (SSF)
release the cellulosic and hemicellulosic sugars. The hexose and pentose sugars are transported by
processes to release the cellulosic and hemicellulosic sugars. The hexose and pentose sugars are
specific hexose and pentose sugar transporters into yeast for further metabolizing processes.
transported by specific hexose and pentose sugar transporters into yeast for further metabolizing
processes.
As a rule of thumb for metabolizing the xylose in most of xylose-fermenting yeasts [4], firstly the
xylose is reduced by xylose reductase (XR) to xylitol. In the second step, the xylitol is oxidized by xylitol
As a rule of thumb for metabolizing the xylose in most of xylose‐fermenting yeasts [4], firstly
dehydrogenase (XDH) to xylulose. Afterward, the xylulose passes into the pentose phosphate pathway
the xylose is reduced by xylose reductase (XR) to xylitol. In the second step, the xylitol is oxidized by
being metabolized into glyceraldehyde-3-P which is further reduced to pyruvate. Finally, the pyruvate
xylitol dehydrogenase (XDH) to xylulose. Afterward, the xylulose passes into the pentose phosphate
is decarboxylated to acetaldehyde which is further reduced to ethanol by alcohol dehydrogenase
pathway being metabolized into glyceraldehyde‐3‐P which is further reduced to pyruvate. Finally,
(Figure 1). Notably, most xylose reductase enzymes have dual cofactor specificity, using both NADH
the pyruvate is decarboxylated to acetaldehyde which is further reduced to ethanol by alcohol+
and NADPH, but typically favor NADPH. However, xylitol dehydrogenase enzymes use NAD
dehydrogenase (Figure 1). Notably, most xylose reductase enzymes have dual cofactor specificity,
specifically as a cofactor, which could cause imbalance between the cofactor’s source for the XR-XDH
using both NADH and NADPH, but typically favor NADPH. However, xylitol dehydrogenase
pathway and xylitol accumulation under uncontrolled oxygen conditions (Figure 2) [10].
enzymes use NAD+ specifically as a cofactor, which could cause imbalance between the cofactor’s
source for the XR‐XDH pathway and xylitol accumulation under uncontrolled oxygen conditions
(Figure 2) [10].
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Figure 2. Schematic model of the central metabolism for bioethanol production from xylose indicating
Figure 2. Schematic model of the central metabolism for bioethanol production from xylose indicating
the rate limiting steps (XR: xylose reductase; XDH: xylitol dehydrogenase, and ADH: alcohol
the rate limiting steps (XR: xylose reductase; XDH: xylitol dehydrogenase, and ADH: alcohol
dehydrogenase) and cofactors demand/balance in most of native xylose metabolizing yeasts. Xylose
dehydrogenase) and cofactors demand/balance in most of native xylose metabolizing yeasts. Xylose
assimilation reactions starts with XR to produce xylitol. The xylitol is further metabolized by XDH to
assimilation reactions starts with XR to produce xylitol. The xylitol is further metabolized by XDH to
produce xylulose, which further metabolized to xylulose-phosphate to enter the glycolysis (indicate by
produce xylulose, which further metabolized to xylulose‐phosphate to enter the glycolysis (indicate
black dotted arrow and summarized in Figure 1) to produce acetaldehyde. The acetaldehyde finally
by black dotted arrow and summarized in Figure 1) to produce acetaldehyde. The acetaldehyde
converted to ethanol by ADH.
finally converted to ethanol by ADH.
One of pioneer xylose fermenting yeasts is Pichia stipites. P. stipitis is heterothallic
One of pioneer xylose fermenting yeasts is Pichia stipites. P. stipitis is heterothallic ascomycetous
ascomycetous yeast, predominantly haploid and related to pentose fermenting yeasts, such as Candida
yeast, predominantly haploid and related to pentose fermenting yeasts, such as Candida shehatae
shehatae [1,4,11–15]. P. stipitis was recently renamed to be Scheffersomyces stipitis [15] and it is natively
[1,4,11–15]. P. stipitis was recently renamed to be Scheffersomyces stipitis [15] and it is natively one of
one of the highest xylose-utilizing and fermenting yeasts. In type culture collections, the P. stipitis
the highest xylose‐utilizing and fermenting yeasts. In type culture collections, the P. stipitis strains
strains are among the best xylose-metabolizing microbes [16]. Under controlled low O2 conditions, P.
are among the best xylose‐metabolizing microbes [16]. Under controlled low O2 conditions, P. stipitis
stipitis is able to consume xylose and produce up to 57 g/L of bioethanol at 30◦ C [4,13,14,17]. Pichia uses
is able to consume xylose and produce up to 57 g/L of bioethanol at 30°C [4,13,14,17]. Pichia uses an
an alternative nuclear genetic code (ANGC) in which CUG encodes for Ser rather than Leu [17], which
alternative nuclear genetic code (ANGC) in which CUG encodes for Ser rather than Leu [17], which
makes the genetic manipulation of Pichia with the commercial drug resistance markers unusually
makes the genetic manipulation of Pichia with the commercial drug resistance markers unusually
problematic because essentially all of these markers are derived from bacteria that use the universal
problematic because essentially all of these markers are derived from bacteria that use the universal
codon system. Moreover, one of classical challenges in fermentation technology is that some of
codon system. Moreover, one of classical challenges in fermentation technology is that some of key
key enzymes of bioethanol production pathway are expressed relatively in low levels [1,4,13,14].
enzymes of bioethanol production pathway are expressed relatively in low levels [1,4,13,14].
Therefore, the metabolic engineering of the bioethanol pathway in yeasts, which can ferment the
Therefore, the metabolic engineering of the bioethanol pathway in yeasts, which can ferment the
sugars of the agriculture biomass with considerable and recoverable bioethanol concentrations, could
sugars of the agriculture biomass with considerable and recoverable bioethanol concentrations, could
enhance the productivity and sustainability of renewable biofuel sources [1,13,14]. To improve
enhance the productivity and sustainability of renewable biofuel sources [1,13,14]. To improve
bioethanol production and xylose metabolism, a stable and manipulatable genetic system that enables
bioethanol production and xylose metabolism, a stable and manipulatable genetic system that
overexpression or deletion of one or more of key enzymes and sugar transporters in xylose-fermenting
enables overexpression or deletion of one or more of key enzymes and sugar transporters in xylose‐
yeast P. stipitis, was developed [1,3,18]. This approach comprises modelling, metabolic and flux
fermenting yeast P. stipitis, was developed [1,3,18]. This approach comprises modelling, metabolic
analysis, quantitative metabolomics and transcriptomics followed by the targeted overexpression or
and flux analysis, quantitative metabolomics and transcriptomics followed by the targeted
deletion of genes of the rate-limiting steps [1,3,4,13,14]. Since there is reasonable information about the
overexpression or deletion of genes of the rate‐limiting steps [1,3,4,13,14]. Since there is reasonable
metabolic capacity of P. stipitis to ferment xylose on various omics levels, this makes it an attractive
information about the metabolic capacity of P. stipitis to ferment xylose on various omics levels, this
model system for metabolic engineering.
makes it an attractive model system for metabolic engineering.
Recently, a new xylose-fermenting yeast Spathaspora passalidarum was discovered, which naturally
Recently, a new xylose‐fermenting yeast Spathaspora passalidarum was discovered, which
co-ferments xylose, glucose, and cellobiose and demonstrates potentials in the effective conversion of
naturally co‐ferments xylose, glucose, and cellobiose and demonstrates potentials in the effective
mixed sugars from hemicellulosic hydrolysates into ethanol [9]. S. passalidarum was initially isolated
conversion of mixed sugars from hemicellulosic hydrolysates into ethanol [9]. S. passalidarum was
from extremely O2 -limited and hemicellulosic sugar rich environments from the gut of a wood-boring
initially isolated from extremely O2‐limited and hemicellulosic sugar rich environments from the gut
beetle (as will be discussed below). Although the anaerobic fermentation of glucose is broadly known,
of a wood‐boring beetle (as will be discussed below). Although the anaerobic fermentation of glucose
the xylose fermentation typically needs a controlled oxygen condition. Uncontrolled oxygen conditions
is broadly known, the xylose fermentation typically needs a controlled oxygen condition.
in another xylose-fermenting yeast, such as P. stipitis, leads to accumulation of xylitol due to insufficient
Uncontrolled oxygen conditions in another xylose‐fermenting yeast, such as P. stipitis, leads to
amounts of NAD+ , and as consequence the xylose metabolism will be blocked (Figure 2) [9,13,14]. To
accumulation of xylitol due to insufficient amounts of NAD+, and as consequence the xylose
solve this problem, precise controlled O2 (very low O2 concentrations) during the xylose fermentation
metabolism will be blocked (Figure 2) [9,13,14]. To solve this problem, precise controlled O2 (very
is required in P. stipitis to generate NAD+ from NADH.
low O2 concentrations) during the xylose fermentation is required in P. stipitis to generate NAD+ from
NADH.
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The bioethanol production from the bioconversion of lignocellulose biomass must be achieved
Therates
at high bioethanol
and yields production from the bioconversion
for economically of lignocellulose
recoverable concentrations. Thebiomass
achieving must be achieved
of such targets
at
forhigh rates bioethanol
efficient and yields production
for economically recoverable
are more difficultconcentrations.
with cellulose and The hemicellulose.
achieving of such Thetargets
major
for efficient
barrier bioethanolutilization
for cellulose production is are more difficult
enzymatic with cellulose
saccharification, while andfor hemicellulose.
hemicelluloseThe major
it is the
barrier for cellulose
utilization of mixedutilization is enzymatic
sugars (hexose sugars: saccharification,
glucose, galactose, while for hemicellulose
mannose, and rhamnose; it is the utilization
and pentose
of mixed
sugars: sugars
xylose and(hexose sugars:
arabinose) glucose,
in the presence galactose,
of ferulicmannose,
and acetic and rhamnose;
acids along with andother
pentose sugars:
byproducts
xylose
of the and arabinose) inpretreatment
thermochemical the presence of of the
ferulic and acetic [13,14].
hydrolysates acids along with other
However, byproductsand
S. passalidarum of the
P.
thermochemical
stipitis yeasts possess pretreatment of the hydrolysates
a set of unique physiological [13,14].
meritsHowever,
that make S. them
passalidarum and P.
very useful stipitis yeasts
biodegradable
possess
organisms a setforof bioconversion
unique physiological merits that make
of lignocellulosic biomass them very useful
[4,9,13,14]. biodegradable
Pichia can utilizeorganisms
and ferment for
bioconversion of lignocellulosic
effectively cellobiose, biomass [4,9,13,14].
glucose, galactose, and mannose Pichia can with
along utilize and high
xylan ferment effectively
oligomeric cellobiose,
sugars xylan
glucose,
and mannan, galactose, and mannose
in addition along withstudied
to its extensively xylan high oligomeric
ability sugarsand
to metabolize xylan and mannan,
ferment the xylose in
[4,13,14].to
addition Theitsprimary
extensively sugar released
studied in enzymatic
ability to metabolizehydrolysis is cellobiose
and ferment the xylose and,[4,13,14].
remarkably, TheP. stipitis
primary
and S.released
sugar passalidarum have the
in enzymatic capability
hydrolysis to utilizeand,
is cellobiose the remarkably, P. stipitis
cellobiose, which and S.
make passalidarum
such yeasts potenthave
organisms for simultaneous saccharification and fermentation (SSF) or hydrolysate, because most
the capability to utilize the cellobiose, which make such yeasts potent organisms for simultaneous
commercially available
saccharification cellulase(SSF)
and fermentation products are oftenbecause
or hydrolysate, deficientmostin commercially
β‐glucosidaseavailable
enzymecellulase
so the
accumulation
products are oftenof cellobiose
deficient in inhibits celluloseenzyme
β-glucosidase activities. Since
so the P. stipitis and
accumulation of S. passalidarum
cellobiose cancellulose
inhibits directly
metabolizeSince
activities. the cellobiose,
P. stipitis they
and S.have the potential
passalidarum cantodirectly
improve SSF processes
metabolize [4,9,10,13,14,19,20].
the cellobiose, they have the
Collectively,
potential to improve theSSFnative ability[4,9,10,13,14,19,20].
processes of P. stipitis and S. passalidarum to metabolize the oligomeric
sugars is of highthe
Collectively, importance as the
native ability of P.mild acidic
stipitis and S.pretreatments
passalidarum to ofmetabolize
agriculturethe waste biomass
oligomeric can
sugars
prevent
is of highthe formationasofthe
importance themild
sugar degradation
acidic pretreatmentsbyproducts, which waste
of agriculture could biomass
inhibit significantly
can prevent the
fermentation
formation process,
of the sugarbut can releasebyproducts,
degradation about 15%–55% whichofcould
soluble oligomeric
inhibit sugars.the
significantly Therefore,
fermentationwith
low cost but
process, andcan highrelease
yield, about
the hemicellulosic
15–55% of solublesugars oligomeric
can be moresugars.
readilyTherefore,
recovered with and underutilized
low cost and
from yield,
high cellulose biomass than sugars
the hemicellulosic glucose. canAlthough such easy
be more readily recoverable
recovered sugars can be
and underutilized from utilized for
cellulose
formation
biomass thanof glucose.
a number of usefulsuch
Although products such as xylitol,
easy recoverable sugarsbutanol, lactic acid,
can be utilized and otherofchemicals,
for formation a number
bioethanol
of is still the
useful products such major product
as xylitol, with the
butanol, largest
lactic acid, potential
and othermarket.
chemicals, Hence, bioethanol
bioethanol production
is still the major
from thewith
product lignocellulosic
the largest biomasses is receiving
potential market. Hence,a lot of attention
bioethanol as a consequence
production of agriculture
from the lignocellulosic
policies and
biomasses energy demands
is receiving to improve
a lot of attention as athe production
consequence of of alternative
agriculture renewable
policies biofuels
and energy and to
demands
reduce
to improveCO2the emissions
production[2,4,13,14].
of alternative renewable biofuels and to reduce CO2 emissions [2,4,13,14].

2. Spathaspora passalidarum
2. Spathaspora passalidarum aa Promising
Promising Genetic
Genetic Source
Source
The
The Spathaspora
Spathasporaclade cladecontains many
contains bioethanol
many producer
bioethanol yeasts, including
producer Spathaspora
yeasts, including arborariae,
Spathaspora
Spathaspora
arborariae, brasiliensis,
SpathasporaSpathaspora gorwiae,
brasiliensis, Spathasporagorwiae,
Spathaspora hagerdaliae, Spathaspora hagerdaliae,
Spathaspora passalidarum, Spathaspora
Spathaspora
roraimanensis, Spathaspora suhii, Spathaspora xylofermentans. They are usually endosymbioticly
passalidarum, Spathaspora roraimanensis, Spathaspora suhii, Spathaspora xylofermentans. They are associated
usually
with wood-boring-beetles
endosymbioticly associated thatwith
occupy rotting wood. Spathaspora
wood‐boring‐beetles passalidarum
that occupy rotting (Figure
wood. 3), the first
Spathaspora
identified
passalidarumspecies of 3),
(Figure genus Spathaspora,
the first identifiedwas isolated
species fromSpathaspora,
of genus the gut of passalid Odontotaenius
beetlefrom
was isolated the gut of
disjunctus [9,21–24]. Notably, S. arborariae, S. gorwiae, S. hagerdaliae, and S. passalidarum
passalid beetle Odontotaenius disjunctus [9,21–24]. Notably, S. arborariae, S. gorwiae, S. hagerdaliae, ferment
and
xylose to produce
S. passalidarum bioethanol,
ferment xylosewhereas
to producethe bioethanol,
rest within the Spathaspora
whereas the restclade
withinare thought
the to be xylitol
Spathaspora clade
producers
are thought[9].
to be xylitol producers [9].

Figure
Figure 3.
3. Spathaspora
Spathaspora passalidarum
passalidarum budding
budding cells
cells with
with characteristic
characteristic curved
curved and
and elongated
elongated ascospore.
Fermentation 2020, 6, 33 5 of 12

S. passalidarum was firstly described in 2006 by Nguyen et al. [21]. The authors speculated that
Spathaspora mainly exists in the beetle’s biosphere rather than the beetle’s gut microbiota, and it may be
only by coincidence that O. disjunctus beetles ingested decaying wood contaminated by yeasts. Later in
2012 and 2017, another 12 strains were described in two independent studies from wood-boring beetles
and wood samples of Amazonian forest in Brazil [23,25]. Among these strains, only one isolate was
obtained from the gut of Popilus marginatus beetle, while the rest of the strains were obtained from the
woody samples inhabited by the beetles [23,25]. In 2014, two more strains were isolated from rotted
wood in China [26]. Additionally, Rodrussamee and colleges in 2018 reported a new thermotolerant
strain, named S. passalidarum CMUWF1–2, which was isolated from Thailand soil [27]. The frequency
of finding S. passalidarum mainly among the woody samples supports the notion that those yeasts are
probably associated with decaying wood niches rather than with the gut microbiota of wood-boring
beetles. However, the fact of the low frequency of finding S. passalidarum among other yeast species
keeps an open possibility that they inhabit mainly the wood-related beetles [9].

3. Fermentation Capability of Spathaspora passalidarum

It is believed that the beetle’s gut is truly anaerobic or microaerobic, therefore it was speculated that
S. passalidarum possess a unique adaptation capability to survive under oxygen-depleted conditions
on mixtures of hemicellulosic sugars in the midgut of wood-boring beetles [10,19]. Currently, S.
passalidarum is among the best xylose-utilizing and fermenting yeasts. Under anaerobic or microaerobic
conditions, S. passalidarum possess rapid utilization and consumption rates for xylose and produces up
to 0.48 g/g bioethanol (near to the maximum theoretical bioethanol production of 0.51 g/g), in contrast to
P. stipitis which can hardly metabolize xylose anaerobically, accumulating xylitol and a very low yield
of bioethanol [10,19,20,28,29]. Under anaerobic conditions, Hou in 2012 showed that S. passalidarum
has a high growth rate with rapid consumption rate of sugars and can ferment xylose into a high yield
of bioethanol with higher production efficiency than P. stipitis [10]. Similarly, Veras and colleges in
2017 showed that under anaerobic conditions, S. passalidarum accumulates 1.5 times more bioethanol
than S. stipitis, while both stains accumulate around 0.44 g/g under O2 limiting conditions [30]. The
previous work by Hou (2012) defined strictly that S. passalidarum can metabolize and ferment xylose
in tightly capped flasks [10]. In contrast to the previous report by Hou (2012) [10], under stringent
O2 limiting conditions, the S. passalidarum was not able virtually to utilize the sugars, indicating that
native wild-type S. passalidarum does not ferment sugars under truly anaerobic conditions [19,20].
Therefore, it is still under debate whether S. passalidarum can ferment xylose truly anaerobically or
whether it requires a controlled microoxygenic condition similar to P. stipitis.
One of the major challenges in fermentation technology is the inability of the majority of known
microbes to co-ferment xylose and glucose, since glucose usually inhibits the metabolization of the
other sugars in lignocellulose hydrolysate, as in the case of P. stipitis [13,14]. Astonishingly, in a recent
study to address the metabolic profiling and fermentation capacity of S. passalidarum, S. passalidarum
was found to co-ferment xylose, cellobiose, and glucose simultaneously with high bioethanol yields
ranging from 0.31 to 0.42 g/g [19,20]. Moreover, an adapted S. passalidarum strain was found to
accumulate up to 39 g/L bioethanol with a 0.37 g/g yield from a lignocellulosic hydrolysate. The
specific production rate of bioethanol on xylose as a carbon source was superior with three times more
than the corresponding rate on glucose, where the flux of glycolytic intermediates was meaningfully
lower on glucose than on xylose and its xylose reductase enzyme had a higher affinity for NADH than
NADPH [19,20]. Thus, the allosteric activation of glycolytic routes associated with the xylose utilization
and the NADH-dependent xylose reductase are most likely the causes for such unique ability of S.
passalidarum to co-ferment mixed sugars [19,23]. Later, such results were confirmed in a metabolic
flux study, where S. passalidarum showed about 1.5–2 times high flux rate in the NADH-dependent
xylose reductase reaction [31], which caused continuous recycling and reduction of xylitol levels. Such
directed high flux rates to glycolytic routes and pentose phosphate pathway was the cause for high
levels of bioethanol production in S. passalidarum [31]. In large scale fed-batch fermentation study, S.
Fermentation 2020, 6, 33 6 of 12

passalidarum was able metabolize around 90% of xylose sugar and all of glucose of sugarcane bagasse
hydrolysate, even so glucose had approximately three-fold higher xylose content; and produced a
high ethanol yield of 0.46 g/g with volumetric productivity of 0.81 g/L/h in contrast to P. stipitis which
produced 0.32 g/g ethanol with productivity of 0.36 g/L/h [32]. In follow up study, S. passalidarum
UFMG-CM-Y473 strain was able to simultaneously utilize and co-ferment about 78% of the released
sugars (xylose, glucose, and cellobiose) of pretreated sugarcane bagasse hydrolysate (delignified and
enzymatically hydrolyzed) to yield up to 0.32 g/g bioethanol with productivity of 0.34 g/L/h without any
nutritional supplementation [33]. Moreover, the new thermotolerant strain, S. passalidarum CMUWF1–2,
was able to co-ferment various sugars (mannose, galactose, xylose, and arabinose) of lignocellulosic
biomass, even in presence of glucose, to accumulate considerable amounts of bioethanol and low
amounts of xylitol at higher temperatures. For example, it was able to accumulate 0.43, 0.40, and
0.20 g/g ethanol per xylose at 30, 37, and 40 ◦ C, respectively [27]. Constant with absence of the glucose
repression effect on the utilization of other sugars, S. passalidarum CMUWF1–2 exhibited a resistance
to 2-deoxy glucose, the nonmetabolizable glucose analog, and tolerance to elevated levels of glucose
(35.0% of w/v) and ethanol (8.0% of v/v) [27]. In contrast, the first discovered S. passalidarum NRRL
Y-27907 strain was sensitive to 2-deoxy glucose, as 2-deoxy glucose suppressed the xylose consumption
under anaerobic conditions. While under aerobic conditions, the 2-deoxy glucose inhibited, only
partially, S. passalidarum NRRL Y-27907 [10]. Therefore, the author speculated that xylose uptake in S.
passalidarum NRRL Y-27907 may take place by different xylose transport systems under aerobic and
anaerobic conditions. Under aerobic conditions, xylose is taken up by means of ATP-dependent/high
affinity xylose-proton symporter and low affinity transporter via facilitated diffusion driven only by
the sugar gradient. While, under anaerobic condition, the yeasts are most likely to use only the low
affinity xylose pump, as the active transport via xylose-proton symporter will deplete the ATP levels.
The inhibitory effect of 2-deoxy glucose on S. passalidarum NRRL Y-27907 can be therefore explained by
(1) blocking of the low-affinity-facilitated diffusion transporters which are occupied with transporting
2-deoxy glucose, and (2) the inhibition xylose active transport due to the depletion of the intracellular
ATP levels to actively phosphorate the 2-deoxy glucose into the non-metabolizable phospho-2-deoxy
glucose [10].

4. Genetic and Physiological Features of Spathaspora passalidarum Emphasis Special Roles for
Xylose Reductase and Xylitol Dehydrogenase
These unusual unique traits of S. passalidarum are very attractive for studying on a molecular level.
The complete genome sequence of xylose-fermenting yeast S. passalidarum was therefore necessary and
it was accomplished and published for first time in 2011 [34]. The comparative genomic, transcriptomic,
and metabolomic analysis between two of the native xylose-fermenting yeasts, the relatively newly
discovered S. passalidarum, and the deeply studied P. stipitis, allowed a better understanding of the
regulatory mechanisms of lignocellulose utilization, and identified the target key genes involved
in xylose metabolism [9,10,19,31,34]. The comparative genomic and phylogenetic analysis clearly
revealed that S. passalidarum is one of the CUG yeast clades, similar to P. stipites [34]. In addition, the
transcriptome analysis indicated upregulation of the genes implicated in transporting carbohydrate
and xylose- and carbohydrate-metabolisms under xylose growth. Several of genes, which are involved
in regulation of redox balance and recycling of NAD(P)H/+ , were upregulated to probably keep
the redox balance during xylose utilization. Additionally, the genes encoding for cellulases and
β-glucosidases were also upregulated, which suggests a positive feedback of xylose on the upstream
genes to activate its own liberation from the higher oligomeric sugars of hemicelluloses by means of
the catalytic activities of cellulases and β-glucosidases [34].
The previously mentioned capabilities of S. passalidarum to co-ferment different sugars and
accumulate high levels of bioethanol with very low concentrations of xylitol, can be explained by the
presence of a set of physiological characters encoded by unique set of genes [10,19,34]. Thus, the high
capacity of xylose fermentation and low levels of xylitol accumulation by S. passalidarum was speculated
Fermentation 2020, 6, 33 7 of 12

to be due to the cofactor’s equilibrium between the intracellular demand and supply of the cofactors
via NADH-favored xylose reductase enzyme and NAD+ -specific xylitol dehydrogenase enzyme, the
key enzymes of the xylose utilization pathway [10,28]. Normally, the xylose metabolization occurs
through the reduction of xylose to xylitol with xylose reductase, which requires NADPH or NADH as
a cofactor with preference for NADPH. Only few NADH-favored xylose reductase enzymes have been
described so far [10,35–37]. Then the xylitol is metabolized further by xylitol dehydrogenase which
is strictly NAD+ dependent (Figure 2). The unbalance between NAD+ supplement and requirement
can block the xylose metabolization and leads to accumulation of xylitol. Later, S. passalidarum
was found to harbor two genes encoding for xylose reductase (SpXYL1.1 and SpXYL1.2) [28]. The
SpXYL1.1 gene product is more equivalent to XYL1 found in other yeasts. The expression levels of
SpXYL1.2 were found to be higher than SpXYL1.1 and bioethanol production in S. passalidarum was
attributed to higher xylose reductase activity with NADH than with NADPH [28]. The SpXYL1.2 was
found to use both NADH and NADPH with preference for NADH, while SpXYL1.1 was stringently
NADPH-dependent. Furthermore, the transformation of S. cerevisiae with SpXYL1.2 of S. passalidarum
enabled the overexpressing S. cerevisiae::SpXYL1.2 strain to grow anaerobically on xylose and to ferment
it to higher ethanol yield than the isogenic S. cerevisiae TMB 3422 strain, which overexpresses P. stipitis
XYL1. While, the S. cerevisiae::SpXYL1.1 overexpressing strain was not able to grow on xylose [28].
Similarly, in the yeast-like fungus Aureobasidium pullulans, the overexpression of SpXYL1.2 xylose
reductase along with S. passalidarum xylitol dehydrogenase encoded by SpXYL2.2 enhanced the xylose
metabolization by 17.76% and improved the fermentation capability and the pullulan production by
97.72% of the overexpressing mutants compared with the parental strain [38].
Finally, a metabolic analysis of S. passalidarum speculated that NADH-preferred xylose reductase
and NAD+ -dependent xylitol dehydrogenase would tend to drive both of xylose assimilation via the
oxidoreductase pathway and the acetaldehyde reduction to ethanol by the alcohol dehydrogenase
enzyme [19]. Recently, a metabolic flux analysis of different xylose-fermenting yeasts confirmed a
better cofactors balance within S. passalidarum cells during xylose catabolism to bioethanol production
than within P. stipitis cells [31], which further supports the growth characteristics of S. passalidarum.
Collectively, those unique and unusual traits of S. passalidarum encourage using it as a source
for genes to improve xylose utilization and bioethanol production from lignocellulosic biomass in
the current xylose fermenting yeasts, such as P. stipites, or to introduce xylose metabolism genes to
develop industrial strains of Saccharomyces cerevisiae capable of co-fermentation of pentose and hexose
sugars. Or alternatively, to domesticate it given the excellent results already accomplished by wild-type
representatives of that species for co-fermentation of mixed sugars. To facilitate that purpose, Li et
al. (2017) developed a stable genetic expression system compatible with the CUG yeasts clade for
genomic integration of Gene Of Interest (GOI) into several yeasts [39]. The developed multi-host
integrative system was functional in several of the xylose-fermenting yeasts including S. passalidarum,
P. stipitis, and Candida jeffriesii and Candida amazonensis, as well as in a hexose metabolizing yeast
Saccharomyces cerevisiae, for heterologous expression of green fluorescent protein (GFP) or lactate
dehydrogenase. For lactate dehydrogenase overexpressing strains, all the engineered yeast strains
were able to metabolize either glucose (in case of S. cerevisiae) or xylose (in case of xylose-fermenting
yeasts) to produce lactate [39].

5. New Adaptive Strains of Spathaspora passalidarum for Potential Industrial Applications

One of the unique features of those xylose metabolizing yeasts, is the ability to use not only
the monomeric hexose and pentose sugars but also the high oligomeric disaccharide sugar in
mixed co-fermentation [9,19], which can be an advantage for large scale industrial applications.
The mild acid pretreatment of agriculture waste biomasses is relatively cheap and prevents the
accumulation of harmful compounds, which inhibits the fermentation processes, but releases the
sugars in higher oligomeric stats. Therefore, the ability of such native xylose fermenting strains, P.
Fermentation 2020, 6, 33 8 of 12

stipitis, and S. passalidarum, to use the high oligomeric sugar can be a great advantage for various
biotechnological applications.
One of the major problems that hinders the use of S. passalidarum for industrial bioethanol
production, even with its remarkable ability for bioethanol production, is the high sensitivity of S.
passalidarum to the chemical inhibitors, such as ferulic and acetic acids, which are released in preparation
of hemicellulosic hydrolysates [9]. Several elaborative studies have focused mainly on improving
the tolerability of S. passalidarum to the hydrolysates inhibitors with keeping in mind the bioethanol
productivity of the strains [19,40–43]. Hou and Yao in (2012) reported a strong strain [40], which is
able to grow on furfurals and many other inhibitors of wheat straw hydrolysate (75%) and able to
accumulate up to 0.40 g/g ethanol. Such strain was generated through hybridization of a S. cerevisiae
and a UV-mutagenized S. passalidarum [40]. In 2012 also, another resistant strain was developed
under O2 limiting conditions through several passage of the wild-type S. passalidarum NRRL Y-27907
on wood hydrolysate, followed by adaptive growth of the strain on corn stover AFEX (ammonia
fiber expansion) hydrolysate [19]. Even with such efforts, the strain was not able to accumulate
significant amounts of ethanol during the fermentation of the AFEX hydrolysate, despite its ability
to grow in AFEX hydrolysate media. When the acetic acid was depleted from AFEX hydrolysate
media, ethanol production was surprisingly observed with a yield of 0.45 g/g and most of the xylose
content was consumed [19]. Later in 2017, Morales and colleagues developed an evolutionary adapted
strain [41] with high tolerance toward the classical inhibitor of the fermentation processes, acetic acid,
and that produces ethanol with a yield of 0.48 g/g. In a non-detoxified hydrolysate of Eucalyptus
globulus, the authors reported also the ability of this strain to co-utilize mixed sugars of xylose, glucose,
and cellobiose under microaerobic conditions [41]. This strain was generated by UV irradiation
followed by successive growing of the strain under elevated acetic acid concentrations [41]. In similar
way, another group also obtained a mutated S. passalidarum strain but via plasma mutagenesis and
continuous cultivation in alkaline liquor pretreated corncob [42]. Under a simultaneous saccharification
and co-fermentation, the obtained strain produced bioethanol with efficiency of 75% [42]. Finally,
Su et al. in 2018 developed an adaptive S. passalidarum strain (named YK208-E11) [43], which is
designated for resistance to AFEX hydrolysate inhibitors, from the wild-type NRRL Y-27907 through
high-throughput screen via combining several approaches of batch adaptation, cell recycling, and cell
mating [43]. The S. passalidarum YK208-E11 strain produced less biomass (about 40% compared to the
wild-type), co-metabolized mixed sugars of xylose, glucose, and cellobiose, and exhibited a three-fold
improvement in the ethanol production rate with a yield of 0.45 g/g. The whole genome sequence of S.
passalidarum YK208-E11 strain revealed a deletion of about 11 kb in this strain. The ORF, which was
deleted in S. passalidarum YK208-E11, is encoding for proteins predicted to be involved in cell division
and respiration. Therefore, the authors speculated that this deletion may account for those unique
adaptive/physiological features of this AFEX-acclimatized S. passalidarum YK208-E11 strain [43].

6. Future Perspective for Engineering New Strains for Better Bioethanol Production
The metabolic engineering approaches involve targeted overexpression and/or deletion of
fermentative key genes that facilities quick and efficient conversion of sugars into bioethanol with
high recoverable yields [1,3]. As we discussed above, S. passalidarum xylose reductase and xylitol
dehydrogenase are among the promising candidates for targeted overexpression. The cumulative
knowledge of the transcriptomics, metabolomics, and comparative genomics studies for P. stipitis and
S. passalidarum, identified other key enzymes controlling the xylose assimilation, rather than XDH
and XR (Figure 2). One of such promising key genes is adh that encodes for fermentative isozyme
alcohol dehydrogenase (ADH), which is vital for production and/or assimilation of ethanol (Figure 2).
Generally, ADH catalyzes the final (rate limiting) step in the yeast glycolytic pathway, the reduction of
acetaldehyde to ethanol and NAD+ , and therefore it accepts NADH as a co-factor [44,45]. However,
ADH enzymes are also able to perform the reverse reaction from ethanol to acetaldehyde, enabling the
yeasts to oxidize and grow on ethanol as a carbon source. In P. stipitis, the ADH fermentative activities
Fermentation 2020, 6, 33 9 of 12

is crucial not only for ethanol production and/or consumption but also for maintenance redox balance
within the yeast cell, so it is considered to be a part of the cofactor balance system in P. stipitis [44].
The sequencing projects of S. passalidarum NRRL Y-27907 and P. stipitis CBS6054 (JGI-MycoCosm)
revealed the presence of several/different alcohol dehydrogenase (ADH) encoding genes. For example,
in P. stipitis, sevem genes were predicted to encode for alcohol dehydrogenase (PsADH1 to PsADH7)
enzymes [13,17]. Among them PsADH1 and PsADH2 were found to be essential for xylose assimilation
and ethanol production [44,46]. Each of the ADH proteins in S. passalidarum and P. stipitis are supposed
to have different kinetic properties. Some of the enzymes could be mainly responsible for producing
ethanol while others might be responsible for oxidizing it. In S. passalidarum, the gene encoding
for SpADH1 was found to be expressed at a very high level during xylose metabolization [34]. In
addition, metabolic analysis and metabolic flux analysis revealed that alcohol dehydrogenase is one
of key enzymes driving ethanol production in S. passalidarum [19,31]. Notably, owing to relative
SpADH1 abundance, the SpADH1 promotor was used to develop a multi-host integrative system for
xylose-fermenting yeast [39].
While in P. stipitis, the function of some ADH enzymes are better understood, in particular PsADH1
and PsADH2 [13,17,44,46–49]. Transcriptomic studies of the P. stipites adh system indicated that the
PsADH activities are correlated with and induced under O2 limited/microaerobic conditions [46,48].
Under xylose fermentation, the PsADH1 was found to be the primary key enzyme among the PsADH
system. The deletion of PsADH1 caused a reduction in P. stipites growth rate and a notable increase in
xylitol accumulation accompanied with a dramatic decrease in ethanol production, due to intracellular
cofactors imbalance [44]. The PsADH2 is not expressed under microaerobic or aerobic conditions
unless PsADH1 is deleted [44,46], which further confirms that the significant role of PsADH1 is in sugar
assimilation and ethanol production. The levels of PsADH1 and PsADH2 transcripts were observed,
however, to be low through xylose metabolism relative to the transcript levels of other fermentative
and glycolytic enzymes [13,17]. In addition, PsADH1 and PsADH2 were able to complement the
growth of the S. cerevisiae ∆adh mutant on ethanol as a sole carbon source [47]. Moreover, PsADH3
to PsADH7 were speculated to keep the balance between the cofactors NADPH and NADH [17].
However, the expression patterns of the other PsADHs on xylose and glucose under microaerobic
conditions, in particular, for PsADH7 and PsADH4 are not fully understood [13]. PsADH5 was
found in proximity to NADPH dehydrogenase, implying a function in maintenance the intracellular
cofactors balance, however, it is not proven yet. Notably, PsADH7 was found to be upregulated under
aerobic growth on xylose [50]. PsADH7 was described as a strictly NADP(H) dependant enzyme with
broad spectrum for substrates-specificity, including variety of aromatic and linear aldehydes (e.g.,
acetaldehyde, butanal, propanal, and furfural) and alcohols (e.g., ethanol, butanol, pentanol, hexanol,
and octanol) for forward and reverse reactions, respectively [50]. Surprisingly, PsADH7 was able to
utilize xylitol as a substrate too with moderate activity. In the same context, the overexpression of
PsADH7 into a P. stipites xylitol dehydrogenase mutant (∆PsXDH) [18], which cannot metabolize xylitol
and therefore cannot grow on xylose as a sole carbon source, was able exclusively to complement
the growth of ∆PsXDH on xylose, in contrast to PsADH1, 2, 4, and 5 [50]. Hence, there is a need to
understand the kinetic characteristics of each of PsADH and SpADH enzymes in order to target the
correct genes for overexpression and/or deletion. Finally, we would like to state that genes encoding
for adh isozymes are worth studying, especially of S. passalidarum, owning to their significant functions
in bioethanol production/consumption and/or intracellular cofactor balance.

7. Conclusions
Taken together, the advances in fermentation performance by S. passalidarum pave the way for
engineering the conventional and the nonconventional fermenting yeasts, such as S. cerevisiae and
P. stipites, for economical fermentation of hexose and pentose sugars in hemicellulosic hydrolysates
on industrial scales. Keeping in mind that the efficient metabolization and fermentation of xylose
is essential for the bioconversion of lignocellulosic biomasses into biofuels and chemicals, but the
Fermentation 2020, 6, 33 10 of 12

conventional wildtype strains like S. cerevisiae cannot use the xylose. Therefore, researchers keep
trying to engineer the xylose utilization pathway into the conventional yeast. The genomes of the
natural xylose-fermenting yeasts, in particular of P. stipitis and S. passalidarum, are of huge importance,
as their genomics features and regulatory patterns can serve as guides and genomic resources for
further genetic engineering development in those native xylose-metabolizing yeasts or to engineer
non-xylose fermenting yeasts. Therefore, S. passalidarum and P. stipitis can be considered as genomic
treasure sources for various genes to engineer the xylose metabolism and to improve the bioethanol
production [1,24,34].

Author Contributions: K.A.S. undertook extensive literature review and wrote the manuscript. S.M.E. and
A.I.E.-D. supervised the research. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: The authors would like to thank Thomas Jeffries (University of Wisconsin Madison), Laura
Willis (Forest Products Laboratory, USDA), Amal rabeá, Ali Selim, and Katerina Peros for the invaluable and
unlimited support to complete this project. K.A.S. received the Parwon scholarship at Institute for Microbial and
Biochemical Technology, FPL, USDA.
Conflicts of Interest: The authors declare no conflict of interest.

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