Applied Microbiology and Biotechnology (2019) 103:5143–5160

https://doi.org/10.1007/s00253-019-09881-1

MINI-REVIEW

Biosynthetic strategies to produce xylitol: an economical venture

Yirong Xu 1 & Ping Chi 1 & Muhammad Bilal 2 & Hairong Cheng 1

Received: 10 January 2019 / Revised: 26 April 2019 / Accepted: 29 April 2019 / Published online: 17 May 2019
# Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract
Xylitol is a natural five-carbon sugar alcohol with potential for use in food and pharmaceutical industries owing to its insulin-
independent metabolic regulation, tooth rehardening, anti-carcinogenic, and anti-inflammatory, as well as osteoporosis and ear
infections preventing activities. Chemical and biosynthetic routes using D-xylose, glucose, or biomass hydrolysate as raw
materials can produce xylitol. Among these methods, microbial production of xylitol has received significant attention due to
its wide substrate availability, easy to operate, and eco-friendly nature, in contrast with high-energy consuming and
environmental-polluting chemical method. Though great advances have been made in recent years for the biosynthesis of xylitol
from xylose, glucose, and biomass hydrolysate, and the yield and productivity of xylitol are substantially improved by metabolic
engineering and optimizing key metabolic pathway parameters, it is still far away from industrial-scale biosynthesis of xylitol. In
contrary, the chemical synthesis of xylitol from xylose remains the dominant route. Economic and highly efficient xylitol
biosynthetic strategies from an abundantly available raw material (i.e., glucose) by engineered microorganisms are on the hard
way to forwarding. However, synthetic biology appears as a novel and promising approach to develop a super yeast strain for
industrial production of xylitol from glucose. After a brief overview of chemical-based xylitol production, we critically analyzed
and comprehensively summarized the major metabolic strategies used for the enhanced biosynthesis of xylitol in this review.
Towards the end, the study is wrapped up with current challenges, concluding remarks, and future prospects for designing an
industrial yeast strain for xylitol biosynthesis from glucose.

Keywords Xylitol . Yarrowia lipolytica . Biosynthetic routes . Metabolic engineering . Synthetic biology

Introduction abandoned plant fermentations (Werpy et al. 2004; Brar et al.

Xylitol is a naturally occurring five-carbon sugar alcohol hav-
ing a similar sweetness as regular sugar, but 40% less caloric.
It is one of the BTop Value-Added^ chemicals from biomass
produced by Department of Energy (DOE), which can utilize
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00253-019-09881-1) contains supplementary
material, which is available to authorized users.
* Muhammad Bilal
bilaluaf@hotmail.com
* Hairong Cheng
chrqrq@sjtu.edu.cn
1
State Key Laboratory of Microbial Metabolism, School of Life
Sciences and Biotechnology, Shanghai Jiao Tong University,
Shanghai 200240, China
2
School of Life Science and Food Engineering, Huaiyin Institute of
Technology, Huaian 223003, China
2016). It has been known to organic chemistry at least since
the 1890s. French and German researchers who succeeded in
isolating the molecule in the 1930s originally discovered xy-
litol. Scientists obviously did not realize the biologic proper-
ties of xylitol until research started to exploit its insulin-
independent nature after World War II. In 1975, the Finnish
company Finnish Sugar Co. Ltd. commenced producing xyli-
tol on an industrial scale. Several unique properties such as
protection from cavities, tooth decay, and demineralization of
enamel render xylitol an interesting candidate for food and
pharmaceutical industries. Xylitol is perfect for diabetic pa-
tients as a good sucrose substitute because of its insulin-
independent metabolic regulation (Mussatto and Roberto
2003; Albuquerque et al. 2014). It is used for parenteral nu-
trition (PN) in infusion therapy due to its inertness to amino
acids. This alternative sweetener can limit obesity, when used
regularly in the diet (Parajó et al. 1998a, b), and can also
promote tooth rehardening and remineralization to prevent
dental caries when used in chewing gums, toothpaste, and
5144
mouthwashes as anticariogenic products (Franceschin et al.
2011; Mussatto and Roberto 2003). Xylitol possesses mois-
ture retention properties and hence is used in toothpaste.
Further, it prevents osteoporosis, ear infections, and in-
flammation in children (Albuquerque et al. 2014). In com-
bination with sugar alcohols and amino acids, xylitol has
been used as a stabilizing agent for proteins during their
extraction from natural membranes, thus avoiding dena-
turation. At 80% weight/volume, it can be used as a sta-
bilizer for fibrinogen pastes for human plasma (Parajó
et al. 1998a). Xylitol has almost equal sweetening power
to sucrose, but with low caloric content (2.4 cal/g), thus is
used as an alternative to sucrose (Mohamad et al. 2015).
Since the 1960s, it has been increasingly used as an alter-
native low-energy sweetener and food additive in the food
industries (Nigam and Singh 1995; Mohamad et al. 2015).
Importantly, xylitol does not undergo detrimental Millard
reaction, so efficiently preserving the nutritional signifi-
cance of food proteins during thermal processing
(Soleimani et al. 2006; Mussatto and Roberto 2003).
Due to the high endothermic heat of solution (34.8 cal/
g), xylitol causes a pleasant cooling effect and fresh sen-
sation in oral and nasal cavities after consumption; there-
fore, it is widely used as a xylitol-coated pharmaceutical,
confectionery products, and dietary complement prepara-
tions. The products’ flexibility and texture improving
properties along with a flavor (Mussatto and Roberto
2003) enable xylitol application (used solely or in combi-
nation with other sugar substitutes) in food industries in
the manufacturing of confectionery products.
Despite a large number of xylitol advantages, it still has a
laxative effect, which could cause diarrhea and gas, if
consumed at high doses (Silveira and Jonas 2002;
Akinterinwa et al. 2008). In the case of extremely high intra-
venous doses, it leads to high uric acid levels in the blood and
alters the liver function tests (LFTs) resulting in kidney stones
and liver stress. Therefore, the expected daily consumption
level is ≤ 15 g/day as recommended by the Food and Drug
Administration (FDA) in the USA (US FDA 2008). Like other
sugar alcohols, D-mannitol and D-sorbitol (D-glucitol) are
popular, which also have wide applications in the food and
pharmaceutical industries.
Naturally, xylitol is found in small amounts in plant fruits,
microorganisms, animal tissues, and the human body
(Washüttl et al. 1973; Pepper and Olinger 1988; Mohamad
et al. 2015). Large-scale xylitol is mainly produced chemically
by reducing D-xylose via a catalytic chemical reaction at high
pressure and temperature conditions, which is expensive,
complicated, time-laborious, and environmentally unfriendly
(Albuquerque et al. 2014). Considering the huge commercial
demand of xylitol bioproduction, researchers have devoted
widespread attention to microbiological methods that con-
sume less energy and cost during the process and gain much
Appl Microbiol Biotechnol (2019) 103:5143–5160
progress (Nigam and Singh 1995; Converti et al. 1999).
Current manufacture strategy mainly employs chemical
methods to produce xylitol from xylose purified from biomass
hydrolysate. In order to make biosynthesis method available
for industrial production, extensive studies are needed to im-
prove the yield of xylitol from a cheaply available material
such as glucose. Regulation of tricarboxylic acid (TCA) cycle
and pentose phosphate pathway or modifying metabolic path-
ways, including knockout and expression of critical genes in
the artificial glucose-to-xylitol pathway, is highly important to
industrialize this method. Identification of novel strains with
tolerance to high concentrations of carbon sources (such as
glucose) and the high glucose flux ratio into the pentose phos-
phate pathway could improve the productivity and yield of
xylitol from glucose. In this review, we summarized the recent
progress in xylitol biosynthesis strategies, ongoing challenges,
and future prospects. After a brief overview of chemical-based
xylitol production, we described the diversity of microorgan-
isms employed for microbial biosynthesis of xylitol with the
main emphasis on molecular approaches applied at the
cellular/molecular level for improving its yield and productiv-
ity. In addition, current challenges, concluding remarks, and
future prospects for designing an industrial strain for xylitol
biosynthesis from glucose are also discussed.
Chemical synthesis of xylitol production
In the 1970s, the commercial-scale chemical process for xyli-
tol production was developed in Finland by acid hydrolysis
reduction of xylan-containing lignocellulosics from
hemicellulosic vegetal materials (such as birch-wood,
corncob, and other rich flora) followed by nickel catalyst hy-
drogenation of D-xylose at 80–140 °C and 8–10 MPa hydro-
gen pressure (Granström et al. 2007a, b; Wang et al. 2016, b)
(Fig. 1, S1). The high temperature, high pressure, expensive
metals, and the use of a toxic catalyst make chemical method
uneconomical, environmentally unsafe, and energy-intensive
production process (Sohyun et al. 2010). Additionally, the
downstream process also relies on various expensive and un-
competitive chromatographic or recrystallization approaches
to separate and purify D-xylose from hemicellulose-xylan hy-
drolysates to remove these by-products such as color, proteins,
metal ions, and other sugars (Silva et al. 1998). In this context,
researchers have rekindled their interest in bio-based xylitol
biosynthesis due to its obvious environmentally friendly and
easy-to-operate advantages.
Biosynthetic methods of xylitol production
In view of serious contamination and safety concerns by the
chemical reduction of xylose, researchers are encouraged to
utilize alternative methods to produce xylitol by microorgan-
isms such as yeast, bacteria, fungus, and molds. These
Appl Microbiol Biotechnol (2019) 103:5143–5160
Fig. 1 Chemical and
biotechnological routes for the
conversion of xylan-containing
lignocellulosics to xylitol
production
bioconversion methods are energy-efficient and ecologically
justified, thus demonstrating a great socioeconomic potential
(Rodrigues et al. 2011). Hallborn et al. (1991) reported the
conversion of xylose to xylitol by an engineered
Saccharomyces cerevisiae with a gene encoding the xylose
reductase (XR, EC 1.1.1.307) of Pichia stipitis CBS 6054
(Hallborn et al. 1991). The recombinant S. cerevisiae strain
transformed over 95% of xylose into xylitol, yield much
higher than the natural xylose-utilizing yeasts. In addition,
Saccharomyces, Candida, Debaryomyces, Pichia,
Torulopsis, Kloeckera, Hansenula, Trichosporon,
Cryptococcus, Rhodotorula, Monilia, Kluyveromyces,
E n t e ro b a c t e r, P a c h y s o l e n , l i q u e f a c i e n s , a n d
Corynebacterium spp. can also produce xylitol (Hong et al.
2014a, b). Depending on a great variety of substrates, several
different strategies have been developed and attempted for the
xylitol biosynthesis.
Bioconversion of xylose to xylitol
Metabolic pathway of xylose
Xylitol can be produced from D-xylose by fermentation with
yeast and fungus. XR (EC 1.1.1.9) and xylitol dehydrogenase
(XDH, EC 1.1.1.9) are the two key enzymes involved in this
process. In yeast, filamentous fungi, and other eukaryotes, the
reduction of D-xylose to xylitol by XR (XYL1, Xyl1p) is the
first step in D-xylose metabolism. After reduction, the xylitol
5145
is oxidized to xylulose by XDH (XYL2, Xyl2p) and incorpo-
rated xylulose-5-phosphate by xylulokinase (XK, EC
2.7.1.17) (XylB in E. coli, Xyl3 in P. stipitis) into the pentose
phosphate metabolic pathway to ethanol and glycerol
(Aranda-Barradas et al. 2000) (Fig. 2). In bacteria, xylose is
first isomerized to xylulose by xylose isomerase (XI, EC
5.3.1.5) and then reduced to xylulose-5-phosphate by XK into
the pentose phosphate pathway (Nigam and Singh 1995).
Xylose can also be metabolized to D-xylonate, a versatile
platform chemical with numerous applications (Toivari et al.
2012), such as its use as a precursor for 1,2,4-butanetriol, co-
polyamides, polyesters, and hydrogel synthesis in the
manufacturing of biomass-derived plastics (Niu et al. 2003;
Toivari et al. 2012). D-Xylose is metabolized to D-xylonate
using NAD(P)+ or pyrroloquinoline quinol (PQQ)-dependent
xylose dehydrogenases (EC 1.1.1.175) or glucose oxidase
(EC 1.1.3.4) (Hardy et al. 1993; Galar and Boiardi 1995). In
some bacteria and archaea, xylose is initially oxidized to D-
xylonolactone with cytoplasmic NAD(P)+-dependent D-
xylose dehydrogenases (Johnsen and Schönheit 2004;
Stephens et al. 2007; Johnsen et al. 2009) and then cleaved
to D-xylonate by lactonase in the cytoplasm. The resulting D-
xylonate is dehydrated to produce 2-keto-3-deoxy-pentanoate,
which is subsequently cleaved by an aldolase or dehydrated
an d r edu ce d to α- k e t o g l u t a r a t e t o p y r u v a t e a n d
glycolaldehyde. Yeast and other fungi also produce D-
xylonic acid by D-xylose dehydrogenase (Kiessling et al.
1962; Suzuki and Onishi 1973; Kanauchi and Bamforth
5146
Fig. 2 Metabolic pathway for xylose utilization; microbial xylitol is produced from xylose via either of two pathways: xylitol is directly converted to
xylitol by NAD(P)H-XR, or it is first isomerized to xylulose by XI and then reduced to xylitol by NADH-XDH
2012) or D-glucose oxidase (Chun et al. 2006; Pezzotti and
Therisod 2006) (Fig. 2). These enzymes require pyridine nu-
cleotide co-factors, i.e., NAD+/NADH or NADP+/NADPH.
The nicotinamide co-factor of XR is a dual dependent
(NADPH and NADH) in some yeasts, and the activity of
NADPH-XR is the highest, whereas XDH is a predominantly
NAD+-dependent enzyme (Rizzi et al. 1989).
Bioconversion of xylose to xylitol by yeast
Yeasts are preferred for xylitol fermentation primarily due to
their high pentose assimilation rates, xylitol productivity, and
stable expression levels of XR and XDH. Xylitol production
by both the wild-type and engineered yeasts have been exten-
sively studied and reported in the literature. Microbial xylitol
is produced from xylose via two steps: xylose is directly con-
verted into xylitol by NAD(P)H-XR, and the resultant xylitol
is then dephosphorylated by NAD+ or NADP+-dependent
XDH. In some strains such as C. tropicalis (Ko et al. 2006),
S. cerevisiae (Kim et al. 1999), and P. stipitis (Kötter et al.
1990), xylitol is accumulated in the xylose-assimilation path-
way. Candida sp. is known to be a potential source of xylitol
production from xylose as a carbon source. It is demonstrated
that xylitol dehydrogenase-deficient mutant of C. tropicalis
produced the xylitol yields of 0.97 g/g with a productivity of
more than 3 g/L/h in shaking flask cultures (Ko et al. 2006).
The NADH-dependent XR and xylitol dehydrogenase of
Candida sp. are usually cloned for heterologous expression.
Appl Microbiol Biotechnol (2019) 103:5143–5160
Certain Candida strains exhibit dual co-enzyme specificity to
utilize phosphorylated as well as unphosphorylated NAD+ as
co-factors. This is particularly advantageous because NADH+
pool is more prevalent in the cell with higher stability as com-
pared to its phosphorylated form (Banta et al. 2002). P. stipitis
is also reported for xylitol biosynthesis; however, it is more
studied for ethanol fermentation due to the presence of active
genes in a pathway downstream to xylitol. Kluyveromyces
marxianus is known to carry out high-temperature fermenta-
tion of both pentoses and hexoses due to its natural xylose
assimilation ability (De Albuquerque et al. 2015).
S. cerevisiae naturally uses glucose rather than xylose as a
substrate and utilizes xylose only after the depletion of glu-
cose. It takes up and assimilates xylose by the hexose trans-
porter system, which has much higher affinity to glucose than
xylose. In this scenario, genetic engineering is a promising
approach to develop xylose-fermenting S. cerevisiae to over-
come the abovementioned drawback (Bae et al. 2004).
Xylose reductase is the key enzyme involved in single-step
reduction of xylose to xylitol. However, the high-level accu-
mulation of xylitol in broth may be hampered due to low
expression levels of the enzyme. Several engineering ap-
proaches have been applied to improve native XR expression
levels in the parent strain. XR genes from Candida and Pichia
were integrated into S. cerevisiae and expressed under the
control of constitutive promoters. The high xylitol yield of
0.9–1.0 g/g with improved xylose assimilation rates in the
recombinant yeasts was attributed to elevated levels of XR
Appl Microbiol Biotechnol (2019) 103:5143–5160
activities (Bae et al. 2004). Similarly, S. cerevisiae overex-
pressing endogenous aldose reductase gene presented im-
proved xylitol titer (22.4 g/L) and volumetric productivity
(0.28 g/L/h) than the wild-type strain, but the values were
lower in contrast to heterologous XRs introduced from
Candida or Pichia (Kogje and Ghoshalkar 2016).
During the yeast metabolic process, ethanol and glycer-
ol production regenerate NAD+ for xylitol oxidation by
XDH, whereas acetate production regenerates NADPH
for xylose reduction by XR (Mohamad et al. 2015).
Several factors including pH, temperature, agitation rate,
inoculum percentage, inoculum age, oxygen level, oxygen
transfer rate, and initial xylose concentration can affect the
biosynthesis of xylitol (Pal et al. 2016). The regeneration
of co-factors is highly dependent on oxygen level and ox-
ygen transfer rates (Furlan et al. 1994). Under oxygen-
deficient conditions, the imbalanced supply of NAD+ in-
hibits the activity of XDH leading to high xylitol accumu-
lation. Under high aeration conditions, NADH is oxidized
to NAD + ; some of the xylitol may be metabolized to
xylulose due to the high NAD+/NADH ratio, thus resulting
in a lesser accumulation of xylitol. Moreover, redox imbal-
ance between NADPH-XR and NAD-XDH in the cell is
one of the main reasons for xylitol accumulation (Kötter
et al. 1990). An oxidative phase of pentose phosphate path-
way (PPP) has been found the most potent NADPH sup-
plier by two key enzymes G6PD and 6-PGD.
Overexpression of these enzymes accompanied by an ex-
ternal secondary substrate addition was used towards
in vivo co-factor regeneration (Uppada et al. 2014). High
expression of endogenous G6PD increased NADPH sup-
ply in recombinant Saccharomyces BJ3505, resulting in
xylitol productivity improvement from 1.6 to 2.0 g/L/h in
glucose-deficient fed-batch fermentations (Kwon et al.
2006). Downregulation of phosphoglucoisomerase (PGI)
gene further increased the glucose flux towards PPP for
NADPH regeneration in S. cerevisiae. The recombinant
strain with simultaneous overexpression of G6PD and re-
duction in PGI activity led to a 1.9-fold increase in specific
xylitol productivity (Oh et al. 2007). S. cerevisiae L2612
transformed with Pichia XR, cellodextrin transporter, and
β-glucosidase gene from N. crassa displayed 85.7% in-
crease in xylitol production from cellobiose and xylose
mixtures in comparison with the glucose and xylose (Zha
et al. 2013a, b). Recently, glycerol has been reported to
enhance the NADPH supply. Disruption of glycerol kinase
and co-expression of P. stipitis glycerol dehydrogenase
genes in C. tropicalis BSXDH-3 under the control of
GAPDH promoter enabled the plentiful supply of
NADPH co-factor and enhanced xylitol productivity
(Ahmad et al. 2013). Galactose can also facilitate
NADPH generation via the Leloir pathway coupled to
PPP without integrating any additional gene (Govinden
5147
et al. 2001). Therefore, the creation of a suitable expression
cassette comprising XR and G6PD gene under the control
of GAL promoter could effectively improve the xylitol titer
by galactose induction.
The knocking out or silence xylitol dehydrogenase gene
(xyl2) to hinder the metabolism of xylitol and cloning of xy-
lose reductase (xyl1) in microbial strains is an important mo-
lecular strategy in improving the final yield of xylitol
(Bruinenberg et al. 1983). Xyl2 genes of C. tropicalis were
disrupted to xyl2-disrupted mutant and then xylitol fermenta-
tion was carried out. By optimization of the process parame-
ters and screening suitable co-substrates, the productivity and
yield of xylitol fermentation by the xyl2-disrupted mutant
were significantly enhanced (Ko et al. 2006). Hong et al.
(2014a, b) knocked out xylitol dehydrogenase gene (xyl2) to
overexpress xylose reductase gene (xyl1) or silenced transfor-
mation of D-xylulokinase gene (XSK or xyl3) to reduce the
expression of the XSK gene at a transcriptional level in
Trichoderma reesei strains, which improved xylitol produc-
tion (Hong et al. 2014a, b). Nevertheless, the xylitol produc-
tion might be limited due to insufficient co-factor (NADPH)
regeneration or transport of xylose (Pal et al. 2013). Likewise,
chemical mutagenesis in Candida varieties induced XDH
point mutation resulting in functional loss of the active en-
zyme with xylitol yield improvement by ∼ 1.2-fold (Kumar
et al. 2010). An XDH-disrupted mutant of K. marxianus ac-
cumulated improved xylitol concentration compared to its
wild-type strain in the presence of glycerol as a co-substrate.
However, xylitol titers of the mutants were significantly lower
than the recombinant Candida spp. (Zhang et al. 2014, b).
Another factor affecting xylose assimilation is the presence
of other carbon sources (mainly glucose), which repressed the
uptake capacity of enzymes to pentoses (Meinander and
Hahnhägerdal 1997). Repeated batch or fed-batch fermenta-
tion-based processes are easier and time-saving with cleaning,
sterilization, and inoculation and had advantages of maintain-
ing glucose at the desired level (Bae et al. 2004). However,
Tochampa et al. (2005) developed a xylitol production model
by the yeast C. mogii using a genetic algorithm to predict the
product formation rate and yield. In xylose fermentation by
the yeast C. mogii ATCC18364, biomass yield and volumetric
productivity were substantially enhanced by an initial glucose/
xylose ratio of 10% as compared to xylose as the sole carbon
source. Wannawilai et al. (2017) reported that furfural and
glucose could enhance the conversion of xylose into xylitol
by C. magnoliae TISTR 5663. Fig. S2 represents the biosyn-
thetic route of xylitol in Yarrowia lipolytica yeast.
Bioconversion of hemicellulose to xylitol by yeast
Lignocellulosic biomass is an abundant and readily available
material in some plants, such as corn cobs (Rivas et al. 2010),
barley bran (Cruz et al. 2000), rice straw (Roberto et al. 1994),
5148
aspen wood hemicellulosic hydrolysate (Pandey et al. 2000),
hybrid poplar wood chips (Dien et al. 1997), corn fiber, and
sugarcane bagasse (Rao et al. 2006). Hemicellulose is a major
component of lignocellulose and is the second most common
polysaccharide in nature (Nigam and Singh 1995).
Hemicelluloses are short-branched chain
heteropolysaccharides with mixed hexosans and pentosans,
especially xylan and arabinan, and are easily hydrolyzed
(Rao et al. 2006). In order to reduce the xylitol production
cost, crude hemicellulose can be used as feedstock for xylitol
biosynthesis as a potential alternative to pure xylose (Rao et al.
2006).
After the hydrolysis of plant hemicellulose by dilute sulfu-
ric acid, the resulting hydrolysates contain the presence of
degradation products with inhibitor components, including
acetic acid, inorganic salt, sugar, hydroxybenzaldehyde
(HBA), syringaldehyde (SGA), vanillin, and
hydroxymethylfurfural (HMF), which have significant influ-
ence on yeast growth and fermentation performance
(Winkelhausen and Kuzmanova 1998; Tsao et al. 2002; Wei
et al. 2010). Therefore, these hydrolysates should be
pretreated to adsorb inhibiting compounds before the micro-
bial cultivation. The bench-scale process includes the follow-
ing pretreatment methods such as activated carbon treatment
(Carvalho et al. 2010), CaO treatment (Silva et al. 1998), ion-
exchange resins (Palmqvist and Hahnhägerdal 2000),
Ca(OH) 2 (Dien et al. 2000), intracellular acidification
(Lohmeier-Vogel et al. 1998), organic solvents (Morita and
Silva 2000; Palmqvist and Hahnhägerdal 2000), yeast strain
variation (Mancilha and Karim 2010), laccase enzyme
(Cassland and Jönsson 1999), recombinant strains (Dien
et al. 1997), overliming (Morita and Silva 2000; Wei et al.
2010), and continuous-effect membrane distillation.
Membrane distillation is a separation process based on evap-
oration through the pores of the hydrophobic membrane. It is
widely to concentrate non-volatile sugars and to eliminate
volatile inhibitors from hydrolysates. For example, Chen
et al. (2013) reported that vacuum membrane distillation re-
moved 98% of furfural and 25% of acetic acid from a ligno-
cellulosic hydrolysate resulting in ethanol yield improvement
by 17.8%. The recovery rate of glucose from the sugar-acid
mixture was greater than 99.12% and the sulfuric acid was
completely removed from the hydrolysate using a
continuous-effect membrane distillation process (Li et al.
2019). Similarly, an aqueous solution of sulfuric acid was also
concentrated by membrane distillation for recycling purposes
(Liu et al. 2016). In conclusion, the continuous-effect mem-
brane distillation treatment can be effectively applied for sul-
furic acid recovery and sugar concentration, so it might im-
prove the economic advantage for the bioconversion of hemi-
cellulose to the xylitol production process. As shown in Fig. 1,
the inhibitor contents can be minimized through detoxification
by pH alteration (Wei et al. 2010).
Appl Microbiol Biotechnol (2019) 103:5143–5160
If the employed yeast strain has good tolerance to toxic
substances, it is not necessary to remove from the hydrolysates
(Peng et al. 2012). Zhang et al. demonstrated that Issatchenkia
occidentalis (Lj-3, CCTCC M 2006097) and I. orienalis (S-7,
CCTCC M 2006098) strains display good activity in
degrading toxic substances, which significantly improves the
xylitol productivity and yield (Zhang et al. 2009b). It effec-
tively reduces the burden of physical and chemical purifica-
tion or detoxification processes. Fonseca et al. used the
potential of the yeast I. occidentalis CCTCC M 206097 as a
detoxification agent of the hemicellulosic hydrolysate to
remove inhibitor compounds (Fonseca et al. 2011). The yeast
can efficiently metabolize furans and phenolic compounds in
hemicellulosic hydrolysates of biomass (Fonseca et al. 2011).
After nutritional supplementation, the detoxified hydrolysates
were used as the source of xylose for xylitol production.
In China, approximately 50,000–80,000 tons of waste xy-
lose mother liquor (WXML) is produced every year that has
become a substantial industrial concern. Regarding composi-
tion, WXML contains about 60–75% total sugars and only
xylose accounts for 50–70% (Cheng et al. 2011). However,
WXML is challenging to recycle due to various sugars and
high content of inhibitors. Zhang et al. (2012) produced 100 g/
L of xylitol from detoxified horticultural waste hemicellulosic
hydrolysates by a new isolate of Candida athensensis SB18.
The furan compounds furfural and 5-hydroxymethylfurfural
(HMF) released from dilute acid hydrolysis were toxic to mi-
croorganisms for the subsequent biotransformation. There are
two biological detoxification approaches to eliminate such by-
product sugars (Fig. 3). The first scheme implicates one
Bperfect^ strain to transform xylose to xylitol and consume
all of the by-product sugars under the stress of inhibitors.
Another is to employ Bcomplementary^ strains, one of which
could either transform xylose to xylitol or consume by-
product sugars or detoxify the inhibitors. Among these routes,
the first scheme seems very simple in terms of processing, but
it is sometimes quite problematic to construct a Bperfect^
strain. In an earlier study, we proposed a technical route in
which biodetoxification, biotransformation, and purification
were integrated using Candida maltosa and recombinant
Bacillus subtilis with a disrupted xylose isomerase gene
xylA. An elevated titer of 213 g/L xylitol was obtained in
medium containing 250 g/L of xylose from WXML in the
lab-scale fermenter (Cheng et al. 2011). Nevertheless, this
technology needs further improvements due to its complicated
operation and large equipment investment. Recently, our
group developed a one-pot biotransformation procedure to
produce xylitol from WXML with C. tropicalis X828 and
B. subtilis Bs12, including detoxification, biotransformation,
and purification. A high level of pure xylitol (95 g/L) was
achieved from the fermentation medium containing 400 g/L
of WXML at a yield of 0.75 g/g xylose with the simultaneous
depletion of by-products including sugars glucose, L-
Appl Microbiol Biotechnol (2019) 103:5143–5160
Fig. 3 Scheme of biotransformation by one perfect strain (a) and
complementary strains (b). S1—main substrate xylose; S2—by-product
sugars; S3—inhibitors; P—product; I and II—complementary strains
arabinose, and galactose (Fig. 4) (Wang et al. 2016, b). The
newly developed one-pot biotransformation process not only
enhances the application of WXML to produce value-added
chemicals at industrial scale but also provides useful evidence
for the efficient biotransformation of other compounds on the
combination of biotechnological processes (Wang et al. 2016, b).
Bioconversion of xylose to xylitol by engineered
E. coli
Although some bacteria possess natural capability to synthe-
size xylitol, little research efforts have focused on using bac-
teria for xylitol production. E. coli is an ideal engineering
Fig. 4 Complementary biotransformation of WXML by C. tropicalis
X828 and B. subtilis Bs12 in shake flasks. a, b Batch biotransformation
and glucose fed-batch biotransformation; the arrow represents the addi-
tion of 15 g/L glucose at 40 h. c Ratio of strain Bs12 and X828 cells in
5149
(adopted from Wang et al. 2016; an open-access article distributed under
the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/))
bacterium for the industrial production of chemicals due to
its ease of manipulation, cheaper cultivation medium require-
ments, and higher growth rates and yields on recombinant
proteins. Because of the presence of endotoxins in E. coli, it
is not a suitable host to produce food additives such as xylitol
(Suzuki et al. 1999). However, E. coli can be used as the host
of NAD(P)H-dependent co-factor, where central metabolism
serves as the co-factor regeneration system (Cirino et al.
2010). It has been reported that the xylose reductases from
C. boidinii (CbXR) (Bolen and Mccracken 1990; Kang et al.
2003), C. tenuis (CtXR) (Häcker et al. 1999; Nidetzky et al.
2015), P. stipitis (PsXR) (Hallborn et al. 1991), and
S. cerivisiae (ScXR) (Ford and Ellis 2001; Jeong et al.
glucose fed-batch biotransformation. d HPLC profiles of xylitol produc-
tion (adopted from Wang et al. 2016; an open-access article distributed
under the terms of the Creative Commons Attribution 4.0 International
License (http://creativecommons.org/licenses/by/4.0/))
5150
2010), and xylitol dehydrogenases from Gluconobacter
oxydans (GoXDH) (Sugiyama et al. 2003) and P. stipitis
(PsXDH) (Cirino et al. 2010) were all functional in
E. coli to varying extents. Suzuki et al. (1999) expressed
xylose reductase gene of C. tropicalis in E. coli and
successfully converted D-xylose to xylitol when D-xylose
(50 g/L) and D-glucose (5 g/L) were added to IPTG-
induced cells. Notably, a higher concentration of 13.3 g/L
of xylitol accumulated after 20 h of cultivation in shake
flask fermentation (Suzuki et al. 1999). Similarly, xylose
reductase gene (xyl1) was cloned from C. tropicalis
SCTCC 300249, which has relatively high xylitol produc-
tion, and expressed heterologously in E. coli BL21(DE3)
and S. cerevisiae W303-1A (Zhang et al. 2009a). Due to
low catalytic activity and the presence operon governing of
prokaryotic cells, some methods are needed to improve the
expression of XR in E. coli to enhance the productivity and
yield of xylitol. These methods are as follows:
Cyclic AMP receptor protein mutation
The cyclic AMP receptor protein (CRP; also known as the
catabolite receptor protein) is an allosteric transcription factor
that regulates over 150 genes in Escherichia coli. Upon bind-
ing to cyclic AMP (cAMP), the homodimeric CRP undergoes
a conformational change whereby two alpha helices reorient
to open a DNA-binding domain, allowing CRP to bind to
DNA and affect transcription (Einav et al. 2018). Cirino
et al. (2010) replaced the native gene encoding CRP with a
mutant CRP (CRP*) by E. coli W3110, which reduced sensi-
tivity to cAMP, to overcome glucose repression of xylose
transporter expression. An alternative strategy was to express
either of the native xylose transporters, D-xylose/proton
symporter (XylE) or the D-xylose ABC transporter
(XylFGH), under the control of a (CRP-independent) tac pro-
moter growing on glucose (Khankal et al. 2008).
Heterologous expression of xylulokinase
Xylulokinase plays an important role in xylose assimilation by
all natural and engineered microorganisms. In bacteria, D-
xylose is first isomerized to xylulose by XI (xylA), which is
then phosphorylated to xylulose-5-phosphate by XK (xylB) in
the pentose phosphate pathway (Nigam and Singh 1995). D-
Xylulokinase from Aerobacter aerogenes has revealed to
phosphorylate xylitol at a low rate (Simpson 1966). This
non-specific activity of xylulokinases led to the buildup of
potentially toxic phosphorylated compounds that are not fur-
ther metabolized. During xylitol production by E. coli, xylitol
phosphate was synthesized by XylB, which is toxic and in-
hibits the use of xylose as the sole carbon source (Akinterinwa
and Cirino 2009). This toxicity can be relieved by a
xylulokinase gene mutation. Inactivation of an E. coli
Appl Microbiol Biotechnol (2019) 103:5143–5160
xylulokinase gene (xylB) and replacement with xylulokinase
(xyl3) gene from P. stipitis results in drastically enhanced
xylitol titers by engineered E. coli strains during growth on
xylose (Akinterinwa and Cirino 2009). It is also revealed that
the xylulokinase activity was similar and showed significantly
more activity on xylitol by XylB compared to Xyl3 when
examined the effects of plasmid-based expression of Xyl3
versus XylB on growth and xylitol production by engineered
E. coli strains (Akinterinwa and Cirino 2009).
Inactivating xylB gene by Red recombinase
technology
The phage lambda-derived Red recombination system
(Brecombineering^) is a simple and versatile technique for
making targeted genetic changes (insertion, deletion, and
point mutations on chromosomal) in E. coli. In the first ho-
mologous recombination, the plasmid pKD46 homologous
recombination system was used to replace the xylB gene with
the chloramphenicol resistance gene. The second homologous
recombination occurred at the flippase recognition target
(FRT) site by recombinase to eliminate the resistance gene.
XylB gene of E. coli strain was successfully knocked out in
the xylose disruption pathway and the resulting constructed
mutant strain improved the xylitol conversion rate (Datsenko
and Wanner 2000; Cirino et al. 2010).
Increase the extracellular supply of NADPH
NADPH is a coenzyme of xylose reductase. Improving the
activity of xylose reductase by increasing NADPH quantity
is a useful strategy to produce high levels of xylitol. Chin and
Cirino (2011) utilized engineered E. coli to produce enhanced
levels of xylitol from glucose- and xylose-mixed fermentation
medium after expressing NADPH-dependent xylose reduc-
tase from C. boidinii (CbXR). In another study, Nidetzky
et al. (2003) constructed a plasmid-encoded gene of xylitol
dehydrogenase from the yeast G. mastotermitis and expressed
in E. coli. The ratio of NADPH/NADP in E. coli was deter-
mined by the deuterium-labeling method. Notably, the amount
of NADP was decreased by increasing the amount of NADPH
in E. coli to increase the yield of xylitol (Nidetzky et al. 2003).
Besides the above strategies, improving the conversion ef-
ficiency of xylose by codon optimization and changing differ-
ent promoters or inactivating the genes of the related enzymes
is an efficient way to improve xylitol titer. For example, xy-
lose isomerase (XylA) converts xylose to xylulose, which is
then phosphorylated to X-5-P by xylulokinase (XylB).
Akinterinwa and Cirino (2009) reported that CRP and the
xylose-inducible regulator XylR arrange xylA and xylB genes
in E. coli in a single operon governed by a promoter.
Subsequent deletion of the xylB gene (encoding xylulokinase)
and expression of xylose reductase from C. boidinii (CbXR)
Appl Microbiol Biotechnol (2019) 103:5143–5160
to construct engineering E. coli resulted in efficient xylitol
production from glucose-xylose mixtures (Akinterinwa and
Cirino 2009).
Bioconversion of xylose to xylitol by other
engineering bacteria
Besides yeasts, a few bacteria are also capable of producing
xylitol, but with a lower yield as compared with the yeasts. In
bacteria, D-xylose is converted to D-xylulose by xylose isom-
erase in a single step; however, the complete transformation of
D-xylose to xylitol is challenging due to its inhibiting effect on
xylose isomerase. M. smegmatis (Izumori and Tuzaki 1988)
and G. oxydans (Suzuki et al. 2002) have also been document-
ed to produce xylitol in small amounts. Izumorim and Tuzaki
reported that immobilized D-xylose isomerase enzyme and
immobilized M. smegmatis with D-xylose as a substrate led
to 80% xylitol yield. Moreover, the media needed D-xylulose
for xylitol production to occur (Izumori and Tuzaki 1988).
Rangaswamy and Agblevor (2002) screened 17 cultures of
facultative bacteria and found that Corynebacterium sp. pro-
duced the highest amount of xylitol (maximum yield 0.57 g/
g), with an initial xylose concentration of 75 g/L within 24 h of
cultivation. Yoshitake et al. (2014) screened Enterobacter
strain and showed that Enterobacter strain no. 553 accumu-
lated 0.35 g/L/h xylitol by an NADPH-dependent XR using
D-xylose as a sole substrate (Yoshitake et al. 2014). It is worth
mentioning that the enzymatic conversion is not confined to
fungi and yeasts. B. subtilis, a safe and aerobe soil bacterium,
can metabolize various sugars, such as xylose, L-arabinose,
glucose, and galactose (Cheng et al. 2011). Table 1 summa-
rizes the production of xylitol from xylose by different
engineered microorganisms.
Bioconversion of glucose to xylitol
Glucose to xylitol bioconversion by a three-step process
The process for xylitol production from D-xylose is econom-
ically not viable because of the high cost of the raw material
xylose (approximately US $2500/t) (Cheng et al. 2014),
which limits the range of current applications in food and
chemical technology. In contrast, D-glucose is an efficient,
readily available, and much cheaper substrate for large-scale
xylitol production. In the late 1960s, Onishi and Suzuki have
produced xylitol from D-glucose, by a three-step fermentation
process as follows. At first, D-glucose is converted into D-
arabitol by some osmophilia yeast strains, such as Pichia,
Zygosaccharomyces, Hansenula, Saccharomyces, Candida,
and Debaryomyces (Escalante et al. 1990). In the second step,
D-arabitol was oxidized to D-xylulose by Acetobacter
suboxydans-derived D-arabitol dehydrogenase (ArDH),
which is subsequently reduced to xylitol by
5151
C. guilliermondii xylitol dehydrogenase (XDH) or by the pu-
rified enzyme in vitro reaction using XDH (Onishi and Suzuki
1969; Mayer et al. 2002). However, compared to the D-xylose
to xylitol reduction, the low productivity and yield of this
method restrict its application to an industrial scale
(Granström et al. 2007c). In addition, the low activity of
XDH limits the ultimate production of xylitol. Sugiyama
and coworkers cloned the NAD-dependent XDH gene from
G. oxydans and expressed it in G. oxydans to increase XDH
activity and improve xylitol yield by 100%. Moreover, this
engineered microorganism reduces the three-step process into
two steps. Unfortunately, the separation process and multi-
fermentation process improvement was still insufficient and
unsatisfactory (Sugiyama et al. 2003; Povelainen and
Miasnikov 2007).
Glucose to xylitol bioconversion by the one-step
fermentation process
In order to reduce the three-step fermentation process, numer-
ous research efforts have been made to construct engineered
microorganisms that directly convert glucose to xylitol. The
osmotolerant yeast species Debaryomyces hansenii (Jovall
et al. 1990), S. mellis (Weimberg 1962), Zygosaccharomyces
rouxii (Saha et al. 2007), and S. rouxii (Ingram and Wood
1965) possess potential ability to convert D-glucose to D-
ribulose-5-PO4 via the pentose phosphate pathway (PPP)
followed by dephosphorylating to D-ribulose. D-Ribulose
can be reduced to D-arabitol by an NADP-dependent pentitol
dehydrogenase or by a second pathway that reduces D-
ribulose-5-P to D-arabitol-5-P and dephosphorylates to D-
arabitol (Wong et al. 1995). In addition, the marine fungus
Dendryphiella salina (Lowe and Jennings 1985) and some
other strains of the yeast S. rouxii (strain P3a) (Blakley and
Spencer 1962) can convert D-glucose to D-xylulose-5-PO4
via PPP and then dephosphorylate to D-xylulose. The
resulting D-xylulose is reduced to D-arabitol by a NAD-
dependent pentitol dehydrogenase or D-xylulose-5-P dephos-
phorylates to D-arabitol after D-xylulose-5-P reduction to D-
arabitol-1-P (Wong et al. 1995). D-Arabitol could be oxidized
to D-xylulose by xylulose-forming dehydrogenase, followed
by the reduction of D-xylulose to xylitol with xylitol dehydro-
genase (Povelainen and Miasnikov 2007; Cheng et al. 2014).
Arabitol-5-P could be transformed to D-xylulose-5-P by a
bacterial D-arabitol-phosphate dehydrogenase and then de-
phosphorylate to xylitol-5-P by XPDH. Further, xylitol-5-P
was dephosphorylated to xylitol (Povelainen and Miasnikov
2007). Figure 5 illustrates the three possible xylitol biosyn-
thetic pathways from glucose that have been described in
engineered yeast cells.
Since D-arabitol is the key intermediate product in the met-
abolic process, increasing its titer by the optimization of the
microbial transformation of D-glucose into D-arabitol is
5152 Appl Microbiol Biotechnol (2019) 103:5143–5160

Table 1 Production of xylitol from xylose by engineered microorganisms

Microorganism Strain descriptions Initial xylose Xylitol Volumetric Yield Reference

concentration (g/L) titer (g/L) productivity (g/L/h) (g/g)

Yeast
Candida intermedia – 20 45.7 0.38 0.57 Wu et al. (2018)
FL023
Candida tropicalis Metabolize glucose and 400 g/L (waste xylose 95 – 0.75 Wang et al.
X828 inhibitors furfural and mother liquor) (2016)
hydroxymethylfurfural
Depletion of by-product sugars
Kluyveromyces Overexpression of transporter 152 150 3.40 0.99 Zhang et al.
marxianus genes KmFPS1, CiGXF1, (2015)
and CiGXS1
Saccharomyces Cloned CDT-1, gh1-1, and 20 19.2 19.2 1.0 Zha et al. (2013a,
cerevisiae XYL1 genes b)
Kluyveromyces Overexpression 100 71.3 4.43 1.01 Zhang et al.
marxianus NcXR gene, Δxyl1 (2014)
Kluyveromyces Expression of XR gene 40 23.4 0.325 0.70 Zhang et al.
marxianus Substitution of Kmxyl1 (2013)
with XR
Candida tropicalis Expressed of xylose 50 50 1.14 1.0 Jeon et al. (2013)
transporter gene
Candida tropicalis Codon optimization of 50 48 1.44 0.59 Jeon et al. (2012)
NcXR gene
Fungi
Ashbya gossypii Overexpression of endogenous 80 22.6 0.086 0.28 Díaz-Fernández
xylose utilization pathway et al. (2017)
Mutant Coniochaeta Two third silencing of XR 3.0 0.002 – 0.59 Nichols and Saha
ligniaria C8100 and XDH (2016)
Trichoderma reesei Overexpressed xyl1 gene 25 3.72 0.026 0.148 Hong et al.
Silenced xyiH gene (2014a, b)
Bacteria
Escherichia coli Co-expression of xylose reductase 250 161.6 8.0 Jin et al. (2018)
and glucose dehydrogenase
Corynebacterium araE integration, ΔldhA, CtXR, 200 166 7.9 0.83 Sasaki et al.
glutamicum ΔptsF, and ΔxylB (2010)
Corynebacterium Overexpressed XYL1 35 30.1 0.77 0.96 Kim et al. (2010)
glutamicum
Lactococcus lactis Expression of xylose reductase 160 70 1.75 0.43 Nyyssölä et al.
and xylose transporter (2005)

considered an important strategy for enhanced xylitol biosyn-
thesis. For instance, Zhu et al. (2010) described a process for
81.2 g/L D-arabitol production by K. ohmeri NH-9 in 72 h
with productivity and yield of 1.278 g/L h and 0.406 g
arabitol/g glucose, respectively. Nozaki et al. (2003) achieved
the highest production of 81.4 g/L with a corresponding yield
and productivity of 0.078 g xylitol/g glucose and 0.701 g D-
arabitol/L h, respectively, by Metschnikowia reukaufii after
116 h of fermentation duration. However, a wide variety of
by-product (polyhydric alcohols) was generated and compet-
ed with the production, when certain osmophilic yeasts are
grown in the presence of a high concentration of glucose.
This accumulation of by-products can be minimized under
controlled temperature and D-glucose feeding environment
(Meng et al. 2014). Some osmophilic yeasts have no
capabilities to transform D-arabitol into D-xylulose due to
the lack of D-arabitol-4-dehydrogenase enzyme. Moreover,
the XDH of G. oxydans expressed in P. pastoris showed
strong xylitol dehydrogenase activity and high efficiency in
reducing D-xylulose to xylitol (Cheng et al. 2014). Harkki
group constructed an engineered Z. rouxii strain, which could
tolerate high concentration of glucose (400 g/L), by express-
ing the D-arabitol dehydrogenase gene from Klebsiella
terrigena and xylitol dehydrogenase gene from P. stipitis to
produce a high level of xylitol from glucose. The resulting
recombinant strain produced about 15 g/L xylitol from
400 g/L glucose in 24 h with yield and productivity of
0.038 g xylitol/g glucose and 0.063 g/L h, respectively
(Harkki et al. 2004). Recently, our laboratory exploited an
engineering P. pastoris for the conversion of glucose to xylitol
Appl Microbiol Biotechnol (2019) 103:5143–5160
Fig. 5 The three metabolic pathway for the conversion of glucose to
xylitol in engineered yeasts. PTS phosphotransferase system, P the
phosphate moiety, PPP pentose phosphate pathway. P1 and P2, the first
pathway of conversion of glucose to arabitol (Wong et al. 1995); P3 and
P4, the second pathway of converting glucose to arabitol (Povelainen and
Miasnikov 2007); arabitol was oxidized to xylulose, followed by the
reduction of D-xylulose to xylitol; P5, the third pathway of conversion
of glucose to D-xylitol-5-phosphate suggested by Povelainen and
Miasnikov (2007). The enzymes are designated as follows: E1, NADP-
dependent glucose 6-P dehydrogenase; E2, NADP-dependent gluconate
6-P dehydrogenase; E3, ribulose-5-P-epimerase; E4, NAD-dependent
by co-expressing XDH from Gluconobacter oxydans and D-
arabitol dehydrogenase (DalD) gene from Klebsiella
pneumoniae in P. pastoris GS2270L in a 3-L fermenter.
Fermentation results showed that recombinant P. pastoris cells
with both DalD and XDH converted glucose to xylitol with
the highest yield of 0.078 g xylitol/g glucose and productivity
of 0.29 g xylitol/L h. The obtained yield was fourfolds higher
than those of Harkki et al. (2004). This was the first report to
convert xylitol from glucose by the pathway of glucose–D-
arabitol–D-xylulose–xylitol in a single process (Cheng et al.
2014). The recombinant yeast could be used as a yeast cell
factory and has the potential to produce xylitol from glucose.
However, in contrast to some other yeasts that achieved a D-
arabitol yield of 20–30% from glucose (Ahmed 2001), less
than 10% of glucose was converted to D-arabitol by
P. pastoris GS225 even when pre-adapted in high glucose
concentration. One of the probable reasons is the low capacity
of the PPP in the GS225 strain because D-arabitol was syn-
thesized via PPP. The yield of D-arabitol from glucose might
be increased by driving glucose through the PPP by
inactivating phosphoglucose isomerase-encoding gene (pgi)
and simultaneously manipulating the cells NADPH demand
by introducing a transhydrogenase reaction. On the other
5153
arabitol 1-P dehydrogenase; E5 and E13, presumptive intracellular (or
membrane-associated) sugar-phosphate phosphatase; E6, xylulokinase;
E7, NAD/NADP-dependent ketose reductase or NADH/NADPH-
dependent polyol dehydrogenase; E8, NADP-dependent xylulose 5-P
reductase; E9, a presumptive intracellular (or membrane-bound) sugar-
phosphate phosphatase; E10, ribulokinase; E11, NAD/NADP-dependent
ketose reductase or NADH/NADPH-dependent polyol dehydrogenase;
E12, ribulose-5P-reductase; E14, NAD-dependent xylulose-forming
arabitol dehydrogenase from K. pneumoniae; E15, NAD-dependent xy-
litol dehydrogenase from P. stipitis or G. oxydans or its native enzyme
hand, overexpression of the glucose-6-phosphate and
gluconate-6-phosphate dehydrogenase genes can also im-
prove the PPP flux from glucose leading to an enhanced yield
of ribulose-5-phosphate. The overexpression of the phosphor-
ylase gene and ribulose reductase gene would then dephos-
phorylate ribulose-5-phosphate to ribulose and reduce
ribulose to D-arabitol, respectively. Further studies are ongo-
ing in our laboratory to improve the pentose phosphate flux
from glucose by P. pastoris GS225.
Enzymatic biosynthesis of xylitol
Enzymatic bioconversion of xylose into xylitol offers a note-
worthy alternative towards the large-scale production of xyli-
tol. Enzyme-mediated catalytic reactions are generally consid-
ered safe, highly reproducible, cost-efficient, and environmen-
tally friendly (Bilal et al. 2017, 2018a, b, c; Wang et al. 2018).
Though enzymatic biosynthesis of xylitol presents several ad-
vantages over fermentative production, lower yields and pre-
requisite of a high-precise control render this approach less
favorable (de Freitas Branco et al. 2012). As described above,
xylose-assimilating yeasts transform D-xylose via two con-
secutive enzymatic oxidoreductive reactions with the
5154
activities of XR and XDH. D-Xylose is reduced to xylitol by
XR-driven catalytic conversion. In these bioprocesses, en-
zymes can be incorporated as biocatalysts to improve or sub-
stitute production steps (Bon et al. 2008). For the first time,
Kitpreechavanich et al. (1984) reported the xylitol production
from xylose using XR from C. pelliculosa accompanied by an
oxidoreductase system of Methanobacterium sp. for NADP
reduction. About 90% xylose was converted to xylitol at pH
7.5 and 35 °C after a 24-h reaction period. The co-enzyme was
successfully regenerated and retained in a membrane reactor
during this process. Later on, Nidetzky et al. (1996) evaluated
the conversion of xylose to xylitol using an NADH-dependent
XR from C. tenuis. For the co-enzyme, they explored a
charged membrane reactor and an enzymatic regeneration
system. In spite of the relatively good result, this technique
did not grasp required attention due to economic problems.
Kroutil et al. (2004) suggested that the system connected to
the substrate using a single enzyme, while simultaneously
converting the substrate as well as co-substrate. However,
the co-substrate must be incorporated in greater quantities
than the substrate to favorably shift the equilibrium of the
reactions. In such cases, a second biocatalyst has to be utilized
to restore the reduced co-enzyme using a co-substrate and
generating a co-product. Formate dehydrogenase (EC
1.2.1.2) from a recombinant strain of Pseudomonas sp. has
shown great potential for the regeneration of NAD(P)H in
the reaction medium. Similarly, the enzymatic biosynthesis
of xylitol by applying the glucose dehydrogenase system to
regenerate NADPH was also found to be interesting because
of cost-effective and environmentally responsiveness. In fu-
ture, the discovery or engineering of a more chemically stable
XR to increase its half-life B) in-house production of co-
enzymes using microbial biomass and the development of
continuous bioconversion processes by the immobilized XR
or using membrane reactors are important to increase the eco-
nomic viability of the enzymatic xylitol process (de Freitas
Branco et al. 2012).
Challenges and future prospects
Xylitol is an important alternative sweetener and widely ap-
plied in the food and pharmaceutical industries. Current man-
ufacture strategy mainly employs chemical methods to pro-
duce xylitol from xylose purified from biomass hydrolysate.
In order to make biosynthesis method available for industrial
production, extensive studies are needed to improve the yield
of xylitol from a cheaply available material such as glucose.
Regulation of TCA cycle and pentose phosphate pathway or
modifying metabolic pathways, including knockout and ex-
pression of critical genes in the artificial glucose-to-xylitol
pathway, is of worth significance to scaling up this technolo-
gy. Discovery of a novel yeast with high tolerance to elevated
concentrations of carbon sources (such as glucose) and the
Appl Microbiol Biotechnol (2019) 103:5143–5160
high glucose flux ratio into pentose phosphate pathway, which
largely produces the xylitol intermediate D-xylulose that is
also an extremely imperative strategy to improve the yield
and productivity of xylitol from glucose. In addition,
engineered microbial strains capable of simultaneously assim-
ilating multiple sugars found in plant biomasses would greatly
increase the perspective of plant biomass as a sustainable and
renewable feedstock for the biosynthesis of xylitol and other
value-added compounds.
Yarrowia lipolytica, an oleaginous yeast, plays a key role in
biosynthesis, metabolic engineering, and biodegradation as
well as biotransformation processes, due to its tremendous
potential to produce a variety of value-added metabolites such
as short-chain fatty alcohols, alkanes as biofuels, polyunsatu-
rated fatty acids, polyhydroxyalkanoates and dicarboxylic
acids, carotenoid-type pigments, and campesterol as a precur-
sor for steroid drugs (Darvishi Farshad et al., 2018). It is gen-
erally regarded as safe by GRAS (generally recognized as
safe) and a safe microorganism by the Food and Drug
Administration (FDA) in the USA (Zinjarde 2014).
Therefore, it is widely applied in the hyperproduction of food
ingredients and other industrially pertinent bioproducts, such
as erythritol (Li et al. 2017; Cheng et al. 2018), lipase, man-
nitol, fatty acids and lipids (Tai and Stephanopoulos 2013;
Gonçalves et al. 2014), and citric acid (Ledesma-Amaro
et al. 2016).
In the case of Y. lipolytica yeast, glucose-glycerol mixture,
carbohydrates, n-alkanes, and different classes of lipids except
for sucrose act as a carbon source in the metabolic process
(Ludwika et al. 2012; Zhu and Jackson 2015). With the recent
improvements in synthetic biology, Y. lipolytica is the most
popular non-conventional organism with a relatively high tol-
erance capacity to elevated concentrations of salts, hydrocar-
bons, and waste carbon (Fickers et al. 2005; Papanikolaou and
Aggelis 2010), and can produce sugar alcohols in response to
increased external high osmotic pressure (Ludwika et al.
2012). Y. lipolytica has achieved a series of success in the
production of lipase (Brígida et al. 2014) and lipids with sub-
stantial engineering efforts. More recently, Y. lipolytica was
used in our laboratory for the synthesis of erythritol using
glucose as the main carbon source (Cheng et al. 2018).
Overexpression of the newly isolated two erythrose reductase
genes (ER10 or ER25) and engineering of NADPH co-factor
metabolism by overexpression of 6-phosphogluconate dehy-
drogenase genes (GND1) and glucose-6-phosphate dehydro-
genase genes (ZWF1) in Y. lipolytica strain CGMCC7326 led
to a considerable increase in erythritol titer compared to the
wild-type strain (Cheng et al. 2018).
The pentose phosphate pathway is the same metabolic pro-
cess between xylitol and erythritol, and the common interme-
diate is D-xylulose. Xylitol can be obtained by reducing
xylulose with xylitol dehydrogenase. Rodriguez et al. (2016)
reported that Y. lipolytica is naturally unable to grow on xylose
Appl Microbiol Biotechnol (2019) 103:5143–5160
as its sole carbon source; however, a small amount of weak
growth was observed on xylitol by a partially active xylose
pathway (Rodriguez et al. 2016). They sought whether there
were xylose reductase (XYR1), xylitol dehydrogenase
(XDH), and xylulose kinase (XKS) genes in the oxidoreduc-
tase pathway of xylose metabolism in Y. lipolytica. Measuring
native mRNA expression and extent of inducibility (ΔΔCT) of
these three candidate genes by quantitative PCR, they found
that the weakened expression of XKS is one of the major
bottlenecks for xylose and xylitol, and metabolism could be
the limited expression of XKS (Rodriguez et al. 2016).
In Y. lipolytica yeast systems, especially for erythritol-
producing strains, the pentose phosphate pathway is the major
metabolic pathway for glucose utilization; however, about
20% of glucose is metabolized by Embden–Meyerhof–
Parnas glycolytic pathway (EMP) to produce valuable
chemicals, including oleochemicals, nutraceuticals, organic
acids, and many other specialty chemicals (Markham and
Alper 2018). Our group observed that the pentose phosphate
pathway is the same metabolic process between xylitol and
erythritol, and D-xylulose is one of the common intermediates
converted from glucose. We attempted to utilize glucose as a
carbon source to improve the yield and productivity of xylitol
by knocking out genes belonging to EMP, which indirectly
affect PPP (Mirończuk et al. 2017) and blocking the synthesis
of erythritol in yeast to obtain xylitol by pathway engineering
and metabolic pathway.
Fig. 6 Schematic representation of two metabolic pathways for the
conversion of glucose to xylitol in engineered Y. lipolytica. PTS
phosphotransferase system. The enzymes are designated as follows: E1,
epimerase; E2, kinase or sugar phosphate phosphatase; E3, NAD-
dependent xylitol dehydrogenase; E4, NADPH transhydrogenase; E5,
5155
In order to restrict EMP pathway and block the synthesis of
erythritol, we deleted transketolase genes (TKL2 and TKL1)
and mannitol dehydrogenase (MDH) gene, which can de-
crease the number of by-products, such as erythritol and man-
nitol. According to Fig. 3, D-ribulose and D-xylulose can be
reduced to D-arabitol and D-arabitol, respectively, using
arabinitol dehydrogenase (Cheng et al. 2009). Disruption of
arabinitol dehydrogenase gene and overexpression of
xylulose-5-P phosphatase gene (convert xylulose-5-P to
xylulose), and XDH gene (reduce xylulose to xylitol) can lead
to the accumulation of xylitol (Fig. 6). Therefore, the
xylulokinase gene (XSK) was inactivated to hinder the con-
version of xylulose to 5-P-xylulose. As 5-P-ribulose, 5-P-
xylulose, and 5-P-ribose are isomers of each other, deletion
of 5-P-ribulose isomerase (RPI) can improve the ratio of 5-P-
ribulose to 5-P-xylulose by hindering the conversion of 5-P-
ribulose to 5-P-ribose. Moreover, the xylulose conversion to
xylitol process is reversible, and substrate inhibition exists,
which limits the accumulation of xylitol. Therefore, the im-
portant step is to transport xylitol to extracellular by xylitol
transporter protein.
Though several microorganisms such as bacteria, fungi,
and yeasts have been reported in the literature for xylitol pro-
duction, yeasts are considered the best xylitol producers.
Among the yeasts, Y. lipolytica has distinct metabolic advan-
tages, such as great tolerance to chemicals, high protein pro-
duction, and the lack of tight metabolic regulation, which
kinase; E6, NAD/NADP-dependent ketose reductase or NADH/
NADPH-dependent arabitol dehydrogenase; E7, NADH/NADPH-
dependent arabitol dehydrogenase; E8, NADH/NADPH-dependent man-
nitol dehydrogenase
5156
make it an attractive host in synthetic biology. However, the
development of competitive genetic tools, improving transfor-
mation efficiency, and utilization of the CRISPR/Cas9 for
gene modification are some major limitations (Markham and
Alper 2018). Moreover, the construction of metabolically ro-
bust and stable strain, identification of new strains with high
tolerance to lignocellulosic hydrolysate inhibitors, scaled-up
bioprocessing, efficient downstream processing technologies
for high product recovery, and economic bioconversion of
lignocellulosic biomass to xylitol are also the challenges that
need to be addressed to make economical production of bio-
xylitol a reality. We believe that engineered Y. lipolytica will
be applied in industrial-scale xylitol production by a one-step
fermentation process from glucose as a carbon source.
Funding This research was supported by the National Natural Science
Foundation of China (No. 21877078).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
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