Mar Biotechnol (2015) 17:511–522
DOI 10.1007/s10126-015-9638-8
ORIGINAL ARTICLE
Genetic Modification of the Marine-Isolated Yeast Aureobasidium
melanogenum P16 for Efficient Pullulan Production from Inulin
Zai-Chao Ma 1 & Nan-Nan Liu 1 & Zhe Chi 1 & Guang-Lei Liu 1 & Zhen-Ming Chi 1
Received: 5 November 2014 / Accepted: 28 April 2015 / Published online: 19 May 2015
# Springer Science+Business Media New York 2015
Abstract In this study, in order to directly and efficiently
convert inulin into pullulan, the INU1 gene from
Kluyveromyces maximum KM was integrated into the geno-
mic DNA and actively expressed in the high pullulan producer
Aureobasidium melanogenum P16 isolated from the man-
grove ecosystem. After the ability to produce pullulan from
inulin by different transformants was examined, it was found
that the recombinant strain EI36, one of the transformants,
produced 40.92 U/ml of inulinase activity while its wild-
type strain P16 only yielded 7.57 U/ml of inulinase activity.
Most (99.27 %) of the inulinase produced by the recombinant
strain EI36 was secreted into the culture. During the 10-l fer-
mentation, 70.57±1.3 g/l of pullulan in the fermented medium
was attained from inulin (138.0 g/l) within 108 h, high
inulinase activity (42.03 U/ml) was produced within 60 h,
the added inulin was actively hydrolyzed by the secreted
inulinase, and most of the reducing sugars were used by the
recombinant strain EI36. This confirmed that the genetically
engineered yeast of A. melanogenum strain P16 was suitable
for direct pullulan production from inulin.
Keywords Metabolic engineering . Pullulan production .
Inulin . Inulinase gene . A. pullulans
* Zhen-Ming Chi
zhenming@sdu.edu.cn
1
Unesco Chinese Center of Marine Biotechnology, Ocean University
of China, Yushan Road, No. 5, Qingdao, China
Introduction
Pullulan which is essentially a linear glucan containing α-1,4
and α-1,6 linkages in the ratio of 2:1 is the water-soluble
homopolysaccharide produced extracellularly by
Aureobasidium spp. The regular alternation of α-1,4 and α-
1,6 bonds results in two distinctive properties of structural
flexibility and enhanced solubility. The unique linkage pattern
also endows pullulan with distinctive physical traits along
with adhesive properties and its capacity to form fibers, com-
pression moldings, and strong oxygen impermeable films.
Consequently, this polysaccharide is of economic importance
with increased application in food, pharmaceutical, agricultur-
al, chemical, and cosmetic industries (Prajapati et al. 2013).
The pullulans also have many physiological functions for the
producers (Ma et al. 2014). It has been reported that pullulan is
synthesized intracellularly at the cell wall or cell membrane
and secreted out to the cell surface to form a loose and slimy
layer. Therefore, a pathway for pullulan biosynthesis was pro-
posed (Li et al. 2013a).
Because the yeast-like cells cannot synthesize and secrete
high level of amylase, inulinase, and cellulase, the pullulan is
only produced from glucose, sucrose, coconut by-products,
beet molasses, agro-industrial wastes, grape skin pulp, starch
waste, olive oil wastes, carob pod, and hydrolysate of inulin
(Prajapati et al. 2013; Ma et al. 2014; Shin et al. 1989).
Therefore, it is very important to widen their substrate ranges
for pullulan production by metabolic engineering of the
producers.
Inulin consists of linear chains of β-2,1-linked D-
fructofuranose molecules terminated by a glucose residue
through a sucrose-type linkage at the reducing end and is
widely presented in the roots and tubers of plants such as
Jerusalem artichoke, chicory, dahlia, and yacon. It has been
reported that the dried materials of the roots and tubers of the
512 Mar Biotechnol (2015) 17:511–522

plants contained over 70 % inulin. So far, inulin and inulin-
containing materials have been used as raw materials for pro-
duction of ethanol, single-cell protein, single cell oil, ultra-
high-fructose syrup, inulooligosaccharide, citric acid, 2,3-
butanediol, lactic acid, sugar alcohols, and other useful prod-
ucts (Chi et al. 2011). The exo-inulinase produced by micro-
organisms catalyzes removal of the terminal fructose residues
from the non-reducing end of the inulin molecule to produce
major fructose and minor glucose. In our previous studies
(Duan et al. 2008), fructose and glucose were found to be
the best sugars for production of pullulan by Aureobasidium
spp. (Fig. 1). As stated above, the hydrolysates of inulin are
also the mixture of glucose (minor) and fructose (major).
Therefore, they may be the best sugars for production of
pullulan. However, as shown in Fig. 1, inulin has not been
used as a material for direct production of pullulan from inulin
because of very low or no inulinase activity of Aureobasidium
spp.
In our previous studies (Ma et al. 2014), Aureobasidium
pullulans var. melanogenum P16 strain isolated from man-
grove system was found to be able to produce high level of
pullulan. In recent studies (Gostinčar et al. 2014), the authors
have thought that the four varieties A. pullulans var. pullulans,
A. pullulans var. subglaciale, A. pullulans var. namibiae, and
A. pullulans var. melanogenum of Aureobasidium spp. are
large enough to justify their redefinition as four separate
Aureobasidium species: A. pullulans, Aureobasidium
m e l a n o g e n u m , A u re o b a s i d i u m s u b g l a c i a l e , a n d
Aureobasidium namibiae. After analysis of the genomic
DNA of A. melanogenum (http://genome.jgi-psf.org/Aurpu_
var_mel1/Aurpu_var_mel1.home.html), it was found that
there was no the gene encoding inulinase in this yeast.

Fig. 1 Inulin hydrolysis and

pullulan biosynthesis by the
engineered Aureobasidium spp.
carrying the INU1 gene. After the
INU1 gene from K. maximum
KM is actively expressed in
Aureobasidium spp., the
produced inulinase is secreted
into medium (Gong et al. 2007).
After the inulin is hydrolyzed into
glucose and fructose by the
secreted inulinase (Chi et al.
2011), the glucose and fructose
are taken up by the cells of the
engineered Aureobasidium spp.
and transformed into pullulan
which is secreted into the medium
(Li et al. 2013a)
Mar Biotechnol (2015) 17:511–522
Therefore, in this study, in order to genetically engineer the
pullulan producer, an inulinase gene (INU1) from
Kluyveromyces maximum KM was actively expressed in A.
melanogenum P16 strain and the recombinant strains which
could produce high inulinase activity were used for efficient
production of pullulan from inulin (Fig. 1).
Materials and Methods
Strains and Cultivation Media
The yeast A. melanogenum strain P16 isolated from the man-
grove systems in Hainan Province of China was used in this
study and stored at −80 °C in this laboratory (Ma et al. 2014).
The medium for growth of the seed culture contained 60.0 g/l
sucrose, 3.0 g/l yeast extract, 5.0 g/l K2HPO4, 0.2 g/l MgSO4·
7H2O, 1.0 g/l NaCl, and 0.6 g/l (NH4)2SO4 (Duan et al. 2008).
The cultivation time and temperature were 48 h and 28 °C,
respectively. The medium for pullulan production consisted of
138.0 g/l inulin, 3.0 g/l yeast extract, 5.0 g/l K2HPO4, 0.2 g/l
MgSO4·7H2O, 1.0 g/l NaCl, and 0.6 g/l (NH4)2SO4 (Duan
et al. 2008). K. maximum KM which is a high inulinase pro-
ducer was grown in the inulinase production medium (Zhou
et al. 2013). The Escherichia coli strain DH5α [F− endA1
hsdR17 (rK_/mK+) supE44 thi− λ−recA1gyr96DlacU169
(j80lacZDM15)] was used in this study and was grown in
Luria-Bertani broth (LB). The E. coli transformants were
grown in LB medium with 100 μg/ml of ampicillin. The yeast
transformants were grown in the YPD medium containing
100 μg/ml of hygromycin B. The inulinase production medi-
um was 10.0 g/l yeast extract, 20.0 g/l polypeptone, and
20.0 g/l inulin. The inulin used in this study was bought from
Haida Biotech Company in Qingdao, China.
Plasmids
The expression vector pAPX13 for over-expression of heter-
ologous gene in Aureobasidium spp. was constructed in this
Table 1 Primers used in this study
Name Sequences
INU-se 5′-GAGCTCGATGGTGACAGCAAGGCCATCACTA-3′ (bold and underlined bases encode SacI restriction site)
INU-an 5′-GTCGACTCAAACGTTAAATTGGGTAACGTTA-3′ (bold bases encode SalI restriction site)
YIF 5′ -AAAACCCCAACTTCGGAAAGGGGTG-3′
YIR 5′ –TGGTGTAAGCCTTGTCGTATGGCGA-3′
INU RAse 5′-CACCGTCACCTCCGAAAAC-3′
INU RAan 5′-GTTGTCGTCGTTGTTACCCTTG-3′
AP26S 5′- AGCCTTCGGGTTCGCATTG-3′
AP26X 5′-CCAGTCACATACGGGATTCTCAC-3′
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laboratory. pMD 19-T simple vectors were purchased from
TaKaRa (Japan).
Isolation of DNA, Restriction Digestions,
and Transformation
The genomic DNAs were isolated from K. maximum KM and
A. melanogenum strain P16 and its transformants, respective-
ly, and DNA manipulations were carried out using the stan-
dard methods (Sambrook et al. 1989). Bacterial plasmid DNA
was purified using TIANprep Mini Plasmid Kit (TIANGEN).
Restriction endo-nuclease digestions and DNA ligations were
performed according to the manufacturer’s recommendations.
E. coli was transformed with plasmid DNA according to
Sambrook et al. (1989). A. melanogenum strain P16 was trans-
formed according to the methods described by Chi et al.
(2012).
Vector Construction for Expression of the INU1 Gene
in A. melanogenum Strain P16
To express the INU1 gene from K. maximum KM in
A. melanogenum strain P16, the primers for amplification of
the INU1 gene encoding the inulinase gene were designed
according to the sequence of the gene (accession no.
AF135594.1) in K. maximum. The forward primer and the
reverse primer were INU-se and INU-an (Table 1 and
Fig. 2). The genomic DNA of K. maximum KM was used as
the template for PCR. The reaction system was 50.0 μl con-
taining 1.0 μl Takara EX Taq, 5.0 μl 10× La PCR buffer II
(Mg2+ Plus), 8.0 μl 2.5 mM dNTPs, 1.0 μl 20.0 μM each
primer, 1.0 μl of 10 ng/ml genomic DNA, and sterile deion-
ized water up to 50.0 μl. The conditions for the PCR amplifi-
cation were initial denaturation at 94 °C for 5 min, denatur-
ation at 94 °C for 45 s, annealing temperature at 60 °C for 45 s,
extension at 72 °C for 1 min 45 s, and final extension at 72 °C
for 5 min. PCR was run for 30 cycles. The PCR products were
separated by agarose gel electrophoresis and ligated into the
plasmid pMD19-T simple vector. The recombinant vector was
514 Mar Biotechnol (2015) 17:511–522

Fig. 2 Construction of the

expression vector for over-
expression of the INU1 gene in
A. melanogenum strain P16. The
DNA fragment encoding the
signal peptide comes from the
gene (accession number
EF198023) encoding alkaline
protease in A. pulllulans HN2-3.
26S rDNA, 18S rDNA, PolyA,
and TEF come from the genomic
DNA in A. pullulans. The plasmid
was digested with the enzymes
EcoRI and SphI. The linear
fragments carrying the INU1 gene
were used for yeast
transformation

transformed into E. coli DH5α. The recombinant vectors car-
rying the PCR products from the E. coli transformants were
extracted and purified. The purified recombinant vectors car-
rying the PCR products were digested with SacI and SalI, and
the digests were ligated into the expression vector pAPX13
digested with the same enzymes (Fig. 2). The resulting plas-
mid carrying the INU1 gene was designated as pAPX13-INU
(Fig. 2).
Transformation and Selection
activity produced by the cells of different positive
transformants was determined as described below, respective-
ly. A. melanogenium strain P16 not to be transformed was
used as a control. After determination of inulinase activity
produced by the cells of over 200 positive transformants, it
was found that inulinase activity produced by the recombinant
strain EI36 among them was the highest. Therefore, the re-
combinant strain EI36 was used as the inulinase producer,
subsequently.

The plasmid pAPX13-INU obtained above was digested with
the enzymes EcoRI and SphI. The linear fragments carrying
the INU1 gene were separated in agarose gel and recovered
using TaKaRa Agarose Gel DNA Purification Kit Ver.2.0,
respectively. The recovered linear fragments carrying the
INU1 gene were transformed into the cells of
A. melanogenum strain P16 by using the methods described
by Chi et al. (2012). The putative transformants were verified
by cultivation on YPD agar containing 100 μg/ml of
hygromycin B. The positive transformants were grown in
the inulinase production medium for 4 days and inulinase
Confirmation of the Integrated INU1 Gene
in the Genomic DNA of the Recombinant Strain EI36
The genomic DNAs in the recombinant strain EI36 and
A. melanogenum strain P16 were extracted as described above
and used as the templates for PCR. The DNA fragments (2,
529 bp) containing the partial sequence of 18S rDNA and the
whole INU1 gene were PCR amplified using the primers YIF
and YIR (Table 1 and Fig. 2). The sizes of the PCR products
were estimated using the Automated Documentation and
Analysis System (Gene-Genius, USA). The PCR products
Mar Biotechnol (2015) 17:511–522
were sequenced by Shanghai Sangon Company (Zhao et al.
2010).
Determination of the Recombinant Inulinase Activity
The recombinant inulinase activity of different positive
transformants including the recombinant strain EI36 obtained
above was determined according to Gong et al. (2007). One
inulinase unit (U) was defined as the amount of enzyme that
produces 1 μmol of reducing sugar per minute under the assay
conditions used in this study. At the same time, the yeast cells
of the recombinant strain EI36 in 5.0 ml of the cultures were
washed three times with sterile distilled water by centrifuga-
tion at 4 °C and 5,000×g for 10 min, respectively. The cell-
bound inulinase activity of the washed yeast cell suspensions
(5.0 ml) was determined as described above. The determina-
tions were carried out in triplicate and all the data were the
average of the three independent experiments.
Inulinase Production from Inulin at Flask Level
The cells of the recombinant strain EI36 and A. melanogenum
strain P16 were transferred to 50.0 ml of the sterile medium
for the seed culture and cultivated at the shaking speed of
180 rpm and 28 °C for 24 h, respectively. Two milliliters of
the culture (2.5×108 cells/ml) was transferred to 50.0 ml of the
sterile inulinase production medium, and the yeast cells were
cultivated at the shaking speed of 180 rpm and 28 °C for 96 h.
The measurements of inulinase activity and cell mass were
performed as described above and below. The cultivations
were carried out in triplicate and all the data were the average
of the three independent experiments.
Fluorescent Real-Time PCR
Fluorescent real-time PCR was performed according to the
methods described by Liu et al. (2011). The seed cultures of
the recombinant strain EI36 and A. melanogenum strain P16
were prepared as described above. The seed cultures were
transferred into 50 ml of the sterile inulinase production me-
dium and the cultures were aerobically grown for 72 h and, at
this time, inulinase activity reached the highest. The cells were
harvested and washed, and the washed cells were used for
RNA extraction and cDNA preparation (Liu et al. 2011). All
the primers used for fluorescent real-time PCR were designed
according to the corresponding gene sequences of
K. maximum and A. melanogenum strain P16. The primers
INU RAse and INU RAan were designed according to the
INU1 gene sequence (GenBank accession no. AF135594.1)
in K. maximum, and the primers Ap26S and Ap26X were
designed according to 26S rRNA gene sequence (GenBank
accession no. EU622575) in A. pullulans (Table 1). The tran-
scriptional level of the INU1 gene in A. melanogenum strain
515
P16 was regarded as 100 %. The determinations were carried
out in triplicate and all the data were the average of the three
independent experiments.
Pullulan Production from Inulin at Flask Level
The seed cultures of the recombinant strain EI36 and
A. melanogenum strain P16 were prepared as described above.
Two milliliters of the culture (2.5 × 10 8 cells/ml)
were transferred to 50.0 ml of the sterile pullulan production
medium, and the yeast cells were cultivated at the shaking
speed of 180 rpm and 28 °C for 96 h. The determination of
pullulan, inulinase activity, reducing sugar, total sugar, and
cell mass was performed as described above and below. The
cultivations were carried out in triplicate and all the data were
the average of the three independent experiments.
Pullulan Production from Inulin by Batch Fermentation
Pullulan production by the recombinant strain EI36 was also
carried out in the 10-l fermentor [BIOQ-6005-6010B,
Huihetang Bio-Engineering Equipment (Shanghai) CO-
LTD]. The seed cultures were prepared as described above.
The fermentation was carried out in the sterile fermentor
equipped with baffles, a stirrer, heating element, oxygen sen-
sor, and temperature sensor. Three hundred milliliters of the
seed culture were transferred into 6.5 l of the sterile pullulan
production media containing 138.0 g/l inulin and 3.0 g/l yeast
extract. The fermentation was performed under the conditions
of agitation speed of 300 rpm, aeration rate of 6.5 l/min, tem-
perature of 28 °C, and fermentation period of 132 h. Only
40.0 ml of the culture was collected in the interval of 12 h
and was centrifuged at 5,000×g and 4 °C for 5 min, and
pullulan, inulinase activity, reducing sugar, and total sugar in
the supernatant obtained were determined as described above
and below. The cell dry weight in 20.0 ml of the culture during
the 10-l fermentation was also determined as described below.
The fermentations were carried out in triplicate and all the data
were the average of the three independent experiments.
Purification and Quantitative Determination of EPS
The cultures obtained during the 10-l fermentation were heat-
ed at 100 °C in water bath for 15 min, then were cooled to
room temperature. The heated cultures were centrifuged at 14,
046×g and 4 °C for 5 min to remove cells and other precipi-
tates. The supernatant (10.0 ml) was transferred into a test
tube, then 20.0 ml of cold ethanol (absolute ethanol or 95 %
ethanol) was added to the test tube and mixed thoroughly and
held at 4 °C for 12 h to precipitate the extracellular polysac-
charide. After removal of residual ethanol, the precipitate was
dissolved in 10.0 ml of deionized water at 80 °C and the
solution was dialyzed against deionized water for 48 h to
516
remove small molecules in the solution. After the EPS was
precipitated again by using 20.0 ml of the cold ethanol, the
precipitate was dried at 80 °C to a constant weight (Lee et al.
2001).
Hydrolysis of the Purified Extracellular Polysaccharide
and Assay of Reducing Sugar
Hydrolysis of the purified extracellular polysaccharide and
assay of reducing sugar were done as described by Ma et al.
(2014).
Fourier-Transform Infrared Analysis of the Purified
Pullulan
The purified pullulan obtained was characterized using
Fourier-transform infrared (FT-IR) spectroscopy (a Nicolet
Nexus FTIR 470 spectrophotometer). Two milligrams of the
purified pullulan sample were mixed with 60 mg of 95 %
potassium bromide powder. The mixture was desiccated over-
night at 50 °C under vacuum. The FT-IR spectra were taken
using potassium bromide pellets of the purified pullulan and
standard pullulan obtained from sigma over arrange of 4,000–
400 cm−1 at a rate of 16 scans with a resolution of 2 cm−1.
Determination of Reducing Sugar and Total Sugar
Reducing sugar in the fermented media was determined by the
Nelson–Somogyi method (Spiro 1966). Residual total sugar
was measured as reduction of sugar after hydrolysis of the
fermented media (10.0 ml of the fermented media, 10.0 ml
of 25 % HCl, and 30 ml of distilled water were mixed and
heated in a boiling water bath for 3 h) (Chi et al. 2001). The
determinations were carried out in triplicate and all the data
were the average of the three independent experiments.
Measurement of Cell Dry Weight
Cell dry weight was measured according to the methods de-
scribed by Chi et al. (2001). The determinations were carried
out in triplicate and all the data were the average of the three
independent experiments.
Statistic Analysis
Statistical tests (two-tailed paired t test and two-tailed two-
sample t test) were performed using Statistical Package for
the Social Sciences (SPSS) (ver.19; IBM corporation; USA)
with significance set at α=0.05. The results of two indepen-
dent experiments, each carried out in duplicate (means±SD),
are presented in the figures.
Mar Biotechnol (2015) 17:511–522
Results
Expression of the INU1 Gene in the High Pullulan
Producing Yeast A. melanogenum Strain P16
In order to genetically engineer the high pullulan producer of
A. melanogenum strain P16, the INU1 gene from K. maximum
KM was integrated into the genomic DNA and actively
expressed in A. melanogenum strain P16 as described in
BMaterials and Methods^ (Fig. 2). After determination of
inulinase activities of the different transformants and
A. melanogenum strain P16, it was found that the recombinnat
strain EI36 among them produced the highest inulinase activ-
ity (40.92 U/ml) as supported by ANOVA analysis (P=0.01
that differences were significant), and the inulinase activities
produced by the 200 transformants were in the range from
11.72±0.25 U/ml to 40.92±0.43 U/ml (Fig. 3). It was strange
that its wild-type strain P16 also produced inulinase activity of
7.57 U/ml (Fig. 3).
At the same time, the transcriptional level of the INU1 gene
in the recombinant strain EI36 was also much higher than that
(almost 0) of the inulinase gene in A. melanogenum strain P16
(Fig. 4) as indicated from ANOVA analysis that found differ-
ences to be significant (P=0.01).
Confirmation of the Integrated INU1 Gene
in the Genomic DNA of the Recombinant Strain EI36
Our data showed that the INU1 gene was amplified from the
genomic DNA in the recombinant strain EI36 (data not
shown). However, no such PCR products were amplified
from the genomic DNA of A. melanogenum strain P16 (data
not shown). This demonstrated that the INU1 gene was indeed
integrated into the genomic DNA in the recombinant strain
EI36, leading to stable occurrence of the gene in the genomic
DNA of the recombinant strain EI36 according to the Fig. 2.
Fig. 3 Inulinase activities of the different transformants including the
recombinant strain EI36 and A. melanogenum strain P16. Data are
given as mean±SD, n=3 *(P<0.05) significant difference compared
with the P16 group; **(P < 0.01) extreme significant difference
compared with the P16 group
Mar Biotechnol (2015) 17:511–522
Fig. 4 Transcriptional level of
the INU1 gene in the recombinant
strain EI36 and A. melanogenum
strain P16. Data are given as
mean±SD, n=3. **(P<0.01)
extreme significant difference
compared with the P16 group
Inulinase Production and Cell Growth
by the Recombinant Strain EI36 and A. melanogenum
Strain P16 at Flask Level
During the recombinant strain EI36 and A. melanogenum
strain P16 were aerobically grown in the inulinase production
medium for 96 h, inulinase activity and cell growth were mea-
sured as described in BMaterials and Methods.^ It can be seen
from the data in Fig. 5 that the recombinant strain EI36 carry-
ing the heterologous INU1 gene grew much better and pro-
duced much higher inulinase activity than its wild-type strain
P16 as shown from ANOVA analysis that found differences to
be significant (P=0.01). For example, the OD600nm value of
the culture of the recombinant strain EI36 reached 9.4 within
60 h of the cultivation while that of the culture of its wild-type
strain P16 was 7.6 within 72 h of the cultivation under the
same conditions. Inulinase activity produced by the recombi-
nant strain EI36 reached to 40.94 U/ml within 72 h of the
cultivation while inulinase activity produced by its wild-type
strain P16 reached to 7.6 U/ml within 72 h of the cultivation
under the same condition. This meant that not only inulinase
Fig. 5 Time courses of inulinase production (filled square) and cell
growth (filled circle) by the recombinant strain EI36 and inulinase
production (empty circle) and cell growth (empty square) by
A. melanogenum strain P16. Data are given as mean±SD, n=3. The
shaking speed of the flask was 180 rpm, the cultivation temperature
was 28 °C, and the cultivation time was 96 h
517
production by the recombinant strain EI36 carrying the heter-
ologous INU1 gene was enhanced but also cell growth of the
recombinant strain EI36 was promoted.
Location of the Produced Inulinase
After determination of the produced inulinase occurred in the
supernatants and cell-bound, the results in Fig. 6 indicated that
most (99.27 %) of the inulinase produced by the recombinant
strain EI36 was secreted into the supernatant as indicated from
ANOVA analysis that found differences to be significant (P=
0.01). This suggested that the signal peptide from the alkaline
protease produced by A. pulllulans HN2-3 could work well in
A. melanogenum strain P16 and A. melanogenum strain P16
was a good host for expression of the heterologous INU1 gene
and secretion of foreign protein and enzyme.
Pullulan Production from Inulin by the Recombinant
strain EI36 and Its Wild-Type Strain P16 at Flask Level
During the recombinant strain EI36 and its wild-type strain
P16 were grown in the pullulan production medium at flask
level, pullulan concentrations, inulinase activity, cell density,
Fig. 6 Distribution of the produced inulinase by the recombinant strain
EI36. Data are given as mean±SD, n=3. **(P<0.01) extreme significant
difference compared with the cell-bound group
518 Mar Biotechnol (2015) 17:511–522

the amount of reducing sugar, and total sugar were tested. Our
results showed that 53.17±0.61 g/l of pullulan produced by
the recombinant strain EI36 was reached within 96 h of the
cultivation when OD600nm value of the cell culture was 12.23
±0.20 while 37.10±0.68 g/l of pullulan produced by the wild-
type strain P16 was reached within 84 h of the cultivation
when OD600nm value of the cell culture was 11.21±0.81 as
indicated from ANOVA analysis that found differences to be
significant (P=0.01) (Fig. 7). Under the conditions, 25.37±
1.83 g/l total sugar and 14.07±0.77 g/l reducing sugar were
left in the culture of the recombinant strain EI36 while 72.53±
0.92 g/l of total sugar and 19.07±0.68 g/l reducing sugar were
left in the culture of the wild-type strain P16 as supported by
ANOVA analysis (P=0.01 that differences were significant)
(Fig. 7). Meanwhile, during the cultivation, inulinase activity
(38.03±0.98 U/ml) was produced by the recombinant strain
EI36 within 48 h and inulinase activity (12.25±0.13 U/ml)
was yielded by the wild-type strain P16 within 60 h, indicating
that when the recombinant strain EI36 and the wild-type strain
P16 were grown in the pullulan production medium in which
nitrogen source was deficient, the inulinase was still synthe-
sized and secreted (Fig. 7).
Pullulan Production from Inulin by the Recombinant
Strain EI36 During 10-l Fermentation
To scale up for EPS production from inulin, 10-l fermentation
was carried out. During the fermentation, EPS yield, cell
growth, and changes in reducing sugar and total sugar con-
centrations were monitored. The results in Fig. 8 showed that
during the 10-l fermentation, 70.57±1.3 g/l of EPS in the
fermented medium was attained from inulin of 13.8 % and
dried cell mass in the culture was 24.0 g/l within 108 h. It also
can be observed from the data in Fig. 8 that the yield of 0.58±
0.04 g/g of the consumed inulin and the productivity of 0.65±
0.1 g/l/h were reached within 108 h of the fermentation.
Furthermore, 1.69 % total sugar and only a small amount of
reducing sugar (1.34 %) were left in the fermented medium
(Fig. 8), suggesting that 87.74 % of the total sugar was used
for EPS production and cell growth by the recombinant strain

Fig. 7 Time course of pullulan

production (empty diamond),
inulinase production (empty
circle), cell growth (empty
square) and changes in reducing
sugar (empty triangle) and total
sugar (filled circle) during the
cultivation of the recombinant
strain EI36 (a) and its wild-type
strain P16 (b) at flask level. Data
are given as mean±SD, n=3
Mar Biotechnol (2015) 17:511–522 519

EI36. It is interesting to note from Fig. 8 that during the 10-l

fermentation, high inulinase activity (42.03 U/ml) was also
produced within 60 h and the added inulin was actively hy-
drolyzed, leading to rapid increase in reducing sugar. This
meant that the recombinant strain EI36 could synthesize and
secrete a large amount of inulinase into medium for inulin
hydrolysis under the fermentation conditions used in this
study.

Characterization of the Purified Pullulan from Inulin

Fig. 8 Time course of pullulan production (filled circle), inulinase
production (filled diamond), cell growth (empty circle) and changes in
reducing sugar (filled square) and total sugar (empty square) during the
cultivation of the recombinant strain EI36 in the fermentor. Data are given
as mean±SD, n=3
After the purified EPS was hydrolyzed by the pullulanase
bought from Sigma, a large amount of reducing sugar was
released from the EPS, demonstrating that the purified EPS
was pullulan (data not shown). It can be clearly observed from
the data in Fig. 9 that FT-IR spectra for the standard pullulan
(Sigma) used as a reference and for the purified EPS produced

Fig. 9 FTIR spectra of the

purified EPS (a) and standard
pullulan (b)
520
by the recombinant strain EI36 were almost identical. Both the
spectra of the standard pullulan and the purified EPS showed a
strong absorption at a stretching frequency of 3,288.40 m−1
which was a characteristic of the presence of repeating units of
OH groups in sugars. The absorption at 2,926.48 cm−1 indi-
cated the presence of sp3 hybridized C–H bonds, a character-
istic of alkane compounds in both the cases. The presence of
α-D-glucopiranoside bond was confirmed by absorption at
848.01 cm−1 whereas absorption at 757.92 cm−1 indicated
the presence of α-(1,4)-D-glucosidic bond, and absorption at
1,017.42 cm−1 proved the presence of α-(1,6)-D-glucosidic
linkages. These absorption patterns at various stretching fre-
quencies confirmed the identity of the purified EPS as
pullulan (Ma et al. 2014; Choudhury et al. 2011).
Discussion
From Fig. 1, it can be seen that inulinase can play an important
role in inulin hydrolysis for direct conversion of inulin into
pullulan by A. melanogenum strain P16 which has been con-
firmed to produce high level of pullulan (Ma et al. 2014). As
the pullulan-producing yeast has very low inulinase activity,
inulin in the medium can not be directly and efficiently trans-
formed into fructose and glucose for pullulan biosynthesis
(Fig. 3) (Shin et al. 1989). So far, it has been reported that
inulinase from different sources has high activity against inu-
lin (Liu et al. 2013), and invertase and β-fructofuranosidase
also have low inulinase activity (Li et al. 2013b; Arrizon et al.
2012). However, after analysis of the genomic DNA sequence
from A. melanogenum (http://genome.jgi-psf.org/Aurpu_var_
mel1/Aurpu_var_mel1.home.html), it was found that the
genomic DNA did not contain the gene encoding inulinase,
but had the gene encoding β-fructofuranosidase
(gi|662010380|gb|AYEN01000002.1|) (Gostinčar et al.
2014). Therefore, the low inulinase activity produced by A.
melanogenum strain P16 may be due to the presence of β-
fructofuranosidase. Many results have shown that different
strains of K. maximum have the highest inulinase activity
(Liu et al. 2013). In this case, we think that it is very important
to genetically engineer the pullulan-producing yeast used in
this study by cloning and active expressing of the INU1 gene
from K. maximum in it. After the INU1 gene from K.
maximum KM was integrated into the genomic DNA and
actively expressed in A. melanogenum strain P16 (Figs. 2, 3,
and 4), it was found that inulinase production by the recom-
binant strain EI36 was greatly enhanced (Fig. 5). So far,
inulinase genes from different sources have been over-
expressed in the Saccharomyces cerevisiae mutant (Δgal80),
Saccharomyces sp. W0, Pichia pastoris, and Yarrowia
lipolytica ACA-DC 50109 (Zhao et al. 2010; Liu et al. 2011;
Wang et al. 2011; Zhang et al. 2010; Liu et al. 2012; He et al.
2014). Therefore, this is the first time to show that the
Mar Biotechnol (2015) 17:511–522
inulinase gene from K. maximum can be actively expressed
in A. melanogenum. This also confirmed that the expression of
the INU1 gene indeed could enhance biosynthesis and secre-
tion of the extracellular inulinase in the recombinant strain
EI36 used in this study.
So far, only some A. pullulans strains deficient in melanin
production were used for pullulan production (Youssef et al.
1998). Therefore, this is the first time to report that the genet-
ically engineered Aureobasidium strain could be used for di-
rect pullulan production from inulin.
The recombinant strain EI36 grown in the inulinase pro-
duction medium yielded 40.94 U/ml within 72 h of the culti-
vation (Fig. 5). In contrast, its wild-type strain P16 produced
only 7.6 U/ml within 72 h of the cultivation under the same
conditions (Fig. 5). Even when the recombinant strain EI36
was grown the pullulan production medium, it could produce
high level of inulinase within the short period (Figs. 7 and 8).
It has been well evidenced that a high ratio of carbon to nitro-
gen in the production medium is crucial for high pullulan
biosynthesis (Ma et al. 2014; Chi and Zhao 2003). However,
such conditions are usually not suitable for enzyme produc-
tion. Moreover, it is very important for the pullulan producer
to have the ability to produce both pullulan and inulinase
during the fermentation in order to directly and efficiently
converse inulin into pullulan (Fig. 1). It also can be seen from
the data in Fig. 6 that most of the inulinase synthesized by the
recombinant strain EI36 was secreted into the medium. All
these results demonstrated that A. melanogenum strain P16
was a good host for metabolic engineering and the recombi-
nant strain EI36 was suitable for direct pullulan production
from inulin. So far, among the yeasts, S. cerevisiae,
P. pastoris, Y. lipolytica, Kluyveromyces lactis, Hansenula
polymorpha, Schizosaccharomyces pombe, and Arxula
adeninivorans have been widely used for expression of the
heterologous genes and secretion of foreign proteins and en-
zymes (Çelik and Çal 2012). Therefore, this is the first time to
report that Aureobasidium sp. also can be a good host for
expression of the heterologous genes and secretion of foreign
proteins and enzymes.
At the same time, the optimal temperature and pH of the
crude inulinase produced by the recombinant strain EI36 were
60 °C and 4.5 (data not shown). Furthermore, the crude
inulinase was stable below 30 °C and the inulinase activity
was positively proportional to inulin concentrations in the
range from 20.0 to 140.0 g/l (data not shown). However, the
optimal temperature and pH of the native inulinase produced
by K. marxianus were 50 °C and 4.5 (Laloux et al. 1991). This
meant that the optimal temperature of the crude inulinase pro-
duced by the recombinant strain EI36 was increased. The
reasons for this are still completely unknown.
During the cultivation at flask level, 53.17±0.61 g/l of
pullulan produced by the recombinant strain EI36 was reached
within 96 h of the cultivation while 37.10±0.68 g/l of pullulan
Mar Biotechnol (2015) 17:511–522
produced by the wild-type strain P16 was reached within 84 h
of the cultivation (Fig. 7). However, 65.3±2.1 g/l pullulan
from sucrose was produced within 120 h of the cultivation
by the wild-type A. melanogenum P16 and the cell mass was
18.7 g/l at the flask level (Ma et al. 2014). Maximum pullulan
concentration (51.5 g/l) was achieved after 120 h of fermen-
tation when A. pullulans P56 (a strain deficient in melanin
production) was grown in the medium containing 8.0 % su-
crose (Youssef et al. 1998). When A. pullulans CGMCC1234
was grown in the medium containing 0.5 % Tween 80, 8.0 %
sucrose, and 0.09 % yeast extract, 55.5 g/l of pullulan was
accumulated in the fermented medium (Shen et al. 2013).
A. pullulans produced the highest pullulan (23.1 g/l) when
the initial ammonium sulfate was at 0.5 % (Cheng et al.
2011). A. pullulans CJ001 isolated from sea mud produced
31.25 g/l of melanin-free pullulan (Wu et al. 2012).
However, when A. pullulans RBF 4A3 isolated from flowers
of Caesulia axillaris was grown in the medium containing
2.0 % corn steep liquor (CSL) and 150 g/l glucose, it produced
88.59 g/l pullulan (Sharma et al. 2013). This meant that the
recombinant strain EI36 obtained in this study still can pro-
duce more EPS than most of the yeast strains.
During the 10-l fermentation, 70.57±1.3 g/l of EPS was
yielded within 108 h, the yield was 0.58±0.04 g/g of the
consumed inulin and the productivity was 0.65±0.1 g/l/ h
(Fig. 8). This meant that the recombinant strain EI36 could
produce much more pullulan during the fermentation than
during the cultivation at flask level due to the fact that the
conditions for pullulan biosynthesis and secretion in the fer-
mentor were better than those in the flask. During the 10-l
fermentation, 67.4±4.5 g/l of EPS in the fermented medium
was attained from sucrose by the wild-type strain P16 and
dried cell mass in the culture was 23.01 g/l within 120 h
(Ma et al. 2014). In the 7-l Applikon fermenter with 5-l work-
ing volume, at the end of 96 h of fermentation, 88.6 g/l of
pullulan was produced by A. pullulans RBF 4A3, and the
yield was 0.6 g/g of the consumed glucose and the productiv-
ity was 0.9 g/l/h (Sharma et al. 2013).
An undesirable characteristic feature of A. pullulans is the
production of a melanin-like compound as the presence of
melanin in the culture will make purification of pullulan more
complex (Chi et al. 2009). Therefore, many native strains or
mutants which are deficient in melanin production were used
for pullulan production (Wu et al. 2012). Fortunately, during
the fermentation, like A. melanogenum P16, the recombinant
strain EI36 used in this study did not produce any melanin
within the period of the cultivation, either (data not shown).
Like A. melanogenum P16, the EPS produced by the re-
combinant strain EI36 was also pullulan (Fig. 9).
Acknowledgments This work was supported by Hi-Tech Research and
Development Program of China (863) (grant no. 2013BAB01B05).
521
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