Bioprocess Biosyst Eng (2015) 38:399–405
DOI 10.1007/s00449-014-1279-1
ORIGINAL PAPER
Increased polysaccharide production and biosynthetic gene
expressions in a submerged culture of Ganoderma lucidum
by the overexpression of the homologous a-phosphoglucomutase
gene
Jun-Wei Xu • Sen-Lin Ji • Huan-Jun Li •
Jiang-Sheng Zhou • Yan-Qing Duan •
Li-Zhi Dang • Ming-He Mo
Received: 29 June 2014 / Accepted: 31 August 2014 / Published online: 14 September 2014
 Springer-Verlag Berlin Heidelberg 2014
Abstract This study aimed to improve the production of
polysaccharide by engineering the biosynthetic pathway in
Ganoderma lucidum through the overexpression of a-
phosphoglucomutase (PGM) gene. PGM is responsible for
the linkage between sugar catabolism and sugar anabolism.
The effects of PGM gene overexpression on intracellular
polysaccharide (IPS) content, extracellular polysaccharide
(EPS) production and transcription levels of three genes
encoding the enzymes involved in polysaccharide biosyn-
thesis, including PGM, UDP-glucose pyrophosphorylase
(UGP), and b-1,3-glucan synthase (GLS), were investi-
gated. The maximum IPS content and EPS production in G.
lucidum overexpressing the PGM gene were 23.67 mg/
100 mg dry weight and 1.76 g/L, respectively, which were
higher by 40.5 and 44.3 % than those of the wild-type
strain. The transcription levels of PGM, UGP and GLS
were upregulated by 4.77-, 1.51- and 1.53-fold, respec-
tively, in the engineered strain, suggesting that increased
polysaccharide biosynthesis may result from a higher
expression of those genes.
J.-W. Xu and S.-L. Ji have contributed equally to this work.
J.-W. Xu (&)  S.-L. Ji  H.-J. Li  J.-S. Zhou
Faculty of Life Science and Technology, Kunming University of
Science and Technology, Kunming 650500, China
e-mail: jwxu@kmust.edu.cn; xjuwei@163.com
Y.-Q. Duan  L.-Z. Dang
Hongyun Honghe Tobacco (Group) Co. Ltd,
Kunming 650202, China
M.-H. Mo (&)
Laboratory for Conservation and Utilization of Bio-resources
and Key Laboratory for Microbial Resources of the Ministry of
Education, Yunnan University, Kunming 650091, China
e-mail: minghemo@163.com
Keywords Polysaccharide  Ganoderma lucidum 
Genetic engineering  a-Phosphoglucomutase (PGM) 
Biosynthetic genes
Introduction
Medicinal mushrooms have attracted much attention as
sources for the development of drugs and nutraceuticals in
recent years [19, 34]. Ganoderma lucidum, a famous
medicinal mushroom, is used as a medication for the pre-
vention and treatment of various human diseases for cen-
turies in Asia [30, 32]. Currently, much interest is now
focused on the polysaccharide fractions containing b-1,3-
and 1,6-glucans produced by G. lucidum due to their
important biological activities such as anti-oxidant, anti-
cancer, anti-inflammatory, and immunomodulatory activi-
ties [28, 29]. The biosynthetic pathways of polysaccharides
involve the biosynthetic pathways for the nucleotide sugar
precursors, assembly of the repeating monosaccharide
units, and the process of polymerization [6, 11, 21, 23].
Among the polysaccharide biosynthetic enzymes, phos-
phoglucomutase (PGM) and UDP-glucose pyrophosphor-
ylase (UGP) are important enzymes in the biosynthetic
pathway of nucleotide sugar precursors. PGM catalyzes the
inter conversion of glucose-6-phosphate into glucose-1-
phosphate; UGP catalyzes the reversible conversion of
glucose-1-phosphate and UTP into UDP-D-glucose. b-1,3-
glucan synthase (GLS) catalyzes the repetitive addition of
glucose from UDP-glucose to growing glycan chains [6,
11, 23].
Submerged cultures offer a promising alternative to
obtain large quantities of polysaccharides and are fast,
cost-effective, and easy to control. Many medicinal
mushroom polysaccharides are being commercially
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produced by submerged cultures [5, 9, 10]. In addition
to intracellular polysaccharide (IPS) obtainable from the
biomass, mycelia cultures also excrete extracellular
polysaccharide (EPS) into the fermentation broth.
Polysaccharides of G. lucidum have gained wide
attention due to their important bioactivities. Thus,
several approaches are adopted to increase its IPS and
EPS production. These include the manipulation of
fermentation conditions [1, 8, 9, 13, 16], addition of
inducers [14, 18, 37, 38], and development of new
bioprocessing strategies [15, 24, 35, 39]. However,
higher yields of polysaccharide are needed for com-
mercial applications. A highly productive strain is the
primary factor to achieve an industrial level polysac-
charide production. One promising approach to increase
polysaccharide yield is to manipulate the expression
levels of the biosynthetic genes [4, 17, 25]. Genetic
manipulation has been successfully used to enhance
exopolysaccharide production in Streptococcus thermo-
philus [17], Lactococcus lactis [4] and Sphingomonas
S7 [25]. An engineered strain for efficient production of
polysaccharide may allow for a sustainable and indus-
trial scale production of these valuable metabolites.
However, no study reported the enhancement of poly-
saccharide production through genetic manipulation of
basidiomycetes.
PGM is a key enzyme in the biosynthetic pathway of
nucleotide sugar precursors. This enzyme catalyzes a step
representing a branch point in carbohydrate metabolism;
glucose-6-phosphate enters catabolic processes to yield
energy and reducing power, whereas glucose-1-phosphate
is a precursor of sugar nucleotides that are used by cells in
the synthesis of various polysaccharides [21]. Expression
of PGM gene had a positive correlation with the polysac-
charide production in several bacteria such as Streptococ-
cus [7], Lactobacillus helveticus [26] and Pediococcus
parvulus [27]. In Streptococcus pneumoniae, deletion of
PGM gene decreased capsule biosynthesis to \10 % [12].
These observations indicated that the PGM gene is a vital
regulatory gene for polysaccharide biosynthesis. In G. lu-
cidum, Tang et al. [23] reported that there is a linear
relationship between the PGM activity and EPS produc-
tion. However, the regulation of polysaccharide biosyn-
thesis by overexpression of the PGM gene remains to be
elucidated.
In this study, we investigated the effects of the
overexpression of PGM gene on cell growth, IPS con-
tent, EPS production and transcription levels of three
biosynthetic genes, namely, PGM, UGP, and GLS, in a
submerged culture of G. lucidum. This study is helpful
for further investigations on the polysaccharide biosyn-
thesis regulation and the development of more efficient
mushroom fermentations for polysaccharide production.
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Bioprocess Biosyst Eng (2015) 38:399–405
Materials and methods
Strain and culture conditions
Ganoderma lucidum CGMCC 5.616 from the China Gen-
eral Microbiological Cultivation Center was maintained on
potato dextrose agar slants and cultured at 30 C for
7 days. The details of preculture medium and conditions
were described earlier [30, 36]. For the shake flask fer-
mentation, a 45-mL medium in a 250-mL flask was inoc-
ulated with 5 mL of second-stage preculture broth [at an
inoculum size of 300–330 mg dry weight (DW)/L]. The
culture was incubated in the dark at 30 C on a rotary
shaker at 120 rpm for 12 days. For plasmid construction,
Escherichia coli strain DH5a was used and grown on
Luria–Bertani (LB) agar plates containing 100 lg/mL
ampicillin. CYM medium (1 % maltose, 2 % glucose,
0.2 % yeast extract, 0.2 % tryptone, 0.05 % MgSO4,
0.46 % KH2PO4, 0.6 M mannitol, 1 % agar) was used for
regeneration of protoplasts from G. lucidum.
Vector construction
The plasmid pJW-EXP [33] was used as a backbone to
construct the vector pJW-EXP-PGM. The G. lucidum PGM
gene was amplified from genome DNA using primers
PGM-NheI-F (50 -GCTAGCATGTCGTACCAGGTCAAG
GAG-30 ) and PGM-SmaI-R(50 -GGGCCCCTACGTGATG
ACGGTCGGC-30 ). The vector pJW-EXP-PGM was made
by digesting pJW-EXP with both NheI and SmaI, and
inserting the NheI-SmaI PCR fragment containing the
G. lucidum PGM gene.
Transformation of G. lucidum and molecular analysis
of the transformants
Protoplasts of G. lucidum were prepared as described by Xu
et al. [31]. The expression vector pJW-EXP-PGM was trans-
ferred into G. lucidum protoplasts according to the method
described by us earlier [33].The transformants were selected
and transferred to fresh CYM medium containing carboxin
(2 lg/mL), which was repeated five times to obtain stable
transformants. The stable transformants were identified for
integration of the fusion fragment of gpd promoter and PGM
gene by PCR using primers gpd-F (50 -GGGTTCCCCTCG
TTCAAGC-30 ) and PGM-R(50 - AGCGCAAGTCAGCATTC
TCATAT-30 ) that should give a 1.37-kb fragment.
Sampling, analysis of cell dry weight, residual sugar
in medium, EPS production and IPS content
Mycelia were harvested by centrifuging a sample at
10,000g for 10 min, and the precipitated cells were washed
Bioprocess Biosyst Eng (2015) 38:399–405
for three times with distilled water and then dried at 50 C
to constant weight. The dry cell weight was measured by
the gravimetric method. The residual sugar was measured
according to the method described by Miller [20]. For the
determination of EPS, after removal of mycelia by centri-
fugation, the crude EPS was precipitated with addition of
95 % (v/v) ethanol by four times of volume, then separated
by centrifugation at 13,000 rpm. The insoluble components
were suspended in 1 M NaOH at 60 C for 1 h, and the
supernatant was measured by phenol–sulfuric acid method.
For the analysis of IPS, the dried mycelia (100 mg) were
extracted by using 1 M NaOH at 60 C (1 h), then the
supernatant was assayed by phenol–sulfuric acid method.
Nucleic acids extraction and cDNA synthesis
Ganoderma lucidum mycelia were harvested, frozen in
liquid nitrogen, and ground to a fine powder with a mortar
and pestle. The genomic DNA was extracted using the
CTAB method, and the total RNA was extracted using
TriZol Reagent (Invitrogen, Carlsbad, CA, USA). The
quality and quantity of DNA and RNA samples were
determined by ethidium bromide stained agarose gel
electrophoresis and spectrophotometric measurements.
Residual genomic DNA in isolated total RNA was removed
using RNase-free DNase I (MBI Fermentas, Canada)
according to the manufacturer’s protocol. Reverse tran-
scription was achieved using total RNA as starting material
and the Superscript RNAase H- First-strand synthesis kit
(Invitrogen, Carlsbad, CA, USA).
Measure of pgm, ugp and gls expression by real-time
quantitative PCR (qRT-PCR)
The transcription levels of the pgm, ugp, and gls (which
encode the enzymes PGM, UGP, and b-1,3-glucan syn-
thase, respectively) were analyzed by qRT-PCR. The
sequences of the primer for amplification of 18S rRNA were
described previously [31]. For pgm, ugp and gls, the fol-
lowing primer sets were used: pgm-forward, 50 -GGGCCT
GAGGAAGAGGGTGA-30 and pgm-reverse, 50 -CGGTTT
CGGGGGAGAAGTAG-30 ; ugp-forward, 50 -TGGTCTCG
GAACTTCTATGGG-30 and ugp-reverse, 50 -CAGTGCTT
CTTCTCGTCCTCA-30 ; gls-forward, 50 -TCGTTTGGGTT
GGGTCTGT-30 and gls-reverse, 50 -GAAGCCCTTGTCGC
TCTGC-30 . The 18S rRNA gene was used as the internal
control gene, because its expression was found to be stable
under our experimental conditions. The expression level of
the different genes was normalized with respect to the 18S
rRNA gene expression level. For each gene, an expression
level of 1 was assigned to the samples from the wild-type
(WT) strain, and the expression levels in G. lucidum over-
expressing the PGM gene are presented as the fold changes
401
relative to this reference level. Post qRT-PCR calculations
to analyze relative gene expression were performed
according to the 2DDCT method.
Statistical analysis
Data are the averages of three independent sample mea-
surements. The error bars indicate the standard deviations
from the means of triplicates. The data were analyzed with
the Student t test. The difference between contrasting
treatments was considered significant when P was\0.05 in
a two-tailed analysis.
Results and discussion
Overexpression of PGM gene in G. lucidum
Recently, the genome sequence of G. lucidum has been
reported [6]. In this study, we successfully amplified the
PGM gene from the genome of G. lucidum. The genomic
sequence was submitted to the DDBJ/EMBL/GenBank
nucleotide sequence database under accession no.
KJ591029. The entire genome DNA of G. lucidum PGM
was 2038-bp long and has an open reading frame of 579
amino acids giving a predicted molecular mass of
63.5 kDa. The 579-amino acid protein product of PGM
showed 85 % identity to PGM of Dichomitus squalens,
79 % identity to PGM of Trametes versicolor, 52 %
identity to PGM of Neurospora crassa, and 51 % identity
of PGM of Agrobacterium tumefaciens. The amino acid
sequences and locations of two peptide segments, which
are useful to identify PGM are shown in Fig S1. The first
and second segments contain the amino acids (G… T.
SHNP and D. D. D, respectively) believed to comprise a
highly conserved site residing in a deep crevice of the
enzyme, which is involved in phosphate transfer. Phylo-
genetic analysis also demonstrated the homology between
G. lucidum PGM and PGMs from various organisms
(Fig. S2).
To better increase the carbon flux to a sufficiently high
level to improve polysaccharide production, the gene
encoding G. lucidum a-phosphoglucomutase, which cata-
lyzes conversion of the glycolytic intermediate glucose
6-phosphate to glucose 1-phosphate was overexpressed.
The expression vector pJW-EXP-PGM, which encodes the
PGM gene from the G. lucidum genome that contained
introns, was constructed (Fig. 1a) and used to transform the
WT strain using a homologous transformation system
developed in our laboratory [33]. The PGM gene was
controlled through the strongly constitutive glycer-
aldehyde-3-phosphate dehydrogenase gene (gpd) promoter
and the iron–sulfur protein subunit of succinate
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402 Bioprocess Biosyst Eng (2015) 38:399–405

Fig. 1 a Structure of the vector pJW-EXP-PGM used for G. lucidum
transformation. See methods section for descriptions on the construc-
tion of this vector. b Identification and characterization of the PGM
gene overexpressed (PGM) strain. Amplification pattern obtained
with primers for the fusion gpd promoter-PGM gene fragment in
genomic DNA isolated from different strains. lane N negative control,
lane WT wild-type strain, Line PGM the PGM gene overexpressed
strain, lane P pJW-EXP-PGM as positive control, lane M DL 2000
DNA marker

dehydrogenase gene (sdhB) terminator of G. lucidum in the

vector pJW-EXP-PGM. The carboxin resistance was used
to select putative transformants. The putative transformants
were transferred to a selective CYM medium after five
rounds of growth on a non-selective medium (without
carboxin antibiotics). All tested transformants continued to
grow on the selective medium. The integration of the PGM
gene in the carboxin-resistant strains was confirmed with
PCR. The PCR product showed a clear band for the fusion
gpd promoter and PGM gene fragment (1.37 kb) in the
PGM transformant. However, no corresponding band was
observed in the WT strain (Fig. 1b). In addition, no mor-
phological difference was observed between the PGM
Fig. 2 Transcriptional levels of three biosynthetic genes, namely,
transformant and WT strain (data not shown). The total
PGM (blank), UGP (gray) and GLS (dark), in the WT strain and the
RNA of different strains was isolated. The qRT-PCR PGM strain. Expression of the samples from the WT strain is defined
analysis was conducted to compare the PGM gene as 1.0, and expression levels in the PGM strain are displayed as the
expression levels in the transformant and WT strain. The fold increase over the reference sample. d days, asterisk indicates
statistical significance (P \ 0.05) compared to the WT
results showed that the PGM gene was overexpressed in the
PGM transformant by about twofolds higher than that of
the control on day 0 (Fig. 2).
EPS production were significantly higher compared with
Enhanced polysaccharide production in G. lucidum that of the WT stain. The results confirmed the consistency
overexpressing the PGM gene of the PGM gene overexpression. One overexpressing
transformant (PGM1) was chosen with the WT strain to
The maximum biomass, IPS content and EPS production study the kinetic profiles of cell growth, sugar consump-
were assayed in the WT and three PGM-overexpressing tion, IPS content, and EPS production. Figure 3 shows that
strains. The maximum biomass of all strains was reached the maximum dry cell weights in the WT and PGM strains
on day 12, and the maximum IPS content and EPS pro- were 7.48 and 8.12 g/L, respectively. Compared to the WT
duction were obtained on days 9 and 12, respectively. The strain, overexpression of the PGM gene only slightly
IPS content and EPS production in the three pJW-PGM affected cell growth. The time profiles of IPS content and
transformed G. lucidum strains, namely, PGM1, PGM2, EPS production in the WT and PGM strains are shown in
and PGM3, were shown in Table 1. The IPS content and Fig. 4. For the PGM strain, IPS content reached the highest

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Bioprocess Biosyst Eng (2015) 38:399–405 403

level on day 9 and then gradually decreased, thereafter, fermentation. The maximum IPS content in the PGM strain
although still at a higher level than the WT. The EPS was 23.67 mg/100 mg DW, which was about 1.4-fold of
production showed similar profiles in the WT and PGM that obtained in the WT strain (13.51/100 mg DW). The
strains. The EPS production increased during the fermen- maximum IPS content in the PGM strain was 1.26 times
tation and reached its maximum value at the end of higher than that reported for a submerged culture of G.
lucidum by simultaneously adopting a strategy of multiple
Table 1 Maximum IPS content and EPS production in different cell Cu2? additions, three-stage light irradiation and multi-
lines pulse feeding of carbon and nitrogen sources [39].The
Cell Biomass IPS content EPS production maximum EPS production in the PGM strain was 1.76 g/L,
line (g/L) (mg/100 mg DW) (g/L) which was 1.44 higher than that obtained in the WT strain.
In Sphingomonas S7, the overexpression of PGM gene
WT 7.53 ± 0.26 14.16 ± 1.03 1.19 ± 0.03*
resulted in a small increase of S-7 polysaccharide pro-
PGM1 8.31 ± 0.31 24.37 ± 2.18* 1.84 ± 0.04*
duction [25]. In S. thermophilus, Levander et al. [17]
PGM2 7.85 ± 0.42 21.21 ± 2.15* 1.72 ± 0.05*
reported that the overexpression of PGM gene in combi-
PGM3 8.02 ± 0.28 23.23 ± 1.80* 1.78 ± 0.02*
nation with UDP-glucose pyrophosphorylase gene
The maximum IPS content and EPS production were reached on days enhanced the EPS synthesis about twofolds, although no
9 and 12, respectively effect could be seen when either gene was overexpressed
mg DW milligram dry weight alone. Our result is the first observation on the enhanced
* Significantly different from value for WT (P \ 0.05) polysaccharide production through the overexpression of
biosynthetic genes in mushroom. The flux-controlling
function of PGM may be dependent on strain and the
produced type of polysaccharide [2, 3, 7]. The overex-
pression of PGM gene resulted in an enhanced accumula-
tions of IPS and EPS, which indicated that PGM is a key
enzyme for polysaccharide biosynthesis in G. lucidum. The
results agree with previous reports, which showed that
PGM activity correlated with production of polysaccharide
produced by S. thermophiles [7] and G. lucidum [23].

Upregulation of polysaccharide biosynthetic gene

Fig. 3 Kinetic profiles of cell growth (circles) and residual sugar
(triangles) in the WT strain (blank) and the PGM strain (dark). The
error bars indicate the standard deviations from three independent
samples. d days, asterisk indicates statistical significance (P \ 0.05)
compared to the WT
expressions in G. lucidum overexpressing the PGM
gene
Previous studies have reported that the increased pro-
duction of polysaccharide accompanied the upregulated

Fig. 4 Kinetic profiles of IPS content (a) and EPS production (b) in samples. d days, asterisk indicates statistical significance (P \ 0.05)
the WT strain (blank circles) and the PGM strain (dark circles). The compared to the WT
error bars indicate the standard deviations from three independent

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expression of the biosynthetic genes [4, 22]. To explore
the underlying molecular mechanism for the higher
polysaccharide production in G. lucidum overexpressing
the PGM gene, the expression kinetics of three important
genes, namely, PGM, UGP, and GLS in the WT and
PGM strains were examined using qRT-PCR. Gene
expression was normalized against the expression level of
the 18S rRNA gene. Figure 2 showed that the overex-
pression of the PGM gene caused the evident accumu-
lation of PGM mRNA throughout the cultivation period.
The transcription levels of PGM gene on days 6 and 12
in the PGM strain were about 4.2 and 4.77 times higher
than that of the WT strain, respectively. In the case of the
UGP gene, the highest transcription level in the PGM
strain was about 1.5 times higher than the control on day
3. For the GLS gene, the largest changes in the tran-
scription profiles were observed on day 6 when it was
upregulated 1.53-fold in the PGM strain. Overexpression
of the PGM gene may lead to an increased accumulation
of intermediates, which may stimulate the transcription of
downstream genes involved in polysaccharide biosynthe-
sis. The mRNA levels of those genes remained much
higher during day 3–day 6 in the PGM strain than that of
the WT strain. These trends were consistent with those
observed for polysaccharide accumulation levels, sug-
gesting the increased transcription level of biosynthesis
genes could contribute to the enhanced polysaccharide
production in the PGM strain.
Conclusions
The results showed that the PGM gene is an important
regulatory gene for the biosynthesis of polysaccharide in G.
lucidum. Overexpression of the PGM gene resulted in an
increased polysaccharide production and upregulation of
downstream genes in a submerged culture of G. lucidum.
The constructed engineered strain represents a suitable
basis for the further development of an industrial process
for the hyperproduction of polysaccharide in G. lucidum.
Acknowledgments Financial support from the National Natural
Science Foundation of China (31360495), the start-up grant from
Kunming University of Science and Technology (KKSY201226107),
Department of Science and Technology of Yunnan Province
(2012BA015), CNTC (110201201009 BR-03) and Hongyun Honghe
Tobacco (Group) Co. Ltd. (HYHH2012HX06) is gratefully
acknowledged.
References
1. Babitskaya VG, Shcherba VV, Puchkova TA, Smirnov DA
(2005) Polysaccharides of Ganoderma lucidum: factors affecting
their production. Appl Biochem Microbiol 41(2):169–173
123
Bioprocess Biosyst Eng (2015) 38:399–405
2. Boels IC, Kleerebezem M, de Vos WM (2003) Engineering of
carbon distribution between glycolysis and sugar nucleotide
biosynthesis in Lactococcus lactis. Appl Environ Microbiol
69(2):1129–1135
3. Boels IC, Ramos A, Kleerebezem M, De Vos WM (2001)
Functional analysis of the Lactococcus lactis galU and galE
genes and their impact on sugar nucleotide and exopolysaccha-
ride biosynthesis. Appl Environ Microbiol 67(7):3033–3040
4. Boels IC, van Kranenburg R, Kanning MW, Chong BF, de Vos
WM, Kleerebezem M (2003) Increased exopolysaccharide pro-
duction in Lactococcus lactis due to increased levels of expres-
sion of the NIZO B40 eps gene cluster. Appl Environ Microbiol
69(8):5029–5031
5. Chen H, Yan M, Zhu J, Xu X (2011) Enhancement of exo-
polysaccharide production and antioxidant activity in submerged
cultures of Inonotus obliquus by lignocellulose decomposition.
J Ind Microbiol Biotechnol 38(2):291–298
6. Chen SL, Xu J, Liu C, Zhu YJ, Nelson DR, Zhou SG, Li CF,
Wang LZ, Guo X, Sun YZ, Luo HM, Li Y, Song JY, Henrissat B,
Levasseur A, Qian J, Li JQ, Luo X, Shi LC, He L, Xiang L, Xu
XL, Niu YY, Li QS, Han MV, Yan HX, Zhang J, Chen HM, Lv
AP, Wang Z, Liu MZ, Schwartz DC, Sun C (2012) Genome
sequence of the model medicinal mushroom Ganoderma lucidum.
Nat Commun 3:913
7. Degeest B, De Vuyst L (2000) Correlation of activities of the
enzymes alpha-phosphoglucomutase, UDP-galactose4-epimer-
ase, and UDP-glucose pyrophosphorylase with exopolysaccha-
ride biosynthesis by Streptococcus thermophilus LY03. Appl
Environ Microbiol 66(8):3519–3527
8. Fang QH, Tang YJ, Zhong JJ (2002) Significance of inoculation
density control in production of polysaccharide and ganoderic
acid by submerged culture of Ganoderma lucidum. Process
Biochem 37(12):1375–1379
9. Fang QH, Zhong JJ (2002) Submerged fermentation of higher
fungus Ganoderma lucidum for production of valuable bioactive
metabolites-ganoderic acid and polysaccharide. Biochem Eng J
10(1):61–65
10. Gao H, Gu WY (2007) Optimization of polysaccharide and
ergosterol production from Agaricus brasiliensis by fermentation
process. Biochem Eng J 33(3):202–210
11. Gastebois A, Clavaud C, Aimanianda V, Latge JP (2009)
Aspergillus fumigatus: cell wall polysaccharides, their biosyn-
thesis and organization. Future Microbiol 4(5):583–595
12. Hardy GG, Caimano MJ, Yother J (2000) Capsule biosynthesis and
basic metabolism in Streptococcus pneumoniae are linked through
the cellular phosphoglucomutase. J Bacteriol 182(7):1854–1863
13. Hsieh C, Tseng MH, Liu CJ (2006) Production of polysaccha-
rides from Ganoderma lucidum (CCRC 36041) under limitations
of nutrients. Enzyme Microb Technol 38(1–2):109–117
14. Huang HC, Chen CI, Hung CN, Liu YC (2009) Experimental
analysis of the oil addition effect on mycelia and polysaccharide
productions in Ganoderma lucidum submerged culture. Bioproc
Biosyst Eng 32(2):217–224
15. Lee KM, Lee SY, Lee HY (1999) Bistage control of pH for
improving exopolysaccharide production from mycelia of
Ganoderma lucidum in an air-lift fermentor. J Biosci Bioeng
88(6):646–650
16. Lee WY, Park Y, Ahn JK, Ka KH, Park SY (2007) Factors
influencing the production of endopolysaccharide and exopoly-
saccharide from Ganoderma applanatum. Enzyme Microb
Technol 40(2):249–254
17. Levander F, Svensson M, Radstrom P (2002) Enhanced exo-
polysaccharide production by metabolic engineering of Strepto-
coccus thermophilus. Appl Environ Microbiol 68(2):784–790
18. Liu GQ, Zhang KC (2007) Enhancement of polysaccharides
production in Ganoderma lucidum by the addition of ethyl acetate
Bioprocess Biosyst Eng (2015) 38:399–405
extracts from Eupolyphaga sinensis and Catharsius molossus.
Appl Microbiol Biotechnol 74(3):572–577
19. Liu YS, Wu JY (2012) Effects of Tween 80 and pH on mycelial
pellets and exopolysaccharide production in liquid culture of a
medicinal fungus. J Ind Microbiol Biotechnol 39(4):623–628
20. Miller GL (1959) Use of dinitrosalicylic acid reagent for deter-
mination of reducing sugars. Anal Chem 31:426–428
21. Sa-Correia I, Fialho AM, Videira P, Moreira LM, Marques AR,
Albano H (2002) Gellan gum biosynthesis in Sphingomonas
paucimobilis ATCC 31461: genes, enzymes and exopolysaccha-
ride production engineering. J Ind Microbiol Biotechnol 29(4):
170–176
22. Spatafora G, Rohrer K, Barnard D, Michalek S (1995) A Strep-
tococcus mutans mutant that synthesizes elevated levels of
intracellular polysaccharide is hypercariogenic in vivo. Infect
Immun 63(7):2556–2563
23. Tang YJ, Zhong JJ (2002) Exopolysaccharide biosynthesis and
related enzyme activities of the medicinal fungus, Ganoderma
lucidum, grown on lactose in a bioreactor. Biotechnol Lett
24(12):1023–1026
24. Tang YJ, Zhang W, Zhong JJ (2009) Performance analyses of a
pH-shift and DOT-shift integrated fed-batch fermentation process
for the production of ganoderic acid and Ganoderma polysac-
charides by medicinal mushroom Ganoderma lucidum. Bioresour
Technol 100(5):1852–1859
25. Thorne L, Mikolajczak MJ, Armentrout RW, Pollock TJ (2000)
Increasing the yield and viscosity of exopolysaccharides secreted
by Sphingomonas by augmentation of chromosomal genes with
multiple copies of cloned biosynthetic genes. J Ind Microbiol
Biotechnol 25(1):49–57
26. Torino MI, Mozzi F, Font de Valdez G (2005) Exopolysaccharide
biosynthesis by Lactobacillus helveticus ATCC 15807. Appl
Microbiol Biotechnol 68(2):259–265
27. Velasco SE, Yebra MJ, Monedero V, Ibarburu I, Duenas MT,
Irastorza A (2007) Influence of the carbohydrate source beta-
glucan production and enzyme activities involved in sugar
metabolism in Pediococcus parvulus 2.6. Int J Food Microbiol
115(3):325–334
28. Wagner R, Mitchell DA, Sassaki GL, Amazonas M (2004) Links
between morphology and physiology of Ganoderma lucidum in
submerged culture for the production of exopolysaccharide.
J Biotechnol 114(1–2):153–164
29. Wang SH, Liang CJ, Weng YW, Chen YH, Hsu HY, Chien HF,
Tsai JS, Tseng YC, Li CY, Chen YL (2012) Ganoderma lucidum
polysaccharides prevent platelet-derived growth factor-stimulated
405
smooth muscle cell proliferation in vitro and neointimal hyper-
plasia in the endothelial-denuded artery in vivo. J Cell Physiol
227(8):3063–3071
30. Xu JW, Xu YN, Zhong JJ (2010) Production of individual gan-
oderic acids and expression of biosynthetic genes in liquid static
and shaking cultures of Ganoderma lucidum. Appl Microbiol
Biotechnol 85(4):941–948
31. Xu JW, Xu YN, Zhong JJ (2012) Enhancement of ganoderic acid
accumulation by overexpression of an N-terminal truncated
3-hydroxy-3-methylglutaryl CoA reductase gene in the basidio-
mycete Ganoderma lucidum. Appl Environ Microbiol 78(22):
7968–7976
32. Xu JW, Zhao W, Zhong JJ (2010) Biotechnological production
and application of ganoderic acids. Appl Microbiol Biotechnol
87(2):457–466
33. Yu X, Ji SL, He YL, Reng MF, Xu JW (2014) Development of an
expression plasmid and its use in genetic manipulation of lingzhi
or reishi medicinal mushroom, Ganoderma lucidum (higher
basidiomycetes). Int J Med Mushrooms 16(2):161–168
34. Zhang BB, Cheung PCK (2011) Use of stimulatory agents to
enhance the production of bioactive exopolysaccharide from
Pleurotus tuber-regium by submerged fermentation. J Agric Food
Chem 59(4):1210–1216
35. Zhang W, Tang YJ (2008) A novel three-stage light irradiation
strategy in the submerged fermentation of medicinal mushroom
Ganoderma lucidum for the efficient production of ganoderic
acid and Ganoderma polysaccharides. Biotechnol Prog 24(6):
1249–1261
36. Zhou JS, Ji SL, Ren MF, He YL, Jing XR, Xu JW (2014)
Enhanced accumulation of individual ganoderic acids in a sub-
merged culture of Ganoderma lucidum by the overexpression of
squalene synthase gene. Biochem Eng J 90:178–183
37. Zhu LW, Tang YJ (2010) Significance of protein elicitor isolated
from Tuber melanosporum on the production of ganoderic acid
and Ganoderma polysaccharides during the fermentation of
Ganoderma lucidum. Bioproc Biosyst Eng 33(8):999–1005
38. Zhu LW, Zhong JJ, Tang YJ (2008) Significance of fungal elic-
itors on the production of ganoderic acid and Ganoderma poly-
saccharides by the submerged culture of medicinal mushroom
Ganoderma lucidum. Process Biochem 43(12):1359–1370
39. Zhu LW, Zhong JJ, Tang YJ (2010) Multi-fed batch culture
integrated with three-stage light irradiation and multiple additions
of copper ions for the hyperproduction of ganoderic acid and
Ganoderma polysaccharides by the medicinal mushroom Gano-
derma lucidum. Process Biochem 45(12):1904–1911
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