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ORIGINAL PAPER
Control of apple blue mold by Pichia pastoris recombinant strainsexpressing cecropin A
Xueyan Ren • Qingjun Kong • Huili Wang •
Ting Yu • Ya-Jie Tang • Wen-Wen Zhou •
Xiaodong Zheng
Received: 26 June 2011 / Accepted: 9 November 2011 / Published online: 23 November 2011
� Springer-Verlag 2011
Abstract Recombinant Pichia pastoris yeasts expressing
cecropin A (GS115/CEC), was evaluated for the control of
the blue mold of apple caused by Penicillium expansum
due to cecropin A peptide’s effective antimicrobial effects
on P. expansum spores by the thiazolyl blue (MTT) assay.
Then, the protein concentration was determined and it was
expressed at high levels up to 14.2 mg/L in the culture
medium. Meanwhile, the population growth was assayed in
vivo. The population growth of recombinant strain GS115/
CEC was higher than that of non-transformed strain GS115
in red Fuji apples wounds. Recombinant yeast strains
GS115/CEC significantly inhibited growth of germinated
P. expansum spores in vitro and inhibited decay develop-
ment caused by P. expansum in apple fruits in vivo when
compared with apple fruits inoculated with sterile water or
the yeast strain GS115/pPIC (plasmid pPIC9k transformed
in GS115). This study demonstrated the potential of
expression of the antifungal peptide in yeast for the control
of postharvest blue mold infections on pome fruits.
Keywords Postharvest disease � Penicillium expansum �Biological control � Apple fruit � Blue mold
Introduction
Fresh apples are stored after harvest to provide nutritious
fruit throughout the year [1]. More than 90 fungal species
can cause decay of apples during storage [2], but posthar-
vest fungal diseases of apples are caused mainly by Peni-
cillium expansum [3]. Traditionally, this disease is
controlled by chemical fungicides. However, the potential
impact on the environment as well as human health limits
their application [4]. It is reported that some microbes
become fungicide resistant [5], and thus fungicides’
application in controlling fungal growth may be greatly
reduced. Considering the human health and pollution risks,
some fungicides are prohibited from use in many devel-
oped countries.
Biological control of postharvest decays (BCPD) with
antagonistic yeasts is a promising strategy for postharvest
diseases control [6] and considerable success has been
achieved by utilizing antagonistic microorganisms for
controlling postharvest diseases on citrus, pome fruits,
stone fruits, seed potatoes, and sweet potatoes [7, 8].
However, the major problems with the use of those
products are their insufficient and inconsistent perfor-
mance under commercial conditions. Consequently, they
are mainly used to control wound pathogens but not for
pathogens invading directly through the cuticle and
causing quiescent infection [9]. The current study was
undertaken to investigate the possibility of enhance bio-
control activity of antagonists against fungal pathogens by
expressing a gene encoding cecropin A in yeast for the
production of an antifungal peptide to produce an
X. Ren � H. Wang � T. Yu � W.-W. Zhou (&) � X. Zheng (&)
School of Biosystems Engineering and Food Science,
Zhejiang University, Hangzhou 310058, Zhejiang, China
e-mail: [email protected]
X. Zheng
e-mail: [email protected]
X. Ren � Q. Kong
Department of Life Science,
Shihezi University, Shihezi 832003, Xinjiang, China
Y.-J. Tang
Key Laboratory of Fermentation Engineering
(Ministry of Education), Hubei University of Technology,
Wuhan 430068, Hubei, China
123
Bioprocess Biosyst Eng (2012) 35:761–767
DOI 10.1007/s00449-011-0656-2
improved organism [10]. Genetic manipulation of antag-
onists shows tremendous potential for improving BCPD.
For example, foreign genes can be transferred to antag-
onists to increase its tolerance to environmental stresses
or to produce antifungal substances [11]. In this way, it
may be feasible to convert microorganisms that can col-
onize fruit but do not exhibit antagonistic activity into
biocontrol agents [9, 12].
Antimicrobial peptides are integral components of the
innate immune system. They can counteract outer
membrane pathogen such as bacteria, fungi, viruses,
protozoa, and so on [13]. Insects produce a large amount
of antimicrobial peptides that play a crucial role in
protecting them from invading microorganisms. Insect
antibacterial peptides are classified into five major
groups: cecropins, insect defensins, glycine-rich peptides,
proline-rich peptides, and lysozymes [13]. Molecular size
of cecropins which are considered as the most potent
antibacterial peptides is 3,500–4,000 Da. Cecropins have
a strong basic amino (N)-terminal part and a long
hydrophobic carboxyl (C)-terminal stretch interrupted by
a hinge region composed of a Gly-Pro sequence [14].
Cecropins were first isolated from Hyalophora cecropia
[15, 16], which have a broad spectrum activity against
Gram-positive, Gram-negative bacteria and fungi, and
they act by destroying the ionic balance of bacterial
membrane by the formation of ionic pores [17]. The
most attractive feature of antibacterial peptides is that
they rarely induce drug resistance [18], which has
become a serious problem with conventional antibiotics.
Therefore, antimicrobial peptides have emerged from a
new class of antibiotics as one of the most promising
candidates.
For years, the heterologous expression system of the
yeast Pichia pastoris has been successfully used for the
production of a variety of proteins from different sources
[6, 19]. The yeast expression system offers many advan-
tages. The yeast growth is fast, low cost and as eukaryotes,
they have the machinery for post-translational modifica-
tions [19]. Recently, many antibacterial peptides have been
expressed in Pichia pastoris, including Pisum sativum
defensin [6], penaeidin [20], and anti-lipopolysaccharide
factor [21].
To study the potential of recombinant strains
expressing cecropin A in inhibiting postharvest decay of
apple fruits caused by P. expansum, as a precondition,
the inhibition effects of this peptide on P. expansum
spores in vitro was evaluated by MTT method, then the
concentration of the expressed protein was determined
and the recombinant strains’ activity against P. expansum
in vivo and its potential long-term efficacy against
postharvest blue mold infections on apple fruit was
reported.
Materials and methods
Medium, fungi, and culture
The yeast medium components of BMGY (1% yeast
extract, 2% peptone, 1.34% YNB, 4 9 10-5% biotin, and
1% glycerol in 100 mmol/L potassium phosphate buffer,
pH 6.0) and BMMY (1% yeast extract, 2% peptone, 1.34%
YNB, 4 9 10-5% biotin, and 0.5% methanol in 100 mmol/
L potassium phosphate buffer, pH 6.0) were referring to Jin
et al. [18].
Penicillium expansum was isolated from decayed red
Fuji apples. The fungus was maintained on potato dextrose
agar (PDA) plates at 4 �C. Arthroconidium suspension was
rubbed from the medium surface with 5 mL of sterile
distilled water. An arthroconidium suspension was deter-
mined by a hemocytometer and adjusted the concentration
of the suspension to 5 9 104 spores/mL.
Material and biocontrol agents
Fuji apple fruit (Malus domestica Borkh.) was harvested at
commercial maturity and selected for uniformity of size.
After being superficially disinfected in 0.1% (vol/vol)
sodium hypochlorite for 5 min, fruit samples were rinsed
with tap water and then air dried prior to wounding.
Biocontrol agents (GS115/CEC) used for controlling
postharvest diseases of apple were constructed in our pre-
vious experiment. The antagonist was cultured in 50 mL of
BMGY medium on a rotary shaker at 200 rpm for 18 h at
30 �C. Then, cells were centrifuged and resuspended in
100 mL of BMMY medium to induce expression of the
recombinant proteins. The culture was supplemented daily
with 0.5% methanol. Three-day yeast culture was centri-
fuged at 7,000g for 10 min, resuspended in sterile distilled
water, and then crushed with acid-washed glass beads for
three times. The samples were centrifuged and resuspended
twice to remove culture media. Suspensions of yeast cells
were adjusted to a final concentration as required with
sterile distilled water according to the method of Liu et al.
[22].
In vitro inhibition effects assay
Antimicrobial effects on P. expansum spores were assayed
using the thiazolyl blue (MTT) method. In MTT assay, the
arthroconidium suspensions (100 lL) at a density of
5 9 104 spores/mL were plated in 96-well microtiter plates
and incubated at 28 �C for 24 h. The peptides cecropin A
(Sigma, Germany) solutions at different concentrations
(0.5, 1, 5, 10, 20, 40, and 80 lL) were added to each well
and then further incubated for 24 h under the same con-
dition and the well without peptides was used as control.
762 Bioprocess Biosyst Eng (2012) 35:761–767
123
Then 20 lL of the MTT solution (5 mg/mL in PBS) was
added to each well and incubated for 4 h. The reaction was
stopped by addition of dimethyl sulfoxide (DMSO, Merck,
Germany) (100 lL/well) and the contents of the wells were
dissolved spontaneously during 2–3 min. Absorbance of
each well was measured spectrophotometrically at 570 nm
using an ELISA plate reader (Awareness, USA). All the
tests were performed in triplicates.
The inhibitory rate of peptides at different concentra-
tions was calculated by the formula: (Acontrol - Atreated)/
Acontrol 9 100%, in which ‘‘A’’ represents absorbance.
Dose–response curves were generated and the half maxi-
mal efficient control concentration (EC50) value of the
peptide was defined as the concentration of compound
required to inhibit conidia proliferation by 50%.
Determination of the expressed protein concentrations
The yeast GS115/CEC was cultured as described above.
One milliliter of the uninduced and 3-day induced
expression culture were taken and centrifuged at 7,000g for
10 min. Then UV absorbance of the supernatant was
measured spectrophotometrically at 280 and 260 nm using
an ELISA plate reader (Awareness, USA). According to
experience formula: protein (mg/mL) = 1.45 9 AOD280 -
0.74 9 AOD260, in which ‘‘A’’ represents absorbance, the
concentration of the expressed protein was defined as the
difference between the protein content of 3-day induced
supernatant and uninduced supernatant. All the tests were
performed in triplicates.
In vitro arthroconidium germination and growth assay
The effect of recombinant strains GS115/CEC on arthroco-
nidium germination was tested in potato dextrose broth
(PDB). The suspension of 1 9 105 arthroconidia/mL was
prepared as described above. The yeast GS115/CEC was
cultured and adjusted to concentrations of 1 9 108 cells/mL
as described above. Aliquots of 100 lL of GS115/CEC
suspensions were added to a 10 mL glass tube containing
5 mL PDB and aliquots of 100 lL of pathogen suspension
were added to each tube. One hundred microliters
1 9 108 cells/mL GS115/pPIC suspension was used as
negative control. After incubation on a rotary shaker
(50 rpm) at 28 �C for 20 h, the samples were examined with
a BH-2 light microscope (Olympus, Japan). Then, at least
200 arthroconidia per replicate were observed microscopi-
cally to determine germination rate and germ tube length.
Growth of recombinant strains in apple fruit wounds
To determine the suitability of recombinant strains GS115/
CEC for biocontrol tests on apple fruit, the growth of
recombinant strains GS115/CEC and non-transformed
strains GS115 in apple fruit tissue were evaluated. The
amount of strains GS115/CEC and GS115 was determined as
described before. Two yeast cultures were inoculated to
achieve 1 9 105 cells/mL and shaked at 200 rpm at 28 �C.
Apple fruit was wounded with a sterile cork-borer (5 mm
diameter 9 3 mm deep), and then injected with 25 lL of the
yeast suspension. Fruit was placed on fruitpack trays in
plastic boxes at 24 �C. The samples were taken after 0, 12, 24,
36, 48, 72, and 96 h of inoculation by using a bigger sterile
cork-borer (10 mm diameter 9 10 mm deep) and grounded
with a mortar pestle in 10 mL of sterile distilled water.
Hemocytometer was used to count GS115/CEC and GS115
yeast cells. There were three replicates of six apple fruits for
each treatment, and experiment was conducted twice.
Biocontrol activity of recombinant strains GS115/CEC
Three-day yeast cultures were harvested, counted, and
repeatedly crushed as described above. Apples were dis-
infected with 2% available chlorine using sodium hypo-
chlorite solution for 2 min, washed with tap water, dried,
and wounded as described above, and the wounds inocu-
lated with 50 lL of an aqueous suspension of yeast GS115/
CEC and GS115/pPIC at 1 9 108 cells/mL and sterile
distilled water as control. Four hours later, the wounds
were inoculated with 20 lL of the conidial suspension of
P. expansum at 5 9 104 spores/mL. Each treatment was
kept in a polyethylene-lined plastic tray to maintain high
relative humidity (about 95%) at 25 �C for 5 days [23].
Infection rate and lesion diameter were examined at
3–4 days after inoculation. Each treatment was recorded
three times with 20 fruits per replicate, and experiment was
repeated twice.
Statistical analysis
All data were analyzed using SAS software ver. 8.0. Mean
separations were performed by one-way analysis of vari-
ance (ANOVA) and Duncan’s multiple range tests. Dif-
ferences at P \ 0.05 were considered as significant.
Data of the population level of GS115/CEC and GS115
yeast (cells per wound) were log-transformed to increase
the homogeneity of variances.
Results
In vitro inhibition effects assay of cecropin A
In order to study the fungicidal activity of cecropin A on P.
expansum spores, MTT method was used to confirm the
inhibitory efficiency of different peptides concentrations
Bioprocess Biosyst Eng (2012) 35:761–767 763
123
against P. expansum spores. Results showed the obvious
dose–response relationship between the inhibitory rate and
doses of peptides cecropin A. Figure 1 showed that the
EC50 of this drug was determined as about 10 lL on these
spores, and when the concentrations of peptides was
80 lL, its inhibitory rate can get to 85.08%.
Concentration of the expressed protein
We have obtained recombinant strain GS115/CEC
expressing cecropin A in our previous experiment.
Through this current experiment, the protein concentration
of the recombinant strain GS115/CEC was determined and
can expressed at high levels up to 14.2 mg/L in the culture
medium.
Spore germination and growth in vitro
Due to the effective antimicrobial effects of cecropin A
peptides on P. expansum spores, in order to determine
whether the recombinant strain GS115/CEC can influence
the arthroconidium germination and germ tube length, the
result was observed by microscopy after 20 h incubation.
Table 1 showed the effect of recombinant strain GS115/
CEC on arthroconidium germination. The suspensions of
recombinant strain GS115/CEC strongly influenced the
arthroconidium germination and germ tube growth of
P. expansum. GS115/pPIC treated group also could inhibit
the germination and germ tube growth of P. expansum.
When the spore germination of control (sterile distilled
water) was 70.4 ± 2.84%, the spore germination treated by
recombinant strain GS115/CEC was only 2.5 ± 0.71%. It
dropped 96% compared with sterile distilled water control
group. The germ tubes were 432.8 ± 2.21 and
30.5 ± 1.1 lm in control groups while it was only
9.8 ± 0.55 lm in recombinant strain treated group.
Growth of recombinant strains in apple fruit wounds
In wounds of apple fruits, recombinant strain GS115/CEC
multiplied more quickly than GS115 (Fig. 2). However, the
growth trend of two yeast strains was similar, population of
two yeast strains (GS115/CEC and GS115) increased rap-
idly. The amount of two yeast strains increased rapidly and
reached approximately four log cycles during the first 48 h
after incubation, and the population of two yeasts
decreased slightly and gradually stabilized after this.
Biocontrol tests
As shown in Fig. 3, the application of transformed yeast
strains was effective on control of blue mold incidence by
more than 48.8%. When blue mold incidence on both
control samples were 85.4 and 81.3%, respectively, the
incidence of GS115/CEC treated apple was 43.7%. Lesion
diameter with GS115/CEC treatment was 4.41 mm and the
Fig. 1 The efficiency control concentrations of cecropin A on the
growth of P. expansum spores. Error bars represent standard error
from the mean. Filled diamonds inhibitory rates under different
concentrations of cecropin A
Table 1 Effects of recombinant strains GS115/CEC on arthroconidia
germination and germ tube elongation of P. expansum
Yeast strains Spore
germination (%)
Germ tube
length (lm)
Control 70.4 ± 2.84a 432.8 ± 2.21a
GS115 5.5 ± 0.71b 30.5 ± 1.10b
GS115/CEC 2.5 ± 0.71b 9.8 ± 0.55c
All the values were means of three replicates ± SD. Different lettersmeant significant differences between different treated groups
(P \ 0.05)
Germination rate and germ tube length were measured after 20 h
incubation at 26 �C in PDB
Fig. 2 Growth rate of Pichia pastoris strains GS115 and recombinant
strain GS115/CEC in wounds of apple fruits. The fruit were wound-
inoculated with both strains and stored on fruitpack trays in plastic
boxes at 24 �C. Error bars represent standard error from the mean.
Filled triangles GS115/CEC; open circles GS115
764 Bioprocess Biosyst Eng (2012) 35:761–767
123
diameter of control apples was 9.8 and 8.0 mm,
respectively.
Discussion
Biological control has in recent years emerged as one of the
most promising alternatives to synthetic fungicides because
it is a healthy and environmental-friendly method [24]. But
the inhibitory effect of single antagonist is not very good
compared to fungicides, as no single method is as consis-
tently effective as fungicides on inhibiting postharvest
diseases of fruit, and promising alternatives such as
improving antagonists through genetic manipulation need
to be evaluated. Genetic manipulation offers tremendous
potential for improving biocontrol effect. Our data showed
that the method using recombinant strains expressing
cecropin A antifungal peptide is a successful strategy to
inhibit postharvest decay on apple fruits under postharvest
storage conditions.
The in vitro inhibition effects of cecropin A on P. ex-
pansum spores was confirmed by MTT assay. These studies
have demonstrated that low concentrations of cecropin A
(10 lL) can inhibit about 50% spores obviously. Results
revealed that cecropin A had significant inhibition effect on
P. expansum spores, and exhibited the potential for further
study of this peptide in inhibiting postharvest decay of
apple fruits caused by P. expansum. This study also showed
that cecropin A can be heterologously expressed in Pichia
pastoris and it expressed at high levels about 14.2 mg/L in
the culture medium. The in vitro assay of arthroconidium
germination and growth of P. expansum spores by
recombinant strains, we obtained the same result, the strain
inhibited spore germination and germ tube growth of
P. expansum in PDB (Table 1). Similar anti-fungal activities
have been reported for cecropins [13, 25]. Jin et al. [18]
reported that the product of a housefly cecropin gene had
high activity inhibiting the growth of the five fungi (Pyri-
cularia oryzae, Botrytis cinerea, Penicillium crustosum,
Valsa mali and Fusarium oxysporum) in vitro, and fungal
growth inhibition was associated with permeation of the
membrane after treatment with recombinant strains
expressing cecropin A antifungal peptide [18]. With these
as the prerequisite, expression of the antifungal peptide in
yeast, therefore, represents a new approach for inhibiting
decay development caused by P. expansum in apple fruits.
The results of the present study indicated that recombinant
strain GS115/CEC suspension obviously inhibited the
growth of P. expansum. Furthermore, the different envi-
ronment bring rise to the different inhibition effect of yeast
GS115/CEC to P. expansum spores in vitro and in vivo.
Biocontrol tests on apple fruits against blue mold decay
revealed significant differences in the effectiveness and
consistency among recombinant strain and controls
(GS115/pPIC and sterile water). The disease incidence of
artificially inoculated apple fruit was reduced about 48.8%
as compared to control (sterile water). With respect to the
population growth of GS115/CEC strain, expressing of
cecropin A gene did not affect the growth of the Pichia
pastoris yeast in wounds of apple, on the other hand,
growth of recombinant strain was faster than empty strain
GS115, and the high growth rate of it was beneficial to its
biocontrol effect although the precise reason of this is not
yet understood.
The results from this study also showed that the
recombinant plasmid significantly inhibited the growth of
P. expansum in vitro and reduced decay indicating the
potential of this yeast for biocontrol. As a yeast, Pichia
pastoris applying in biocontrol was mainly due to the
competition for space and nutrients [26], and the similar
results were found in some other previous reports, because
other Pichia spp. have been reported to control various
Fig. 3 Inhibition of P. expansum on artificially inoculated and
wounded apple fruits by recombinant strain GS115/CEC. Water was
used as control (CK). Bars represent standard errors of three
replications. Significant differences (P \ 0.05) between means were
indicated by different letters above histogram bars
Bioprocess Biosyst Eng (2012) 35:761–767 765
123
diseases. For example, Pichia guilermondii controlled gray
mold on strawberries in the field [27], Pichia guillermondi
controlled postharvest decays on apple fruit [28], and
Pichia membranefaciens controlled gray mold of grapevine
[29] and postharvest disease caused by B. cinerea in tomato
fruit [30], while Pichia anomala could control spoilage of
moist feed grain in long-term storage [31].
Developing new biocontrol agents or improving bio-
control effect through genetic manipulation against post-
harvest fruit diseases and using them as a delivery system
for various biocontrol traits isolated from other microor-
ganisms or other foreign genes responsible for antifungal
activity have been suggested in the past [12]. Efficient
control of the fruit wound invading pathogens was con-
sidered to be a prerequisite for a potential microbe candi-
date. Jones and Prusky used this approach to express an
antifungal peptide in Saccharomyces a biocontrol agent by
cloning cecropin-based gene. This genetically modified
yeast expressed the cloned gene and controlled postharvest
decay caused by Colletotrichum coccodes on tomatoes [9].
Janisiewicz et al. [6] expressed PsdI antifungal peptide, a
plant defensin, with Pichia pastoris recombinant strains to
inhibit blue mold decay of apple caused by P. expansum.
Wisniewski et al. [11] cloned and expressed in Pichia
pastoris a defensin gene isolated originally from peach
bark that had antifungal activity against B. cinerea and
P. expansum in vitro.
As we all known, the most appealing feature of anti-
bacterial peptides is that they rarely induce bacterial
resistance [18]. Therefore, antimicrobial peptides have
become one of the most promising candidates for a new
group of antibiotics. The use of antagonists producing
antibacterial peptides on consumable products, apart from
other regulatory issues, will have to overcome the hurdles
of potential allergic reactions and mammalian toxicity
before approval can be anticipated. Antimicrobial peptide
cecropin have been reported to display inhibition activity
toward Gram-positive, Gram-negative bacteria and fungus
and to be virtually non-toxic to human cells [13]. Never-
theless, all these cases confirmed the validity of this
approach in inhibiting postharvest disease of fruits. The
Pichia system has an additional attraction because this
yeast has been approved to feed animals as a dietary sup-
plement [58 FR 59170, November 8, 1993; 21 CFR
Chapter 1 (4-1-02 Ed.) Section 573.750], and moreover,
the result of the safety test in our previous experiment
(acute toxicity to mice (food standard)) also confirmed the
safety of the recombinant strain GS115/CEC (data not
show).
Our results show a potential of recombinant yeast in
applying to control blue mold infections in pome fruit.
However, to optimize expression of cecropin A under a
variety of conditions (e.g., fruit wounds, storage
conditions, etc.), take some strategy to improve foreign
protein expression in Pichia pastoris, and understand its
exact mechanism of action against Penicillium spp. to
develop a suitable formulation for its application on apple
appears to be the next major challenge in determining the
commercial potential of this system.
Acknowledgments This research was partially supported by the
Foundation for the Author of National Excellent Doctoral Disser-
tation of People’s Republic of China (201061), the Program for
Key Innovative Research Team of Zhejiang Province
(2009R50036), the Ph.D. Programs Foundation of Ministry of
Education of China (20090101120079, 20100101110087), the Open
Foundation from Top Key Discipline of Modern Agricultural
Biotechnology and Biological Control of Crop Diseases in Zhejiang
Provincial Colleges (2010KFJJ006), the National Natural Science
Foundation of China (20906060), Open Project Program for Key
Laboratory of Fermentation Engineering (Ministry of Education)
(2009KFJJ02), and the Fundamental Research Funds for the Cen-
tral Universities.
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