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Mutagenesis of Monascus purpureus for Higher/Reduced Red Pigment
Production, and Phenotypic/Genetic Analyses on the Relevant Mutants
SASTIA PRAMA PUTRI*, HIROSHI KINOSHITA and TAKUYA NIHIRA
International Center for Biotechnology, Osaka University, 2-1 Yamadaoka, Suita, Osaka
565-0871, Japan
Monascus purpureus is a fungus well-known for its ability to produce
pigments which are widely used as natural food colorant. However, the
knowledge about pigment biosynthesis in this fungus is very limited
especially in molecular level. In this paper, 9 mutant strains which exhibit
alteration in pigment production were acquired by treatment with UV and
chemical mutagens from a wild-type Monascus purpureus NBRC30873.
Mutants with increased and decreased pigment production, orange pigment
production and albino mutants were obtained and characterized. These
mutants were compared with the parental strain in terms of growth, citrinin
production and pigment production. Mutagenesis had no recognizable effect
on the growth of the mutants. Mutants obtained by UV mutagenesis showed
increased citrinin production while those by chemical mutagenesis showed
reduced citrinin production. Pigment profiles of the mutant strains
determined by column chromatography and HPLC analysis revealed the
absence of all pigments in albino mutant, absence of red pigments in the
orange-mutant and increased pigment production in the hyper-pigmented
mutant. The orange-mutant was judged to be a good host for elucidating the
mechanism/pathway of red pigment biosynthesis since it only lacked the
ability to produce red pigment while still retaining the capability to
synthesize main structure giving yellow and orange colors.
Keywords: � UV mutagenesis, chemical mutagenesis, mutants, pigment
production, Monascus purpureus.
Research work in the “UNESCO Postgraduate Inter-University Course in Biotechnology”
supported by the Japanese Government and Japanese National Commission for UNESCO.
*
Present address: International Center for Biotechnology, Osaka University, 2-1 Yamadaoka,
Suita, Osaka 565-0871, Japan; Faculty of Mathematics and Natural Sciences, Bandung
Institute of Technology, Indonesia.
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Introduction
Monascus purpureus is a homothallic fungus found on red rice that is commonly used
as a natural food colorant. From a medical standpoint, it is also important since it produces
monacolins which inhibit cholesterol biosynthesis [1,2] and GABA which possess several
physiological functions including neurotransmitting, hypotensive, and diuretic effects [3,4].
This fungus also produces an antibacterial compound, citrinin, usually together with
pigment compounds through polyketide pathway [5].
The major importance of this strain lies within the ability to produce broad spectrum
of pigments which are a group of fungal metabolites, called azaphilones, which have
similar molecular structures as well as chemical properties (yellow pigments, ankaflavin
and monascin; orange pigments, rubropunctatin and monascorubrin; and red pigments;
rubropunctamine and monascorubramine, the nitrogen analogues of the orange pigments)
[6,7]. The pigment molecule consists of two parts condensed by esterification, which are
short chain fatty acid (C6 or C8) synthesized via fatty acid synthesis pathway and
hexaketide chromophore which is derived from the condensation of acetate and malonate
synthesized by the polyketide pathway. Yellow pigments are produced by reduction of the
orange pigments, whereas amination of orange pigments with NH3 units give rise to red
pigments [8].
Most researches on Monascus have focused on the study of applicable growth
substrates and optimization of fermentation conditions to enhance pigmentation. Although
numerous mutant strains with enhanced ability for pigment production have been isolated
previously, the mechanism of pigment biosynthesis in Monascus is not clearly understood
[9]. Production of mutants that are non-pigmented or with altered pigment production by
mutagenesis and comparative phenotypic and genetic analyses of these mutants against the
wild-type strain could provide valuable information in elucidating the mechanism of
pigment biosynthesis in Monascus.
Fig. 1. Major pigments produced by Monascus during culture production [10].
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In this study, UV and chemical mutagenesis were carried out in order to obtain
pigment mutants of Monascus purpureus. Comparative phenotypic analysis was also
performed on the relevant mutants.
Materials and Methods
Fungal strain and growth conditions
Wild type strain Monascus purpureus NBRC30873 was used in this study. Cultivation
was performed at 28�C for 7d on solid Henneberg medium (50 g/l glucose, 7.5 g/l
polypeptone, NH4H2PO4 2 g/l, MgSO4.7H2O 0.5 g/l, CaCl2.2H2O 0.1 g/l, 2 g/l KNO3).
Mutagenesis
To prepare the spore suspension, wild-type strain was cultivated in Henneberg
medium and Power medium (Czapek Dox medium [sucrose 3 g/l, NaNO3 2 g/l, KCl 0.5 g/l,
magnesium glycerolphosphate 0.5 g/l, FeSO4 0.1g/l, K2SO4 0.35g/l] and PM1 [30 g/l
lactose, 2 g/l bactopeptone, 0.5 g/l corn steep solid, 4 g/l NaCl, 0.001 g/l CuSO4.7H2O, 0.03
g/l FeCl3.6H2O, 0.06 KH2PO4, 0.05 g/l MgSO4.7H2O] with 1:1 ratio) and the fungal spores
were collected by washing Monascus mycelial mat with sterilized spore buffer (0.1%
Tween 80, 0.8% NaCl), centrifuged (3000 rpm for 5 min) and washed twice with sterile
distilled water.
UV treatment
One milliliter of spore suspension (about 1�106 spores/ml) was irradiated with UV
light for 0; 0.5; 1; 1.5; 2; 2.5; 3; 4; 5; 6; 7 min with a distance of 59 cm. Irradiated spores
were incubated in the dark at 28�C for 1 hour. The cells were then serially diluted and
plated on Henneberg medium and were incubated at 28�C until colonies appeared. Survival
rate was determined from the ratio between number of colonies of mutagenized cells and
that of untreated cells. Colonies which exhibited alteration in pigmentation were isolated
and were passed three times on Henneberg medium to verify the stability of gained
character.
NTG and EMS treatment
One milliliter of EMS or NTG (0, 25, 50, 75, 100 �l/ml, in 0.2 M, citrate buffer
solution, pH 5.0) was added to 1 ml of spore suspension (about 1�106 spores/ml). After it
was incubated at 28�C for 1 hour, the mixture was centrifuged and washed twice with
buffer containing 5% Na2S2O3. The spores were then serially diluted and plated on
Henneberg medium and incubated at 28�C until colonies appeared.
Morphological and cultural characterization
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Morphological and cultural characteristics of mutants were compared with those of
wild-type strain on solid and liquid Henneberg, Power, Czapek Dox and Conidiation
medium (sucrose 100 g/l, KH2PO4 10 g/l, MgSO4.7H2O 0.5 g/l, NaNO3 2g/l, KCl 0.5g/l,
FeSO4.7H2O 0.001g/l, yeast extract 3 g/l, casamino acid 5 g/l). Cultivation was performed
at 28�C for 10 days and qualitative observation of mycelial growth, pigmentation, colony
size, shape and color was performed daily.
Evaluation of secondary metabolites production
Citrinin
Dried mycelium (1 g) was ground to fine powder and extracted with 40 ml of 70%
ethanol at room temperature, with shaking at 80 spm for 3 hrs. The extracts were passed
through a 0.20-�m filter and were analyzed by HPLC on a 250 mm�10 mm ODS C18
column under the following conditions: 55% a CH3CN + 0.1% trifluoro acetic acid (TFA)
as the mobile phase at a flow rate of 1.0 ml/min with detection of fluorescence (excitation
at 330 nm and emission at 500 nm).
Pigment
Sample is diluted 50 times with 70% ethanol (pH 8.0). Pigment concentration was
determined spectrophotometrically by measuring the absorbance of crude mycelium extract
at 500 nm.
Pigment profile analysis
Pigments were isolated from mycelium and culture broth. Dried mycelium (1 g) was
extracted with 40 ml of 70% ethanol for 3 hrs under stirring. The extracts were evaporated
in vacuo at 50�C and suspended in H2O. The suspensions were extracted three times with
ethyl acetate (EtOAc). As for culture broth, EtOAc extraction was carried out with 1:1 ratio
(v/v). The EtOAc solution was evaporated in vacuo at 50�C to yield the crude EtOAc
extract. The extract was chromatographed on SepPak silica gel, eluted successively with
solvent of increasing polarity (n-hexanes-EtOAc =1:0, 9:1, 4:1, 1:1, 3:7, 1:9, 0:1; EtOAcMeOH) 9:1, 1:1, 0:1; v/v) to provide pigment fractions, following the procedure described
by Akihisa et al [11]. To obtain whole production profile in mutant strains, the crude EtOAc
extract of culture broth was also suspended in CH3CN and analyzed by HPLC on a 250
mm�10 mm ODS C18 column under the following conditions: 55% CH3CN + 0.1% TFA as
the mobile phase at a flow rate of 1.0 ml/min using UV detector in 370 nm. Determination
of the wavelength used in HPLC analysis was based on UV spectrophotometer
measurement of crude extract sample from 250 nm to 700 nm.
Results
Isolation of mutants with altered pigmentation
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UV and chemical mutagenesis generate different types of mutation; i.e UV forms
pyrimidine dimers whereas NTG and EMS cause alkylation in DNA [14], and therefore
both were adopted to obtain mutants with altered pigment production. Survival rate and
frequency of pigment mutants obtained from UV, NTG and EMS mutagenesis were
summarized in Figure 2. Survival rate decreased as the dose of irradiation or the amount of
mutagen increased. However, correlation between frequency of pigment mutant and dose of
mutagen was not found. Frequency of mutants obtained both from UV and chemical
mutagenesis are relatively high. However, after serially passed to verify the stability of their
phenotype, most mutants recovered the pigment production. In addition, mutants generated
from treated spore suspension which were cultivated in Henneberg medium tend to show
instability in respect to pigmentation compared to those cultivated in Power medium, while
the reason remained unclear (data not shown). This result suggested that cultivation in
Henneberg medium was not suitable for collection of spores used for mutagenesis.
Cultivation in Power medium could give more stable mutants in respect to pigmentation
and therefore was used to collect spores for further mutagenesis. Mutants with shorter
exposure time of UV or which were exposed to lower concentration of EMS and NTG were
preferable in mutant isolation because they possibly have lower number of point mutation.
120
percentage (%)
100
percentage (%)
100
80
60
40
20
80
60
40
20
0
0
0
0
1
2
3
4
5
6
7
75
survival rate EMS
frequency of mutants EMS
survival rate NTG
Frequency of mutants NTG
time of exposure (min)
survival rate
25
50
concentration (ug/ml)
frequency of mutants
A. UV mutagenesis
B. Chemical mutagenesis
Fig. 2. Survival rate and frequency of mutants obtained from mutagenesis.
Five mutants with altered pigment production, which are mutants lacking the ability to
produce pigments, designated as albino mutants, and a mutant lacking the ability to produce
red pigments but still retained the ability to synthesize yellow and orange pigments,
designated as orange-pigment mutant, were obtained from UV mutagenesis. EMS and NTG
mutagenesis also generated four mutants which showed increased, decreased and no
pigment production.
Characterization of mutants
Morphological features, i.e mycelial growth, colony size, shape and color of all
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mutants were observed and were compared to those of the wild-type. As a result, there was
no significant difference among both strains, suggesting that mutants have low point
mutation and the morphological and cultural features were not altered. Mutants were also
compared with wild-type strain in terms of growth, citrinin production and pigmentation.
Citrinin production in the mutants was measured to select mutants having mutations only in
pigment biosynthesis because the change of production pattern in citrinin which is also a
polyketide compound is usually accompanied with that in pigment compounds [5].
Mutagenesis had no recognizable effect on the growth of the mutants (data not shown).
Pigmentation, citrinin and pigment concentration of the mutants are shown in Table 1.
Mutants obtained by UV mutagenesis (PM3U, AM0.5U, AM1Ua, AM1Ub, AM3U) showed
increased citrinin production while those by chemical mutagenesis showed reduced citrinin
production. All mutants in exception of a hyper-pigmented mutant showed decreased
pigment production whereas hyper-pigmented mutant showed 1.75-fold increase in pigment
production compared with that of the wild-type strain. The presence of pigments in albino
mutants, which is shown by absorbance at 500 nm might be caused by other colored
compounds in the cell than Monascus pigments. This assumption was confirmed by
pigment profiles determined by column chromatography and HPLC (Table 2 and Fig. 3).
Table 1. Pigmentation, citrinin and pigment concentration of mutants in liquid
Henneberg medium at 28�C, 120 spm, inoculated with 2.5�105 spore
suspension.
Strain
Pigmentation
Citrinin concentration
(ppm)
Pigment concentration
(OD500/g)
5 day
7 day
9 day
5 day
7 day
9 day
2.10
0.32
3.25
1.92
6.03
0.50
0.70
0.45
0.95
1.65
2.45
4.30
18.6
0.05
0.23
71.92
0.025
0.11
30.16
0.11
0.05
0.45
0.1
0.80
0.78
0.25
1.00
0.80
0.25
0.95
AM0.5U
10.21
51.04
17.98
0.40
0.55
0.80
AM1Ua
10.67
21.45
13.81
0.35
0.55
0.50
11.25
20.28
58
0.45
0.50
0.50
8.82
55.10
30.16
0.2
0.25
0.35
-
-
-
0.15
0.15
0.20
WT
HPM75E*
Wild type
PM3U
PM75E
PM75N
Orange pigmented
AM1Ub
AM3U
AM75E
Hyperpigmented
Low pigmented
Albino
(non pigmented)
*The last capital letter stands for by which mutation the strain was obtained. U: UV, E: EMS
and N: NTG.
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A
B
C
D
Fig. 3. HPLC analysis of pigment profile of wild-type (A), hyper-pigmented mutant (B),
orange-pigmented mutant (C), albino mutant (D).
Table 2. Number of pigment fractions obtained from column chromatography.
Culture broth
Mycelium
Strain
Yellow
Orange Red
Yellow
Red
Orange
WT
1
2
5
1
PM3U
1
5
1
AM0.5U
Pigment profiles of mutant strains
In order to clarify the pigmentation character of the mutants, pigment extracts from 10
days culture of wild-type (WT), orange-pigment and albino mutant were fractionated by
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column chromatography and further analyzed by HPLC. Pigment compounds were more
abundant in culture broth compared to mycelium as indicated in table 4. It is possible that
after 10 days of cultivation, pigments react with amino acids in medium and was excreted
outside the cells to form water-soluble pigments [15].
Yellow, orange and red pigment fractions were obtained from culture broth of WT
whereas no red pigment fractions were obtained from that of orange-pigment mutant. From
mycelium, only red fraction and orange fraction was obtained in WT and orange-pigment
mutant, respectively. No pigment fraction was obtained from albino mutant from both
culture broth and mycelium.
Orange and yellow fractions from orange-pigment mutant (PM3U) were further
analyzed by HPLC and compared with those obtained from wild-type strain (WT). Identical
pattern was observed in yellow and orange fractions from WT and PM3U, suggesting that
PM3U still retain the ability to synthesize orange and yellow pigments.
Crude pigment extract from culture broth of wild-type, hyper-pigmented mutant,
orange-pigment mutant and albino mutant was analyzed by HPLC and UV spectrum was
measured, following the procedures described in Materials and Methods. Pigment profiles
of mutant strains revealed the absence of all pigments in albino mutant, absence of red
pigments in the orange-mutant and increased pigment production in the hyper-pigmented
mutant (Fig. 3).
Discussion
Monascus pigments have been isolated and their chemical structures have been
studied for more than 40 years [16]. However, the genes involved in pigment biosynthesis
of Monascus are still unknown. Because the pigments are widely used in food industry as
natural colorant, knowledge about the mechanism of pigment biosynthesis is of interest.
To gain more insight into genes involved in red pigment biosynthesis, mutagenesis
was carried out using UV and chemical mutagens, and led to isolation of nine mutant
strains which exhibit altered pigment production. Among all pigment mutants obtained in
this study, orange pigment-producing mutant gained more interest because it was judged to
be a good host in elucidating the mechanism of red pigment biosynthesis since it only
lacked the ability to produce red pigment while still retaining the ability to synthesize main
structure giving orange and yellow colors (Table 2).
The orange pigments, monascorubrin and rubropunctatin, are synthesized in the
cytosol from acetyl coenzyme A through a multi-enzymatic polyketide synthase complex.
Reactions with amino acids lead to formation of water-soluble red pigments,
monascorubramine and rubropunctamine (Fig. 4). It is assumed that this strain only
underwent partial mutation in pigmentation, suspected only in the genes encoding the
modifying enzyme which converts orange pigment to red pigment or interfere the reaction
with amino acids via Schiff base formation and dehydration [15] and therefore will be
easier to complement compared to albino mutant strain. At present, molecular basis of
orange-pigment mutant characterized here has not yet been analyzed with regard to
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pigmentation. However, complementation analysis of this strain could provide valuable
material to identify genes involved in pigmentation of Monascus.
1 acetate + 5 malonates
1 acetate + 3 malonates
Polyketide synthase
Fatty acid synthase
Octanoid acid
Hexaketide
Acetyl-CoA
COOH
CH3
+
COSCOA
O
O
COOH
OH
�-ketoacid
O
Polyketide chromophore
Esterification
O
O
O
O
O
Monascorubrine
amino acids
O
O
NH
O
O
monascorubramine
Fig. 4. Scheme of the metabolic routes leading to the formation of red pigment
in Monascus purpureus [5].
Acknowledgement
The authors are grateful to the Japanese Government and UNESCO for their
financial support and with grateful to Dr. Shigeru Kitani and all the members of the
Laboratory of Molecular Microbiology for their useful discussion and kind assistance.
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Table of Contents
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