Download Sampling techniques and comparative extraction procedures for

FEMS Microbiology Letters 164 (1998) 195^200
Sampling techniques and comparative extraction procedures for
quantitative determination of intra- and extracellular metabolites
in ¢lamentous fungi
H. Hajjaj, P.J. Blanc, G. Goma, J. Franc°ois *
Deèpartement de Geènie Biochimique et Alimentaire, Centre de Bioingenieèrie Gilbert Durand, UMR-CNRS 5504, LA. INRA,
Institut National des Sciences Appliqueèes, Complexe Scienti¢que de Rangueil, 31077 Toulouse Cedex 04, France
Received 11 November 1997; revised 25 February 1998; accepted 4 March 1998
Abstract
Two methods for rapid sampling and three procedures for extraction of metabolites from the filamentous fungus Monascus
ruber were compared. It is shown that arrest of metabolism by either dropping the mycelial cultures in liquid nitrogen or by
spraying them on a 60% solution of methanol kept at 340³C followed by rapid centrifugation at 310³C were equally effective.
Metabolites were extracted from mycelia using different procedures including acid and alkaline treatments, permeabilization by
cold chloroform and extraction by boiling buffered ethanol, to demonstrate that the latter method gave the best results both in
terms of recovery and stability of metabolites. In addition, this method is very simple to handle and allows the use of very low
amounts (i.e. 10^20 mg dry mass) of cellular material since the removal of ethanol by evaporation after extraction results in a
concentration step of metabolites. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science
B.V. All rights reserved.
Keywords : Monascus ruber; Secondary metabolite
1. Introduction
Filamentous fungi are good candidates for industrial production of heterologous proteins [1], organic
acids [2] and pigments [3,4]. However, the poor
knowledge of metabolic activities and intermediate
metabolites with respect to cultivation conditions of
these organisms precludes any rational metabolic
strategy to improve their performance. Together
with knowledge of the kinetic properties of enzymes,
* Corresponding author. Tel.: +33 05 61559492;
Fax: +33 05 61559400; E-mail: [email protected]
metabolite levels can be used to quantify £ux distribution in metabolic pathways and to determine factors which control metabolic £ux in vivo.
To obtain this information, fast sampling methods
and reliable metabolite extraction procedures are required, since more than enzymes, metabolites are
prone to fast changes induced by the culture conditions or, worst, by the way the samples are harvested
from the culture vessel. Fast sampling methods have
been recently developed for yeast [4^6]. However, the
increasing interest of ¢lamentous fungi as cell factories to produce high-added-value molecules has also
prompted the development of reliable methods for
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
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metabolite determination. However, because the
physiology and the morphology of these organisms
are quite di¡erent from those of yeast, we still need
to demonstrate that methods developed for yeast can
be used for the ¢lamentous fungi.
We recently became interested in the regulation of
metabolic pathways leading to the production of organic acids and secondary metabolites in the ¢lamentous fungus Monascus ruber [3,7]. We thus considered the methodology of Ruijter and Visser [8] to
determine key intermediates in this organism. This
method was actually identical to the yeast sampling
technique devised by De Koning and Van Dam [5].
However, in our hands, this procedure was unsatisfactory, quite tedious and, more importantly, not
reproducible. We have therefore systematically investigated di¡erent methods for fast sampling and compare di¡erent protocols for metabolite extraction.
We demonstrate that the boiling bu¡ered ethanol
method developed previously for yeast [6] is the
most reliable and e¤cient extraction method that
should be therefore applicable to any other ¢lamentous fungal species.
2. Materials and methods
2.1. Materials
Enzymes and biochemicals were purchased from
Sigma (St. Louis, USA) or Boehringer (Mannheim,
Germany). All ¢ne chemicals were of analytical
grade and obtained from Merck (Darmstadt, Germany). Pure ethanol and HPLC-grade methanol
was used for the quenching and extraction procedures.
2.2. Microorganism and growth conditions
The strain used was a high pigment-producing
strain, Monascus ruber, classi¢ed as ATCC 96218.
The synthetic-de¢ned fermentation medium developed in previous studies [3] contained per liter of
distilled water: glucose, 8 g; monosodium glutamate,
5 g; K2 HPO4 , 5 g; KH2 PO4 , 5 g; CaCl2 , 0.1 g;
MgSO4 7H2 O, 0.5 g; FeSO4 7H2 O, 0.01 g; ZnSO4
7H2 O, 0.01 g; MnSO4 H2 O, 0.03 g. The initial pH of
the medium was adjusted to 6.5 with phosphoric
acid. The stock culture was kept on Potato Dextrose
Agar (PDA, Difco). Spores of strains were prepared
by growth on PDA slants for 10 days at 30³C.
Spores were washed with sterile water. A suspension
of 108 spores was used to inoculate a 1-l ba¥ed
Erlenmeyer £ask containing 0.2 l of synthetic medium which was incubated at 30³C for 2 days. This
inoculum was then transferred to a 2-l fermenter
containing 1.6 l of synthetic medium. The culture
was incubated at 30³C, at an aeration rate of 0.65 l
min31 . The agitation speed was increased from 250
to 600 rpm to maintain dissolved oxygen near 40%
of saturation. Fungal biomass was determined by
gravimetric analysis after ¢ltration of cell samples
through preweighed nylon ¢lters (45 mm diameter,
0.8 Wm porosity) and dried to constant weight at
60³C under partial vacuum (200 mm Hg).
2.3. Quenching methods
2.3.1. Quenching in liquid nitrogen
A 15-ml mycelial sample taken during a fast
growth period was transferred directly from the fermenter into liquid nitrogen and stored at 325³C.
The sampling time was less than 3 s. The frozen
suspension was lyophilized at 320³C in a lyophilizator (Usifroid SMH 13, Maurepas, France).
2.3.2. Quenching in 340³C bu¡ered methanol
A volume of mycelial sample (containing approximately 20 mg mycelial dry weight) was dropped directly in 5 volumes of a solution of 60% (v/v) methanol (taken from 380³C deep freezer) and 10 mM
HEPES, pH 7.5 and kept at 340³C in a dry ice/
ethanol bath. The mixture was allowed to cool
down for 3^5 min to allow temperature to return
to 340³C, as checked with a digital thermometer.
The mixture was centrifuged at 5000Ug for 6 min
in a Sorvall RC5B set at 310³C. Following exactly
this procedure, it was veri¢ed that the temperature of
the mixture remained below 320³C after the centrifugation period.
2.4. Extraction of metabolites
2.4.1. By acid or alkaline solution
The lyophilized sample or the cell pellet obtained
after methanol quenching was resuspended in 6.4 ml
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197
Table 1
Extraction of metabolites from Monascus ruber by boiling bu¡ered ethanol after quenching in liquid nitrogen
Metabolite concentrations (Wmol ml31 )
G6P
F6P
ATP
NAD
NADH
NADP
NADPH
Pyruvate
Total extract (cell extract+medium)
In medium
2.6 þ 0.17
0.3 þ 0.02
0.4 þ 0.08
4.3 þ 0.30
0.6 þ 0.08
1.6 þ 0.30
0.4 þ 0.06
4.1 þ 0.03
0.2
b.d.
b.d.
b.d.
b.d.
b.d.
b.d.
1.6 þ 0.08
A suspension (6 ml) of Monascus ruber cultures cultivated on glucose synthetic medium was directly dropped into liquid nitrogen. The frozen
samples were lyophilised and extracted in boiling bu¡ered ethanol as described in Section 2. Metabolites were determined in both total
extracts (corresponding to cell extract and the medium) and in the culture medium and were expressed as mole per ml of intracellular cell
volume or of medium. Data are averages of three di¡erent extractions from the same culture. b.d. means below detection.
of 10% (v/v) cold HClO4 and 0.6 ml of 1 M Imidazole. Acid extraction was performed by three freezethaw cycles with vigourous shaking between each
cycle. After centrifugation for 5 min at 8000Ug at
0³C, the supernatant was neutralized with 10 M
KOH and the KClO4 precipitate was removed by
centrifugation. The supernatant was stored at
325³C until use. Alkaline extraction was performed
by addition of 3 ml of 0.25 M KOH to the lyophilized sample or the cell pellets, followed by 15 min
incubation at room temperature. After centrifugation at 4³C for 8 min at 8000 rpm, NADPH and
NADH were immediately assayed in the supernatant.
2.4.2. By boiling bu¡ered ethanol mixture
The lyophilized mycelial samples or the cell pellets
recovered after methanol quenching were extracted
in 5 ml of a 75% (v/v) boiling ethanol containing 10
mM HEPES, pH 7.1 for 5 min at 80³C. After cooling down the mixture on ice for 4 min, the volume
was reduced by evaporation at 45³C using a rotavapor apparatus (Buëchi Rotavapor R-134, Bioblock,
France). The vacuum was progressively adjusted to
0.05 bars using a manometrically controlled pump.
The rate of evaporation was about 0.5 ml min31 .
The residue was resuspended in a ¢nal volume of
2 ml distilled water and centrifuged 10 min at
5000Ug at 4³C to remove the insoluble particles.
The supernatant was stored at 325³C until use.
2.5. Determination of metabolites
Unless otherwise stated, metabolites (ATP, Glc6P,
Fru6P, Fru1,6P2 , pyruvate) were determined essentially as described in Bergmeyer [9] in 50 mM triethanolamine pH 7.6, containing 7.5 mM MgSO4
and 2 mM EDTA, by coupling appropriate enzymes
with a £uorimetric detection of NADH or NADPH.
Table 2
Extraction of metabolites from Monascus ruber by boiling buffered ethanol after quenching in cold methanol
Metabolite concentrations (Wmol ml31 )
G6P
F6P
ATP
NAD
NADH
NADP
NADPH
Pyruvate
In the cell extract
In supernatant
3.0 þ 0.35
0.3 þ 0.06
0.7 þ 0.02
5.3 þ 0.21
0.7 þ 0.06
1.5 þ 0.06
0.3 þ 0.04
2.2 þ 0.26
b.d.
b.d.
b.d
b.d.
b.d.
b.d
b.d.
1.8 þ 0.25
A suspension (6 ml) of Monascus ruber cultures cultivated on glucose synthetic medium was sprayed into 5 volumes of 60% methanol solution kept at 340³C in a dry ice/ethanol bath. After centrifugation, the pellet was extracted into 8 ml of a solution of 75%
(volume/¢nal volume) boiling bu¡ered ethanol for 3 min at 80³C.
The supernatant and the ethanol extract were then concentrated to
dryness by evaporation and resuspended into 2 ml of sterile water.
Metabolites were determined as described in Section 2. Measurements are the average þ standard deviation of three extractions
made from the same culture. b.d. means below detection.
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Table 3
Metabolite recovery after extraction in perchloric acid in the presence of Monascus ruber mycelia
G6P
ATP
Pyruvate
NAD
Metabolite measured
in cell extract
(Wmol g31 dry mass)
Amount of metabolites
added before extraction
(Wmol g31 dry mass)
Metabolite measured in extract with
metabolites added before extraction
(Wmol g31 dry mass)
Recovery (%)
3.2 þ 0.30
0.2 þ 0.09
4.7 þ 0.65
1.0 þ 0.43
20
10
30
15
22.8 þ 0.40
6.2 þ 0.80
33.5 þ 0.50
5.8 þ 0.70
98
60
96
32
Exogenous metabolites were added to a suspension of Monascus ruber cultivated on glucose synthetic medium, just before harvesting. The
suspension was sprayed on to 10% (¢nal volume) of ice-cold HClO4 . Extraction and metabolite assays were performed as described in Section
2. Measurements are the average þ standard deviation of three extracts from the same culture.
Emission was measured at 450 or 435 nm after excitation at 350 nm using a £uorescence spectrophotometer (Hitachi F-2000). NADP‡ was determined
in the presence of 100 mM Glc6P by the addition
of 2 U ml31 glucose 6-phosphate dehydrogenase.
NADPH was measured in the presence of 100 mM
of glutathione disul¢de by addition of 20 U ml31
glutathione reductase. NADH was determined in
the presence of 200 mM pyruvate by addition of
2.0 U ml31 lactate dehydrogenase. NAD‡ was determined in a pyrophosphate bu¡er (NaPPi 250 mM,
pH 8.8 containing 12 g l31 of semicarbazide) in the
presence of 0.1 M ethanol by addition of 4 U ml31
of alcohol dehydrogenase. Metabolites are expressed
in mol per g dry weight. According to Ruijter and
Visser [8] and to our unpublished results, conversion
into molar concentration can be done assuming a
value of 1.2 ml of intracellular volume per g dry
mass.
3. Results and discussion
3.1. Advantages of the spectro£uorometric method
Industrial relevance of ¢lamentous fungi lies in
their production of secondary metabolites, such as
pigments. However, these compounds heavily absorb
in a range between 340 and 450 nm, which seriously
hampers the determination of metabolites by coupling with spectrophotometric detection of
NAD[P]H at 340 nm. To circumvent this interference, a spectro£uorometric method for metabolite
determination was used with some modi¢cations.
The emission wavelength was set at 435 nm instead
of 400 nm, reducing almost completely the interference caused by red pigments produced by Monascus
ruber. This modi¢cation still allowed this method to
be 10 times more sensitive than a spectrophotometric
detection of NAD[P]H, with a detection limit of 0.1
nmol NAD[P]H ml31 .
3.2. Quenching methods
Because cultures of ¢lamentous fungi are highly
viscous, heterogeneous and not easy to collect, two
methods of sampling have been tested and compared
with respect to an immediate arrest of metabolism
and metabolites recovery. The ¢rst method was a
direct quenching of mycelia in liquid nitrogen, while
the second was dropping one volume of the cultures
into 5 volumes of 60% methanol kept at 340³C, as
described previously [5,6] for yeast. Tables 1 and 2
show that both sampling protocols were equally e¤-
Table 4
Metabolite recovery after extraction in KOH in the presence of Monascus ruber mycelia
NADPH
NADH
Metabolite measured
in cell extract
(Wmol g31 dry mass)
Amount added
before extraction
(Wmol g31 dry mass)
Metabolite measured in extract with
metabolites added before extraction
(Wmol g31 dry mass)
Recovery (%)
0.5 þ 0.08
0.4 þ 0.06
10
20
8.5 þ 0.80
11.2 þ 0.60
80
54
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199
Table 5
Metabolite recovery after extraction in bu¡ered boiling ethanol in the presence of mycelia of Monascus ruber
NADPH
G6P
ATP
Pyruvate
NADH
NAD
Metabolite measured
in extract
(Wmol g31 dry mass)
Amount of metabolites added
before extraction
(Wmol g31 dry mass)
Metabolite measured in extract
with metabolites added before
extraction (Wmol g31 dry mass)
Recovery (%)
0.5 þ 0.08
3.1 þ 0.21
0.5 þ 0.02
4.9 þ 0.04
0.7 þ 0.10
5.1 þ 0.37
10
20
10
30
20
15
8.7 þ 0.70
23.3 þ 0.50
8.8 þ 0.25
34.3 þ 0.55
16.5 þ 0.60
19.5 þ 0.90
82
101
83
98
79
96
Experiment was performed with the same Monascus ruber culture as in Table 3, except that the cell suspension (6 ml) was dropped into 75%
(volume/¢nal volume) of boiling bu¡ered ethanol. Extraction and metabolite assays were performed as described in Section 2. Measurements
are the average þ standard deviation of three extracts from the same culture.
cient in arresting metabolism since the levels of glycolytic intermediates, ATP and pyridine coenzymes
as measured after extraction in boiling bu¡ered ethanol were identical in both conditions. With the exception of pyruvate, none of these metabolites was
detected in the supernatant after centrifugation of
the methanol-quenched cultures, indicating that
methanol does not alter the integrity of the mycelial
cultures.
The choice for one of the two sampling procedures
should be guided by the type of experiments to be
done and by the metabolites to be measured.
Quenching in liquid nitrogen could allow fast and
repeated sampling under subsecond time scales, providing the metabolites to be tested are not present in
the medium. Alternatively, quenching in cold methanol can be done when metabolites are present both
intracellularly and extracellularly. However, this latter method should need some technical adaptation to
be used for short time experiments, especially when
samples have to be collected within a time window of
milliseconds.
3.3. Comparative procedures for metabolite extraction
As emphasized in previous reports [4^6], the basic
requirements for extraction method are that (i) metabolite levels do no change by enzymatic or chemical conversion during extraction, (ii) they are completely extracted and (iii) they are not destroyed
throughout the extraction procedure. Acidic and alkaline solutions are commonly used as extractable
agents for acid- and alkaline-stable compounds, respectively. Recently, we have developed a procedure
for metabolite extraction in yeast using boiling bu¡ered ethanol solution [6]. Therefore, it was of interest
to compare these di¡erent extraction protocols to
search for the most simple, more e¤cient and more
reliable method applicable to ¢lamentous fungi. The
results of these comparative experiments are illustrated in Tables 3^5. Extraction of metabolites by
either acidic (Table 3) or alkaline (Table 4) solution
as carried out previously for Aspergillus [10] was
clearly not complete and caused destruction of metabolites, like NAD‡ in perchloric acid and NADH
in alkaline solution. In addition, much lower
amounts of metabolites were recovered when the extraction was carried out by thawing-freezing in
HClO4 or 0.1 M HCl, followed by a 8 min incubation at 50³C (not shown). Similar loss of alkalinestable metabolites was observed after extraction in
0.25 M KOH for 10 min at 80³C. In contrast, the
recovery of acid- and alkali-stable metabolites after
extraction in boiling bu¡ered ethanol ranged between 83 and 100%.
From this comparative study, it becomes quite
clear that the extraction in boiling bu¡ered ethanol
gave the best results both in terms of recovery and
stability of metabolites. It can be noticed that the
extraction of acid- or alkali-stable molecules in the
corresponding acid or alkaline solution was even
lower than in bu¡ered boiling ethanol. An additional
advantage of using boiling bu¡ered ethanol procedure is that the quenching and extraction steps are
followed by evaporation of the solvent, allowing the
concentration of metabolites in a minimal volume.
Hence, quanti¢cation of metabolites can be performed with a much lower amount of cellular mate-
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rial than with traditional methods based on acidic or
alkaline treatments of cells pellets.
Although no extensive metabolic analysis has been
presented here, the method described in this paper
presents the opportunity to investigate changes in
metabolic activities of fungi under very short time
periods and under various culture conditions. Work
currently in progress in our laboratory has illustrated
the validity of this method, and has shown that intracellular levels of TCA cycle intermediates and
amino acids can be quantitatively measured under
this extraction condition (F. Allemand, V. Guillou,
H. Hajjaj and J. Franc°ois, unpublished data).
Acknowledgments
[2]
[3]
[4]
[5]
[6]
This work was supported in part by grant no.
9507430 from Region-Midi Pyreèneèes. We thanks
Benjamin Gonzalez and Estelle Barbier for their contribution to this work. H.H. is supported by a fellowship from INRA (Institut National de la Recherche Agronomique, France).
[7]
[8]
[9]
[10]
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