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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. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 1 9 1 - 8 FEMSLE 8199 29-6-98 196 H. Hajjaj et al. / FEMS Microbiology Letters 164 (1998) 195^200 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 FEMSLE 8199 29-6-98 H. Hajjaj et al. / FEMS Microbiology Letters 164 (1998) 195^200 197 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. FEMSLE 8199 29-6-98 198 H. Hajjaj et al. / FEMS Microbiology Letters 164 (1998) 195^200 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 FEMSLE 8199 29-6-98 H. Hajjaj et al. / FEMS Microbiology Letters 164 (1998) 195^200 199 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- FEMSLE 8199 29-6-98 200 H. Hajjaj et al. / FEMS Microbiology Letters 164 (1998) 195^200 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] References [1] van den Hondel, C.A.M.J.J., Punt, P.J. and van Gorcom, R.F.M. (1991) Heterologous gene expression in ¢lamentous fungi. In: More Gene Manipulations in Fungi (Bennett, J.W. and Lasure, L.L., Eds.), pp. 396^428. Academic Press, San Diego. Kubicek, C.P. and Roëhr, M. (1986) Citric acid fermentation. CRC Crit. Rev. Biotechnol. 3, 331^373. Hajjaj, H., Klaebe, A., Loret, M.O., Tzedakis, T., Goma, G. and Blanc, P.J. (1997) Production and identi¢cation of N-glucosylrubropunctamine and N-glucosylmonascorubramine from Monascus ruber and the occurrence of electron donoracceptor complexes in these red pigments. Appl. Environ. Microbiol. 63, 2671^2678. Theobald, U., Mailinger, W., Reuss, M. and Rizzi, M. (1993) In vivo analysis of glucose-induced fast glycolytic changes in yeast adenine nucleotide pool applying a rapid sampling technique. Anal. Biochem. 214, 31^37. De Koning, W. and Van Dam, K. (1992) A method for the determination of changes of glycolytic metabolites in yeast on a subsecond time scale using extraction at neutral pH. Anal. Biochem. 204, 118^123. Gonzalez, B., Franc°ois, J. and Renaud, M. (1997) A rapid and reliable method for metabolite extraction in yeast using boiling bu¡ered ethanol. Yeast 13, 1347^1356. Blanc, P.J., Loret, M.A. and Goma, G. (1995) Production of citrinin by various species of Monascus. Biotechnol. Lett. 17, 291^294. Ruijter, G.J.G. and Visser, J. (1996) Determination of intermediary metabolites in Aspergillus niger. J. Microbiol. Methods 25, 295^302. Bergmeyer, H.U. (1985) Methods of Enzymatic Analysis, Vols. VI and VII, VCH Publishers. Kuwahara, M., Ishida, Y. and Miyagawa, Y. (1982) Biosynthesis of pyridine coenzymes by Aspergillus fungi. J. Ferment. Technol. 60, 399^404. FEMSLE 8199 29-6-98