MIRACLE: mass isotopomer ratio analysis of U-13C-labeled extracts. A new method for accurate quantification of changes in concentrations of intracellular metabolites

First, we report the application of stable isotope dilution theory in metabolome characterization of aerobic glucose limited chemostat culture of S. cerevisiae CEN.PK 113-7D using liquid chromatography-electrospray ionization MS/MS (LC-ESI-MS/MS). A glucose-limited chemostat culture of S. cerevisiae was grown to steady state at a specific growth rate (mu)=0.05 h(-1) in a medium containing only naturally labeled (99% U-12C, 1% U-13C) carbon source. Upon reaching steady state, defined as 5 volume changes, the culture medium was switched to chemically identical medium except that the carbon source was replaced with 100% uniformly (U) 13C labeled stable carbon isotope, fed for 4 h, with sampling every hour. We observed that within a period of 1 h approximately 80% of the measured glycolytic metabolites were U-13C-labeled. Surprisingly, during the next 3 h no significant increase of the U-13C-labeled metabolites occurred. Second, we demonstrate for the first time the LC-ESI-MS/MS-based quantification of intracellular metabolite concentrations using U-13C-labeled metabolite extracts from chemostat cultivated S. cerevisiae cells, harvested after 4 h of feeding with 100% U-13C-labeled medium, as internal standard. This method is hereby termed "Mass Isotopomer Ratio Analysis of U-13C Labeled Extracts" (MIRACLE). With this method each metabolite concentration is quantified relative to the concentration of its U-13C-labeled equivalent, thereby eliminating drawbacks of LC-ESI-MS/MS analysis such as nonlinear response and matrix effects and thus leads to a significant reduction of experimental error and work load (i.e., no spiking and standard additions). By coextracting a known amount of U-13C labeled cells with the unlabeled samples, metabolite losses occurring during the sample extraction procedure are corrected for.     

An optimized protocol for metabolome analysis in yeast using direct infusion electrospray mass spectrometry

A method for the global analysis of yeast intracellular metabolites, based on electrospray mass spectrometry (ES-MS), has been developed. This has involved the optimization of methods for quenching metabolism in Saccharomyces cerevisiae and extracting the metabolites for analysis by positive-ion electrospray mass spectrometry. The influence of cultivation conditions, sampling, quenching and extraction conditions, concentration step, and storage have all been studied and adapted to allow direct infusion of samples into the mass spectrometer and the acquisition of metabolic profiles with simultaneous detection of more than 25 intracellular metabolites. The method, which can be applied to other micro-organisms and biological systems, may be used for comparative analysis and screening of metabolite profiles of yeast strains and mutants under controlled conditions in order to elucidate gene function via metabolomics. Examples of the application of this analytical strategy to specific yeast strains and single-ORF yeast deletion mutants generated through the EUROFAN programme are presented.     

The development of metabolomic sampling procedures for Pichia pastoris, and baseline metabolome data

Metabolic profiling is increasingly being used to investigate a diverse range of biological questions. Due to the rapid turnover of intracellular metabolites it is important to have reliable, reproducible techniques for sampling and sample treatment. Through the use of non-targeted analytical techniques such as NMR and GC-MS we have performed a comprehensive quantitative investigation of sampling techniques for Pichia pastoris. It was clear that quenching metabolism using solutions based on the standard cold methanol protocol caused some metabolite losses from P. pastoris cells. However, these were at a low level, with the NMR results indicating metabolite increases in the quenching solution below 5% of their intracellular level for 75% of metabolites identified; while the GC-MS results suggest a slightly higher level with increases below 15% of their intracellular values. There were subtle differences between the four quenching solutions investigated but broadly, they all gave similar results. Total culture extraction of cells + broth using high cell density cultures typical of P. pastoris fermentations, was an efficient sampling technique for NMR analysis and provided a gold standard of intracellular metabolite levels; however, salts in the media affected the GC-MS analysis. Furthermore, there was no benefit in including an additional washing step in the quenching process, as the results were essentially identical to those obtained just by a single centrifugation step. We have identified the major high-concentration metabolites found in both the extra- and intracellular locations of P. pastoris cultures by NMR spectroscopy and GC-MS. This has provided us with a baseline metabolome for P. pastoris for future studies. The P. pastoris metabolome is significantly different from that of Saccharomyces cerevisiae, with the most notable difference being the production of high concentrations of arabitol by P. pastoris.     

Optimization of cold methanol quenching for quantitative metabolomics of Penicillium chrysogenum

A sampling procedure for quantitative metabolomics in Penicillium chrysogenum based on cold aqueous methanol quenching was re-evaluated and optimized to reduce metabolite leakage during sample treatment. The optimization study included amino acids and intermediates of the glycolysis and the TCA-cycle. Metabolite leakage was found to be minimal for a methanol content of the quenching solution (QS) of 40% (v/v) while keeping the temperature of the quenched sample near -20°C. The average metabolite recovery under these conditions was 95.7% (±1.1%). Several observations support the hypothesis that metabolite leakage from quenched mycelia of P. chrysogenum occurs by diffusion over the cell membrane. First, a prolonged contact time between mycelia and the QS lead to a somewhat higher extent of leakage. Second, when suboptimal quenching liquids were used, increased metabolite leakage was found to be correlated with lower molecular weight and with lower absolute net charge. The finding that lowering the methanol content of the quenching liquid reduces metabolite leakage in P. chrysogenum contrasts with recently published quenching studies for two other eukaryotic micro-organisms. This demonstrates that it is necessary to validate and, if needed, optimize the quenching conditions for each particular micro-organism. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0367-3) contains supplementary material, which is available to authorized users.     

Quantitative analysis of the microbial metabolome by isotope dilution mass spectrometry using uniformly 13C-labeled cell extracts as internal standards

A novel method was developed for the quantitative analysis of the microbial metabolome using a mixture of fully uniformly (U) (13)C-labeled metabolites as internal standard (IS) in the metabolite extraction procedure the subsequent liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. This mixture of fully U (13)C-labeled metabolites was extracted from biomass of Saccharomyces cerevisiae cultivated in a fed-batch fermentation on fully U (13)C-labeled substrates. The obtained labeled cell extract contained, in principle, the whole yeast metabolome, allowing the quantification of any intracellular metabolite of interest in S. cerevisiae. We have applied the labeled cell extract as IS in the analysis of glycolytic and tricarboxylic acid (TCA) cycle intermediates in S. cerevisiae sampled in both steady-state and transient conditions following a glucose pulse. The use of labeled IS effectively reduced errors due to variations occurring in the analysis and sample processing. As a result, the linearity of calibration lines and the precision of measurements were significantly improved. Coextraction of the labeled cell extract with the samples also eliminates the need to perform elaborate recovery checks for each metabolite to be analyzed. In conclusion, the method presented leads to less workload, more robustness, and a higher precision in metabolome analysis.     

13C-labeled gluconate tracing as a direct and accurate method for determining the pentose phosphate pathway split ratio in Penicillium chrysogenum

In this study we developed a new method for accurately determining the pentose phosphate pathway (PPP) split ratio, an important metabolic parameter in the primary metabolism of a cell. This method is based on simultaneous feeding of unlabeled glucose and trace amounts of [U-13C]gluconate, followed by measurement of the mass isotopomers of the intracellular metabolites surrounding the 6-phosphogluconate node. The gluconate tracer method was used with a penicillin G-producing chemostat culture of the filamentous fungus Penicillium chrysogenum. For comparison, a 13C-labeling-based metabolic flux analysis (MFA) was performed for glycolysis and the PPP of P. chrysogenum. For the first time mass isotopomer measurements of 13C-labeled primary metabolites are reported for P. chrysogenum and used for a 13C-based MFA. Estimation of the PPP split ratio of P. chrysogenum at a growth rate of 0.02 h(-1) yielded comparable values for the gluconate tracer method and the 13C-based MFA method, 51.8% and 51.1%, respectively. A sensitivity analysis of the estimated PPP split ratios showed that the 95% confidence interval was almost threefold smaller for the gluconate tracer method than for the 13C-based MFA method (40.0 to 63.5% and 46.0 to 56.5%, respectively). From these results we concluded that the gluconate tracer method permits accurate determination of the PPP split ratio but provides no information about the remaining cellular metabolism, while the 13C-based MFA method permits estimation of multiple fluxes but provides a less accurate estimate of the PPP split ratio.     

Generating short-term kinetic responses of primary metabolism of Penicillium chrysogenum through glucose perturbation in the bioscope mini reactor

A first study of the in vivo kinetic properties of primary metabolism of Penicillium chrysogenum is presented. Dynamic metabolite data have been generated by rapidly increasing the extracellular glucose concentration of cells cultivated under well-defined conditions in an aerobic glucose-limited chemostat followed by measurement of the fast dynamic response of the primary metabolite levels (glucose pulse experiment). These experiments were carried out directly in the chemostat as well as in a mini plug flow reactor (BioScope) outside the chemostat. The results of the glucose pulse experiments carried out in the chemostat and the Bioscope were highly similar. During the 90 s time window of the pulse experiment, the glucose consumption rate increased to a value twice as high as in the steady state, a much lower increase than observed for the fermenting yeast Saccharomyces cerevisiae under similar conditions. Although the observed metabolite patterns in P. chrysogenum were comparable to S. cerevisiae large differences in the magnitude of the dynamic behavior were observed between both organisms. During the pulse experiment the level of glycolytic and TCA cycle intermediates, and adenine nucleotides changed between two- and five-fold. Furthermore, a highly similar five-fold increase in the cytocolic NADH/NAD ratio could be calculated from two independent equilibrium assumptions (fructose 1,6 bis-phosphate to the pool of 2 and 3PG and oxaloacetate to fumarate with glutamate transaminase). It was also found that the C4 pool (aspartate, fumarate, and malate) became much more reduced due to this increase in NADH/NAD ratio. Equilibrium conditions were confirmed to exist in the hexose-P pool, the glycolysis between F16bP and 2+3PG and in the C4 pool of the TCA cycle (fumarate, malate, oxaloacetate and aspartate).     

Metabolic-flux analysis of Saccharomyces cerevisiae CEN.PK113-7D based on mass isotopomer measurements of (13)C-labeled primary metabolites

Metabolic-flux analyses in microorganisms are increasingly based on (13)C-labeling data. In this paper a new approach for the measurement of (13)C-label distributions is presented: rapid sampling and quenching of microorganisms from a cultivation, followed by extraction and detection by liquid chromatography-mass spectrometry of free intracellular metabolites. This approach allows the direct assessment of mass isotopomer distributions of primary metabolites. The method is applied to the glycolytic and pentose phosphate pathways of Saccharomyces cerevisiae strain CEN.PK113-7D grown in an aerobic, glucose-limited chemostat culture. Detailed investigations of the measured mass isotopomer distributions demonstrate the accuracy and information-richness of the obtained data. The mass fractions are fitted with a cumomer model to yield the metabolic fluxes. It is estimated that 24% of the consumed glucose is catabolized via the pentose phosphate pathway. Furthermore, it is found that turnover of storage carbohydrates occurs. Inclusion of this turnover in the model leads to a large confidence interval of the estimated split ratio.     

Correlation of cell volume fractions with cell concentrations in fermentation media

Cell volume fractions and cell concentrations were measured in submerged cultures of Saccharomyces cerevisiae, Escherichia coli, and Penicillium chrysogenum. Correlations for cell volume fractions with cell concentrations in fermentation media of the microorganisms were established accordingly. Other key properties of microorganisms, such as cell water content, wet cell density, and dry cell density, can also be obtained with the use of the current method. The results are in good agreement with data available in the literature.     

The biosynthesis of low-molecular-weight nitrogen-containing secondary metabolite-alkaloids by the resident strains of Penicillium chrysogenum and Penicillium expansum isolated on the board of the Mir space station

The analysis of the absorption spectra of the low-molecular-weight nitrogen-containing secondary metabolites--alkaloids--of 4 Penicillium chrysogenum strains and 6 Penicillium expansum strains isolated on board the Mir space station showed that all these strains synthesize metabolites of alkaloid origin (roquefortine, 3,12-dihydroroquefortine, meleagrin, viridicatin, viridicatol, isorugulosuvin, rugulosuvin B, N-acetyl-tryptamine, and a "yellow metabolite" containing the benzoquinone chromophore).     

链霉菌S01菌株几丁质酶对植物病原真菌的拮抗作用

纯化后的链霉菌S01菌株几丁质酶用环柱法在PDA平板上对杨树腐烂病菌（Valsasordida）、葡萄孢菌（Botrytissp．AS3．266）、黄瓜黑腥病菌（Cladosporiumcucumerinrm）、辣椒疫病菌（Phytophlhoracopsici）、棉花黄萎病菌（Verliilliumalbo-atrum)、立枯丝核菌（Rhizoctonasolani）等植物病原真菌及产黄青霉（Penlcillumc     

Simultaneous quantification of free nucleotides in complex biological samples using ion pair reversed phase liquid chromatography isotope dilution tandem mass spectrometry

A new sensitive and accurate analytical method has been developed for quantification of intracellular nucleotides in complex biological samples from cultured cells of different microorganisms such as Saccharomyces cerevisiae, Escherichia coli, and Penicillium chrysogenum. This method is based on ion pair reversed phase liquid chromatography electrospray ionization isotope dilution tandem mass spectrometry (IP-LC-ESI-ID-MS/MS. A good separation and low detection limits were observed for these compounds using dibutylamine as volatile ion pair reagent in the mobile phase of the LC. Uniformly ¹³C-labeled isotopes of nucleotides were used as internal standards for both extraction and quantification of intracellular nucleotides. The method was validated by determining the linearity, sensitivity, and repeatability.     

Subcellular localization of the homocitrate synthase in<Emphasis Type="Italic"> Penicillium chrysogenum</Emphasis>

There are conflicting reports regarding the cellular localization in Saccharomyces cerevisiae and filamentous fungi of homocitrate synthase, the first enzyme in the lysine biosynthetic pathway. The homocitrate synthase (HS) gene (lys1) of Penicillium chrysogenum was disrupted in three transformants (HS(-)) of the Wis 54-1255 pyrG strain. The three mutants named HS1(-), HS2(-) and HS3(-) all lacked homocitrate synthase activity and showed lysine auxotrophy, indicating that there is a single gene for homocitrate synthase in P. chrysogenum. The lys1 ORF was fused in frame to the gene for the green fluorescent protein (GFP) gene of the jellyfish Aequorea victoria. Homocitrate synthase-deficient mutants transformed with a plasmid containing the lys1-GFP fusion recovered prototrophy and showed similar levels of homocitrate synthase activity to the parental strain Wis 54-1255, indicating that the hybrid protein retains the biological function of wild-type homocitrate synthase. Immunoblotting analysis revealed that the HS-GFP fusion protein is maintained intact and does not release the GFP moiety. Fluorescence microscopy analysis of the transformants showed that homocitrate synthase was mainly located in the cytoplasm in P. chrysogenum; in S. cerevisiae the enzyme is targeted to the nucleus. The control nuclear protein StuA was properly targeted to the nucleus when the StuA (targeting domain)-GFP hybrid protein was expressed in P. chrysogenum. The difference in localization of homocitrate synthase between P. chrysogenum and S. cerevisiae suggests that this protein may play a regulatory function, in addition to its catalytic function, in S. cerevisiae but not in P. chrysogenum.     

Integrated sampling procedure for metabolome analysis

Metabolome analysis, the analysis of large sets of intracellular metabolites, has become an important systems analysis method in biotechnological and pharmaceutical research. In metabolic engineering, the integration of metabolome data with fluxome and proteome data into large-scale mathematical models promises to foster rational strategies for strain and cell line improvement. However, the development of reproducible sampling procedures for quantitative analysis of intracellular metabolite concentrations represents a major challenge, accomplishing (i) fast transfer of sample, (ii) efficient quenching of metabolism, (iii) quantitative metabolite extraction, and (iv) optimum sample conditioning for subsequent quantitative analysis. In addressing these requirements, we propose an integrated sampling procedure. Simultaneous quenching and quantitative extraction of intracellular metabolites were realized by short-time exposure of cells to temperatures < or =95 degrees C, where intracellular metabolites are released quantitatively. Based on these findings, we combined principles of heat transfer with knowledge on physiology, for example, turnover rates of energy metabolites, to develop an optimized sampling procedure based on a coiled single tube heat exchanger. As a result, this sampling procedure enables reliable and reproducible measurements through (i) the integration of three unit operations into a one unit operation, (ii) the avoidance of any alteration of the sample due to chemical reagents in quenching and extraction, and (iii) automation. A sampling frequency of 5 s(-)(1) and an overall individual sample processing time faster than 30 s allow observing responses of intracellular metabolite concentrations to extracellular stimuli on a subsecond time scale. Recovery and reliability of the unit operations were analyzed. Impact of sample conditioning on subsequent IC-MS analysis of metabolites was examined as well. The integrated sampling procedure was validated through consistent results from steady-state metabolite analysis of Escherichia coli cultivated in a chemostat at D = 0.1 h(-)(1).     

PcMtr, an aromatic and neutral aliphatic amino acid permease of Penicillium chrysogenum

The gene encoding an aromatic and neutral aliphatic amino acid permease of Penicillium chrysogenum was cloned, functionally expressed and characterized in Saccharomyces cerevisiae M4276. The permease, designated PcMtr, is structurally and functionally homologous to Mtr of Neurospora crassa, and unrelated to the Amino Acid Permease (AAP) family which includes most amino acid permeases in fungi. Database searches of completed fungal genome sequences reveal that Mtr type permeases are not widely distributed among fungi, suggesting a specialized function.     

Simple and robust method for estimation of the split between the oxidative pentose phosphate pathway and the Embden-Meyerhof-Parnas pathway in microorganisms

The flux through the oxidative pentose phosphate (PP) pathway was estimated in Bacillus clausii, Saccharomyces cerevisiae, and Penicillium chrysogenum growing in chemostats with [1-(13)C]glucose as the limiting substrate. The flux calculations were based on a simple algebraic expression that is valid irrespective of isotope rearrangements arising from reversibilities of the reactions in the PP pathway and the upper part of the Embden-Meyerhof-Parnas pathway. The algebraically calculated fluxes were validated by comparing the results with estimates obtained using a numerical method that includes the entire central carbon metabolism. Setting the glucose uptake rate to 100, the algebraic expression yielded estimates of the PP pathway flux in B. clausii, S. cerevisiae, and P. chrysogenum of 20, 42, and 75, respectively. These results are in accordance with the results from the numerical method. The information on the labeling patterns of glucose and the proteinogenic amino acids were obtained using gas chromatography / mass spectrometry, which is a very sensitive technique, and therefore only a small amount of biomass is needed for the analysis. Furthermore, the method developed in this study is fast and readily accessible, as the calculations are based on a simple algebraic expression.     

Penicillium persicinum, a new griseofulvin, chrysogine and roquefortine C producing species from Qinghai province, China

A unique Penicillium isolate from Chinese soil with terverticillate penicilli and ellipsoidal to cylindrical smooth-walled conidia, produces, in addition to the common metabolite ergosterol, copious amounts of an unknown peach-red pigment and the following secondary metabolites: griseofulvin, dechlorogriseofulvin, lichexanthone, roquefortine C, roquefortine D, chrysogine, 2-pyrovoylaminobenzamide, 2-acetyl-quinazolin-4(3H)-one. This isolate, CBS 111235, is described as Penicillium persicinum sp. nov., which belongs to subgenus Penicillium section Chrysogena but is morphologically similar to P. italicum. On the basis of the production of secondary metabolites it resembles P. griseofulvum and P. coprophilum. Sequence data using part of the beta-tubulin gene showed that it is phylogenetically related to P. chrysogenum and P. aethiopicum in section Chrysogena with which it shares both secondary metabolites and ability to grow at 37 degrees C.     

Autophagy deficiency promotes beta-lactam production in Penicillium chrysogenum

We have investigated the significance of autophagy in the production of the β-lactam antibiotic penicillin (PEN) by the filamentous fungus Penicillium chrysogenum. In this fungus PEN production is compartmentalized in the cytosol and in peroxisomes. We demonstrate that under PEN-producing conditions significant amounts of cytosolic and peroxisomal proteins are degraded via autophagy. Morphological analysis, based on electron and fluorescence microscopy, revealed that this phenomenon might contribute to progressive deterioration of late subapical cells. We show that deletion of the P. chrysogenum ortholog of Saccharomyces cerevisiae serine-threonine kinase atg1 results in impairment of autophagy. In P. chrysogenum atg1 cells, a distinct delay in cell degeneration is observed relative to wild-type cells. This phenomenon is associated with an increase in the enzyme levels of the PEN biosynthetic pathway and enhanced production levels of this antibacterial compound.     

Automated fast filtration and on‐filter quenching improve the intracellular metabolite analysis of microorganisms

To reliably determine intracellular metabolite concentrations in microorganisms, accurate sampling and sample inactivation strategies are crucial. Here, we present a method for automated fast filtration and on‐filter quenching of microbial samples to overcome metabolite leakage induced by cold shock and significantly reduce the sampling and treatment time compared to manual filtration methods. The whole process of sampling, sample filtration, filter wash, and quenching of the filter with liquid nitrogen was finished in less than 6–15 s, depending on the experimental setup. By integration into an automated fast sampling device, we compared our method to the conventional methanol quenching method and showed that intracellular amino acid contents in Escherichia coli were significantly increased (≥75%) with our fast filtration and on‐filter quenching method. Furthermore, we investigated different filter types for the fast filtration and the efficiency of metabolite extraction from cells held on filters. Additionally, we found that the fast filtration behaves considerably different during exponential and nonexponential growth, probably due to variations of cell morphologies. Overall, we demonstrated that the automation of the fast filtration method significantly reduces the time for filtration and quenching and hence enlarge the number of metabolites that can be quantified with this leakage‐free sampling method.