Fast Sampling of the Cellular Metabolome.

Obtaining meaningful snapshots of the metabolome of microorganisms requires rapid sampling and immediate quenching of all metabolic activity, to prevent any changes in metabolite levels after sampling. Furthermore, a suitable extraction method is required ensuring complete extraction of metabolites from the cells and inactivation of enzymatic activity, with minimal degradation of labile compounds. Finally, a sensitive, high-throughput analysis platform is needed to quantify a large number of metabolites in a small amount of sample. An issue which has often been overlooked in microbial metabolomics is the fact that many intracellular metabolites are also present in significant amounts outside the cells and may interfere with the quantification of the endo metabolome. Attempts to remove the extracellular metabolites with dedicated quenching methods often induce release of intracellular metabolites into the quenching solution. For eukaryotic microorganisms, this release can be minimized by adaptation of the quenching method. For prokaryotic cells, this has not yet been accomplished, so the application of a differential method whereby metabolites are measured in the culture supernatant as well as in total broth samples, to calculate the intracellular levels by subtraction, seems to be the most suitable approach. Here we present an overview of different sampling, quenching, and extraction methods developed for microbial metabolomics, described in the literature. Detailed protocols are provided for rapid sampling, quenching, and extraction, for measurement of metabolites in total broth samples, washed cell samples, and supernatant, to be applied for quantitative metabolomics of both eukaryotic and prokaryotic microorganisms.

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.

Fast sampling of the cellular metabolome.

Obtaining meaningful snapshots of the metabolome of microorganisms requires rapid sampling and immediate quenching of all metabolic activity, to prevent any changes in metabolite levels after sampling. Furthermore, a suitable extraction method is required ensuring complete extraction of metabolites from the cells and inactivation of enzymatic activity, with minimal degradation of labile compounds. Finally a sensitive, high-throughput analysis platform is needed to quantify a large number of metabolites in a small amount of sample. An issue which has often been overlooked in microbial metabolomics is the fact that many intracellular metabolites are also present in significant amounts outside the cells, and may interfere with the endometabolome measurements. Attempts to remove the extracellular metabolites with dedicated quenching methods often induce release of intracellular metabolites into the quenching solution. For eukaryotic microorganisms, leakage can be minimized by adaptation of the quenching method. For prokaryotic cells this had not yet been accomplished, so the application of a differential method whereby metabolites are measured in the culture supernatant as well as in total broth samples, to calculate the intracellular levels by subtraction, seems to be the most suitable approach. Here we present an overview of different sampling, quenching, and extraction methods developed for microbial metabolomics, described in the literature. Detailed protocols are provided for rapid sampling, quenching, and extraction for measurement of metabolites in total broth samples, washed cell samples and supernatant, to be applied for quantitative metabolomics of both eukaryotic and prokaryotic microorganisms.

Novel insights in transport mechanisms and kinetics of phenylacetic acid and penicillin-G in Penicillium chrysogenum.

Although penicillin-G (PenG) production by the fungus Penicillium chrysogenum is a well-studied process, little is known about the mechanisms of transport of the precursor phenylacetic acid (PAA) and the product PenG over the cell membrane. To obtain more insight in the nature of these mechanisms, in vivo stimulus response experiments were performed with PAA and PenG in chemostat cultures of P. chrysogenum at time scales of seconds to minutes. The results indicated that PAA is able to enter the cell by passive diffusion of the undissociated acid at a high rate, but is at the same time actively excreted, possibly by an ATP-binding cassette transporter. This results in a futile cycle, dissipating a significant amount of metabolic energy, which was confirmed by increased rates of substrate and oxygen consumption, and carbon dioxide production. To estimate the kinetic properties of passive import and active export of PAA over the cell membrane, a dynamic mathematical model was constructed. With this model, a good description of the dynamic data could be obtained. Also, PenG was found to be rapidly taken up by the cells upon extracellular addition, indicating that PenG transport is reversible. The measured concentration gradient of PenG over the cell membrane corresponded well with facilitated transport. Also, for PenG transport, a dynamic model was constructed and validated with experimental data. The outcome of the model simulations was in agreement with the presence of a facilitated transport system for PenG.

Degeneration of penicillin production in ethanol-limited chemostat cultivations of Penicillium chrysogenum: A systems biology approach.

BACKGROUND: In microbial production of non-catabolic products such as antibiotics a loss of production capacity upon long-term cultivation (for example chemostat), a phenomenon called strain degeneration, is often observed. In this study a systems biology approach, monitoring changes from gene to produced flux, was used to study degeneration of penicillin production in a high producing Penicillium chrysogenum strain during prolonged ethanol-limited chemostat cultivations. RESULTS: During these cultivations, the biomass specific penicillin production rate decreased more than 10-fold in less than 22 generations. No evidence was obtained for a decrease of the copy number of the penicillin gene cluster, nor a significant down regulation of the expression of the penicillin biosynthesis genes. However, a strong down regulation of the biosynthesis pathway of cysteine, one of the precursors of penicillin, was observed. Furthermore the protein levels of the penicillin pathway enzymes L-α-(δ-aminoadipyl)-L-α-cystenyl-D-α-valine synthetase (ACVS) and isopenicillin-N synthase (IPNS), decreased significantly. Re-cultivation of fully degenerated cells in unlimited batch culture and subsequent C-limited chemostats did only result in a slight recovery of penicillin production. CONCLUSIONS: Our findings indicate that the observed degeneration is attributed to a significant decrease of the levels of the first two enzymes of the penicillin biosynthesis pathway, ACVS and IPNS. This decrease is not caused by genetic instability of the penicillin amplicon, neither by down regulation of the penicillin biosynthesis pathway. Furthermore no indications were obtained for degradation of these enzymes as a result of autophagy. Possible causes for the decreased enzyme levels could be a decrease of the translation efficiency of ACVS and IPNS during degeneration, or the presence of a culture variant impaired in the biosynthesis of functional proteins of these enzymes, which outcompeted the high producing part of the population.

Intracellular metabolite determination in the presence of extracellular abundance: Application to the penicillin biosynthesis pathway in Penicillium chrysogenum.

Important steps in metabolic pathways are formed by the transport of substrates and products over the cell membrane. The study of in vivo transport kinetics requires accurate quantification of intra- and extracellular levels of the transported compounds. Especially in case of extracellular abundance, the proper determination of intracellular metabolite levels poses challenges. Efficient removal of extracellular substrates and products is therefore important not to overestimate the intracellular amounts. In this study we evaluated two different rapid sampling methods, one combined with cold filtration and the other with centrifugation, for their applicability to determine intracellular amounts of metabolites which are present in high concentrations in the extracellular medium. The filtration-based method combines fast sampling and immediate quenching of cellular metabolism in cold methanol, with rapid and effective removal of all compounds present outside the cells by means of direct filtration and subsequent filtration-based washing. In the centrifugation-based method, removal of the extracellular metabolites from the cells was achieved by means of multiple centrifugation and resuspension steps with the cold quenching solution. The cold filtration method was found to be highly superior to the centrifugation method to determine intracellular amounts of metabolites related to penicillin-G biosynthesis and allowed the quantification of compounds of which the extracellular amounts were 3-4 orders of magnitude higher than the intracellular amounts. Using this method for the first time allowed to measure the intracellular levels of the side chain precursor phenylacetic acid (PAA) and the product penicillin-G of the penicillin biosynthesis pathway, compounds of which the transport mechanism in Penicillium chrysogenum is still far from being sufficiently understood.

Dynamic gene expression regulation model for growth and penicillin production in Penicillium chrysogenum.

As is often the case for microbial product formation, the penicillin production rate of Penicillium chrysogenum has been observed to be a function of the growth rate of the organism. The relation between the biomass specific rate of penicillin formation (q(p)) and growth rate (mu) has been measured under steady state conditions in carbon limited chemostats resulting in a steady state q(p)(mu) relation. Direct application of such a relation to predict the rate of product formation during dynamic conditions, as they occur, for example, in fed-batch experiments, leads to errors in the prediction, because q(p) is not an instantaneous function of the growth rate but rather lags behind because of adaptational and regulatory processes. In this paper a dynamic gene regulation model is presented, in which the specific rate of penicillin production is assumed to be a linear function of the amount of a rate-limiting enzyme in the penicillin production pathway. Enzyme activity assays were performed and strongly indicated that isopenicillin-N synthase (IPNS) was the main rate-limiting enzyme for penicillin-G biosynthesis in our strain. The developed gene regulation model predicts the expression of this rate limiting enzyme based on glucose repression, fast decay of the mRNA encoding for the enzyme as well as the decay of the enzyme itself. The gene regulation model was combined with a stoichiometric model and appeared to accurately describe the biomass and penicillin concentrations for both chemostat steady-state as well as the dynamics during chemostat start-up and fed-batch cultivation.

An engineered yeast efficiently secreting penicillin.

This study aimed at developing an alternative host for the production of penicillin (PEN). As yet, the industrial production of this beta-lactam antibiotic is confined to the filamentous fungus Penicillium chrysogenum. As such, the yeast Hansenula polymorpha, a recognized producer of pharmaceuticals, represents an attractive alternative. Introduction of the P. chrysogenum gene encoding the non-ribosomal peptide synthetase (NRPS) delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) in H. polymorpha, resulted in the production of active ACVS enzyme, when co-expressed with the Bacillus subtilis sfp gene encoding a phosphopantetheinyl transferase that activated ACVS. This represents the first example of the functional expression of a non-ribosomal peptide synthetase in yeast. Co-expression with the P. chrysogenum genes encoding the cytosolic enzyme isopenicillin N synthase as well as the two peroxisomal enzymes isopenicillin N acyl transferase (IAT) and phenylacetyl CoA ligase (PCL) resulted in production of biologically active PEN, which was efficiently secreted. The amount of secreted PEN was similar to that produced by the original P. chrysogenum NRRL1951 strain (approx. 1 mg/L). PEN production was decreased over two-fold in a yeast strain lacking peroxisomes, indicating that the peroxisomal localization of IAT and PCL is important for efficient PEN production. The breakthroughs of this work enable exploration of new yeast-based cell factories for the production of (novel) beta-lactam antibiotics as well as other natural and semi-synthetic peptides (e.g. immunosuppressive and cytostatic agents), whose production involves NRPS's.

Controlling light-use by Rhodobacter capsulatus continuous cultures in a flat-panel photobioreactor.

The main bottleneck in scale-up of phototrophic fermentation is the low efficiency of light energy conversion to the desired product, which is caused by an excessive dissipation of light energy to heat. The photoheterotrophic formation of hydrogen from acetate and light energy by the microorganism Rhodobacter capsulatus NCIMB 11773 was chosen as a case study in this work. A light energy balance was set up, in which the total bacterial light energy absorption is split up and attributed to its destinations. These are biomass growth and maintenance, generation of hydrogen and photosynthetic heat dissipation. The constants defined in the light energy balance were determined experimentally using a flat-panel photobioreactor with a 3-cm optical path. An experimental method called D-stat was applied. Continuous cultures were kept in a so-called pseudo steady state, while the dilution rate was reduced slowly and smoothly. The biomass yield and maintenance coefficients of Rhodobacter capsulatus biomass on light energy were determined at 12.4 W/m(2) (400-950 nm) and amounted to 2.58 x 10(-8) +/- 0.04 x 10(-8) kg/J and 102 +/- 3.5 W/kg, respectively. The fraction of the absorbed light energy that was dissipated to heat at 473 W/m(2) depended on the biomass concentration in the reactor and varied between 0.80 and 0.88, as the biomass concentration was increased from 2.0 to 8.0 kg/m(3). The process conditions were estimated at which a 3.7% conversion efficiency of absorbed light energy to produced hydrogen energy should be attainable at 473 W/m(2).