Robust identification of metabolic control for microbial l-methionine production following an easy-to-use puristic approach.

The identification of promising metabolic engineering targets is a key issue in metabolic control analysis (MCA). Conventional approaches make intensive use of model-based studies, such as exploiting post-pulse metabolic dynamics after proper perturbation of the microbial system. Here, we present an easy-to-use, purely data-driven approach, defining pool efflux capacities (PEC) for identifying reactions that exert the highest flux control in linear pathways. Comparisons with linlog-based MCA and data-driven substrate elasticities (DDSE) showed that similar key control steps were identified using PEC. Using the example of l-methionine production with recombinant Escherichia coli, PEC consistently and robustly identified main flux controls using perturbation data after a non-labeled 12C-l-serine stimulus. Furthermore, the application of full-labeled 13C-l-serine stimuli yielded additional insights into stimulus propagation to l-methionine. PEC analysis performed on the 13C data set revealed the same targets as the 12C data set. Notably, the typical drawback of metabolome analysis, namely, the omnipresent leakage of metabolites, was excluded using the 13C PEC approach.

Evolution of pyruvate kinase-deficient Escherichia coli mutants enables glycerol-based cell growth and succinate production.

AIMS: The aim of this study was to engineer Escherichia coli strains that efficiently produce succinate from glycerol under anaerobic conditions after an aerobic growth phase. METHODS AND RESULTS: We constructed E. coli strain ss195 with deletions of pykA and pykF, which resulted in slow growth on glycerol as sole carbon source. This growth defect was overcome by the selection of fast-growing mutants. Whole-genome resequencing of the evolved mutant ss251 identified the mutation A595S in PEP carboxylase (Ppc). Reverse metabolic engineering by introducing the wild-type allele revealed that this mutation is crucial for the described phenotype. Strain ss251 and derivatives thereof produced succinate with high yields above 80% mol mol(-1) from glycerol under nongrowth conditions. CONCLUSIONS: The results show that during the aerobic growth of ss251, the formation of pyruvate proceeds via the proposed POMP pathway, starting with the carboxylation of PEP by Ppc. The resulting oxaloacetate is reduced by malate dehydrogenase (Mdh) to malate, which is then decarboxylated back to pyruvate by a malic enzyme (MaeA or MaeB). Mutation of ppc is crucial for fast growth of pykAF mutants on glycerol. SIGNIFICANCE AND IMPACT OF STUDY: An E. coli mutant that is capable of achieving high yields of succinate (a top valued-added chemical) from glycerol (an abundant carbon source) was constructed. The identified ppc mutation could be applied to other production strains that require strong PEP carboxylation fluxes.

Systems biology for industrial strains and fermentation processes--example: amino acids.

Systems biology is attracting significant interest finding applications not only in pharmaceutical development but also for basic studies on microbial systems. The latter often concentrate on the quantitative understanding of global regulation phenomena. So far, these activities are dominated by academic groups basically mirroring the necessity to prepare the sound scientific understanding first, before industrial applications can be derived later. However, this short-term view may not be sufficient because systems biology already offers numerous benefits for industrial applications, provided that special constraints are considered. This contribution indicates some of the constraints worth noticing when industrial systems biology projects are carried out. Consequently, differences in project structure and goals between purely academic and industrial systems biology projects are outlined.

Model-based analysis and optimization of an ISPR approach using reactive extraction for pilot-scale L-phenylalanine production.

Based on experimental data from fermentation runs, as well as from L-phenylalanine (l-Phe) separation studies, a simple model is presented that describes the total ISPR approach for on-line L-Phe separation. While fermentation process modeling via a macrokinetic model revealed an L-Phe inhibition constant of 20 +/- 1.35 g/L using recombinant E. coli cells, the reactive-extraction process modeling identified the L-Phe cation diffusion in the aqueous donor film and the transport of the lowly soluble carrier/L-Phe complex in the aqueous acceptor film as the most dominant transfer steps. The corresponding mass transfer coefficients were estimated as k(PheD) = 128 x 10(-7) cm/s (extraction) and k(CPheA) = 178 x 10(-5) cm/s (back-extraction). Simulation studies were performed for the total ISPR approach, which gave hints for strategies of further process optimization.

Pulse experiments as a prerequisite for the quantification of in vivo enzyme kinetics in aromatic amino acid pathway of Escherichia coli.

Glucose pulse experiments were performed to elucidate their effects on the carbon flux into the aromatic amino acid pathway in different Escherichia coli strains. Using a 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP, aroB(-))-producing strain, a fed-batch fermentation strategy specialized for glucose pulse experiments was developed and further applied for 3-dehydroshikimate (DHS, aroE(-))- and shikimate 3-phosphate (S3P, aroA(-))-producing E. coli strains. The strains overexpress a feedback-resistant DAHP synthase and additional enzymes to prevent rate-limiting steps in the aromatic amino acid pathway. Changes of carbon flux into the aromatic amino acid pathway were determined via extracellular metabolite accumulations using (1)H NMR and HPLC measurements. As an important result, a close relationship between pulse intensity and aromatic metabolite formation rates was identified. The more downstream an aromatic pathway intermediate was located, the stronger the glucose pulse intensity had to be in order to detect significant changes in product formation. However, with the experimental conditions chosen, changes after pulse were detected even for shikimate 3-phosphate, the most downstream accumulating metabolite of this experimental series. Hence glucose pulse experiments are assumed to be a promising tool even for the analysis of final pathway products such as, for example, L-phenylalanine.

Stimulation, monitoring, and analysis of pathway dynamics by metabolic profiling in the aromatic amino acid pathway.

Using a concerted approach of biochemical standard preparation, analytical access via LC-MS/MS, glucose pulse, metabolic profiling, and statistical data analysis, the metabolism dynamics in the aromatic amino acid pathway has been stimulated, monitored, and analyzed in different tyrosine-auxotrophic L-phenylalanine-producing Escherichia coli strains. During the observation window from -4 s (before) up to 27 s after the glucose pulse, the dynamics of the first five enzymatic reactions in the aromatic amino acid pathway was observed by measuring intracellular concentrations of 3-deoxy-d-arabino-heptulosonate 7-phosphate DAH(P), 3-dehydroquinate (3-DHQ), 3-dehydroshikimate (3-DHS), shikimate 3-phosphate (S3P), and shikimate (SHI), together with the pathway precursors phosphoenolpyruvate (PEP) and P5P, the lumped pentose phosphate pool as an alternative to the nondetectable erythrose 4-phosphate (E4P). Provided that a sufficient fortification of the carbon flux into the pathway of interest is ensured, respective metabolism dynamics can be observed. On the basis of the intracellular pool measurements, the standardized pool velocities were calculated, and a simple, data-driven criterion--called "pool efflux capacity" (PEC)--is derived. Despite its simplifying system description, the criterion managed to identify the well-known AroB limitation in the E. coli strain A (genotype delta(pheA tyrA aroF)/pJF119EH aroF(fbr) pheA(fbr) amp) and it also succeeded to identify AroL and AroA (in strain B, genotype delta(pheA tyrA aroF)/pJF119EH aroF(fbr) pheA(fbr) aroB amp) as promising metabolic engineering targets to alleviate respective flux control in subsequent L-Phe producing strains. Furthermore, using of a simple correlation analysis, the reconstruction of the metabolite sequence of the observed pathway was enabled. The results underline the necessity to extend the focus of glucose pulse experiments by studying not only the central metabolism but also anabolic pathways.

Human chymotrypsinogen B production from Pichia pastoris by integrated development of fermentation and downstream processing. Part 2. Protein recovery.

The purification of human chymotrypsinogen B (hCTRB) after expression and secretion by the yeast Pichia pastoris is described based on two different approaches using integrated initial recovery. Extraction employing aqueous two-phase systems (ATPS) from poly(ethylene glycol) and sodium sulfate allows direct processing of cell containing yeast suspensions of 50% wet weight. The target protein is obtained partially purified in the top phase while cells and cell debris are partitioned to the bottom phase of the system. hCTRB is further purified by adsorption from the top phase to the cation exchanger SP Sepharose Big Beads and elution in a salt step. The single step isolation of hCTRB is possible by expanded bed adsorption (EBA) using a fluidized cation exchanger (Streamline SP XL). A design strategy is shown taking both target protein binding and stable fluidization of the stationary phase in cell containing suspensions into consideration. For the example of hCTRB isolation from cell containing P. pastoris suspensions, a successful use of this strategy is demonstrated. Both initial recovery strategies deliver a product that can be further purified and formulated by ultrafiltration/diafiltration followed by lyophilization, resulting in a homogeneous product. Scale-up to 30-90 L of culture suspension was shown for both methods, resulting in a product of similar quality. Comparing both strategies reveals that the two-step ATPS route is better suited for high cell density cultures, while the single step EBA method is preferred for cultures of moderate cell density. This is due to the fact that application of EBA is restricted to suspensions of 10-12.5% wet weight cell concentration, thus necessitating dilution of the original broth prior to sample application. The data presented show that integrated recovery operations are a valuable alternative to traditional processing for systems that are problematic during initial solid-liquid separation.

Human chymotrypsinogen B production with Pichia pastoris by integrated development of fermentation and downstream processing. Part 1. Fermentation.

Based on an integrated approach of genetic engineering, fermentation process development, and downstream processing, a fermentative chymotrypsinogen B production process using recombinant Pichia pastoris is presented. Making use of the P. pastoris AOX1-promotor, the demand for methanol as the single carbon source as well as an inducer of protein secretion enforced the use of an optimized feeding strategy by help of on-line analysis and an advanced controller algorithm. By using an experimental system of six parallel sparged column bioreactors, proteolytic product degradation could be minimized while also optimizing starting conditions for the following downstream processing. This optimization of process conditions resulted in the production of authentic chymotrypsinogen at a final concentration level of 480 mg.L(-)(1) in the whole broth and a biomass concentration of 150 g.L(-)(1) cell dry weight, thus comprising a space-time yield of 5.2 mg.L(-)(1).h(-)(1). Alternatively to the high cell density fermentation approach, a continuous fermentation process was developed to study the effects of reduced cell density toward oxygen demand, cooling energy, and biomass separation. This development led to a process with a highly increased space-time yield of 25 mg.L(-)(1).h(-)(1) while reducing the cell dry weight concentration from 150 g.L(-)(1) in fed-batch to 65 g.L(-)(1) in continuous cultivation.

Scale-up of microbial processes: impacts, tools and open questions.

Industrial biotechnological production is developing rapidly worldwide. Consequently, more and novel bioprocesses need to perform optimally not only in small lab- but also in large production scales. This article shortly reviews typical impacts found when cells are exposed to micro environmental heterogeneities typically occurring in poorly mixed large scale production reactors. The current state-of-the-art of tool development is presented for analyzing these phenomena. Finally, still open questions are formulated and needs for future research are outlined to further support the expansion of biotech industries by successful research results.

Quantification of intracellular metabolites in Escherichia coli K12 using liquid chromatographic-electrospray ionization tandem mass spectrometric techniques.

The quantitative comprehension of microbial metabolic networks is a prerequisite for an efficient rational strain improvement ("metabolic engineering"). It is therefore necessary to accurately determine the concentration of a large number of reactants (i.e., metabolites, nucleotides, cofactors) in order to understand "in vivo" reaction kinetics. Quantification of intracellular concentrations of glycolytic intermediates and nucleotides in Escherichia coli K12 using a perchloric acid extraction and an LC-ESI-MS method was achieved. Intracellular metabolites (e.g., glucose 6-phosphate, fructose 1,6-bisphosphate, 6-phospho gluconate, acetyl-CoA, adenine nucleotides) were quantified under defined (glucose-limited steady-state) growth conditions. The method was verified by comparing the intracellular metabolite concentrations measured via LC-ESI-MS with enzymatic determinations. It is thus possible to identify and quantify more than 15 intracellular metabolites in parallel with a minimal amount of sample volume.

Experimental design for the identification of macrokinetic models and model discrimination.

An experimental design method for the identification of macrokinetic models was developed applying an extended D-optimal design criterion. The D-optimal design criterion was modified to consider variable measurement variances as well as multivariate macrokinetic models. The macrokinetics of formate dehydrogenase (FDH) production with Candida boidinii were thus identified within 10 steady state experiments in a labscale continuous stirred tank reactor (10 model parameters). Closed loop control (nutristat) was applied to set-up the operating states suggested by this experimental design method. After each set of steady state experiments the quality of macrokinetic parameters was characterized statistically. For model discrimination a parameter discrimination algorithm based on entropy formulations was adapted. Again a multivariate criterion considering variable measurement variances was developed. This discrimination algorithm was applied to discriminate the macrokinetic model of FDH production with Candida boidinii out of 10 different macrokinetic approaches. An unequivocal discrimination result could be obtained calculating model specific probabilities. These were compared with commonly used sum of squares values. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 564-576, 1997.

Modeling of the pyruvate production with Escherichia coli in a fed-batch bioreactor.

A family of 10 competing, unstructured models has been developed to model cell growth, substrate consumption, and product formation of the pyruvate producing strain Escherichia coli YYC202 ldhA::Kan strain used in fed-batch processes. The strain is completely blocked in its ability to convert pyruvate into acetyl-CoA or acetate (using glucose as the carbon source) resulting in an acetate auxotrophy during growth in glucose minimal medium. Parameter estimation was carried out using data from fed-batch fermentation performed at constant glucose feed rates of q(VG)=10 mL h(-1). Acetate was fed according to the previously developed feeding strategy. While the model identification was realized by least-square fit, the model discrimination was based on the model selection criterion (MSC). The validation of model parameters was performed applying data from two different fed-batch experiments with glucose feed rate q(VG)=20 and 30 mL h(-1), respectively. Consequently, the most suitable model was identified that reflected the pyruvate and biomass curves adequately by considering a pyruvate inhibited growth (Jerusalimsky approach) and pyruvate inhibited product formation (described by modified Luedeking-Piret/Levenspiel term).

Automated sampling device for monitoring intracellular metabolite dynamics.

An automated sampling device coupled to a stirred tank reactor was developed for monitoring intracellular metabolite dynamics. Sample flasks fixed in transport magazines were moved by a step engine in a way that each sample flask was filled within 220 ms, resulting in a sampling rate of 4.5 s-1. Rapid inactivation of the metabolism was achieved by spraying the samples into 60% methanol at -50 degrees C. After centrifugation of the quenched cells at -20 degrees C the metabolites were extracted with perchloric acid and analyzed biochemically or with HPLC. The automated sampling device was applied for investigation of the intracellular metabolite dynamics of glycolysis in Escherichia coli after rapid glucose addition to a glucose-limited steady-state culture. For the first time oscillations of intracellular metabolite concentrations like glucose-6-phosphate, phosphoenolpyruvate, glyceraldehyde 3-phosphate, dihydroxyacetonphosphate, 3-phosphoglycerate, and pyruvate were quantified on a subseconds to seconds scale in E. coli. As an example, the kinetics of the decomposition of fructose 1, 6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetonphosphate were investigated by use of a well-known mechanistic kinetic model and the measured in vivo metabolite dynamics.

Serial flux mapping of Corynebacterium glutamicum during fed-batch L-lysine production using the sensor reactor approach.

Using our recently developed sensor reactor approach, lysine-producing, nongrowing Corynebacterium glutamicum MH20-22B cells were subjected to serial (13)C-labeling experiments for flux analysis during the leucine-limited fed-batch production phase in a 300-L bioreactor. Based on two-dimensional (2D) nuclear magnetic resonance (NMR) measurements of (13)C-labeling patterns of cytoplasmic free metabolites, metabolic flux distributions in the central metabolism were successfully determined. Focusing on the highly concentrated metabolite L-glutamate, the working hypothesis was validated that the equilibration of labeling patterns in intracellular pools was much faster (up to 9.45 min) than the labeling period (3 h) used in the experiments. Analysis of anaplerotic reactions revealed that highly selective lysine production was accompanied by a significant reduction of decarboxylating reactions from 10 mol% to only 2 mol%, whereas PEP/pyruvate-carboxylating fluxes remained constant at about 40 mol% of consumed glucose. These results support the conclusion that an optimized C. glutamicum L-lysine producer should possess increased PEP carboxylase and/or pyruvate carboxylase activity combined with downregulated, decarboxylating fluxes consuming oxaloacetate/malate. The findings also illustrate the usefulness of the sensor reactor approach in the study of industrial fermentations.

Process control for enhanced L-phenylalanine production using different recombinant Escherichia coli strains.

A novel fed-batch approach for the production of L-phenylalanine (L-Phe) with recombinant E. coli is presented concerning the on-line control of the key fermentation parameters glucose and tyrosine. Two different production strains possessing either the tyrosine feedback resistant aroF(fbr) (encoding tyrosine feedback resistant DAHP-synthase (3-desoxy-D-arabino-heptusonate-7-phosphate)) or the wild-type aroF(wt) were used as model systems to elucidate the necessity of finding an individual process optimum for each genotype. With the aid of tyrosine control, wild-type aroF(wt) could be used for L-Phe production achieving higher final L-Phe titers (34 g/L) than the aroF(fbr) strain (28 g/L) and providing higher DAHP-synthase activities. With on-line glucose control, an optimum glucose concentration of 5 g/L could be identified that allowed a sufficient carbon supply for L-Phe production while at the same time an overflow metabolism leading to acetate by-product formation was avoided. The process approach is suitable for other production strains not only in lab-scale but also in pilot-scale bioreactors.

Integrated L-phenylalanine separation in an E. coli fed-batch process: from laboratory to pilot scale.

Pilot-scale reactive-extraction technology for fully integrated L-phenylalanine (L-Phe) separation in Escherichia coli fed-batch fermentations was investigated in order to prevent an inhibition of microbial L-Phe production by-product accumulation. An optimal reactive-extraction system, consisting of an organic kerosene phase with the cation-selective carrier DEHPA (di-2-ethylhexyl phosphonic acid) and an aqueous stripping phase including sulphuric acid, was found particularly efficient. Using this system with two membrane contactors, mass-transfer coefficients of up to 288 x 10(-7) cm s(-1) for the aqueous/organic and 77 x 10(-7) cm s(-1) for the organic/stripping phase were derived from experimental data using a simple modelling approach. Concentration factors higher than 4 were achieved in the stripping phase as compared to the aqueous donor phase. Reactive extraction enabled a 98% cation portion of L-Phe in the stripping phase, leading to final product purity higher than 99% after L-Phe precipitation. A doubling of L-Phe/glucose yield was observed when kerosene/DEHPA was added to the fermentation solution in the bioreactor to experimentally simulate a fully integrated L-Phe separation process.

Production process monitoring by serial mapping of microbial carbon flux distributions using a novel sensor reactor approach: I--Sensor reactor system.

A novel Sensor Reactor technology is presented which permits 13C labeling experiments for metabolic flux analysis during large-scale, semi-industrial, (fed-) batch fermentation processes deriving a series of flux maps that document fermentation courses in detail. The small-scale Sensor Reactor can be inoculated within 1.50-1.20s via a special inoculation unit with an inoculation volume accuracy of 1.025+/-0.021 L. The large-scale production reactor (here: 300 L) and the Sensor Reactor were run in parallel master/slave modes to control the current pH, temperature, pressure and dissolved oxygen values as changing set points for the Sensor Reactor. Using an automated pulsing technology, glucose pulses of 5 g/L could be realized within 0.51 s. The similarity of fermentations in the Sensor Reactor with the production process was demonstrated by studying L-lysine production with C. glutamicum during multiple, 'simulated' labeling experiments each lasting 2.5h. 'Real' labeling experiments are presented in Part II.

Fully integrated L-phenylalanine separation and concentration using reactive-extraction with liquid-liquid centrifuges in a fed-batch process with E. coli.

A novel in situ product recovery (ISPR) approach for the (fully) integrated separation of L-phenylalanine (L-phe) from a 20 l fed-batch process with the recombinant L-tyrosine auxotrophic strain E. coli F-4/pF81 is presented. The strain was rationally constructed for the production of the aromatic amino acid. Glucose and tyrosine control is used. A reactive extraction system consisting of kerosene, the cation-selective carrier D(2)EHPA and sulphuric acid, all circulating in liquid-liquid centrifuges, is applied for the on-line L-phe separation from cell- and protein-free permeate. Permeate is drained off from the bioreactor bypass. Using the novel ISPR approach, a significantly extended product formation period at 0.25 mmol/(g*h) together with a reduced by-product formation and a 28% relative glucose/L-phe yield increase is observed. Thus, the ISPR approach is superior to the reference non-ISPR process and even offers extraction rates approximately three times higher than the published membrane-based process.

Enhanced pilot-scale fed-batch L-phenylalanine production with recombinant Escherichia coli by fully integrated reactive extraction.

A fully integrated process for the microbial production and recovery of the aromatic amino acid L-phenylalanine is presented. Using a recombinant L-tyrosine (L-Tyr) auxotrophic Escherichia coli production strain, a fed-batch fermentation process was developed in a 20-l-scale bioreactor. Concentrations of glucose and L-Tyr were closed-loop-controlled in a fed-batch process. After achieving final L-phenylalanine (L-Phe) titres >30 g/l the process strategy was scaled up to 300-l pilot scale. In technical scale fermentation L-phenylalanine was continuously recovered via a fully integrated reactive extraction system achieving a maximum extraction rate of 110 g/h (final purity >99%). It was thus possible to increase L-Phe/glucose selectivity from 15 mol% without to 20.3 mol% with integrated product separation.

Production process monitoring by serial mapping of microbial carbon flux distributions using a novel Sensor Reactor approach: II--(13)C-labeling-based metabolic flux analysis and L-lysine production.

Corynebacterium glutamicum is intensively used for the industrial large-scale (fed-) batch production of amino acids, especially glutamate and lysine. However, metabolic flux analyses based on 13C-labeling experiments of this organism have hitherto been restricted to small-scale batch conditions and carbon-limited chemostat cultures, and are therefore of questionable relevance for industrial fermentations. To lever flux analysis to the industrial level, a novel Sensor Reactor approach was developed (El Massaoudi et al., Metab. Eng., submitted), in which a 300-L production reactor and a 1-L Sensor Reactor are run in parallel master/slave modus, thus enabling 13C-based metabolic flux analysis to generate a series of flux maps that document large-scale fermentation courses in detail. We describe the successful combination of this technology with nuclear magnetic resonance (NMR) analysis, metabolite balancing methods and a mathematical description of 13C-isotope labelings resulting in a powerful tool for quantitative pathway analysis during a batch fermentation. As a first application, 13C-based metabolic flux analysis was performed on exponentially growing, lysine-producing C. glutamicum MH20-22B during three phases of a pilot-scale batch fermentation. By studying the growth, (co-) substrate consumption and (by-) product formation, the similarity of the fermentations in production and Sensor Reactor was verified. Applying a generally applicable mathematical model, which included metabolite and carbon labeling balances for the analysis of proteinogenic amino acid 13C-isotopomer labeling data, the in vivo metabolic flux distribution was investigated during subsequent phases of exponential growth. It was shown for the first time that the in vivo reverse C(4)-decarboxylation flux at the anaplerotic node in C. glutamicum significantly decreased (70%) in parallel with threefold increased lysine formation during the investigated subsequent phases of exponential growth.