The effects of feed and intracellular pyruvate levels on the redistribution of metabolic fluxes in Escherichia coli

In a previous study, an Escherichia coli strain lacking the key enzymes (acetate kinase and phosphotransacetylase, ACK-PTA) of the major acetate synthesis pathways reduced acetate accumulation. The ackA-pta mutant strain also exhibits an increased lactate synthesis rate. Metabolic flux analysis suggested that the majority of excessive carbon flux was redirected through the lactate formation pathway rather than the ethanol synthesis pathway. This result indicated that lactate dehydrogenase may be competitive at the pyruvate node. However, a 10-fold overexpression of the fermentative lactate dehydrogenase (ldhA) gene in the wild-type parent GJT001 was not able to divert carbon flux from acetate. The carbon flux through pyruvate and all its end products increases at the expense of flux through biosynthesis and succinate. Intracellular pyruvate measurements showed that strains overexpressing lactate dehydrogenase (LDH) depleted the pyruvate pool. This observation along with the observed excretion of pyruvate in the ackA-pta strain indicates the significance of intracellular pyruvate pools. In the current study, we focus on the role of the intracellular pyruvate pool in the redirection of metabolic fluxes at this important node. An increasing level of extracellular pyruvate leads to an increase in the intracellular pyruvate pool. This increase in intracellular pyruvate affects carbon flux distribution at the pyruvate node. Partitioning of the carbon flux to acetate at the expense of ethanol occurs at the acetyl-CoA node while partitioning at the pyruvate node favors lactate formation. The increased competitiveness of the lactate pathway may be due to the allosteric activation of LDH as a result of increased pyruvate levels. The interaction between the reactions catalyzed by the enzymes PFL (pyruvate formate lyase) and LDH was examined.     

Expression of the pfl gene and resulting metabolite flux distribution in nuo and ackA-pta E. coli mutant strains

Our laboratory previously studied the interaction between nuo and the acetate-producing pathway encoded by ackA-pta in Escherichia coli. We examined metabolic patterns, particularly the ethanol and acetate production rates, of several mutant strains grown under anaerobic growth conditions. Since the pyruvate formate-lyase (PFL) pathway is the major route for acetyl-CoA and formate production under anaerobic conditions, we examined the effects of nuo and ackA/pta mutations on the expression of pyruvate formate-lyase (pfl) under anaerobic conditions. The ackA-pta mutant has a pfl::lacZ expression level much higher than that of the wild-type strain, and cultures also exhibit the highest ethanol production. Real-time PCR demonstrated that the adhE gene expression in the ack-pta mutant strain was approximately 100 fold that of the same gene in the ackA-pta nuo mutant strain. This result correlates with the observed ethanol production rates in cultures of the strain. However, the lack of exact correlation between the ethanol production rates and the RT-PCR data suggests additional regulation actions at the posttranslation level. In addition, the activity of the pfl gene as indicated by mRNA levels was also considerably greater in theack-pta mutant. We can conclude that deletions of nuo and ack/pta can partially affect the expression of the genes encoding adhE and pfl under anaerobic conditions.     

Effect of variation of Klebsiella pneumoniae acetolactate synthase expression on metabolic flux redistribution in Escherichia coli

Escherichia coli strains carrying the Bacillus subtilis acetolactate synthase (ALS) gene were previously shown to produce less acetate with higher ATP yields. Metabolic flux analysis was used to show that excess pyruvate was channeled into the less inhibitory product, acetoin. To further understand the role of intrinsic enzymatic properties and the effect of variations in enzyme levels in the alternation of metabolic fluxes, we constructed a chromosomal integrant of the Klebsiella pneumoniae ALS gene. The reported in vitro Michaelis-Menten constants (K(m)) for the Bacillus and the Klebsiella ALS are 13.0 mM and 8.0 mM, respectively. Furthermore, expression of the Klebsiella ALS is under the control of an inducible trp promoter system. Shake-flask experiments showed a linear induction response (the ALS activity changes from about 9 to 223 U/mg of protein when the inducer concentration [IAA] varied from 0 to 40 mg/L). Chemostat experiments showed a similar induction response. Interactions between the branched reactions catalyzed by the PFL, LDH, and the ALS enzymes at the pyruvate node were examined. The results indicate the importance of in vivo enzyme activities in the redistribution of metabolic fluxes.     

Effect of Escherichia coli biomass composition on central metabolic fluxes predicted by a stoichiometric model

The amino acid composition of proteins and the fatty acid composition of the cell membranes were measured in Escherichia coli growing exponentially in batch culture on glucose, succinate, glycerol, pyruvate, and acetate, and growing under continuous culture conditions on glucose at dilutions rates equivalent to the growth rates of the batch cultures. Although the fatty acid composition of the membranes did change significantly with carbon source and dilution rate, the amino acid content of proteins did not change significantly under either condition. A previously developed stoichiometric model of metabolism was used to calculate the fluxes through the metabolic reactions and to determine their sensitivity to changes in fatty acid and amino acid composition.     

Effect of different levels of NADH availability on metabolic fluxes of Escherichia coli chemostat cultures in defined medium

Escherichia coli overexpressing a NAD(+)-dependent formate dehydrogenase (FDH) from Candida boidinii was grown in chemostat culture on various carbon sources at 0.05 h(-1) dilution rate, under anaerobic conditions using defined medium and compared to a control without the heterologous FDH pathway. Metabolic fluxes, NADH/NAD(+) ratios and NAD(H/(+)) levels were determined under a range of intracellular NADH availability. The effect of NADH manipulation on the distribution of metabolic fluxes in E. coli was assessed under steady-state conditions. The heterologous FDH pathway converts 1 mol of formate into 1 mol of NADH and carbon dioxide, in contrast with the native FDH where no cofactor involvement is present. Previously, we found that this NADH regeneration system doubled the maximum yield of NADH from 2 to 4 mol NADH/mol glucose consumed and reached 4.6 mol NADH/mol of substrate when sorbitol was used as a carbon source in a complex medium. In the current study, it was found that higher NADH yields and NADH/NAD(+) ratios were achieved with our in vivo NADH regeneration system compared to a control lacking the new FDH pathway in the three carbon sources (glucose, gluconate and sorbitol) examined suggesting a more reduced intracellular environment. The total NAD(H/(+)) amounts were very similar for all the combinations studied. It was also found that the ethanol to acetate ratio increased with increased NADH availability. This ratio increased from 1.05 for the control strain in glucose to 9.45 for the strain expressing the heterologous NAD(+)-dependent FDH in sorbitol.     

Redistribution of metabolic fluxes in the central aerobic metabolic pathway of E. coli mutant strains with deletion of the ackA-pta and poxB pathways for the synthesis of isoamyl acetate

Although the bacterium E. coli is chosen as the host in many bioprocesses, products derived from the central aerobic metabolic pathway often compete with the acetate-producing pathways poxB and ackA-pta for glucose as the substrate. As such, a significant portion of the glucose may be excreted as acetate, wasting substrate that could have otherwise been used for the desired product. The production of the ester isoamyl acetate from acetyl-CoA by ATF2, a yeast alcohol acetyl transferase, was used as a model system to demonstrate the beneficial effects of reducing acetate production. All strains tested for ester production also overexpressed panK, a native E. coli gene that previous studies have shown to increase free intracellular CoA levels when fed with pantothenic acid. A recombinant E. coli strain with a deletion in ackA-pta produces less acetate and more isoamyl acetate than the wild-type E. coli strain. When both acetate-producing pathways were deleted, the acetate production was greatly reduced. However, pyruvate began to accumulate, so that the overall ester production remained largely unchanged. To produce more ester, a previously established strategy of increasing the flux from pyruvate to acetyl-CoA was adopted by overexpressing pyruvate dehydrogenase. The ester production was then 80% higher in the poxB, ackA-pta strain (0.18 mM) than that found in the single ackA-pta mutant (0.10 mM), which also overexpressed PDH.     

Redistribution of metabolic fluxes in Escherichia coli with fermentative lactate dehydrogenase overexpression and deletion

Under anaerobic conditions, competition for pyruvate between the branch point enzymes pyruvate formate lyase (PFL, Km = 2 mM) and fermentative lactate dehydrogenase (LDH, Km = 7.2 mM) determines the partition of carbon flux. Two Escherichia coli mutant strains, one deficient in ackA, pta, and ldhA and the other overexpressing LDH, were constructed to systematically analyze the effects of these perturbations in the existing pathways on the redistribution of carbon fluxes. Deletion of the lactate and acetate synthesis pathways was detrimental to cell growth. Carbon flux is forced through ethanol and formate production pathways, resulting in a concomitant increase in those fluxes. In addition, overexpression of LDH simultaneously increases the common flux as well as the flux to the competing acetyl-CoA branch. Overexpression of lactate dehydrogenase (ldhA) in the parent strain increases the lactate synthesis rate from 0.19 to 0.40 mmol/g-biomass-h when the LDH activities increases from 1.3 to 15.3 units. Even an increase of more than 10 times in the LDH activity fails to divert a large fraction of the carbon flux to lactate; the majority of the flux still channels through the acetyl-CoA branch. Overexpression of LDH in the parent strain simultaneously increases the common flux as well as the flux through the acetyl-CoA branch. Subsequently, the flux amplification factors (or deviation indices which can be related to the flux control coefficients) are positive for all three fluxes occurring at the pyruvate node.     

Effect of suspension media on nonthermal inactivation of Escherichia coli

AIMS: To investigate the influence of suspension media on the survival of Escherichia coli M23 exposed to nonthermal, lethal stresses. METHODS AND RESULTS: Populations of E. coli M23 suspended in minimal medium (MM) or in different nutrient-rich broths were exposed to water activity 0.90 and/or pH 3.5 and inactivation was determined by culture-based enumeration. In response to the osmotic or acid challenges, E. coli M23 displayed enhanced survival in MM rather than in complex broth. That trend was reversed when populations were exposed to low water activity in combination with low pH. Comparison of microbial survival in three complex media indicated that even relatively small differences in composition influenced inactivation. In most media the combination of lethal stresses resulted in a synergism, which enhanced bacterial inactivation; however, an exception (tryptone soya broth) was observed. CONCLUSIONS: The suspension medium strongly influences the inactivation of E. coli M23 by osmotic and/or acid stresses. This should be considered when comparing studies of microbial survival that use different media and when broth-derived data are intended to represent specific environments (e.g. food matrices). SIGNIFICANCE AND IMPACT OF THE STUDY: The specific effects of synthetic media need to be appreciated when studying bacterial inactivation in conditions relevant to food-manufacturing regimes.     

Cofactor engineering of intracellular CoA/acetyl-CoA and its effect on metabolic flux redistribution in Escherichia coli

Coenzyme A (CoA) and its thioester derivatives are important cofactors participating in over 100 different reactions in intermediary metabolism of microorganisms. The time profiles of intracellular CoA and acetyl-CoA levels were studied in an aerobic batch reactor. The CoA level starts at a high value and falls off gradually over the exponential and stationary growth phases, reaching negligible levels at the end of 24h. The acetyl-CoA level, on the other hand, increases initially reaching a maximum and decreases gradually reaching negligible levels after 24h. Overexpressing one of the upstream rate-controlling enzyme the pantothenate kinase with simultaneous supplementation of the precursor pantothenic acid to the culture medium increased the intracellular CoA/acetyl-CoA levels. It was found that supplementation of the precursor pantothenic acid is essential to increase CoA/acetyl-CoA levels. A 10-fold increase in CoA level was observed upon this overexpression in complex medium. Acetyl-CoA levels also increased (5-fold) but not as much as CoA, leaving much of the CoA in free unacetylated form. The increase in intracellular CoA/acetyl-CoA levels led to an increase in carbon flux to the acetate production pathway leading to formation of more acetate in complex medium, whereas no such change in metabolite redistribution was observed in minimal medium.     

Quantitative analysis of metabolic fluxes in Escherichia coli, using two-dimensional NMR spectroscopy and complete isotopomer models.

Complete isotopomer models that simulate distribution of label in 13C tracer experiments are applied to the quantification of metabolic fluxes in the primary carbon metabolism of E. coli under aerobic and anaerobic conditions. The concept of isotopomer mapping matrices (IMMs) is used to simplify the formulation of isotopomer mass balances by expressing all isotopomer mass balances of a metabolite pool in a single matrix equation. A numerically stable method to calculate the steady-state isotopomer distribution in metabolic networks in introduced. Net values of intracellular fluxes and the degree of reversibility of enzymatic steps are estimated by minimization of the deviations between experimental and simulated measurements. The metabolic model applied includes the Embden-Meyerhof-Parnas and the pentose phosphate pathway, the tricarboxylic acid cycle, anaplerotic reaction sequences and pathways involved in amino acid synthesis. The study clearly demonstrates the value of complete isotopomer models for maximizing the information obtainable from 13C tracer experiments. The approach applied here offers a completely general and comprehensive analysis of carbon tracer experiments where any set of experimental data on the labeling state and extracellular fluxes can be used for the quantification of metabolic fluxes in complex metabolic networks.     

Optimal selection of metabolic fluxes for in vivo measurement. II. Application to Escherichia coli and hybridoma cell metabolism.

A method of analysis was presented in part I of this series for determining the fluxes in a biochemical network that are the optimal choices for experimental measurement. This algorithm is applied to two important biological models: Escherichia coli and a hybridoma cell line (167.4G5.3). Our results show that potentially poor choices for in vivo measurement of metabolic fluxes exist for both model systems. For the subset of reactions in E. coli that was studied, the condition number of the augmented stoichiometric matrix reveals that a 60-fold amplification of experimental error during computations is possible. The biochemical network of the hybridoma cell is more complex than the E. coli system, and thus results in much larger possible error amplification--up to 100,000-fold. The physiological situations appear to have sensitivities that are less than 1/4 to 1/10 of those estimated by the condition number, and the maximum sensitivities are proportional to the condition number. These maximum sensitivities calculated using estimates of the fluxes and the worst possible error vector are upper bounds on the system's actual sensitivity. By examining the effect of measurement error on the sensitivity, the most probable sensitivity is calculated. These results indicate that an approximate two-fold increase in sensitivity of the E. coli system is likely when the worst set of fluxes are measured rather than the best set. The most likely sensitivity of the hybridoma system can range three orders of magnitude, depending on the set of fluxes that are measured. The propagation of experimental error during computations can be diminished for both systems by increasing the number of flux measurements over and above the minimum number of experimental measurements. The findings from these two model systems indicate that the calculation of the condition number can be a useful method for efficient experimental design, and that the usefulness of this method increases as the order of the system increases.     

Tools for metabolic engineering in Escherichia coli: inactivation of panD by a point mutation

l-Aspartate-alpha-decarboxylase (PanD) catalyzes the decarboxylation of aspartate to produce beta-alanine, a precursor of Coenzyme A (CoA). The pyruvoyl-dependent enzyme from Escherichia coli is activated by self-cleavage at serine 25 to generate a 102-residue alpha subunit with the pyruvoyl group at its N terminus and a 24-residue beta subunit with a hydroxy at its C terminus. A mutant form of the panD gene from E. coli in which serine 25 was replaced with an alanine (S25A) was constructed. Assays conducted in vitro and in vivo confirmed that the mutant version was completely inactive and was incapable of undergoing self-cleavage to generate the active form of the enzyme. The S25A panD mutant was used to replace the chromosomal copy of panD in BAP1, a strain of E. coli modified for polyketide production. Comparison of this strain with panD2 mutant strains derived from E. coli SJ16 showed an equivalent dependence on exogenous beta-alanine for growth in liquid medium. Unlike the undefined and leaky panD2 mutation, the panD S25A mutation is defined and tight. The panD S25A E. coli strain enables analysis of intracellular acyl-CoA pools in both defined and complex media and is a useful tool in metabolic engineering studies that require the manipulation of acyl-CoA pools for the heterologous production of polyketides.     

Assay of metabolic superoxide production in Escherichia coli.

Superoxide production has been measured in subcellular fractions of SOD-deficient Escherichia coli provided with physiological reductants. Although cytosolic enzyme(s) do generate O2-., the larger portion is produced by autoxidation of components of the respiratory electron-transport chain. At 37 degrees C and with pO2, NADH, and NAD+ levels matching those in vivo, respiring membrane vesicles generate 3 O2-./10,000 electrons transferred. This corresponds to intracellular O2-. production, in glucose-fed cells, of 5 microM/s. The high SOD content of normal cells restricts O2-. accumulation to 2.10(-10) M, with a moderate gradient from the membrane to the center of the cell. SOD-deficient mutants achieve a much higher steady-state content of O2-.. Rates of superoxide-mediated inactivation of certain enzymes are sufficiently rapid that even 10(-10) M O2-. imposes a significant oxidative stress.     

Catabolite inactivation of biodegradative threonine dehydratase of Escherichia coli.

Incubation of Escherichia coli cells with glucose, pyruvate, and certain other metabolites led to rapid inactivation of inducible biodegradative threonine dehydratase. Analysis with several mutant strains showed that pyruvate, and not a metabolite derived from pyruvate, was capable of inactivating enzyme, and that glucose acted indirectly after being converted to pyruvate. Some other alpha-keto acids such as oxaloacetate and alpha-ketobutyrate (but not alpha-ketoglutarate) were also effective. Inactivation of threonine dehydratase by pyruvate was also observed with purified enzyme preparations. The rates of enzyme inactivation increased with increased concentrations of pyruvate and decreased with increased levels of AMP. Increasing protein concentrations lowered the rates of enzyme inactivation. Dithiothreitol had a large effect on the maximum extent of inactivation of the enzyme by pyruvate; high concentrations of AMP and DTT almost completely counteracted the effect of pyruvate. Gel filtration data showed that pyruvate influenced the oligomeric state of the enzyme by altering the association-dissociation equilibrium in favor of dissociation; the Stokes' radius of the pyruvate-inactivated enzyme was 32 A as compared to 42 A for the untreated enzyme. Reassociation of the dissociated form of the enzyme was achieved by removal of excess free pyruvate by dialysis against buffer supplemented with AMP and DTT. Incubation of threonine dehydratase with [14-C]pyruvate revealed apparent covalent attachment of pyruvate to the enzyme. Strong protein denaturants such as guanidine, urea, and sodium dodecyl sulfate failed to release bound radioactive pyruvate; the molar ratio of firmly bound pyruvate was approximately 1 mol/150,000 g of protein. Pretreatment of the enzyme with p-chloromercuribenzoate and 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) did not reduce the binding of [14-C]pyruvate suggesting no active site SH was involved in the pyruvate-enzyme linkage. Titration of active and pyruvate-inactivated enzyme with Nbs2 indicated that the loss in enzyme activity was not due to oxidation of essential sulfhydryl groups on the enzyme. Based on these data we propose that the mechanism of enzyme inactivation by pyruvate involves covalent attachment of pyruvate to the active oligomeric form of the enzyme followed by dissociation of the oligomer to yield inactive enzyme.     

Soft sensor control of metabolic fluxes in a recombinant Escherichia coli fed-batch cultivation producing green fluorescence protein

A soft sensor approach is described for controlling metabolic overflow from mixed-acid fermentation and glucose overflow metabolism in a fed-batch cultivation for production of recombinant green fluorescence protein (GFP) in Escherichia coli. The hardware part of the sensor consisted of a near-infrared in situ probe that monitored the E. coli biomass and an HPLC analyzer equipped with a filtration unit that measured the overflow metabolites. The computational part of the soft sensor used basic kinetic equations and summations for estimation of specific rates and total metabolite concentrations. Two control strategies for media feeding of the fed-batch cultivation were evaluated: (1) controlling the specific rates of overflow metabolism and mixed-acid fermentation metabolites at a fixed pre-set target values, and (2) controlling the concentration of the sum of these metabolites at a set level. The results indicate that the latter strategy was more efficient for maintaining a high titer and low variability of the produced recombinant GFP protein.