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M M Ataai

Publications and source records attributed to M M Ataai.

At least 19 recordsLinked to original sources
PubMed2005

Engineering of Bacillus subtilis for enhanced total synthesis of folic acid.

We investigated whether the yield of the B vitamin folic acid could be elevated in Bacillus subtilis. Strategies for increasing the folic acid yield were investigated by employing computer-aided flux analysis and mutation. Controlling the activity of the enzyme pyruvate kinase by placing it under inducible control was one strategy devised to elevate yield while insuring that a rapid growth rate results. Other single mutation strategies included amplifying the expression of the genes in the folate operon and overexpressing the Escherichia coli aroH gene, which encodes 2-dehydro-3-deoxyphosphoheptonate aldolase. The latter could conceivably elevate the abundance of the folic acid precursor, para-aminobenzoic acid. Strains that combined two or more mutations were also constructed. Overall, a strain possessing inducible pyruvate kinase, overexpressed aroH, and increased transcription and translation of genes from the folic operon exhibited the best yield. The yield was eightfold higher than that displayed by the parent B. subtilis 168 strain.

Bacillus subtilis↗
PubMed2003

A metabolic network analysis & NMR experiment design tool with user interface-driven model construction for depth-first search analysis.

A Windows program for metabolic engineering analysis and experimental design has been developed. A graphical user interface enables the pictorial, "on-screen" construction of a metabolic network. Once a model is composed, balance equations are automatically generated. Model construction, modification and information exchange between different users is thus considerably simplified. For a given model, the program can then be used to predict all the extreme point flux distributions that optimize an objective function while satisfying balances and constraints by using a depth-first search strategy. One can also find the minimum reaction set that satisfies different conditions. Based on the identified flux distributions or linear combinations, the user can simulate the NMR and GC/MS spectra of selected signal molecules. Alternately, spectra vectorization allows for the automated optimization of labeling experiments that are intended to distinguish between different, yet plausible flux extreme point distributions. The example provided entails predicting the flux distributions associated with deleting pyruvate kinase and designing 13C NMR experiments that can maximally discriminate between the flux distributions.

Computer Simulation↗
PubMed2001

A MILP-based flux alternative generation and NMR experimental design strategy for metabolic engineering.

A mixed-integer linear program (MILP) is described that can enumerate all the ways fluxes can distribute in a metabolic network while still satisfying the same constraints and objective function. The multiple solutions can be used to (1) generate alternative flux scenarios that can account for limited experimental observations, (2) forecast the potential responses to mutation (e.g., new reaction pathways may be used), and (3) (as illustrated) design (13)C NMR experiments such that different potential flux patterns in a mutant can be distinguished. The experimental design is enabled by using the MILP results as an input to an isotopomer mapping matrices (IMM)-based program, which accounts for the network circulation of (13)C from a precursor such as glucose. The IMM-based program can interface to common plotting programs with the result that the user is provided with predicted NMR spectra that are complete with splittings and Lorentzian line-shape features. The example considered is the trafficking of carbon in an Escherichia coli mutant, which has pyruvate kinase activity deleted for the purpose of eliminating acetate production. Similar yields and extracellular measurements would be manifested by the flux alternatives. The MILP-IMM results suggest how NMR experiments can be designed such that the spectra of glutamate for two flux distribution scenarios differ significantly.

Algorithms↗
PubMed2000

Production of viral vectors for gene therapy applications.

Advances in cell culture engineering, cell metabolism, bioreactor design and operation, and downstream processing will all positively impact the bioprocessing of viral vectors. Design of appropriate vectors and tailoring of packaging cells to support more productive infections will be of paramount importance for production of high-titer and high-quality vectors. Furthermore, quantitative analysis of the infection parameters during virus propagation, such as time of infection, multiplicity of infection, the length of replication cycle, virus half-life, and burst size, will also be important to the process optimization. Finally, procedures for separation, purification and formulation of vector preparations have to be further developed.

Adenoviridae↗
PubMed2000

Characterization of growth and acid formation in a Bacillus subtilis pyruvate kinase mutant.

Based on measurements and theoretical analyses, we identified deletion of pyruvate kinase (PYK) activity as a possible route for elimination of acid formation in Bacillus subtilis cultures grown on glucose minimal media. Evidence consistent with the attenuation of PYK flux has come from metabolic flux calculations, metabolic pool and enzymatic activity measurements, and a series of nuclear magnetic resonance experiments, all suggesting a nearly complete inhibition of PYK activity for glucose-citrate fed cultures in which the amount of acid formation was nearly zero. In this paper, we report the construction and characterization of a pyk mutant of B. subtilis. Our results demonstrate an almost complete elimination of acid production in cultures of the pyk mutant in glucose minimal medium. The substantial reduction in acid production is accompanied by increased CO(2) production and a reduced rate of growth. Metabolic analysis indicated a dramatic increase in intracellular pools of phosphoenolpyruvate (PEP) and glucose-6-P in the pyk mutant. The high concentrations of PEP and glucose-6-P could explain the decreased growth rate of the mutant. The substantial accumulation of PEP does not occur in Escherichia coli pyk mutants. The very high concentration of PEP which accumulates in the B. subtilis pyk mutant could be exploited for production of various aromatics.

Bacillus subtilis↗
PubMed1999

Metabolic fluxes, pools, and enzyme measurements suggest a tighter coupling of energetics and biosynthetic reactions associated with reduced pyruvate kinase flux.

In this study, it is found that, for Bacillus subtilis, citrate-glucose cometabolism leads to zero acid production over a wide range of growth rates and nearly theoretical carbon yield. Experimental results are presented that point to pyruvate kinase (PYK) as a site of citrate-mediated glycolytic flux attenuation. First, the measured fluxes show that, compared with cultures grown on glucose, the PYK flux drops by more than tenfold when citrate is added. Second, relative to cultures metabolizing glucose, the phosphoenolpyruvate (PEP) pool elevates substantially, whereas the pyruvate pool drops, when citrate is present. Finally, our modeling results indicate that maximizing carbon yield corresponds to nearly eliminating pyruvate kinase (PYK) flux and that the pyruvate supplied by the PEP-consuming glucose transport system can supply the biosynthetic requirements. A literature review suggests some mechanisms for how PYK attenuation by citrate addition can occur. At this juncture, we hypothesize that direct PYK inhibition occurs which, in turn, also leads to phosphofructokinase inhibition via the elevated PEP pool. These two inhibition events combine to throttle glycolytic flux; minimize acid formation; and substantially increase cellular, product, and energetic yields.

Bacillus subtilis↗
PubMed1999

Vector engineering anomalies: impact on fusion protein purification performance.

Recombinant protein purification using IMAC is often carried out by protein fusion to affinity tags. We have identified several tags useful for protein purification on Zn(II)-IDA columns. These tags were fused to the green fluorescent protein (rGFPuv) using the vector pGFPuv distributed by Clontech Lab (Palo Alto, CA) and analyzed for purification on Zn(II)-IDA. Each fusion protein exhibited elution heterogeneity (elution in two distinct pHs) from Zn(II)-IDA columns This led us to believe that two populations of fluorescent proteins were being expressed: one without the tag coeluting with Escherichia coli proteins at pH 7.5 and one bearing the tag eluting at a pH lower than pH 7.5. Assessment of the constructs revealed the possibility of a ribosomal binding site and start codon between the fusion tag and the rGFPuv sequence which might be used as a secondary translation start site. This hypothesis was confirmed by changing the second ATG (methionine) codon to an ACG (threonine) codon. The protein produced from this new construct eluted in a single fraction from a Zn(II)-IDA column. Thus, vector irregularities (along with other possibilities) should be examined when searching for the cause of elution heterogeneity of a target protein.

Chromatography, Affinity↗
PubMed1997

Selection of optimum affinity tags from a phage-displayed peptide library. Application to immobilized copper(II) affinity chromatography.

Immobilized metal affinity chromatography (IMAC) is a versatile tool for the purification of proteins with affinity for immobilized metals. Moreover, this technique has also been used for the separation of proteins that do not exhibit significant metal affinity in the native form, by their fusion to a short metal-binding peptide (a tail), most commonly, a sequence consisting of six adjacent histidine residues (His6). A phage-displayed random hexamer library is used to select for peptides with affinity for immobilized copper. The study follows our previous investigation in which a stringent selection protocol led to the selection of only one copper-binding peptide containing two histidines. The less stringent conditions employed in this work resulted in the selection of a more diverse population of peptides, but again, dominated by peptides containing two histidines (13 out of 19). The prevalence of peptides with two histidines, in contrast to peptides with a higher number of histidines (e.g. His6 or HHHMVH), is explained based on the differences in the pH dependence of their affinity for copper. As discussed, the selected peptides with two histidines will be superior affinity tails than peptides with a higher histidine content (e.g. His6). Moreover, a peptide with a single histidine but with a very high copper affinity, is also identified. Its high copper affinity is related to the presence of several hydrophobic residues in the neighborhood of histidine. Chromatography of human interleukin-1 beta (hIL-1 beta) and several other proteins containing a single surface-exposed histidine surrounded by several hydrophobic residues confirmed that such a sequence could also serve as a very effective metal binding domain for protein purification using immobilized copper(II) columns.

Affinity Labels↗
PubMed1997

Site accessibility and the pH dependence of the saturation capacity of a highly cross-linked matrix. Immobilized metal affinity chromatography of bovine serum albumin on chelating Superose.

Immobilized metal ion affinity chromatography has shown promise for isolating desired proteins from a mixture based on their affinity for chelated metal ions. Using frontal analysis, the pH dependence of the saturation capacity of chelating Superose matrix for bovine serum albumin (BSA) is examined over a broad pH range. A significant increase in the capacity was observed near the elution pH of BSA (pH 4.5) from a Cu-imminodiacetic acid column. The results of several experiments indicated that this apparently abnormal variation may reflect the low degree of accessibility of a large portion of copper sites inside chelating Superose. In a broader sense, these results suggest that during frontal analysis, the assumption of column saturation based on a plateau in the exit concentration that is almost at the same level as the input concentration could be misleading for highly cross-linked matrices and relatively large sized proteins. That is, the relatively less accessible copper sites may become difficult to be reached due to high levels of protein adsorption in the more accessible regions and thus give the appearance of a plateau in the breakthrough curve prior to complete column saturation. This is likely to be the case at high pH where BSA demonstrates very high affinity for immobilized copper or at high input concentrations where the equilibrium coverage is expected to be high. The results demonstrate that the estimated saturation capacity could be significantly smaller than the actual capacity.

Binding Sites↗
PubMed1997

Supply-Side Analysis of Growth of Bacillus subtilis on Glucose-Citrate Medium: Feasible Network Alternatives and Yield Optimality.

Our prior work revealed that compared to the case for glucose metabolism, increased carbon yield and nil acid formation result when Bacillus subtilis grows on glucose medium containing citrate. To scrutinize further how citrate addition may alter metabolic flux regulation and the degree that the observed carbon yield corresponds to the maximal value, experimental (by least-squares analysis) and optimal (by linear programming) fluxes and yields were contrasted. Networks with differing reaction routes, directionality constraints, and transhydrogenase activities were examined. To attain an elevated carbon yield, citrate-glucose utilization need not alleviate any stoichiometric constraints that can sometimes interfere with the attainment of network objectives. Rather, the high carbon yield and nil acid formation attained may be linked to restriction of glycolytic capacity, particularly at the level of pyruvate kinase, which is consistent with a hypothesized effect of coupled metal-citrate uptake. Allowing for malic enzyme activity, hexose monophosphate pathway cycling, and transhydrogenase activity may also lead to the flux distributions underlying the high carbon yield observed. Finally, the observed carbon yield corresponded well to the maximum yield provided by all the network alternatives examined. Collectively, these results suggest that (i) the observed carbon yield is essentially equal to the maximal values associated with plausible networks and (ii), as suggested by others, nonoptimal flux regulation may contribute significantly to apparent cellular maintenance requirements.

Journal Article↗
PubMed1994

Flux adaptations of citrate synthase-deficient Escherichia coli.

The results from the experiments performed with a mutant deficient in citrate synthase activity can be summarized as follows. (1) Totally blocking entry into the TCA cycle did not appreciably alter the cellular ATP yield. The unchanged yield suggests that for growth on abundant glucose, the sensitivity of ATP yield to TCA cycle flux is low. ATP production in the mutant is altered, in part, by modulating the relative amounts of formate and acetate produced. (2) The in vivo operation of pyruvate-formate lyase and malic enzyme corresponds to proposals developed from in vitro studies. Namely, pyruvate activates the former, and acetyl CoA inhibits the latter. Overall, the diversion of pyruvate to formate under aerobic conditions constitutes an adaptation of the mutant to the enzymatic lesion. The low alpha-ketoglutarate dehydrogenase flux estimated for the mutant indicates that the enzyme is highly repressed in cells growing rapidly on glucose, which is in accord with prior induction-repression studies. Moreover, the lack of a change in uptake flux during the bulk of batch growth is consistent with prior induction-repression studies. (3) The mutant exhibits a heightened sensitivity to CO2 as compared to wild-type counterparts. Growth rate is increased, and the production of formate, malate, glycerate, and pyruvate is reduced. This sensitivity illustrates that citrate synthase is more than an expendable component in an amphibolic pathway. Its presence in wild-type cells "immunizes" against the effect of CO2 fluctuations. (4) The effects of CO2 can be tentatively explained by assuming that the PEP carboxylase-catalyzed reaction is stimulated.

Adenosine Triphosphate↗
PubMed1986

Mathematical model for the control of ColE1 type plasmid replication.

A mathematical model for the molecular events controlling replication of ColE1 type plasmids is described. All the model parameters can be evaluated independently. The model simulates plasmid replication and accurately predicts the copy-number of ColE1 plasmids carrying a variety of regulatory mutations. The model is used to test the plausibility of hypotheses concerning the interactions of regulatory elements involved in the replication apparatus. The model favorably supports the mechanism proposed by Tomizawa and co-workers concerning the nature of RNA-RNA interactions and that the Rom protein increases the binding between the two RNA species. The hypothesis that the interactions of RNA I-II increases the susceptibility of RNA II to the action of endonucleases is not a plausible mechanism.

DNA Replication↗
PubMedDate not supplied

13C NMR evidence for pyruvate kinase flux attenuation underlying suppressed acid formation in Bacillus subtilis.

When batch and continuous Bacillus subtilis cultures are provided with a small amount of citrate, acid production ceases, carbon yield increases by more than 2-fold, and the productivity of recombinant protein increases. It has been hypothesized that pyruvate kinase activity is attenuated, which in turn lowers glucose flux and minimizes the acid overflow prompted by low Krebs cycle capacity. To complement existing enzyme activity, linear programming, and metabolite pool studies, (13)C NMR studies were performed. Atom mapping and isotopomer mapping matrix methods were used to select the best glucose label. "Best" was defined such that the NMR spectra of glutamate associated with metabolizing labeled glucose via the different candidate metabolic trafficking scenarios would differ considerably in fine structure (e.g., relative singlet intensities). When experiments were performed with 1-(13)C glucose, the observed NMR spectra corresponded well to the one predicted to arise when the metabolic trafficking occurs according to a pyruvate kinase attenuation scenario. This evidence further fortifies the prospects for successfully basing a metabolic engineering strategy on reducing pyruvate kinase activity to better match glycolytic and Krebs cycle capacities.

Bacillus subtilis↗
PubMedDate not supplied

Effect of protease inhibitors on yield of HSV-1-based viral vectors.

The ability to obtain high titer replication-defective herpes simplex virus (HSV) recombinant vectors will dramatically affect their use in gene therapy clinical trials. A variety of techniques and reagents have been employed to increase the overall yield of the vector. The effects of protease inhibitors on the yield of an HSV-1-based viral vector were examined. Experiments were conducted using a commercial protease inhibitor cocktail typically used in mammalian cell culture for protein production. Contrary to our expectation for enhanced vector yield, the results showed a dramatic reduction in vector yield. Moreover, it was found that AEBSF is the only component in the protease cocktail responsible for the low vector yield. On the basis of our hypothesis regarding the mode of action of AEBSF, we suggest that it should not be included in protease inhibitor cocktails designed for use in cultures aimed at production of viral vectors derived from HSV-1 or possibly several other vectors.

Culture Media↗