Biochemical engineering. Bridging the gap between gene and product.
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Animals↗
Biomedical subjects
G Stephanopoulos
Publications and source records attributed to G Stephanopoulos.
Mitochondrial membrane potential differentiates cells resistant to apoptosis in hybridoma cultures.
Previous research has implicated mitochondrial physiology and, by extension, respiratory capacity in the initiation and progress of apoptosis of cells in culture and tissue environments. This hypothesis was tested by separating a hybridoma cell population into subpopulations of varying mitochondrial membrane potential (MMP) using Rhodamine 123 stain and fluorescence-activated cell sorter analysis and subjecting them to two apoptosis inducers, rotenone and staurosporin. Apoptotic death was characterized morphologically through the determination of apoptosis-related chromatin condensation and biochemically through the measurement of caspase-3 enzymatic activity. We found dramatic differences in the apoptotic death kinetics for the subpopulations, with the high MMP cells showing higher resistance to apoptotic death. After incubation with 30 microM rotenone, the low MMP cells exhibited one-third of the viability of the high MMP cells and a three-fold increase in the capsase-3 enzymatic activity. No changes were observed in the DNA content or the cell cycle distributions of the two cell subpopulations, which maintained their mean MMP difference after 20 generations. These results suggest that heterogeneity exists in mammalian cell populations with respect to mitochondrial physiology, which correlates with resistance to apoptotic death.
Amino Acid Chloromethyl Ketones↗
Identification and analysis of the polyhydroxyalkanoate-specific beta-ketothiolase and acetoacetyl coenzyme A reductase genes in the cyanobacterium Synechocystis sp. strain PCC6803.
Synechocystis sp. strain PCC6803 possesses a polyhydroxyalkanoate (PHA)-specific beta-ketothiolase encoded by phaA(Syn) and an acetoacetyl-coenzyme A (CoA) reductase encoded by phaB(Syn). A similarity search of the entire Synechocystis genome sequence identified a cluster of two putative open reading frames (ORFs) for these genes, slr1993 and slr1994. Sequence analysis showed that the ORFs encode proteins having 409 and 240 amino acids, respectively. The two ORFs are colinear and most probably coexpressed, as revealed by sequence analysis of the promoter regions. Heterologous transformation of Escherichia coli with the two genes and the PHA synthase of Synechocystis resulted in accumulation of PHAs that accounted for up to 12.3% of the cell dry weight under high-glucose growth conditions. Targeted disruption of the above gene cluster in Synechocystis eliminated the accumulation of PHAs. ORFs slr1993 and slr1994 thus encode the PHA-specific beta-ketothiolase and acetoacetyl-CoA reductase of Synechocystis and, together with the recently characterized PHA synthase genes in this organism (S. Hein, H. Tran, and A. Steinbüchel, Arch. Microbiol. 170:162-170, 1998), form the first complete PHA biosynthesis pathway known in cyanobacteria. Sequence alignment of all known short-chain-length PHA-specific acetoacetyl-CoA reductases also suggests an extended signature sequence, VTGXXXGIG, for this group of proteins. Phylogenetic analysis further places the origin of phaA(Syn) and phaB(Syn) in the gamma subdivision of the division Proteobacteria.
Acetyl-CoA C-Acyltransferase↗
Effect of glutamine limitation on the death of attached Chinese hamster ovary cells.
The effect of glutamine depletion on the death of attached Chinese hamster ovary (CHO) cells was investigated. Experiments were performed using an anchorage dependent CHO cell line expressing gamma-IFN and a second cell line obtained by transfection of that cell line with the human bcl-2 (hbcl-2). Either cell line could grow in media devoid of glutamine with minimal cell death due to endogenous glutamine synthetase activity that allowed cells to synthesize glutamine from glutamic acid in the medium. However, compared to control cultures in glutamine-containing media, the cell growth rate in glutamine-free media was slower with an increased fraction of cells distributed in the G0/G1 phase. The slower rate of cell cycling apparently protected the cells from entering apoptosis when they were stimulated to proliferate in an environment devoid of other protective factors, such as serum or over-expressed hbcl-2. The depletion of both glutamine and glutamic acid did cause cell death, which could be mitigated by hbcl-2 over-expression.
Animals↗
Metabolic flux analysis of hybridoma continuous culture steady state multiplicity.
Physiological state multiplicity was observed in continuous cultures of the hybridoma cell line ATCC CRL-1606 cultivated in glutamine-limited steady state chemostats. At the same dilution rate (0.04 h-1), two physiologically different cultures were obtained which exhibited similar growth rates and viabilities but drastically different cell concentrations (7.36 x 10(5) and 1.36 x 10(6) cells/mL). Metabolic flux analysis conducted using metabolite and gas exchange rate measurements revealed a more efficient culture for the steady state with the higher cell concentration, as measured by the fraction of pyruvate carbon flux shuttled into the TCA cycle for energy generation. The low-efficiency steady state was achieved after innoculation by growing the cells in a nutrient rich environment, first in batch mode followed by a stepwise increase of the dilution rate to its set point at 0.04 h-1. The high-efficiency steady state was achieved by reducing the dilution rate to progressively lower values to 0.01 h-1 resulting in conditions of stricter nutrient limitation. The high energetic efficiency attained under such conditions was preserved upon increasing the chemostat dilution rate back to 0.04 h-1 with a higher nutrient consumption, resulting in approximate doubling of the steady state cell concentration. This metabolic adaptation is unlikely due to favorable genetic mutations and could be implemented for improving cell culture performance by inducing cellular metabolic shifts to more efficient flux distribution patterns.
Animals↗
Analysis of the pathway structure of metabolic networks.
Metabolic networks comprise a multitude of enzymatic reactions carrying out various functions related to cell growth and product formation. Although such reactions are occasionally organized into biochemical pathways, a formal procedure is desired to identify the independent pathways in a bioreaction network and the degree of engagement of each individual reaction in these pathways. We present a procedure for the identification of the independent pathways of bioreaction networks of any size and complexity. The method makes use of the steady-state internal metabolite stoichiometry matrix and defines the independent pathways through the reaction membership of its kernel matrix. Examples from the aromatic amino acid biosynthetic pathway and central carbon metabolism of cells in culture are provided to illustrate the method. Applications to the analysis of the control structure of bioreaction networks are also discussed.
Adenosine Triphosphate↗
Metabolite and isotopomer balancing in the analysis of metabolic cycles: II. Applications.
In a previous paper (Klapa et al., 1999), we presented a model for the analysis of isotopomer distributions of the TCA cycle intermediates resulting from 13C (or 14C) labeling experiments. Results allow the rigorous determination of the degree of enrichment at specific carbon atoms of metabolites, of the molecular weight distribution of metabolite isotopomers, as well as of the fine structure of NMR spectra in terms of a small number of metabolic fluxes. In this paper we validate the model by comparing model predictions with experimental data and then apply it to the analysis of metabolic networks that have been investigated in previous studies. The results have allowed us to conclude that: (1) there is no evidence of propionyl-CoA carboxylase pathway in Escherichia coli; and (2) the possibility that acetone utilization in mammals occurs solely via the "lactate/methylglyoxal" pathway is consistent with available labeling data. The presented modeling framework provides additional constraints that must be satisfied by experimental data in a biochemical network structure and therefore enhances the power of labeling methods for resolving in vivo metabolic fluxes.
Acetic Acid↗
Metabolic effects on recombinant interferon-gamma glycosylation in continuous culture of Chinese hamster ovary cells.
Asparagine linked (N-linked) glycosylation is an important modification of recombinant proteins, because the attached oligosaccharide chains can significantly alter protein properties. Potential glycosylation sites are not always occupied with oligosaccharide, and site occupancy can change with the culture environment. To investigate the relationship between metabolism and glycosylation site occupancy, we studied the glycosylation of recombinant human interferon-gamma (IFN-gamma) produced in continuous culture of Chinese hamster ovary cells. Intracellular nucleotide sugar levels and IFN-gamma glycosylation were measured at different steady states which were characterized by central carbon metabolic fluxes estimated by material balances and extracellular metabolite rate measurements. Although site occupancy varied over a rather narrow range, we found that differences correlated with the intracellular pool of UDP-N-acetylglucosamine + UDP-N-acetylgalactosamine (UDP-GNAc). Measured nucleotide levels and estimates of central carbon metabolic fluxes point to UTP depletion as the cause of decreased UDP-GNAc during glucose limitation. Glucose limited cells preferentially utilized available carbon for energy production, causing reduced nucleotide biosynthesis. Lower nucleoside triphosphate pools in turn led to lower nucleotide sugar pools and reduced glycosylation site occupancy. Subsequent experiments in batch and fed-batch culture have confirmed that UDP-sugar concentrations are correlated with UTP levels in the absence of glutamine limitation. Glutamine limitation appears to influence glycosylation by reducing amino sugar formation and hence UDP-GNAc concentration. The influence of nucleotide sugars on site occupancy may only be important during periods of extreme starvation, since relatively large changes in nucleotide sugar pools led to only minor changes in glycosylation.
Animals↗
Metabolism of peptide amino acids by Chinese hamster ovary cells grown in a complex medium.
Metabolic flux analysis is a useful tool for unraveling relationships between metabolism and cell function. Material balancing can be used to provide estimates of major metabolic pathway fluxes, provided all significant metabolite uptake and production rates are measured. Potential sources of metabolizable material in many serum-free media formulations are low molecular weight digests of biological material such as yeast extracts and plant or animal tissue hydrolysates. These digests typically contain large amounts of peptides, which may be utilized as amino acids. This article demonstrates the need for accounting for amino acids liberated from peptides in order to accurately estimate pathway fluxes in Chinese hamster ovary cells grown in a complex (hydrolysate containing) medium. A simplified model of central carbon metabolism provides the framework for analyzing external metabolite measurements. Redundant measurements are included to ensure the consistency of data and assumed biochemistry by comparing redundant measurements with their predicted values from a minimum data set, and by expressing the degree of agreement using a statistical "consistency index." The consistency index tests whether redundancies are satisfied within expected experimental error. For chemostat steady states of CHO cultures grown in a hydrolysate-supplemented medium, consistent data were obtained only when amino acids liberated from peptides were taken into account.
Amino Acids↗
Metabolite and isotopomer balancing in the analysis of metabolic cycles: I. Theory.
Proper analysis of label distribution in metabolic pathway intermediates is critical for correct interpretation of experimental data and strategic experimental design. While, for example, 13C nuclear magnetic resonance (NMR) spectroscopy is usually limited to the measurement of degrees of 13C enrichment, more information about metabolic fluxes can be extracted from the fine structure of NMR spectra, or molecular weight distributions of isotopomers of metabolic intermediates (measured by gas chromatography-mass spectrometry). For this purpose, rigorous accounting for the contribution of all pathways to label distribution is required, especially contributions resulting from multiple turns of metabolic cycles. In this paper we present a mathematical model developed to analyze isotopomer distributions of tricarboxylic acid cycle (TCA) intermediates following the administration of 13C (or 14C) labeled substrates. The theory presented provides the basis to analyze 13C NMR spectra and molecular weight distributions of metabolites. In a companion paper (Park et al., 1999), the theory is applied to the analysis of several cases of biological significance. Copyright 1999 John Wiley & Sons, Inc.
Journal Article↗
Metabolic fluxes and metabolic engineering.
Metabolic engineering is the directed improvement of cellular properties through the modification of specific biochemical reactions or the introduction of new ones, with the use of recombinant DNA technology. As such, metabolic engineering emphasizes metabolic pathway integration and relies on metabolic fluxes as determinants of cell physiology and measures of metabolic control. The combination of analytical methods to quantify fluxes and their control with molecular biological techniques to implement genetic modifications is the essence of metabolic engineering. Strategies for metabolic flux determination are reviewed in this paper and it is shown how metabolic fluxes can be used in the systematic elucidation of metabolic control in the framework of reaction grouping and top-down metabolic control analysis.
Animals↗
Flux through the tetrahydrodipicolinate succinylase pathway is dispensable for L-lysine production in Corynebacterium glutamicum.
The N-succinyl-LL-diaminopimelate desuccinylase gene (dapE) in the four-step succinylase branch of the L-lysine biosynthetic pathway of Corynebacterium glutamicum was disrupted via marker-exchange mutagenesis to create a mutant strain that uses only the one-step meso-diaminopimelate dehydrogenase branch to overproduce lysine. This mutant strain grew and utilized glucose from minimal medium at the same rate as the parental strain. In addition, the dapE- strain produced lysine at the same rate as its parent strain. Transformation of the parental and dapE- strains with the amplified meso-diaminopimelate dehydrogenase gene (ddh) on a plasmid did not affect lysine production in either strain, despite an eightfold amplification of the activity of the enzyme. These results indicate that the four-step succinylase pathway is dispensable for lysine overproduction in shake-flask culture. In addition, the one-step meso-diaminopimelate dehydrogenase pathway does not limit lysine flux in Corynebacterium under these conditions.
Acyltransferases↗
Isolation and characterization of indene bioconversion genes from Rhodococcus strain I24.
Rhodococcus strain 124 is able to convert indene into indandiol via the actions of at least two dioxygenase systems and a putative monooxygenase system. We have identified a cosmid clone from 124 genomic DNA that is able to confer the ability to convert indene to indandiol upon Rhodococcus erythropolis SQ1, a strain that normally can not convert or metabolize indene. HPLC analysis reveals that the transformed SQ1 strain produces cis-(1R,2S)-indandiol, suggesting that the cosmid clone encodes a naphthalenetype dioxygenase. DNA sequence analysis of a portion of this clone confirmed the presence of genes for the dioxygenase as well as genes encoding a dehydrogenase and putative aldolase. These genes will be useful for manipulating indene bioconversion in Rhodococcus strain 124.
Amino Acid Sequence↗
Metabolic engineering.
Metabolic engineering is the science that combines systematic analysis of metabolic and other pathways with molecular biological techniques to improve cellular properties by designing and implementing rational genetic modifications. As such, metabolic engineering deals with the measurement of metabolic fluxes and elucidation of their control as determinants of metabolic function and cell physiology. A novel aspect of metabolic engineering is that it departs from the traditional reductionist paradigm of cellular metabolism, taking instead a holistic view. In this sense, metabolic engineering is well suited as a framework for the analysis of genome-wide differential gene expression data, in combination with data on protein content and in vivo metabolic fluxes. The insights of the integrated view of metabolism generated by metabolic engineering will have profound implications in biotechnological applications, as well as in devising rational strategies for target selection for screening candidate drugs or designing gene therapies. In this article we review basic concepts of metabolic engineering and provide examples of applications in the production of primary and secondary metabolites, improving cellular properties, and biomedical engineering.
Biomedical Engineering↗
Experimental determination of group flux control coefficients in metabolic networks
Grouping of reactions around key metabolite branch points can facilitate the study of metabolic control of complex metabolic networks. This top-down Metabolic Control Analysis is exemplified through the introduction of group (flux, as well as concentration) control coefficients whose magnitudes provide a measure of the relative impact of each reaction group on the overall network flux, as well as on the overall network stability, following enzymatic amplification. In this article, we demonstrate the application of previously developed theory to the determination of group flux control coefficients. Experimental data for the changes in metabolic fluxes obtained in response to the introduction of six different environmental perturbations are used to determine the group flux control coefficients for three reaction groups formed around the phosphoenolpyruvate/pyruvate branch point. The consistency of the obtained group flux control coefficient estimates is systematically analyzed to ensure that all necessary conditions are satisfied. The magnitudes of the determined control coefficients suggest that the control of lysine production flux in Corynebacterium glutamicum cells at a growth base state resides within the lysine biosynthetic pathway that begins with the PEP/PYR carboxylation anaplorotic pathway. Copyright 1998 John Wiley & Sons, Inc.
Journal Article↗
Effect of reversible reactions on isotope label redistribution--analysis of the pentose phosphate pathway.
The pentose phosphate pathway plays several key roles in metabolism including supply of biosynthetic carbon skeletons and reducing power. Previous research has focused on determining the fluxes through the reactions of this pathway using carbon-labeled substrates and models that make certain assumptions about the reversibility of the transketolase and transaldolase reactions in the nonoxidative pathway. These assumptions, however, have resulted in inconsistencies between the predicted carbon label distributions using these models and those determined experimentally. A general metabolic reaction network model developed in this paper and applied to the pentose phosphate pathway not only incorporates reaction reversibility but also accounts for the effect of individually varying extents of reaction reversibility on labeled carbon fractional enrichment values for intermediate metabolites. In addition, an algorithm is presented that can be used to calculate the three individual transaldolase and transketolase extents of reversibility. The results of this method show that varying extents of reaction reversibility have an observable effect on the metabolite carbon label distributions which can in turn affect flux calculation for other parts of the metabolic network such as the tricarboxylic acid cycle. In addition, the observability of reversibility extent and accuracy of flux calculations depend on the particular choice of metabolite carbon enrichments measured. In particular, [6-13C]hexose 6-phosphate and [4-13C]erythrose 4-phosphate carbon enrichment values resulting from [1-13C]glucose feeding contained more information as compared to those from ribose 5-phosphate. This analysis was applied to literature data of metabolite carbon labeling that resulted from supplying either 13C- or 14C-enriched substrates to several cell types growing under various conditions. The specific activities of metabolite carbon atoms taken from rat epididymal adipose tissue, goosefish islet cells, Corynebacterium glutamicum, and Escherichia coli supplied with either [2-14C]glucose or [1-13C]glucose demonstrate how reversibility is present in the pentose phosphate pathway and the extents of reversibility can be estimated from labeled carbon data sets.
Adipose Tissue↗
Growth factor and bcl-2 mediated survival during abortive proliferation of hybridoma cell line.
Cultures of the CRL-1606 hybridoma (ATCC) have been reported to undergo continuous proliferation with simultaneous death during nutrient limited fed-batch fermentations. The bcl-2 proto-oncogene has been shown to prevent cell death under a variety of otherwise death inducing conditions. We were interested in elucidating the nature of the massive death observed in cultures of CRL-1606, specifically with respect to the possible environmental causes, and the ability of overexpressed human bcl-2 (hbcl-2) to mitigate cell death. Abortive proliferation, or continuous proliferation in the presence of continuous death, could be induced in serum free cultures of CRL-1606 through the withdrawal of insulin provided the culture was competent for cell proliferation. Culture competency for proliferation was found to be solely determined by the presence of cell culture nutrients. Abortive proliferation was defective in cultures transfected with hbcl-2 and the enhanced viability observed resulted from an increased viable cell population and at the expense of the nonviable cell population normally found in untransfected cultures. Abortive proliferation was also observed in serum containing cultures upon serum shiftdowns. Like the insulin-supplemented serum free culture system, hbcl-2 transfected cultures exhibited defects in the abortive proliferation process. These results suggest that the massive death observed during nutrient-limited fed-batch fermentation originate, in part, from growth or survival factor limitations. Hence, approaches to design cell culture media that account for the cell's proliferation requirements without accounting for the cell's survival requirements may represent a cell death sentence. Given the transformed nature of the hybridomas, we conclude that the abortive proliferation of CRL-1606 is a consequence of inappropriate cell cycle entry in a survival factor limited environment.
Animals↗
Sequence of the Corynebacterium glutamicum pyruvate carboxylase gene.
Pyruvate carboxylase is an important anaplerotic enzyme replenishing oxaloacetate consumed for biosynthesis during growth, or lysine and glutamic acid production in industrial fermentations. We used regions of homology from pyruvate carboxylase sequences of 12 different species (corresponding to the ATP- and pyruvate-binding sites), to design polymerase chain reaction (PCR) primers for amplifying a fragment of the pyruvate carboxylase (pc) gene from C. glutamicum genomic DNA. This 850-base-pair fragment was used to probe a C. glutamicum cosmid library and four candidate pc cosmids were identified. The fragment was sequenced and the sequence of the complete gene was obtained by several rounds of primer synthesis, PCR on one of the positive cosmids, and sequencing. The C. glutamicum pc sequence shows 64% homology with the pc gene of Mycobacterium tuberculosis and 44% homology with the human pc gene. Regions of ATP, pyruvate and biotin binding have also been identified.
Amino Acid Sequence↗