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Biomedical subjects

G Stephanopoulos

Publications and source records attributed to G Stephanopoulos.

12 recordsLinked to original sources
PubMed1995

Flow cytometric study of differentiating cultures of Bacillus subtilis.

We report on 1) the development of a flow cytometry-based technique for detecting beta-galactosidase in differentiating cultures of Bacillus subtilis and 2) the application of this technique in the study of early developmental gene expression. The problems associated with generating detectable signals (despite the small size of B. subtilis cells) have been overcome using the fluorogenic substrate 5-octanolyaminofluorescein di-beta-D-galactopyranoside (C8-FDG). Additionally, to control for background fluorescence during the staining process, we included a control population in the C8-FDG staining mixture that consists of cells devoid of the lacZ gene prestained with another dye, PKH26. The distinct emission spectra of C8-fluorescein and PKH26 allow nonspecific C8-FDG staining in this control population to be monitored using two-color analysis. This technique has been applied in the study of developmental gene expression in sporulating cultures of B. subtilis, and it has been found that such cultures are heterogeneous, comprising two cell populations. One population is induced for expression of early sporulation genes, which is determined using lacZ fusions, whereas the other remains uninduced. These results have allowed us to understand better the patterns of gene expression exhibited by wild-type and mutant cultures early during the development process of spore formation.

Bacillus subtilis↗
PubMed1995

Production of isoleucine by overexpression of ilvA in a Corynebacterium lactofermentum threonine producer.

Overproduction of isoleucine, an essential amino acid, was achieved by amplification of the gene encoding threonine dehydratase, the first enzyme in the threonine to isoleucine pathway, in a Corynebacterium lactofermentum threonine producer. Threonine overproduction was previously achieved with C. lactofermentum ATCC 21799, a lysine-hyperproducing strain, by introduction of plasmid pGC42 containing the Corynebacterium homdr and thrB genes (encoding homoserine dehydrogenase and homoserine kinase respectively) under separate promoters. The pGC42 derivative, pGC77, also contains ilvA, which encodes threonine dehydratase. In a shake-flask fermentation, strain 21799(pGC77) produced 15 g/l isoleucine, along with small amounts of lysine and glycine. A molar carbon balance indicates that most of the carbon previously converted to threonine, lysine, glycine and isolecine was incorporated into isoleucine by the new strain. Thus, in our system, simple overexpression of wild-type ilvA sufficed to overcome the effects of feedback inhibition of threonine dehydratase by the end-product, isoleucine.

Acetolactate Synthase↗
PubMed1995

Effect of inducible thrB expression on amino acid production in Corynebacterium lactofermentum ATCC 21799.

Amplification of the operon homdr-thrB encoding a feedback-insensitive homoserine dehydrogenase and a wild-type homoserine kinase in a Corynebacterium lactofermentum lysine-producing strain resulted in both homoserine and threonine accumulation, with some residual lysine production. A plasmid enabling separate transcriptional control of each gene was constructed to determine the effect of various enzyme activity ratios on metabolite accumulation. By increasing the activity of homoserine kinase relative to homoserine dehydrogenase activity, homoserine accumulation in the medium was essentially eliminated and the final threonine titer was increased by about 120%. Furthermore, a fortuitous result of the cloning strategy was an unexplained increase in homoserine dehydrogenase activity. This resulted in a further decrease in lysine production along with a concomitant increase in threonine accumulation.

Corynebacterium↗
PubMed1994

The effect of protein synthesis inhibitors on the glycosylation site occupancy of recombinant human prolactin.

The relationship between synthesis and N-linked glycosylation site occupancy of recombinant human prolactin produced from C127 cells was studied with the aid of a battery of protein synthesis inhibitors. Non-lethal concentrations of sodium fluoride, gougerotin, puromycin, anisomycin, and emetine did not alter site occupancy, but low concentrations (< 10 micrograms ml-1) of cycloheximide increased the fraction of secreted prolactin bearing oligosaccharide from 20% to 80% of the total. Cycloheximide is an inhibitor of the elongation step of protein synthesis. The observed increase in glycosylation site occupancy upon addition of cycloheximide is consistent with the current opinion that the initial glycosylation event occurs cotranslationally during a limited time period. Cycloheximide may extend this time period by reducing elongation rate. However, the absence of any effect from treatment with other inhibitors of elongation suggests that cycloheximide is unique in its behavior on this system.

Amidohydrolases↗
PubMed1994

Metabolic engineering.

Metabolic engineering is not another form of classic manipulation of intermediary metabolism; rather, it is the purposeful design of metabolic networks. The metabolic engineering approach examines biochemical reactions in their entirety, rather than individually, and is concerned with the construction of novel pathways, the thermodynamic feasibility of pathways, and the location of limiting branch-point(s) and enzymatic reaction(s) in a reaction network. The ultimate goal is the construction of strains with superior yield and productivity obtained through a convergent evolutionary process. Progress has been made toward this objective within the framework of Metabolic Control Analysis through the development of methodologies for the experimental determination of elasticities and flux control coefficients.

Biotechnology↗
PubMed1993

Metabolic engineering--methodologies and future prospects.

Attempts to improve the productivity of cellular systems or to increase metabolite yield often require radical alteration of the flux through primary metabolic pathways. However, achieving the desired result often proves difficult because the control architectures at key branch points have evolved to resist flux changes. Identification and characterization of these metabolic nodes is a prerequisite to rational metabolic engineering.

Biomedical Engineering↗
PubMed1991

Network rigidity and metabolic engineering in metabolite overproduction.

In order to enhance the yield and productivity of metabolite production, researchers have focused almost exclusively on enzyme amplification or other modifications of the product pathway. However, overproduction of many metabolites requires significant redirection of flux distributions in the primary metabolism, which may not readily occur following product deregulation because metabolic pathways have evolved to exhibit control architectures that resist flux alterations at branch points. This problem can be addressed through the use of some general concepts of metabolic rigidity, which include a means for identifying and removing rigid branch points within an experimental framework.

Carbon Dioxide↗
PubMed1990

Multiple episodes of induced secretion of human growth hormone from recombinant AtT-20 cells.

Recombinant AtT-20 cells expressing human growth hormone (hGH) secreted the hormone at a constant, basal rate of 0.3-0.5 ng/10(5) cells-hour when exposed to medium without secretagogues. When triggered with 8 bromo-cyclic AMP, cells secreted hGH at an initial rate of 1.7 ng/10(5) cells-hour while intracellular hGH declined sharply. Upon extended exposure to secretagogue, secretion decreased gradually to the basal rate and intracellular hGH stabilized at a value 40% the initial. In cells switched from secretion to growth medium, the total rate of hGH accumulation intracellularly and in medium was 2.2 times that observed with cells never exposed to secretagogue; however, only a fraction of the hormone was stored intracellularly and the rest was secreted. When cells were exposed alternately to growth and secretion medium, induced cells secreted at rates at least two times higher than uninduced controls during the first five cycles. The induced response deteriorated with time, however, in parallel with outgrowth of attached cells by foci of round cells, and by the eighth cycle induced secretion did not occur. Operational modifications that may improve the performance of cycling schemes are discussed.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1981

Microbial competition.

Populations of microorganisms inhabiting a common environment complete for nutrients and other resources of the environment. In some cases, the populations even excrete into the environment chemicals that are toxic or inhibitory to their competitors. Competition between two populations tends to eliminate one of the populations from their common habitat, especially when competition is focused on a single resource and when the populations do not otherwise interact. However, a number of factors mitigate the severity of competition and thus competitors often coexist.

Bacterial Physiological Phenomena↗