Recombinant Microorganisms Research Articles

The 34th OHOLO Conference Novel Strategies in Production and Recovery of Biologicals from Recombinant Microorganisms and Animal Cells: Eilat, Israel 23–27 April 1990

The 34th OHOLO Conference Novel Strategies in Production and Recovery of Biologicals from Recombinant Microorganisms and Animal Cells: Eilat, Israel 23–27 April 1990

Drug products of recombinant DNA technology

Presented are the steps in creating a recombinant DNA molecule, examples of recombinant drug products, a description of DNA fingerprinting methods for diagnosing diseases, a discussion of the patenting of recombinant drugs, and a look to the future of this revolutionary biotechnology. Constructing a recombinant DNA molecule involves cutting the DNA into fragments with restriction endonucleases and rejoining the fragments in novel arrangements with ligase. Propagating the molecule in a microorganism, or cloning, is necessary to increase the number of gene copies to facilitate detection of the gene of interest and to produce the protein it encodes. Recombinant DNA drug products have been developed that represent the communicator, structural, and modifier classes of proteins. Recombinant communicator proteins include interferons alfa-2a and alfa-2b and granulocyte-macrophage colony-stimulating factor (immune system modulators); epidermal growth factor and erythropoietin (tissue repair promoters); and human insulin, growth hormone, and atrial peptide (metabolism modulators). Recombinant structural proteins include hepatitis B virus vaccine and CD4 protein, and recombinant modifier proteins include tissue plasminogen activator and superoxide dismutase (agents that split or splice organic molecules). In the future, gene defects associated with genetic diseases will be unraveled, leading to the production of new therapeutic agents designed to counteract or actually reverse those defects. Recombinant protein drugs will be further tailored to enhance their activity and specificity. These advances are so novel and momentous that patent protection has been extended not only to recombinant drugs but to the recombinant microorganisms in which they are manufactured. In cloning genes, investigators directly use the protein designs that occur naturally. Basic research will soon lead to the engineering of novel proteins with specified functions.

Compartment model concept used in the design of fermentation with recombinant microorganisms

Simple models are of great value in order to simulate cell dynamics, and a class of potentially useful models are the so-called compartment models, which are a simple extension of the unstructured approach. Compartment models have earlier been used with great success to describe cell dynamics. In this article the authors present a 4-compartment model that is able to describe a majority of the observations that have been made through fermentations with recombinant microorganisms. The compartment model is especially suited for description of dynamic changes in plasmid copy number, e.g., runaway replication. Simulation of batch fermentation with runaway conditions corresponds very well with experimental results. Finally the model is used in the design of an optimal fermentation strategy in a CSTR.

Competitive yeast fermentation with selective flocculation and recycle

Continuous fermentations were carried out involving competition between two strains of Saccharomyces cerevisiae. One of the strains has a lower specific growth rate and is very flocculent, whereas the faster-growing strain is nonflocculent. The product stream from the chemostat was fed into an inclined settler where the flocculent strain was partially separated from the nonflocculent strain as a result of the higher sedimentation rate of the flocculent cells. The underflow from the inclined settler, which was concentrated and enriched with flocculent cells, was recycled to the chemostat. When no recycle was used, the faster-growing, nonflocculent yeast rapidly overtook the culture. With selective recycle, however, the experiments demonstrated that the slower-growing flocculent yeast could be maintained as the dominant species. A theoretical development is also presented in order to describe the competition between two strains in the bioreactor-settler system. The concept of selective recycle via selective flocculation and sedimentation offers a possible means of maintaining unstable recombinant microorganisms in continuous fermentations.

Superoxide dismutase for therapeutic use: clinical experience, dead ends and hopes.

The clinical trials performed with bovine superoxide dismutase (SOD) are reviewed. SOD, applied intraarticularly at a dosage of 2-16 mg, proved to be effective in osteoarthritis of the knee joint in three placebo-controlled and one steroid-controlled double-blind trials. Its efficacy in other inflammatory joint disorders is documented by uncontrolled trials. Similarly, some controlled and many open studies support the efficacy of locally injected SOD in periarticular inflammation. Systemic treatment of rheumatoid arthritis by SOD at the dosages indicated yielded disappointing results. Well documented, though open uncontrolled studies demonstrated beneficial effects of locally administered SOD in radiation cystitis, interstitial cystitis and Peyronie's disease. Tolerance is good, but allergic reactions at low incidence have to be anticipated. Human SOD derived from recombinant microorganisms is being developed to explore its therapeutic potential particularly in ischemia-reperfusion damage, adult respiratory distress or similar conditions.

Carboxysomes and Ribulose Bisphosphate Carboxylase/Oxygenase

Publisher Summary This chapter focuses on recent advances made in the knowledge of the occurrence, composition, properties, and possible functions of carboxysomes. It also discusses the rapidly expanding field of research on Ribulose 1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) enzymes. The chapter compares the RuBisCO enzymes of prokaryotes that contain carboxysomes with those that do not. Insight into carboxysome function also may be gained by comparison with other prokaryotic inclusions and with the compartmentation of RuBisCO in chloroplasts. The knowledge of carboxysome composition is necessary to provide an understanding of the functions of the organelles and their contribution to the physiology of the cell. The carboxysomes isolated from the different physiological groups of autotrophs are similar in the possession of two polypeptides that coincide in terms of molecular mass with the large (L) subunits and small (S) subunits of their respective RuBisCO enzymes. The use of carboxysomes as ecophysiological markers and the prospects of producing RuBisCO-containing inclusion bodies in recombinant micro-organisms is also discussed in the chapter.

8705025 Cloned streptococcal genes encoding protein G and their use to construct recombinant microorganisms to produce protein G: Stephen R Fahnestock, Stephen R Fahnestock assigned to Genex Corporation

8705025 Cloned streptococcal genes encoding protein G and their use to construct recombinant microorganisms to produce protein G: Stephen R Fahnestock, Stephen R Fahnestock assigned to Genex Corporation

The use of the immobilization of whole living cells to increase stability of recombinant plasmids in escherichia coli

Immobilization of whole living cells was used as an experimental approach to enhance plasmid stability in cultured recombinant micro-organisms. pTG201 plasmid which is very unstable in continuous cultures with free cells, was found to be extremely stable in continuous cultures with immobilized cells. To elucidate the mechanism by which immobilization increases the plasmid stability, we analyzed the growth of pTG201-containing E. coli W3101 cells within the gel beads. We found that in immobilized continuous culture, plasmid-free segregants were not detected even after 240 generations. This appears to be due to the mechanical properties of the gel-bead system that allow only a limited number of cell divisions (10–16) to occur in each clone of cells before the clone escapes from the gel bead. This number of generations is not sufficient for the plasmid-free cells to appear within the cavities compared to what was observed in a free-system (plasmid-free segregants were detected after a lag period of approximately 25–30 generations). Even when they appear, they cannot overcome the culture. From the data described in this paper we conclude that cells released from the gel beads at any time during continuous culture are cells which are issued from cells grown in the cavities for only 10–16 generations.

Scale-up of fermentation processes using recombinant microorganisms.

Annals of the New York Academy of SciencesVolume 469, Issue 1 p. 73-82 Scale-up of Fermentation Processes Using Recombinant Microorganisms DEWEY D. Y. RYU, DEWEY D. Y. RYU University of California Davis, California 95616Search for more papers by this authorR. SIEGEL, R. SIEGEL University of California Davis, California 95616Search for more papers by this author DEWEY D. Y. RYU, DEWEY D. Y. RYU University of California Davis, California 95616Search for more papers by this authorR. SIEGEL, R. SIEGEL University of California Davis, California 95616Search for more papers by this author First published: May 1986 https://doi.org/10.1111/j.1749-6632.1986.tb26485.xCitations: 8AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume469, Issue1Biochemical Engineering IVMay 1986Pages 73-82 RelatedInformation

A Novel Bioreactor‐Cell Precipitator Combination for High‐Cell Density, High‐Flow Fermentations

A novel bioreactor design has been investigated for carrying out in a single unit a fermentation and cell recycle process simultaneously. The reactor consisted of a typical fermentor and a side-arm attached at a certain fixed angle to the fermentor. Due to the enhanced sedimentation in the inclined side-arm, the cells precipitate quickly and flow back into the fermentor. This allowed an essentially cell-free fermentation broth to be withdrawn at high rates through the sidearm while maintaining a high cell density culture in the fermentor. Continuous flow fermentation runs of S. cerevisiae demonstrated these features and showed that many fold increases in the steady-state cell density could be achieved using high flowrates, which in an ordinary chemostat would lead to washout. Ethanol productivities were high and can further be increased several-fold through a straightforward scale-up of the precipitator sidearm to sustain even larger flow rates. Discussed also are various other reactor features such as the enhanced resistance to contamination, possible reduction of the plasmid instability problems of recombinant microorganisms, potential applications with flocculent strains, steady-state coexistence of competing populations in mixed culture, and the possibility of carrying out fermentations with stationary cultures under continuous flow conditions.

Instability kinetics of trp operon plasmid ColEl‐trp in recombinant Escherichia coli MV 12[pVH5] and MV 12trp R [pVH5

In studying plasmid instability in recombinant microorganisms, Escherichia coli MV 12[p VH5] and MV 12 trpR[p VH5] harboring trp operon were used as experimental model systems. The host with the trp repression system was partially derepressed by 3-beta-indoleacrylic acid. The results from kinetic analysis of plasmid instability showed that the stability of p VH5 and the growth rate of MV 12[p VH5] decreased rapidly in presence of 3-beta-indoleacrylic acid at a concentration higher than 10 mug/mL, but beyond 30 g/mL no significant change was observed. This suggests that Trp(-) variants from MV 12[p VH5] could be produced from the host cells at a different frequency depending on the physiological condition. In another system, MV 12 trpR[p VH5] which was constructed by conjugation of E. coli MV 12[p VH5] with E. coli CSH61, the plasmid stability was much lower and the frequency of Trp(-) cell production was about 10 times higher as compared with the MV 12[p VH5] when treated with 3-beta-indoleacrylic acid. A kinetic model representing the plasmid instability was derived, and a fairly good agreement with the experimental results was found. The fraction of plasmid-free cell (or negative variant) shows a different time course profile depending on the segregation coefficient (a, production rate of negative variants from positive cells during one generation), growth ratio (G, the ratio of growth rates of negative variants to positive cells), and other parameters. The negative variant fraction continues to increase ("run away" type) or it approaches to a finite value ("settling" type) depending on the relative values of a and G.