Factors affecting plasmid production in Escherichia coli from a resource allocation standpoint

<p>Abstract</p> <p>Background</p> <p>Plasmids are being reconsidered as viable vector alternatives to viruses for gene therapies and vaccines because they are safer, non-toxic, and simpler to produce. Accordingly, there has been renewed interest in the production of plasmid DNA itself as the therapeutic end-product of a bioprocess. Improvement to the best current yields and productivities of such emerging processes would help ensure economic feasibility on the industrial scale. Our goal, therefore, was to develop a stoichiometric model of <it>Escherichia coli </it>metabolism in order to (1) determine its maximum theoretical plasmid-producing capacity, and to (2) identify factors that significantly impact plasmid production.</p> <p>Results</p> <p>Such a model was developed for the production of a high copy plasmid under conditions of batch aerobic growth on glucose minimal medium. The objective of the model was to maximize plasmid production. By employing certain constraints and examining the resulting flux distributions, several factors were determined that significantly impact plasmid yield. Acetate production and constitutive expression of the plasmid's antibiotic resistance marker exert negative effects, while low pyruvate kinase (Pyk) flux and the generation of NADPH by transhydrogenase activity offer positive effects. The highest theoretical yield (592 mg/g) resulted under conditions of no marker or acetate production, nil Pyk flux, and the maximum allowable transhydrogenase activity. For comparison, when these four fluxes were constrained to wild-type values, yields on the order of tens of mg/g resulted, which are on par with the best experimental yields reported to date.</p> <p>Conclusion</p> <p>These results suggest that specific plasmid yields can theoretically reach 12 times their current experimental maximum (51 mg/g). Moreover, they imply that abolishing Pyk activity and/or transhydrogenase up-regulation would be useful strategies to implement when designing host strains for plasmid production; mutations that reduce acetate production would also be advantageous. The results further suggest that using some other means for plasmid selection than antibiotic resistance, or at least weakening the marker's expression, would be beneficial because it would allow more precursor metabolites, energy, and reducing power to be put toward plasmid production. Thus far, the impact of eliminating Pyk activity has been explored experimentally, with significantly higher plasmid yields resulting.</p

Providing Application-Level QoS In 3G/4G Wireless Systems: A Comprehensive Framework Based On Multi-Rate CDMA

The emerging applications for 3G and 4G wireless systems typically require highly heterogeneous and time-varying quality of service from the underlying protocol layers. The wireless links, however, provide only an unreliable communication channel that suffers from temporal outages. As a consequence, protocol mechanisms are needed, that based on the unreliable wireless links provide the different service qualities required by the emerging applications. In this paper we identify the emerging IP based applications for 3G and 4G wireless systems and categorize their QoS requirements. We discuss the wireless access mechanisms that show promise of being the basis for supporting these applications. We than propose a set of protocol mechanisms, that based on the discussed wireless access mechanisms, provide the required QoS for the different application categories

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

New Strategies for Designing Inexpensive but Selective Bioadsorbants for Environmental Pollutants: Selection of specific Ligands and Their Cell Surface Expression

The Broad, long term objective of the research plan is to develop exquisitely selective polypeptide metal chelators for the remediation of aqueous systems. A variety of polypeptide chelators will be developed and optimized ranging from antibodies to small peptides. Then, through unique molecular engineering approaches developed in our laboratories, the polypeptide chelators will be anchored directly on the surface of the cells that produce them. Thus, instead of using isolated biomolecules we will employ inexpensive genetically engineered whole cell adsorbents. Following a simple, easily scaleable treatment, the engineered cells can be used to manufacture an inexpensive, particulate adsorbent for metal removal

Pyruvate Kinase-Deficient Escherichia coli Exhibits Increased Plasmid Copy Number and Cyclic AMP Levels▿

Previously established consequences of abolishing pyruvate kinase (Pyk) activity in Escherichia coli during aerobic growth on glucose include reduced acetate production, elevated hexose monophosphate (HMP) pathway flux, elevated phosphoenolpyruvate carboxylase (Ppc) flux, and an increased ratio of phosphoenolpyruvate (PEP) to pyruvate. These traits inspired two hypotheses. First, the mutant (PB25) may maintain more plasmid than the wild type (JM101) by combining traits reported to facilitate plasmid DNA synthesis (i.e., decreased Pyk flux and increased HMP pathway and Ppc fluxes). Second, PB25 likely possesses a higher level of cyclic AMP (cAMP) than JM101. This is based on reports that connect elevated PEP/pyruvate ratios to phosphotransferase system signaling and adenylate cyclase activation. To test the first hypothesis, the strains were transformed with a pUC-based, high-copy-number plasmid (pGFPuv), and copy numbers were measured. PB25 exhibited a fourfold-higher copy number than JM101 when grown at 37°C. At 42°C, its plasmid content was ninefold higher than JM101 at 37°C. To test the second hypothesis, cAMP was measured, and the results confirmed it to be higher in PB25 than JM101. This elevation was not enough to elicit a strong regulatory effect, however, as indicated by the comparative expression of the pGFPuv-based reporter gene, gfpuv, under the control of the cAMP-responsive lac promoter. The elevated cAMP in PB25 suggests that Pyk may participate in glucose catabolite repression by serving among all of the factors that tighten gene expression

Chemie der mikrobiellen Kohleverfluessigung Abschlussbericht

The authors investigated structural changes in coals and in their humic acids and macromolecular matrix induced by coal-degrading microorganisms. An extensive structural chemical characterisation of the bioconversion products is attempted. Apart from the classic methods of structural analysis, new methods were developed especially for identification of the oxygen functional groups in the small volumes of bioconversion products availableDie Aufgabenstellung umfasst die Aufklaerung der strukturellen Veraenderung der Einsatzkohle sowie ihrer Komponenten Huminsaeuren und makromolekulare Matrix durch die Einwirkung kohleabbauender Mikroorganismen. Die Biokonversionsprodukte sollen umfassend strukturchemisch charakterisiert werden. Die komplexe Problemstellung erfordert neben dem Einsatz von bekannten Methoden der Kohlestrukturanalytik die Entwicklung von Analysenverfahren, insbesondere fuer die Bestimmung der sauerstoffunktionellen Gruppen, die den geringen Mengen der von den mikrobiologischen Arbeitsgruppen bereitgestellten Biokonversionsprodukten angepasst sind. (orig.)SIGLEAvailable from TIB Hannover: F00B375 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung und Forschung (BMBF), Bonn (Germany)DEGerman