High-cell-density cultivation of Escherichia coli.

High-cell-density cultivations of Escherichia coli in glucose-mineral-salt media produce more than 100 g dry cells litre-1 in special fed-batch modes with feeding of glucose and ammonia only. The specific growth rate can be adjusted to allow optimum recombinant protein generation.

High cell density fermentation of recombinant Escherichia coli with computer-controlled optimal growth rate.

In recent years recombinant DNA technology has enabled us to produce various proteins of therapeutic importance with microorganisms. As an appropriate host organism, E. coli plays a dominant role. Yields of E. coli dry cell mass in shaker flask culture range from 1-2 g/L, whereas in fermentors up to 10 g dry cells/L can be achieved. ZIMET and GBF have developed a high cell density fermentation process that produces E. coli (on a glucose/mineral salt medium) up to more than 100 g dry cells/L in a special fed-batch mode. This cultivation strategy prevents oxygen limitation and hence the accumulation of acetate and other metabolic byproducts. The specific growth rate can be adjusted so that product formation reaches its optimum value. An example of the production of alpha1-interferon is presented. The high cell density fermentations were realized in 30- and 450-L Chemap fermentors (ZIMET) and in a three-stage bioreactor scale-up system (72, 300, and 1,500 L) developed in cooperation with GBF and B. Braun Melsungen AG. Multiloop controllers were used to control the process variables.

The appearance of cytoplasmic membranes of Escherichia coli cells in freeze-fracture electron microscopy after stringent and relaxed response.

Guanosine-5'-diphosphate-3'-diphosphate (ppGpp), an effector for many metabolic pathways, is synthesized by the relA gene product after amino acid limitation. Studies of stringent controlled Escherichia coli CP78 (relA+) and relaxed controlled E. coli CP79 (relA-) were carried out to test whether these strains differ in the appearance of their cytoplasmic membranes after induction of stringent and relaxed response. Cytoplasmic membrane structures of the cells were investigated by freeze-fracture electron microscopy after cooling the cells. The obtained micrographs showed a net-like distribution of the particles in the cytoplasmic membranes of relaxed controlled cells whereas such a pattern was not detectable in the stringent controlled counterparts.

The use of elements of the E. coli Ntr-system for the design of an optimized recombinant expression system for high cell density cultivations.

The inducible glnA promoter 2 of the E. coli glutamine synthetase gene is suitable as an expression unit for the production of recombinant proteins at low and high cell densities. It is active when the concentration of ammonium as the sole nitrogen source in the culture medium is below 1 mM. This nitrogen regulatory system was optimized by introduction of expression cassettes consisting of additional elements of the ntr-system. These artificial constructions result in enhanced recombinant gene expression in the production phase. Furthermore, the basic recombinant protein level during the growth phase is reduced due to a tighter promoter control. A three- to four-fold higher accumulation of chloramphenicol-acetyltransferase (as reporter protein) and of anti-EGF-receptor miniantibodies was achieved by increasing the amount of the final regulator molecule NtrC approximately P via plasmidal co-expression of the ntrC gene. The introduction of a modified glnA promoter 1 inverse to glnAp2 lowered the basic activity of glnAp2 to about one half. It is assumed that under nitrogen excess conditions sigma 70-RNA polymerase binds at glnAp1 and thereby prevents most of the binding of sigma 54-RNA polymerase at glnAp2. The optimized expression systems were successfully applied in low and high cell density cultivations. In the fed-batch phase of high cell density cultivations recombinant protein formation was induced through external nitrogen limitation under FIA-controlled concentration of glucose as carbon source.

Formation of recombinant proteins in Escherichia coli under control of a nitrogen regulated promoter at low and high cell densities.

The use of a modified Escherichia coli glnAP2 promoter results in the formation of both homologous and heterologous, cytoplasmic and periplasmic recombinant proteins in a nitrogen concentration dependent manner. As in the E. coli nitrogen regulatory system, glnAP2 controlled gene expression is induced when ammonium concentration in the growth medium is below 1 mM (nitrogen limitation), otherwise only extremely low expression of recombinant genes is observed. Both high cell density cultivations (HCDC) and low cell density cultivations (LCDC) gave similar results for inducibility and formation of the following recombinant proteins: chloramphenicol-acetyltransferase, phosphorylcholine binding mini-antibodies (scFv-dhlx of McPC603) and K1-streptokinase. Recombinant proteins were formed in quantities of about 2-3% of total cellular protein. At low cell densities, slightly higher quantities resulted under partial nitrogen limitations than under total nitrogen limitation. In contrast, high cell density cultivations resulted in lower product concentrations at partial nitrogen limitation compared with total nitrogen limitation. These lowered product concentrations were probably due to the very high amounts of K+ or Na+ ions which accumulated during pH-regulation, thereby disturbing growth. HCDC under partial nitrogen limitation decreased proteolysis of recombinant proteins, therefore reduced amounts of proteases are considered to be responsible.

High cell density cultivation of Escherichia coli at controlled specific growth rate.

A high cell density cultivation (HCDC) for growth of Escherichia coli in an especially designed glucose/mineral salt medium is proposed. The HCDC essentially starts as a batch process which is followed by a two-phase fed-batch cultivation. After unlimited growth at mu max = 0.45 h-1 in the batch part, growth was controlled at a reduced specific growth rate (mu = 0.11 h-1 less than mu max) over a period of 3 doubling times in which the biomass concentration increased from 12 to 95 g 1(-1) (phase 1 of fed-batch cultivation). Control of growth (mu) was realized by a PO2 control loop (by variation of glucose feeding) and a mu control loop (by variation of agitation speed N) while the actual mu was calculated from the off-gas composition. If the agitation rate cannot be increased anymore the mu controller is switched off (end of phase 1). In the following phase 2, mu declines, however, the still acting pO2 (glucose) controller guarantees sufficient O2 supply till the end of the cultivation with a biomass concentration of 110 g 1(-1) (dry mass). The proposed HCDC suppresses generation of inhibitory by-products and the high yield coefficients indicate the economy of the process.

Influence of phospholipid composition on excretion of beta-lactamase from a stringent/relaxed Escherichia coli K 12 strain pair.

In comparison to stringent (relA+) controlled Escherichia coli cells, relaxed (relA) controlled E. coli cells excreted more recombinant beta-lactamase into the culture medium. The analysis of the composition of phospholipid fractions of the cells yielded increased levels of phosphatidylserine in relaxed controlled cells. We added various phospholipid vesicles to growing cells in order to influence the excretion rate via their incorporation in the membranes. The addition of vesicles to phosphatidic acid, phosphatidylglycerol and phosphatidylserine reduced the excretion of beta-lactamase whereas vesicles of phosphatidylethanolamine and phosphatidylcholine decreased or increased the excretion of beta-lactamase in dependence on the individual fatty acid residues of the added phospholipids. The lower the degree of saturation of the added phospholipids the more permeable was the cell envelope for beta-lactamase.

Release of miniantibodies from E. coli cells into the supernatant at low and high cell densities.

By chemical permeabilization of E. coli cells with detergents and membrane active peptides considerable amounts of the periplasmic anti-PC-miniantibodies can be released. Releases of active miniantibodies of 31%, respectively of 38%, were obtained by exposing low cell density suspensions to nonionic detergents like Triton X-100 and tetraethyleneglycolmonodecylether. At high cell densities releasing levels of 40% and 56% were observed for these detergents. The addition of cationic, membrane active peptides (PMBN, polylysine 115, magainin II and melittin) led to release of up to 20% of active miniantibodies at low cell densities. The excretion of miniantibodies at low cell densities was increased up to 35% by phospholipase A2. Interestingly, the membrane associating properties of the anti-PC-miniantibodies influenced the permeability of the outer membrane and the excretion of beta-lactamase.

Co-operative effects of protein engineering and vector optimization on high yield expression of functional bivalent miniantibodies in Escherichia coli.

The volumetric yield of functional phosphocholine-binding miniantibodies could be increased in E. coli fermentations by the combination of the following approaches: Firstly, miniantibody mutants with amino acid exchanges in the VH chain leading to improved folding were expressed. Secondly, the expression vector was stabilized by an efficient suicide system to prevent plasmid loss. Thirdly, the cells were grown to high cell densities in a stirred tank reactor.

Expression of soluble, recombinant alphavbeta3 integrin fragments in Escherichia coli.

No prokaryotic expression of integrin alphavbeta3 has been reported so far. We report here the expression of C-terminally truncated alphavbeta3 receptors in E. coli considering the known features required for dimerization and ligand binding. The expressed protein was insoluble despite of the addition of 'solubilizers' to the culture medium. Osmotic stress conditions combined with added exogenous solutes resulted in a small part of soluble receptor. The alphavbeta3 variants were purified from inclusion bodies or from soluble cytoplasmic maltose binding protein fusions. Heterodimerization of the subunits was proved by immunoprecipitation assays. Receptor-ligand binding was found to depend on the concentration. A competition assay with RGD peptides referred to unspecific receptor-ligand interaction. The latter fact was consistent with the finding that soluble receptors did not bind on RGD peptide-coupled sepharose (GRGDSPK sepharose).

Diagnostic decision support systems.

Diagnostic decision support systems are ready for prime time. We used them in a general medical clinic and found that they could suggest new diagnostic possibilities, focus thinking about clinical problems, and serve as a tool for recertification preparation. In addition, we have found diagnostic decision support systems useful for the novice clinician (fourth-year medical students and interns). These tools serve as a reminder system for learners, suggesting questions to ask the patient, physical exam components to complete, and tests to order. The novice clinician may also use these systems in preparing case presentations. The systems reviewed vary in the ways we described earlier, and there is no one "best buy." Which program is right for you depends on how much detail you want, whether you prefer a CD or internet format, and in what setting you practice. Demos are available from most vendors: Try them out, make a choice, and get on with the business of enhanced diagnostic decision making.

Improved bivalent miniantibodies, with identical avidity as whole antibodies, produced by high cell density fermentation of Escherichia coli.

The combination of single-chain Fv-fragments (scFv) with a C-terminal, flexible linking region followed by a designed or natural dimerization domain provides a versatile system for targeted association of functional fragments in the periplasmic space of Escherichia coli. For homodimerization in vivo, two scFv fragments with a C-terminal hinge followed by a helix-turn-helix motif form "miniantibodies" with significantly higher avidity than in the case of leucine zipper containing constructs. The favorable design probably results in an antiparallel four-helix bundle and brings the homodimer to the same avidity as the whole IgA antibody, from which the binding site was taken. The molecular weight of the bivalent miniantibody is almost the same as that of a monovalent Fab fragment. We report here a high-cell density fermentation of E. coli producing these miniantibodies and a work-up procedure suitable for large scale production. Without any need of subsequent chemical coupling in vitro, approximately 200 mg/l of functional dimeric miniantibodies can be directly obtained from the E. coli culture.

A radioimmunoassay for (p)ppGpp and its application to Streptomyces hygroscopicus.

Aradioimmunoassay for (p)ppGpp was set up and applied to Streptomyces hygroscopicus. The (p)ppGpp specific antibodies obtained from rabbits immunized with ppGpp-human serum albumin could not distinguish between guanosine-5'-triphosphate-3'-diphosphate (pppGpp) and guanosine-5'-diphosphate-3'-diphosphate (ppGpp) whereas the cross reacitivity against structurally related nucleotides was negligible. Therefore, the antibodies were used for determination of (p)ppGpp, i.e. the sum of pppGpp and ppGpp. The pretreatment of biological samples for quantification of (p)ppGpp included formic acid extraction of cell pool followed by step gradient elution on DEAE-Sephadex A25 (Cl-) column for separation of (p)ppGpp from nucleotides with three or less phosphate groups. The very sensitive radioimmunoassay allowed determination of very low amounts: 1--50 pmoles (p)ppGpp per assay tube. The application of the RIA to S. hygroscopicus revealed a basal level in exponentially growing mycelium of about 2 pmoles (p)ppGpp per mg dry weight. The equilibrated pool size after induction of stringent response with serine hydroxamate was found to be about 30 pmoles (p)ppGpp per mg dry weight.

Isolation and characterization of prototrophic relaxed mutants of Klebsiella pneumoniae.

A new selection procedure has been developed for isolating prototrophic relaxed mutants of Klebsiella pneumoniae. Two mutants were isolated. One of them showed a fully relaxed phenotype, while the other one behaved in a semi-relaxed way. The wild-type strain, as well as the rel mutants exerted similar patterns to their E. coli counterparts in RNA, protein, ppGpp and pppGpp accumulation during amino starvation, carbon source shift-down and nitrogen starvation. Both mutants became stringent after introducing an F'-factor carrying the relA+ allele from Escherichia coli. The relaxed phenotype could be recovered by curing the F'-factor. Some of the pleiotropic consequences of rel mutations found in E. coli are present in the Klebsiella mutants also while some of them are absent. The mutants are defective in dinitrogen fixation after the exhaustion of limiting ammonium from the culture medium. However, their merodiploid derivatives, carrying the E. coli relA+ allele, showed the wild-type level of nitrogenase activity under the same conditions.

High-cell-density cultivation of microorganisms.

High-cell-density cultivation (HCDC) is required to improve microbial biomass and product formation substantially. An overview of HCDC is given for microorganisms including bacteria, archae and eukarya (yeasts). Problems encountered by HCDC and their possible solutions are discussed. Improvements of strains, different types of bioreactors and cultivation strategies for successful HCDC are described. Stirred-tank reactors with and without cell retention, a dialysis-membrane reactor, a gas-lift reactor and a membrane cyclone reactor used for HCDC are outlined. Recently modified traditional feeding strategies and new ones are included, in particular those for unlimited growth to very dense cultures. Emphasis is placed on robust fermentation control because of the growing industrial interest in this field. Therefore, developments in the application of multivariate statistical control, artificial neural networks, fuzzy control and knowledge-based supervision (expert systems) are summarized. Recent advances using Escherichia coli--the pioneer organism for HCDC--are outlined.

Dependence of macromolecular composition and morphology of Streptomyces hygroscopicus on specific growth rate.

The dependence of macromolecular composition and morphology of Streptomyces hygroscopicus on specific growth rate micron was investigated. The percentage of DNA on dry weight (%DNA) is constant, % protein is also nearly independent of micron whereas %RNA rises considerably with increasing micron, regarding mycelia grown in glucose-limited and ammonium-limited continuous cultures as well as in discontinuous cultures with various carbon sources. It is probable that the overall synthesis of DNA, RNA and protein is regulated in the mycelium-forming bacterium S. hygroscopicus by the same mechanisms found in unicellular bacteria like Escherichia coli because of the qualitatively similar dependence of %DNA, %RNA and %protein on micron. But differences exist in quantitative regard whereby %DNA, %RNA and %protein of S. hygroscopicus are much smaller at low micron and, with increasing micron, approach those of unicellular bacteria. The hypothesis about the increase of the hyphal regions showing high synthesis activity in S. hygroscopicus mycelia grown in glucose-limited continuous cultures with increasing micron -- derived from comparison of macromolecular composition of S. hygroscopicus and unicellular bacteria -- was confirmed autoradiographically with respect to protein synthesis. The increase of the part of mycelial regions showing high cytoplasmic activity results in an increase of mean hyphal diameter, of mean relative apical growth rate alpha and/or mean relative branching rate beta. Beta depends sigmoidally and alpha inverses sigmoidally on micron. Therefore, the morphology of the mycelium determined by alpha and beta also depends on micron. The hyphal growth unit L/N, the distance from apex to first branch Lp and the mean distance between neighbouring branches Ln decline with increasing micron and reach a minimum at micron = 0.32 (1/h). A further rise of micron is accompanied with an increase of L/N, Lp and Ln. This means that mycelia growing slowly or very quickly have a loose form whereas quickly growing mycelia are characterized by a more compact form. The complicated dependence of alpha, beta, L/N, Lp and Ln on micron indicates that the morphology is regulated by different mechanisms depending on the specific growth rate.