ABSTRACT:In an effort to develop robust Chinese Hamster Ovary host cell lines, a variety of anti-apoptotic genes were over-expressed, either singly or in combination, followed by screening of transfectants for improved cell growth, extended longevity, reduced caspase 3/7 activity, and enhanced mitochondrial membrane potential (MMP). Two particular cell lines, one containing two anti-apoptotic genes, E1B-19K and Aven (EA167), and another containing three, E1B-19K, Aven, and a mutant of XIAP (EAX197), exhibited a reduction in caspase 3 activity of at least 60% and a 170% enhancement in mitochondrial membrane potential compared to controls when treated with staurosporine. In batch cell growth experiments, the peak viable cell densities and viabilities were higher resulting in a 186% increase in integrated viable cell densities. Analyses of metabolite utilization and formation of waste products indicated that the apoptotic resistant cell lines depleted all the lactate when grown in commercially available CD-CHO medium while significant levels (>1.8 g/L) accumulated in the host cell lines. When the lactate level was replenished daily in the apoptotic resistant cell lines, the cell lines consumed lactate and the culture longevity was extended up to four additional days compared to control cell lines. Furthermore, the anti-apoptosis cell lines also accumulated lower levels of ammonia. The ability of the apoptotic resistant cell lines to consume lactate was exploited by cultivating them in a ''high'' glucose medium containing 15 g/L (60 mM glucose) in which apoptotic resistant cell lines exhibited lower maximum lactate (1.8 g/L) compared to control cell lines which accumulated concentrations of lactate (2.2 g/L) that appeared to be deleterious for growth. The shaker flask titer of a therapeutic antibody product expressed in an apoptotic resistant cell line in ''high'' glucose medium reached 690 mg/ L compared to 390 mg/L for a cell line derived from a control host cell line. These results represent to our knowledge the first example in the literature in which manipulation of the apoptosis pathway has altered the nutrient consumption profile of mammalian cells in culture; findings that underscore the interdependence of the apoptotic cellular machinery and metabolism and provide greater flexibility to mammalian bioreactor process development.

Background: A small "sigma-like" protein, AfsS, pleiotropically regulates antibiotic biosynthesis in Streptomyces coelicolor. Overexpression of afsS in S. coelicolor and certain related species causes antibiotic stimulatory effects in the host organism. Although recent studies have uncovered some of the upstream events activating this gene, the mechanisms through which this signal is relayed downstream leading to the eventual induction of antibiotic pathways remain unclear.

In a continuous culture with cell retention the perfusion rate must be adjusted dynamically to meet the cellular demand. An automated mechanism of adjusting the perfusion rate based on real-time measurement of the metabolic load of the bioreactor is important in achieving a high cell concentration and maintaining high viability. We employed oxygen uptake rate (OUR) measurement as an on-line metabolic indicator of the physiological state of the cells in the bioreactor and adjusted the perfusion rate accordingly. Using an internal hollow fiber microfiltration system for total cell retention, a cell concentration of almost 10(8) cells/mL was achieved. Although some aggregates were formed during the cultivation, the viability remained high as examined with confocal microscopy after fluorescent vital staining. The results demonstrate that on-line OUR measurement facilitates automated dynamic perfusion and allows a high cell concentration to be achieved.

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SummaryThe DNA-binding capability of a key secondary metabolite regulatory element (CcaR) in the Streptomyces clavuligerus cephamycin C pathway was investigated by gel mobility retardation and DNase I footprinting analysis. These results revealed that CcaR specifically binds to the promoter region of the lysine-1-aminotransferase gene (lat). Green fluorescent protein (GFP) was subsequently used as a reporter to analyse in vivo expression of CcaR. The corresponding isogenic strain containing ccaR::gfp in the chromosome produced cephamycin C at levels similar to those of wild-type S. clavuligerus. Confocal laser scanning microscopy revealed that expression of CcaR in liquid culture was temporally dynamic and spatially heterogeneous in S. clavuligerus mycelia. The highly fluorescent seed culture mycelia quickly lost fluorescence upon inoculation into fresh culture medium. The characteristic green colour reappeared in a small portion of mycelia during mid-exponential growth phase. As the culture aged, the population expressing CcaR expanded, and the expression level increased. This was followed by a reduction in the CcaR-expressing population towards the end of the culture period. During peak expression, CcaR was distributed uniformly in mycelia, but became localized distal to the chromosome when the culture entered stationary phase. In solid phase analysis, abundant CcaR expression was evident in the substrate mycelia, but was completely absent in aerial hyphae. These results show regulatory linkage between ccaR and lat, whose expression profile showed a similar spatial decoupling between morphogenesis and antibiotic production. In addition, visualizing CcaR within S. clavuligerus mycelia demonstrates a distinct pattern of localization over the course of physiological differentiation.

The biosynthesis of secondary metabolites is closely linked to primary metabolism via the supply of precursors, cofactors, and cellular energy. The availability of these precursors and cofactors can potentially be rate-limiting for secondary metabolism. A combined experimental and kinetic modeling approach was used to examine the regulation of flux in the cephamycin biosynthetic pathway in Streptomyces clavuligerus. The kinetic parameters of lysine 6-aminotransferase (LAT), the first enzyme leading to cephamycin biosynthesis and one which was previously identified as being a rate-limiting enzyme, were characterized. LAT converts lysine to alpha-aminoadipic acid using alpha-ketoglutarate as a cosubstrate. The K(m) values for lysine and alpha-ketoglutarate were substantially higher than those for their intracellular concentrations, suggesting that lysine and alpha-ketoglutarate may play a key role in regulating the flux of cephamycin biosynthesis. The important role of this precursor/cosubstrate was supported by simulated results using a kinetic model. When the intracellular concentrations and high K(m) values were taken into account, the predicted intermediate concentration was similar to the experimental measurements. The results demonstrate the controlling roles that precursors and cofactors may play in the biosynthesis of secondary metabolites.

Recombinant human kidney cells, 293, were cultivated in serum-free fed-batch cultures for the production of Protein C. By coupling the feeding of concentrated medium to pH control based on established stoichiometric relations, the titer of Protein C increased by more than ten fold as compared to batch culture, even though the total cell concentration increased only by less than two fold. Such a fed-batch culture is a simple system for enhancing the productivity of mammalian cells in culture.

Linkage between structural and regulatory genes implies that a direct correlation should exist between the spatio-temporal distribution of their expression. Green fluorescent protein (GFP) and cyan fluorescent protein (CFP) were used as reporters to analyze simultaneously expression of lysine-epsilon-aminotransferase (LAT) and its corresponding genetic regulator, CcaR. The isogenic strain containing lat::gfp and ccaR::cfp in the chromosome produced cephamycin C at levels similar to wild type Streptomyces clavuligerus. Confocal laser scanning microscopy revealed that expression of both LAT and CcaR in liquid culture was temporally dynamic and spatially heterogeneous in S. clavuligerus mycelia. During the early culture stage only a part of the mycelia began to express LAT and CcaR at low levels. As the culture aged, expression levels and the population of mycelia expressing LAT and CcaR increased and were followed late in the growth cycle by a reduction of the mycelia population expressing LAT and CcaR. The approach provides a precise simultaneous temporal-spatial expression profile and corroborates the regulatory linkage between ccaR and lat in S. clavuligerus.