Characterization and complementation of a cephalosporin-deficient mutant of Streptomyces clavuligerus NRRL 3585

Summary We have characterized a mutant of Streptomyces clavuligerus NRRL 3585 which is almost completely blocked in cephalosporin biosynthesis and exhibits depressed activities of both the delta(l-alpha-aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetase and cyclase enzymes of the cephalosporin pathway. A wild-type DNA region was cloned which partially restores antibiotic production, ACV synthetase and cyclase activities to this mutant. The recombinant plasmid exhibits a variable copy number in different transformants. Hybridization experiments indicate that sequences homologous to the cloned region are present in various -lactam-producing Streptomyces spp. but absent in species which are not known to produce this class of antibiotics. Furthermore, the chromosomal copy of the cloned region lies in close proximity to a gene coding for the isopenicilin N synthase gene of the cephalosphorin pathway.Offprint requests to: J. Piret     

Phage-mediated cloning of bldA, a region involved in Streptomyces coelicolor morphological development, and its analysis by genetic complementation.

Streptomyces coelicolor bald (bld) mutants form colonies of vegetative substrate mycelium, but do not develop aerial hyphae or spore chains. The bldA strains form none of the four antibiotics known to be produced by the parent strain. With a vector derived from the temperate bacteriophage phi C31, a 5.6-kilobase fragment of wildtype DNA was cloned which restored sporulation to five independent bldA mutants when lysogenized with the recombinant phage. The cloned gene(s) was dominant over the mutant alleles. Phage integration by recombination of the cloned bldA+ DNA with the bldA region of each mutant produced mainly sporulating colonies, presumably heterozygous bldA+/bldA partial diploids for the insert DNA. However, a minority of these primary transductants were bald and were apparently homozygous bldA/bldA mutant partial diploids, formed by some homogenetization process. The phages released from the bald lysogens carried bldA mutations and were used to show that bldA+ sequences had been cloned and that fine mapping of the region could be performed.     

Optical analysis of perfusion bioreactor cell concentration in an acoustic separator

Automated monitoring of cell concentration in perfusion bioprocesses facilitates the maintenance of constant cell specific perfusion rates. However, most on-line measuring devices are relatively complex and foul as the culture progresses. A simple external optical sensor was developed using the transparent glass walls of acoustic separators for automated optical analysis of their contents. For each measurement, the separator was filled by an automated pumping system with triplicate representative bioreactor samples that were optically analyzed and the device returned to perfusion operation within approximately 1 or 2 min. Chinese hamster ovary cell concentrations, ranging from 5 x 10(5) to 2 x 10(7) cells/mL, were highly correlated (R(2) = 0.99) with the 90 degrees scattered light response. Since the device was operated externally, it did not complicate bioreactor sterilization or cleaning. Viability was not optically analyzed, but this information was not required between manual samples of a properly operated perfusion process. Using single-point recalibration based on routine off-line samples, this external optical system remained effective during a 4-month perfusion run, thus providing a non-invasive and easily maintained on-line cell concentration monitoring system to improve the control of perfusion bioreactors.     

Differential stability of proteolytically active and inactive recombinant metalloproteinase in Chinese hamster ovary cells

Stability of heterologous protein expression during production is critical for regulatory approval of vaccine and therapeutic products. Leishmania GP63, a zinc metalloproteinase that is a potential vaccine candidate, has been expressed on the surface of Chinese hamster ovary (CHO) cells. Flow cytometry was used to follow the stability of GP63 expression. Expression of proteolytically active GP63 (GP63WT) was unstable whether or not methotrexate (MTX) selection was maintained. In contrast, expression of an active site mutant (GP63E265D) was stable under MTX selection. In the absence of selection, the decline in GP63E265D expression was more gradual than the loss of GP63WT expression. Different molecular mechanisms accounted for these losses and resulted in higher growth rate nonproducer populations. A dynamic population model was used to calculate the conversion rates of GP63WT producers to nonproducers. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 594-600, 1997.     

Cellular determinants affecting the rate of cytokine in cultures of human hematopoietic cells

The present study was undertaken to define parameters that may limit the cytokine-mediated expansion of primitive hematopoietic cells in stirred suspension cultures of normal human marrow cells. In a first series of experiments, parallel measurements of the rate and extent of progenitor expansion and cytokine depletion from the medium were made for such cultures in which the cells were exposed to different cytokine concentrations. Supplementation of the medium with 2 ng/mL of interleukin-3 (IL-3), IL-6 and IL-11 plus 10 ng/mL of Flt-3 ligand (FL) and Steel factor (SF) allowed a 45-fold expansion of directly clonogenic cell (CFC) numbers within 2 weeks along with a 2.5-fold expansion of their precursors, detectable as longterm culture-initiating cells (LTC-IC). The addition of 5-fold higher levels of these cytokines enhanced the 2 week output of both CFC and LTC-IC numbers (to 66-fold and 9-fold above input respectively). However, this was also associated with an increase in the individual average rates of depletion of immunoreactive IL-3, SF and FL. As a result, even biweekly addition of fresh medium supplemented with the highest concentrations of cytokines tested failed to prevent a continuing decline in their levels relative to the input medium levels. A similar dependence of the IL-3 depletion rate on its extracellular concentration was demonstrable in suspension cultures of Mo7e cells, an IL-3-dependent human leukemic cell line.Additional experiments with various highly purified marrow cell fractions showed that the rate of cytokine depletion varied according to the type of responding cell as well as the specific cytokine. CD34(+)CD38(-) cells exhibited the greatest average cell-specific cytokine depletion rates (35-fold higher than unseparated bone marrow cells). These findings establish new principles that will be important for the optimization of hematopoietic cell bioreactors. In addition, they suggest that cytokine depletion may provide a novel feedback control mechanism in vivo which would contribute to the control of primitive hematopoietic cell proliferation and differentiation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 58-66, 1997.     

Certain chromosomal regions in Streptomyces glaucescens tend to carry amplifications and deletions

Summary Streptomycetes are subject to a high degree of genetic instability. One manifestation of this phenomenon is the occurrence of tandemly reiterated DNA stretches within the chromosome. We describe the analysis of ten reiterated sequences observed in various ethidium bromide-treated streptomycin-sensitive and melanin-negative mutant strains of Streptomyces glaucescens. The repeated DNA units were 2.9 to 35 kb in lenght. No two sequences were identical. The amplified sequences occupied up to 45% of the total genomic DNA. Structural analysis of the cloned repeated DNA stretches by means of restriction enzymes and by cross hybridization revealed the presence of two chromosomal areas rich in DNA reiterations. In some cases reiterated regions were accompanied by nearby rearrangements.     

Novel guanidine-based inhibitors of inosine monophosphate dehydrogenase

A series of novel guanidine-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for this new series of inhibitors is given.     

Metabolic flux analysis of CHO cells in perfusion culture by metabolite balancing and 2D [13C, 1H] COSY NMR spectroscopy

The physiological state of CHO cells in perfusion culture was quantified by determining fluxes through the bioreaction network using ¹³C glucose and 2D-NMR spectroscopy. CHO cells were cultivated in a 2.5 L perfusion bioreactor with glucose and glutamine as the primary carbon and energy sources. The reactor was inoculated at a cell density of 8×10⁶ cells/mL and operated at ～10×10⁶ cells/mL using unlabeled glucose for the first 13 days. The second phase lasted 12 days and the medium consisted of 10% [U-¹³C]glucose, 40% labeled [1-¹³C]glucose with the balance unlabeled. After the culture attained isotopic steady state, biomass samples from the last 3 days of cultivation were considered representative and used for flux estimation. They were hydrolyzed and analyzed by 2D [¹³C, ¹H] COSY measurements using the heteronuclear single quantum correlation sequence with gradients for artifacts suppression. Metabolic fluxes were determined using the 13C-Flux software package by minimizing the residuals between the experimental and the simulated NMR data. Normalized residuals exhibited a Gaussian distribution indicating good model fit to experimental data. The glucose consumption rate was 5-fold higher than that of glutamine with 41% of glucose channeled through the pentose phosphate pathway. The fluxes at the pyruvate branch point were almost equally distributed between lactate and the TCA cycle (55% and 45%, respectively). The anaplerotic conversion of pyruvate to oxaloacetate by pyruvate carboxylase accounted for 10% of the pyruvate flux with the remaining 90% entering the TCA cycle through acetyl-CoA. The conversion of malate to pyruvate catalyzed by the malic enzyme was 70% higher than that for the anaplerotic reaction catalyzed by pyruvate carboxylase. Most amino acid catabolic and biosynthetic fluxes were significantly lower than the glycolytic and TCA cycle fluxes. Metabolic flux data from NMR analysis validated a simplified model where metabolite balancing was used for flux estimation. In this reduced flux space, estimates from these two methods were in good agreement. This simplified model can routinely be used in bioprocess development experiments to estimate metabolic fluxes with much reduced analytical investment. The high resolution flux information from 2D-NMR spectroscopy coupled with the capability to validate a simplified metabolite balancing based model for routine use make ¹³C-isotopomer analysis an attractive bioprocess development tool for mammalian cell cultures.     

Meta-analysis of differentiating mouse embryonic stem cell gene expression kinetics reveals early change of a small gene set

Stem cell differentiation involves critical changes in gene expression. Identification of these should provide endpoints useful for optimizing stem cell propagation as well as potential clues about mechanisms governing stem cell maintenance. Here we describe the results of a new meta-analysis methodology applied to multiple gene expression datasets from three mouse embryonic stem cell (ESC) lines obtained at specific time points during the course of their differentiation into various lineages. We developed methods to identify genes with expression changes that correlated with the altered frequency of functionally defined, undifferentiated ESC in culture. In each dataset, we computed a novel statistical confidence measure for every gene which captured the certainty that a particular gene exhibited an expression pattern of interest within that dataset. This permitted a joint analysis of the datasets, despite the different experimental designs. Using a ranking scheme that favored genes exhibiting patterns of interest, we focused on the top 88 genes whose expression was consistently changed when ESC were induced to differentiate. Seven of these (103728_at, 8430410A17Rik, Klf2, Nr0b1, Sox2, Tcl1, and Zfp42) showed a rapid decrease in expression concurrent with a decrease in frequency of undifferentiated cells and remained predictive when evaluated in additional maintenance and differentiating protocols. Through a novel meta-analysis, this study identifies a small set of genes whose expression is useful for identifying changes in stem cell frequencies in cultures of mouse ESC. The methods and findings have broader applicability to understanding the regulation of self-renewal of other stem cell types.