Transcriptional and metabolic response of recombinant Escherichia coli to spatial dissolved oxygen tension gradients simulated in a scale-down system.

Escherichia coli, expressing recombinant green fluorescent protein (GFP), was subjected to dissolved oxygen tension (DOT) oscillations in a two-compartment system for simulating gradients that can occur in large-scale bioreactors. Cells were continuously circulated between the anaerobic (0% DOT) and aerobic (10% DOT) vessels of the scale-down system to mimic an overall circulation time of 50 s, and a mean residence time in the anaerobic and aerobic compartments of 33 and 17 s, respectively. Transcription levels of mixed acid fermentation genes (ldhA, poxB, frdD, ackA, adhE, pflD, and fdhF), measured by quantitative RT-PCR, increased between 1.5- to over 6-fold under oscillatory DOT compared to aerobic cultures (constant 10% DOT). In addition, the transcription level of fumB increased whereas it decreased for sucA and sucB, suggesting that the tricarboxylic acid cycle was functioning as two open branches. Gene transcription levels revealed that cytrochrome bd, which has higher affinity to oxygen but lower energy efficiency, was preferred over cytochrome bO3 in oscillatory DOT cultures. Post-transcriptional processing limited heterologous protein production in the scale-down system, as inferred from similar gfp transcription but 19% lower GFP concentration compared to aerobic cultures. Simulated DOT gradients also affected the transcription of genes of the glyoxylate shunt (aceA), of global regulators of aerobic and anaerobic metabolism (fnr, arcA, and arcB), and other relevant genes (luxS, sodA, fumA, and sdhB). Transcriptional changes explained the observed alterations in overall stoichiometric and kinetic parameters, and production of ethanol and organic acids. Differences in transcription levels between aerobic and anaerobic compartments were also observed, indicating that E. coli can respond very fast to intermittent DOT conditions. The transcriptional responses of E. coli to DOT gradients reported here are useful for establishing rational scale-up criteria and strain design strategies for improved culture performance at large scales.

[1]  C. McFarlane,et al.  Scale-down model to simulate spatial pH variations in large-scale bioreactors. , 2001, Biotechnology and bioengineering.

[2]  D. Georgellis,et al.  Retarded RNA turnover in Escherichia coli: a means of maintaining gene expression during anaerobiosis , 1993, Molecular microbiology.

[3]  Mark R. Marten,et al.  Using Computational Fluid Dynamics Software to Estimate Circulation Time Distributions in Bioreactors , 2003, Biotechnology progress.

[4]  J. Bourne,et al.  Fermenter Scale-Up Using an Oxygen-Sensitive Culture , 1988 .

[5]  A Lübbert,et al.  Bioreactor performance: a more scientific approach for practice. , 2001, Journal of biotechnology.

[6]  J. Cronan,et al.  Tricarboxylic Acid Cycle and Glyoxylate Bypass. , 2005, EcoSal Plus.

[7]  G. Bennett,et al.  Redistribution of metabolic fluxes in Escherichia coli with fermentative lactate dehydrogenase overexpression and deletion. , 1999, Metabolic engineering.

[8]  Sidney R. Kushner,et al.  mRNA Decay in Escherichia coli Comes of Age , 2002, Journal of bacteriology.

[9]  S. Kjelleberg,et al.  The role of RNA stability during bacterial stress responses and starvation. , 2000, Environmental microbiology.

[10]  N. W. F. Kossen,et al.  Regime analysis and scale-down: Tools to investigate the performance of bioreactors , 1987 .

[11]  K. Hellingwerf,et al.  Effects of Limited Aeration and of the ArcAB System on Intermediary Pyruvate Catabolism in Escherichia coli , 2000, Journal of bacteriology.

[12]  G. W. Hatfield,et al.  Global gene expression profiling in Escherichia coli K12. The effects of integration host factor. , 2000, The Journal of biological chemistry.

[13]  H. Bremer.  Modulation of Chemical Composition and Other Parameters of the Cell by Growth Rate , 1999 .

[14]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[15]  E. Galindo,et al.  Effect of Oscillating Dissolved Oxygen Tension on the Production of Alginate by Azotobactervinelandii , 2001, Biotechnology progress.

[16]  P. Neubauer,et al.  Influence of controlled glucose oscillations on a fed-batch process of recombinant Escherichia coli. , 2000, Journal of biotechnology.

[17]  D. Clark,et al.  The fermentation pathways of Escherichia coli. , 1989, FEMS microbiology reviews.

[18]  M. Reuss,et al.  Application of Computational Fluiddynamics (CFD) to Modeling Stirred Tank Bioreactors , 2000 .

[19]  N.M.G. Oosterhuis.  Scale-up of bioreactors: A scale-down approach , 1984 .

[20]  S. Enfors,et al.  Glucose overflow metabolism and mixed-acid fermentation in aerobic large-scale fed-batch processes with Escherichia coli , 1999, Applied Microbiology and Biotechnology.

[21]  Ching-Ping Tseng,et al.  Oxygen- and Growth Rate-Dependent Regulation ofEscherichia coli Fumarase (FumA, FumB, and FumC) Activity , 2001, Journal of bacteriology.

[22]  J. Yates,et al.  IChemE research event , 1997 .

[23]  C. Hewitt,et al.  Physiological responses to mixing in large scale bioreactors. , 2001, Journal of biotechnology.

[24]  M. Hecker,et al.  Monitoring of stress responses , 2004 .

[25]  P Neubauer,et al.  Influence of substrate oscillations on acetate formation and growth yield in Escherichia coli glucose limited fed‐batch cultivations , 1995, Biotechnology and bioengineering.

[26]  N. Oosterhuis,et al.  Dissolved oxygen concentration profiles in a production-scale bioreactor. , 1984, Biotechnology and bioengineering.

[27]  F. Neidhart.  Escherichia coli and Salmonella. , 1996 .

[28]  K. Schügerl.  Comparison of different bioreactor performances , 1993 .

[29]  Enrique Galindo,et al.  Effects of dissolved oxygen tension on the production of recombinant penicillin acylase in Escherichia coli , 2003 .

[30]  Francisco Bolívar,et al.  Culture of Escherichia coli under dissolved oxygen gradients simulated in a two-compartment scale-down system: metabolic response and production of recombinant protein. , 2005, Biotechnology and bioengineering.

[31]  Enrique Galindo,et al.  Production of β-galactosidase by Kluyveromyces marxianus under oscillating dissolved oxygen tension , 2005 .

[32]  C. Hewitt,et al.  Studies related to the scale-up of high-cell-density E. coli fed-batch fermentations using multiparameter flow cytometry: effect of a changing microenvironment with respect to glucose and dissolved oxygen concentration. , 2000, Biotechnology and bioengineering.

[33]  Matthias Reuss,et al.  Integration of physiology and fluid dynamics. , 2003, Advances in biochemical engineering/biotechnology.

[34]  P. K. Namdev,et al.  Effect of oxygen fluctuations on recombinant Escherichia coli fermentation , 1993, Biotechnology and bioengineering.

[35]  D. Touati,et al.  Transcriptional and posttranscriptional regulation of manganese superoxide dismutase biosynthesis in Escherichia coli, studied with operon and protein fusions , 1988, Journal of bacteriology.

[36]  Angélica Meneses-Acosta,et al.  Heterogeneous conditions in dissolved oxygen affect N‐glycosylation but not productivity of a monoclonal antibody in hybridoma cultures , 2004, Biotechnology and bioengineering.

[37]  John D. Storey,et al.  Precision and functional specificity in mRNA decay , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[38]  G. W. Hatfield,et al.  Global Gene Expression Profiling in Escherichia coli K12 , 2003, Journal of Biological Chemistry.

[39]  J J Valdes,et al.  Quorum signaling via AI-2 communicates the "Metabolic Burden" associated with heterologous protein production in Escherichia coli. , 2001, Biotechnology and bioengineering.

[40]  J. Cronan,et al.  Expression of Escherichia coli pyruvate oxidase (PoxB) depends on the sigma factor encoded by the rpoS(katF) gene , 1994, Molecular microbiology.

[41]  Alvin W Nienow,et al.  Further studies related to the scale‐up of high cell density escherichia coli fed‐batch fermentations: , 2003, Biotechnology and bioengineering.

[42]  S. Lee,et al.  Combined transcriptome and proteome analysis of Escherichia coli during high cell density culture. , 2003, Biotechnology and bioengineering.

[43]  J. Cronan,et al.  Two-Carbon Compounds and Fatty Acids as Carbon Sources , 2005, EcoSal Plus.

[44]  R. Carlson,et al.  Fundamental Escherichia coli biochemical pathways for biomass and energy production: creation of overall flux states , 2004, Biotechnology and bioengineering.

[45]  S. Enfors,et al.  Scale down of recombinant protein production: a comparative study of scaling performance , 1999 .

[46]  Julie Varley,et al.  The response of GS-NS0 myeloma cells to single and multiple pH perturbations. , 2002, Biotechnology and bioengineering.

[47]  John O. Konz,et al.  Effects of Oxygen on Recombinant Protein Expression , 1998, Biotechnology progress.

[48]  M. Eiteman,et al.  Physiological response of central metabolism in Escherichia coli to deletion of pyruvate oxidase and introduction of heterologous pyruvate carboxylase. , 2005, Biotechnology and bioengineering.

[49]  S. Enfors,et al.  A scale-down two-compartment reactor with controlled substrate oscillations: Metabolic response of Saccharomyces cerevisiae , 1993 .

[50]  Matthias Reuss,et al.  Structured Modelling of Bioreactors , 1994 .

[51]  Francisco Bolívar,et al.  Adaptation for fast growth on glucose by differential expression of central carbon metabolism and gal regulon genes in an Escherichia coli strain lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system. , 2005, Metabolic engineering.

[52]  L. A. Palomares,et al.  Bioreactor Scale-Down , 2003 .

[53]  Alvin W. Nienow,et al.  Mixing in large-scale vessels stirred with multiple radial or radial and axial up-pumping impellers: modelling and measurements , 2000 .

[54]  Alvin W. Nienow,et al.  Mixing in the Fermentation and Cell Culture Industries , 2004 .

[55]  Alfredo Martínez,et al.  Role of Pyruvate Oxidase in Escherichia coli Strains Lacking the Phosphoenolpyruvate:Carbohydrate Phosphotransferase System , 2005, Journal of Molecular Microbiology and Biotechnology.

[56]  R. Gunsalus,et al.  Effect of microaerophilic cell growth conditions on expression of the aerobic (cyoABCDE and cydAB) and anaerobic (narGHJI, frdABCD, and dmsABC) respiratory pathway genes in Escherichia coli , 1996, Journal of bacteriology.

[57]  M. Hecker,et al.  Monitoring of genes that respond to process-related stress in large-scale bioprocesses. , 1999, Biotechnology and bioengineering.

[58]  N. Oosterhuis,et al.  Scale‐down and optimization studies of the gluconic acid fermentation by Gluconobacter oxydans , 1985, Biotechnology and bioengineering.

[59]  R. Kolter,et al.  Sensing starvation: a homoserine lactone--dependent signaling pathway in Escherichia coli. , 1994, Science.

[60]  B Sonnleitner,et al.  Biochemical engineering science. , 1997, Journal of biotechnology.

[61]  A. Veide,et al.  Influence of scale-up on the quality of recombinant human growth hormone. , 2000, Biotechnology and Bioengineering.

[62]  E. Collet,et al.  Substrate gradient formation in the large-scale bioreactor lowers cell yield and increases by-product formation , 1998 .