Metabolic adaptation of Escherichia coli during temperature-induced recombinant protein production: 2. Redirection of metabolic fluxes.

The impact of temperature-induced synthesis of human basic fibroblast growth factor (hFGF-2) in high-cell-density cultures of recombinant Escherichia coli was studied by estimating metabolic flux variations. Metabolic flux distributions in E. coli were calculated by means of a stoichiometric network and linear programming. After the temperature upshift, a substantially elevated energy demand for synthesis of hFGF-2 and heat shock proteins resulted in a redirection of metabolic fluxes. Catabolic pathways like the Embden-Meyerhof-Parnas pathway and the tricarboxylic acid (TCA) cycle showed significantly enhanced activities, leading to reduced flux to growth-associated pathways like the pentose phosphate pathway and other anabolic pathways. Upon temperature upshift, an excess of NADPH was produced in the TCA cycle by isocitrate dehydrogenase. The metabolic model predicted the involvement of a transhydrogenase generating additional NADH from NADPH, thereby increasing ATP regeneration in the respiratory chain. The influence of the temperature upshift on the host's metabolism was investigated by means of a control strain harboring the "empty" parental expression vector. The metabolic fluxes after the temperature upshift were redirected similarly to the production strain; the effects, however, were observed to a lesser extent and with different time profiles.

[1]  Jens Nielsen,et al.  A simple and reliable method for the determination of cellular RNA content , 1991 .

[2]  J. Keasling,et al.  Stoichiometric model of Escherichia coli metabolism: incorporation of growth-rate dependent biomass composition and mechanistic energy requirements. , 1997, Biotechnology and bioengineering.

[3]  Gregory Stephanopoulos,et al.  Carbon Flux Distributions at the Glucose 6‐Phosphate Branch Point in Corynebacterium glutamicum during Lysine Overproduction , 1994 .

[4]  S. Stirdivant,et al.  Physiological effects of TGFα‐PE40 expression in recombinant Escherichia coli JM109 , 1992 .

[5]  A. H. Stouthamer,et al.  Utilization of energy for growth and maintenance in continuous and batch cultures of microorganisms. A reevaluation of the method for the determination of ATP production by measuring molar growth yields. , 1973, Biochimica et biophysica acta.

[6]  P. Davies,et al.  Function of energy-dependent transhydrogenase in Escherichia coli. , 1972, Biochemical and biophysical research communications.

[7]  P. Çalık,et al.  Metabolic flux analysis for serine alkaline protease fermentation by Bacillus licheniformis in a defined medium: Effects of the oxygen transfer rate , 1999 .

[8]  J. Guest,et al.  Metabolic engineering in Escherichia coli: lowering the lipoyl domain content of the pyruvate dehydrogenase complex adversely affects the growth rate and yield. , 1995, Microbiology.

[9]  U. Rinas,et al.  Metabolic adaptation of Escherichia coli during temperature-induced recombinant protein production: 1. Readjustment of metabolic enzyme synthesis. , 2002, Biotechnology and bioengineering.

[10]  K. Andersen,et al.  Charges of nicotinamide adenine nucleotides and adenylate energy charge as regulatory parameters of the metabolism in Escherichia coli. , 1977, The Journal of biological chemistry.

[11]  B. Palsson,et al.  Metabolic Capabilities of Escherichia coli II. Optimal Growth Patterns , 1993 .

[12]  A P Ison,et al.  Classification and sensitivity analysis of a proposed primary metabolic reaction network for Streptomyces lividans. , 1999, Metabolic engineering.

[13]  J. Boudrant,et al.  Amino acid utilization during batch and continuous cultures of Escherichia coli on a semi-synthetic medium , 1994 .

[14]  R. Sauer,et al.  Induction of a heat shock-like response by unfolded protein in Escherichia coli: dependence on protein level not protein degradation. , 1989, Genes & development.

[15]  J. Heijnen,et al.  A metabolic network stoichiometry analysis of microbial growth and product formation , 1995, Biotechnology and bioengineering.

[16]  A. Themmen,et al.  Why are two different types of pyridine nucleotide transhydrogenase found in living organisms? , 1983, European journal of biochemistry.

[17]  A. Zeng,et al.  A method to estimate the efficiency of oxidative phosphorylation and biomass yield from atp of a facultative anaerobe in continuous culture. , 1990, Biotechnology and bioengineering.

[18]  G. Stephanopoulos,et al.  Metabolic flux distributions in Corynebacterium glutamicum during growth and lysine overproduction , 2000, Biotechnology and bioengineering.

[19]  W. Bentley,et al.  Enhancement of recombinant protein synthesis and stability via coordinated amino acid addition , 1993, Biotechnology and bioengineering.

[20]  R. Wallace,et al.  Maintenance coefficients and rates of turnover of cell material in Escherichia coli ML308 at different growth temperatures , 1986 .

[21]  R. Hanson,et al.  Isolation and partial characterization of a mutant of Escherichia coli lacking pyridine nucleotide transhydrogenase. , 1978, Archives of biochemistry and biophysics.

[22]  U. Rinas,et al.  Kinetics of Heat‐Shock Response and Inclusion Body Formation During Temperature‐Induced Production of Basic Fibroblast Growth Factor in High‐Cell‐Density Cultures of Recombinant Escherichiacoli , 2000, Biotechnology progress.

[23]  J Tramper,et al.  Metabolite-balancing techniques vs. 13C tracer experiments to determine metabolic fluxes in hybridoma cells. , 1998, Biotechnology and bioengineering.

[24]  C. Kurland,et al.  Gratuitous overexpression of genes in Escherichia coli leads to growth inhibition and ribosome destruction , 1995, Journal of bacteriology.

[25]  A. Bonsignore,et al.  Regulatory Properties of Glucose-6-Phosphate Dehydrogenase , 1972 .

[26]  U. Rinas,et al.  On-line estimation of the metabolic burden resulting from the synthesis of plasmid-encoded and heat-shock proteins by monitoring respiratory energy generation. , 2001, Biotechnology and bioengineering.

[27]  N. Bruce,et al.  The udhA Gene of Escherichia coli Encodes a Soluble Pyridine Nucleotide Transhydrogenase , 1999, Journal of bacteriology.

[28]  W. Holms,et al.  The central metabolic pathways of Escherichia coli: relationship between flux and control at a branch point, efficiency of conversion to biomass, and excretion of acetate. , 1986, Current topics in cellular regulation.

[29]  A. Villaverde,et al.  Polylinker‐Encoded Peptides Can Confer Toxicity to Recombinant Proteins Produced in Escherichia coli , 1996, Biotechnology progress.

[30]  B. Palsson,et al.  Toward Metabolic Phenomics: Analysis of Genomic Data Using Flux Balances , 1999, Biotechnology progress.

[31]  J Tramper,et al.  Metabolic flux analysis of hybridoma cells in different culture media using mass balances , 1996, Biotechnology and bioengineering.

[32]  G. Stephanopoulos,et al.  FLUX DETERMINATION IN CELLULAR BIOREACTION NETWORKS: APPLICATIONS TO LYSINE FERMENTATIONS , 1990 .

[33]  B. Palsson,et al.  Stoichiometric flux balance models quantitatively predict growth and metabolic by-product secretion in wild-type Escherichia coli W3110 , 1994, Applied and environmental microbiology.

[34]  J. Rydström,et al.  Energy-linked nicotinamide nucleotide transhydrogenases. , 1977, Biochimica et biophysica acta.