Metabolic oscillations in an E. coli fermentation.
Recombinant E. coli fermentations were observed to undergo regular, reproducible oscillations in oxygen uptake for several hours during a controlled fermentation process. Culture growth slowed during the period of oscillations, delaying induction of recombinant protein production. The oscillations were similar in 10-L and 1,000-L fermentors and also occurred with different feed control algorithms. Both observations support the hypothesis that the oscillations are metabolic in nature. Analysis of amino acid, ATP, and GTP pools suggests that the oscillations result from aberrant regulation of isoleucine biosynthesis leading to repeated starvation events in which protein synthesis and growth are impaired. Both a nutritional solution, isoleucine feeding, and a genetic solution, repair of an ilvG frameshift mutation in E. coli K-12 strains, were found to eliminate the oscillations, further supporting the proposed mechanism for the behavior. These results illustrate the interesting and complicated physiological behavior which can be displayed in metabolic networks and provide another example of surprising problems that can arise in growing recombinant organisms in fermentors.
Andersen
D C
DC
Cell Culture & Fermentation Research & Development, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA. andersen@gene.com
Swartz
J
J
Ryll
T
T
Lin
N
N
Snedecor
B
B
D000255:Adenosine Triphosphate / Q000032:analysis; D000465:Algorithms; D000596:Amino Acids / Q000032:analysis; D001683:Biological Clocks / Q000235:genetics / Q000502:physiology*; D019149:Bioreactors; D001709:Biotechnology; D002452:Cell Count; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D005285:Fermentation; D016368:Frameshift Mutation; D005798:Genes, Bacterial; D005947:Glucose / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000032:analysis; D007532:Isoleucine / Q000096:biosynthesis*; D007700:Kinetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D013997:Time Factors; D014633:Valine / Q000378:metabolism / Q000633:toxicity*
D000596:Amino Acids; D011994:Recombinant Proteins; D007532:Isoleucine; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D014633:Valine; D005947:Glucose