Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1990 May;56(5):1296–1302. doi: 10.1128/aem.56.5.1296-1302.1990

Improved expression of human interleukin-2 in high-cell-density fermentor cultures of Escherichia coli K-12 by a phosphotransacetylase mutant.

K A Bauer 1, A Ben-Bassat 1, M Dawson 1, V T de la Puente 1, J O Neway 1
PMCID: PMC184398  PMID: 2187412

Abstract

A fluoroacetate-resistant mutant of Escherichia coli K-12 (MM-294) accumulated less acetate in the medium during growth to high cell density in fermentor cultures and was shown to be defective in its phosphotransacetylase activity. The mutant had an improved ability to continue growing during induction of interleukin-2 (IL-2) synthesis, and in fermentor cultures it gave a higher level of specific IL-2 accumulation than its parent during expression under control of the temperature-sensitive pL promoter. In flask cultures at lower cell density, the mutant again produced less acetate than the parent, although both showed a much lower level of acetate accumulation than that seen in fermentors at high cell density. Both showed a higher specific expression level of IL-2 in flask cultures, and there was a greater difference between the mutant and its parent in the final extent of specific IL-2 accumulation in fermentor cultures compared with flask cultures. Thus, the concentration of acetate in the medium, which was much higher in fermentor cultures (greater than or equal to 300 mM after 5 h of induction) than in flask cultures (less than or equal to mM) of the parent organism, was a significant factor in limiting expression of the heterologous protein product, IL-2. The acetate kinase-phosphotransacetylase pathway was therefore a major source of acetate formation in these cultures. Blocking this pathway improved accumulation of IL-2 and did not slow growth.

Full text

PDF
1296

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bauer S., Shiloach J. Maximal exponential growth rate and yield of E. coli obtainable in a bench-scale fermentor. Biotechnol Bioeng. 1974 Jul;16(7):933–941. doi: 10.1002/bit.260160707. [DOI] [PubMed] [Google Scholar]
  2. Brown T. D., Jones-Mortimer M. C., Kornberg H. L. The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli. J Gen Microbiol. 1977 Oct;102(2):327–336. doi: 10.1099/00221287-102-2-327. [DOI] [PubMed] [Google Scholar]
  3. Chang Y. Y., Cronan J. E., Jr Genetic and biochemical analyses of Escherichia coli strains having a mutation in the structural gene (poxB) for pyruvate oxidase. J Bacteriol. 1983 May;154(2):756–762. doi: 10.1128/jb.154.2.756-762.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chang Y. Y., Cronan J. E., Jr Mapping nonselectable genes of Escherichia coli by using transposon Tn10: location of a gene affecting pyruvate oxidase. J Bacteriol. 1982 Sep;151(3):1279–1289. doi: 10.1128/jb.151.3.1279-1289.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fox D. K., Roseman S. Isolation and characterization of homogeneous acetate kinase from Salmonella typhimurium and Escherichia coli. J Biol Chem. 1986 Oct 15;261(29):13487–13497. [PubMed] [Google Scholar]
  6. Holms W. H. 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. Curr Top Cell Regul. 1986;28:69–105. doi: 10.1016/b978-0-12-152828-7.50004-4. [DOI] [PubMed] [Google Scholar]
  7. LeVine S. M., Ardeshir F., Ames G. F. Isolation and Characterization of acetate kinase and phosphotransacetylase mutants of Escherichia coli and Salmonella typhimurium. J Bacteriol. 1980 Aug;143(2):1081–1085. doi: 10.1128/jb.143.2.1081-1085.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. MacDonald H. L., Neway J. O. Effects of medium quality on the expression of human interleukin-2 at high cell density in fermentor cultures of Escherichia coli K-12. Appl Environ Microbiol. 1990 Mar;56(3):640–645. doi: 10.1128/aem.56.3.640-645.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Meselson M., Yuan R. DNA restriction enzyme from E. coli. Nature. 1968 Mar 23;217(5134):1110–1114. doi: 10.1038/2171110a0. [DOI] [PubMed] [Google Scholar]
  10. Suzuki T. Phosphotransacetylase of Escherichia coli B, activation by pyruvate and inhibition by NADH and certain nucleotides. Biochim Biophys Acta. 1969;191(3):559–569. doi: 10.1016/0005-2744(69)90349-0. [DOI] [PubMed] [Google Scholar]
  11. Wang A. M., Creasey A. A., Ladner M. B., Lin L. S., Strickler J., Van Arsdell J. N., Yamamoto R., Mark D. F. Molecular cloning of the complementary DNA for human tumor necrosis factor. Science. 1985 Apr 12;228(4696):149–154. doi: 10.1126/science.3856324. [DOI] [PubMed] [Google Scholar]
  12. Wong E. M., Muesing M. A., Polisky B. Temperature-sensitive copy number mutants of CoIE1 are located in an untranslated region of the plasmid genome. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3570–3574. doi: 10.1073/pnas.79.11.3570. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES