Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1982 Feb;43(2):289–297. doi: 10.1128/aem.43.2.289-297.1982

New approach to tryptophan production by Escherichia coli: genetic manipulation of composite plasmids in vitro.

S Aiba, H Tsunekawa, T Imanaka
PMCID: PMC241821  PMID: 7036897

Abstract

For the purpose of studying the production of L-tryptophan by Escherichia coli, the deletion mutants of the trp operon (trpAE1) were transformed with mutant plasmids carrying the trp operon whose anthranilate synthase and phosphoribosyl anthranilate transferase (anthranilate aggregate), respectively, had been desensitized to tryptophan inhibition. In addition to release of the anthranilate aggregate from the feedback inhibition required for plasmids such as pSC101 trp.I15, the properties of trp repression (trpR) and tryptophanase deficiency (tnaA) were both indispensable for host strains such as strain Tna (trpAE1 trpR tnaA). The gene dosage effects on tryptophan synthase activities and on production of tryptophan were assessed. A moderate plasmid copy number, approximately five per chromosome, was optimal for tryptophan production. Similarly, an appropriate release of the anthranilate aggregate from feedback inhibition was also a necessary step to ward off the metabolic anomaly. If the mutant plasmid pSC101 trp-I15 was further mutagenized (pSC101 trp.I15.14) and then transferred to Tna cells, an effective enhancement of tryptophan production was achieved. Although further improvement of the host-plasmid system is needed before commercial production of tryptophan can be realized by this means, a promising step toward this goal has been established.

Full text

PDF
289

Selected References

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

  1. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cabello F., Timmis K., Cohen S. N. Replication control in a composite plasmid constructed by in vitro linkage of two distinct replicons. Nature. 1976 Jan 29;259(5541):285–290. doi: 10.1038/259285a0. [DOI] [PubMed] [Google Scholar]
  3. DICKMAN S. R., CROCKETT A. L. Reactions of xanthydrol. IV. Determination of tryptophan in blood plasma and in proteins. J Biol Chem. 1956 Jun;220(2):957–965. [PubMed] [Google Scholar]
  4. Falkow S., Guerry P., Hedges R. W., Datta N. Polynucleotide sequence relationships among plasmids of the I compatibility complex. J Gen Microbiol. 1974 Nov;85(1):65–76. doi: 10.1099/00221287-85-1-65. [DOI] [PubMed] [Google Scholar]
  5. Gibson F., Pittard J. Pathways of biosynthesis of aromatic amino acids and vitamins and their control in microorganisms. Bacteriol Rev. 1968 Dec;32(4 Pt 2):465–492. [PMC free article] [PubMed] [Google Scholar]
  6. Guerry P., van Embden J., Falkow S. Molecular nature of two nonconjugative plasmids carrying drug resistance genes. J Bacteriol. 1974 Feb;117(2):619–630. doi: 10.1128/jb.117.2.619-630.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hickson F. T., Roth T. F., Helinski D. R. Circular DNA forms of a bacterial sex factor. Proc Natl Acad Sci U S A. 1967 Oct;58(4):1731–1738. doi: 10.1073/pnas.58.4.1731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hiraga S. Operator mutants of the tryptophan operon in Escherichia coli. J Mol Biol. 1969 Jan 14;39(1):159–179. doi: 10.1016/0022-2836(69)90340-4. [DOI] [PubMed] [Google Scholar]
  9. Imanaka T., Tanaka T., Tsunekawa H., Aiba S. Cloning of the genes for penicillinase, penP and penI, of Bacillus licheniformis in some vector plasmids and their expression in Escherichia coli, Bacillus subtilis, and Bacillus licheniformis. J Bacteriol. 1981 Sep;147(3):776–786. doi: 10.1128/jb.147.3.776-786.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Imanaka T., Tsunekawa H., Aiba S. Phenotypic stability of trp operon recombinant plasmids in Escherichia coli. J Gen Microbiol. 1980 May;118(1):253–261. doi: 10.1099/00221287-118-1-253. [DOI] [PubMed] [Google Scholar]
  11. Ito J., Yanofsky C. Anthranilate synthetase, an enzyme specified by the tryptophan operon of Escherichia coli: Comparative studies on the complex and the subunits. J Bacteriol. 1969 Feb;97(2):734–742. doi: 10.1128/jb.97.2.734-742.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. Nagahari K., Tanaka T., Hishinuma F., Kuroda M., Sakaguchi K. Control of tryptophan synthetase amplified by varying the numbers of composite plasmids in Escherichia coli cells. Gene. 1977 Mar;1(2):141–152. doi: 10.1016/0378-1119(77)90025-7. [DOI] [PubMed] [Google Scholar]
  14. Pabst M. J., Kuhn J. C., Somerville R. L. Feedback regulation in the anthranilate aggregate from wild type and mutant strains of Escherichia coli. J Biol Chem. 1973 Feb 10;248(3):901–914. [PubMed] [Google Scholar]
  15. Rood J. I., Sneddon M. K., Morrison J. F. Instability in tyrR strains of plasmids carrying the tyrosine operon: isolation and characterization of plasmid derivatives with insertions or deletions. J Bacteriol. 1980 Nov;144(2):552–559. doi: 10.1128/jb.144.2.552-559.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. SMITH O. H., YANOFSKY C. 1-(o-Carboxyphenylamino)-1-deoxyribulose 5-phosphate, a new intermediate in the biosynthesis of tryptophan. J Biol Chem. 1960 Jul;235:2051–2057. [PubMed] [Google Scholar]
  17. Tanaka T., Weisblum B. Construction of a colicin E1-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid. J Bacteriol. 1975 Jan;121(1):354–362. doi: 10.1128/jb.121.1.354-362.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Tribe D. E., Pittard J. Hyperproduction of tryptophan by Escherichia coli: genetic manipulation of the pathways leading to tryptophan formation. Appl Environ Microbiol. 1979 Aug;38(2):181–190. doi: 10.1128/aem.38.2.181-190.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. WASHKO M. E., RICE E. W. Determination of glucose by an improved enzymatic procedure. Clin Chem. 1961 Oct;7:542–545. [PubMed] [Google Scholar]
  20. Widera G., Gautier F., Lindenmaier W., Collins J. The expression of tetracycline resistance after insertion of foreign DNA fragments between the EcoRI and HindIII sites of the plasmid cloning vector pBR 322. Mol Gen Genet. 1978 Jul 25;163(3):301–305. doi: 10.1007/BF00271959. [DOI] [PubMed] [Google Scholar]

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

RESOURCES