Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1996 Dec;62(12):4345–4351. doi: 10.1128/aem.62.12.4345-4351.1996

l-Isoleucine Production with Corynebacterium glutamicum: Further Flux Increase and Limitation of Export

S Morbach, H Sahm, L Eggeling
PMCID: PMC1388995  PMID: 16535457

Abstract

The synthesis of l-isoleucine with Corynebacterium glutamicum involves 11 reaction steps, in at least five of which activity or expression is regulated. We used four genes and alleles encoding feedback-resistant enzymes (Fbr) in various combinations to assay flux increase through the sequence. During strain construction, the order of genes overexpressed was important. Only when ilvA(Fbr) was first overexpressed could hom(Fbr) be introduced. This succession apparently prevents the toxic accumulation of biosynthesis intermediates. The best strain constructed (SM13) was characterized by high-level expression of hom(Fbr), thrB, and ilvA(Fbr). With this strain a yield of 0.22 g of l-isoleucine per g of glucose was obtained, with a maximal specific productivity of 0.10 g of l-isoleucine per g (dry weight) per h. In strain SM13, with the high metabolite flux through the reaction sequence, effects on (i) other enzyme levels, (ii) time-dependent variations with process time, and (iii) concentrations of cytosolic intermediates were quantified. Most importantly, the intracellular l-isoleucine concentration is always higher at all process times than the extracellular concentration. The intracellular concentration rises to 110 mM, whereas extracellularly only 60 mM is accumulated. Also the immediate l-isoleucine precursor 2-ketomethyl valerate accumulates in the cell. Therefore, in the high-level l-isoleucine producer SM13, the export of this amino acid is the major limiting reaction step and therefore is a new target of strain design for biotechnological purposes.

Full Text

The Full Text of this article is available as a PDF (236.5 KB).

Selected References

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

  1. Archer J. A., Solow-Cordero D. E., Sinskey A. J. A C-terminal deletion in Corynebacterium glutamicum homoserine dehydrogenase abolishes allosteric inhibition by L-threonine. Gene. 1991 Oct 30;107(1):53–59. doi: 10.1016/0378-1119(91)90296-n. [DOI] [PubMed] [Google Scholar]
  2. Bröer S., Eggeling L., Krämer R. Strains of Corynebacterium glutamicum with Different Lysine Productivities May Have Different Lysine Excretion Systems. Appl Environ Microbiol. 1993 Jan;59(1):316–321. doi: 10.1128/aem.59.1.316-321.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Colón G. E., Jetten M. S., Nguyen T. T., Gubler M. E., Follettie M. T., Sinskey A. J., Stephanopoulos G. Effect of inducible thrB expression on amino acid production in Corynebacterium lactofermentum ATCC 21799. Appl Environ Microbiol. 1995 Jan;61(1):74–78. doi: 10.1128/aem.61.1.74-78.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Colón G. E., Nguyen T. T., Jetten M. S., Sinskey A. J., Stephanopoulos G. Production of isoleucine by overexpression of ilvA in a Corynebacterium lactofermentum threonine producer. Appl Microbiol Biotechnol. 1995 Jul;43(3):482–488. doi: 10.1007/BF00218453. [DOI] [PubMed] [Google Scholar]
  5. Cremer Josef, Eggeling Lothar, Sahm Hermann. Control of the Lysine Biosynthesis Sequence in Corynebacterium glutamicum as Analyzed by Overexpression of the Individual Corresponding Genes. Appl Environ Microbiol. 1991 Jun;57(6):1746–1752. doi: 10.1128/aem.57.6.1746-1752.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ebbighausen H., Weil B., Krämer R. Transport of branched-chain amino acids in Corynebacterium glutamicum. Arch Microbiol. 1989;151(3):238–244. doi: 10.1007/BF00413136. [DOI] [PubMed] [Google Scholar]
  7. Eikmanns B. J., Eggeling L., Sahm H. Molecular aspects of lysine, threonine, and isoleucine biosynthesis in Corynebacterium glutamicum. Antonie Van Leeuwenhoek. 1993;64(2):145–163. doi: 10.1007/BF00873024. [DOI] [PubMed] [Google Scholar]
  8. Eikmanns B. J., Kleinertz E., Liebl W., Sahm H. A family of Corynebacterium glutamicum/Escherichia coli shuttle vectors for cloning, controlled gene expression, and promoter probing. Gene. 1991 Jun 15;102(1):93–98. doi: 10.1016/0378-1119(91)90545-m. [DOI] [PubMed] [Google Scholar]
  9. Eikmanns B. J., Metzger M., Reinscheid D., Kircher M., Sahm H. Amplification of three threonine biosynthesis genes in Corynebacterium glutamicum and its influence on carbon flux in different strains. Appl Microbiol Biotechnol. 1991 Feb;34(5):617–622. doi: 10.1007/BF00167910. [DOI] [PubMed] [Google Scholar]
  10. Follettie M. T., Shin H. K., Sinskey A. J. Organization and regulation of the Corynebacterium glutamicum hom-thrB and thrC loci. Mol Microbiol. 1988 Jan;2(1):53–62. doi: 10.1111/j.1365-2958.1988.tb00006.x. [DOI] [PubMed] [Google Scholar]
  11. Jäger W., Schäfer A., Pühler A., Labes G., Wohlleben W. Expression of the Bacillus subtilis sacB gene leads to sucrose sensitivity in the gram-positive bacterium Corynebacterium glutamicum but not in Streptomyces lividans. J Bacteriol. 1992 Aug;174(16):5462–5465. doi: 10.1128/jb.174.16.5462-5465.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Keilhauer C., Eggeling L., Sahm H. Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon. J Bacteriol. 1993 Sep;175(17):5595–5603. doi: 10.1128/jb.175.17.5595-5603.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kisumi M., Komatsubara S., Chibata I. Enhancement of isoleucine hydroxamate-mediated growth inhibition and improvement of isoleucine-producing strains of Serratia marcescens. Appl Environ Microbiol. 1977 Dec;34(6):647–653. doi: 10.1128/aem.34.6.647-653.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kisumi M., Komatsubara S., Sugiura M., Chibata I. Isoleucine accumulation by regulatory mutants of Serratia marcescens: lack of both feedback inhibition and repression. J Bacteriol. 1972 May;110(2):761–763. doi: 10.1128/jb.110.2.761-763.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kisumi M., Sugiura M., Chibata I. Biosynthesis of norvaline, norleucine, and homoisoleucine in Serratia marcescens. J Biochem. 1976 Aug;80(2):333–339. doi: 10.1093/oxfordjournals.jbchem.a131281. [DOI] [PubMed] [Google Scholar]
  16. Liebl W., Bayerl A., Schein B., Stillner U., Schleifer K. H. High efficiency electroporation of intact Corynebacterium glutamicum cells. FEMS Microbiol Lett. 1989 Dec;53(3):299–303. doi: 10.1016/0378-1097(89)90234-6. [DOI] [PubMed] [Google Scholar]
  17. Menkel E., Thierbach G., Eggeling L., Sahm H. Influence of increased aspartate availability on lysine formation by a recombinant strain of Corynebacterium glutamicum and utilization of fumarate. Appl Environ Microbiol. 1989 Mar;55(3):684–688. doi: 10.1128/aem.55.3.684-688.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Miyajima R., Otsuka S., Shiio I. Regulation of aspartate family amino acid biosynthesis in Brevibacterium flavum. I. Inhibition by amino acids of the enzymes in threonine biosynthesis. J Biochem. 1968 Feb;63(2):139–148. doi: 10.1093/oxfordjournals.jbchem.a128754. [DOI] [PubMed] [Google Scholar]
  19. Miyajima R., Shiio I. Regulation of aspartate family amino acid biosynthesis in Brevibacterium flavum. VI. Effects of isoleucine and valine on threonine dehydratase activity and its formation. J Biochem. 1972 Jun;71(6):951–960. doi: 10.1093/oxfordjournals.jbchem.a129866. [DOI] [PubMed] [Google Scholar]
  20. Morbach S., Sahm H., Eggeling L. Use of Feedback-Resistant Threonine Dehydratases of Corynebacterium glutamicum To Increase Carbon Flux towards l-Isoleucine. Appl Environ Microbiol. 1995 Dec;61(12):4315–4320. doi: 10.1128/aem.61.12.4315-4320.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Möckel B., Eggeling L., Sahm H. Functional and structural analyses of threonine dehydratase from Corynebacterium glutamicum. J Bacteriol. 1992 Dec;174(24):8065–8072. doi: 10.1128/jb.174.24.8065-8072.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Möckel B., Eggeling L., Sahm H. Threonine dehydratases of Corynebacterium glutamicum with altered allosteric control: their generation and biochemical and structural analysis. Mol Microbiol. 1994 Sep;13(5):833–842. doi: 10.1111/j.1365-2958.1994.tb00475.x. [DOI] [PubMed] [Google Scholar]
  23. Powers S. G., Snell E. E. Ketopantoate hydroxymethyltransferase. II. Physical, catalytic, and regulatory properties. J Biol Chem. 1976 Jun 25;251(12):3786–3793. [PubMed] [Google Scholar]
  24. Pátek M., Krumbach K., Eggeling L., Sahm H. Leucine synthesis in Corynebacterium glutamicum: enzyme activities, structure of leuA, and effect of leuA inactivation on lysine synthesis. Appl Environ Microbiol. 1994 Jan;60(1):133–140. doi: 10.1128/aem.60.1.133-140.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Reinscheid D. J., Eikmanns B. J., Sahm H. Analysis of a Corynebacterium glutamicum hom gene coding for a feedback-resistant homoserine dehydrogenase. J Bacteriol. 1991 May;173(10):3228–3230. doi: 10.1128/jb.173.10.3228-3230.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Reinscheid D. J., Kronemeyer W., Eggeling L., Eikmanns B. J., Sahm H. Stable Expression of hom-1-thrB in Corynebacterium glutamicum and Its Effect on the Carbon Flux to Threonine and Related Amino Acids. Appl Environ Microbiol. 1994 Jan;60(1):126–132. doi: 10.1128/aem.60.1.126-132.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sakurai N., Imai Y., Komatsubara S. Instability of the mutated biotin operon plasmid in a biotin-producing mutant of Serratia marcescens. J Biotechnol. 1995 Nov 21;43(1):11–19. doi: 10.1016/0168-1656(95)00103-9. [DOI] [PubMed] [Google Scholar]
  28. Schäfer A., Kalinowski J., Simon R., Seep-Feldhaus A. H., Pühler A. High-frequency conjugal plasmid transfer from gram-negative Escherichia coli to various gram-positive coryneform bacteria. J Bacteriol. 1990 Mar;172(3):1663–1666. doi: 10.1128/jb.172.3.1663-1666.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Shiio I., Miyajima R. Concerted inhibition and its reversal by end products of aspartate kinase in Brevibacterium flavum. J Biochem. 1969 Jun;65(6):849–859. doi: 10.1093/oxfordjournals.jbchem.a129089. [DOI] [PubMed] [Google Scholar]
  30. UMBARGER H. E., BROWN B. Isoleucine and valine metabolism in Escherichia coli. VIII. The formation of acetolactate. J Biol Chem. 1958 Nov;233(5):1156–1160. [PubMed] [Google Scholar]
  31. Vrljic M., Kronemeyer W., Sahm H., Eggeling L. Unbalance of L-lysine flux in Corynebacterium glutamicum and its use for the isolation of excretion-defective mutants. J Bacteriol. 1995 Jul;177(14):4021–4027. doi: 10.1128/jb.177.14.4021-4027.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wehrmann A., Eggeling L., Sahm H. Analysis of different DNA fragments of Corynebacterium glutamicum complementing dapE of Escherichia coli. Microbiology. 1994 Dec;140(Pt 12):3349–3356. doi: 10.1099/13500872-140-12-3349. [DOI] [PubMed] [Google Scholar]
  33. Zittrich S., Krämer R. Quantitative discrimination of carrier-mediated excretion of isoleucine from uptake and diffusion in Corynebacterium glutamicum. J Bacteriol. 1994 Nov;176(22):6892–6899. doi: 10.1128/jb.176.22.6892-6899.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES