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. 1992 Oct;174(19):6076–6086. doi: 10.1128/jb.174.19.6076-6086.1992

Identification, sequence analysis, and expression of a Corynebacterium glutamicum gene cluster encoding the three glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and triosephosphate isomerase.

B J Eikmanns 1
PMCID: PMC207673  PMID: 1400158

Abstract

To investigate a possible chromosomal clustering of glycolytic enzyme genes in Corynebacterium glutamicum, a 6.4-kb DNA fragment located 5' adjacent to the structural phosphoenolpyruvate carboxylase (PEPCx) gene ppc was isolated. Sequence analysis of the ppc-proximal part of this fragment identified a cluster of three glycolytic genes, namely, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene gap, the 3-phosphoglycerate kinase (PGK) gene pgk, and the triosephosphate isomerase (TPI) gene tpi. The four genes are organized in the order gap-pgk-tpi-ppc and are separated by 215 bp (gap and pgk), 78 bp (pgk and tpi), and 185 bp (tpi and ppc). The predicted gene product of gap consists of 336 amino acids (M(r) of 36,204), that of pgk consists of 403 amino acids (M(r) of 42,654), and that of tpi consists of 259 amino acids (M(r) of 27,198). The amino acid sequences of the three enzymes show up to 62% (GAPDH), 48% (PGK), and 44% (TPI) identity in comparison with respective enzymes from other organisms. The gap, pgk, tpi, and ppc genes were cloned into the C. glutamicum-Escherichia coli shuttle vector pEK0 and introduced into C. glutamicum. Relative to the wild type, the recombinant strains showed up to 20-fold-higher specific activities of the respective enzymes. On the basis of codon usage analysis of gap, pgk, tpi, and previously sequenced genes from C. glutamicum, a codon preference profile for this organism which differs significantly from those of E. coli and Bacillus subtilis is presented.

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Selected References

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

  1. Alber T., Kawasaki G. Nucleotide sequence of the triose phosphate isomerase gene of Saccharomyces cerevisiae. J Mol Appl Genet. 1982;1(5):419–434. [PubMed] [Google Scholar]
  2. Alefounder P. R., Perham R. N. Identification, molecular cloning and sequence analysis of a gene cluster encoding the class II fructose 1,6-bisphosphate aldolase, 3-phosphoglycerate kinase and a putative second glyceraldehyde 3-phosphate dehydrogenase of Escherichia coli. Mol Microbiol. 1989 Jun;3(6):723–732. doi: 10.1111/j.1365-2958.1989.tb00221.x. [DOI] [PubMed] [Google Scholar]
  3. Banks R. D., Blake C. C., Evans P. R., Haser R., Rice D. W., Hardy G. W., Merrett M., Phillips A. W. Sequence, structure and activity of phosphoglycerate kinase: a possible hinge-bending enzyme. Nature. 1979 Jun 28;279(5716):773–777. doi: 10.1038/279773a0. [DOI] [PubMed] [Google Scholar]
  4. Banner D. W., Bloomer A. C., Petsko G. A., Phillips D. C., Pogson C. I., Wilson I. A., Corran P. H., Furth A. J., Milman J. D., Offord R. E. Structure of chicken muscle triose phosphate isomerase determined crystallographically at 2.5 angstrom resolution using amino acid sequence data. Nature. 1975 Jun 19;255(5510):609–614. doi: 10.1038/255609a0. [DOI] [PubMed] [Google Scholar]
  5. Bennetzen J. L., Hall B. D. Codon selection in yeast. J Biol Chem. 1982 Mar 25;257(6):3026–3031. [PubMed] [Google Scholar]
  6. Biesecker G., Harris J. I., Thierry J. C., Walker J. E., Wonacott A. J. Sequence and structure of D-glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus. Nature. 1977 Mar 24;266(5600):328–333. doi: 10.1038/266328a0. [DOI] [PubMed] [Google Scholar]
  7. Birnboim H. C. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 1983;100:243–255. doi: 10.1016/0076-6879(83)00059-2. [DOI] [PubMed] [Google Scholar]
  8. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  9. Branlant C., Oster T., Branlant G. Nucleotide sequence determination of the DNA region coding for Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase and of the flanking DNA regions required for its expression in Escherichia coli. Gene. 1989 Jan 30;75(1):145–155. doi: 10.1016/0378-1119(89)90391-0. [DOI] [PubMed] [Google Scholar]
  10. Branlant G., Branlant C. Nucleotide sequence of the Escherichia coli gap gene. Different evolutionary behavior of the NAD+-binding domain and of the catalytic domain of D-glyceraldehyde-3-phosphate dehydrogenase. Eur J Biochem. 1985 Jul 1;150(1):61–66. doi: 10.1111/j.1432-1033.1985.tb08988.x. [DOI] [PubMed] [Google Scholar]
  11. Börmann E. R., Eikmanns B. J., Sahm H. Molecular analysis of the Corynebacterium glutamicum gdh gene encoding glutamate dehydrogenase. Mol Microbiol. 1992 Feb;6(3):317–326. doi: 10.1111/j.1365-2958.1992.tb01474.x. [DOI] [PubMed] [Google Scholar]
  12. Conway T., Ingram L. O. Phosphoglycerate kinase gene from Zymomonas mobilis: cloning, sequencing, and localization within the gap operon. J Bacteriol. 1988 Apr;170(4):1926–1933. doi: 10.1128/jb.170.4.1926-1933.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Conway T., Sewell G. W., Ingram L. O. Glyceraldehyde-3-phosphate dehydrogenase gene from Zymomonas mobilis: cloning, sequencing, and identification of promoter region. J Bacteriol. 1987 Dec;169(12):5653–5662. doi: 10.1128/jb.169.12.5653-5662.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cosloy S. D., Oishi M. The nature of the transformation process in Escherichia coli K12. Mol Gen Genet. 1973 Jul 31;124(1):1–10. doi: 10.1007/BF00267159. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Davies G. J., Littlechild J. A., Watson H. C., Hall L. Sequence and expression of the gene encoding 3-phosphoglycerate kinase from Bacillus stearothermophilus. Gene. 1991 Dec 20;109(1):39–45. doi: 10.1016/0378-1119(91)90586-z. [DOI] [PubMed] [Google Scholar]
  17. Eikmanns B. J., Follettie M. T., Griot M. U., Sinskey A. J. The phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum: molecular cloning, nucleotide sequence, and expression. Mol Gen Genet. 1989 Aug;218(2):330–339. doi: 10.1007/BF00331286. [DOI] [PubMed] [Google Scholar]
  18. Eikmanns B. J., Kircher M., Reinscheid D. J. Discrimination of Corynebacterium glutamicum, Brevibacterium flavum and Brevibacterium lactofermentum by restriction pattern analysis of DNA adjacent to the hom gene. FEMS Microbiol Lett. 1991 Aug 1;66(2):203–207. doi: 10.1016/0378-1097(91)90333-6. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Fabry S., Heppner P., Dietmaier W., Hensel R. Cloning and sequencing the gene encoding 3-phosphoglycerate kinase from mesophilic Methanobacterium bryantii and thermophilic Methanothermus fervidus. Gene. 1990 Jul 2;91(1):19–25. doi: 10.1016/0378-1119(90)90157-m. [DOI] [PubMed] [Google Scholar]
  22. Fabry S., Lang J., Niermann T., Vingron M., Hensel R. Nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase gene from the mesophilic methanogenic archaebacteria Methanobacterium bryantii and Methanobacterium formicicum. Comparison with the respective gene structure of the closely related extreme thermophile Methanothermus fervidus. Eur J Biochem. 1989 Feb 1;179(2):405–413. doi: 10.1111/j.1432-1033.1989.tb14568.x. [DOI] [PubMed] [Google Scholar]
  23. Fairbrother W. J., Walker P. A., Minard P., Littlechild J. A., Watson H. C., Williams R. J. NMR analysis of site-specific mutants of yeast phosphoglycerate kinase. An investigation of the triose-binding site. Eur J Biochem. 1989 Jul 15;183(1):57–67. doi: 10.1111/j.1432-1033.1989.tb14896.x. [DOI] [PubMed] [Google Scholar]
  24. Gribskov M., Devereux J., Burgess R. R. The codon preference plot: graphic analysis of protein coding sequences and prediction of gene expression. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 2):539–549. doi: 10.1093/nar/12.1part2.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Grosjean H., Fiers W. Preferential codon usage in prokaryotic genes: the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes. Gene. 1982 Jun;18(3):199–209. doi: 10.1016/0378-1119(82)90157-3. [DOI] [PubMed] [Google Scholar]
  26. Hitzeman R. A., Hagie F. E., Hayflick J. S., Chen C. Y., Seeburg P. H., Derynck R. The primary structure of the Saccharomyces cerevisiae gene for 3-phosphoglycerate kinase. Nucleic Acids Res. 1982 Dec 11;10(23):7791–7808. doi: 10.1093/nar/10.23.7791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hocking J. D., Harris J. I. D-glyceraldehyde-3-phosphate dehydrogenase. Amino-acid sequence of the enzyme from the extreme thermophile Thermus aquaticus. Eur J Biochem. 1980 Jul;108(2):567–579. doi: 10.1111/j.1432-1033.1980.tb04752.x. [DOI] [PubMed] [Google Scholar]
  28. Hoekema A., Kastelein R. A., Vasser M., de Boer H. A. Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression. Mol Cell Biol. 1987 Aug;7(8):2914–2924. doi: 10.1128/mcb.7.8.2914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Holland J. P., Holland M. J. The primary structure of a glyceraldehyde-3-phosphate dehydrogenase gene from Saccharomyces cerevisiae. J Biol Chem. 1979 Oct 10;254(19):9839–9845. [PubMed] [Google Scholar]
  30. LENNOX E. S. Transduction of linked genetic characters of the host by bacteriophage P1. Virology. 1955 Jul;1(2):190–206. doi: 10.1016/0042-6822(55)90016-7. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Maquat L. E., Chilcote R., Ryan P. M. Human triosephosphate isomerase cDNA and protein structure. Studies of triosephosphate isomerase deficiency in man. J Biol Chem. 1985 Mar 25;260(6):3748–3753. [PubMed] [Google Scholar]
  33. Minard P., Bowen D. J., Hall L., Littlechild J. A., Watson H. C. Site-directed mutagenesis of aspartic acid 372 at the ATP binding site of yeast phosphoglycerate kinase: over-expression and characterization of the mutant enzyme. Protein Eng. 1990 May;3(6):515–521. doi: 10.1093/protein/3.6.515. [DOI] [PubMed] [Google Scholar]
  34. Nowak K., Wolny M., Banaś T. The complete amino acid sequence of human muscle glyceraldehyde 3-phosphate dehydrogenase. FEBS Lett. 1981 Nov 16;134(2):143–146. doi: 10.1016/0014-5793(81)80587-x. [DOI] [PubMed] [Google Scholar]
  35. O'Regan M., Thierbach G., Bachmann B., Villeval D., Lepage P., Viret J. F., Lemoine Y. Cloning and nucleotide sequence of the phosphoenolpyruvate carboxylase-coding gene of Corynebacterium glutamicum ATCC13032. Gene. 1989 Apr 30;77(2):237–251. doi: 10.1016/0378-1119(89)90072-3. [DOI] [PubMed] [Google Scholar]
  36. Pichersky E., Gottlieb L. D., Hess J. F. Nucleotide sequence of the triose phosphate isomerase gene of Escherichia coli. Mol Gen Genet. 1984;195(1-2):314–320. doi: 10.1007/BF00332765. [DOI] [PubMed] [Google Scholar]
  37. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  38. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schläpfer B. S., Branlant C., Branlant G., Zuber H. Nucleotide sequence of the phosphoglycerate kinase gene from Bacillus megaterium. Nucleic Acids Res. 1990 Nov 11;18(21):6423–6423. doi: 10.1093/nar/18.21.6423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shields D. C., Sharp P. M. Synonymous codon usage in Bacillus subtilis reflects both translational selection and mutational biases. Nucleic Acids Res. 1987 Oct 12;15(19):8023–8040. doi: 10.1093/nar/15.19.8023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Skarzyński T., Moody P. C., Wonacott A. J. Structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus at 1.8 A resolution. J Mol Biol. 1987 Jan 5;193(1):171–187. doi: 10.1016/0022-2836(87)90635-8. [DOI] [PubMed] [Google Scholar]
  42. Sørensen M. A., Kurland C. G., Pedersen S. Codon usage determines translation rate in Escherichia coli. J Mol Biol. 1989 May 20;207(2):365–377. doi: 10.1016/0022-2836(89)90260-x. [DOI] [PubMed] [Google Scholar]
  43. Tinoco I., Jr, Borer P. N., Dengler B., Levin M. D., Uhlenbeck O. C., Crothers D. M., Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. doi: 10.1038/newbio246040a0. [DOI] [PubMed] [Google Scholar]
  44. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  45. Watson H. C., Walker N. P., Shaw P. J., Bryant T. N., Wendell P. L., Fothergill L. A., Perkins R. E., Conroy S. C., Dobson M. J., Tuite M. F. Sequence and structure of yeast phosphoglycerate kinase. EMBO J. 1982;1(12):1635–1640. doi: 10.1002/j.1460-2075.1982.tb01366.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yoshihama M., Higashiro K., Rao E. A., Akedo M., Shanabruch W. G., Follettie M. T., Walker G. C., Sinskey A. J. Cloning vector system for Corynebacterium glutamicum. J Bacteriol. 1985 May;162(2):591–597. doi: 10.1128/jb.162.2.591-597.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. von der Osten C. H., Barbas C. F., 3rd, Wong C. H., Sinskey A. J. Molecular cloning, nucleotide sequence and fine-structural analysis of the Corynebacterium glutamicum fda gene: structural comparison of C. glutamicum fructose-1,6-biphosphate aldolase to class I and class II aldolases. Mol Microbiol. 1989 Nov;3(11):1625–1637. doi: 10.1111/j.1365-2958.1989.tb00148.x. [DOI] [PubMed] [Google Scholar]

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