Cloning and Expression of the Rhodobacter capsulatus hemA Gene in E. coli for the Production of S-Aminolevulinic Acid

  • Published : 2004.12.01

Abstract

The hemA gene encoding 5-aminolevulinic acid synthase (ALAS) was cloned from Rhodobacter capsulatus, and its nucleotide sequence was determined. DNA sequencing data revealed one open reading frame coding for a protein with 401 amino acids that displayed high similarity to the amino acid sequences of other known ALASs. The hemA gene was then cloned and expressed under the control of constitutive promotor in E. coli. The recombinant E. coli strain was able to accumulate 5-aminolevulinic acid to 21 mM in the liquid medium supplemented with 45 mM glycine and 120 mM succinate. In addition, a marked effect of the pH of the culture medium on ALA production was observed, and the optimum pH for culture medium was determined to be 5.8-6.3.

Keywords

References

  1. Adams, C. W., M. E. Forrest, S. N. Cohen, and T. Beatty. 1989. Structural and functional analysis of transcriptional control of the Rhodobacter capsulatus puf operon. J. Bacteriol. 171: 473-482
  2. Atri, N. and L. C. Rai. 2003. Differential responses of three cyanobacteria to UV-B and Cd. J. Microbiol. Biotechnol. 13(4): 544-551
  3. Avissar, Y. J. and S. I. Beale. 1989. Identification of the enzymatic basis for $\delta$-aminolevulinic acid auxotrophy in a hemA mutant of Escherichia coli. J. Bacteriol. 171: 2919-2924
  4. Beale, S. J. and P. A. Castelfranco. 1974. The biosynthesis of $\delta$-aminolevulinic acid in higher plants. II. Formation of $^14$C-$\delta$-aminolevulinic acid from labeled precursors in greening plant tissue. Plant Physiol. 53: 297-303
  5. Bunke, A., H. Schmid, G. Burmeister, H. P. Merkle, and B. Gander. 2000. Validation of a capillary electrophoresis method for determination of 5-aminolevulinic acid and degradation products. J. Chromatogr. 883: 285-290
  6. Choi, C., B.-S. Hong, H.-C. Sung, H.-S. Lee, and J.-H. Kim. 1999. Optimization of extracellular 5-aminolevulinic acid production from Escherichia coli transformed with ALA synthase gene of Bradyrhizobium japonicum. Biotechnol. Lett. 21: 551-554
  7. Choi, H.-P., H.-J. Kang, H.-C. Seo, and H.-C. Sung. 2002. Isolation and identification of photosynthetic bacterium useful for wastewater treatment. J. Microbiol. Biotechnol. 12(4): 643-648
  8. Choi, K.-M., W.-J. Lim, and S.-Y. Hwang. 1993. Influence of C5-precursors on $\delta$-aminolevulinic acid biosynthesis in Rhodocyclus gelatinosus KUP-74. Kor. J. Appl. Microbiol. Biotechnol. 21: 527-533
  9. Elliott, T. and J. R. Roth. 1989. Heme-deficient mutants of Salmonella typhimurium: Two genes required for ALA synthesis. Mol. Gen. Genet. 216: 303-314
  10. Ferreira, G. and J. Gong. 1995. 5-Aminolevulinate synthase and the first step of heme biosynthesis. J. Bioenerg. Biomembr. 27: 151-159
  11. Gadmar O. B., J. Moan, E. Scheie, L.-W. Ma, and Q. Peng. 2002. The stability of 5-aminolevulinic acid in solution. J. Photochem. Photobiol. 67: 187-193
  12. Granick, S. and S. I. Beale. 1978. Hemes, chlorophylls, and related compound: Biosynthesis and metabolic regulation. Adv. Enzymol. 46: 33-203
  13. Grimm, B., A. Kumar, P. Talmage, and D. Shemin. 1991. Structural gene of glutamate-1-semialdehyde aminotransferase for porphyrin synthesis in a cyanobacterium and Escherichia coli. Mol. Gen. Genet. 225: 1-10
  14. Hotta, Y. and K. Watanabe. 1999. Plants growth-regulating activities of 5-aminolevulinic acid. Syokubutu-no-Kagaku- Tyousetu (Chemical regulation of plants) 34: 85-96
  15. Hunter, G. A. and G. C. Ferreira. 1999. Lycine-313 of 5-aminolevulinate synthase acts as a general base during formation of the quinonoid reaction intermediates. Biochemistry 38: 3711-3718
  16. Kikuchi, G., A. Kumor, P. Talmage, and D. Shemin. 1958. The enzymatic synthesis of $\delta$-aminolevulinic acid. J. Biol. Chem. 233: 1214-1219
  17. Kim, N.-J., I. S. Suh, B.-K. Hur, and C.-G. Lee. 2002. Simple monodimensional model for linear growth rate of photosynthetic microorganisms in flat-plate photobioreactors. 12(6): 962-971
  18. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 680-685 https://doi.org/10.1038/227680a0
  19. Leong, S. A., P. H. Williams, and G. S. Ditta. 1985. Analysis of the 5' regulatory region of the gene for 5-aminolevulinic acid synthetase of Rhizobium meliloti. Nucleic Acids Res. 13: 5965-5976 https://doi.org/10.1093/nar/13.16.5965
  20. Lim, W.-J., K.-M., Choi, and S.-Y. Whang. 1993. Optimization of an intact cell system of Rhodobacter gelatinosus KUP-74 for $\delta$-aminolevulinic acid production. J. Microbiol. Biotechnol. 3: 244-251
  21. Mak, Y. M. and K. K. Ho. 1992. An improved method for isolation of chromosomal DNA from various bacteria and cyanobacteria. Nucleic Acids Res. 20: 4101-4102
  22. Mauzerall, D. and S. Granick. 1956. The occurrence and determination of 5-aminolevulinic acid and porphobilinogen in urine. J. Biol. Chem. 219: 435-446
  23. Nishkawa, S., K. Watanabe, T. Tanaka, N. Miyachi, Y. Hotta, and Y. Murooka. 1999. Rhodobacter sphaeroides mutants which accumulate 5-aminolevulinic acid under aerobic and dark conditions. J. Biosci. Bioeng. 87: 798-804
  24. Poo, H., J. J. Song, S.-P. Hong, Y.-H. Choi, S. W. Yun, J.-H. Kim, S. C. Lee, S.-G. Lee, and M. H. Sung. 2002. Novel high-level constitutive expression system, pHCE vector, for a convenient and cost-effective soluble production of human tumor necrosis factor. Biotechnol. Lett. 24: 1185-1189 https://doi.org/10.1023/A:1016107230825
  25. Rebeiz, C. A., A. Montazer-Zouhool, H. Hopen, and S. M. Wu. 1984. Photodynamic herbicides. I. Concepts and phenomenology. Enzyme Microb. Technol. 6: 390-401
  26. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, New York, U.S.A
  27. Sasaki, K., M. Watanabe, T. Tanaka, and T. Tanaka. 2002. Biosynthesis, biotechnological production and applications of 5-aminolevulinic acid. Appl. Microbiol. Biotechnol. 58: 23-29
  28. Sasaki, K., S. Ikeda, Y. Nishizawa, and M. Hayashi. 1987. Production of 5-aminolevulinic acid by photosynthetic bacteria. J. Ferment. Technol. 65: 511-515
  29. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673-4680
  30. Werf, M. and J. G. Zeikus. 1996. 5-Aminolevulinate production by Escherichia coli containing the Rhodobacter sphaeroides hemA gene. Appl. Environ. Microbiol. 62: 3560-3566
  31. Zucconi, A. P. and J. T. Beatty. 1988. Posttranscriptional regulation by light of the steady-state levels of mature B800- 850 light-harvesting complexes in Rhodobacter capsulatus. J. Bacteriol. 170: 877-882