Article | Published:

Semisynthetic production of unnatural L-α-amino acids by metabolic engineering of the cysteine-biosynthetic pathway

Nature Biotechnology volume 21, pages 422427 (2003) | Download Citation

Subjects

Abstract

There is an increasing demand for peptide-mimicking molecules to modulate the interactions between proteins of pharmaceutical and agrochemical interest and their target polypeptides. Unnatural L-α-amino acids differing from the 20 naturally proteinogenic amino acids only in their side chain are ideal for this purpose, but their chemical synthesis is complex. Here we describe a fermentation-based approach for biosynthesis of unnatural amino acids after re-engineering the cysteine-biosynthetic pathway in Escherichia coli. O-acetylation of serine, the committed step of the pathway, was released from feedback inhibition by mutating the serine acetyltransferase gene. Next, the naturally broad substrate specificity of O-acetylserine sulfhydrylase was exploited for the direct in vivo incorporation of an unnatural side chain in a semisynthetic fermentation process comparable to the production of β-lactams. O-acetyl-L-serine extruded from the cells by way of the O-acetylserine efflux protein was amenable to further biotransformations.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    Building fine chemicals muscle with amino acids. Chem. Market Reporter June 14 (1999), pp. 9–11.

  2. 2.

    Chiral amino acids: a versatile tool in the synthesis of pharmaceuticals and fine chemicals. Chimia 51, 310–315 (1997).

  3. 3.

    Peptides: a boom in the making. Chem. Eng. News January 8 (2001), pp. 11–15.

  4. 4.

    et al. Viracept (nelfinavir mesylate, AG1343): a potent, orally bioavailable inhibitor of HIV-1 protease. J. Med. Chem. 40, 3979–3985 (1997).

  5. 5.

    , & Studies on the synthesis of the Fe-S cluster of dihydroxy-acid dehydratase in Escherichia coli crude extract. J. Biol. Chem. 271, 16053–16067 (1996).

  6. 6.

    & Enzymatic synthesis of non-protein β-substituted alanines and some higher homologues in plants. Phytochemistry 35, 1089–1104 (1994).

  7. 7.

    & Pyridoxal 5′-phosphate-dependent α,β-elimination reactions: mechanism of O-acetylserine sulfhydrylase. Acc. Chem. Res. 34, 49–59 (2001).

  8. 8.

    , & Enzymatic proof for the identity of the S-sulfocysteine synthase and cysteine synthase B of Salmonella typhimurium. J. Bacteriol. 158, 112–1127 (1984).

  9. 9.

    , , & Cysteine biosynthesis pathway in the archeon Methanosarcina barkeri encoded by acquired genes? J. Bacteriol. 182, 143–145 (2000).

  10. 10.

    , & Enantiospecific synthesis of quisqualic acid. Tetrahedron 4, 2041–2046 (1993).

  11. 11.

    & The enzymatic synthesis of L-cysteine in Escherichia coli and Salmonella typhimurium. J. Biol. Chem. 241, 4955–4956 (1966).

  12. 12.

    & L-cysteine biosynthesis in Escherichia coli: nucleotide sequence and expression of serine acetyltransferase (cysE) gene from the wild type and a cysteine-excreting mutant. J. Gen. Microbiol. 133, 515–525 (1987).

  13. 13.

    & Process for preparing O-acetylserine, L-cysteine and L-cysteine-related products. PCT patent WO97/15673 (1996).

  14. 14.

    , , & Identification of a major facilitator protein from Escherichia coli involved in efflux of metabolites of the cysteine pathway. Mol. Microbiol. 36, 1101–1112 (2000).

  15. 15.

    & Cysteine, even at low concentrations, induces transient amino acid starvation. J. Bacteriol. 173, 5244–5246 (1991).

  16. 16.

    & Bacterial amino acid transport proteins: occurrence, functions, and significance for biotechnological applications. Appl. Microbiol. Biotechnol. 58, 265–274 (2002).

  17. 17.

    , & Process for preparing O-acetylserine by fermentation. German patent application DE A 10107002 (2001).

  18. 18.

    , & Cyclization of cysteinylglycine under Pummer reaction conditions. Can. J. Chem. 57, 2412–2425 (1972).

  19. 19.

    et al. Process for S-aryl-L-cysteine and derivatives. European patent application EP 0968997A2 (1998).

  20. 20.

    Gene-expression tools for metabolic engineering of bacteria. Trends Biotechnol. 17, 452–460 (1999).

  21. 21.

    , , & Expanding the genetic code of Escherichia coli. Science 292, 498–500 (2001).

  22. 22.

    et al. Enlarging the amino acid set of Escherichia coli by infiltration of the valine coding pathway. Science 292, 501–504 (2001).

  23. 23.

    , , , & Incorporation of an unnatural amino acid in the active site of porcine pancreatic phospholipase A2. Substitution of histidine by 1,2,4-triazole-3-alanine yields an enzyme with high activity at acidic pH. Protein Eng. 9, 345–352 (1996).

  24. 24.

    , , , & Biosynthesis of complex polyketides in a metabolically engineered stain of E. coli. Science 291, 1790–1792 (2001).

  25. 25.

    , & Molecular breeding of carotenoid biosynthetic pathways. Nat. Biotechnol. 18, 750–753 (2000).

  26. 26.

    , , , & Novel hydroxycarotenoids with improved antioxidative properties produced by gene combination in E. coli. Nat. Biotechnol. 18, 843–846 (2000).

  27. 27.

    The Escherichia coli K-12 “wild types” W3110 and MG1655 have an rph frameshift mutation that leads to pyrimidine starvation due to low pyrE expression levels. J. Bacteriol. 175, 3401–3407 (1993).

  28. 28.

    et al. Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res. 17, 3468–3479 (1989).

  29. 29.

    & Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. Bacteriol. 134, 1141–1156 (1978).

  30. 30.

    A spectrophotometric method for the direct determination of cysteine in the presence of naturally occurring amino acids. Biochem. J. 104, 627–633 (1967).

Download references

Acknowledgements

The author thanks the entire Consortium team for support in many different ways, but especially Renate Flinspach, Alex Leinauer, and Benedikt Reschka for excellent technical assistance. I am also indebted to Carsten Gaebert for valuable discussions and for analytical measurements. Moreover, August Boeck, Tobias Dassler, and Guenter Wich are acknowledged for critical reading of the manuscript.

Author information

Affiliations

  1. Wacker Chemie GmbH Consortium für elektrochemische Industrie GmbH (Corporate Research Facility of Wacker-Chemie), Zielstattstr. 20, 81379 Munich, Germany.  thomas.maier@wacker.com

    • Thomas H. P. Maier

Authors

  1. Search for Thomas H. P. Maier in:

Competing interests

The author declares no competing financial interests.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nbt807

Further reading