Expression of a functional non-ribosomal peptide synthetase module in Escherichia coli by coexpression with a phosphopantetheinyl transferase

Jung Ku, Raghu Mirmira, Lu Liu, Daniel V. Santi

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

Background: Non-ribosomal peptide synthetases (NRPSs) found in bacteria and fungi are multifunctional enzymes that catalyze the synthesis of a variety of biologically important peptides. These enzymes are composed of modular units, each responsible for the activation of an amino acid to an aminoacyl adenylate and for the subsequent formation of an aminoacyl thioester with the sulfhydryl group of a 4'-phosphopantetheine moiety. Attempts to express these modules in Escherichia coli have resulted in recombinant proteins deficient in 4'-phospho-pantetheine. The recent identification of a family of phosphopantetheinyl transferases (P-pant transferases) associated with NRPS have led us to investigate whether coexpression of NRPS modules with P-pant transferases in E. coli would lead to the incorporation of 4'-phosphopantetheine. Results: A truncated module of gramicidin S synthetase, PheAT(His6), was expressed as a His6 fusion protein in E. coli with and without Gsp, the P-pant transferase associated with gramicidin S synthetase. Although PheAT(His6) expressed alone in E. coli catalyzed Phe-AMP formation from Phe and ATP, 80% of the PheAT(His6) that was coexpressed with Gsp could form the Phe thioester in the presence of Phe and ATP. Conclusions: Our finding indicates the presence of an almost equimolar amount of 4'-phosphopantetheine covalently bound to the NRPS module PheAT(His6), and that the functional expression of NRPS modules in E. coli is possible, provided that they are coexpressed with an appropriate P-pant transferase.

Original languageEnglish (US)
Pages (from-to)203-207
Number of pages5
JournalChemistry and Biology
Volume4
Issue number3
StatePublished - Mar 1997
Externally publishedYes

Fingerprint

Peptide Synthases
Escherichia coli
Gramicidin
Ligases
Pantetheine
Adenosine Triphosphate
Multifunctional Enzymes
Aminoacylation
Escherichia coli Proteins
Adenosine Monophosphate
Fungi
Recombinant Proteins
Bacteria
Fusion reactions
Chemical activation
phosphopantetheinyl transferase
Amino Acids
Peptides
Enzymes
4'-phosphopantetheine

Keywords

  • coexpression
  • gramicidin S synthetase
  • module
  • non-ribosomal peptide synthetase
  • phosphopantetheine

ASJC Scopus subject areas

  • Organic Chemistry

Cite this

Expression of a functional non-ribosomal peptide synthetase module in Escherichia coli by coexpression with a phosphopantetheinyl transferase. / Ku, Jung; Mirmira, Raghu; Liu, Lu; Santi, Daniel V.

In: Chemistry and Biology, Vol. 4, No. 3, 03.1997, p. 203-207.

Research output: Contribution to journalArticle

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abstract = "Background: Non-ribosomal peptide synthetases (NRPSs) found in bacteria and fungi are multifunctional enzymes that catalyze the synthesis of a variety of biologically important peptides. These enzymes are composed of modular units, each responsible for the activation of an amino acid to an aminoacyl adenylate and for the subsequent formation of an aminoacyl thioester with the sulfhydryl group of a 4'-phosphopantetheine moiety. Attempts to express these modules in Escherichia coli have resulted in recombinant proteins deficient in 4'-phospho-pantetheine. The recent identification of a family of phosphopantetheinyl transferases (P-pant transferases) associated with NRPS have led us to investigate whether coexpression of NRPS modules with P-pant transferases in E. coli would lead to the incorporation of 4'-phosphopantetheine. Results: A truncated module of gramicidin S synthetase, PheAT(His6), was expressed as a His6 fusion protein in E. coli with and without Gsp, the P-pant transferase associated with gramicidin S synthetase. Although PheAT(His6) expressed alone in E. coli catalyzed Phe-AMP formation from Phe and ATP, 80{\%} of the PheAT(His6) that was coexpressed with Gsp could form the Phe thioester in the presence of Phe and ATP. Conclusions: Our finding indicates the presence of an almost equimolar amount of 4'-phosphopantetheine covalently bound to the NRPS module PheAT(His6), and that the functional expression of NRPS modules in E. coli is possible, provided that they are coexpressed with an appropriate P-pant transferase.",
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N2 - Background: Non-ribosomal peptide synthetases (NRPSs) found in bacteria and fungi are multifunctional enzymes that catalyze the synthesis of a variety of biologically important peptides. These enzymes are composed of modular units, each responsible for the activation of an amino acid to an aminoacyl adenylate and for the subsequent formation of an aminoacyl thioester with the sulfhydryl group of a 4'-phosphopantetheine moiety. Attempts to express these modules in Escherichia coli have resulted in recombinant proteins deficient in 4'-phospho-pantetheine. The recent identification of a family of phosphopantetheinyl transferases (P-pant transferases) associated with NRPS have led us to investigate whether coexpression of NRPS modules with P-pant transferases in E. coli would lead to the incorporation of 4'-phosphopantetheine. Results: A truncated module of gramicidin S synthetase, PheAT(His6), was expressed as a His6 fusion protein in E. coli with and without Gsp, the P-pant transferase associated with gramicidin S synthetase. Although PheAT(His6) expressed alone in E. coli catalyzed Phe-AMP formation from Phe and ATP, 80% of the PheAT(His6) that was coexpressed with Gsp could form the Phe thioester in the presence of Phe and ATP. Conclusions: Our finding indicates the presence of an almost equimolar amount of 4'-phosphopantetheine covalently bound to the NRPS module PheAT(His6), and that the functional expression of NRPS modules in E. coli is possible, provided that they are coexpressed with an appropriate P-pant transferase.

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