Expression of a functional non-ribosomal peptide synthetase module in Escherichia coli by coexpression with a phosphopantetheinyl transferase.
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'-phosphopantetheine. 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, <1% was converted to the Phe thioester. In contrast, >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.
Ku
J
J
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143 0448, USA.
Mirmira
R G
RG
Liu
L
L
Santi
D V
DV
D000591:Amino Acid Isomerases / Q000096:biosynthesis / Q000235:genetics; D064753:Ammonium Hydroxide; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors; D006878:Hydroxides / Q000494:pharmacology; D010453:Peptide Synthases / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D017855:Transferases (Other Substituted Phosphate Groups) / Q000096:biosynthesis* / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006878:Hydroxides; D064753:Ammonium Hydroxide; D017855:Transferases (Other Substituted Phosphate Groups); C021364:holo-(acyl-carrier-protein) synthase; D000591:Amino Acid Isomerases; C022343:phenylalanine racemase (ATP-hydrolyzing); D010453:Peptide Synthases