Post-translational modification of heterologously expressed Streptomyces type II polyketide synthase acyl carrier proteins

verfasst von: Russell J. Cox, Timothy S. Hitchman, Kate J. Byrom, I. Stuart C. Findlow, Julian A. Tanner, John Crosby, Thomas J. Simpson
Abstract: Expression in Escherichia coli of Streptomgces acyl carrier proteins (ACPs) associated with polyketide biosynthesis using the pT7-7 expression system of Tabor and Richardson led to the production predominantly of inactive ape-proteins lacking the 4'-phosphopantetheinyl prosthetic group essential for polyketide synthase activity. Modification of growth conditions led to an increase of production of active holo-protein for the actinorhodin (act) ACP, but this technique was ineffective for oxytetracycline (otc) and griseusin (gris) ACPs. Labelling experiments revealed that a low level of otc ACP expressed prior to induction was produced mainly as active holo-protein, while post-induction ¹⁵N-labelled protein was almost exclusively in the apo-ACPP form. Limiting endogenous holo-acyl carrier protein synthase (ACPS) concentration was implicated as responsible for low apo-ACP to holo-ACP conversion, rather than limiting substrate (coenzyme A) and cofactor (Mg²⁺) concentrations. Co-expression of act and gris ACPs with ACPS in E. coli led to high levels of production of active holo-ACPs and ACPS. We have also made the significant observation that AGES is able to transfer acylated CoA moieties to act apo-ACP.
Externe Organisation(en): University of Bristol
Typ: Artikel
Journal: FEBS letters
Band: 405
Seiten: 267-272
Anzahl der Seiten: 6
ISSN: 0014-5793
Publikationsdatum: 01.04.1997
Publikationsstatus: Veröffentlicht
Peer-reviewed: Ja
ASJC Scopus Sachgebiete: Biophysik, Strukturelle Biologie, Biochemie, Molekularbiologie, Genetik, Zellbiologie
Elektronische Version(en): https://doi.org/10.1016/S0014-5793(97)00202-0 (Zugang: Unbekannt)

BibTeX

@article{0932beebba42462c914ad92467dac960,
title = "Post-translational modification of heterologously expressed Streptomyces type II polyketide synthase acyl carrier proteins",
abstract = "Expression in Escherichia coli of Streptomgces acyl carrier proteins (ACPs) associated with polyketide biosynthesis using the pT7-7 expression system of Tabor and Richardson led to the production predominantly of inactive ape-proteins lacking the 4'-phosphopantetheinyl prosthetic group essential for polyketide synthase activity. Modification of growth conditions led to an increase of production of active holo-protein for the actinorhodin (act) ACP, but this technique was ineffective for oxytetracycline (otc) and griseusin (gris) ACPs. Labelling experiments revealed that a low level of otc ACP expressed prior to induction was produced mainly as active holo-protein, while post-induction 15N-labelled protein was almost exclusively in the apo-ACPP form. Limiting endogenous holo-acyl carrier protein synthase (ACPS) concentration was implicated as responsible for low apo-ACP to holo-ACP conversion, rather than limiting substrate (coenzyme A) and cofactor (Mg2+) concentrations. Co-expression of act and gris ACPs with ACPS in E. coli led to high levels of production of active holo-ACPs and ACPS. We have also made the significant observation that AGES is able to transfer acylated CoA moieties to act apo-ACP.",
keywords = "4'-Phosphopantetheine, Acyl carrier protein, Heterologous expression, Polyketidc, Post-translational modification, Streptomyces",
author = "Cox, {Russell J.} and Hitchman, {Timothy S.} and Byrom, {Kate J.} and Findlow, {I. Stuart C.} and Tanner, {Julian A.} and John Crosby and Simpson, {Thomas J.}",
note = "Funding information: Financial assistance from the BBSRC, EPSRC and the University of Bristol is gratefully acknowledged. We thank Dr Len Hall, Department of Biochemistry, University of Bristol, for oligonucleotide synthesis and DNA sequencing and Dr W. Peter Revill and Mrs Maureen J. Bibb of the John Innes Centre, Norwich, for helpful discussions and the generous provision of pIJ5235.",
year = "1997",
month = apr,
day = "1",
doi = "10.1016/S0014-5793(97)00202-0",
language = "English",
volume = "405",
pages = "267--272",
journal = "FEBS letters",
issn = "0014-5793",
publisher = "Wiley-Blackwell",
number = "3",
}