Details
| Original language | English |
|---|---|
| Pages (from-to) | 267-272 |
| Number of pages | 6 |
| Journal | FEBS letters |
| Volume | 405 |
| Issue number | 3 |
| Publication status | Published - 1 Apr 1997 |
| Externally published | Yes |
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
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biophysics
- Biochemistry, Genetics and Molecular Biology(all)
- Structural Biology
- Biochemistry, Genetics and Molecular Biology(all)
- Biochemistry
- Biochemistry, Genetics and Molecular Biology(all)
- Molecular Biology
- Biochemistry, Genetics and Molecular Biology(all)
- Genetics
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology
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In: FEBS letters, Vol. 405, No. 3, 01.04.1997, p. 267-272.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Post-translational modification of heterologously expressed Streptomyces type II polyketide synthase acyl carrier proteins
AU - Cox, Russell J.
AU - Hitchman, Timothy S.
AU - Byrom, Kate J.
AU - Findlow, I. Stuart C.
AU - Tanner, Julian A.
AU - Crosby, John
AU - Simpson, Thomas J.
N1 - 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.
PY - 1997/4/1
Y1 - 1997/4/1
N2 - 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.
AB - 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.
KW - 4'-Phosphopantetheine
KW - Acyl carrier protein
KW - Heterologous expression
KW - Polyketidc
KW - Post-translational modification
KW - Streptomyces
UR - http://www.scopus.com/inward/record.url?scp=0030936596&partnerID=8YFLogxK
U2 - 10.1016/S0014-5793(97)00202-0
DO - 10.1016/S0014-5793(97)00202-0
M3 - Article
C2 - 9108302
AN - SCOPUS:0030936596
VL - 405
SP - 267
EP - 272
JO - FEBS letters
JF - FEBS letters
SN - 0014-5793
IS - 3
ER -