Post-translational modification of heterologously expressed Streptomyces type II polyketide synthase acyl carrier proteins.
Expression in Escherichia coli of Streptomyces 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 apo-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-ACP 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 ACPS is able to transfer acylated CoA moieties to act apo-ACP.
Cox
R J
RJ
School of Chemistry, University of Bristol, UK. r.j.cox@bristol.ac.uk
Hitchman
T S
TS
Byrom
K J
KJ
Findlow
I S
IS
Tanner
J A
JA
Crosby
J
J
Simpson
T J
TJ
D000213:Acyl Carrier Protein / Q000378:metabolism*; D001059:Apoproteins / Q000378:metabolism; D003065:Coenzyme A / Q000378:metabolism; D004220:Disulfides; D004926:Escherichia coli / Q000201:enzymology; D009097:Multienzyme Complexes / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D013045:Species Specificity; D013302:Streptomyces / Q000201:enzymology
D000213:Acyl Carrier Protein; D001059:Apoproteins; D004220:Disulfides; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D003065:Coenzyme A