Polyketide synthase acyl carrier proteins from Streptomyces: expression in Escherichia coli, purification and partial characterisation.
Acyl carrier proteins (ACPs) of the type II polyketide synthases for the aromatic antibiotics actinorhodin, granaticin, frenolicin and oxytetracycline were expressed in Escherichia coli downstream of an inducible phage T7 promoter. For the act and otc genes, several of the first eight codons were changed to synonymous codons used in highly expressed E. coli genes. Correlated with these changes, the amounts of the act and otc ACPs purified from the recombinant E. coli cultures were an order of magnitude greater than for the gra and fren ACPs expressed from the unmodified genes. Electrospray mass spectrometry (ESMS) of the purified proteins confirmed their calculated M(r) based on the DNA sequences while also revealing that, in the act and gra ACP samples, some 2% and 30% of the holo-form of the protein was present (i.e., carrying the 4'-phosphopantetheine prosthetic group), with the remainder (and 100% of the otc and fren samples) being in the apo-form. Increasing incubation time post heat induction led to an increase in act holo-ACP. The recombinant act and gra ACPs could function in vitro as substrates for an S. coelicolor malonyl CoA:ACP acyl transferase, as measured by the coupling of a labelled malonyl unit to the ACP; their quantitative abilities to do so correlated with the proportions of deduced holo form in the two samples.
Crosby
J
J
School of Chemistry, University of Bristol, UK.
Sherman
D H
DH
Bibb
M J
MJ
Revill
W P
WP
Hopwood
D A
DA
Simpson
T J
TJ
D000213:Acyl Carrier Protein / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D013058:Mass Spectrometry; D009097:Multienzyme Complexes / Q000378:metabolism*; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism
D000213:Acyl Carrier Protein; D009097:Multienzyme Complexes