Expression and characterization of polyketide synthase module involved in the late step of cephabacin biosynthesis from Lysobacter lactamgenus.
The cephabacins produced by Lysobacter lactamgenus are beta-lactam antibiotics composed of a cephem nucleus, an acetate residue, and an oligopeptide side chain. In order to understand the precise implication of the polyketide synthase (PKS) module in the biosynthesis of cephabacin, the genes for its core domains, beta-ketoacyl synthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), were amplified and cloned into the pET-32b(+) expression vector. The sfp gene encoding a protein that can modify apo-ACP to its active holo-form was also amplified. The recombinant KS, AT, apo-ACP, and Sfp overproduced in the form of His6-tagged fusion proteins in E. coli BL21(DE3) were purified by nickel-affinity chromatography. Formation of stable peptidyl-S-KS was observed by in vitro acylation of the KS domain with the substrate [L-Ala-L-Ala-LAla- L-3H-Arg] tetrapeptide-S-N-acetylcysteamine, which is the evidence for the selective recognition of tetrapeptide produced by nonribosomal peptide synthetase (NRPS) in the NRPS/ PKS hybrid. In order to confirm whether malonyl CoA is the extender unit for acetylation of the peptidyl moiety, the AT domain, ACP domain, and Sfp protein were treated with 14C-malonyl-CoA. The results clearly show that the AT domain is able to recognize the extender unit and decarboxylatively acetylated for the elongation of the tetrapeptide. However, the transfer of the activated acetyl group to the ACP domain was not observed, probably attributed to the improper capability of Sfp to activate apo-ACP to the holo-ACP form.
Lee
Ji Seon
JS
Faculty of Pharmacy and 2Faculty of Biotechnology, Yeungnam University, Gyongsan 712-749 Korea.
Vladimirova
Miglena G
MG
Demirev
Atanas V
AV
Kim
Bo Geum
BG
Lim
Si Kyu
SK
Nam
Doo Hyun
DH
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000213:Acyl Carrier Protein / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002511:Cephalosporins / Q000096:biosynthesis*; D015870:Gene Expression; D005822:Genetic Vectors; D053528:Lysobacter / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D048630:Polyketide Synthases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D002511:Cephalosporins; D011993:Recombinant Fusion Proteins; D048630:Polyketide Synthases; C044529:cephabacins; D000217:Acyltransferases; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase