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KMID : 1094720180230060693
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Biotechnology and Bioprocess Engineering
2018 Volume.23 No. 6 p.693 ~ p.703
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Biosynthesis of Methoxymalonyl-acyl Carrier Protein (ACP) as an Extender Unit for Bafilomycin Polyketide in Streptomyces griseus DSM 2608
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Hanh Nguyen Phan Kieu
Hwang Jae-Yoon
Nam Doo-Hyun
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Abstract
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Bafilomycins belong to the 16-membered macrolactone family plecomacrolide antibiotics. In the bafilomycin biosynthetic gene cluster of Streptomyces griseus DSM 2608, five polyketide synthase (PKS) genes coding 12 PKS modules responsible for bafilomycin polyketide backbone were found. Based on the chemical structure of bafilomycin polyketide having two methoxy side chains, it was assumed that PKS module 6 and module 12 can utilize the methoxymalonyl-acyl carrier protein (ACP) as an extender unit to incorporate methoxy group. At the downstream of PKS genes, five putative genes responsible for the generation of methoxymalonyl-ACP from 1,3-bis-phosphoglycerate were located. In order to confirm the biochemical role of those genes, five putative genes (bafAI, bafAII, bafAIII, bafAIV, and bafAV) were amplified, cloned into the pET-32a(+) plasmid, and expressed in Escherichia coli Rosetta(DE3). The produced soluble recombinant proteins were purified through nickel-affinity column chromatography, and the biochemical reactions of recombinant proteins were investigated. Firstly the purified BafAII protein, a putative apo-ACP, was converted to active form by attaching 4’-phosphopantetheinyl group of coenzyme A (CoA) by the action of Bacillus subtilis Sfp protein. The linking of glyceryl group on functional BafAII, a holo-ACP, from 1,3-bis-phosphoglycerate (1,3-BPG) was confirmed by the reaction of the recombinant BafAIV protein, a putative glyceryl-ACP synthase. The next oxidation step of glyceryl-BafAII to 2-hydroxy-3-oxopropanoyl- BafAII was identified, which was catalyzed by the recombinant BafAI, a putative glyceryl-ACP dehydrogenase, in the presence of NAD+. Further oxidation of 2-hydroxy- 3-oxopropanoyl-BafAII to 2-hydroxymalonyl-BafAII was also verified by the recombinant BafAIII, a putative 3- oxoacyl-ACP dehydrogenase, in the presence of FAD. The final methylation of 2-hydroxymalonyl-BafAII by the recombinant BafAV, a putative 2-hydroxymalonyl-ACP O-methyltransferase, was proven to be mediated using S-adenosylmethionine as a methyl donor. Throughout checking the biochemical characteristics of five putative genes, the biosynthetic pathway of methoxymalonyl-ACP as an extender unit for bafilomycin polyketide synthesis was clearly evidenced.
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KEYWORD
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methoxymalonyl-ACP, glyceryl-ACP, bafilomycin, Streptomyces griseus
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