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Direct transfer of starter substrates from type I fatty acid synthase to type III polyketide synthases in phenolic lipid synthesis Akimasa Miyanaga, Nobutaka Funa, Takayoshi Awakawa, and Sueharu Horinouchi* Department of Biotechnology, Graduate School of Agriculture and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Edited by Arnold L. Demain, Drew University, Madison, NJ, and approved November 28, 2007 (received for review October 16, 2007) Alkylresorcinols and alkylpyrones, which have a polar aromatic ring and a hydrophobic alkyl chain, are phenolic lipids found in plants, fungi, and bacteria. In the Gram-negative bacterium Azo- tobacter vinelandii , phenolic lipids in the membrane of dormant cysts are essential for encystment. The aromatic moieties of the phenolic lipids in A. vinelandii are synthesized by two type III polyketide synthases (PKSs), ArsB and ArsC, which are encoded by the ars operon. However, details of the synthesis of hydrophobic acyl chains, which might serve as starter substrates for the type III polyketide synthases (PKSs), were unknown. Here, we show that two type I fatty acid synthases (FASs), ArsA and ArsD, which are members of the ars operon, are responsible for the biosynthesis of C 22 –C 26 fatty acids from malonyl-CoA. In vivo and in vitro recon- stitution of phenolic lipid synthesis systems with the Ars enzymes suggested that the C 22 –C 26 fatty acids produced by ArsA and ArsD remained attached to the ACP domain of ArsA and were trans- ferred hand-to-hand to the active-site cysteine residues of ArsB and ArsC. The type III PKSs then used the fatty acids as starter substrates and carried out two or three extensions with malonyl- CoA to yield the phenolic lipids. The phenolic lipids in A. vinelandii were thus found to be synthesized solely from malonyl-CoA by the four members of the ars operon. This is the first demonstration that a type I FAS interacts directly with a type III PKS through substrate transfer. Azotobacter vinelandii alkylresorcinol alkylpyrone long-chain fatty acid cyst A zotobacter vinelandii is a Gram-negative nitrogen-fixing soil bacterium that differentiates into metabolically dormant cysts under adverse environmental conditions (1). During en- cystment, a considerable portion of the membrane phospholipids are replaced by phenolic lipids, alkylresorcinols and alkylpy- rones, which consist of polar aromatic rings and hydrophobic alkyl chains (Fig. 1 A ) (2, 3). The amphiphilic nature of the phenolic lipids contributes to the formation of stable monomo- lecular layers in vitro (4), and thus the phenolic lipids presumably allow the cysts to resist desiccation and heat. We previously revealed that the ars operon is essential for the biosynthesis of phenolic lipids in A. vinelandii (5). The ars operon directs the synthesis of two type III PKSs, ArsB and ArsC, and two putative type I FASs, ArsA and ArsD (Fig. 1 B ). Type III PKSs are simple homodimeric proteins that synthesize aromatic polyketides in plants, fungi, and bacteria (6, 7). ArsB and ArsC
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