Isolation of New Polyketide Synthase Gene Fragments and a Partial Gene Cluster from East China Sea and Function Analysis of a New Acyltransfrase |
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Authors: | Yu-Liang Jiao Liang-Hua Wang Xiao-Yi Dong Yu-Feng Chen Ying Zong Yun Gao Na Ren Ai-Yun Guo Xin-Qun Zhang Bing-Hua Jiao |
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Affiliation: | (1) Department of Biochemistry and Molecular Biology, College of Basic Medical Science, 800 Xiangyin Road, Shanghai, 200433, People’s Republic of China;(2) Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai, 200433, People’s Republic of China |
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Abstract: | Using the consensus-degenerate hybrid oligonucleotide primer polymerase chain reaction method, 26 new ketoacyl synthase (KS) fragments were isolated from a marine sediment sample in the East China Sea (ECS) and analyzed by construction of a phylogenetic tree. With a digoxigenin-labeled KS gene fragment used as a probe, a partial polyketide synthase (PKS) gene cluster was isolated and identified by hybridization screening of a marine sediment sample metagenome fosmid library constructed for this study. A new acyltransferase (AT) gene was cloned from the PKS gene cluster and heterogeneously expressed as a protein fused to maltose-binding protein (MBP). Ultraviolet spectrophotometry was used to study the binding of the MBP–AT fusion protein and single AT domain to substrates using MBP and bovine serum albumin as control proteins. Binding constants (Ka, per micromolar) were calculated and used to analyze the substrate specificity of the acyltransferase. We concluded that there are many unrevealed new PKS gene clusters in marine sediments in the ECS. The acyltransferase is presumably an acetyltransferase from a new PKS gene cluster. |
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Keywords: | Acetyltransferase Acyltransferase Ketoacyl synthase Polyketide synthase Substrate specificity |
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