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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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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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Abbreviations

CODEHOP PCR:

consensus-degenerate hybrid oligonucleotide primers PCR

BSA:

bovine serum albumin

PKS:

polyketide synthase

ECS:

the East China Sea

CoA:

coenzyme A

DIG:

digoxigenin

KS:

ketoacyl synthase

PFGE:

pulsed-field gel electrophoresis

AT:

acyltransferase

ACP:

acyl carrier protein

MBP:

maltose-binding protein

PBS:

phosphate-buffered saline

UV:

ultraviolet

SDS-PAGE:

sodium dodecyl sulfate polyacrylamide gel electrophoresis

IPTG:

isopropyl β-d-1-thiogalactopyranoside

NBT/BCIP:

nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate

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Acknowledgment

This work was sponsored by the Natural Science Funds of the People’s Republic of China (grant number 30670043) and the National High Technology Research and Development Program of China (863 Program; grant number 2006AA09Z434).

Jiao, Wang and Dong contributed equally to this work.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, College of Basic Medical Science, 800 Xiangyin Road, Shanghai, 200433, People’s Republic of China

    Yu-Liang Jiao, Liang-Hua Wang, Xiao-Yi Dong, Ying Zong, Yun Gao, Na Ren, Ai-Yun Guo, Xin-Qun Zhang & Bing-Hua Jiao

  2. Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai, 200433, People’s Republic of China

    Yu-Feng Chen

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  1. Yu-Liang Jiao
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  2. Liang-Hua Wang
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Correspondence to Bing-Hua Jiao.

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Jiao, YL., Wang, LH., Dong, XY. et al. Isolation of New Polyketide Synthase Gene Fragments and a Partial Gene Cluster from East China Sea and Function Analysis of a New Acyltransfrase. Appl Biochem Biotechnol 149, 67–78 (2008). https://doi.org/10.1007/s12010-007-8053-7

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  • DOI: https://doi.org/10.1007/s12010-007-8053-7

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