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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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.
KeywordsAcetyltransferase Acyltransferase Ketoacyl synthase Polyketide synthase Substrate specificity
- CODEHOP PCR
consensus-degenerate hybrid oligonucleotide primers PCR
bovine serum albumin
the East China Sea
pulsed-field gel electrophoresis
acyl carrier protein
sodium dodecyl sulfate polyacrylamide gel electrophoresis
nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate
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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