Isolation of new polyketide synthase gene fragments and a partial gene cluster from East China Sea and function analysis of a new acyltransferase.
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.
Jiao
Yu-Liang
YL
Department of Biochemistry and Molecular Biology, College of Basic Medical Science, 800 Xiangyin Road, Shanghai 200433, People's Republic of China.
Wang
Liang-Hua
LH
Dong
Xiao-Yi
XY
Chen
Yu-Feng
YF
Zong
Ying
Y
Gao
Yun
Y
Ren
Na
N
Guo
Ai-Yun
AY
Zhang
Xin-Qun
XQ
Jiao
Bing-Hua
BH
D000217:Acyltransferases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002681:China; D004247:DNA / Q000235:genetics; D016521:Electrophoresis, Gel, Pulsed-Field; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D016678:Genome; D019015:Geologic Sediments / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D009792:Oceans and Seas; D010802:Phylogeny; D048630:Polyketide Synthases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D012623:Seawater / Q000032:analysis*; D016415:Sequence Alignment; D013379:Substrate Specificity
D048630:Polyketide Synthases; D004247:DNA; D000217:Acyltransferases