title: Cloning, characterization, and expression analysis of acyl-acyl carrier protein (ACP)-thioesterase B from seeds of Chinese Spicehush (Lindera communis).
abstract: Acyl-acyl carrier protein (ACP) thioesterases (TE EC 3.1.2.14) are fatty acid biosynthesis key enzymes that determine fatty acid carbon chain length in most plant tissues. A full-length cDNA corresponding to one of the fatty acyl-ACP thioesterase (Fat) genes, designated LcFatB, was isolated from developing Lindera communis seeds using PCR and RACE with degenerate primers based on conserved sequences of multiple TE gene sequences obtained from GenBank. The 1788 bp cDNA had an open reading frame (ORF) of 1260 bp encoding a protein of 419 amino acids. The deduced amino acid sequence showed 61-73% identity to proteins in the FatB class of plant thioesterases. Real-time quantitative PCR analysis revealed that LcFatB was expressed in all tissues of L. communis, with the highest expression in the developing seeds 75days after flowering. Recombinant pET-MLcFatB was constructed using the pET-30 a vector and transformed into Escherichia coli BL21(DE3)△FadE, a strain that deleted the acyl-CoA dehydrogenase (FadE). SDS-PAGE analysis of proteins isolated from pET-MLcFatB E. coli cells after induction with IPTG revealed a protein band at ~40.5kDa, corresponding to the predicted size of LcFatB mature protein. The decanoic acid and lauric acid contents of the pET-MLcFatB transformant were increased significantly. These findings suggest that an LcFatB gene from a non-traditional oil-seed tree could be used to function as a saturated acyl-ACP thioesterase and could potentially be used to modify the fatty acid composition of seed oil from L. communis or other species through transgenic approaches.
authors.affiliation: Lab of Systematic Evolution and Biogeography of Woody Plants, College of Nature Conservation, Beijing Forestry University, Beijing 100083, PR China.
authors.forename: Shubin
authors.initials: S
authors.lastname: Dong
authors.affiliation: Baoshan City Forestry Bureau, Baoshan 678000, China.
authors.forename: Jiacong
authors.initials: J
authors.lastname: Huang
authors.affiliation: Lab of Systematic Evolution and Biogeography of Woody Plants, College of Nature Conservation, Beijing Forestry University, Beijing 100083, PR China.
authors.forename: Yannan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Lab of Systematic Evolution and Biogeography of Woody Plants, College of Nature Conservation, Beijing Forestry University, Beijing 100083, PR China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Lab of Systematic Evolution and Biogeography of Woody Plants, College of Nature Conservation, Beijing Forestry University, Beijing 100083, PR China.
authors.forename: Shanzhi
authors.initials: S
authors.lastname: Lin
authors.affiliation: Lab of Systematic Evolution and Biogeography of Woody Plants, College of Nature Conservation, Beijing Forestry University, Beijing 100083, PR China. Electronic address: zxzhang@bjfu.edu.cn.
authors.forename: Zhixiang
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003652:Decanoic Acids / Q000378:metabolism; D020869:Gene Expression Profiling; D007850:Lauric Acids / Q000378:metabolism; D027443:Lindera / Q000201:enzymology*; D008969:Molecular Sequence Data; D010802:Phylogeny; D012639:Seeds / Q000201:enzymology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013869:Thiolester Hydrolases / Q000096:biosynthesis / Q000145:classification / Q000235:genetics* / Q000378:metabolism
chemical_list: D003652:Decanoic Acids; D007850:Lauric Acids; C030358:lauric acid; C031071:decanoic acid; D013869:Thiolester Hydrolases; C044320:thioesterase B; C018748:oleoyl-(acyl-carrier-protein) hydrolase
keywords: E. coli; FatB class thioesterases; Fatty acid; Heterologous expression; Lindera communis