title: Functional characterization of an ACCase subunit from the diatom Phaeodactylum tricornutum expressed in Escherichia coli.
abstract: The marine diatom Phaeodactylum tricornutum, a widely used forage species, has a storage lipid content of up to 30% dry cell weight. To explore the mechanism behind the high storage lipid accumulation in this diatom, acetyl-CoA carboxylase (ACCase), which catalyzes the first committed step of the fatty acid biosynthetic pathway, was characterized in this study. A homogeneous type of ACCase (PtACC) was identified from P. tricornutum by homology searches. The first exon of the ACCase gene (PtACC-1) was cloned. PtACC-1 was fused with a Myc epitope tag and cloned into plasmid pMD18 driven by the LacZ promoter and expressed in Escherichia coli. The expression of the PtACC-1-Myc protein was verified by Western blot. The neutral lipid content in transformed E. coli increased substantially by twofold as determined by Nile red fluorescent dye staining. Concomitantly, ACCase activity increased by 1.72-fold. The fatty acid composition, analyzed by GC-MS, demonstrated a significant difference in the ratio of saturated fatty acids and monounsaturated fatty acids (MUFAs). MUFAs of PtACC-1 expressing cells increased by 13%. This study represents the first characterization of the key domains of ACCase from a diatom and demonstrates high neutral lipid accumulation in E. coli expressing PtACC-1, providing an additional genetic resource with the potential for biodiesel development.
authors.affiliation: Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Jinan University, Guangzhou, PR China.
authors.forename: Wei-Hong
authors.initials: WH
authors.lastname: Xie
authors.forename: Fei
authors.initials: F
authors.lastname: Pang
authors.forename: Ying-Fang
authors.initials: YF
authors.lastname: Niu
authors.forename: Meng-Han
authors.initials: MH
authors.lastname: Zhang
authors.forename: Wei-Dong
authors.initials: WD
authors.lastname: Yang
authors.forename: Jie-Sheng
authors.initials: JS
authors.lastname: Liu
authors.forename: Dao-Guang
authors.initials: DG
authors.lastname: Yan
authors.forename: Hong-Ye
authors.initials: HY
authors.lastname: Li
mesh_terms: D000103:Acetyl-CoA Carboxylase; D003001:Cloning, Molecular / Q000379:methods; D017377:Diatoms / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005091:Exons / Q000235:genetics; D005227:Fatty Acids / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression / Q000235:genetics; D010802:Phylogeny; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits / Q000235:genetics* / Q000378:metabolism
chemical_list: D005227:Fatty Acids; D021122:Protein Subunits; D000103:Acetyl-CoA Carboxylase