title: [Expression, purification and characterization of a novel fatty acid synthase from Rhodosporidium toruloides].
abstract: Fatty acid synthase (FAS) catalyses the reaction between acetyl-CoA and malonyl-CoA to produce fatty acids. It is one of the most important enzyme in lipid biosynthesis. FAS of the oleaginous yeast Rhodosporidium toruloides has two acyl carrier protein (ACP) domains and a distinct subunit composition compared with FASs of other species. As ACP is a protein cofactor crucial for fatty acid chain elongation, more ACPs in the FAS may facilitate the reaction. To study the biochemical and structural properties of this novel FAS from R. toruloides, plasmids were constructed and transformed into Escherichia coli BL21 (DE3). The strain ZWE06 harboring plasmids pET22b-FAS1 and pET24b-FAS2 could co-overexpress the two subunits. The recombinant FAS was purified by sequentially using ammonium sulphate precipitation, sucrose density gradient centrifugation and anion exchange chromatography. The specific activity of the recombinant FAS was 548 mU/mg. The purified complex would be used to study enzyme kinetics and protein structure of FAS, and heterogeneous expression and purification will facilitate revealing the mechanism of this novel FAS with double ACPs.
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Zhu
authors.forename: Sufang
authors.initials: S
authors.lastname: Zhang
authors.forename: Xinping
authors.initials: X
authors.lastname: Lin
authors.forename: Wujun
authors.initials: W
authors.lastname: Liu
authors.forename: Zongbao K
authors.initials: ZK
authors.lastname: Zhao
mesh_terms: D000213:Acyl Carrier Protein; D001487:Basidiomycota / Q000201:enzymology*; D002845:Chromatography; D004926:Escherichia coli / Q000378:metabolism; D064429:Fatty Acid Synthases / Q000096:biosynthesis* / Q000235:genetics; D005227:Fatty Acids / Q000096:biosynthesis; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000213:Acyl Carrier Protein; D005227:Fatty Acids; D011994:Recombinant Proteins; D064429:Fatty Acid Synthases