title: [Expression of Arabidopsis thaliana thioesterase gene in Pichia pastoris].
abstract: Thioesterase catalyzes the hydrolysis of acyl-ACP and saturated fatty acyl chain. It plays a key role in the accumulation of medium chain fatty acids in vivo. In this study, to construct an engineering strain to produce MCFAs, the Arabidopsis acyl-ACP thioesterase gene AtFatA was amplified by PCR from cDNA of arabidopsis and double digested by EcoR I/Xba I, then linked to the plasmid digested with same enzymes to get the recombinant plasmid pPICZaA-AtFatA. We transformed the gene into Pichia pastoris GS115 by electroporation and screened positive colonies by YPD medium with Zeocin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the recombinant enzyme had a molecular of 45 kDa band which was consistent with the predicted molecular mass and we constructed the expression system of gene AtFatA in fungus for the first time. Under shake-flask conditions, Gas Chromatograph-Mass Spectrometer-computer results indicated that recombinant strain produced 51% more extracellular free MCFAs than the wild and its yield reached 28.7% of all extracellular fatty acids. This figure is 10% higher than the control group. The result provides a new way to produce MCFAs.
authors.forename: Zhaocheng
authors.initials: Z
authors.lastname: Hao
authors.forename: Tengfei
authors.initials: T
authors.lastname: Wang
authors.forename: Zhongkui
authors.initials: Z
authors.lastname: Li
authors.forename: Zikai
authors.initials: Z
authors.lastname: Hao
authors.forename: Kun
authors.initials: K
authors.lastname: Dai
authors.forename: Ruiming
authors.initials: R
authors.lastname: Wang
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000096:biosynthesis*; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation; D010843:Pichia / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D013869:Thiolester Hydrolases / Q000096:biosynthesis*; D014170:Transformation, Genetic
chemical_list: D029681:Arabidopsis Proteins; D018076:DNA, Complementary; D011994:Recombinant Proteins; D013869:Thiolester Hydrolases; C018748:oleoyl-(acyl-carrier-protein) hydrolase