Expression of Arabidopsis thaliana thioesterase gene in Pichia pastoris.
- Author:
Zhaocheng HAO
;
Tengfei WANG
;
Zhongkui LI
;
Zikai HAO
;
Kun DAI
;
Ruiming WANG
- Publication Type:Journal Article
- MeSH:
Arabidopsis;
genetics;
Arabidopsis Proteins;
biosynthesis;
DNA, Complementary;
Electrophoresis, Polyacrylamide Gel;
Electroporation;
Pichia;
metabolism;
Plasmids;
Polymerase Chain Reaction;
Recombinant Proteins;
biosynthesis;
Thiolester Hydrolases;
biosynthesis;
Transformation, Genetic
- From:
Chinese Journal of Biotechnology
2015;31(1):115-122
- CountryChina
- Language:Chinese
-
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.
