Expression of Arabidopsis thaliana thioesterase gene in Pichia pastoris / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 115-122, 2015.
Article
in Chinese
| WPRIM
| ID: wpr-345521
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Pichia
/
Plasmids
/
Thiolester Hydrolases
/
Transformation, Genetic
/
Recombinant Proteins
/
Polymerase Chain Reaction
/
Arabidopsis
/
Electroporation
/
DNA, Complementary
/
Arabidopsis Proteins
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2015
Type:
Article
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