Expression of delta6-fatty acid desaturase gene from Mortierella alpina in Pichia pastoris / 生物工程学报

ABSTRACT

Gamma-linolenic acid (GLA, C183delta6 ,9,12), an essential polyunsaturated fatty acid, plays an important role in hormone regulation and fatty acid metabolization. Delta6-fatty acid desaturase (D6D) is the rate-limiting enzyme of the desaturation of linoleic acid (C182delta9,12) in the production of gamma-linolenic acid. A deficiency of GLA may have occurred when delta6-fatty acid desaturase activity decreases in aging, stress, diabetes, eczema, and some infections. To establish a new expression system for delta6-fatty acid desaturase gene in Pichia pastoris, which is an increasingly popular heterologous gene expression system, a gene encoding delta6-fatty acid desaturase from Mortieralla alpina was isolated by PCR amplification. The PCR product was then digested by EcoR I and Not I and subcloned into the intracellular expression vector pPIC3.5K to generate the recombinant vector pPIC3.5K-MA6. The resulting vector was linearized by Sac I and electroporated into P. pastoris SMD1168 (his- pep-) host cells. After electroporation, aliquots were spreaded on the MDS plates and incubated at 30 degrees C for three days until colonies appeared. Those transformants were subsequently screened for clones with high copy number by using the YPD plates containing G418. To identify the D6D constructs that were produced, chromosomal DNA of the transformants were prepared and used as template for PCR with the primer 5' AOX and 3' AOX. The PCR product of Mut+ recombinants was shown as a band of 1.38 kb of D6D gene and the product of 2.2 kb of AOX1 gene, while the product of Mut(s) transformants only was shown as a band of 1.38 kb of the D6D gene.To further confirm the transformants containing a functional D6D gene, the positive clones were selected and induced by methanol for expression. Those induced cultures were taken for analyses of the intracellular fatty acid composition by GC. The resultant chromatograms of fatty acid methyl esters showed that a novel peak was detected, which was not apparent in the case of control. Comparisons of the retention times of the newly yielded peaks with those of authentic standards have anticipated that the fatty acid is GLA. And this prospects was positively supported by definitive assignments of the compounds by GCMS analyses. Thus, the active delta6-fatty acid desaturase was expressed intracellularly in P. pastoris and gamma-linolenic acid reached 16.26% of the total fatty acid in recombinant P. pastoris strains. It was the first report about the expression of Mortieralla alpina D6D gene in P. pastoris.