Expression In Yeast Of△5Fatty Acid Desaturase Gene From Mortierella Isabellina Mutant Strain

Arachidonic acid (ARA) is synthesized by dehydrogenation and carbon chain extendingreaction of linoleic acid and α-linolenic acid, and that’s the process△5-fatty acid dehydrogenasecatalyzed the reaction had desaturation of big effect, controlled the enzyme reaction rate for ARAsynthesizing. This study uses pBR322as cloning vector, the Mortierella isabellina total genomicRNA was extracted,△5-fatty acid desaturase gene was cloned, and then pGAPZαA asexpression vector recombined with△5-fatty acid desaturase gene and recombined plasmid wasconverted into the Pichia-SMD1168is expression of Pasteur, and the verification test was carriedout using DHGLA as substrate to measure conversion rate. For the subsequent to fatty aciddehydrogenase functional expression and new strains build research theory basis.In this study, the cloned gene fragment was1364nucleotides. This gene fragment and thepublished Mortierella isabellina△5desaturase gene (AF067654) was highly homologous, Thereis a complete open reading frame (1341BP), encoding446amino acids. In the series, there are3highly conserved histidine clusters of unique fatty acid dehydrogenase. The results showed that,the gene fragments for△5-fatty acid desaturase gene.The transformation was induced with recombinant△5-fatty acid desaturase geneengineering yeast strain by electroporation, and identified, the molecular weight of the expressedproduct was about50kDa by SDS-PAGE electrophoresis and Western blot identification, andpossessed biological activity. By fermentation validation test,GC using recombinant yeastfermentation liquid engineering analysis of fatty acids，it was confirmed that the engineeringstrain capable of producing arachidonic acid, Arachidonic acid in the fermentation liquid wasaccounted total fatty acid content for9.53%and the substrate of high gamma linolenic acidconversion rate reached51.7%.