Clone And Functional Verification Of △9-and △15-Fatty Acid Desaturase Gene From Lipomyces Kononenkoae

Fatty acids are important nutrient of human. Among them, polyunsaturated fatty acids (PUFAs) are particularly important for human physical activities. Linoleic acid (LA) andα- linoleic acid (ALA) are substrates of the PUFAs, which are essential fatty acids (EFA) for human. Microbial could proliferate rapidly by lot cost materials without much environmental impaction, produce PUFAs by oleaginous microbial fermentation could be lots of advantages. So that it could be good prospects to develop genetically modified (GM) oleaginous microbial strains for very long chain PUFAs production.In this study, the coding sequences ofΔ9- andΔ15-fatty acid desaturase gene (lkfad9 and lkfad15) were cloned from Lipomyces kononenkoae and heterologous expressed in yeast expression systems.The main results of this study are as follows:1. The fermentation condition of Lipomyces kononenkoae was optimized by medium composition and culture condition optimization to product high yield polyunsaturated fatty acids.2. The core DNA sequence of lkfad9 and lkfad15 were amplified using degenerate primers according to the conservative amino acid sequences. And then, the total coding sequences of these two genes were cloned using Genome-Walking and reverse transcript PCR techniques. Bioinformatic analysis results showed that these two genes are members of the fatty acid desaturase superfamily which reveal high homology to the corresponding desaturase genes of the other yeasts.3. The coding sequence of lkfad15 was transformed into Pichia pastoris GS115 and Saccharomyces cerevisiae INVScI respectively. The recombinant yeasts were cultured and induced to express lkfad15. Fatty acids components of the recombinant yeasts were analyzed by gas chromatography. The results shown that Ikfad15 could increase the ALA content (4.68% to 21.93%) in GS115 by transform LA to ALA, and it even could produce both LA and ALA in INVScI use only oleic acid as substrate. It reveals that LKFAD15 have the functions of bothΔ12- andΔ15 -desaturases.This work proved the LKFAD15 has the ability of bothΔ12- andΔ15 -desaturase, it is the basis work for productω-3 PUFAs in GM organisms using bi-functional desaturase LKFAD15.