Cloning And Identification Of Δ6Fatty Acid Desaturase Gene From Isochrysis Sphaerica And Fermentation Process Optimization Of Oleaginous Yeast Lipomyces Kononenkoae

Polyunsaturated fatty acids (PUFAs) have some useful biological functions,such as regulation andcontrol of lipid metabolism, anti-tumor and enhancing immunity of human. Marine algae is rich inPUFAs. There is a lot of problems such as being affected by environment and unstable yield in makinguse of microalgae to produce PUFAs.As people realize the importance of PUFAs and the deficit of PUFAs, more researchers try tochange the metabolize pathway by genetic engineering technology in order to get more PUFAs.AΔ6-fatty acid desaturase gene from Isochrysis sphaerica is a good gene resourse for constructing.Oleaginous microorganisms which are good host for fatty acid gene expressing has advantages inproducing oil such as short production cycle, low cost, easily being cultivated in large scale.In this research, a Δ6-fatty acid desaturase gene from Isochrysis sphaerica was cloned andidentified; the oil-producing conditions of Lipomyces kononenkoae on flask fermentation level.Afoundation is going to be built by transforming Δ6-fatty acid desaturase gene to oil-producing yeastthrough so as to combine high lipid conten and Polyunsaturated fatty acids. The results of this study aresummarized as follows:(1) A Δ6-fatty acid desaturase gene which named isfad6was first cloned from Isochrysis sphaericavia degenerate PCR, chromosome walking and RACE. isfad6was1398bp in length, encoding465amino acid.(2) by transforming isfad6to Pichia pastori GS115, the recombinant Pichia pastori fatty acidswere analysised.(3) The oil-producing conditions of Lipomyces kononenkoae were optimized on flask fermentationlevel by the single-factor experiment. The highest lipid yield was obtained with ammonium sulfate andglucose as nitrogen source and carbon source, respectively. The optimum culture conditions fortemperature, inoculation, initial pH value and C/N ratio were28℃,10%,7.0and99, respectively. Batchculture result showed that the biomass, lipid content and lipid yiled of Lipomyces kononenkoae couldreach to7.01g/L,15.91%,1.12g/L after optimization, respectively. The results of flask fed-batchcultures indicated the dry biomass and lipid yiled of Lipomyces kononenkoae could further reach to24.13g/L and3.06g/L respectively, increasing372%and363%respectively.