Recombinant Expression Of ω3Fatty Acid Desaturase From Mortierella Alpina

The main source of long chain polyunsaturated fatty acids is the fish oil, but with theincreasing of world population and the pollution of marine environment the ω-3PUFAsprovided by marine fishery is withstanding huge stress and potential risks. Single cell oil as akind of economic security ω-3PUFAs alternative resource is gradually become the researchhotspot in the field of PUFAs. Mortieralla alpina is an industrial AA producing filamentousfungi with oil content up to50%of its dry cell weight. According to the PUFAs metabolicpathway in M.alpina, the ω3fatty acid desaturase is the key enzyme that catalyze the ω-6PUFAs to the ω-3PUFAs. Therefore, to understand the structure and function of this enzymeplays an important role for increasing ω-3PUFAs production.In recent years, membrane-bound fatty acid desaturases have been expressed in varioushosts, but none of the crystal structure have been identified. The reasons are as follows: first,the expression level of membrane protein is very low,which can only be used for qualitativeresearch; second, membrane protein is hard to purify thus makes is difficult to get highamount of purified protein for enzyme characteristics study; third, there is no mature in vitromembrane-bound fatty acid desaturase activity test system. As a consequence, it is veryurgent to find an efficient system for high amount and purity membrane-bound proteinexpression that paves the way for subsequent research. Cell free protein synthesis system asan open protein expression system is rapidly used to efficiently express membrane proteins. Itonly takes16~24h to get the proteins after the exogenous gene adding to the system, andbecause of avoiding the constraints of the cell membrane, the production of protein wassignificantly increased. Furthermore, exogenous addition of membrane component such asliposomes makes the purification step much more easy. Thus, cell free protein synthesissystem can be used as an important means of ω3fatty acid desaturase expression.In this paper, we choose the ω3fatty acid desaturase gene FADS15from M.alpinaATCC#32222as subject, using PichiaPinkTMexpression system and Wheat-germ cell freeprotein synthesis system respectively express the FADS15gene, then explored and identifiedthe conditions for FADS15soluble expression, at last make a study of the in vitro activityidentification system for the purified protein.Firstly, We use PichiaPinkTMexpression system and Wheat-germ cell free synthesissystem to in vitro and in vivo express ω3fatty acid desaturase,finally get0.13mg/mL and1.86mg/mL crude protein respectively.Secondly, by optimizing expressions method and liposome addition strategy to realizesoluble expression of ω3fatty acid desaturase, and finally determined that liposomes addingat10.5h with the final concentration of1.2mg/mL was the best strategy. The amount ofprotein can reach1.8mg/mL, wherein the soluble part is0.23mg/mL.Thirdly, the in vivo and in vitro expressed proteins were repectively purified through Histag affinity purification and Density gradient ultracentrifugation,8μg/mL and59μg/mLFADS15with90%purity were obtained, the relative yield is6.15%and25.65%.