Construction Of The Very Long-chain Monounsaturated Fatty Acid Biosynthetic Pathway

The neivonic acid(C24:1)is a tetracosenic acid with a double bond on C15 and it is known as the double effect in the world which can repair damaged nerve fibers and regenerate nerve fiber.Some research data indicate that neivonic acid can delay nerve aging and promote development of brain.It plays an important role in the processing of information between nerve cells,and has a good effect on cardiovascular and cerebrovascular diseases,immune system diseases,and certain skin diseases.However,it is difficult for the human body to synthesize nervonic acid and it is mainly ingested from the outside world.However,the production of nervonic acid is low and the market demand is large at present.Therefore,the development of biosynthesis methods for nervonic acid has become an important research field.In this study,Saccharomyces cerevisiae YS58 was selected as the starting strain,and the genetic engineering strains involved in the elongation of monounsaturated fatty acids,including FAE1 and KCS,were constructed.The effect of different types of promoters on the expression of foreign genes was explored and the fuinction of the KCS and FAE1 was studied.The extraction method of very long chain monounsaturated fatty acids from Saccharomyces cerevisiae was determined.At the same time,the transcription factor Mga2 was used to regulate the synthesis of unsaturated fatty acids of Saccharomyces cerevisiae.The functions of FAEI and KCS genes were clarified through the fermentation,and the FAEe-iCS engineering strain was established.The FAEI-KCS strain eventually achieved biosynthesis of nervonic acid in Saccharomyces cerevisiae.1.The single-gene engineering strains of FAE1 and KCS were constructed,and the function of two genes in the process of the elongation of very long chain mo no unsaturated fatty acids were preliminarily determined through the fermentation result.The FAE1 and KCS engineering strains were constructed using the constitutive promoter PGK and the galactose-inducible promoter GAL1.The fermentation results showed that the constitutive promoter PGK can promote the expression of FAEl and KCS more compared with the galactose-inducible promoter GAL L.The FAE1 engineering strain eventually promotes the synthesis of C20:1 and C22:1,while KCS engineering strain can promote the synthesis of C20:1,C22:1 and C24:0,and the content of C20:1 and C22:1 were lower than that of FAE1 engineering strain,indicating that FAE1 plays a major role in the extension of C20:1 and C22:1,while KCS gene may play a major role in the extension of C24.2.The effect of multi-gene combination on the synthesis of nervonic acid was explored,and according the synthetic route of nervonic acid,a double-gene engineering strain of PTEF1-FAE1-TCYC-PPGK-KCS-TcYC was constructed by the fennentation results of single-gene engineering strain to explore the use of two-gene linkage for the enlongation of very long chain mo no unsaturation fatty acids.In addition,the Mga2 transcription factor was used for regulating the synthesis of unsaturated fatty acids of Saccharomyces cerevisiae to enhance the synthesis of nervonic acid.Fermentation results showed that,at a culture temperature of 220C for 4 days,PTEF1-FAE1-TCYC、PPGK-KCS-TCYC engineering strain and PTEF1-Mga2-TCYC-PPGK-FAE1-TCYC-PPGK-KCS-TCYC engineering strain eventually were detected production of nervonic acid,but the level of nervonic acid in the PTEF1-Mga2-TCYC-PPGK-FAE1-TCYC-PPGK-KC S-TCYC engineering strain did not increase significantly compared with PPGK-FAE1-TCYC-PPGK-KCS-TCYC engineering strain.3.The culture conditions were optimized to promote the synthesis of very long chain monounsaturated fatty acids,through the preliminary exploration the synthesis of nervonic acid by exogenously adding different fatty acids,malic acid,citric acid and bio tin,we found that the external addition of oleic acid,Malic acid and biotin had a good promoting effect on the synthesis of nervonic acid.Through single factor optimization,we determined that the final concentration of oleic acid was 1.5 mM,malic acid was 0.2%,and biotin concentration was 1.5 μg/mL,and the content of C22:1 ultimately increased by 31%,77%,and 75%,respectively,and the content of C24:lincreased by 206%,140%,and 143%respectively.By using segmented culture,it was finally determined that the strains were cultured at 28℃ for 48 hours and then were transferred to 22℃ and cultured for 4 days.The contents of C22:1 and C24:1 were increased by 38%and 42%,respectively.Through the optimization of fermentation time,it was determined that the content of C22:1 and C24:1 had a significant increase on the sixth day,which was increased by 57%and 200%,respectively.