Study On Screening And Cloning And Expression Of Key Enzyme Genes In Fatty Acid Synthesis Of Functional Lipid-producing Endophytic Fungus From Wild Chinese Torreya Grandis

In modern society,research and development of new functional lipids has become an effective way to improve the health of human beings.Torreya grandis is a rare tree species of economic value,its seeds have a high lipid content with a high proportion of unsaturated fatty acids.However,Torreya seeds require 3 years from blossoming to maturation,and they are scarce and expensive.It is difficult to obtain of the special nutrients in seeds of Torreya grandis by traditional methods.Recent studies demonstrated that certain endophytic fungi can produce metabolites similar to those produced by the host plant.In this study,the fatty acid composition of functional lipids produced by endophytic fungi isolated from wild Chinese Torreya grandis,fatty acid and unsaponifiable matter composition were determined.Experimental results proved that the functional lipids of endophytic fungi could be applied to production instead of Torreya grandis.Through this study,it was confirmed that the endophytic fungus could produce lipids to those produced by the host and acetyl coenzyme A is an important factor to affect the synthesis of fatty acids.The purpose of improving the lipid content was achieved.The goal of the present work was the microorganism fermentation techniques could be used for the production of lipids through optimization of fermentation conditions,comparison of lipid production in the culture medium with cellulose,and the function of ACC and inhibitors mechanism.The following is the main research results:(1)Isolation,identification and lipid content of lipid-producing endophytic fungus from Torreya grandis.106 strains endophytic fungal were isolated from wild Chinese Torreya grandis.Among these,47 strains can product lipids by means of Sultan black dye.The XF-38 strain had the highest lipid content at 17.68%.The morphology and internal transcribed spacer rDNA of high lipid production strains were determined,and the phylogenetic tree of ITS was built.The results showed that the ability of lipid production was related to tree age,species and region.The most important factor is tree age,the most kinds of high lipid production endophytic fungus were all in the oldest tree.The similar phylogenetic consisted in the endophytic fungus from the same tree,7 strains whose lipid content above 7.5%were isolated from 500-year-old Torreye tree.(2)Fatty acid and unsaponifiable matter composition of 15 endophytic fungus were determined by GC-MS.Comparative analysis of the fatty acid composition of lipids produced by endophytic fungus and Torreya seeds showed that the two sources shared some of the same components,unsaturated fatty acids were the main components,including linoleic acid,oleic acid,and so on.In addition,sciadonic acid(5,11,14-eicosatrienoicacid)which has special activity function was discoved in fatty acid samples.Squalene,ergosterol and ?-sitosterin were the main unsaponifiable components and the total content was below 0.01%.The results showed that the lipids fromendophytic fungus could be applied to production practice as functional lipids.Functional fatty acids such as inoleic acid and sciadonic acid were successfully separated by silica gel column chromatography and HPLC.(3)Improve the functional lipid production of XF-38 strain(Endophytic fungi from Torreya grandis had the highest functional lipid content including sciadonic acid),through optimization of fermentation conditions and comparison of lipid production in the culture medium with cellulose,which is the basis for practical application.The optimized fermentation conditions were determined with 28?,pH6.5,180 rpm,3.0%glucose and 0.5%yeast powder,and the lipid production was 3.05 g/L.Next,1.5%glucose was added to the fermentation liquid after 60 h culture,the lipid production was 3.35 g/L,which was increased 9.8%than before.XF-38 strain could produce lipid in the culture medium with Torreya leaves and maize stalk,and the lipid production was reached 2.01 g/L with Torreya leaves by cellulase.Sciadonic acid could be up to 0.22 g/L with the optimum culture conditions.(4)Acetyl-CoA carboxylase(ACC)and fatty acid dehydrogenase are the most important enzyme in fatty acid synthesis.Fatty acid composition showed that the fatty acid dehydrogenase was not the main factor affecting the lipid production.So the acetyl-CoA carboxylase(ACC)of the endophytic fungi XF-38 strain was discussed as the important factor on lipid-producing.The ACC enzyme activities of Torreya seed and XF-38 strain were determined,and the results were 2259 U/L ? 1027 U/L.One of the metabolites named benzoic acid 2,2,6,6-tetramethyl-4-piperidinylester was confirmed the inhibition of ACC,when the concentration reached 500 mg/L,the ACC was inactivated.Finally,cDNA sequence of CT functional domain of XF-38 strain was cloned and introduced into the prokaryotic expression vector PKC1139 and successfully expressed in Escherichia coli BL21(DE3),recombinant protein was obtained,and it could be decomposed by benzoic acid 2,2,6,6-tetramethyl-4-piperidinylester.This result provided a valuable material to study the function of ACC and inhibitors mechanism.