Study On Biosynthesis Of Campesterol In Recombinant Yarrowia Lipolytica

Phytosterols,an important steroid compound with physiological activity,play a crucial roles in lowering total plasma cholesterol,anti-cancer,anti-oxidation and parhormone.Phytosterols and their derivatives have been widely used in health foods,medicines and cosmetics.Yeast produces more ergosterol naturally which has similar chemical structure to phytosterols,and ergosta-5,7-dienol is a common precursor of ergosterol and phytosterol.Therefore,it's feasible to synthesize phytosterols based on the ergosterol synthesis pathway in yeast through genetic engineering technology.This study developed a method for the determination of sterols in microorganisms by GC-MS/SIM;Yarrowia lipo lipolytica of was selected as the host to construct a producing campesterol strain YL-PE,and the fermentation product was identified by GC-MS;As a kind of oleaginous organism,Y.lipolytica lipid droplets provides more storage for the accumulation of steroid compounds,and relieves the adverse effect of cell membrane fluidity caused by excess accumulation of sterols.However,the synthesis of fatty acid and campesterol share common precursors such as acetyl-CoA.That is,the intracellular lipids have both promoting and competing effects on the synthesis of campesterol.Therefore,how to make full use of the favorable factors for the synthesis of sterols by lipid accumulation to further increasing the sterols content is a basic theoretical problem.In this study,we constructed the campesterol synthesis pathway in different lipid yield strains.The relationship between lipid content and campesterol accumulation was studied,the multicopy plasmids of DHCR7 were constructed and transformed to improve campesterol content.Finally the fermentation of the optimum strain in a 5 L fermenter was studied.The main results were as follows:(1)Based on the sterol mixed standards and Y.lipolytica polf fermentation broth,we developed and validated a GC-MS with SIM method for the determination of sterols in microorganisms.The results showed that the resolution R between chromatographic peaks was greater than 1.2;The tested components had a good linear relationship between 3-200 ?g/mL in SIM mode,and the detection limit of each component was greater than 5.0 ?g/mL;The results of precision,stability,and spike recovery indicated that the RSD was 8.14%when the samples were determinated repeatedly,RSD was 6.96%within 48 h,and the recoveries of different gradients were between 80.69-91.41%.The method validation results demonstrated that this method has excellent resolution,linear range,repeatability,stability and accuracy,and can meet the detection requirements of sterol compounds in yeast.(2)Based on the host Y.lipolytica polf,the YL-PE strain capable of synthesizing campesterol was constructed,by knocking out the ERG5 gene in the ergosterol synthesis pathway and expressing the optimized DHCR7 gene from Xenopus laevis.After identification fermentation product of the control strains polf,polf-ERG5' and the recombinant strain YL-PE by GC-MS,it was found that knocking out ERGS prevented the synthesis of ergosterol and inhibited the growth of the host,expressing DHCR7 genes converted ergosta-5,7-dienol to campesterol.The strain YL-PE was cultivated and 485?g/g DCW campesterol was achieved in shake flask;the maximum yield of campesterol in 5 L Bioflo fermentor was 15.2 mg/L.(3)Based on three strains(polf,polf-mfe",polf-pex10--mfe-)of different lipid content,the effect of lipid on the synthesis of campesterol was studied,and multi-copy plasmid DHCR7 were expressed to further enhance campesterol content,.The results shown that,with the increasing of lipid content,the campesterol content were 757.8,480 and 589.9 ?g/g in these three strains(polf-mfe--ERG5-?polf-ERG5-?polf-pex10--mfe--ERG5')respectively.The highest campesterol content 757.8 ?g/g was obtained in strain polf-mfe--ERG5-which had the least lipid content 42.3 mg/g.Therefore,there was no direct correlation between lipid and campesterol content.It might due to that the sterols content was relatively small,and sterols not stored in lipid droplets as the form of esterified,the effect of lipid to increase sterols content was not fully reflected.At the same time,campesterol content was higher in the strain with MFE knocking out than the control strain polf-ERG5-.The cells involved in the repair of cell membranes by enhancing the synthesis of campesterol,and alleviated the damage caused by knocking out MFE.In addition,increasing the copy number of DHCR7 enhanced the relative transcript level of ERG4 and DHCR7,allowing the carbon metabolism to flow more towards campesterol,as a result the campesterol content increased significantly.The strains po1f-pex10--mfe--ERG5-with expressing the multi-copy of DHCR7 obtained the highest campesterol content,1062 ?g/g DCW,the yield 49.2 mg/L was achieved in 5 L Bioflo fermentor.In conclusion,this study developed a method for the determination of sterols in microorganisms by GC-MS/SIM,constructed a strain YL-PE for the synthesis of campesterol.We found that knocking out the MFE gene and increasing the copy number of DHCR7 were effective ways to increase the biosynthesis of campesterol in Yeast.This study laid an application basis for the production of campesterol in recombinant yeast.