| ?-carotene is a kind of natural carotenoid which is widely distributed in green vegetables,such as carrots and papaya.It is found primarily in the all-trans configuration in the nature,which has 11 conjugated carbon-carbon double bonds.P-carotene has predominantly functions of scavenging of free radicals,resisting oxidation,and quenching singlet oxygen,and it can act great roles on preventing cancer and anti-aging.Yarrowia lipolytica as a kind of oleaginous organism,it has a clear genetic background,abundant lipid,rich ?-carotene synthesis precursors in cell,higher safety.In the fields of the ?-carotene transgenic research,Y.lipolytica has many advantages and obtains more and more attentions and applications.Blakeslea trispora produces ?-carotene as the final product and also accumulates lycopene as an intermediate.In this work,several carotenogenic genes-phytoene dehydrogenase gene(carB),phytoene synthase/lycopene cyclase gene(carRA)of B.trispora were colned and expressed in Y.lipolytica and ?-carotene biosynthesis pathway was researched.The main contents and results are as follows.(1)Construction of ?-carotene biosynthetic pathway in Y.lipolytica.CarRA and carB cDNAs of B.tripora,as well as the GGS1 of Y.lipolytica was cloned and inserted into pJN44 to construct the expression plasmids native pJN44-carRA/carB,native pJN44-carRA/carB/GGS1,which were expressed in Y.lipolytica.The codon usage of exogenous carRA and carB genes was optimized.And the expression plasmids opt pJN44-carRA/carB,opt pJN44-carRA/carB/GGS1 were co-constructed and co-expressed with GGS1 in Y.lipolytica.The biomass was determined by shaking flask fermentation,and ?-carotene was extracted and analyzed.HPLC results showed that there was no p-carotene in native AB and native ABG strains,the content of?-carotene was 0.0089 mg/g DCW in opt AB strain,and the content of ?-carotene was 6.16 mg/g DCW in opt ABG strain.(2)The co-expression of opt carRA/carB,GGS1 and the key gene tHMG of MVA pathway.Bioinformatics analysis of HMGR and tHMG(HMGR catalytic domain fraction)were performed by ExPASy software.The results of bioinformatics analysis showed that the coding region sequence contains a complete ORF(3000 bp),the first half of HMGR had more transmembrane regions.Secondary structure of HMGR and tHMG protein were composed of a-helix and random coil.And its tertiary structure indicated that both proteins exist in the state of homotetramer,and the catalytic domain is in the 441-875 amino acids residues.The results of laser conf'ocal microscopy showed that the fusion protein pJN44-tHMG/GFP had green fluorescence at 488 nm,which indicating that the tHMG gene has been expressed and has a function.On the basis of the expression plasmid opt pJN44-carRA/carB/GGS1,the expression plasmid opt pJN44-carRA/carB/GGS1/tHMG,which containing tHMG gene,was constructed and transf'ormed into Y.lipolytica to obtain recombinant opt ABGtH strain.The content of ?-carotene was 26.03 mg/g DCW in opt ABGtH strain by shaking flask experiment.(3)The optimum strain(opt ABGtH strain)was cultured by fermentor,and isolated and purified.The results of full-wavelength scanning showed that the maximum absorption peak of the recrystallized product was at 450 nm,which was consistent with the ?-carotene standard.The results of HPLC showed that the peak time(Rt = 20.307 min)of the recrystallized product was consistent with that of?-carotene standard(Rt = 20.233 min).The results of IR analysis showed that the characteristic peak position of recrystallized product(3408 cm-1,1622 cm-1,1452 cm-1,962 cm-1,611 cm-1).Finally,the fermentation product was determined to be?-carotene.In this study,three strains of recombinant Y.lipolytica with ?-carotene synthesis ability were constructed,which provided the basis for further study on how to improve the yield of P-carotene,and also provided conditions for other carotenoids synthesis in Y.lipolytica. |
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