Optimization Of Fermentation Conditions For The Red Pigment Branch Of Sporidiobolus Pararoseus And Key Enzyme Gene Transcription Level Analysis

Carotenoid is a kind of isoprene substance widely existing in nature.It has important biological functions and can be used as food colorant.It also has medicinal values such as antioxidant,anti-cancer and reducing cataract.The carotenoid synthesis pathway of Sporidiobolus pararoseus includes the yellow branch(β-carotene)and the red branch(torulene and turolarhodin).Among them,torulene and turolarhodin belong to the unique carotenoid of fungi,which has the characteristics of strong polarity,bright color,etc.,and its antioxidant capacity is better than that of β-carotene.Therefore,in this study,by optimizing the medium formulation and culture conditions,the contents of torulene and turolarhodin in S.pararoseus were increased.The key carotenoids synthetic enzyme gene of S.pararoseus : crt E,crt YB and crt I.The regulation of these three gene expression leads to the accumulation of the final pigment synthesis in different directions.CrtE is a GGPP synthetase that synthesizes the secondary metabolite precursor GGPP.CrtI is an octahydrolycopene dehydrogenase,which has a dehydrogenation function during carotenoid synthesis.Whether the cyclase domain in CrtYB(phytoene synthase-lycopene cyclase)participates in the synthesis of torulene is not yet clear.This study attempts to analyze the key sites and catalytic domains of the enzyme by truncation experiments.The main conclusions are as follows:(1)The optimized medium formula is glucose 20.0 g,ammonium sulfate 5.0 g,magnesium sulfate 0.5 g,yeast dip powder 0.2 g,potassium dihydrogen phosphate 1.0 g,culture temperature 28℃,0.1 M potassium dihydrogen phosphate-phosphoric acid Hydrogen dipotassium buffer was used to control p H 6.0,and torulene extract content was 747.94 μg/L,and turolarhodin content was 783.41 μg/L.(2)The expression of genes crt E,crt YB and crt I in the YPD,YAD and YAD optimized medium was different in the S.pararoseus NGR.The crt I gene expression in YAD-optimized was 17% more than YAD,but the expression level of crt YB was reduced to 21% of YAD,and the content of torulene and turolarhodin in YAD optimized medium was increased compared with YAD.Thus,when the ratio of crt I to crt YB reaxhed 1.48,the pigment synthesis proceeds to the red branch pathway,so the yeast cell accumu;ated more red pigments.(3)Bioinformatics analysis of CrtYB protein sequence of S.pararoseus NGR using BLAST revealed that there were six transmembrane hydrophobic regions at its amino terminus,two CrtY domains were predicted,and a CrtB functional hydrophilic domain waspredicted at the carboxy terminus.(4)PCR-amplified crt YB-145-597 and crt YB-286-597 truncated fragments containing Bam HI restriction sites;the gel recovery products crt YB-145-597 and crt YB-286-597 were ligated into the cloning vector p MD18 T.The recombinant plasmids p MD18T-crt YB-145-597 and p MD18T-crt YB-286-597 were successfully constructed and transformed into E.coli DH5α.The expression vector p ET30 a was successfully ligated,and the target fragment was successfully verified by sequencing.