ORF2 Gene Of Sulfolobus Promoting Polyunsaturated Fatty Acids And Carotenoids Produced By Rhodosporidium Kratochvilovae

Polyunsaturated fatty acids?PUFAs?and carotenoids have received great attention due to their extensive role in the food and medical fields.Currently,the application of microbial fermentation to produce PUFAs and carotenoids has many advantages.Due to rapid production and high biomassaccumlation,the use of yeasts to produce PUFAs and carotenoids has become a focus of attention.It is also promising to modify yeasts to produce PUFAs and carotenoids by metabolical engineering methods.Our previous studies showed that the STSV2-encoded protein ORF2 could significantly up-regulate the transcription of virus and host genes during viral infection,and may be a potential transcriptional activator.In addition,the oleaginous yeast Rhodosporidium kratochvilovae strain YM25235 can biosynthesize both PUFAs and carotenoids.Therefore,in this study,ORF2 gene will be overexpressed in YM25235,with the aim to analyze its effects on the growth of YM25235and the biosynthesis of PUFAs and carotenoids.The ORF2 gene was sucloned into the vector pRH2304 to construct the recombinant expression plasmid pRH2034ORF2,which was transformed into YM25235 by Agrobacterium-mediated transformation method.Then,the effect of ORF2 gene on the growth of YM25235 was analyzed.The results showed that the expression of ORF2 gene increase the resistence of YM25235 to salt,osmolarity and low temperature stresses,as well as the biosynthesis glycerol in transgenic strains.The results of fatty acid analysis showed that the contents of linoleic acid?LA?and linoleic acid?ALA?increased from 16.97%and 3.42%in the YM25235 to 22.39%and 5.88%in the transgenic strain,with an increase of 1.32 times and 1.72 times,respectively.Analysis results also showed that the expression of ORF2 gene promoted the mRNA expresson of the?^12/15-desaturase gene RKD12 by 1.89 time,whose coding product converts oleic acid?OA?into LA and ALA in YM25235.The level of transcription was increased s.Further analysis of EMSA showed that the ORF2protein heterologously expressed in Escherichia coli BL21 intereacted the RKD12gene promoter RKD12P was analyzed.These results indicated that the ORF2gene-encoded protein bind to the promoter sequence RKD12P of the RKD12 gene and increase the mRNA expression level of RKD12,thus promoting the biosynthesis of PUFAs in YM25235 and the cold adaptation of the YM25235.The changes in the content of lipid and carotenoids in the ORF2-transformed YM25235 were analyzed to study the effects of ORF2 gene expression on the biosynthesis of lipid and carotenoids in YM25235.The results showed that the lipid content in the transgenic strain was 101 mg/g,which was 1.53 times higher than that in the wild-type strain.The carotenoid content in the transgenic strain was 2.29 mg/g,which was about 1.72 times higher than that in the wild-type strain.Further analysis showed that the mRNA expression levels of lipid and carotenoid synthesis-related genes in the transgenic strain was increased globally,confirming the conclusion of ORF2-encoded protein is a universal regulator that can promote gene trancsription.In summary,expression of the gene ORF2 from Sulfolobus virus STSV2 in YM25235 promoted the resistance of host to high salinity,high osmotic stress,and low temperature growth adaptability,and also significantly increased the biosynthesis of PUFAs,lipid and carotenoids in the transgenic strain.This study will provide a theoretical basis for the theproduction of PUFAs,lipids and carotenoids of Rhodosporidium sp.YM25235 strain by metabolic engineering,and also provided a reference for further analysis of the structure and function of ORF2 protein.