Cloning,Expression And Functional Analysis Of Key Enzyme Genes Involved In Carotenoids Biosynthesis Of Sporidiololus Pararoseus

Carotenoids, as a group of over 750 valuable unsaturated terpene compounds classified as carotene and xanthophyll families, are endowed with high nutritional value, for instance, they can be the precursor of vitamin A. Phytoene desaturase, phytoene synthase and lycopene cyclase are the key rate-limiting enzyme in carotenoids biosynthesis pathway. They are involved in catalyzing the formation of phytoene which is the structure basis of carotenoids and the convertion from the colorless hydrocarbon phytoene to other pigmented carotenoids, play an essential central regulation role.Sporidiobolus genus is a kind of basidiomycota fungi, can catapult spores and accumulate mostly β-carotene, torulene and torularhodin. In this study, we got the transcriptome information of the Sporidiobolus pararoseus CGMCC 2.5280 (WT, named NGR) by RNA-sequencing. Subsequently, the intact open reading frames of phytoene desaturase gene crtI and lycopene cyclase/phytoene synthase gene crtYB were obtained. The crtI full-length genomic DNA and cDNA is 2330bp and 1683bp respectively, contain nine introns, its GenBank accession no. is KR108014. By the TLC, HPLC and MS analysis combined with heterogenous complementation detection in E. coli allowed us to confirm that the S. pararoseus CrtI is responsible for both four- and five-step dehydrogenation of phytoene, namely lycopene and 3,4-didehydrolycopene are the major end products of the S. pararoseus CrtI. The crtYB full-length genomic DNA and cDNA is 2409bp and 1791bp respectively, contain eight introns, its GenBank accession no. is KR108013. By TLC and HPLC analysis combined with heterologous complementary detection in E. coli allowed us to confirm that the enzymatic conversion of GGPP to phytoene as well as lycopene to β-carotene are catalyzed by this S. pararoseus CrtYB.In our previous study, we discovered that low-concentration NaCl or high-temperature stress promotes the accumulation of torulene and torularhodin in S. pararoseus NGR, while low-temperature stress facilitates the formation of β-carotene. The analysis results of intracellular antioxidant enzyme revealed that the mechanism of carotenoids resisting NaCl or high-temperature stress is similar to SOD/CAT clear up superoxide anion. The qPCR was used for measuring the transcriptional level of crtI and crtYB gene in cDNA under the NaCl or high-temperature stress. Results revealed that both crtI and crtYB gene were up-regulated observably, and the up-regulated level of crtI gene is higher than crtYB gene. These results suggested that the crtI gene play a key role in resisting NaCl and high-temperature stress. However, the transcriptional level of crtl and crtYB gene in cDNA under low-temperature stress was no difference compared with normal conditions. To date, the related genes involved in carotenoids biosynthesis pathway of S. pararoseus was not clear, and the whole genome sequencing is expected to combine with transcriptome sequencing to comprehensive analysis the mechanism of carotenoids biosynthesis pathway in NGR. This study will provide insights into the the mechanism of resist kinds of stress in NGR and may facilitate the optimization of carotenoids production in genetic engineering strategy.