Research On The Function Of Phytoene Synthase/Lycopene Cyclase (CrtYB) In Sporidiololus Pararoseus

Carotenoid is a class of more than 700 kinds of terpene groups of unsaturated compounds in general,which is commonly found in animals,plants,algae,fungi and bacteria.It has an important biological function,and its bright color and anti-oxidation are widely used in food,medicine,cosmetics and feed and other industries.Sporidiobolus pararoseus is a class of three main carotenoids(?-carotene,torulene and torularhodin),and can catapult spores.Phytoene synthase/lycopene cyclase is a key enzyme in the carotenoid biosynthetic pathway,which is encoded by a bifunctional gene crtYB in S.pararoseus.It catalyzes Geranylgeranyl-pyrophosphate(GGPP,colorless)synthesis of phytoene(colorless)and catalyzes cyclization of lycopene(pink)to form ?-carotene(yellow),which is an essential catalytic enzyme for the conversion of carotenoids from colorless to color.The cDNA of gene crtYB is 1791 bp in length and encoded 596 amino acids.The N-terminal of CrtYB encodes lycopene cyclase,the C-terminal encoding phytoene synthase.In this paper,researching on the function of CrtYB in S.pararoseus by mutating CrtYB,and using the method of real-time qPCR.The main conclusions are as follows:(1)The position 322th spartate is mutated into tryptophan for phytoene synthase domain,and confirming by the heterologous complementary assay of Escherichia coli.It is finally determined that the function of mutated gene crtYBD322W encoding phytoene synthase is lost,and weakened the functional activity of lycopene cyclase.It proves that the 322th mutation site is a fundamental functional site of CrtYB.(2)At the same time,it finds that crtYBD322W could accumulate another carotenoid in Escherichia coli carrying the recombinant plasmid,which presumes that the intermediate product y-carotene produced by one cyclization reaction of lycopene,only a small amount of?-carotene was accumulated.Indicating that mutant site at position 322 of CrtYB protein not only inactivated the function of C-terminal phytoene synthase,but also affected the function of N-terminal lycopene cyclase.(3)The components and contents of carotenoids in the wild type NGR and the yellow mutant strain Y9 were analyzed by high performance liquid chromatography(HPLC).The results show:the accumulation of ?-carotene in the mutant strain Y9 is 683.05?g/g,which is higher than that of the wild type NGR(624.39?g/g).But the accumulation of torulene in the NGR is 249.59?g/g,which is higher than that of the mutant strain Y9 of 146.61?g/g.(4)Compared with the wild type NGR,the crtYB gene is up-regulated obvious and the crtl gene is up-regulated and crtE gene is down-regulated under normal culture conditions,but there is no significant difference in the crtl gene and crtE gene.This result indicates that the up-regulation of crtYB gene is related to the increase in the amount of ?-carotene accumulation in the yellow mutant strain Y9,and the up-regulation of the expression of the crtYB gene makes the synthesis of carotenoids biased towards the ?-carotene side.Functional research of CrtYB in S.pararoseus provides a new idea for the construction of engineering bacteria synthesis of carotenoids.