Study On The Genes Which Encode Key Enzymes For The Biosynthesis Of Carotenoids In Rhodosporidium Kratochvilovae:Pdh And Lc

Carotenoid,a kind of fat-soluble pigments,is widely distributed in the nature and has many important biological functions,such as anti-cancer,oxidation reistance and resistance to disease of heart head blood-vessel.At present,carotenoids is widely applied to the food,feed,heath care products and cosmetics industries,and attracting more and more anention.Although many researchers on how to improve the production of carotenoids have been done,the output of carotenoids can not meet people's needs.Nowadays,the methods of carotenoids production can be categorized into two groups,chemical synthesis and biotransformation.Chemical synthesis is inexpensive but low-activity,and restricted to use due to toxicity.By contrast,biotransformation has more advantages,such as high-quality productions.AS the research object,Rhodosporidium kratochvilovae YM25235 has a lot of advantages,such as short production cycle,genetic stability,safety and so on.There are some strains of Saccharomyces that have ability to product carotenoids rely on themselves,such as Phaffia rhodozyma and Rhodotorula glutinis.Farnesyl pyrophosphate(FPP)is a precursor of the carotenoids' synthesis in Saccharomyces.The main enzymes of this process,from FPP to carotenoids,are GGPP synthetase(encoded by CrtE gene),phytoene dehydrogenase(encoded by CrtI gene)and phytoene synthetase/lycopene cyclase(encoded by CrtYB gene).Under the action of these enzymes,FPP turn into phytoene,lycopene,?-carotene and other carotenoids.The ?-carotene is an important end product in carotenoids' synthesis,which served as an index in many research.Therefore,the emphasis of our research is some key enzyme gene in Rhodosporidium kratochvilovae YM25235.Besides,There are some studies,at home and abroad,have showed that carotenoids' synthesis in plants and microorganisms affected by low temperature stress.And,the color of Rhodosporidium kratochvilovae YM25235 was enhanced when cultured in low temperature,so we predict there are some connection between Rhodosporidium kratochvilovae YM25235 and cold adaption.First,we extracted pigment from Rhodosporidium kratochvilovae YM25235 and we found there are two carotenoids,lycopene and ?-carotene,by separation and identification of pigment.It suggests that the Rhodosporidium kratochvilovae YM25235 can product carotenoids by itself.In order to find out the gene that encoded key enzymes for the synthesis of carotenoids in Rhodosporidium kratochvilovae YM25235.On the basic of sequencing,we found two nucleic acid sequences that may encode key enzymes for the synthesis of carotenoids in Rhodosporidium kratochvilovae YM25235,called Pdh and Lc respectively.So we take the above two cDNA fragments and vector pRH2304 to construct recombinant expression plasmid pRHPdh and pRHLc.Then two expression plasmids were transformed to Rhodosporidium kratochvilovae YM25235 respectively.In this situation,we detected gene expression level of Pdh and Lc and ?-carotene's production for these over-expression strains,compared with the reference strain(the YM25235 strains including the empty plasmid pRH2304).The results show that Pdh gene expression level of overexpression-strain pRHPdh is as 1.8 times as the reference strains,and Lc expression level is as 1.2 times as reference strains.Besides,the yield of ?-carotenen in overexpression-strain pRHPdh is as 1.38 times as reference strains;Pdh gene expression level of overexpression-strain pRHLc is as 1.4 times as the reference strains,and Lc expression level is as 3.9 times as reference strains.Besides,the yield of ?-carotenen in overexpression-strain pRHLc is as 1.90 times as reference strains.It suggests that proteins encoded by Pdh and Lc gene can promote the synthesis of?-carotene.It suggests that proteins encoded by Pdh and Lc gene can promote the synthesis of ?-carotene.There are three genes(HisK2301,Rkhogl and MSN4)for cold adation in Rhodosporidium kratochvilovae YM25235.In 15 ? cultivation condition,the relation between cold adaption and cold adaption is unknown.So we also detected gene expression level of Pdh and Lc and ?-carotene's production for over-expression strain pRHHisK2301,over-expression strain pRHRkhog1 and over-expression strain pRHMSN4 compared with the reference strain.The results suggest:Pdh gene expression level of overexpression-strain pRHHisK2301 is as 2.5 times as the reference strains,and Lc expression level is as 2.4 times as reference strains.Besides,the yield of ?-carotenen in overexpression-strain pRHHisK2301 is as 1.37 times as reference strains;Pdh gene expression level of overexpression-strain pRHRkhogl is as 3.4 times as the reference strains,and Lc expression level is as 2.9 times as reference strains.Besides,the yield of ?-carotenen in overexpression-strain pRHhog is as 1.89 times as reference strains;Pdh gene expression level of overexpression-strain pRHMSN4 is as 2.6 times as the reference strains,and Lc expression level is as 2.3 times as reference strains.Besides,the yield of ?-carotenen in overexpression-strain pRHmsn is as 2.00 times as reference strains.All of HisK2301,Rkhog1 and MSN4 gene's over-expression can promote the synthesis of carotenoids in Rhodosporidium kratochvilovae YM25235.And carotenoids's synthesis in Rhodosporidium kratochvilovae YM25235 is stimulated by cod adaption in low temperature.In conclusion,this study has obtained two key enzyme genes responsible for the biosynthesis of carotenoids in Rhodosporidium kratochvilovae YM25235,Pdh and Lc.The proteins encoded by Pdh and Lc can promote the biosynthesis of carotenoids.The biosynthesis of carotenoids in Rhodosporidium kratochvilovae YM25235 relate to cold adaption.This mechanism can promote the biosynthesis of carotenoids in Rhodosporidium kratochvilovae YM25235.This research helps to further revealing the pathway for the carotenoids' biosynthesis,laying a foundation for research and application in the future.