Construction Of Crocin Biosynthesis Pathway

The plant glycoside crocin is naturally present in the crocin stigma and has sedative,antioxidant and anti-cancer effects.One molecule of crocin is formed by the condensation of one molecule of crocetin and four molecules of uridine diphosphate glucose(UDPG).According to the position and number of condensed UDPG,its glycosylation products can be divided into crocin-5,crocin-4,crocin-3,crocin-2 and crocin.At present,crocin is mainly obtained by plant extraction,but the yield and purity are low.The de novo synthesis of crocin by microorganisms is an important supplement to the existing manufacturing method,which has not yet been realized in heterologous eukaryotic hosts.Our laboratory has successfully constructed a Saccharomyces cerevisiae strain for crocetin synthesis.This strain can be used as the host for crocetin production.In this study,by introduction glycosyltransferase UGT75L6 and UGT94E5 from Gardenia jasminoides into the host and reducing the fermentation temperature to 20?,the synthesis of the primary glycosylation product crocin-5(one side plus 1 UDPG)and the secondary glycosylation product crocin-2(one side plus 1 UDPG,and the other side plus 2 UDPG)were firstly achieved in S.cerevisiae.Screening six combinations of primary and secondary glycosyltransferases from Crocus sativus L.,Stevia rebaudiana and Bacillus subtilis 168,the synthesis of crocin-5 and crocin-2 were all achieved by all the combinations except those containing crocin source UGT2.The strain harboring glycosyltransferase Yoj K/Yji C/Ydh E from B.subtilis produced the highest level of crocin-5(up to 6.08 mg/L at shake-flask level).By up-regulating the yeast endogenous UDPG synthesis pathway via overexpressing phosphomutase(PGM1),glucose-1-uridine transferase(UGP1)as well as nucleoside diphosphate kinase(YNK1),only the strain with Yoj K/Yji C/Ydh E achieved the synthesis of crocin with a titer of 0.263 mg/L.In the meanwhile,the strain containing the glycosyltransferase UGT75L6/UGT94E5 obtained the highest crocin-5 titer of 8.8 mg/L among those combinations.The supply of UDPG is a rate-limiting step in the synthesis of crocin.Crocin biosynthesis pathway and cell growth compete for UDPG.If we can build synthetic microbial consortia of S.cerevisiae and Escherichia coli to realize the division of crocetin production and glycosylation processes,there will be sufficient UDPG for crocin synthesis.This study proves that crocetin can be enriched in extracellular culture of S.cerevisiae.Crocetin added to the fermentation medium can be transported into the cells of E.coli.And BL21(DE3)has higher crocetin transport efficiency than E.coli MG1655.Therefore,E.coli BL21(DE3)was selected as the starting strain,and its UDPG hydrolase gene(ush A)was knocked out,while PGM1 and phosphoglucomutase(Gal U)were both overexpressed to enlarge UDPG supply.Four glycosyltransferase combinations UGT75L6/UGT94E5,UGT75L6/UGT76G1,Yoj K,and Yoj K/Yji C/Ydh E were introduced into the host,respectively.By feeding crocetin,the strain containing glycosyltransferase Yoj K/Yji C/ Ydh E realized the highest crocin-5titer of 1.81 mg/L,and also the synthesis of crocin-2,among the tested combinations.However,no crocin has been detected in this strain,which probably due to that glycosyltransferases expressed in E.coli mostly exist in the form of inclusion bodies,severely limiting the accumulation of crocin-5 and crocin.This study is the first time to realize de nove biosynthesis of crocin in S.cerevisiae,which will be benefit to achive higher crocin titer in future.At the same time,E.coli strains capable of producing crocin-2 and crocin-5 were also constructed,which can be used to construct the synthetic microbial consortia for crocin synthesis.