| Hyperoside,also known as quercetin-3-O-galactose,is a class of important secondary metabolites found in plants,which exists in Guttiferae,Rosaceae,Polygonaceae,Malvaceae,Asteraceae and many other plants.It has lots of pharmacological effects such as anti-cancer,anti-oxidation,anti-inflammatory,and anti-depression.The research results on its production show that the method of biosynthesis has a good application prospect.This study focuses on the mixed culture of engineered Escherichia coli and engineered Saccharomyces cerevisiae to produce hyperoside.By distributing a metabolic pathway of hyperoside among a microbial consortium of E.coli and S.cerevisiae,the objective of this study is to establish a mixed culture system with naringin and sucrose as substrates for the efficient preparation of hyperoside.1.Pathway enzyme screening and functional characterizationThe biosynthesis pathway of hyperoside using naringenin and sucrose as substrates containing multiple enzymes,which are hydroxylase,flavanone 3-hydroxylase,flavonol synthase,sucrose synthase,UDP-glucose isomerase and galactosyltransferase.These genes were transferred into corresponding host cells for screening and functional characterization.And then selected pathway enzymes with better activities are obtained for subsequent pathway reconstruction and microbial cell factories construction,including CsF3’H,CsF3Ha,AcFLSHRB,FtFLS1,OcSUS1,OcUGE1,PhUGT and DkFGT.2.Construction of engineered E.coli yielding kaempferolFlavanone 3-hydroxylase and flavonol synthase were constructed into prokaryotic expression vectors,and then transformed into E.coli expression competent cells BL21(DE3)to form engineered E.coli strains BL21(DE3)-aA and BL21(DE3)-aF.With naringin as substrate,different kinds of media and different volume culture systems were selected for whole cell reaction,and kaempferol were found after the reaction.In 100 mL LB culture medium systems,the yields of kaempferol were 0.52 mg/L and 0.64 mg/L.In 300 mL LB culture medium systems,the yields of kaempferol were 1.56 mg/L and 1.26 mg/L.In 100 mL TB culture medium systems,the yields of kaempferol were 59.15 mg/L and 54.77 mg/L.In 300 mL TB culture medium systems,the yields of kaempferol were 79.80 mg/L and 72.59 mg/L.By detecting the distribution of products,the yields of kaempferol in pellet were higher than in supernatant.In order to increase the yield of products in the supernatant to facilitate the next reaction,different concentrations of Tween-80 were added to the culture medium.The results showed that different concentrations of Tween-80 had a certain effect on the increase of product content in supernatant compared with that without adding Tween-80,among which 1%Tween-80 had a more significant effect.3.Construction of engineered S.cerevisiae producing hyperosideThe hydroxylase,sucrose synthase,UDP-glucose epimerase and galactosyltransferase were constructed into eukaryotic expression vectors and then transformed into S.cerevisae competent cell W303B to form engineered S.cerevisae strains W303B-HFSU1,W303BHFSU4,W303B-HLFSU4,W303B-HLFCSU1 and W303B-HLFCSU4.The whole cell reaction was carried out with kaempferol and sucrose as substrates,and hyperoside appeared after the reaction.The yields of hyperoside for 120 hours of reaction were 16.16 mg/L,16.71 mg/L,40.35 mg/L,12.85 mg/L,21.42 mg/L and 15.06 mg/L respectively.4.Construction of co-culture system of engineered E.coli and engineered S.cerevisiae generating hyperosideThe engineered E.coli strains BL21(DE3)-aA and BL21(DE3)-aF were mixed with engineered S.cerevisae strains W303B-HFSU1 and W303B-HFSU4 respectively to form four mixed culture systems,namely aA-HFSU1,aA-HFSU4,aF-HFSU1 and aF-HFSU4.With naringin and sucrose as substrates,the target product hyperoside appeared.After 120 hours of reaction,the yields of hyperoside were 0.774 mg/L,0.499 mg/L,0.873 mg/L and 0.384 mg/L.In addition,the yields of other product,trifolin,were 28.070 mg/L,21.295 mg/L,27.082 mg/L and 20.786 mg/L。... |
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