Identifying Flavanone Hydroxylases And Constructing And Enhancing Strain Of Dihydroquercetin Synthesis

Dihydroquercetin,widely found in fruits,tea,vegetables,and other daily food and drink,has very high antioxidant activity.Dihydroquercetin has important application value in food,medical treatment,health care,cosmetics,etc.For example,dihydroquercetin has been approved by the National Health Commission as an additive material for food.The aim of this study was to screen high-efficiency flavanone hydroxylase and construct a microbial engineering strain with high efficiency to synthesize dihydroquercetin,so as to achieve high-efficiency biosynthesis of dihydroquercetin and promote its industrial application.Young leaves of Ampelopsis grossedentata are rich in dihydromyricetin,which is the product of hydroxylation of 5’ carbon atoms of dihydroquercetin.Therefore,A.grossedentata has the potential of high efficiency flavanone hydroxylase.In this study,the key hydroxylase genes involved in the synthesis of dihydroquercetin were excavated from A.grossedentata.Then heterogeneously expressed in Saccharomyces cerevisiae to verify their catalytic functions and efficiency.An engineered strain for de novo synthesis of dihydroquercetin was constructed,and the production of dihydroquercetin was further improved by regulating the metabolic flux and optimizing the expression of cofactor producing genes.Subsequently,in order to solve the problem of intermediate product accumulation,a new synthetic pathway of dihydroquercetin was designed and verified.The key catalytic enzymes were explored to realize the efficient synthesis of dihydroquercetin,and the feasibility of the scheme was verified.In this study,the genes mining method of highly active plant enzymes,the strengthening strategy of bacterial metabolic cofactors,and the construction of a new metabolic pathway have important guiding significance for the construction of strains biosynthesizing other flavonoids.The specific research contents are as follows:1)Analysis of transcriptome data and mining of flavanone hydroxylase gene in A.grossedentata.By detecting the temporal and spatial distribution of dihydromyricetin in A.grossedentata,it was found that there was a high content in young leaves.By analyzing the transcriptome sequencing data of five tissues of A.grossedentata,12 dihydroflavonol synthesis pathway genes with the full length of CDS were predicted.The high expression of chalcone synthetase(AgCHS)and flavanone 3’5’hydroxylase(AgF3’5’H)was the reason for the high content of dihydromyricetin.2)The biocatalytic activity of flavanone hydroxylase from A.grossedentata was analyzed,and the metabolic process of producing dihydromyricetin from naringin was analyzed.Flavanone 3hydroxylase had a substrate preference,which preferentially uses eriodictyol.Cytochrome P450 reductase 1 had high activity and good application value.The bioactivity of flavanone 3’-hydroxylase was higher,but the substrate specificity was poor.The 3’ hydroxylation activity of flavanone 3’5’hydroxylase was stronger than that of 5’.Dihydromyricetin was synthesized from naringin in A.grossedentata mainly through naringin-eriodictyol-dihydroquercetin-dihydromyricetin metabolic pathway.3)The engineering strain of S.cerevisiae was constructed and strengthened.The synthesis of dihydroquercetin was achieved by integrating high-activity F3H,F3’H,and CPR into a naringin de novo synthetic engineering strain.The fusion of flavanone 3-hydroxylase and flavanone 3’hydroxylase with liker could not improve the conversion of the substrate.First discovered overexpression of NADPH dependent isocitrate dehydrogenase can improve the titer of dihydroquercetin to 126.27 mg·L-1.F3H overexpression can improve the naringenin conversion rate and the titer of dihydroquercetin to 170.45 mg·L-1.This was the highest titer of dihydroquercetin synthesized at the shaker level to date.4)A novel biosynthetic pathway of dihydroquercetin was designed and validated.The functional activity of a new flavonoid reductase(FLR)catalyzed quercetin to dihydroquercetin was verified.The activity of FuFLR was found to be the highest by screening FLRs from different sources.Optimiziing the fermentation conditions can make the FuFLR catalyzing 200 mg·L-1 quercetin generated 147.17 mg·L-1 dihydroquercetin,and the substrate conversion efficiency reached 74%.Using the N-terminal fusion XXA labeling strategy increased FLR solubility,while the construction of a cofactor supply pathway based on glycerol metabolism did not increase the substrate conversion rate.