Characterization Of Flavonoid 3′-Hydroxylase From Tea Plant And Its Application In Metabolic Engineering

Catechins are important determinants of tea quality and taste, which belong to flavonoids compounds and process beneficial health effects. Based on the hydroxylation pattern in the B-ring, catechins can be categorized in B-3′,4′-dihydroxylated catechins and B-3′,4′,5′-trihydroxylated catechins. In flavonoids biosynthesis, flavonoid 3′-hydroxylase(F3′H) hydroxylates the B-ring of naringenin and dihydrokaempferol at the 3′-postion to form eriodictyol and dihydroquercetin, respectively, the two important intermediates for the biosynthesis of B-3′,4′-dihydroxylated catechins in plants. Investigation on this key enzyme in flavonoids metabolic pathway will contribute to regular biosynthesis of catechins through appropriate measures and will provide insights into the development of innovative strategies to enhance tea quality and taste. Meanwhile it will provide an gene pool to microbial metabolic engineering. We isolated a Cs F3′H gene from tea plant by homologous cloning and characterized Cs F3′H by enzymatic analysis firstly. Then the expression profiles of Cs F3′H and other related genes involved in flavonoids biosynthesis were examined and the concentrations of catechins were determined in different samples, which included tea seedling during the growth period, tea seedling suffered nitrogen deprived and tea plant with purple leaf. Finally the application of Cs F3′H in metabolic engineering were explored. The results were shown as following.A gene encoding F3′H, designated as Cs F3′H, was isolated from Camellia sinensis with RACE cloning technique and deposited in the Gen Bank(Gen Bank ID: KT180309). The full transcript has 1706 nucleotides with a 5′-untranslated region(UTR) of 26 bp, an open reading frame(ORF) of 1557 bp and a 3′-URT of 12 bp. Cs F3′H was predicted to encode a protein with 518 amino acids, a theoretical moleclar weight of 57.07 KDa and a calculated isoelectric point of 6.82. Bioinformatic analysis revealed that Cs F3′H was highly homologous with the characterized F3′Hs from other plant species. Four conserved cytochrome P450-featured motifs( PPGPNPWP; FGAGRRISAG; E-R-R; AGTDTS) and three F3′H-specific conserved motifs( VVVAAS; GGEK; ADVRG) were discovered in the protein sequence of Cs F3′H. Enzymatic analysis of the heterologously expressed Cs F3′H in yeast demonstrated that Cs F3′H catalyzed the 3′-hydroxylation of naringenin, dihydrokaempferol and kaempferol. Apparent Km values for these substrates were 17.08, 143.64 and 68.06 μM, and their apparent Vmax values were 0.98, 0.19 and 0.44 p M· min-1, and their apparent kcat values were 49.09, 9.86 and 21.88 p M· min-1· mg-1 microsome, respectively. Among these three compounds, naringenin was shown to be the optimal substrate.During the developmental process of tea seedling the changes in catechin(C), epigallocatechin gallate(EGCG), gallocatechin gallate(GCG), B-3′,4′-dihydroxylated catechins, B-3′,4′,5′-trihydroxylated catechins and Total catechins, showed similar patterns, with a gradual increase from S1 to S3, then decrease in S4. Cs F3′H and other flavonoids biosynthetic genes showed similar variation pattern during the plant growth. Their expression level gradually increased to the maximum by S3 and decreased rapidly in S4. During tea seed germination, the expression levels of Cs F3′H and other flavonoids biosynthetic genes correlated positively with B-3′,4′-dihydroxylated catechins, B-3′,4′,5′-trihydroxylated catechins and Total catechins accumulation pattern in leaves.The concentration of B-3′,4′-dihydroxylated catechins, B-3′,4′,5′-trihydroxylated catechins and Total catechins on day 12 was 1.33-, 1.18- and 1.21-fold higher, respectively, in nitrogen deprived plants than in the plants given full nutrient solution. From day 8 to day 12, B-3′,4′-dihydroxylated catechins, B-3′,4′,5′-trihydroxylated catechins and Total catechins showed an increasing trend in response to nitrogen deprivation. The expression of Cs F3′H and ten other genes( PAL, CHS, CHI, F3 H, F3’5’H, DFR, LAR, ANS, ANR1 and ANR2) on day 12 was 2.61-, 2.59-, 2.03-, 2.83-, 2.98-, 1.61-, 4.66-, 4.31-, 5.33-, 1.66- and 4.02-fold higher, respectively, in the plants deprived of nitrogen as compared to that in the plants given full nutrient solution. All genes showed a general increase in their expressions in tea plants in response to nitrogen deprivation from day 8 to day 12.We employed a tea plant with purple leaf and a tea plant with green leaf both from MeiTang-Tai-Cha, which had significant difference on leaf color. The purple leaf had higher concentration of anthocyanidins than the green leaf, while the green leaf was richer in Total catechines. The expression levels of Cs F3′H and other related genes( PAL, CHS, CHI, F3 H, DFR, ANS, ANR1, ANR2 and F3’5’H) were up-regulated in purple leaves as compared with those in the green leaves. In purple leaves, the expression levels of related genes( except LAR) were highly correlated with both concentrations of Total catechines( r=0.84~0.99) and anthocyanidins( r= 0.72~1.00). By contrast, there was only a high correlation between the expression levels of related genes( except LAR and Cs F3′H) and the concentration of Total catechines( r= 0.64~0.77) in green leaves.For eridictyol, dihydroquercetin and quercetin production, E.coli and S. cerevisiae were employed as fermention strains. To express Cs F3′H in prokaryote, four articifical P450 constructs harboring Cs F3′H and ATR1(or ATR2) were introduced into E. coli straints TOP10, DH5α and BL21 respectively. The strain TOP10 transformed with the construct SUMO-Cs F3?H[28-518]::ATR1[49-688] 3 AA, which fused Cs F3′H gene sequence encoding amino acids 28 to 518 with ATR1 gene sequence encoding amino acids 49 to 688 by a linker gene sequence encoding three amino acids, generated highest bioconversion efficiency at 25 ℃ among all E. coli strains. Supplemented with 1 000 μM naringenin, dihydrokaempferol and kaempferol, this bacteria strain produced 287.93 μM eriodictyol, 131.76 μM dihydroquercetin and 188.62 μM quercetin respctively. The plasmid p YES-Dest52-Cs F3′H harboring Cs F3′H gene was introduced into the yeast S. cerevisiae WAT11. Supplemented with 1 000 μM naringenin, dihydrokaempferol and kaempferol, the maximum amounts of eriodictyol, dihydroquercetin and quercetin produced by this yeast strain were 734.32 μM, 446.07 μM and 594.64 μM respctively.For recombinant-kaempferol production, six SUMO plasmids were constructed by fusing F3 H gene with FLS gene by a linker gene sequence encoding three amino acids, which were then introduced into E. coli strain BL21. The bacteria strain BL21 transformed with SUMO-Cs F3H::At FLS 3AA(three amino acids linker) generated 1 579.41μM kaempferol, which was the highest production among these six E.coli strains. To compare the bioconversion efficiency of coexpression proteins with that of fusion protein, plasmids p ETDuet-Cs F3H::At FLS 3 AA, p RSFDuet-Cs F3H::At FLS 3 AA, p ETDuet-Cs F3 H and p RSFDuet-At FLS were constructed. The coexpression strain transformed with p ETDuetCs F3 H and p RSFDuet-At FLS could transform 37.79% substrate naringenin into end-product kaempferol. The strain transformed with p ETDuet-Cs F3H::At FLS 3 AA could transform 35.52% substrate naringenin into end-product kaempferol, while the strain transformed with p RSFDuet-Cs F3H::At FLS 3 AA could transform 26.64% substrate naringenin into endproduct kaempferol only. The amount of intermediate product dihydrokaempfeol generated by the strain transformed with p ETDuet-Cs F3 H and p RSFDuet-At FLS was 2.85-fold higher than that of the strain transformed with p ETDuet-Cs F3H::At FLS 3 AA. To explore the effect of the amino acids linker length between Cs F3 H and At FLS on the bioconversion efficiency of fusion proteins, plasmid SUMO-Cs F3H::At FLS 9AA was constructed with a linker gene sequence encoding nine amino acids. The strain transformed with SUMO-Cs F3H::At FLS 9 AA generated 3 133.09 μM end-product kaempferol, which was 1.98-fold than that of the strain transformed with SUMO-Cs F3H::At FLS 3 AA. While the amount of intermediate product dihydrokaempferol generated by the strain transformed with SUMO-Cs F3H::At FLS 9 AA was 21.33% of that of the strain transformed with SUMO-Cs F3H::At FLS 3 AA. Enzymatic analysis demostrated that the bioconversion efficiency of fusion proteins Cs F3H::At FLS 9 AA on substrate naringenin(or dihydrokaempferol) was higher than that of fusion proteins Cs F3H::At FLS 3 AA. This results indicated that the acitivity of fusion proteins Cs F3H::At FLS was affected by the length of the amino acids linker.For recombinant-quercetin, plasmids p ES-URA-Cs F3′H and p ES-HIS- Cs F3H::At FLS 9 AA were constructed, which were then co-introduced into the yeast S. cerevisiae WAT11. Supplemented with 5 m M naringenin, the maximum amounts of eriodictyol, kaempferol, quercetin, dihydroquercetin and dihydrokaempferol produced by this yeast strain were 1412.16 μM, 490.25 μM, 445.75 μM, 66.75 μM and 73.50 μM during 36 h~48 h fermentaion.