Research Of The Biosynthesis Of Ortho-hydroxylated Flavonoids Using 4-hydroxyphenylacetate 3-hydroxylase Complex(HpaBC) Of Escherichia Coli

Flavonoids are important plant metabolites,their derivatives exhibit various physiological and pharmaceutical functions.Here,efficient hydroxylase complex,HpaBC were selected to construct ortho-hydroxylated flavonoids pathway.However,the insertion site of HpaC leads to instability in the catalytic efficiency of hydroxylase complexes for substrates.To solve this problem,microbial cocultures were designed to minimize the effect of HPAC on bacterial activity.With the optimal conditions(optimization of induction temperature,substrate delay time,substrate concentration and media),46.84±2.85 mg·L-1 of eriodictyol was produced,which is nearly 1.48 times higher than that of the monoculture.The same strategy was used for catechin and caffeic acid production,the highest titer was 29.81±2.66 mg·L-1 with 28.91 ± 1.77 mg·L-1.We also show the evidence of in vivo HpaBC activity towards the kaempferol with quercetin product titers of 20.05±1.48 mg·L-1 and towards the dihydrokaempferol with dihydroquercetin product titers of 20.14±0.75 mg·L-1.To the best of our knowledge,this is the first report about HpaBC production of quercetin and dihydroquercetin.The main results are as follows:1.Cloning of HpaB and HpaC genesThe ORFs of HpaB encoding the monooxygenase component(ORF no.7156703)and HpaC encoding the oxidoreductase component(ORF no.7155545)were cloned into the expression vector pETDuet and pRSFDuet vectors(Novagen,Carlsbad,CA,USA),respectively.These results indicate that HpaB and HpaC were successfully recombinantly expressed in E.coli cells.SDS-PAGE analysis revealed the presence of major bands corresponding to HpaB(58.5.kDa)and HpaC(18.6 kDa)in samples prepared from the soluble fractions of E.coli cells.2.Comparison of catalytic activity of the strains carrying different recombinant plasmidsFour recombinant plasmids(P1):pRSFDuet-B-C,(P2):pRSFDuet-C-B,(P3):pETDuet-B-C,(P4):pETDuet-C-B were constructed and two co-transformed plasmids(P1&4):pRSFDuet-B and pETDuet-C-B,(P2&3):pRSFDuet-C-B and pETDuet-B-C also were constructed.These plasmids were introduced into BL21*(DE3)cells by transformation.Finally,the expressed strains BL21-P1,BL21-P2,BL21-P3,BL21-P4,BL21-P1&4 and BL21-P2&3 were obtained.The 200 mg·L-1 of N as substrate and generation of E was detected.3.Optimization of fermentation conditions for ortho-hydroxylated flavonoidsIn order to further increase the yield of p-hydroxylated flavonoids,ortho-hydroxylated flavonoids were synthesized.We optimized the substrate concentration(40 mg·L-1,60 mg·L-1,80 mg·L-1,100 mg·L-1,120 mg·L-1)and culture medium(TB,LB,M9),induction temperature(20℃,28℃ and 37℃)and substrate delay time(4 H,6 H,8 H),respectively.The yield of E was 46.84±2.85 mg·L-1 with naringin 80 mg·L-1 as substrate and induced for 6 hours at 28℃ in M9 medium,the conversion rate of N to E was 58%.4.Substrate diversity analysis of the HpaBC complexTo further investigate the diversity of the substrate,in addition to flavanone(N),the monohydroxylated phenolic acid(p-coumaric acid,p-CA),dihydroflavonol(DHK),flavonol(K),flavan-3-ol(afzelechin,Af),and anthocyanin(pelargonidin,PEL)were fed under the optimal condition,and the fermentation products were detected by HPLC and HPLC-MS method.The previous researches suggested that the HpaBC complex had the in vivo activity towards p-CA,N,and Af with high catalytic efficiency.However,the activity of the substrate PEL was not detected.