Development And Optimization Of An In Vitro Multienzyme Synthetic System For Production Of Flavonols

Flavonols,also known as 3-hydroxyflavones,are the most common and widely distributed flavonoids,which are commonly found in the form of glycosylation.They possess multiple pharmacological properties,including anti-oxidant,anti-tumor,and anti-inflammation activities,prevention of cardiovascular diseases and diabetes,and protection of the nervous system,and thus have a wide range of applications in food,cosmetics,and biomedicine.Currently,the major approaches for deriving flavonols include organic solvent extraction from plant tissues,chemical synthesis,and microbial metabolic engineering.The organic solvent extraction has many disadvantages.For example,it is time-and labor-consuming and needs a large amount of organic solvent.It greatly depends on the source of plant tissues.The chemical synthesis usually requires toxic reagents,extreme reaction conditions,and multiple steps.Another challenge is to separate and purify the target molecules synthesized in the reaction.Compared with these two approaches.the microbial metabolic engineering is cost-effective.It is easy to scale up for mass production of target molecules with a relatively high yield.It is also easy to purify the molecules of interest.However,not all engineered microbes can produce desired products because of the complexity of the cellular system,incompatibility of artificially synthesized genetic element and hosts,inhibition of the host cells by target products,and instability of an engineered biosystem.This study aimed to establish a method for enzymatic synthesis of flavonols in vitro to overcome the disadvantages of the above three methods.Firstly,a pathway was designed for enzymatic synthesis of flavonols from flavanones which were sequentially catalyzed by flavanone 3-hydroxylase(F3H)and flavonol synthase(FLS1).The recombinant plasmids preserved in the laboratory were transformed into Escherichia coli BL21(DE3)and the induced fusion proteins were purified by affinity chromatography.SDS-PAGE analysis indicated that the purified fusion proteins showed a high purity of more than 90%,which were pure enough for the subsequent experiments in this study.Then,the enzyme activities of F3H and FLS1 from both Glycine max and Arabidopsis thaliana were determined and compared.The reaction products were analyzed by polyamide thin layer chromatography and high performance liquid chromatography-mass spectrometry.The result showed that the activities of both enzymes from A.thaliana were higher than those from G.max in our system and His-AtF3H and His-AtFLS1 possessed Michaelis-Menten constants of 0.081 ± 0.025 mM and 0.072 ± 0.052 mM,respectively,at 30℃.Next,an in vitro multienzyme synthetic system was established and optimized for production of flavonols and kaempferol was successfully synthesized from naringenin in this system.After optimization,the system contained 0.1 M Tris-HCl(pH 7.2),0.4%ascorbic acid,10%glycerol,8.2 mM α-ketoglutaric acid,0.01 mM ferrous ion,0.5 mM naringenin,25 μg/mL His-AtF3H,and 25 μg/mL His-AtFLS1.The optimized reaction parameters include a temperature of 40℃ and a reaction time of 40 min with a shake speed of 600 rpm.The yield of kaempferol increased by 1.88 folds after optimization and reached up to 37.55± 1.62 mg/L.The substrate conversion rate was 55.89%±2.74%when the concentration of naringenin was 0.5 mM.Finally,quercetin and galangin were successfully synthesized from eriodictyol and pinocembrin,respectively,in the multienzyme synthetic system.The conversion rate of eriodictyol was 62.85%±0.48%with a quercetin yield of 25.56±0.38 mg/L when the concentration of eriodictyol was 0.35 mM.In the synthesis of galangin from pinocembrin,the conversion rate of pinocembrin was 13.58%± 0.60%.The yield of galangin was 0.58±0.01 mg/L when the concentration of pinocembrin was 0.5 mM.In summary,this study established a method for enzymatic synthesis of flavonols in vitro.This method can efficiently produce flavonols from flavanones and thus pave a new way for the production of flavonols.It is beneficial for wide application of flavonols in biomedicine,food and health care industries and also provided a new idea for the development and utilization of other secondary metabolites.