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Prenyl groups increase the lipophilicity of flavonoids, endowing them with a special activity, selectivity, and pharmacological properties by prenylation. Herein, a novel prenyltransferase (ShFPT) gene from Streptomyces sp. NT11 was expressed in Escherichia coli, and its biochemical characteristics were determined. ShFPT exhibited high selectivity to prenylate naringenin at C-6 to generate 6-prenylnaringenin. The optimal activity was observed at pH 6.0 and 55 °C. The Kcat and Km for naringenin were 0.0095 s-1 and 0.20 mM, respectively. Several promiscuous kinase and isopentenyl phosphate kinase genes were screened to develop the most efficient dimethylallyl diphosphate (DMAPP) synthesis pathway for 6-prenylnaringenin synthesis in E. coli. The 6-prenylnaringenin production was improved by changing the induction strategies and optimizing the bioconversion conditions. Finally, 6-prenylnaringenin production reached the highest yield of 69.9 mg/L with average productivity of 4.0 mg/L/h after 16 h incubation, which is the highest yield for any prenylated flavonoid reported to date in E. coli. Therefore, this study provides an efficient method for 6-prenylnaringenin production and reveals the DMAPP synthesis pathway.

UDP-rhamnose is the main type of sugar donor and endows flavonoids with special activity, selectivity, and pharmacological properties by glycosylation. In this study, several UDP-glucose synthesis pathways and UDP-rhamnose synthases were screened to develop an efficient UDP-rhamnose biosynthesis pathway in Escherichia coli. Maximal UDP-rhamnose production reached 82.2 mg/L in the recombinant strain by introducing the cellobiose phosphorolysis pathway and Arabidopsis thaliana UDP-rhamnose synthase (AtRHM). Quercitrin production of 3522 mg/L was achieved in the recombinant strain by coupling the UDP-rhamnose generation system with A. thaliana rhamnosyltransferase (AtUGT78D1) to recycle UDP-rhamnose. To further increase UDP-rhamnose supply, an NADPH-independent fusion enzyme was constructed, the UTP supply was improved, and NADPH regenerators were overexpressed in vivo. Finally, by optimizing the bioconversion conditions, the highest quercitrin production reached 7627 mg/L with the average productivity of 141 mg/(L h), which is the highest yield of quercitrin and efficiency of UDP-rhamnose supply reported to date in E. coli. Therefore, the method described herein for the regeneration of UDP-rhamnose from cellobiose may be widely used for the rhamnosylation of flavonoids and other bioactive substances.

Prenyl groups increase the lipophilicity of flavonoids, endowing them with a special activity, selectivity, and pharmacological properties by prenylation. Herein, a novel prenyltransferase (

UDP-rhamnose is the main type of sugar donor and endows flavonoids with special activity, selectivity, and pharmacological properties by glycosylation. In this study, several UDP-glucose synthesis pathways and UDP-rhamnose synthases were screened to develop an efficient UDP-rhamnose biosynthesis pathway in

Hyperoside exhibits many biological properties and is more soluble in water than quercetin. A uridine 5′-diphosphate (UDP) galactose regeneration system and one-pot synthesis of hyperoside was described herein. Glycine max sucrose synthase (GmSUS) was coupled with Escherichia coli UDP-galactose 4-epimerase (GalE) to regenerate UDP-galactose from sucrose and UDP. Petunia hybrida glycosyltransferase (PhUGT) with high activity toward quercetin was used to synthesize hyperoside via the UDP-galactose regeneration system. The important factors for optimal synergistic catalysis were determined. Through the use of a fed-batch operation, the final titer of hyperoside increased to 2134 mg/L, with a corresponding molar conversion of 92% and maximum number of UDP-galactose regeneration cycles (RCmax) of 18.4 under optimal conditions. Therefore, the method described herein for the regeneration of UDP-galactose from UDP and sucrose can be widely used for the glycosylation of flavonoids and other bioactive substances.

Astragalin (kaempferol 3-O-glucoside) is used as a standard to assess the quality of Radix astragali and has exhibited a number of biological properties. In this work, we screened several UDP-dependent glycosyltransferases (UGT) for their potential as efficient biocatalysts for astragalin synthesis. The highest astragalin production with 285 mg/L was detected in the recombinant strain expressing UGT from Arabidopis thaliana (AtUGT78D2). To further improve astragalin production, an efficient UDP-glucose synthesis pathway was reconstructed in the recombinant strain by introducing sucrose permease, sucrose phosphorylase, and uridylyltransferase. On the basis of those results, a recombinant strain, BL21-II, was constructed to produce astragalin. By optimizing conversion conditions, astragalin production was increased from 570 to 1708 mg/L. The production was scaled up using a fed-batch fermentation, and maximal astragalin production was 3600 mg/L, with a specific productivity of 150 mg/L/h after 24 h incubation and a corresponding molar conversion of 91.9%, the highest yield reported to date.

Isoorientin is a C-glycosylated derivative of luteolin and exhibits a number of biological properties. In this study, multiple strategies were adopted to improve isoorientin production from luteolin in Escherichia coli. Isoorientin production was improved substantially by adjusting induction strategies and controlling acetic acid accumulation, with maximum isoorientin production reaching 826 mg/L. Additionally, a novel UDP-glucose synthesis pathway was reconstructed in E. coli through cellobiose phosphorylase-catalyzed phosphorolysis of cellobiose for the production of glucose 1-phosphate, which serves as a precursor in UDP-glucose formation. The results from two mechanisms of UDP-glucose formation in E. coli, cellobiose phosphorolysis and sucrose phosphorolysis, were compared. Increasing the UDP-glucose supply resulted in maximal isoorientin production reaching 1371 mg/L. Finally, isoorientin (1059 mg) was obtained from 1 L of fermentation broth by simple purification steps with a yield of 81.5%. Therefore, this study provides an efficient method for isoorientin production and a novel UDP-glucose synthesis pathway.

This paper presents a prefermentation treatment method involving fungi to improve flavonoid extraction from the leaves of Ginkgo biloba . The fungi employed for this treatment were screened from the soil present under an ancient ginkgo tree. Seventy-six strains belonging to 23 genera were isolated and identified by a molecular identification method employing 18S rDNA sequences. Thirty-three strains grew well using ginkgo leaves as the growth medium. One strain, Gyx086, with higher extracted yield of flavonoids and more similar to the control, was finally selected for prefermentation processing. The major fermentation factors were optimized by response surface methodology. The optimal conditions for the highest total falvonoid yield were 27.8 °C for temperature, 64.2% for moisture content, and 61 h for fermentation time. Under the optimal condition, a actual total flavonoid yield of 27.59 ± 0.52 mg/g dry weight culture sample was obtained, which was about 70% higher than that of unfermented gingko leaf samples.