Functional Characterization and Screening of Promiscuous Kinases and Isopentenyl Phosphate Kinases for the Synthesis of DMAPP via a One-Pot Enzymatic Cascade.

Dimethylallyl diphosphate (DMAPP) is a key intermediate metabolite in the synthesis of isoprenoids and is also the prenyl donor for biosynthesizing prenylated flavonoids. However, it is difficult to prepare DMAPP via chemical and enzymatic methods. In this study, three promiscuous kinases from Shigella flexneri (SfPK), Escherichia coli (EcPK), and Saccharomyces cerevisiae (ScPK) and three isopentenyl phosphate kinases from Methanolobus tindarius (MtIPK), Methanothermobacter thermautotrophicus str. Delta H (MthIPK), and Arabidopsis thaliana (AtIPK) were cloned and expressed in Escherichia coli. The enzymatic properties of recombinant enzymes were determined. The Kcat/Km value of SfPK for DMA was 6875 s-1 M-1, which was significantly higher than those of EcPK and ScPK. The Kcat/Km value of MtIPK for DMAP was 402.9 s-1 M-1, which was ~400% of that of MthIPK. SfPK was stable at pH 7.0-9.5 and had a 1 h half-life at 65 °C. MtIPK was stable at pH 6.0-8.5 and had a 1 h half-life at 50 °C. The stability of SfPK and MtIPK was better than that of the other enzymes. Thus, SfPK and MtIPK were chosen to develop a one-pot enzymatic cascade for producing DMAPP from DMA because of their catalytic efficiency and stability. The optimal ratio between SfPK and MtIPK was 1:8. The optimal pH and temperature for the one-pot enzymatic cascade were 7.0 and 35 °C, respectively. The optimal concentrations of ATP and DMA were 10 and 80 mM, respectively. Finally, maximum DMAPP production reached 1.23 mM at 1 h under optimal conditions. Therefore, the enzymatic method described herein for the biosynthesis of DMAPP from DMA can be widely used for the synthesis of isoprenoids and prenylated flavonoids.

Screening ß-glucosidase and α-rhamnosidase for biotransformation of naringin to naringenin by the one-pot enzymatic cascade.

Naringenin is a kind of flavonoid with many kinds of pharmacological activities, and is also a key intermediate metabolite in the flavonoid synthesis pathway. In this study, three α-rhamnosidases from Thermotoga petrophia DSM 13995 (TpeRha), Alternaria sp. L1 (AsRha), and Aspergillus mulundensis (AmRha), and three ß-glucosidases from T. thermarum DSM 5069 T (BGLI-Tt and BGLII-Tt), and A. niger NL-1 (BGL-NL) were cloned, expressed, and characterized. The Kcat/Km value of AmRha for naringin was 2.389 s-1mM-1 which was 796-fold and 26-fold of TpeRha and AsRha. The Kcat/Km value of BGL-NL for prunin was 0.946 s-1mM-1, which was about 4.4-fold and 4.6-fold of BGLI-Tt and BGLII-Tt. According to the catalytic efficiency, expression level, and reaction condition compatibility, AmRha was coupled with BGL-NL to construct a one-pot enzymatic cascade for preparing naringenin from naringin. The effects of the ratio and dosage of the enzyme, the naringin concentration, and reaction conditions on naringenin production were optimized. At a dosage of 200 U/L AmRha and 1000 U/L BGL-NL, a temperature of 50 °C and pH 5.0, 30 mM naringin was transformed into 29.3 mM naringenin for 24 h reaction with a corresponding molar conversion of 97.6%. Therefore, this study provides an efficient enzymatic cascade to meet the large-scale and low cost preparation of naringenin from naringin.

Screening and characterizing flavone synthases and its application in biosynthesizing vitexin from naringenin by a one-pot enzymatic cascade.

C-glycosylated flavonoids are important structural derivatives of flavonoids and have a variety of physiological activities. Flavone synthase is a key enzyme for producing C-glycosylated flavonoids. In this study, three flavone synthase genes were cloned, overexpressed and characterized in E. coli. By analyzing the enzymatic properties of the enzymes, Aethusa cynapium flavone synthase (AcFNS) was better than Apium graveolens flavone synthase (AgFNS) and Petroselinum crispum flavone synthase (PcFNS) in terms of catalytic ability, organic solvent tolerance and stability. Then, a one-pot enzymatic cascade was developed to synthesize vitexin from naringenin by using AcFNS, C-glycosyltransferase (TcCGT) from Trollius chinensis, and sucrose synthase (GmSUS) from Glycine max. The effects of enzyme ratios, substrate concentrations, cofactors, and reaction conditions on vitexin production were determined. The highest vitexin production reached 935.6 mg/L with a corresponding molar conversion of 78.7 % for (2 S)-naringenin. Thus, this is the first report of a one-pot enzymatic cascade for vitexin production from naringenin in vitro.