Optimization of the Biosynthesis of B-Ring Ortho-Hydroxy Lated Flavonoids Using the 4-Hydroxyphenylacetate 3-Hydroxylase Complex (HpaBC) of Escherichia coli

Abstract

Flavonoids are important plant metabolites that exhibit a wide range of physiological and pharmaceutical functions. Because of their wide biological activities, such as anti-inflammatory, antioxidant, antiaging and anticancer, they have been widely used in foods, nutraceutical and pharmaceuticals industries. Here, the hydroxylase complex HpaBC was selected for the efficient in vivo production of ortho-hydroxylated flavonoids. Several HpaBC expression vectors were constructed, and the corresponding products were successfully detected by feeding naringenin to vector-carrying strains. However, when HpaC was linked with an S-Tag on the C terminus, the enzyme activity was significantly affected. The optimal culture conditions were determined, including a substrate concentration of 80 mg·L^-1, an induction temperature of 28 °C, an M9 medium, and a substrate delay time of 6 h after IPTG induction. Finally, the efficiency of eriodictyol conversion from P2&3-carrying strains fed naringin was up to 57.67 ± 3.36%. The same strategy was used to produce catechin and caffeic acid, and the highest conversion efficiencies were 35.2 ± 3.14 and 32.93 ± 2.01%, respectively. In this paper, the catalytic activity of HpaBC on dihydrokaempferol and kaempferol was demonstrated for the first time. This study demonstrates a feasible method for efficiently synthesizing in vivo B-ring dihydroxylated flavonoids, such as catechins, flavanols, dihydroflavonols and flavonols, in a bacterial expression system.

Keywords: 4-hydroxyphenylacetate 3-hydroxylase; B-ring ortho-hydroxylation; Escherichia coli; biosynthesis; flavonoids.

Figure 1

SDS-PAGE of the proteins HpaB and HpaC. The protein expression of different plasmids in BL21 cells. P1: pRSFDuet-HpaBC; P2: pRSFDuet-HpaCB; P3: pETDuet-HpaBC; P4: pETDuet-HpaCB; P2&3: co-expression of P2 and P3; and P1&4: co-expression of P1 and P4. The locations of the HpaB and HpaC proteins are indicated by the arrows on the right. The molecular weights of the marker proteins (180 kDa, 100 kDa, 70 kDa, 40 kDa, 35 kDa and 15 kDa) are also shown.

Figure 2

Construction strategy for all engineered Duet vectors (grey shadowed area) with HpaB and HpaC genes (colored boxes). The ortho-hydroxylation activities of different strains; (a): pRSFDuet-HpaBC (P1), (b): pRSFDuet-HpaCB (P2), (c): pETDuet-HpaBC (P3), (d): pETDuet-HpaCB (P4), (e): co-expression of P2 and P3, and (f): co-expression of P1 and P4. ‘His His His’ represents the three amino acid composition of His-Tag, and’ Lys Phe Ser ‘represents the label composition of S-Tag. Final substrate concentration of 200 mg·L⁻¹, n = 3.

Figure 3

Production of E from the corresponding substrate, N. The substrate (final concentration of 200 mg·L⁻¹) was added to the cell culture in LB medium. (a): Conversion efficiency of E at different induction temperatures. The strains were induced for 8 h at 20 °C, 28 °C or 37 °C. (b): Conversion efficiency of E at different substrate delay times after IPTG induction. Bacterial culture medium was induced for 4 h, 6 h or 8 h at 28 °C. Data are shown as the means ± s.d.s (n = 3).

Figure 4

Growth curve of bacterial culture at different substrate concentrations and the conversion efficiency of E at different incubation times. The hollow boxes show the growth curve of bacterial cells, and the solid circles represent the titer of E at different incubation times. The IPTG induction time is shown by a red arrow, and the red squares indicate the substrate addition time. (a,b): Substrate (200 mg·L⁻¹) in LB medium; (c,d): Substrate (80 mg·L⁻¹) in LB medium. Data are shown as the means ± s.d.s (n = 3).

Figure 5

Conversion efficiency of E in different media (LB, TB and M9) and substrate concentrations (substrate concentrations from 40 mg·L⁻¹ to 120 mg·L⁻¹). (a): the conversion efficiency of E of the P2-carrying strain in LB, TB and M9 media. (b): the conversion efficiency of E of the P2&3-carrying strain in LB, TB and M9 media. Data are shown as the means ± s.d.s (n = 3).

Figure 6

HPLC analysis of the enzymatic products of the HpaBC complex, when feeding with different substrates. HPLC chromatogram (left) and standard compound (right) analyses of the enzymatic reaction. N, E, K, Q, DHK, DHQ, C and Af were monitored at 280 nm, and p-CA and CA were monitored at 340 nm. The substrates and corresponding products were detected by HPLC and LC-MS. The ortho-hydroxylation activities of (a): p-CA to CA; (b): N to E; (c): Af to C; (d): K to Q; and (e): DHK to DHQ. Final substrate concentration of 80 mg·L⁻¹, n = 3.

Figure 7

The catalytic process of HpaBC with different substrates. The red color is the first discovered catalytic activity in this study.