Biosynthetic pathway from DHA to 10-epi PDX via 10 R -HDHA by serial reactions of 8 S - and 15 S -LOXs
10-Epi PDX (10R,17S-12E,13Z,15E-triene) can be produced from DHA via two intermediates 10R- and 17S-HDHAs by combined reactions of ARA 8R- and 15S-LOXs. P. homomalla 8R-LOX was selected and used to produce 10-epi PDX from DHA via 17S-HDHA and 10R-HDHA with 15S-LOX because the enzyme is the only reported 8R-LOX. E. coli expressing P. homomalla 8R-LOX showed activity for DHA but not for 17S-HDHA (Table 1), indicating that P. homomalla 8R-LOX using DHA was only available for 10-epi PDX production. Thus, the enzyme was first used to convert DHA into 10R-hydroperoxy-DHA, which was then reduced to 10R-HDHA in the presence of a reducing agent such as cysteine (Fig. 2). The formed 10R-HDHA was sequentially converted into 10-epi PDX (10R,17S-DiHDHA) via 10R-hydroxy-17S-hydroperoxy-DHA by A. violaceum 15S-LOX in the presence of the reducing agent.
Table 1
Specific activities of E. coli expressing 8R- and E. coli expressing 15S-LOX towards DHA or HDHA.
Substrate | Product | E. coli expressing LOX | Specific activity (mmol/min/mg-cells) |
DHA | 10R-HDHA | P. homomalla 8R-LOX | 20 ± 0.2 |
| 17S-HDHA | A. violaceum 15S-LOX | 510 ± 2.1 |
| 17S-HDHA | B. thailandensis 15S-LOX | 243 ± 1.7 |
| 17S-HDHA | F. oxysporum 15S-LOX | 168 ± 1.4 |
| 17S-HDHA | N. seriolae 15S-LOX | 209 ± 1.1 |
17S-HDHA | | P. homomalla 8R-LOX | NDa |
10R-HDHA | 10-epi PDX | A. violaceum 15S-LOX | 280 ± 1.3 |
| 10-epi PDX | B. thailandensis 15S-LOX | 132 ± 0.9 |
| 10-epi PDX | F. oxysporum 15S-LOX | 58 ± 0.2 |
| 10-epi PDX | N. seriolae 15S-LOX | 106 ± 0.5 |
a ND: not detected. |
Selection of 15 S -LOX for the efficient biotransformation of 10 R -HDHA into 10-epi PDX
The putative LOXs from F. oxysporum and N. seiolae were identified as ARA 15S-LOX by characterizing their ARA-derived reaction product as 15S-hydroxyeicosatetraenoic acid (data not shown). The specific activities of E. coli expressing 15S-LOXs from A. violaceum, B. thailandensis, F. oxysporum, and N. seiolae towards 10R-HDHA were compared for their efficiency in the biotransformation of 10R-HDHA into 10-epi PDX. The specific activities followed the order E. coli expressing A. violaceum 15S-LOX > B. thailandensis 15S-LOX > N. seiolae 15S-LOX > F. oxysporum 15S-LOX (Table 1). E. coli expressing A. violaceum 15S-LOX showed the highest specific activity was selected as a biocatalyst to produce 10-epi PDX using the 10R-HDHA obtained after the reaction of E. coli expressing P. homomalla 8R-LOX using DHA.
Identification of the products obtained from the conversion of DHA and 10 R -HDHA by P. homomalla 8R-LOX and A. violaceum 15S-LOX
The product obtained from the conversion of DHA by P. homomalla 8R-LOX was identified as 10R-HDHA by comparing the retention time with that of the 10-HDHA standard using HPLC with reversed-phase column (Figure S1) and the 10S-HDHA standard and 10(±)-HDHA with chiral-phase column (Fig. 3A). The total molecular mass of the product was indicated by the peak at mass per charge (m/z) value of 343.8 [M − H]− in the LC-MS spectrum (Figure S2A), which was the same as that of 10-HDHA (MW = 344.5) with a difference of m/z 0.3. The critical peaks at m/z 161.2 and 325.3 in the LC-MS/MS spectrum of the compound (Fig. 4A) were the same as those originating from cleavage between C10 and C11 near the hydroxyl group and the loss of H2O from the total molecular mass of 10-HDHA with differences of m/z 0.2, respectively.
The product obtained from the conversion of 10R-HDHA by A. violaceum 15S-LOX was identified as 10,17-DiHDHA by comparing the retention time with that of the 10,17-DiHDHA standard using HPLC with reversed-phase column (Figure S3) In HPLC with normal-phase column, the retention times of the product 10,17-DiHDHA and PDX (10S,17S-DiHDHA) were different (Fig. 3B). It was due to the different R- and S-forms at C10. The retention times of PDX and 10-epi PDX also differed in the LC/MS chromatograms (Serhan et al., 2006). 15S-LOX converted 10S-HDHA into PDX (10S,17S-DiHDHA) (Shin et al., 2022), indicating that the product obtained from the conversion of 10R-HDHA by A. violaceum 15S-LOX was 10-epi PDX (10R,17S-DiHDHA).
In the LC-MS spectrum, the peak at m/z 359.8 indicated the total molecular mass of the product, corresponding to that of DiHDHA (MW = 360.5) with a difference of m/z 0.3 (Figure S2B). The fragment peaks at m/z 153.1, 177.1, and 261.2 in the LC-MS/MS spectrum of the product resulted from cleavages between C9 and C10, C10 and C11, and C16 and C17 near the hydroxyl groups with differences within m/z 0.2, respectively (Fig. 4B). The fragment peak at m/z 341.3 in the LC-MS/MS spectrum was formed by the loss of H2O from the total molecular mass with a difference of m/z 0.2. These results indicate that the product is 10,17-DiHDHA.
The chemical structure of the product obtained from the conversion of 10R-HDHA by A. violaceum 15S-LOX was determined as 10,17-DiHDHA by 1D and 2D NMR analysis (Table S1 and Figure S4). The coupling constants J11 − 12 and J15 − 16 were 15.1 Hz, indicating that the double bonds had E geometry at resonance frequencies of 5.68 − 5.70 and 6.68 − 6.69 ppm. The three double bonds of H4 − 5, H7 − 8, and H19 − 20 were Z geometry at 5.30 − 5.46 ppm. J13 − 14 was 10.0 Hz, and H13 and H14 had the same resonance frequency of 5.93 ppm, indicating that the double bond was Z geometry. These results indicate that the product is 10R,17S-4Z,7Z,11E,13Z,15E,19Z-DiHDHA.
Biotransformation of DHA into 10 R -HDHA by E. coli expressing P. homomalla 8R-LOX
The effects of temperature and pH on the conversion of DHA as a substrate into 10R-HDHA were investigated by E. coli expressing P. homomalla 8R-LOX. The maximal activity was observed at 30°C and pH 8.0 as an optimal temperature and pH, respectively (Figure S5). The effects of the concentration of DHA and cells on 10R-HDHA production was examined, and the maximal production was observed at 2.0 mM DHA and 4.0 g/L cells (Figure S6).
The optimal reaction conditions for 10R-HDHA production from DHA by E. coli expressing P. homomalla 8R-LOX were 30°C, pH 8.0, 2.0 mM DHA, 4.0 g/L cells, and 5% (v/v) ethanol in the presence of 200 mM cysteine. Under these conditions, the cells produced 1.4 mM (482 mg/L) 10R-HDHA from (657 mg/L) 2.0 mM DHA for 40 min (Fig. 5A), with a molar conversion of 70%, specific productivity of 8.8 µmol/min/g-cells, and volumetric productivity of 2.1 mM/h.
Biotransformation of 10 R -HDHA into 10-epi PDX by E. coli expressing A. violaceum 15S-LOX
The effects of temperature and pH on the conversion of 10R-HDHA as a substrate into were examined by E. coli expressing A. violaceum 15S-LOX. The maximal activity was observed at 20°C and pH 8.5 as an optimal temperature and pH, respectively (Figure S7). The optimal cell concentration for 10-epi PDX production by E. coli expressing A. violaceum 15S-LOX with 1.4 mM 10R-HDHA was investigated (Figure S8). 10-Epi PDX production was increased with increasing cell concentration up to 1.0 g/L and reached a plateau above this cell concentration. Therefore, the optimal cell concentration for the maximal production of 10-epi PDX was determined to be 1.0 g/L.
10R-HDHA obtained from the conversion of DHA by E. coli expressing P. homomalla 8R-LOX was purified, and the purified 10R-HDHA was used as a substrate to biosynthesize 10-epi PDX. The optimal reaction conditions for the production of 10-epi PDX from 10R-HDHA by E. coli expressing A. violaceum 15S-LOX were 20°C, pH 8.5, 1.4 mM 10R-HDHA, 1.0 g/L cells, and 5% (v/v) ethanol in the presence of 200 mM cysteine. Under these conditions, E. coli expressing A. violaceum 15S-LOX converted 1.4 mM (482 mg/L) 10R-HDHA into 1.2 mM (433 mg/L) 10-epi PDX in 30 min (Fig. 5B), with a molar conversion of 86%, specific productivity of 30.0 µmol/min/g−cells, and volumetric productivity of 2.4 mM/h (865 mg/L/h).
Biotransformation of DHA into 10-epi PDX via 10 R -HDHA by serial reactions of E. coli expressing P. homomalla 8R-LOX and E. coli expressing A. violaceum 15S-LOX
Under each optimal conditions, the serial reactions of E. coli expressing P. homomalla 8R-LOX and E. coli expressing A. violaceum 15S-LOX were performed for 90 min. DHA at 2.0 mM (657 mg/L) was converted into 1.2 mM (433 mg/L) 10-epi PDX via 1.4 mM (482 mg/L) 10R-HDHA in 80 min (Fig. 5C), with a molar conversion of 60% and volumetric productivity of 0.9 mM/h (319 mg/L/h). In the serial reactions, E. coli expressing P. homomalla 8R-LOX converted 2.0 mM DHA into 1.4 mM 10R-HDHA in 40 min, with a molar conversion of 70% and volumetric productivity of 2.1 mM/h. After 40 min, E. coli expressing P. homomalla 8R-LOX was removed and 1 g/L of E. coli expressing A. violaceum 15S-LOX was added to the cell-removed solution and incubated for additional 50 min. E. coli expressing A. violaceum 15S-LOX converted 1.4 mM 10R-HDHA into 1.2 mM 10-epi PDX for 40 min, with a molar conversion of 86% and volumetric productivity of 1.8 mM/h (649 mg/L/h). The productivities of 10-epi PDX in the serial reactions were 1.3-fold lower than those using 1.4 mM purified 10R-HDHA due to the presence of DHA. The residual DHA derived from the first whole-cell reaction was converted into byproducts, including 0.21 mM 17S-HDHA, 0.07 mM RvD5, and 0.04 mM 16,17-hepoxilin B5, by E. coli expressing A. violaceum 15S-LOX.