Screening experiments
Nostoc sp. strain PCC 7120, Nostoc punctiforme PCC 73102, and Anabaena variabilis ATCC 29413 possess six, ten, and four P450s, respectively. We screened the biocatalytic functions of these P450s using 47 small molecules that contain flavonoids, sesquiterpenes, low-molecular-weight drugs, naphthalene derivatives, and other chemicals with benzene rings [ Additional file 1 Figure S3)]. E. coli BL21 (DE3) cells carrying each P450 gene inserted into the pRED vector were co-cultured with the substrates and possible bioconversion products were analyzed by HPLC. Consequently, CYP110E1 of Nostoc PCC 7120 was found to be promiscuous for the substrate range mediating the biotransformation of various small molecules. The CYP110E1 enzyme that is C-terminally fused to RhFRed was confirmed to constitute the active P450 form by CO difference spectral analysis [ Additional file 1Figure S4]. Thus, cells of E. coli BL21 (DE3) carrying plasmid pCYP110E1-Red were used for the following experiments.
Bioconversion of flavanones by E. coli (pCYP110E1-Red)
Naringenin was biotransformed to a product (F-1) with a conversion ratio of 31.5% (Figure 2) through co-cultivation with cells of E. coli (pCYP110E1-Red). F-1 was identified as apigenin (4’,5,7-trihydroxyflavone) by its comparison with an authentic sample on HPLC analysis. Flavanone (RT 18.2 min) was converted to products F-2 (RT 15.9 min; 5.7%) and F-3 (RT 17.2 min; 2.1%), which were identified as 3-hydroxyflavanone and flavone, respectively, by their comparison with authentic samples on HPLC analysis. Figure 3 shows their production rate curves. Since this P450 was thought to biotransform various flavanones, we further examined 6-hydroxyflavanone (RT 15.9 min) and 7-hydroxyflavanone (RT 15.4 min). As a result, these hydroxyflavanones were converted to products F-4 (RT 15.1 min; 12.1%) and F-5 (RT 14.5 min; 1.4%), which were identified as 6-hydroxyflavone and 7-hydroxyflavone, respectively, by their comparison with authentic samples on HPLC analysis.
Bioconversion of a sesquiterpene by E. coli (pCYP110E1-Red)
Only zerumbone among the examined terpenes [ Additional file 1: Figure S3] was biotransformed through co-cultivation with cells of E. coli (pCYP110E1-Red). The crude ethyl acetate (EtOAc) extract (152.0 mg) from this bioconversion mixture (200 ml), subjected to silica gel column chromatography (hexane-EtOAc = 2:1), yielded 5.6 mg (12.7%) of S-1 (fr. 10–12), 3.2 mg (6.8%) of S-2 (fr. 19–26), and 1.5 mg (3.2%) of S-3 (fr. 30–40). These spectroscopic data are shown in Additional file 2.
The molecular formula of S-1 was determined to be C15H24O (zerumbone + 2 H) by HREI-MS. Consistent with its molecular formula, S-1 was proposed to be a product obtained by the reduction of a double bond in the substrate. The reduced double bond was determined to be 2,3Δ by the observation of a doublet methyl signal (δH 1.05, H-12), and the 1H-13C long range coupling from this doublet methyl to the ketone carbon (δC 205.1, C-1). The identity of S-1 was thus determined as (6E,10E)-2,6,9,9-tetramethylcycloundeca-2,6-dien-1-one (Figure 4) [16]. Zerumbone was found to be converted to S-1 with nontransformed E. coli BL21 (DE3) cells (data not shown). It was therefore thought that S-2 and S-3 were the genuine products by CYP110E1-Red.
The molecular formula of S-2 was determined to be C15H24O2 by HREI-MS. Consistent with its molecular formula and 1H-NMR spectrum, the introduction of an alcoholic OH group in S-1 was proposed. The position of the alcoholic OH group was clarified to be C-13 by the observation of an oxymethylene signal (δH 3.92 and δH 4.02, H-13) and the 1H-13C long range coupling from this oxymethylene to C-5 (δC 35.0), C-6 (δC 140.1), and C-7 (δC 126.0). The identity of S-2 was thus determined as (6Z,10E)-6-hydroxymethyl-2,9,9-trimethylcycloundeca-2-ene-1-one (Figure 4). This product (S-2) was a novel compound according to the CAS database.
The molecular formula of S-3 was determined to be C15H24O2 by HREI-MS. Consistent with its molecular formula and 1H-NMR spectrum, the introduction of an alcoholic OH group in S-1 was proposed. The position of the alcoholic OH group was clarified to be C-8 by the observation of an oxymethylene signal (δH 4.24, H-8) and the 1H-13C long range coupling from H-14 (δH 1.12) and H-15 (δH 1.26) to C-8 (δC 75.5). The identity of S-3 was thus determined as (6E,10E)-8-hydroxy-2,6,9,9-tetramethylcycloundeca-2,6-dien-1-one (Figure 4). This product (S-3) was a novel compound according to the CAS database.
Bioconversion of aryl compounds by E. coli (pCYP110E1-Red)
A variety of aryl compounds, which include naphthalene derivatives and low-molecular-weight drugs, were biotransformed through co-cultivation with cells of E. coli (pCYP110E1-Red). Converted compounds were identified by chromatographic and spectroscopic analyses. Spectroscopic data are shown in Additional file 2.
Compounds converted from 1-methoxynaphthalene
The crude EtOAc extract (156.5 mg) from the bioconversion mixture (200 ml) with 1-methoxynaphthalene and E. coli (pCYP110E1-Red), subjected to silica gel column chromatography (hexane-EtOAc = 6:1), yielded 2.6 mg (7.5%) of A-1 (fr. 13–15), 0.4 mg (1.1%) of A-2 (fr. 19–22), and 0.7 mg (2.0%) of A-3 (fr. 28–35).
The molecular formula of A-1 was determined to be C22H18O4 by HRAPCI-MS. Consistent with its molecular formula and 1H-NMR spectrum, A-1 was proposed to be a secondary product (dimer) obtained by a phenol oxidation coupling reaction of the direct conversion product. The structure of A-1 was determined to be 4,4'-dimethoxy-[2,2'-binaphthalene]-1,1'-diol (Figure 5) by the observation of 1H-13C long range couplings from H-2 (2’) (δ 6.92) to C-1 (1’) (δ 149.3), C-3 (3’) (δ 120.4), and C-4 (4’) (δ 142.1), and 1H vicinal spin network of H-5 (5’) (δ 8.30) – H-6 (6’) (δ 7.54) – H-7 (7’) (δ 7.49) - H-8 (8’) (δ 8.20) [18].
The molecular formula of A-2 was determined to be C11H10O2 by HRAPCI-MS. Consistent with its molecular formula and 1H-NMR spectrum, the introduction of one phenolic OH group in the aromatic ring was proposed. The position of this phenolic OH group was determined to be C-5 by the observation of 1H-1H vicinal spin networks of H-2 (δ 6.84) – H-3 (δ 7.39) – H-4 (δ 7.74) and H-6 (δ 6.84) – H-7 (δ 7.30) – H-8 (δ 7.85), and an NOE observed between H-8 and H-9 (δ 3.99). The identity of A-2 was thus determined to be 5-methoxynaphthalen-1-ol (Figure 5) [17].
The molecular formula of A-3 was determined to be C11H10O2 by HRAPCI-MS. Consistent with its molecular formula and 1H-NMR spectrum, the introduction of one phenolic OH group in the aromatic ring was proposed. The position of this phenolic OH group was determined to be C-2 by the observation of 1H-1H vicinal spin coupling of H-3 (δ 7.23, d, J = 8.5 Hz) and H-4 (δ 7.57, d, J = 8.5 Hz) and 1H-13C long range couplings from H-4 to C-2 (δ 145.4) and C-5 (δ 128.3). The identity of A-3 was thus determined to be 1-methoxynaphthalen-2-ol (Figure 5) [19].
Compounds converted from 1-ethoxynaphthalene
The crude EtOAc extract (89.3 mg) from the bioconversion mixture (200 ml) with 1-ethoxynaphthalene and E. coli (pCYP110E1-Red), subjected to silica gel column chromatography (hexane-EtOAc = 6:1), yielded 1.8 mg (4.8%) of A-4 (fr. 10–12) and 1.5 mg (4.0%) of A-5 (fr. 14–16).
The molecular formula of A-4 was determined to be C12H12O2 by HRAPCI-MS. Consistent with its molecular formula and 1H-NMR spectrum, the introduction of one phenolic OH group in the aromatic ring was proposed. The position of this phenolic OH group was determined to be C-4 by the observation of 1H-1H vicinal spin coupling of H-2 (δ 7.55, d, J = 8.7 Hz) and H-3 (δ 7.23, d, J = 8.7 Hz) and the 1H-13C long range couplings from H-5 (δ 7.78) to C-4 (δ 145.8). The identity of A-4 was thus determined to be 4-ethoxynaphthalen-1-ol (Figure 5) [20].
The molecular formula of A-5 was determined to be C12H12O2 by HRAPCI-MS. Consistent with its molecular formula and 1H-NMR spectrum, the introduction of one phenolic OH group in the aromatic ring was proposed. The position of this phenolic OH group was determined to be C-5 by the observation of 1H-1H vicinal spin networks of H-2 (δ 6.83) – H-3 (δ 7.37) – H-4 (δ 7.71), and H-6 (δ 6.85) – H-7 (δ 7.29) – H-8 (δ 7.89), and the 1H-13C long range couplings from H-3 to C-1 (δ 154.8) and C-4a (δ 125.4) and from H-7 to C-5 (δ 151.2) and C-8a (δ 127.3). The identity of A-5 was thus determined to be 5-ethoxynaphthalen-1-ol (Figure 5) [21].
Compounds converted from 2-methylnaphthalene
The crude EtOAc extract (175.0 mg) from the bioconversion mixture (200 ml) with 2-methylnaphthalene and E. coli (pCYP110E1-Red), subjected to silica gel column chromatography (hexane-EtOAc = 5:1), yielded 1.2 mg (3.8%) of A-6 (fr. 11–13) and 1.5 mg (4.7%) of A-7 (fr. 20–24).
The molecular formula of A-6 was determined to be C11H10O by HRAPCI-MS. Consistent with its molecular formula and 1H-NMR spectrum, the introduction of one phenolic OH group in the aromatic ring was proposed. The position of this phenolic OH group was determined to be C-4 by the observation of two singlet sp2 methines (δ 6.67 and δ 7.22) and the 1H-1H vicinal spin networks of H-5 (δ 8.10) – H-6 (δ 7.40) – H-7 (δ 7.43) - H-8 (δ 7.71). The identity of A-6 was thus determined to be 3-methylnaphthalen-1-ol (Figure 6). A-7 was identified as naphthalene-2-ylmethanol (Figure 6) with HPLC analysis by its comparison with an authentic sample extracted from co-culture with 2-methylnaphthalene and E. coli BL21 cells carrying plasmid pUCRED-Balk, which expressed the CYP153A13a gene [13].
Compounds converted from 1,6-dimethylnaphthalene
The crude EtOAc extract (95.7 mg) from the bioconversion mixture (200 ml) with 1,6-dimethylnaphthalene and E. coli (pCYP110E1-Red), subjected to silica gel column chromatography (hexane-EtOAc = 6:1), yielded 1.9 mg (5.5%) of A-8 (fr. 12–15) and 1.0 mg (2.9%) of A-9 (fr. 21–26).
The molecular formula of A-8 was determined to be C12H12O by HRAPCI-MS. Consistent with its molecular formula and 1H-NMR spectrum, the introduction of one phenolic OH group in the aromatic ring was proposed. The position of this phenolic OH group was determined to be C-4 by the observation of 1H-1H vicinal spin couplings of H-2 (δ 7.05, d, J = 7.9 Hz) and H-3 (δ 6.70, d, J = 7.9 Hz), and H-7 (δ 7.37, d, J = 8.6 Hz) and H-8 (δ 7.84, d, J = 8.6 Hz), and 1H-13C long range couplings from H-9 (δ 2.58) to C-1 (δ 126.6), C-2 (δ 125.1), and C-8a (δ 131.7). The identity of A-8 was thus determined to be 4,7-dimethylnaphthalen-1-ol [17]. A-9 was identified as (5-methylnaphthalen-2-yl)methanol (Figure 6) with HPLC analysis by its comparison with an authentic sample extracted from co-culture with 1,6-dimethylnaphthalene and E. coli BL21 (pUCRED-Balk) [13].
A compound converted from 2-bromophenol
The EtOAc extract from the bioconversion mixture (0.5 ml) with 2-bromophenol and E. coli (pCYP110E1-Red) was subjected to HPLC to yield a product (A-10). A-10 was identified as 2-bromobenzene-1,4-diol (Figure 6) with HPLC by its comparison with an authentic sample extracted from co-culture with 2-bromophenol and E. coli BL21 (pUCRED-Balk) [13].
A compound converted from 4-methylbiphenyl
The EtOAc extract from the bioconversion mixture (0.5 ml) with 4-methylbiphenyl and E. coli (pCYP110E1-Red) was subjected to HPLC to yield a product (A-11). A-11 was identified as [1,1’-biphenyl]-4-ylmethanol (Figure 6) with HPLC by its comparison with an authentic sample extracted from co-culture with 4-methylbiphenyl and E. coli BL21 (pUCRED-Balk) [13].
A compound converted from 7-ethoxycoumarine
The EtOAc extract from the bioconversion mixture (0.5 ml) with 7-ethoxycoumarine and E. coli (pCYP110E1-Red) was subjected to HPLC to yield a product (A-12). A-12 was identified as 6-hydroxy-2 H-chromen-2-one (Figure 6) with HPLC by its comparison with an authentic sample.
A compound converted from 2-(p-tolyl)pyridine
The crude EtOAc extract (103.7 mg) from the bioconversion mixture (200 ml) with 2-(p-tolyl)pyridine and E. coli (pCYP110E1-Red), subjected to silica gel column chromatography (CH2Cl2-MeOH = 20:1), yielded 3.1 mg (8.4%) of A-13 (fr. 23–27). The molecular formula of A-13 was determined to be C12H11NO by HREI-MS. The 1H- and 13C-NMR spectra showed the methyl group in the substrate was oxidized to the corresponding primary alcohol. The identity of A-13 was thus determined to be (4-(pyridin-2-yl)phenyl)methanol (Figure 6), which was also produced through co-culture with 2-(p-tolyl)pyridine and E. coli BL21 (pUCRED-Balk) [13].
A compound converted from ibuprofen methylester
The crude EtOAc extract (225.0 mg) from the bioconversion mixture (200 ml) with ibuprofen methylester and E. coli (pCYP110E1-Red), subjected to silica gel column chromatography (hexane-EtOAc = 4:1), yielded 1.1 mg (2.3%) of A-14. The molecular formula of A-14 was determined to be C14H20O3 by HREI-MS. Consistent with its molecular formula and 1H-NMR, the introduction of one alcoholic OH group in the substrate was proposed. The position of this alcoholic OH group was determined to be C-11 because all signals of H-10, H-12, and H-13 were observed to be singlet. The identity of A-14 was thus determined to be methyl 2-(4-(2-hydroxy-2-methylpropyl)phenyl)propanoate (Figure 7) [22].
Compounds converted from flurbiprofen methylester
The crude EtOAc extract (254.7 mg) from the bioconversion mixture (200 ml) with flurbiprofen methylester and E. coli (pCYP110E1-Red), subjected to silica gel column chromatography (hexane-EtOAc = 4:1), yielded 1.5 mg (2.7%) of A-15 (fr 10–12) and 1.8 mg (3.3%) of A-16 (fr 23–28).
The molecular formula of A-15 was determined to be C16H15FO3 by HREI-MS. Consistent with its molecular formula and 1H-NMR spectrum, the introduction of one alcoholic OH group in the substrate was proposed. The position of this alcoholic OH group was determined to be C-2 because the signal of H-3 (δ 1.80) was observed to be singlet. The identity of A-15 was thus determined to be methyl 2-(2-fluoro-[1,1'-biphenyl]-4-yl)-2-hydroxypropanoate (Figure 7).
The molecular formula of A-16 was determined to be C16H15FO3 by HRAPCI-MS. Consistent with its molecular formula and 1H-NMR spectrum, the introduction of one phenolic OH group in the aromatic ring was proposed. The position of this phenolic OH group was determined to be C-13 because the signals of H-12 and H-14 were observed to be doublet (J = 8.6 Hz) and at high field (δ 6.89). The identity of A-16 was thus determined to be methyl 2-(2-fluoro-4'-hydroxy-[1,1'-biphenyl]-4-yl)propanoate (Figure 7).