(R)-reticuline production by methyltransferases

Previously, we achieved the total biosynthesis of (S)-reticuline, a precursor of thebaine, from glycerol in a shake-flask culture (yield, 103 μM) using an engineered E. coli strain15. Previous studies have shown that the synthesis of the R-form of reticuline should be the initial production step (Fig. 1 and Supplementary Fig. 1)5. To produce (R)-reticuline, we examined the combination of three methyltransferases derived from Coptis japonica: 6-O-methyltransferase (6OMT)16, coclaurine N-methyltransferase (CNMT)17 and 4′-O-methyltransferase (4′OMT)18; these would theoretically synthesize racemic forms of reticuline from (R,S)-THP (Fig. 2a). However, only the S-form is produced by the strain that expresses the three methyltransferases (AN1752) cultured in (R,S)-THP-supplemented medium (Fig. 2b,c). After extensive empirical trials, we found that the CNMT- and 4′OMT-expressing strain (AN1600) could produce R-form reticuline (Fig. 2d). Because it has been reported that 4′OMT has 6OMT activity towards norcoclaurine3, we speculated that 4′OMT has 6OMT activity towards THP as well as norcoclaurine. To verify this, an enzyme assay was conducted with crude extract from 6OMT- or 4′OMT-expressing strains (AN1472 or AN1028, respectively). When 4′OMT was incubated with THP, both 6-O-methyl THP and 4′-O-methyl THP were synthesized, whereas only 6-O-methyl THP was detected in the reaction containing the 6OMT crude extract (Supplementary Fig. 2a,b). Indeed, 6,4′-O-dimethyl THP was synthesized by 4′OMT only (Supplementary Fig. 2c). These results suggested that 4′OMT has 6OMT activity towards THP, and consequently, reticuline could be produced without 6OMT (Fig. 2d). However, it was not clear why the R-form of reticuline was produced by the CNMT and 4′OMT expression strains, and further investigations are required to resolve this issue (described in the Supplementary Note 1).

Figure 1: Total biosynthesis of thebaine using four-step culture. The brackets indicate the reactions in the individual strain of each culture step. ATR2, NADPH-cytochrome P450 reductase 2; CNMT, coclaurine N-methyltransferase; DODC, dopa decarboxylase; MAO, monoamine oxidase; SalSNcut, N-terminally-truncated salutaridine synthase; SalR, salutaridine reductase; SalAT, salutaridinol acetyltransferase; TYR, tyrosinase; 3,4-DHPAA, 3,4-dihydroxyphenylacetaldehyde; 4′OMT, 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase. Full size image

Figure 2: Total biosynthesis of (R,S)-reticuline. (a) (R,S)-Reticuline synthetic pathway from (R,S)-THP. (b) The chirality analysis of pure (R,S)-reticuline. (c) The products from the culture of AN1752. (d) The products from the culture of AN1600. Experiments in b,c were conducted three times, and same tendency was observed. (e) (R,S)-Reticuline production following the addition of various concentrations of (R,S)-THP to the AN1600 culture. The error bar indicates the standard deviation of three independent experiments. Full size image

When (R,S)-reticuline was produced from various concentrations of (R,S)-THP by the two methyltransferases expression strain (AN1600), (S)-reticuline production increased as (R,S)-THP increased, whereas (R)-reticuline formation decreased slightly, presumably owing to an S-form preference by CNMT and/or 4′OMT (Fig. 2e). The maximum amount of (R)-reticuline was obtained using fermentatively produced (R,S)-THP at 99.2 μM in the AN1600 culture. The (R,S)-reticuline yield reached 48±11 μM (16±3.6 mg l−1), including 15±4.2 μM (4.9±1.4 mg l−1) of the R-form. Thus, (R,S)-THP was used at ∼100 μM to produce downstream compounds in subsequent experiments.

Selection of a suitable cytochrome P450 reductase (CPR)

During the establishment of the conditions necessary to produce (R,S)-reticuline using E. coli with 4′OMT and CNMT expression, the enzyme that converts (S)-reticuline to (R)-reticuline, known as STORR, was identified (Supplementary Fig. 1)8,19,20. Therefore, we also examined the efficiency of STORR in E. coli. As STORR contains a P450 enzymatic domain (CYP80Y2), and requires the reductase partner of P450 enzymes, CPR, we searched for a suitable CPR for P450s in E. coli. We had CPRs of Arabidopsis thaliana (ATR2), Papaver somniferum (PsCPR) and Rattus norvegicus (RnCPR) as laboratory stocks. Because the N-terminal deletion mutant of ATR2 is functional in E. coli21, a 45-amino-acid deletion of ATR2 (ATR2Ncut) was also prepared. The CPR activity towards bovine cytochrome c was verified using the crude extract from CPR-expressing strains22. The crude extract contained endogenous E. coli enzymes that catalyse nicotinamide adenine dinucleotide phosphate (NADPH); therefore, NADPHase activity was observed, even in the control sample (Supplementary Table 1). Although the activity of PsCPR was almost identical to that of the control, others had significant activity. Because ATR2 activity was strongest, it was used as a reductase partner of P450 enzymes in opiate production by E. coli.

Functional expression of STORR in E. coli

Generally, P450 expression is difficult in E. coli. Because some P450 enzymes are successfully expressed in E. coli only after the deletion of their N-terminus23,24,25,26, N-terminal-deleted STORR (STORRNcut) was constructed in addition to full-length STORR. These STORRs were expressed with ATR2 and cultured in medium containing (S)-reticuline, which was biosynthesized from glycerol as described previously15. SDS–polyacrylamide gel electrophoresis (SDS–PAGE) analysis showed that the deletion of N-terminus was effective for protein expression (Supplementary Fig. 3a). However, neither STORR expression strain produced detectable R-form reticuline (Supplementary Fig. 3b–d). Because P450 is a haemoprotein, the addition of 5-aminolevulinic acid (5-ALA), a precursor of haeme, is sometimes required for functional expression in E. coli27. Therefore, 5-ALA was added to the culture medium at the same time as isopropyl-β-D-thiogalactopyranoside (IPTG) induction. As a result, small amount of (R)-reticuline was detected in the culture medium of the STORRNcut expression strain, indicating that STORR was functional in E. coli, although the N-terminal deletion and 5-ALA addition were required (Supplementary Fig. 3e,f). However, (R)-reticuline production by STORR was low (Supplementary Fig. 3f, less than 10 μM) and required the addition of an expensive compound, 5-ALA; therefore, it is not applicable for the practical production of opiates in current conditions. Thus, to construct an opiate production system in E. coli, we attempted to develop (R,S)-reticuline production using two methyltransferases from (R,S)-THP.

Functional expression of SalS in E. coli

The next step required for the production of opiates is the conversion of (R)-reticuline to salutaridine, which is catalysed by the P450 enzyme salutaridine synthase (SalS; Fig. 1, Supplementary Fig. 1)28. Human P450s, CYP2D6 and CYP3A4 also have SalS activity, and have been successfully expressed in E. coli27,29,30. However, because these P450s produce undesirable by-products29, they are unsuitable for the practical production of opiates. Thus, we attempted functional expression of SalS in E. coli. We constructed two SalS variants, full-length SalS (SalS) and N-terminus deleted (SalSNcut), as in the case of STORR. These SalS proteins were expressed with ATR2. SDS–PAGE analysis showed that SalSNcut was expressed in E. coli, whereas the wild-type SalS was not, suggesting that the N-terminus sequence negatively affected SalS expression in E. coli, similar to STORR (Supplementary Figs 2a and 4a). These strains were grown on medium containing pure (R,S)-reticuline, and we measured the production of salutaridine. According to liquid chromatography mass spectrometry (LC-MS) analysis, the specific peak was observed in the medium of the SalSNcut-expressing strain, and the tandem mass spectrometry (MS/MS) fragment pattern of the peak was almost identical to that of pure salutaridine (Supplementary Fig. 4b,c), indicating that SalSNcut had salutaridine synthetase activity in E. coli. In the medium of the full-length SalS-expressing strains, a significant peak was observed at the same retention time as the salutaridine standard (an asterisk in Supplementary Fig. 4b); however, we could not confirm their identity because the MS/MS fragment patterns of the peak was ambiguous. In contrast to STORR, the addition of 5-ALA was not required for salutaridine synthetic activity of SalS (Supplementary Fig. 5), indicating that this property of SalSNcut is adequate for the practical production of opiates. Thus, we decided to use SalSNcut for the total biosynthesis of opiates.

Thebaine production from authentic (R,S)-reticuline

To validate the activities of salutaridine reductase (SalR)31 and salutaridinol 7-O-acetyltransferase (SalAT)32, the last two enzymes in the thebaine synthetic pathway (Fig. 1 and Supplementary Fig. 1), their corresponding genes were expressed in the salutaridine-producing E. coli strain AN1096 to generate the thebaine-producing strain AN1829. Measurement of thebaine production from authentic (R,S)-reticuline showed a thebaine-specific peak at m/z=312 (Fig. 3a). The MS/MS fragment pattern of the peak was almost identical to that of pure thebaine (Fig. 3b), indicating that both SalR and SalAT were functional in E. coli. The thebaine yield was 57±4.6 μM using pure (R,S)-reticuline at 200 μM, containing the R-form at ∼100 μM.

Figure 3: Thebaine production from pure (R,S)-reticuline or (R,S)-reticuline synthesized by total biosynthesis. (a) LC-MS analysis of thebaine (m/z=312) from the culture of the parental strain (salutaridine producer, AN1096; upper panel), the thebaine producer (AN1829; mid panel) and the thebaine standard (lower panel). (b) MS/MS fragment pattern of the products of AN1829 (upper panel) and the thebaine standard (lower panel). Experiments in a and b were conducted at least three times, and same tendency was observed. (c) Time-course analysis of the total biosynthesis of thebaine from fermentatively produced (R,S)-reticuline in the four-step culture. (R,S)-Reticuline synthesized by total biosynthesis was added to the fourth step of the culture at time zero. The error bar indicates the standard deviation of three independent experiments. Full size image

We determined that glucose was essential for thebaine production from (R,S)-reticuline (Supplementary Fig. 5). In thebaine production, acetyl-CoA is required to convert salutaridinol to salutaridinol 7-O-acetate (Supplementary Fig. 1), and acetyl-CoA is synthesized during glycolysis. Furthermore, SalS and SalR require NADPH as a cofactor, which is mainly synthesized in the pentose phosphate pathway during glucose metabolism. These observations explain why glucose is essential for thebaine production.

Stepwise culture strategy for opiates production

For optimal (R)-reticuline production, the amount of (R,S)-THP should be limited (Fig. 2e). Therefore, the thebaine production pathway should be divided into two: the total biosynthesis of (R,S)-THP, and thebaine production from (R,S)-THP. Although tyrosinase is essential for dopamine production in our system (Fig. 1), tyrosinase degrades THP12. To avoid the undesirable action of tyrosinase, a stepwise culture method was employed using the dopamine production strain AN1126 and (R,S)-THP production strain AN1055. Using this method, a total biosynthesis system for (R,S)-THP was successfully constructed12. Thus, for total biosynthesis of thebaine, the thebaine production pathway should be divided into three: dopamine production, conversion of dopamine to (R,S)-THP and thebaine production from (R,S)-THP. These pathways were individually constructed in three strains, resulting in the dopamine producer AN1126, a strain that produces (R,S)-THP from dopamine (AN1055) and AN1998, which produces thebaine from (R,S)-THP. Although we attempted thebaine production using three-step cultures, thebaine was not produced by the system, regardless of the presence/absence of IPTG. Although IPTG negatively affects (R,S)-reticuline production (Supplementary Fig. 6), the titre of thebaine was increased by adding IPTG (Supplementary Fig. 5); therefore, we speculated that opposing effects of IPTG on (R,S)-reticuline and thebaine productions resulted in the low productivity of AN1998 strain. Hence, (R,S)-reticuline production step would better be separated from the thebaine production step. Thus, we decided to construct the total biosynthesis system using a four-step culture method using four strains: AN1126, AN1055, the (R,S)-reticuline producer AN1600 and AN1829, which produces thebaine from (R,S)-reticuline (Fig. 1 and Supplementary Fig. 7).

Thebaine biosynthesis using a four-step culture system

For the first and second steps, total biosynthesis of (R,S)-THP was performed as described previously12, and the (R,S)-THP yield was 983 μM (282 mg l−1). In the third step, a one-eighth volume of (R,S)-THP (109 μM) was added to the (R,S)-reticuline production culture, yielding 16.9 μM (R)-reticuline. In the fourth step, the same volume of (R,S)-reticuline-containing supernatant (final concentration, 8.5 μM) was added to the culture of the thebaine producer. A thebaine yield of 6.8±0.67 μM (2.1±0.21 mg l−1) was obtained 15 h post initiation of the fourth step (Fig. 3c). The yields achieved were a 300-fold improvement compared with those of the latest yeast system8.

Hydrocodone biosynthesis using a four-step culture system

Hydrocodone is one of the most frequently used opioids and can be produced from thebaine using two enzymes, thebaine 6-O-demethylase and morphinone reductase (MorB;33 Fig. 4a). To evaluate the ability of the thebaine total biosynthesis system to produce medical opioids, we constructed a hydrocodone producer by introducing the genes encoding the two enzymes into the thebaine producer, resulting in strain AN1942 (Fig. 4a). The four-step culture system was used for hydrocodone production. A specific peak in the AN1942 culture was observed, and the MS/MS fragment pattern was identical to that of the hydrocodone standard (Fig. 4b,c). The hydrocodone-producing culture yielded 1.2±0.50 μM (0.36±0.15 mg l−1) codeine equivalents of hydrocodone, indicating that the thebaine total biosynthesis system was applicable for the biosynthesis of opioids.