Fermentative production of tetrahydropapaverine and its
derivatives using Escherichia coli.
Akira NAKAGAWA

Research Institute for Bioresources and Biotechnology,
Ishikawa Prefectural University

Research aims
Benzylisoquinoline alkaloids (BIAs) have powerful physiological effects and arewidely used as pharmaceuticals. They include drugs such as morphine (analgesics),berberine (stegnotic), and magnoflorine (anticancer). BIAs have complex structureswith asymmetric carbons, making chemical synthesis of BIAs particularly difficult.
Recently, the potential for microbial production of BIAs has attracted attention.
The antispasmodic drug atracurium is listed in the World Health Organization’s (WHO)Model List of Essential Medicines, although it is relatively expensive compared to otherlisted drugs. Therefore, it is important to develop a cost-effective method for atracurium
production. Atracurium is manufactured from tetrahydropapaverine (THPP), itself atype of BIA. To potentially develop a new supply of inexpensive atracurium, we
attempted to construct a microbial production system for THPP. Intermediates of THPP
production, such as methylated norlaudanosolines (NLSs), are also importantcompounds that can be used or converted
6

into pharmaceuticals. An additional aim ofthe project is to catalogue a methylated NLS

7library, in concert with developing THPP

3'4'
Norlaudanosoline (NLS)

Tetrahydropapaverine (THPP)

Fig.1 Chemical formulae of NLS and THPP

production. This research will not only
contribute to the supply of inexpensive
atracurium, but also affect drug discovery
using BIAs.

Methods
We have previously shown BIA production from simple carbon sources such as
glucose and glycerol, using Escherichia coli (E. coli). Tools developed by us during thisresearch will be applied to THPP production. THPP can be obtained from NLS bymethylation of four hydroxyl groups (Fig. 1). Four enzymes were used to perform themethylation; coclaurine 6-O-methyltransferase (6OMT) from Coptis japonica (C.
japonica) for 6-O methylation, reticuline 7-O-methyltransferase (7OMT) fromEschscholzia californica for 7-O methylation, human catechol O-methyltransferase(COMT) for 3ʹ-O methylation, and 3ʹ-hydroxy-N-methylcoclaurine methyltransferase(4'OMT) from C. japonica for 4ʹ-O methylation.
After confirming expression of these enzymes in E. coli, the properties of eachenzyme were analyzed in vitro with crude extracts. Subsequently, the possible reaction
orders suitable for THPP production were investigated. Finally, THPP production wasattempted using multiple strains and a stepwise culture method to control the order ofreactions.
Results
1. Synthesis of various methylated NLSs using different enzyme combinations
In total, there are fifteen possible combinations to mix the four enzymes; this creates
fifteen different methylated NLSs. Initially,

products were analyzed in the reaction

mixtures containing combinations of enzymes. Twelve of the fifteen NLSs could be
identified in these mixtures (Table 1).

Table 1 Products when NLS was reacted with various combinations ofmethyltransferases

3ʹ, 4ʹ-O-dimethyl NLS was not

Combination of methyltransferases

Methylated positions of products

64'

C

6
○ ○ ○
7
○
3'
○
4'
○
67
63'
○
64'
○ ○
73'
○
74'
○
3'4'
673'
674'
63'4'
73'4'
673'4'

7

6+
4'

6+C

6+ 4'+ 4'+ C+ 6+ 6+ 6+ 4'+ 6+
7 C 7 7 4'+ 4'+ C+ C+ 4'+
C 7 7 7 C+
7○ ○ ○ ○ ○ ○
○
○
○
○
○
○ ○
○
○
○
○
○ ○
○ ○ ○
○
○
○
○
○
○

○ ○ ○ ○
○
○
○
○ ○ ○
○
○ ○
○ ○ ○ ○○○○○

6,3ʹ,4ʹ- and 7,3ʹ,4ʹ-O-trimethylated
NLSs because of the fact that a○
○
○

○
○ ○
○ ○ ○
○ ○ ○ ○ ○ ○ ○ ○○detected in any reactions. It was notpossible to discriminate between

○ ○
○ ○

suitable LC-MS protocol to separate
these NLS derivatives could not bedetermined. Surprisingly, THPP could
be detected in the reaction mixture
without 6OMT. Subsequently, it was
shown that 4ʹOMT possesses some
6OMT activity and THPP can beproduced even without 6OMT. As the amount of THPP in the reaction with 6OMT was
higher than that without 6OMT, 6OMT was used for THPP production in furtherexperiments. Additionally, the 6OMT-like activity of 4ʹOMT can produce the R-form ofthe NLS derivative (R)-reticuline, which is a very important intermediate chemical in
the production of opiates1). Although this is not directly related to this study, it is an
important discovery for BIA production in general.
2. Optimization of reaction order for THPP production
Although THPP could be produced by simultaneous reactions using all four enzymes,
productivity was low. The substrate recognition of some methyltransferases is affectedby positions of the methoxy group in methylated NLSs. Therefore, optimizing thereaction order will be important for THPP production. When COMT was reacted with
NLS as the first reaction, productivities were relatively high (Fig. 2A). Furthermore,
after COMT reaction, subsequent reactions still occurred efficiently using the threeremaining enzymes mixed at the same time (Fig. 2B). These data suggest that efficient
THPP production could be developed using a 2-step culture method, with an initial stepA

7

Busing a strain that

612Signal intensity (×106 cps)

Signal intensity (×106 cps)

14

10
8

expresses COMT,
and a second strain

54

that simultaneously

2

expresses 4ʹOMT-

2

1

6OMT-7OMT .

00

47→4'→6→C
7→4'→C→6
7→C→4'→6
7→C→6→4'
7→6→4'→C
7→6→C→4'4'→7→C→64'→7→6→C
4'→C→7→64'→C→6→7
4'→6→7→C
4'→6→C→7
C→7→4'→6
C→7→6→4'C→4'→7→6
C→4'→6→7
C→6→4'→7C→6 →7→4'
6→7→4'→C
6→7→C→4'6→4'→7→C
6→4'→C→7
6→C→7→4'
6→C→4'→7

C＋4'→6→7
C＋4'＋6→7
C＋4'＋6＋7
C→4'＋6＋7
C→4'＋6→7
C→4'→6＋7
C→4'＋6＋7

3

6Fig.2 Suitable reaction order for THPP synthesis in in vitro

A. THPP synthesis by four-step reaction. B．Detail investigation of C→4'→7→6．→and＋ indicate stepwise and simultaneous, respectively.

3. THPP production by 2-step culture
The COMT gene was introduced into an NLS production strain. The strain wascultured in a glucose-containing medium. After incubation, methylated NLSs could bedetected. THPP production was conducted by adding these NLSs in solution to theculture of the 4'OMT-6OMT-7OMT expressing strain. Unfortunately, THPP was notdetected in the second culture. However, trimethylated-NLSs were detected, suggestingthat part of the pathway is functional in the second strain.

Conclusions
In this study, reaction order was shown to be important for THPP production. Howand which enzymes determine the importance of the reaction order requires furtherinvestigation. Although THPP was successfully synthesized using in vitro experiments,
it was not replicated in vivo. This study suggests that COMT reacts with dopamine prior
to NLS synthesis, reducing NLS productivity. Furthermore, dimethyl-NLSs could bedetected in the second culture step, indicating that activities of 4ʹOMT, 6OMT, and/or7OMT may be reduced in comparison to the in vitro experiments. Fully understanding
why this reduced activity is observed will be important for further development of
THPP production systems.
References1) Nakagawa A, Matsumura E, Koyanagi T, Katayama T, Kawano N, Yoshimatsu K,Yamamoto K, Kumagai H, Sato F, and Minami H (2016) Total biosynthesis ofopiates by stepwise fermentation using engineered Escherichia coli

Nat Commun 7:10390