Fermentation Production of Plant Alkaloids via Synthetic Biology
and Development of Drug Discovery
Hiromichi MINAMI
Research Institute for Bioresources and Biotechnology,
Ishikawa Prefectural University
Research objective
Plant secondary metabolites such as alkaloids, with a wide variety of pharmacological
functions, are widely used in our lives and mainly produced via extraction from plants.
However, details on them are not clear owing to their low content, and many of them aredifficult to utilize on their own. If specific alkaloids can be produced efficiently, it would bepossible to find new bioactivities that have not been revealed due to low content or foreign
substances, and this could be an effective tool for drug discovery research.
The purpose of this study was to develop a practical alkaloid production system via a microbial
fermentation that does not require the addition of expensive substrates and establish an
efficient method for utilizing rare active ingredients of herbal medicines. In particular, we
conducted a practical production of morphinan alkaloids (thebaine), an important
pharmaceutical raw material, using a microbial fermentation method. In addition, we produced
large amounts of rare alkaloids, such as magnoflorine and sanguinarine, which are active
ingredients of crude drugs, and attempted to develop new drugs by discovering new
bioactivities.
Furthermore, by combining biosynthetic genes (combinatorial biosynthesis) and constructing
a unit-type production system, a practical production system can be established to produce
novel biologically active substances in a short time and at a low cost.
Methods
To date, we have been able to construct an alkaloid production system using a microbial
fermentation method and succeeded in producing 165.9 mg/L of (S)-reticuline, which is an
important intermediate of isoquinoline alkaloids and contains only trace amounts in plants,from inexpensive glucose1. However, the production system that we constructed used threeplasmids of biosynthetic enzymes, which cause cellular instability. To construct a more stableproduction system, an alkaloid biosynthetic pathway was constructed on the Escherichia coli
chromosome. Each of the biosynthetic genes up to (S)-reticuline (12 genes) was sequentiallyinserted into the chromosome together with the T7 promoter to construct a (S)-reticuline
production strain. By optimizing the culture conditions using a jar fermentor, we establisheda practical production system with a production volume in g/L.

Afterward, we investigated the production of morphinan alkaloids and rare alkaloids such as
magnoflorine, which are important pharmaceutical raw materials. The biosynthetic enzymes
for each alkaloid from (S)-reticuline were successfully expressed in E. coli using a plasmid.
However, where it was difficult to construct a biosynthetic pathway on the chromosome, weconsidered constructing it on a plasmid.
Results
Each of the biosynthetic genes (12 types) up to (S)-reticuline was sequentially inserted into
the E. coli chromosome along with the T7 promoter to establish a reticuline biosynthetic
pathway from glucose (Fig. 1). As a result of optimizing the culture condition using a jar
fermentor, 3 g/L of (S)-reticuline production was successfully achieved.
The production of the morphinan alkaloid thebaine was examined (Fig. 1). Eightbiosynthetic genes from (S)-reticuline to thebaine were inserted into the constructed (S)reticuline-high-producing E. coli chromosome to establish the thebaine biosynthetic pathway
from glucose. However, the production of thebaine could not be confirmed. Therefore, weconstructed a thebaine-producing strain by incorporating eight biosynthetic genes into threeplasmids and introducing them into (S)-reticuline-high-producing E. coli cells. As a result,

we succeeded in producing 40.6 mg/L of thebaine.
Furthermore, the (S)-reticuline-producing strain was used as a platform for the production ofvarious isoquinoline alkaloids (Fig. 2). Therefore, by introducing two magnoflorine
biosynthesis genes into the (S)-reticuline-producing strain, we investigated the production ofmagnoflorine from glucose and succeeded in producing 10 mg/L.

Conclusion
Microbial fermentation production of isoquinoline alkaloids ((S)-reticuline, thebaine, andmagnoflorine) was successfully achieved using E. coli cells. This production system is a modelcase for introducing 20 levels of exogenous genes and enables us to obtain unique and superior

data and improve the accuracy of development. The production of 3 g/L of (S)-reticuline was
successfully achieved, and the improvement in the production efficiency of other compounds
is expected to lead to a wide range of practical production of various secondary metabolites
via the microbial fermentation method, which has been difficult to achieve so far.
References
1) Matsumura E. et al. (2018) Microbial production of novel sulphated alkaloids for drug
discovery. Scientific Reports 8: Article number 7980.