CHAPTER

Production of pharmaceuticals in a
specially designed plant factory
18 Eiji Goto
Graduate School of Horticulture, Chiba University, Matsudo, Chiba, Japan

18.1 Introduction
Plant factories with artificial lighting (PFALs), where leafy vegetables are cultivated until harvest in
closed plant production systems with artificial lighting (CPPSs), were proposed, developed, and
implemented in Japan during the 1980s. During the 1990s, the products from these factories received
high evaluations by the food service industry, to which they primarily catered. During the 2000s,
commercial production of nursery plants of fruits and vegetables, flowers, and ornamental plants was
initiated in CPPSs or PFALs (see Chapter 22). PFALs also provide good cultivation systems for the
production of plants for pharmaceutical use.
Recently, genetically modified (GM) plants have attracted much attention because of their use in
the production of valuable materials, such as pharmaceutical proteins, for humans or livestock.
Pharmaceutical products that have been or are being developed include oral vaccines for humans or
livestock, agents that prevent lifestyle-related diseases, substances with antimicrobial or antiin-
flammatory effects, and functional proteins and secondary metabolites.
The production of pharmaceutical materials by using GM plants (plant-made pharmaceuticals,
PMPs) has advantages over conventional production methods using animals or microorganisms
(Daniell et al., 2001; Ma et al., 2003), such as (1) low risk of contracting infectious diseases from
animals or microorganisms used, (2) elimination of the need to maintain a cold chain, (3) low pro-
duction cost, (4) ease of large-scale production, and (5) utilization as functional food.
Food crops can be utilized for the production of valuable materials because their edible compo-
nents have a low risk of transmitting infectious diseases. In Japan, research groups have introduced
functional protein-encoding genes into rice, potato, soybean, lettuce, tomato, and strawberry plants
(Table 18.1) [for oral vaccine see Nochi et al. (2007); for miraculin see Sugaya et al. (2008) and Kim
et al. (2010)]. Expression of the inserted DNA and accumulation of the required protein in the target
plant organ are significantly influenced by plant and climatic conditions (Stevens et al., 2000).
Therefore, a highly controlled environmental system is required to achieve an annual production of a
valuable material with consistent quality.
A CPPS is ideal for the production of medicinal substances that have been approved by pharma-
ceutical law. Advantages of the CPPS over open fields include: (1) stable plant production under
complete environmental control, (2) efficient use of water and CO2 gas, (3) fulfillment of the good

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251
252 Chapter 18 Production of pharmaceuticals

Table 18.1 Pharmaceutical materials developed or being developed in Japan.

Type Target Material Host plant

Oral vaccine Human Vaccine for cholera Rice

Human Influenza vaccine Rice
Livestock Vaccine for swine Lettuce
edema disease
Livestock Bird influenza vaccine Potato
Pharmaceutical material Human Human thioredoxin Lettuce
Pet animal Canine interferon for Strawberry
periodontal disease
Functional protein Human Lactoferrin Strawberry
Human Miraculin Tomato

The materials were selected from research projects subsidized by the METI as “Development of fundamental technologies for
production of high-value materials using transgenic plants” (2006e10).

manufacturing practice (GMP) specifications, and (4) low risk of gene diffusion. Because responses of
GM plants to environmental conditions have not been investigated, it is necessary to research the
physiological characteristics of the plants and establish suitable cultivation methods for each plant
under artificial conditions.
The author’s research group developed a CPPS for stable accumulation of high concentrations of
desired functional proteins in the edible components of strawberry and rice plants. The GM strawberry
plant accumulates a functional protein in the achenes and the fruit, which is known to prevent lifestyle-
related diseases. The GM rice plant accumulates an oral vaccine in the protein body of the seed. This
chapter summarizes the production system and appropriate environmental conditions for plant pro-
duction of valuable materials in large quantities.

18.2 Candidate crops for PMPs

Considering the selection of plant species, established genetic engineering technology for the plants is
primarily important, and the mechanism by which a plant accumulates the target material should also
be investigated. For commercialization of a large-scale production system, reduction in labor in terms
of cultivation management and easy installation of mechanization and automation are also of
importance.
We have tested a variety of food crops in the CPPS and calculated the production efficiency of
photoassimilates per unit light energy for rice, strawberry, lettuce, and tomato from our experimental
data (Table 18.2). Leafy vegetables and fruits are cultivated in commercial PFALs and greenhouses.
However, from the viewpoint of productivity, the production efficiency of photoassimilates per unit
light energy does not differ significantly among food crops. Therefore, besides vegetables, crops such
as cereals, pulses, and potatoes are candidate crops. For example, cereal and pulse crops are suitable
for the production of valuable materials derived from carbohydrates and proteins, and cereal crops and
potatoes can be stored for long periods at normal temperatures. Leafy vegetables have a high harvest
 18.3 Construction of GM plant factories 253

Table 18.2 Comparison of candidate crops for PMPs based on experimental data.
Rice Strawberry Lettuce Tomato

PPF mmol m s 2 1
800 300 200 600
Light period h day 1 12 16 16 12
Daily PPF (DLI) mol m 2 day 1
34.6 17.3 11.5 25.9
Integrated PPF mol m 2 year 1
12,440 6220 4150 9330
Cultivation period Days 100 90 30 90
Edible biomass per gFW m 2 800 3200 2500 7500
cultivationa
Harvest index 0.5 0.5 0.9 0.5
Dry matter ratio % 85 10 5 6
2
Total dry biomass per gDW m 1360 640 139 900
cultivation
Number of cultivations 3.6 4 12 4
2 1
Total dry biomass per gDW m year 4896 2560 1667 3600
year
1
Production efficiency of g mol 0.39 0.41 0.40 0.39
photoassimilates per
unit light energy
a
Highest production was calculated for optimal cultivation management at a light condition based on our experimental data.

index and a short cultivation period, and we do not need to focus on the diffusion of pollen because
they are harvested before flowering. Fruits originally have functional compounds such as polyphenols;
therefore, GM fruit plants may have multiple positive effects if plant modification is designed to
accumulate a valuable material that can be used for the prevention of lifestyle diseases.

18.3 Construction of GM plant factories

We constructed two CPPSs with artificial lighting for stable accumulation of high concentrations of
desired functional proteins in the edible components of strawberry and rice plants. Fig. 18.1 shows the
facility design and air and water flow of a closed rice production system. The production system is an
advanced model of a commercial PFAL using only artificial light. However, the design is unique from
the viewpoint of transgenic crop management. The system must have facilities to prevent diffusion of
plant parts, such as pollen, and to inactivate the unused parts of the crop. If the system specifications
meet the standards, an ideal environment for GM plant production can be provided.
With regard to environmental control, spatial uniformity of light, temperature, humidity, and gas in
a plant canopy is crucial to the production of a material bearing consistent quality. The CPPS can be
used to manipulate not only the aerial conditions but also the root zone conditions. By using hydro-
ponics, the nutrient solution can be optimized for each growth stage. Therefore, the system can provide
the plant with a variety of artificial stimuli and physiological stresses to maximize accumulation of the
target material. Because the system provides appropriate growth conditions and an agrochemical-free
254 Chapter 18 Production of pharmaceuticals

FIGURE 18.1
Facility design and air and water flow in the typical cultivation room of a CPPS.

FIGURE 18.2
Cultivation of GM strawberry plants in a CPPS.

environment by blocking pest invasion, agronomic plant traits to overcome natural environment-
related problems are not required.
We developed a prototype of a strawberry cultivation room (20 m2) equipped with multilayer
shelves with white fluorescent lamps (FHF32-EX-N-H; Panasonic Electric Works Co., Ltd.) and a
commercial NFT hydroponic system for GM strawberry production (Fig. 18.2). We used non-GM
everbearing strawberry (Fragaria  ananassa Duch. cv. “HS138”) plants for the cultivation
 18.4 Optimization of environment conditions for plant growth 255

FIGURE 18.3
Cultivation of GM rice plants in a CPPS. The day is an example of a standard cultivation condition.

experiment. The seedlings were derived from the micropropagation of stem meristems (Hikosaka
et al., 2009; Miyazawa et al., 2009) and were then cultivated in the room. Air temperature, relative
humidity, and CO2 concentration in the room could be regulated to suitable levels. In the flowering
stage, pollination was carried out manually every 2e3 days.
We developed a prototype of a rice cultivation room with a lighting system by using a high-
intensity light source for GM rice production (Fig. 18.3). This prototype has a hydroponic cultiva-
tion area of about 15 m2, a height of about 2 m, and is equipped with 56 ceramic metal halide lamps
(M400FCEH-W/BUD/H0; Iwasaki Electric Co., Ltd.). The maximum light intensity is about
1200 mmol m 2 s 1 of photosynthetic photon flux (PPF) at 1 m above the floor surface. It is possible to
vary the light intensity to the required level within a range of 65%e100% by using a light control
system. Furthermore, the thermal conditions of the cultivation area can be controlled by separating the
lamp and cultivation spaces with glass and circulating air exhausted from the cultivation space to
the lamp space. Non-GM rice (Oryza sativa L., Nipponbare) plants were used for material plants in the
cultivation experiment.

18.4 Optimization of environment conditions for plant growth

18.4.1 Strawberry
We adopted the same approach as that of the rice plants to establish optimal light conditions for the
strawberry plants in the CPPS. According to the results of the growth experiment using various air
temperatures and CO2 concentrations, we set the average daily temperature at 23 C and the CO2
concentration at 1000 mmol mol 1. Fig. 18.4 shows the fruit yield of an individual plant for 35 days
from when the first flower was fertilized. A DLI (daily light integral) of 19.4 mol m 2 day 1 produced
380 g of fruits. With three cultivations per year and a planting density of five plants m 2, the annual
yield would be 42% greater than the average yield of greenhouses. The results indicate that suitable
256 Chapter 18 Production of pharmaceuticals

FIGURE 18.4
Fruit yield of strawberry plants grown in the CPPS under different PPF, light period, and DLI conditions.

cultivation conditions in the CPPS enhance photosynthesis of strawberry plants and subsequently
facilitate maximum fruit production.
We studied the effects of light quality on plant growth and concentration of human adiponectin
(hAdi) in transgenic everbearing strawberry (Fragaria  ananassa Duch. “HS 138”) (Hikosaka et al.,
2013). The hAdi plants in the flowering stage were exposed to three different light qualities (white,
blue, and red) for a 16-h light period under fluorescent lamps until the fruits were harvested. The hAdi
concentration on a fresh-weight basis in plants grown under red light condition was significantly
highest among all treatments.

18.4.2 Tomato
Kato et al. (2010) created a new variety of dwarf tomato suitable for high productivity of fruits that
contain the target pharmaceutical material in a closed cultivation system under stable environmental
conditions. We investigated the effects of light intensity on tomato fruit yield and the accumulation of
recombinant miraculin, a taste-modifying glycoprotein, in transgenic tomato fruits (Kato et al., 2011).
Plants were cultivated at various PPFs of 100e400 mmol m 2 s 1 under white fluorescent lamps in the
CPPS. Miraculin production per unit of energy used was the highest at 100 mmol m 2 s 1; however,
miraculin production per unit area was the highest at 300 mmol m 2 s 1. The commercial productivity
of recombinant miraculin in transgenic tomato fruits largely depended on light conditions in the PFAL.

18.4.3 Rice
To determine the optimal light conditions for rice plant production, we analyzed light conditions of the
conventional rice cultivation method in paddy fields and established a new lighting strategy. In Hikone
City, Shiga Prefecture, located in the center of Japan’s main island, the DLI exceeds 65 mol m 2 day 1
on a clear and sunny day in mid-June. However, the average DLI in paddy fields for approximately
5 months from transplanting to harvest is 43 mol m 2 day 1 (Fig. 18.5). In the CPPS, it is not
necessary to recreate a typical sunlight curve of 43 mol m 2 day 1 with a maximum intensity of
 18.4 Optimization of environment conditions for plant growth 257

2500 Clear and Average FIGURE 18.5

sunny day for 5 months Light conditions of conventional cultivation of rice in
2000
PPF (µmol m−2 s−1)

paddy fields and a lighting strategy specialized for a

1500 CPPS.

1000

500 GM rice room

DLI = 43 mol m−2 / day
0
4 8 12 16 20
Time (h)

1400 mmol m 2 s 1 because initial costs of lamps and ballasts are high and air temperature and hu-
midity control becomes relatively complicated with large changes in light intensity. Consequently, we
set light intensity at 1000 mmol m 2 s 1 PPF and light period at 12 h day 1 so the DLI reached
43 mol m 2 day 1. The air temperature in the light period was controlled at 27 C to maximize both
photosynthetic and growth rates. The air temperature in the dark period was set at 23 C, so the average
daily air temperature was 25 C. The relative humidity was set at 70%, and the CO2 concentration was
set at 400 mmol mol 1.
Rice plants were cultivated under various planting densities and DLI conditions. The results
showed that the yield of rice grown in the room was 40%e60% greater than the average yield of the
cultivar grown in paddy fields in Japan (Fig. 18.6). The main reason was probably that there were no

Productivity Light utilization efficiency FIGURE 18.6

1200 200 Comparison of productivity and light utilization ef-
180 ficiency of rice plants grown in the CPPS and in
1000 160
Rice yield (g/m2)

paddy fields.
LUE (g/kmol)

800 140
120
600 100
80
400 60
200 40
20
0 0
1000 1000 1200 Paddy field
PPF
−2
(µmol m s ) −1

Planting 90 120 120

density (m−2)
DLI 41 41 49 43
(mol m−2 day−1)
Avg temp. 25 25 25 24
(°C)