Arora R. (ed.) Medicinal Plant Biotechnology

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24 Plant cell cultures for production of medicinal compounds

Case: Functional genomics to discover alkaloid biosynthesis in two Solanaceae species

In our studies, we aimed to improve understanding of the regulation of alkaloid

biosynthesis in two Solanaceae species, Nicotiana tabacum and Hyoscyamus muticus,by

establishing a functional genomics-based technology. We applied cDNA-AFLP transcript

profiling in combination with targeted metabolite analysis in order to map the biosynthetic

genes involved in the tobacco alkaloid pathway. In these studies, Nicotiana tabacum BY-2

cell suspension culture was used and the alkaloid production was elicited in these cultures

with methyl jasmonate (Goossens et al., 2003). Altogether 459 unique genes were found to

be differentially regulated by methyl jasmonate, and homology searches performed with

these genes revealed that 58% of the genes displayed similarity with already known genes,

whereas no homology was found for 26% of the genes. Alkaloid accumulation took place

12 h after methyl jasmonate application with varying kinetics, and in addition to common

tobacco alkaloids such as nicotine, anabasine and anatabine, anatalline was also shown to

accumulate in tobacco cell culture after elicitation (Häkkinen et al., 2004). Interestingly,

nicotine was only a minor alkaloid accumulating in these cultures, whereas anatabine and

anatalline formed the main alkaloid pool. However, the limiting step in nicotine

biosynthesis was not observed to be very early in the pathway, since N-methylputrescine

was produced in high amounts. This result suggested that the limiting step in nicotine

biosynthesis occurred after N-methylputrescine. These results were supported by the recent

findings of Shoji and Hashimoto (2008), who showed that BY-2 cell cultures, contrary to

the case in tobacco hairy roots, exhibit very low activity for MPO (N-methylputrescine

oxidase), the next step in the pathway resposible for N-methylputrescine conversion.

Functional testing of the selected genes found from tobacco BY-2 was performed by

overexpressing these genes both in tobacco and in the related species Hyoscyamus muticus.

Two novel genes were found to activate tobacco alkaloid metabolism either in

undifferentiated cultures or in differentiated hairy root cultures (Häkkinen et al., 2007).

Interestingly, one of the genes also caused a marked increment in tropane alkaloid

derivative production in H. muticus hairy roots. This result suggests that this gene might

have a broader function in alkaloid metabolism in Solanaceae. On the other hand, when the

tropane alkaloid gene H6H was overexpressed in Nicotiana tabacum hairy roots, 45% of

the hyoscyamine supplied to the culture was converted into scopolamine (Häkkinen et al.,

2005). Most interestingly, up to 85% of the produced scopolamine was secreted out of the

cells. This is noteworthy, since tropane alkaloids are commonly retained in the root tissue

in hairy roots of endogenous tropane alkaloid producers (Hashimoto et al., 1993;

Jouhikainen et al., 1999). The transport mechanism of scopolamine in Hyoscyamus and

Nicotiana is not known, although it is likely that different transporter proteins are involved

in the two species because scopolamine is an endogenous product in the former but a

foreign metabolite in the latter. However, recently the set of tobacco BY-2 genes, which we

discovered earlier by the cDNA-AFLP analysis, revealed the presence of a MATE-type

transporter involved in alkaloid transport and it was co-regulated by methyl jasmonate in a

similar manner as other tobacco alkaloid biosynthetic genes (Morita

et al., 2009). Earlier, it

has been shown that the alkaloid berberine is transported by an MDR-type transporter in

Coptis japonica (Shitan et al., 2003), for which berberine is an endogenous metabolite,

whereas in Arabidopsis another type of transporter MATE is involved (Li et al., 2002).

Thus, due to its ability to take up and convert exogenously applied hyoscyamine, Nicotiana

could be of interest when designing a candidate production host for tropane alkaloids or

Plant cell cultures for production of medicinal compounds 25

possibly for other secondary compounds which are currently still being extracted from

whole plants for commercial production.

Case: Production of recombinant collagen and gelatin-related proteins with

predetermined composition and structure

Collagen and its denaturated form gelatin are used extensively in various applications in the

pharmaceutical industry. Annually over 50 thousand metric tonnes of gelatin are processed

to capsules and tablets, plasma expanders, stabilizers for biologics, and to structural

components for tissue engineering purposes. Traditionally gelatin is extracted from animal

tissues resulting in inhomogeneous products consisting of molecules having different

molecular weights and gelling properties. Particularly in the case of medical applications it

is important to have collagen-related products that are homogeneous and have a defined

composition; thus there is a need to apply recombinant technology for their manufacturing.

The production of recombinant collagen and collagen fragments with designed and

predetermined characteristics has been demonstrated in mammalian cells, insect cell

cultures, yeast, milk of transgenic animals and in tobacco plants (e.g. Geddis and Prockop,

1993; Vuorela et al., 1997; Nokelainen et al., 1998; John et al., 1999; Ruggiero et al., 2000;

Olsen et al., 2003). In our studies, we aimed to produce a recombinant full-length collagen

type I B1 (rCIa1) and a related 45-kDa rCIa1 fragment in barley (Ritala et al., 2008;

Eskelin et al., 2009). We selected barley to be the production host because barley is an

important crop plant with well established agronomic, harvesting, transport, storage and

processing practices. Barley has been adapted to a wider variety of climates than any other

cereal, from sub-Arctic to sub-tropical areas. Barley is a self-pollinating plant and our

earlier study has verified that gene flow through pollen dispersal is extremely low (Ritala et

al., 2002). In addition, barley stores large amounts of protein in its seed endosperm and

desiccated mature seeds can be stored for several years without substantial protein

degradation or loss of activity.

Our first study (Ritala et al., 2008) was designed to demonstrate the feasibility of barley

cell culture as a rapid and efficient means of prototype characterization. Both transient and

stable expression modes were applied to predict whether barley is a suitable host for

production of recombinant

collagen and gelatin. The rCIa1 accumulation was targeted to

the endoplasmic reticulum (ER) by fusing the CIa1 gene to an ER-directing signal peptide

sequence and an ER retention signal HDEL. The gene delivery was carried out by particle

bombardment into immature barley half embryos or barley cell suspension. Preliminary

evaluation of the suitability of barley as a production host for rCIa1 was obtained by using

a transient expression system. Less than 1 week was required to determine expression,

avoiding the time-consuming and labour-intensive production of stably transformed

suspension cell lines and genetically modified plants. After the preliminary transient

testing, stable transgenic barley cell lines were produced and grown as suspension cultures

in flasks and in a Wave bioreactor. The best producing cell line synthesized rCIa1 similar

to that purified from the yeast Pichia pastoris based on Western blotting, pepsin resistance

and mass spectroscopy analyses. However, the barley cell culture-derived CIa1

intracellular accumulation levels were low, ranging from 2 to 9 g/l. The other published

barley cell culture production system reached a level of 0.5 mg/l secreted heat-stable endo-

C-glucanse (Aspegren et al., 1995). It is difficult to compare productivities of different

systems since e.g. the nature of the product itself, factors regulating the gene expression,

26 Plant cell cultures for production of medicinal compounds

integration sites and copy numbers all determine the overall outcome. Nevertheless, the

obtained production levels illustrated the need for further process improvement in order to

use this technology to supply material for product development activities.

The promising results obtained using barley cell culture as a production host for rCla1

motivated us to carry on to produce a related 45-kDa rCIa1 fragment in barley seeds (Eskelin

et al., 2009). The 45-kDa rCIa1 fragment expression, also targeted to the ER, was

controlled by three different promoters (a constitutive maize ubiquitin, seed endosperm-

specific rice glutelin and germination-specific barley alpha-amylase fusion) in order to

compare their effects on 45-kDa rCIa1 fragment accumulation. Highest accumulation of

the 45-kDa rCIa1 was obtained with the glutelin promoter (140 mg/kg seed), whereas the

lowest accumulation was obtained with the alpha-amylase promoter (2 mg/kg seed). The

study of Qu and Takaiwa (2004) showed that the glutelin promoter GluB1 also used in our

experiments was among the weakest. Thus it is possible to increase the expression level by

selecting a stronger promoter carrying high transcriptional activity. Mass spectroscopy and

amino acid composition analysis of the purified 45-kDa rCIa1 fragment revealed that a

small percentage of prolines were hydroxylated, with no additional detectable post-

translational modifications. In conclusion, we successfully expressed recombinant collagen

and gelatin in barley. With the obtained accumulation levels we calculated that by

dedicating 10% of the land area under barley cultivation in Finland we could fulfill the

estimated global annual need of specialized collagens.

Discussion

From the point of view of the pharma-industry, plants are and will remain an important

source of new drugs, and also have a significant role as a source of lead compounds for the

design and development of novel pharmaceuticals for research and development (Wang,

2008). The plant kingdom possesses a remarkable collection of efficient and highly active

medicinal compounds, the majority of them yet uncharacterized. More than 200,000

secondary metabolite structures have hitherto been discovered from plants (Pichersky and

Gang, 2000). The majority of plant-derived compounds currently undergoing clinical trials

are directed to the oncological area, including new analogs of known anticancer drugs

based on the camptothecin-, taxane-, podophyllotoxin-, or vinblastine-type skeletons

(Butler, 2005; 2008; Arora et al., 2008; Arora, 2010). It remains to be seen whether the

current global economic crisis will slow down the breakthrough of biopharmaceuticals, but

it has been forecast that their total value will be $70 billion in 2010 (Walsh, 2006).

However, biopharmaceuticals account for 32% of all new medical entities. Approximately

165 biopharmaceuticals have been approved and about half of them belong to the five

major target categories of cancer, diabetes, growth disturbances, haemophilia and hepatitis.

Erythropoietin has paved the way as one of the flagships of biopharmaceuticals, with sales

exceeding $10 billion in 2005 (Walsh, 2006). The drug discovery process has made great

advances as a result of modern automated high-throughput technology and data handling,

which allows thousands of plant extracts to be screened in a few days. Plant cell cultures

have been shown to be good alternatives for production of high-value compounds, which

are produced in whole plants often in very minute amounts. Paclitaxel (Taxol

), an

important anticancer drug, is a good example of the importance of plant-based compounds,

as well as of their complexity and the necessity of finding alternative sources for their

Plant cell cultures for production of medicinal compounds 27

production. Theoretically, in order to produce 2.5 kg of taxol, 27,000 t of Taxus brevifolia

bark is required and altogether 12,000 trees must be cut down (Rates, 2001). Paclitaxel is

currently produced by plant cell cultures (Tabata, 2004).

Major advantages in plant cell-based production platforms include low production

costs, easy scale-up and particularly the general safety attributed to these methods. Due to

these factors, plant cell-based systems might one day outweigh other production systems,

especially those using animal cells as hosts (Ma et al., 2003). Furthermore, plant cells are

capable of carrying out many desirable post-translational modifications for recombinant

protein production, which cannot be obtained by e.g. microbial hosts to the same extent.

However, the limitations faced by plant cell cultivation technology still include the

generally low product yields and variable production rates. The production of plant-derived

compounds sometimes requires the use of two-phase cultivation, since the biosynthesis of

secondary metabolites is often linked to growth stage. Advances in bioreactor technology

have led to a wide variety of different types of reactors suitable for plant cell and tissue

cultures (Eibl and Eibl, 2008), and nowadays the cultivation of fragile and demanding plant

cells, traditionally considered rather challenging, can no longer be considered to be the

major limiting factor in the development of production processes. However, difficulties

with downstream processing and the presence of non-authentic glycan residues in

recombinant human proteins have caused complications in regulatory and approval issues

concerning production of plant-based biopharmaceuticals (Miele, 1997). Coping with these

factors is essential for the overall production process, since for example downstream

processing may account for even 80% of all production costs (Hellwig et al., 2004). In the

case of downstream processing, plant cell cultures offer a good choice for easier and

economically more viable production, since the formation of by-products, such as waxes

and phenolics, is typically lower in cell cultures than in differentiated cultures or whole

plants. In plant cell culture-based platforms, an essential prerequisite for a viable system is

the banking of the production cell line (Hellwig et al., 2004). In the case of plant cell

cultures this means that cryopreservation standard operation protocols must be drawn up

and a storage system with back-ups must be established. The cryopreservation operates in

two basic modes, either by slow-freezing or via rapid cooling of the cell line by

vitrification or encapsulation-dehydration. Both modes require empirical studies,

optimization and the use of cryoprotectants. However, rapid cooling can be considered

favourable, if successful, due the simplicity of the process. Furthermore, this system does

not require expensive or highly specialized equipment (e.g. Winkelmann et al., 2004;

Grout, 2007). As decisions concerning the manufacturing process are taken at an early

stage, considerable emphasis must be placed on development of the storage of the

production lines in question. This is a stage that cannot be neglected and should be

included in calculations when making profitability estimations and overall calculations of

product costs.

The regulatory process concerning plant-made pharmaceuticals (PMPs) still requires

development. The main problem is that the existing guidelines of the Food and Drug

Administration (FDA, 2002) and the European Agency for the Evaluation of Medicinal

Products (CPMP, 2002) are focused on production in whole plants, making the regulatory

issues concerning plant cell cultures rather obscure. Contained production using plant cell

cultures will fall under different regulations than those for field-grown products. Thus it is

expected that the regulation can be less stringent (Spök et al., 2008). From the regulatory

point of view, the whole production chain must comply both with the guidelines

concerning GM crops and with the requirements set for the production of pharmaceuticals

28 Plant cell cultures for production of medicinal compounds

(Sparrow et al., 2007; Spök et al., 2008). Currently, the roles of regulatory authorities are

overlapping both in the USA and in the EU. Of the applicant point of view, a clarified

guidance would be beneficial to support the application process and speed up the launching

of plant-derived pharmaceuticals.

With regard to general public approval, in March 2006 the EU comission published a

eurobarometer tracking European opinions in biotechnology. On the basis of this query

biotechnology related both to medicine and to industrial use, e.g. bioplastics and

bioproduction of pharmaceuticals, are widely supported in EU countries. On the other

hand, opinions towards biotechnology related to agriculture are still sceptical, with 58% of

the respondents having a negative attitude towards agricultural exploitation of

biotechnology. Directed to the public, and especially to patients worldwide, in 2005 the

International Alliance of Patients Organizations (IAPO) launched a Briefing Paper on the

topic of plant-made pharmaceuticals, providing accurate and independent information

based on evidence-based research. It is clear that when developing modern

biotechnological applications, especially those exploiting genetic engineering, it is of great

importance to consider the ethical issues relating to them. Particularly in the case of the

agricultural and food sector, environment and healthcare, biotechnological applications are

connected to new processes and ethical questions, which should be discussed in open

forums. In addition, it should be considered that prevailing values are dependent on the

cultural background and may vary considerably between countries.

The hopefully soon-to-be success stories of insulin and glucocerebrosidase will clear

the way for other plant-made biopharmaceuticals. Safflower-produced insulin was shown

to be bioequivalent to already marketed recombinant human insulin (Eli-Lilly’s Humulin)

in Phase III clinical trials in the UK in 2009 (www.sembiosys.com). The demand for

insulin is expected to grow exponentially in coming years as incidences of diabetes are

expected to reach 380 million cases in 2025. In addition, the development of new nasal and

inhaled administrations requiring high doses and frequent dosing will further increase the

consumption of insulin (Nykiforuk et al., 2006). Fortunately, it is estimated that the

safflower-based production system is so efficient that two middle-sized Canadian farms

could provide sufficient insulin for global markets (Moloney, 2007). The new

glucocerebrosidase (GCD) production system expected to be approved in the near future

utilizes carrot cells as manufacturing host (Shaaltiel et al., 2007). Recently, in clinical

Phase I studies six healthy volunteers were treated with plant cell-produced recombinant

GCD (prGCD) by intravenous infusion. The prGCD was well tolerated, non-immunogenic

and even showed a prolonged half-life when compared to a commercial enzyme

manufactured in mammalian cells. Phase III clinical trials are currently in progress

(Aviezer et al., 2009). These examples are very welcome, especially as they will cause the

regulatory approval process to take shape. These products will pave the way for plant- and

plant cell culture-manufactured biopharmaceuticals. The global demand for new drugs is

constantly increasing as the world’s population continues to grow. This global burden

makes sustainable and renewable drug production processes even more valuable, even

essential. Although not yet fully exploited, plant cell culture-based production offers a huge

potential to overcome diseases and to contribute to human well-being.

Plant cell cultures for production of medicinal compounds 29

Acknowledgements

We thank Kirsi-Marja Oksman-Caldentey for critically reviewing the manuscript and

Michael Bailey for revising the language of this chapter.

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