Biotech. Adv. Vol. 4, pp. 279-288, 1986 0734-9750/86 $0.00+.

50
Printed in Great Britain. All rights reserved. Pergamon Journals Ltd.

PRODUCTION OF PLANT SECONDARY

METABOLITES WITHOUT PLANTS: A PERSPECTIVE

B. E. ELLIS

Department of Chemistry and Biochemistry, University of Guelph, Guelph,

Ontario, Canada, NIG 3WI

ABSTRACT

Efforts to commercially exploit native plant secondary metabolite produc-

tion patterns in cell culture systems have been largely thwarted by the
repression of secondary metabolism under growth-orlented culture condl-
tlons. Where expression can be obtained by selection or ellcitatlon, the
difficulties of large scale cultlvatlon/processlng still make the cost
effectiveness of c e l l culture systems dubious except where a very high
value market niche can be identified.

The long range prospect for efficiently utilizing the catalytic genius of
higher plants resides in the transfer of appropriate genetic information to
microbial systems for whom the fermentation expertise and industrial faci-
lities already exist.

INTRODUCTION

In vitro culture of plant cells is now a mature technology with several

decades of success in dealing with dicotyledonous somatic tissue. One of
the suggestions for application of culture systems, however, the commercial
production of plant secondary metabolites in vitro (4,29,41,49), has not
materialized on a significant scale. The purpose of this review is to
examine the reasons behind this apparent failure and to indicate future
research directions that should allow us to overcome, or bypass, the exis-
ting problems.

Reviews on the topic of secondary metabolltes in plant cell cultures have

become a perennial feature of the cell culture literature. Authoritative
descriptions of the advances in this field are therefore readily available
(5,9,29,39) and the reader is referred to these for details of the plant
species and compounds which have been investigated.

279
280 B . E . ELLIS

SOME ECONOMIC REALITIES

Plant cell cultures offer very real advantages over intact plants for basic
studies of metabolism, growth and cell-environment interactions (2,18,30).
Perhaps because of the immense commercial potential associated with indus-
trial applications of the new tools of biotechnology, discussions of secon-
dary metabolite production in plant cell cultures have often tended to
revolve around the potential of culture systems for displacing current
methods of production (I,4,49). Factors frequently cited are the commercial
importance of plant secondary metabolites (20,49), the instability and/or
inadequacy of supply from present whole plant sources and, on the other
hand, the potential flexibility of cell culture production processes with
respect to timing and scale of production, i.e. a presumably better ability
to respond to market demand (22).

Without denying the valuable role of plant products in the chemical arsenal
of the medical profession, it should be pointed out that the plant contri-
bution sometimes quoted (20) is dominated by the "enormous sales of oral
contraceptive steroids. While the source of the steroid skeleton in these
products is the plant triterpene diosgenin, there is no prospect of plant
cell culture fermentations competing economically with the cultivation and
extraction of Dioseorea tubers. Although the remaining non-steroid products
still command a substantial sum each year at the retail level, the poten-
tial market for which cell culture-derived pharmaceuticals might realisti-
cally be targeted is considerably smaller than has been suggested (49).
Predictions of the price for a given commodity which will have to be
matched by a cell culture production process are also on uncertain ground.
The long term pattern of supplier/distributor relationships which exists in
the comparatively small market for plant derived pharmaceuticals does not
encourage competitive pricing. Alternative bioteehnological sources will
therefore not simply have to match the manufacturing costs of current
arrangements but will have to bring ma~or cost advantages before they will
be acceptable.

The question of supply instability due to impact of disease, climate o r

political change may well be more effectively addressed by the "low techno-
logy" approaches of plant strain optimization, selection for disease resis-
tance, development of modern husbandry practices and diversification in the
geographical distribution of plantations. Not only are the tools for these
approaches already at hand, but the economic return would thereby be re-
tained and enhanced in sectors of the world economy badly in need of
stability and stimulation.

If, for political or indirect economic reasons (e.g., development of a

national research capability), the in vitro production of plant products is
nevertheless to be pursued, how severe are the constraints which must be
overcome? This can be considered from two perspectives: first, the chal-
lenge of large-scale culturing of plant cells, and second, the problem of
gaining access to the genetic resource which the plant genome represents.

LARGE-SCALE CULTURES

Plant cell suspension cultures can potentially be grown on essentially the

same scale as microbial fermentations, with at least one successful run
using tobacco cells in a 20,OOOL fermentor (27). There are, nevertheless,
significant engineering problems associated with large)scale operations,
related primarily to the slow growth rate and comparative fragility of
 PLANT SECONDARYMETABOLITES 281

plant cells (22,43). Residence times of weeks are not unusual, if one takes
into account the growth of the primary (I0-20L) and intermediate (I00-500L)
inocula needed to launch a multi-thousand litre run (6). Not only does the
transfer between vessels and the prolonged cultivation period greatly
increase the risk of contamination, but, in addition, standard microbial
fermentation hardware is not optimal for large-scale plant cell cultivation
(28,44). This means a major investment in novel or modified fermentor
designs which may well have to remain dedicated to plant cultures.

Some of these factors have been incorporated into a detailed analysis of

the industrial costs associated with plant cell culture fermentations (25).
Using a model involving two-stage fermentations with subsequent harvest and
extraction, as well as a number of assumptions concerning rate of product
synthesis, a manufacturing cost as low as $17 US/kE product was estimated
for an annual output of 106 kg.. £t this cost, a number of plant-derived
products would seem to come into serious consideration as commercial goals.
There is no indication that this is occurring, however, and the true costs
associated with current technology and available culture characteristics
must therefore be much higher.

Significantly, the only compound obtained commercially by plant cell cul-

ture fermentation at present, shikonin (23,29), is not a high-volume pro-
duct aimed at a conventional world market of the sort that is usually
promoted as a biotechnology target. Instead, it is used as a high-added-
value supplement in a new cosmetic within a specific local market.

This type of low volume (102-104 kg/yr) production of plant specific

metabolites could develop for other industrial niches where the cell cul-
ture process can be cost effectively integrated into a total production and
marketing package. High volume production systems, however, presently seem
to be a very remote possibility.

THE GENETIC RESOURCE

The genetic potential of higher plants for production of a vast array of

novel chemicals has been exploited for centuries. The same biochemical
treasure trove has, however, been found to remain largely inaccessible when
plant cells are cultured in vitro (8,42). The reasons for the general lack
of expression of plant secondary metabolism under typical culture condi-
tions are still not well understood, but the phenomenon is probably a
function of the developmental and physiological status imposed upon the
cells by the supply of exogenous growth factors, perhaps in combination
with the rich nutrient regime.

That the metabolic ~otipotency of cultured cells is retained, but repres-

sed, in vitro has been demonstrated by regeneration of plantlets which once
again produce the spectrum of secondary metabolites typical for the species
(26). It is also emphasized by numerous observations of the induction of
specific biosynthetic pathways in cultured cells subjected to an
appropriate stimulus (e.g., biotic or abiotic stresses or elicitors)(]5).
For the most part, the molecular mechanisms which underlie rapid elicita-
tion of a previously repressed facet of plant secondary metabolism are
unknown. The elicitation phenomenon does demonstrate, however, that it is
possible to transiently override at least part of the down-regulation which
in vitro culture conditions can impose in cells of most species. Since it
has recently been shown that elicitation may only be effective within a
certain period of the culture cycle (I0,24), and in one case this restric-
282 B.E. ELLIS

tion has been correlated with the intraoellular concentration of a key

Lmetabolite (17), it is clear that an interplay exists between the elicitor
mechanism and the physiological state of the cell. Unfortunately, so
little is known about the regulation Of secondary metabolism in plant cells
that ellcitation of d e novo, or enhanced, synthesis of a desired end-
product remains a matter of empirical screening and serendipity.

The successful recovery of quantitative biochemical variants from popula-

tions of cultured plant cells can also be viewed as evidence that the
repression of secondary metabolism by culture conditions is not necessarily
a "tight" control pattern. Indeed, when techniques are employed which
allow evaluation of the cell-to-cell variability in product synthesis
(11,13), a striking range of levels of eommittment to secondary metabolism
is observed within the cell population. Considering the presumed homogene-
ity of the culture environment, at least in suspension cultures, the orl-
gins of this variation are not at all clear.

It is possible that it reflects the presence of a range of stable regula-

tory variants, generated, perhaps, by the rapid genomic alteration proces-
ses (somaclonal variation) known to Occur in cell cultures (33,38). While
repeated selection and cloning has been successful in isolating stable
high-producing lines in some specific instances (32,46), however, other
systems have proven unstable (14,16). Before these results can be rationa-
lized, more must be known about the molecular events which comprise regula-
tion of expression in a given secondary metabolic pathway. Attempts to
establish a correlation between high levels of product synthesis and the
levels of extractable activity for putative rate-limiting enzymes, for
example, have had mixed results. Enhanced levels of phenylpropanoid con3u-
gate production in cell cultures have been found to be associated with
elevated phenylalanine ammonia-lyase activity in tobacco cultures (7), but
not in Anchusa officinalis (19) or Rosmarlnus officinalis (45) cultures.
High production in R. offlclnalis lines, on the other hand, did show a
strong correlation with the levels of chorismate mutase activity (45).
Eight cell lines of Catharanthus roseus resistant to 4-methyltryptophan
displayed both elevated levels of tryptophan decarboxylase and of its
product, tryptamine, but only two of these lines also accumulated the
indole alkaloids which form the pathway end-product (36).

There are, therefore, a number of indications that it should be possible to

overcome the repression of secondary metabolism in cell cultures. What is
required before we can rationally design ways of selectively derepressing a
desired pathway is a major research commlttment in natural product enzymo-
logy and molecular biology. Programs such as those undertaken to characte-
rize the enzymes of indole (47) and isoqulnoline (48) alkaloid biosynthesis
are examples of the kind of focus that is badly needed. It is only on such
an information base that rational experiments can be formulated to probe
the associated regulatory mechanisms (21).

DOING WITHOUT PLANTS

Once we truly begin to understand, at the enzymic level, how plant secon-
dary metabolltes are formed and to discover where, and by what means, the
regulatory systems Operate, the question remains whether it will be neces-
sary to continue pursuing industrial applications of this information in a
plant cell culture context. If the goal is to produce a specific plant
metabollte as cheaply and efficiently as possible, the ideal system must
take advantage of the catalytic specificity which has evolved in the plant
 PLANT SECONDARY METABOLITES 283

genome and combine this with a cost effective delivery system. An approp-
riate high-producing cell culture line would clearly have both the neces-
sary enzyme catalysts and the metabolic machinery needed to organize and
support the biosynthetic process. As pointed out earlier, however, such
cultures do not represent cost effective production systems. The low abun-
dance (<0.15 of cellular protein) of the enzymes of secondary metabolism in
plants and in high-producing cell lines (3,31,37) is an indication of how
much "industrially extraneous" biochemical machinery must be supported in
plant cells. Even at high biomass accumulation levels and product accumula-
tion to the hypothetical limit (49) of approximately 25% of dry biomass,
the slow growth rate, vacuolar sequestration and special culture require-
ments of plant cells will continue to make their large-scale handling and
processing too costly for any but the most valuable products.

The obvious alternative is to t r y transferring the necessary plant-derived

catalysts to an efficient production milieu. The latter could take the form
of immobilized enzyme bioreaotors, or use microbes engineered to express
a plant biosynthetic pathway. Chemical production based on immobilized
enzymes is an established industrial technology and can employ either
non-living permeabllized cells or specific partially purified enzymes. The
feasibility of employing plant catalysts in this mode has already been
demonstrated (34,35). It seems that employment of an immobilized form of a
unique plant enzyme could comprise a key element in an overall synthetic
strategy combining enzyme technology and chemical modifications. Reactions
which require costly co-factors (e.g., S-adenosyl methionine fop methyl-
transferases) would not be realistic candidates for this approach, however,
nor is the technology presently available for efficient operation of mul-
tiple linked reaction steps.

Finally, even a limited application of this sort assumes a reliable, inex-

pensive supply of the plant enzyme in order to produce replacement reactors
at regular intervals. Given the low abundance of the enzymes of secondary
metabolism, this could be a serious constraint.

Expression of the structural gone for a plant enzyme in a microbial species

such as B. subtilis or E. coli could, therefore, be valuable at two
levels. First, it would offer a rich, easily-controlled source of the
enzyme which could be harvested and employed as a biosynthetic catalyst in
another context. In addition to guaranteeing a supply of the catalyst,
incorporation of the gene into the readily accessible microbial genome
would open the way for modification of the plant enzyme by site-directed
mutagenesis. It is very likely that a number of features of the native gene
product which are presently optimized for activity within a plant cell
could eventually be eliminated or modified to yield a "stripped down" enzyme
protein with improved stability but full catalytic function. It may also be
possible to modify the mlcro-environment at the active site and thus permit
conversion of modified substrates to novel products.

Expression of plant structural genes in both yeast (12) and bacterial (qO)
systems has recently been described, although the genes involved coded for
non-catalytic proteins. It remains to be demonstrated that a catalytically
functional plant protein can be obtained in this fashion.

Ultimately, it would be desirable to be able to forego the manipulations

involved in recovery of a plant protein from the microbial fermentation and
its incorporation into bloreactors. Will it prove feasible to not only
obtain expression of the plant enzyme structural gene within microbial
284 B E. ELLIS

cells, but also to allow the enzyme to function catalytically within that
compartment? Such a system would potentially bring considerable advantages.

It would:
I. allow utilization of existing fermentation facilities and mini-
mize the need for cell and protein processing;
2. make it possible to exploit biosynthetic reactions which must be
supported with a supply of eofactor(s);
3. allow precise control of the timing of expression of the plant
gene(s) by use of appropriate promoters.
4. take advantage of the microbial cell's inclination to excrete
over-produced metabolites, thus simplifying product isolation and poten-
tially prolonging the productivity of the bioreactor.

It might also be possible to improve the overall efficiency by working in a

microbial over-producer genetic background which would channel an inexpen-
sive fermentaion substrate into, for example, an amino acid needed as
primary substrate for the plant biosynthetic sequence.

There are many questions to be explored before such a concept would become
a reality. Do plant enzymes require some organizational or regulatory
infrastructure, unique to plant cells, in order to operate efficiently?
How long can a plant protein avoid proteolytic degradation within a hetero-
logous cytoplasm? Can a genetically engineered microbe produce significant
quantities of alkaloids or terpenoids and avoid autotoxioity?

Examination of relatively simple model systems involving a single plant

enzyme which can catalyze a unique reaction on a normal microbial metabo-
lite should begin to provide some insight into these and other problems.
Developing efficient solutions and employin E the results industrially will
not only provide exciting research challenges for many years but also
promises to finally allow the biochemical of potential plants to be fully
exploited.

REFERENCES

I. A.W. Alfermann and E. Reinhard (Eds.), Production of Natural Compounds

by Cell Culture Methods, Ges.Strahlen- und Umweltforsch.mbH (19?8)

2. L.A. Anderson, J.D. Phillipson and M.F. Roberts, Biosynthesis of secon-

dary products by cell cultures of higher plants, in Adv.Blochem.
Engin./Biotech. voi.31, Plant Cell Culture, Fiechter (Ed.) Springer-
Verlag, 1-36 (1985)

3. K.L. Bajaj, V. de Luca, H. Khouri and R.K. Ibrahim, Purification and

properties of flavonol-ring B gluoosyltransferase from Chr~sosplenium
americanum, Plant Physiol.72, 891-896 (1983)

4. W. Barz and B.E. Ellis, Potential of plant cell cultures for pharma-
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Reinhard (Eds.), Hippokrates-Verlag, 471-507 (1981)

5. J. Berlin, Secondary products from plant cell cultures, in Biotechno-

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(1986)
 PLANT S E C O N D A R Y METABOLITES 285

6. J. Berlin, Plant cell cultures-a future source of natural products?

Endeavour 8, 5-8 (1984)

7. J. Berlin, K.-H. Knobloch, G.Hofle and L.Witte, Biochemical characteri-

zation of two tobacco cell lines with different levels of cinnamoyl
putrescines, J. Nat.Prod. 45, 83-87 (1982)

8. H. Bohm, The formation of secondary metabolites in plant tissue and

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Cell and Tissue Culture, Vasil (Ed.), Academic, 183-208 (1980)

9. D.N. Butcher, Secondary products in tissue cultures, in Applied and

Fundamental Aspects of Plant, Cell, Tissue and Organ Culture, Reinert
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10. C.C.S. Chapple, Phenylpropanoid metabolism in Syrln~a vul~aris L.

tissue cultures, M. So. thesis, Univ. of Guelph (1984)

11. N. Chaprin and B.E. Ellis, Microspectrophotometric evaluation of

rosmarinic acid accumulation in single cultured plant cells, Can. J.
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12. I. Coraggio, C. Comagno, E. Martegani, B.M. Rnazi, E. Sala, L.

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13. B. Deus and M.H. Zenk, Exploitation of plant cells for the production
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16. D.K. Dougall, J.M. Johnson and G.H. Whitten, A clonal analysis of
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17. U. Eilert, V. DeLuca, F. Constabel and W.G.W. Kurz, Elicitor mediated

induction of tryptophan decarboxylase and strictosidine synthase
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19. B.E. Ellis, Characterization of single-cell clonal lines of Anchusa

officinalis, J. Plant Physiol. 119, 149-158 (1985)
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48. H.H. Zenk. Enzymologyof benzylisoquinolinealkaloidformation,in The

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