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Plant cells sell plants
Plant cells sell plants Under clear, late-summer skies, 106 pamcipants from 21 countries gathered along the pine-covered shores of the sparkling Baltic at Kallvik in Finland to assess progress in biotechnological applications of plant-cell biology*. The conference site, a financial institution’s training centre, was appropriate, because the meeting ranged from basic cellular functions to the breeding and marketing of genetically-engineered plant products. P. Brodelius (University of Lund, Lund, provided an Skveden) overview, in the first session, of the production of secondary metabolites in plant-cell culture. Progress in understanding the biosynthesis of secondary compounds has been helped by such culture systems, o\ving to the comparative ease of isolating precursors, inducing or treating with exogenous substances, and generating cl1NA libraries from the large quantities of relatively uniform cells. Nevertheless, many marketable seconda? compounds are produced only in differentiated cells. hindering their commercial production in culture. Brodelius gave several examples of commercially valuable secondary compounds that can be produced in culture. Among the terpenoids, the sesquiterpene derivative artemisinin can be produced in hair-v-root cultures ofiltienriria 111~1114~ established by infection with &u&uctcvizrr~l rhizqems. Various terpene cyclases are now being cloned for the eventual goal of increasing the yield of specific terpenes in transformed cells. A cDNA clone is now available for taxadiene synthase, which catalyses the cyclization of geranylgeranyl diphosphate in the first step in tasol biosynthesis. Taxol. an important anticancer drug, is currently isolated in low y$d from the Pacific yew (Tux~ls brevlfolia), and commercially viable schemes for
Copyright
0 1998,
Elswer
Science
taxol production in culture are currently being established. A conceptually similar approach is being taken for the production of alkaloids such as dopamine derivatives, hyoscamme and scopolamme in culture. The scheme involves elucidating the synthetic pathway, identifying rate-limiting steps and producing cI1NA clones for the critical enzymes, followed by efforts to direct the biosynthetic pathway towards the desired product in cultured cells. J. Memelink (Leiden University, Leiden, The Netherlands) discussed his group’s efforts to produce terpenoid indole alkaloids (TIA) in periwinkle (Cutlzurmdw moms) cell cultures. These compounds are medically important, and include the anticancer drugs vinblastine and vinc&tine, as well as ajmalicine, which is used in treating circulatory disorders. The first two compounds are extremely expensive, can only be isolated at low yield from plants and lie at the end of a pathway involving some twenty enzymatic steps. Memelink and co-workers are studying ways to increase the flux into TIA, based on improved transformation of the C. rclseus cultures and the use of fungal elicitors, which induce the key biosynthetic enzymes strictosidine synthase and tryptophan decarbox-)/lase. In culture, it has so far been possible to increase the levels of the enzymes but not of TIA production. Many of the secondary-metabolic enzymes currently attracting interest belong to the cytochrome-P45( 1 superfamily, which has several hundred member. M. Petenen (HeinrichHeine University, Diisseldorf, Germany) described the role of some that have been already characterized in the synthesis of a diversity of compounds, ranging from flavour com-ponents of vanilla, spearmint and peppermint, through jasmonic acid and phytoalexins, to phenylpropanoid derivatives such as lignin and subarin, and various alkaloids. The general problems and prospects for the use of cell cultures in the commercial production of marketable compounds were addressed in a workshop [chaired by W. Barz (Westfalische Wilhelms University, Miinster, Germany) and R. Fischer (Rheinisch-Westfilische Technische Hochschule, Aachen,
Ltd. All rights reserved.
0167
- 7799/98/$19.00.
PII: SO167-7799(97)01144-X
Germany)] and in a lecture by H. ten Hoopen (Delft University of Technology, Dell?, The Netherlands). The great promise is that commercially valuable metabolites, found in exotic or difficult-to-cultivate plants and only at very low concentrations, may be efficiently harvested from large-scale cultures. Ill ten Hoopen’s hypothetical example, only 60 X 500 ml flasks of C. roseus cultures, each doubling approximately every 12 hours, adding 0.6 g day’ of cell mass, and synthesizing vincristine at 1% dry weight, would yield approximately US$l million worth of vincristine over the course of a year! Productivity on this level has, unfortunately, not yet been obtained. Assuming that the desired compound can be obtained from culture at sufficient yields, problems including scale-up. maintenance of uniform cultures, provision of sufficient aeration. growth at highenough density and general process control tnust be overcome. Some species (e.g. Etl~inaiea) have been cultured on a large scale (in this example, 60 000 1). Hairy-root and other organ cultures present special difflculties in this regard, and those of hairy roots were addressed by R. Eible (Ingenieurschule Widen&l, Widenswil, Switzerland) and R. Cusidi, (University of Barcelona, Barcelona, Spain). Although industry remains very interested in plant-cell culture, owing to its potential for the production of rare compounds, only a few species are currently cultured commercially for phamlaceuticals Parlnx ,$rlser;e (roots and extracts). Lithospevmrrn eryhdi;on (shikonin), Colrlrr bllrmei (rosmarinic acid) and Coptir jupunica (berberine) among them. Engineering the metabolism of plants, rather than of cell cultures, also received some attention. K. Saito (Chiba University, Chiba. Japan) described the accumulation of high levels of cysteine through overexpression of cysteine synthase, and presented work on serine acetyltransferase and its expression in transgenie plants. I’. Shewry (Bristol University, Bristol, UK) detailed the role of specific high molecular weight glutelin subunits in determining bread quality as related to glutelin tertiary structure. A. Schulman (University of Helsinki, Helsinki, Finland) described approaches to the alteration of the biosynthesis, and hence the structure, of the other main grain component, starch.
Meeting yeport
TIBTECH JANUARY 1998
~((
(VOL 16)
pii-
2 f
Whereas starch and glutelin are important primarily in human nutrition, plants are also valued for more aesthetic reasons. As discussed by T. Teeri (University of Helsinki), much of the attraction of flowers derives from the activity of the anthocyanin biosynthetic pathway. Genetic engineering of floral colour is practical because the enzymes of the flavanoid pathway, which lead to anthocyanin biosynthesis, are well characterized, and the chemical basis ofthe final pigment colours is understood. White flowers have been produced in several species by antisense suppression of chalcone synthase, the first dedicated enzyme on the pathway. Teeri’s group has taken this approach in Gerbera hybrida, for which they have a transformation system, and achieved reduction in the level of floral pigmentation. Shades ofred, blue, pink and purple in anthocyanins relate to the degree and type of hydroxylation and glycosylation of the aromatic B-ring; the final pigment in the flower depends on enzyme specificity for modified intermediates. For example, the dihydroflavanol reductase (DFR) of petunia cannot convert dihydrokaempferol to pelagonidin (orange), whereas the DFRs of maize and gerbera can. Thus, transgenic petunia plants expressing either the maize or gerbera DFR are able to overcome this block and produce orange flowers. Floral colour has also been altered by modulation of the underlying regulatory genes. Manipulation of anthocyanin pigmentation either by traditional or modern means is clearly relevant commercially, because the annual retail value for cut flowers worldwide approaches US96130 billion. Anthocyanins also find their way into more traditional products of
Meeting report
orum
biotechnology, such as wine, and many industries are still based on plant extracts. R. Carle (University of Hohenheim, Stuttgart, Germany) described the extraction of essential oils and the development of a continuous production process for camomile oil. K. Jokinen (Cultor Finnsugar Bioproducts, Helsinki, Finland) explained how glycinebetaine, important in plant salinity and drought tolerance, was formerly viewed as an unwanted byproduct in sucrose production from sugar beet. Today, however, glycinebetaine is being extracted and targeted at a range of applications in fermentation, food processing, cosmetics and agriculture. M. Kiel (Boehringer Ingelheim, Ingelheim, Germany) discussed the vast range of fine chemicals, including the alkaloids atropine, scopolamine and hyoscyamine, that are still being extracted from wild and cultivated plants. The emphasis at Boehringer Ingelheim is on the breeding of high-yielding varieties for plantation production rather than on collection from the wild. Significantly improved scopolamine levels in transgenic Hyoscyamtrs muficzrr hairy-root cultures, as presented in a poster by K-M. Oksman-Caldentey and colleagues (University of Helsinki) may presage its production in reactors rather than in the field. Exhaustive screening for new biologically active compounds is constantly underway, and L-P. MoUeyres (Novartis, Basel, Switzerland) explained the vast discovery programme taking place at Novartis. For example, the new herbicides bialophos and hydantocidin (from Streptom yces hygrorfopicus) and coniexistin (from Paecilomyces vmiotii) have been identified from fungi.
However, the synthesis of hydantocidin requires eight steps and, in this case, fermentation or extraction may remain economically viable. Production of novel substances in the plant, with the field serving as the ‘bioreactor’, was the subject of one session. D. Larrick (Planet Biotechnology, Mountain View, CA, USA), and R. Fischer presented the wide range of projects involving the production of secretory immunoglobulin A (IgA) and other antibodies in plants. Up to 12 kg hectare-r of IgA can be produced in tobacco. Furthermore, if antibodies can be expressed efficiently m the cytoplasm, plant protection by this means may become possible. A session on gaining the approval of the regulatory agencies in Europe and the acceptance of the public at large rounded off the programme. W. Schuch (Zeneca Plant Science, Bracknell, UK) discussed the long road to the market by describing their experience with transgenic tomato paste. This extended from the basic research on ripening carried out in the mid-1980s to the development of the successful antisense transformants, early processing trials, market research, development of marketing partnerships, settling of regulatory issues, consumer research and, finally, the UK product launch in February 1996. We are now begin ning to move from such pioneering efforts to a more routine application of plant and plant-cell transgenic strategies for the production of commercial products.
Alan H. Schulman
correlation coefficient between the variation in mRNA levels and that in protein levels was only 0.48, even for tissues with few secreted proteins, providing a powerful argument for moving beyond gene-expression profiles. Clearly, genome technology is now sufficiently powerful to push the biological limits of its own relevance. The conference was less certain about where to go next, except that it was beyond genes into the domain of proteins and their function, the biological context in which genes, and proteins, operate. Paired
Genes to proteins in context Is the genome project the ultimate reductionist description of biology? A recent conference* answered this with an emphatic ‘No’, with *Beyond
prom
Germ
the Human Genome Prtqccr: 10 Proteins was organized by
Cambridge
Hralthtrch
MA,
USA)
and held
9-10
June
Institute at Nob
(Cambndgr, Hill.
CA,
USA,
1997.
TBTECH JANUARY 1998 NOL 161
Copyright
examples ranging from the almostidentical genomes of man and chimp (W. Bains, Merlin Ventures, London, UK) to the completely identical genomes of caterpillar and butterfly (W. Blackstock, Glaxo Wellcome, Stevenage, UK). More critically, N. L. Anderson [Large Scale Biology (LSB), Rockville, MD, USA] pointed out that the
0 1998,
Elsevter Science
Ltd. All rights reserved.
0167
- 7799/98/$19.00.
PII: SO167-7799(97)01133-5
Copyright
0 1998,
Elswer
Science
taxol production in culture are currently being established. A conceptually similar approach is being taken for the production of alkaloids such as dopamine derivatives, hyoscamme and scopolamme in culture. The scheme involves elucidating the synthetic pathway, identifying rate-limiting steps and producing cI1NA clones for the critical enzymes, followed by efforts to direct the biosynthetic pathway towards the desired product in cultured cells. J. Memelink (Leiden University, Leiden, The Netherlands) discussed his group’s efforts to produce terpenoid indole alkaloids (TIA) in periwinkle (Cutlzurmdw moms) cell cultures. These compounds are medically important, and include the anticancer drugs vinblastine and vinc&tine, as well as ajmalicine, which is used in treating circulatory disorders. The first two compounds are extremely expensive, can only be isolated at low yield from plants and lie at the end of a pathway involving some twenty enzymatic steps. Memelink and co-workers are studying ways to increase the flux into TIA, based on improved transformation of the C. rclseus cultures and the use of fungal elicitors, which induce the key biosynthetic enzymes strictosidine synthase and tryptophan decarbox-)/lase. In culture, it has so far been possible to increase the levels of the enzymes but not of TIA production. Many of the secondary-metabolic enzymes currently attracting interest belong to the cytochrome-P45( 1 superfamily, which has several hundred member. M. Petenen (HeinrichHeine University, Diisseldorf, Germany) described the role of some that have been already characterized in the synthesis of a diversity of compounds, ranging from flavour com-ponents of vanilla, spearmint and peppermint, through jasmonic acid and phytoalexins, to phenylpropanoid derivatives such as lignin and subarin, and various alkaloids. The general problems and prospects for the use of cell cultures in the commercial production of marketable compounds were addressed in a workshop [chaired by W. Barz (Westfalische Wilhelms University, Miinster, Germany) and R. Fischer (Rheinisch-Westfilische Technische Hochschule, Aachen,
Ltd. All rights reserved.
0167
- 7799/98/$19.00.
PII: SO167-7799(97)01144-X
Germany)] and in a lecture by H. ten Hoopen (Delft University of Technology, Dell?, The Netherlands). The great promise is that commercially valuable metabolites, found in exotic or difficult-to-cultivate plants and only at very low concentrations, may be efficiently harvested from large-scale cultures. Ill ten Hoopen’s hypothetical example, only 60 X 500 ml flasks of C. roseus cultures, each doubling approximately every 12 hours, adding 0.6 g day’ of cell mass, and synthesizing vincristine at 1% dry weight, would yield approximately US$l million worth of vincristine over the course of a year! Productivity on this level has, unfortunately, not yet been obtained. Assuming that the desired compound can be obtained from culture at sufficient yields, problems including scale-up. maintenance of uniform cultures, provision of sufficient aeration. growth at highenough density and general process control tnust be overcome. Some species (e.g. Etl~inaiea) have been cultured on a large scale (in this example, 60 000 1). Hairy-root and other organ cultures present special difflculties in this regard, and those of hairy roots were addressed by R. Eible (Ingenieurschule Widen&l, Widenswil, Switzerland) and R. Cusidi, (University of Barcelona, Barcelona, Spain). Although industry remains very interested in plant-cell culture, owing to its potential for the production of rare compounds, only a few species are currently cultured commercially for phamlaceuticals Parlnx ,$rlser;e (roots and extracts). Lithospevmrrn eryhdi;on (shikonin), Colrlrr bllrmei (rosmarinic acid) and Coptir jupunica (berberine) among them. Engineering the metabolism of plants, rather than of cell cultures, also received some attention. K. Saito (Chiba University, Chiba. Japan) described the accumulation of high levels of cysteine through overexpression of cysteine synthase, and presented work on serine acetyltransferase and its expression in transgenie plants. I’. Shewry (Bristol University, Bristol, UK) detailed the role of specific high molecular weight glutelin subunits in determining bread quality as related to glutelin tertiary structure. A. Schulman (University of Helsinki, Helsinki, Finland) described approaches to the alteration of the biosynthesis, and hence the structure, of the other main grain component, starch.
Meeting yeport
TIBTECH JANUARY 1998
~((
(VOL 16)
pii-
2 f
Whereas starch and glutelin are important primarily in human nutrition, plants are also valued for more aesthetic reasons. As discussed by T. Teeri (University of Helsinki), much of the attraction of flowers derives from the activity of the anthocyanin biosynthetic pathway. Genetic engineering of floral colour is practical because the enzymes of the flavanoid pathway, which lead to anthocyanin biosynthesis, are well characterized, and the chemical basis ofthe final pigment colours is understood. White flowers have been produced in several species by antisense suppression of chalcone synthase, the first dedicated enzyme on the pathway. Teeri’s group has taken this approach in Gerbera hybrida, for which they have a transformation system, and achieved reduction in the level of floral pigmentation. Shades ofred, blue, pink and purple in anthocyanins relate to the degree and type of hydroxylation and glycosylation of the aromatic B-ring; the final pigment in the flower depends on enzyme specificity for modified intermediates. For example, the dihydroflavanol reductase (DFR) of petunia cannot convert dihydrokaempferol to pelagonidin (orange), whereas the DFRs of maize and gerbera can. Thus, transgenic petunia plants expressing either the maize or gerbera DFR are able to overcome this block and produce orange flowers. Floral colour has also been altered by modulation of the underlying regulatory genes. Manipulation of anthocyanin pigmentation either by traditional or modern means is clearly relevant commercially, because the annual retail value for cut flowers worldwide approaches US96130 billion. Anthocyanins also find their way into more traditional products of
Meeting report
orum
biotechnology, such as wine, and many industries are still based on plant extracts. R. Carle (University of Hohenheim, Stuttgart, Germany) described the extraction of essential oils and the development of a continuous production process for camomile oil. K. Jokinen (Cultor Finnsugar Bioproducts, Helsinki, Finland) explained how glycinebetaine, important in plant salinity and drought tolerance, was formerly viewed as an unwanted byproduct in sucrose production from sugar beet. Today, however, glycinebetaine is being extracted and targeted at a range of applications in fermentation, food processing, cosmetics and agriculture. M. Kiel (Boehringer Ingelheim, Ingelheim, Germany) discussed the vast range of fine chemicals, including the alkaloids atropine, scopolamine and hyoscyamine, that are still being extracted from wild and cultivated plants. The emphasis at Boehringer Ingelheim is on the breeding of high-yielding varieties for plantation production rather than on collection from the wild. Significantly improved scopolamine levels in transgenic Hyoscyamtrs muficzrr hairy-root cultures, as presented in a poster by K-M. Oksman-Caldentey and colleagues (University of Helsinki) may presage its production in reactors rather than in the field. Exhaustive screening for new biologically active compounds is constantly underway, and L-P. MoUeyres (Novartis, Basel, Switzerland) explained the vast discovery programme taking place at Novartis. For example, the new herbicides bialophos and hydantocidin (from Streptom yces hygrorfopicus) and coniexistin (from Paecilomyces vmiotii) have been identified from fungi.
However, the synthesis of hydantocidin requires eight steps and, in this case, fermentation or extraction may remain economically viable. Production of novel substances in the plant, with the field serving as the ‘bioreactor’, was the subject of one session. D. Larrick (Planet Biotechnology, Mountain View, CA, USA), and R. Fischer presented the wide range of projects involving the production of secretory immunoglobulin A (IgA) and other antibodies in plants. Up to 12 kg hectare-r of IgA can be produced in tobacco. Furthermore, if antibodies can be expressed efficiently m the cytoplasm, plant protection by this means may become possible. A session on gaining the approval of the regulatory agencies in Europe and the acceptance of the public at large rounded off the programme. W. Schuch (Zeneca Plant Science, Bracknell, UK) discussed the long road to the market by describing their experience with transgenic tomato paste. This extended from the basic research on ripening carried out in the mid-1980s to the development of the successful antisense transformants, early processing trials, market research, development of marketing partnerships, settling of regulatory issues, consumer research and, finally, the UK product launch in February 1996. We are now begin ning to move from such pioneering efforts to a more routine application of plant and plant-cell transgenic strategies for the production of commercial products.
Alan H. Schulman
correlation coefficient between the variation in mRNA levels and that in protein levels was only 0.48, even for tissues with few secreted proteins, providing a powerful argument for moving beyond gene-expression profiles. Clearly, genome technology is now sufficiently powerful to push the biological limits of its own relevance. The conference was less certain about where to go next, except that it was beyond genes into the domain of proteins and their function, the biological context in which genes, and proteins, operate. Paired
Genes to proteins in context Is the genome project the ultimate reductionist description of biology? A recent conference* answered this with an emphatic ‘No’, with *Beyond
prom
Germ
the Human Genome Prtqccr: 10 Proteins was organized by
Cambridge
Hralthtrch
MA,
USA)
and held
9-10
June
Institute at Nob
(Cambndgr, Hill.
CA,
USA,
1997.
TBTECH JANUARY 1998 NOL 161
Copyright
examples ranging from the almostidentical genomes of man and chimp (W. Bains, Merlin Ventures, London, UK) to the completely identical genomes of caterpillar and butterfly (W. Blackstock, Glaxo Wellcome, Stevenage, UK). More critically, N. L. Anderson [Large Scale Biology (LSB), Rockville, MD, USA] pointed out that the
0 1998,
Elsevter Science
Ltd. All rights reserved.
0167
- 7799/98/$19.00.
PII: SO167-7799(97)01133-5