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Commercial Success of ECLAIR Programme
AGRE-0021: Optimisation of lignin in crop and industrial plants through genetic engineering (OPLIGE) |
AGRE-0021: Optimisation of lignin in crop and industrial plants through genetic engineering (OPLIGE)
Science Background
Wood, agricultural residues and many other plant materials which can be used for feed or paper production consist largely of lignocellulose (a composite of mainly cellulose, hemicellulose and lignin). In the production of paper pulp (which is mainly cellulose), the hemicellulose and much of the lignin is removed using alkali. To produce bright paper pulp may require further treatments to remove the lignin residues. Breakdown products of lignin contribute to the highly coloured effluents produced by pulping. Lignin also has a negative effect on the digestibility of forage crops limiting the ability of microorganisms to break down the carbohydrate constituents (cellulose and hemicellulose). These carbohydrates would also be valuable as a source of sugar in fermentation of ‘green chemicals’. However, the need to separate the lignin in order to achieve effective hydrolysis (saccharification), is again costly. Hence, it is of interest to design plants with a modified lignin content, adapted to specific agricultural and industrial uses. Advances in plant genetic engineering, particularly the use of antisense technology, in theory makes this possible if the constitutive and regulatory genes were known. At the time this work was started no genes involved in the later stages of lignification had been characterized.
Objectives
This project aimed to use a plant genetic engineering approach to modify lignin content. Antisense RNA technology would be used to interfere with the regulation of three genes coding for enzymes involved in the lignification process. The genes of interest were those encoding proteins with the following enzyme activities: O-methyltransferase (OMT) which controls the degree of methylation of lignins; cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD), both of which are involved in monomer synthesis and hence control the flux of lignin production. First these genes, which had never previously been characterized, had to be obtained through screening of cDNA libraries in order characterize genes responsible for the enzyme activities of interest. Once these had been isolated and cloned, transformation procedures would be applied to model plants (tobacco) and target plants of economic interest (poplar and alfalfa), as well as other potential target plants (tall fescue, Eucalyptus). The chemical and phenotypic characteristics of the transformed plants would be investigated for digestibility and pulping quality.
Significant changes and results since end of ECLAIR
The main benefits of this project were the characterization of the genes encoding some of the stages of lignin biosynthesis and the ability to modify these in model plant systems. The results demonstrated the feasibility of a molecular approach to improving the pattern of lignin in plants in order to achieve commercial benefits. It also established the suitability of the OMT and CAD genes as primary targets for genetic manipulation and the applicability of the antisense technology. This project encouraged participants to continue research with funding successful applications to both AIR and FAIR. At the same time, the commercial partners, Zeneca (UK) continued to the next stage, that is the development of cost effective transformation processes for major pulpwood trees and its integration with clonal propagation systems. Field trials of these clones would be necessary to demonstrate that transformed trees are safe and that they offer economic benefits to the pulping industry. A system should then be set up to produce and supply modified plants on a commercial basis. This has been initiated with outlicensing agreements with major multinationals and a number of companies worldwide (North America, South America, Africa and Asia). Several patents were filed during or as a result of this project.
Results
At end of this ECLAIR project
The activities carried out under this project, coordinated by Université Paul Sabatier (France), showed that down regulation (blocking) of certain lignification genes could result in marked changes in lignin composition and reactivity. These induced changes improved pulping characteristics and animal feed utilization. These modifications did not induce significant changes in growth, structure or morphology of the plants. This indicated that a degree of variability in the lignin polymer has no adverse affect on the physiology of the plant and hence this technique could be used to improve commercial varieties or lines. For example transformed forest trees with more easily extractable lignins would be of value to the paper pulp industry, since smaller quantities of pulping chemicals would have to be used. While it was considered that longer field trials of these plants were required, for instance to determine their resistance to disease and climate variations, possibilities of commercial production were under consideration by the industrial partner, Zeneca (UK). Several patents were filed.
Current position
The participant from Universiteit Gent, Lab of Genetics (Belgium) went on to participate in AIR2-CT93-1661: Structure, function and industrial applications of plant laccases and peroxidases, coordinated by University of Edinburgh (UK). This included studies of the isoenzymes (xylem-specific isoperoxidases from poplar) principally involved in lignin biosynthesis. They then went on to participate in FAIR1-CT95-0424: Tree improvement based on lignin engineering (TIMBER) also coordinated by the ECLAIR project coordinator Université Paul Sabatier and including many of the participants from this project, including the industrial partner, Zeneca. This project is expected to genetically transform woody materials in order to decrease lignin content and thus increase pulp yield and facilitate delignification. It will also attempt to increase lignin in order to produce better fuel wood. Transgenic poplars with modified activities for enzymes that play crucial roles in lignin biosynthesis have been grown for some time. Tissue-specific expression conferred by two different enzyme promoters has been analysed in transgenic poplar.
Future research will investigate the chemical modification of transgenic lignin, stability of transgenes in long-living woody plants and industrial advantages at the mill scale of genetically modified plants. Attempts will be made to fine tune lignin content and composition by simultaneous modification of two or three target genes. The effects of genetic manipulation on normal growth and development, as well as response to stress, will be assessed. While much of the work discussed from the viewpoint of the extension of the ECLAIR project involved reduction in lignin, now that lignin levels can be regulated, there are also possibilities of overexpressing the same genes leading to higher lignin levels. This would increase the strength, pest and disease resistance and calorific value of the wood. This in turn would increase its value as construction material, reduce the need for toxic wood preservatives and increase the unit calorific value of the biofuel.
One of the main limitations is the lack of efficient transformation technology for many species, particularly gymnosperms. However, techniques are constantly improving. The ectopic expression of different lignification gene constructs in trees used in agroforesty will also enable a better understanding of the control of lignification. This will enable researchers to optimize strategies for tree improvement through classical genetic approaches complementary to transgenesis.
In summer 1999, anti-GMO activists caused considerable damage to trials in the UK.
Impact
Commercial
The main purpose of pulping is to remove lignin resulting in pulp of low colour. Hence, a decrease in the lignin content of the raw material used would increase pulp yield and aid delignification. This would enable a reduction in the amounts of chemicals used as well as use of milder alternative (enzyme and/or solvent) processes. For forage crops this would increase the digestibility and energy value of animal feed. In contrast, production of woody materials with a greater percentage of lignin than normal should result in stronger wood products, as well as improve the fuel value. In addition it should be possible to exploit the techniques of genetic transformation developed for various trees species of economic importance further, in future, improving traits other than those associated with lignification.
Several patents were filed during or as a result of this project including:
As reported by Zeneca (Boudet, 1997), cost-effective transformation processes for the major pulpwood trees, notably Eucalyptus, but also pine, spruce, acacia, poplar, etc. are of commercial benefit. This transformation needs to be integrated with clonal propagation systems and field trials are required to demonstrate that the transformed trees do not have adverse agronomic properties. It is also necessary to show that the expected changes in pulping characteristics result in economic benefits in the actual industrial application. A business could then be established to produce and supply modified plants to industry. Over the last few years, Zeneca has developed this concept, negotiating out-licensing rights for the lignin modification technology, to provide an intellectual property platform from which to develop such business. Initial development is focused on short lifecycle trees such as Eucalyptus, acacia and poplar. Market opportunities have been identified in South and Central America, southern Africa, Iberia, the Far East and Australia. Zeneca has signed agreements with Shell (which has an advance Eucalyptus transformation programme and plantations in South America, Africa and Australia). Agreements have also been reached with Nippon Paper Industries (the largest paper manufacturer in Japan and the second largest in the world, owning an international spread of plantations). Further agreements have been reached with a Brazilian company. Zeneca is building relationships with other plantation companies to create an international transformation service that could introduce the desired genetic modification into clonal lines. These negotiations have led to large scale replication of results obtained in ECLAIR and FAIR.
Associated
IBMP (France) is researching the molecular basis governing active plant defense mechanisms against pathogens. During the period 1991-95 their main experimental model system has been the hypersensitive response (HR) seen in tobacco resulting from the interaction between the N-resistance gene and tobacco mosaic virus. Participation in this ECLAIR project has enabled them to make significant progress in understanding genetics of the phenylpropanoid pathway and its activation in tobacco leaves undergoing HR. The phenylpropanoid enzymes involved in lignification were identified and will be manipulated by genetic engineering. Crosses between transgenic plants with highly reduced activity for different types of phenylpropanoid enzymes should enable researchers to assess their role in disease resistance.
Participants from another ECLAIR project investigating biopulping and biobleaching participated in AIR2-CT93-1291: Biological delignification in paper manufacture: optimisation of enzyme mixtures for treating cereal straw and other non-wood materials (see AGRE-0047).
A recent announcement indicated that Nippon Paper Industries and two other Japanese paper mills, have independently produced Eucalyptus with reduced lignin content by genetic modification of different genes. The three companies have plantations in Australia and hope to use transgenic Eucalyptus in Europe in due course.
References
Boudet, A.-M. (1997) Tree improvement through lignin engineering. In: Proceedings of the European Conference on Pulp and Paper Research: The present and the future (Eds: A. Arabatzis, L. Eriksson, I. Seone) pp336-347
Contacts
Author
CNRS
UMR UPS-CNRS 5546
University Edinburgh
University Gent
Zeneca Agrichemicals
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