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The globalization of the pulp and paper industry is a relatively new phenomenon that has been a harbinger of change and opportunities. Today’s paper industry employs advanced chemicaland mechanical-based technologies to provide high-quality consumer products that are in worldwide demand and support the lifestyles of our new global economy. And yet, from these successes arise many of the current and future difficulties of the industry. Various paper industry leaders have stated that the capital requirements of manufacturing paper products are too high and are limiting creativity and the entrepreneurial spirit of the industry. Coupled with these challenges, the emergence of low-cost fiber resources outside the Northern Hemisphere has contributed to further pressures on the paper industry to significantly reduce its manufacturing costs through a major redesign of its core manufacturing technologies. Within these difficulties are disguised but unparalleled opportunities for researchers to efficaciously develop new biotechnology-based processes for our industry. These new technologies must reduce the capital costs of pulp production, be readily implemented in today’s mill, and provide exceptional return for the resources invested if they are to be commercially feasible. The pathway to the future will entail the development of new global alliances between industry, governments, and research organizations. These new research partnerships will be linked together by the Web, coordinated by industry and government, and will utilize the best research expertise and facilities available in the world. These collaborative efforts will generate higher value fiber resources, lower total manufacturing costs, and develop new materials from which new products can be designed and manufactured. In summary, the business of pulp and paper has provided R&D a challenge for change and biotechnology is destined to address this opportunity. Introduction In 1879, the kraft pulping process was invented by the German chemist C. F. Dahl. This discovery was broadcast on Samuel F. B. Morris’ “information superhighway” of the day, the telegraph. Interestingly, the telegraph system was patented 30 years before Dahl’s discovery. The discoveries of the telegraph and the kraft process were, for its time, quantum leaps in technology that contributed to the industrial revolution. These and other technological advances of the day significantly altered the development of Western civilization. Now, at the beginning of a new millennium, we are experiencing another dramatic change in modern society. The advent of inexpensive, powerful personnel computers, broadband telecommunications and other information technologies has begun to dramatically redefine our concepts of business, life-styles, education, and government. Just as Morris’ telegraph developed into today’s information technology revolution, recent events now necessitate the development of new breakthrough manufacturing technologies for the pulp and paper industry. These breakthrough technologies need to be revolutionary in design and operation and must positively impact: (1) the raw material costs, (2) manufacturing costs, (3) energy costs, (4) environmental performance and (5) the production of high-quality products demanded by the consumer. In 1986, Foster analyzed the lifecycle of technologies and proposed that most technologies follow an S-curve relationship between productivity and investment of resources. The basic premise was that the older, more established technologies have upper performance limits that are determined by a combination of physical, chemical and regulatory rules. As a mature technology approaches the top part of this curve, major 8 International Conference on Biotechnology in the Pulp and Paper Industry June 4-8, 2001, Finlandia Hall, Helsinki, Finland 16 investments are required for only marginal gains in performance. The key to improving the return on investment is to identify and develop new technologies that develop along a new S-curve. This challenge represents a unique opportunity for our scientific research community to discover a new Scurve that will provide a new set of “winning” bio-manufacturing technologies for the pulp and paper industry. For example, a doubling of the growth rates of Northern Hemisphere hardwoods and softwoods would decrease wood furnish costs by 10 –15%. Breakthroughs in biopulping and biobleaching technologies would reduce capital requirements for these operations by 50% or more. Finally, enzymatic fiber modification of pulp would provide pulp manufacturers new tailor-designed fibers that reduce the amount of fiber and energy needed to manufacture paper products. These few examples provide only a glimpse of what could be accomplished in the future. Certainly, the biotechnology revolution that is occurring in textiles, detergents, food and other mature industries suggests that we do not fully appreciate the potential of biotechnology in the pulp and paper industry. Nonetheless, it is well appreciated that enzymatic systems are catalytic, highly selective, and are operable under mild temperature and pressures. These features alone indicate that the development of new bio-manufacturing technologies for pulp and papermaking will substantially reduced capital and operating cost requirements while yielding products with improved performance. In addition, enzymatic treatments offer the potential to selectively modify pulp fiber surfaces to yield new products that cannot be manufactured via chemical and/or mechanical methods. The ability to tailor the surface of pulp fibers will provide pulp manufacturers new opportunities to develop differentiated, intellectually protected, high-value-added products for the consumer. The development of these new biotechnologies will require high-risk, breakthrough research programs that will necessitate the development of new alliances and partnerships between industry, government, universities, research institutions and researchers. As in any endeavour of excellence, the team that possesses the best expertise and resources is most likely to be successful. Although a superficial examination of the research requirements needed to develop these new bio-manufacturing processes may suggest that this is a Herculean task, in reality, the resources and expertise are available but dispersed worldwide and are diffuse in their research efforts. Fortunately, the revolution in information technology now facilitates the development of national/international, cross-functional project-based teams that can answer these challenges. Clearly, the last component in this vision is the need for research funding. Although this issue appears to be a daunting challenge, I believe that this task is readily possible for an industry that provides employment for several million people in North America and Europe and is a major contributor to the GDP of many of these nations. These societal benefits, coupled with the industry’s environmental stewardship and exemplary management of renewable resources, strongly fosters the development of partnerships between industry and government to fund breakthrough research in pulp and paper. In the U.S., the Department of Energy’s Office of Industrial Technology has been at the forefront of supporting collaborative R&D projects that have emerged from an industry-driven solicitation process titled “Agenda 2020: Forest Products Industries of The Future.” This is just one example of the development of new alliances that will propel R&D developments of new breakthrough technologies for our industry. Conclusions In summary, it appears that the history of pulp manufacturing is about to repeat itself. A confluence of scientific and engineering accomplishments in the late 1800’s set the stage for the discovery of the basic pulp manufacturing technologies currently employed. In this new millennium, we now have unprecedented developments in genomics, biotechnology, telecommunications, artificial intelligence, material science and engineering. These advanced technologies and the researchers in this audience have the potential to provide breakthrough manufacturing technologies and products for the pulp and paper industry, the world’s premier renewable industry. 8 International Conference on Biotechnology in the Pulp and Paper Industry June 4-8, 2001, Finlandia Hall, Helsinki, Finland 17 O1/3 CHALLENGES IN LIGNIN GENETIC ENGINEERING A-M. Boudet UMR CNRS/UPS 5546, Signaux et Messages Cellulaires chez les Végétaux, Pôle de Biotechnologie Végétale, 24 chemin de Borderouge, B.P. 17 Auzeville, F-31326 CASTANET-TOLOSAN France, E-mail: [email protected] Despite their importance in the adaptive strategies of tracheophytes (pteridophytes, gymnosperms and angiosperms), the occurrence of lignins in plants dramatically affect their agro-industrial uses. The digestibility and dietary conversion of grasses are altered by the presence of lignins and in the pulp industry lignins are undesirable components : their high proportion in wood species involves complex, expensive and polluting processes to extract this phenolic polymer in order to recover cellulose, the main component of pulp. In the first part of this presentation I will highlight the contribution of lignin genetic engineering experiments towards a better understanding of biosynthesis and spatio-temporal deposition of lignins and towards potential improvement of plant biomass. Specific examples from my laboratory will underline the chemical flexibility of lignins and the specific behavior of different cell types within the xylem. Strategies aiming to reduce the lignin content of plants may impact plant development and induce biochemical changes at the level of the cell wall concerning cell wall polysaccharides and non-lignin phenolic components. However, the ectopic expression of a specific transgene may have a different impact depending on the genetic background. In this way, we will report data showing that different cell types within the xylem have independent and precise regulatory mechanisms of lignin synthesis. This is particularly illustrated by the distinctive characteristics of fibers and vessels in tobacco plants simultaneously down-regulated for both CCR and CAD activities which despite a severe reduction in lignin content may undergo normal development. These results suggest that induced, targeted modification of lignin synthesis in specific cell types should be possible in the future for biotechnological applications. In addition to individual cell walls composition the cohesion and interactions between cells may be important for the properties of plant products. We will present recent data concerning the role of individual laccase genes on phenolic metabolism and cell wall structure. Transgenic poplars down-regulated for a specific laccase gene (lac 3) exhibited a normal phenotype but a 2-3 fold increase in total soluble phenolic content. In addition microscopic observation of transgenic stem and root cross sections indicated that lac3 gene suppression led to a dramatic alteration of xylem fiber cell walls. Individual fiber cells were severely deformed, with modifications in fluorescence emission at the primary wall/middle lamella region. Biomechanical properties of this material and the results of simulated digestibility experiments will be reported. At the moment, it is clear that the number of significant modifications of lignin profiles obtained through genetic engineering is already impressive. In addition, recent results have indicated that modulation of transcription factors involved in lignin biosynthesis can also be an efficient strategy for producing plants with a reduced lignin content. New target genes or combinations of genes should also be exploited in the future. Consequently, the main problems we are faced with are not related to a lack of potential target genes but: 1) to an extensive evaluation of the effects of the individual transformations on various aspects of plant metabolism and development and 2) to the transfer of the best technologies to crops and woody species of major interest. More widely the results of lignin genetic engineering induce new possibilities and new problems I will discuss in the second part of this presentation. 8 International Conference on Biotechnology in the Pulp and Paper Industry June 4-8, 2001, Finlandia Hall, Helsinki, Finland 18 For example, it is clear that transformed plants with unusual lignin profiles may help to better identify the chemical characteristics of lignocelluloses which are beneficial in wood processing and to re-evaluate the criteria important for the efficiency of the pulping/bleaching process (in addition to the classical S/G ratio). Morever, the chemical flexibility of lignin induced by ectopic modifications of lignification genes may generate in the future new lignins or new by-products with direct practical applications. The metabolic side-effects of lignin manipulation are not well known at present but since lignins are the major carbon sink in phenolic metabolism, a decrease in lignin content should make upstream precursors available for the synthesis of other cell wall and soluble components. The impact of these changes on the industrial or nutritional properties of the transformed lines should be carefully evaluated in the future as well as their potential effects on plant environment interactions. Micro arrays for probing gene expression and metabolic profiling of different classes of biochemical compounds will reveal the extent of such secondary effects which have to be clearly identified for the acceptance of the transgenic products. One of the main concerns about genetically modified trees is indeed their acceptance by public opinion. The fact that these transformed plants are non-food products designed for industrial purposes should facilitate public acceptability. A careful examination of these new transgenic products is then absolutely necessary in order to evaluate the potential impacts, if any, which could outweigh the already demonstrated advantages for competitiveness of the pulp industry. The results of these studies should be clearly demonstrated it will be necessary to highlight the advantages of the transformed plants in terms of environmental benefits, energy savings and economic competitivity. The support of the European Community through the OPLIGE and TIMBER projets is gratefully aknowledged. 8 International Conference on Biotechnology in the Pulp and Paper Industry June 4-8, 2001, Finlandia Hall, Helsinki, Finland 19 O1/4 NOVEL ENZYMES FOR FIBRE MODIFICATION T. T. Teeri, H. Aspeborg, K. Blomqvist, H. Brumer, S. Denman, M. Hertzberg, E. Mellerowicz, P. Nilsson, S. Raza and Björn Sundberg Wallenberg Wood Biotechnology Center, Department of Biotecnology, KTH, SE-10044 Stockholm, Sweden Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, SLU, SE-90183, Umeå, Sweden Wood and pulp fibres constitute a renewable raw material, which can be processed using enzymes. In addition to microbial enzymes, which are already used in pulp processing, trees constitute a largely unexplored source of fibre active enzymes. We have taken up the hybrid aspen, Populus tremula L. x tremuloides Michx., as a model species to study xylogenesis and to identify new fibre active enzymes for use in controlled modification of fibre properties. The hybrid aspen EST-database We have earlied published an EST database of 4809 EST-sequences, corresponding to 2988 unique transcripts from the cambial tissues of poplar. The likely function of many of these sequences could be identified by similarity searches. These known fiber active enzymes include for example cellulases, xylanases, expansins, putative cellulose synthases and enzymes involved in lignin biosynthesis. However, the function of about 20% of the EST-sequences could not be identified using similarity searches. Some of these are homologous to genes of unknown function in Arabidopsis while others are not yet available in any sequence databases. Since ours is a database consisting primarily of wood related sequences, many of these unknown genes are also likely to code for novel wood specific functions. cDNA expression profiling Wood formation is a developmental process involving both up and down regulation of specific genes. mRNA expression profiling can be used to identifiy genes with stage specific expression indicating their involvement in the different stages of wood development. The formation of secondary xylem in poplar is highly organised, resulting in easily recognized and distinct boundaries between the different developmental zones. Owing to the large physical size of the vascular meristem in trees, tissues at defined developmental stages can be obtained by tangential cryosectioning. We have sampled 30 μm thick sections of tissues highly enriched in cambial derivatives in specific developmental stages including A) meristematic cells, B) early expansion, C) late expansion, D) secondary wall formation and E) late cell maturation or progammed cell death. cDNA prepared from these sections has been used for transcript profiling of the poplar cambial unigene set. For enzymes involved in fiber formation, the analysis of the expression patterns is focused on the zones C, D and E. Enzymes along the pathways of sugar metabolism are essential for the maintenance and synthesis of the main cell wall components. Among the known genes we have identified two members of the cellulose synthase family, CESA1 and CESA3 , which were up-regulated in zones C and D with clearly reduced expression in zone E. Interestingly, a cellulase isoenzyme homologous to plant Family 9 endoglucanases was similarly upregulated in cells forming secondary walls (zones C-E). Sucrose synthase is responsible for channelling sucrose into UDP-glucose, which is the sole precursor of cellulose. The expression of one of the sucrose synthase genes in our EST-library was also synchronised with the CESA1/CESA3 pair. Finally, ten out of the fourteen different ESTs coding for tubulins were strongly up-regulated during late expansion consistent with the proposed role of microtubules in defining the cellulose microfibril angle in the secondary cell wall. 8 International Conference on Biotechnology in the Pulp and Paper Industry June 4-8, 2001, Finlandia Hall, Helsinki, Finland 20 Figure 1. An example of the expression patterns of genes upregulated during late expansion, zone C (left panel), the secondary cell wall formation in zone D (middle panel) and during programmed cell death in zone E (right panel). 15 most strongly upregulated genes are shown for each zone. As shown above, expression profiling is a useful tool to identify the key players in the different stages of wood development thus helping us to focus our efforts on the enzymes dedicated to the wood fiber biosynthesis and modification. This first step of selection is followed by in vivo functional analyses in Arabidopsis and in poplar for the genes of unknown function as well as structure function studies and engineeirng of selected known enzymes. Industrial significance Owing to the difficulties of protein extraction from woody tissues, the enzymatic processes involved in wood formation have remained poorly characterized. An improved understanding of the biochemistry of the cell wall biosynthesis provides new means for altering the structure and chemistry of wood fibres either during their growth or during the post-harvest processing. Such approaches have already been used elsewhere to produce wood with more easily extractable lignin and can now be extended to the carbohydrate moiety of wood fibers. References 1. Sterky F, Regan S, Karlsson J, Hertzberg M, Rhode A, Holmberg A, Amini A, Bhalerao R, Larsson M, Villarroel R, Montagu M, Sandberg G, Olsson O, Teeri TT, Boerjan W, Gustafsson P, Uhlen M, Sundberg B & Lundeberg J. 1998. Proc. Natl. Acad. Sci. USA 95,13330-13335. 2. Uggla C, Moritz T, Sandberg G and Sundberg B. 1996. Proc. Natl. Acad. Sci. US., 93: 9282-9286. 3. Hertzberg M, Aspeborg H, Schrader J, Andersson, A, Blomqvist, K, Bhalerao, R, Rahman D, Marchant A, Bennett M, Uhlén M, Teeri TT, Lundeberg J, Sundberg B, Nilsson P and Sandberg G. 2001. Manuscript in preparation. -10 -5 0 5 10 15 20 25