QLK5-2000-00443 Enzyme discovery in hybrid aspen for fibre engineering

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Biological Conversion : Crops for Paper/Pulp : Fibre : Plant Genetics : Quality of Life - 5.3.1 Multifunctional Management of Forests

Contract No:	QLK5-2000-00443
Source:	First Progress Report Summary - 2002

First Progress Report Summary
October 2002

Introduction

The natural fibres extracted from trees by pulping provide an excellent raw material for e.g. paper, cardboard and packaging materials as well as for many potential future products. The cell wall formation in trees and the resulting fibre structure and properties are a result of a strictly controlled expression and activity of a number of different enzyme systems. An understanding of the biochemistry of the cell wall biosynthesis provides the basis for understanding the chemistry and structure of wood fibres. Detailed understanding of the individual enzymes involved in cell wall formation opens the prospects for controlled modification of the fibre properties either during their growth or during the post-harvest processing. The EDEN project will use functional genomics to identify and characterise a number of new fibre-active enzymes. Enzymes are natural catalysts that are used increasingly to replace environmentally harmful chemicals and to achieve better catalytic selectivity during pulp and paper processing. The main objective of this project is to identify novel plant enzymes for deeper understanding of the process of fibre formation and for future improvement of the quality parameters of wood fibres. Specific modification of the fibre characteristics will facilitate their use as raw materials for the current pulp and paper industries and will lay the basis for the development of completely novel products.

The project is based on a database of over 8000 ESTs (Expressed Sequence Tags) from the wood-forming tissues of hybrid aspen. Enzyme discovery and fibre engineering are carried out by stepwise selection of enzymes, which have high expression levels during wood formation and which have demonstrated effects on the fibre composition and cheniistry. Influence of the altered fibre characteristics on the final product properties will be evaluated by micropulping experiments of juvenile transgenic lines of the hybrid aspen.

Objectives

The main objective is to modify the quality parameters of wood fibres to facilitate their use as raw materials for pulp and paper products. This is done by stepwise selection of enzymes, from an initial pool of several thousand wood-related ESTs, that have a demonstrated effect on the fibre composition and chemistry and are therefore anticipated to influence final product properties. The specific objectives are

• mRNA expression profiling to identify enzymes expressed during wood formation,
• bioinformatics for tentative identification of the key enzymes,
• enzymatic characterisation of the key enzymes,
• use of Arabidopsis reverse genetics
• transgenic aspen in combination with state-of-the-art microscopic and spectroscopic methods to correlate the activities of selected enzymes with specific fibre characteristics, and finally,
• micropulping experiments are carried out for few key enzymes for evaluating their impact on final product characteristics.

Activities

The project is based on the EST libraries of the wood specific sequences of the hybrid poplar. A large number of the sequenced cDNAs code for proteins, which - based on similarity to genes in other organisms - are known to be involved in cell wall synthesis. The second category of ESTs consists of sequences for which homologous counterparts have been found in other organisms but the function of which is not known, or unique genes not found in any other databases available today. Enzyme discovery among these sequences will here be carried out by a stepwise selection scheme:

Step 1. High throughput expression analysis is carried out for thousands of wood specific ESTs spotted on microarrays, while a high-resolution cryosectioning method - is used to prepare specific cDNA from tissue samples in different developmental stages of fibre formation. Screening the microarray by with these cDNAs will give a catalogue of genes involved in the secondary cell wall formation - thereby determining the fibre structure and chemistry. More specific searches of new genes related to cell wall biogenesis will be done by hybridising the microanays with cDNA from unique sets of previously made transgenic poplar plants downregulated for the key enzymes for lignin biosynthesis .

Step 2. Genome mining. Novel bioinformatics tools are then used to predict functions of the wood specific sequences through detection of distant evolutionary relationships to sequences in public databases. These tools also permit identification of the modular structures typical for different glycosyltransferases, glycoside hydrolases and transglycosylases, thereby facilitating the task of selecting the key enzymes and assigning functions for them. Knowledge of the modular structures will also facilitate design of strategies for the heterologous expression, assay development and functional analysis of the selected enzymes.

Step 3. Enzyme production. Protein expression for antibody production for subsequent imunolocatization will be in E. coli using the high-throughput expression system for ESTs previously developed. Full-length clones are generated for selected enzymes which are expressed in the methylotrophic yeast, Pichia pastoris the proteins will then be purified and characterised. Enzymes naturally embedded in membranes will be subjected to modular analysis and the extracellular, likely functional domains will be expressed in E. coli or yeast.

Step 4. Enzymology. The sequences with detectable homology to other enzymes with known functions will be classified in glycosidases, transglycosylases and glycosyltransferases (OTs).

Results and Milestones

The first milestone of the project "Identification of a number of novel fibre specific enzymes in hybrid aspen by expression profiling" has been achieved as planned at 12 months of the project duration. Within a wood specific EST library of 3000 genes, over 200 were found to be specifically activated during xylogenesis (Hertzberg et al, 2001). Extensive bioinformatic analyses of the EST, and selected full-length sequences revealed several enzymes of previously known functions such as cellulose synthases, expansins, cellulases, xylanases and xyloglucan endotransglycosylases (XET). Further analysis revealed 14 novel carbohydrate active enzymes and 2 novel carbohydrate-binding modules- In addition, over 60 genes were found to encode proteins with previously unknown functions.

In order to study the localization and function of the discovered proteins, protein production has been carried out in bacteria to allow generation of specific antibodies. Protein production for enzymatic studies has been carried out for selected proteins in yeast. Substrate synthesis and subsequent assay development has been carried out for detailed enzymatic characterization of the key enzymes. During the first year, expression has been achieved for two isoenzymes of XET and combined imrnunolocalization and in situ activity assays have revealed a novel function for this enzyme during wood development (Bourguin et al, 2002).

Determination of the function of the genes with previously unknown functions in analyzed by using Arabidopsis knock-out mutantrs of corresponding genes and by up and down regulated expression in hybrid aspen. So far, 6 homozygous knock-out lines have been identified for further work in Arabidopsis. The functional studies in hybrid aspen have so far concentrated in the analysis of the genes with known functions, including expansins, cellulases and XET. Benefits and Beneficiaries

Benefits and Beneficiaries

The information of potential public interest so far obtained has been published in scientific journals, as follows:

Hertzberg, M., Aspeborg, H., Schrader, J., Andersson, A., Erlandsson, A., Blomqvist. K., Bhalerao, A., Uhlén, M., Teen, T., Lundeberg, J., Sundberg, B., Nilsson, P. and Sandberg, 3. A transcriptional roadmap to wood formation. Proc NatI Acad Sd USA, 98, 14732-1 4737 (2001).

Bourquin, V, Mshikubo, N, Abe, N., Brurner, H, Denman, S, Eklund, M, Christiernin . M, Teen U, Sundberg, B and Mellerowicz, E. Xyloglucan endotransglycosylases have a function during the formation of secondary cell walls of vascular tissues. In press, Plant Cell, Dec 2002.

Future Actions

The second year of the project will focus on functional analysis of the known wood specific enzymes. Experiments will be designed for their exploitation in wood fibre modification. Further, phenotypic analyses and spectroscopic charatcterization of mutated cell lines targeting the genes with previously unknown functions will be carried out in Arabidopsis and hybrid aspen. Expression profiling of tension wood as well as previously generated cell lines mutated in lignin biosynthetic genes will be carried out in hybrid aspen.