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Upgrading of sugar beet pectins by enzymatic modification and molecular farming

   
Project

QLK3-1999-00089

Cell factory area

3.3.2

EU Contribution

2 478 778 Euro

Duration

36 months

Type

Research project

Starting date

01-02-2000

Keywords
arabidopsis
enzymatic modification
pectin
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ABSTRACT
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More than two million tons of sugar beet pulp containing 20-20% pectin are generated annually in the EU. Because of a poor functional property the pectin cannot be used as a high added-value product. To solve this European problem we want to improve the pectin by enzymatic modification. On a medium term basis we want to control the quality of the pectin product by introducing proteins and enzymes, which can modify the pectin in the plant cell wall. On a long-term basis we want to isolate the biosynthetic machinery producing the pectin polymer, by purifying the membrane-bound enzymes involved in the polygalacturonan synthesis, its methylation and acetylation, and by using Arabidopsis genetics for the discovery of new genes involved in pectin biosynthesis. In addition we want to determine the structure-functional relationship of the new pectin product.

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OBJECTIVES
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The specific research, application and exploitation objectives are:

  • To upgrade low quality raw materials from the agricultural sector, using new methods for pectin extraction and down-stream processing, and to obtain high molecular weights and well-controlled degrees and distribution of acetylation.
  • To use molecular design to generate new improved enzymes for use in the down-stream processing.
  • To isolate enzymes involved in the biosynthesis of the homogalacturonan polymer, its methylation and acetylation, and to identify new genes involved in pectin biosynthesis using mutant libraries in Arabidopsis and a new high throughput screening system.
  • To generate genetically modified crop plants with genes involved in biosynthesis or modification of pectin.
  • To determine pectin structure-function relationship and to exploit the technology by producing pectins with improved functional properties and introduce them into the market place.
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DESCRIPTION OF THE WORK
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In the present proposal "Upgrading of sugar beet pectin by enzymatic modification and molecular farming", we are using several strategies to achieve our objectives. On a short-term basis the pectin from sugar beet will be modified in vitro during down-stream processing by enzymatic treatment to achieve better functional properties. The existing enzymes, such as polygalacturonase, acetyl esterase and pectin lyase, available for this in vitro process, are not ideal for this purpose. It is therefore necessary to improve the quality of these enzymes by 3-D structural analysis and genetic engineering. Special emphasis is given to improving specificity. On a medium term basis we want to control the quality of the pectin products by transforming plants with proteins and enzymes which may modify the pectin polymer in the cell wall of the plants. The target proteins include the polygalacturonase inhibiting proteins (PGIP), and the phagedisplay antibody PAM1, which binds to homogalacturonan blocks, and the acetyl esterase which can reduce the acetylation of pectin. On a long-term basis we want to isolate enzymes and genes involved in the biosynthesis of homogalacturonan, including its methyl esterification and acetylation and express these genes in transgenic plants. Two complementary approaches will be examined. The first is to complete the purification, characterisation of gene coding for the membrane- bound enzymes involved in the polygalacturonan synthesis, its methylation and acetylation. The second is to use Arabidopsis genetics for the discovery of new genes involved in pectin synthesis. This involves the screening of a large number of T-DNA and EMS mutants in Arabidopsis using a newly developed high throughput system whereby 40,000 mutants can be analysed in a few weeks. To monitor changes in pectin structure and functional properties a set of advanced analytical tools will be developed.

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DELIVERABLES
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During the course of this project a number of deliverables will be produced, these include:

  • The 3D-structural elucidation of Polygalacturonase PGI, and the PGIP-PG complex, which will be delivered after six and 24 months, respectively.
  • Isolation and characterisation of homogalacturonan synthase and the methyl transferase (12months) and acetyl transferase (18 months), and isolation of the encoding genes (24 and 36 months, respectively).
  • Screening, characterisation and cloning of genes from Arabidopsis mutants defect in the pectin biosynthesis and deposition (36 months).
  • The structure and functional relationship of pectin will be elucidated within the first 30 months.

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CONSORTIUM
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COORDINATOR
  Dr. Jørn Dalgaard Mikkelsen
Danisco Biotechnology
1 Langebrogade
1001 Copenhagen K, Denmark
Tel: +45 32662232
Fax: +45 32662167
G7JDM@Danisco.com

PARTNERS
  Prof. Felice Cervone
Dipartimento di Biologia Vegetale Universitá "La Sapienza"
Piazzale Aldo Moro 5
00185 Rome, Italy
Tel: +39 649912436
Fax: +39 649912446

Grippo@axrma.uniromal.it

Dr. Herman Höfte
Institut National de la Recherche Agronomique
Laboratoire de Biologie Cellulaire
Cr de Versailles - Grignon / RD 10/ Route de St. Cyr
78026 Versailles Cedex, France
Tel: +33 130833000
Fax: +33 130833099
hofte@versailles.inra.fr

Dr. Paul Knox
Centre for Plant Sciences
University of Leeds
LS2 9JT Leeds, United Kingdom
Tel: +44 1132333169
Fax: +44 1132333144
j.p.knox@leeds.ac.uk

Prof. Grant Reid
University of Stirling
Dpt. of Biological Sciences
FK9 4LA Stirling, United Kingdom
Tel: +44 1786467755
Fax: +44 1786464994
j.s.g.reid@stir.ac.uk

Prof. Henrik Scheller
Plant Biochemistry Laboratory,
Department of Plant Biology,
Royal Veterinary and Agricultural University
40 Thorvaldsensvej
1871 Frederiksberg, Copenhagen, Denmark
Tel: +45 35283354
Fax: +45 35283333
scheller@biobase.dk

Director Jean-Francois Thibault
Institut National de la Recherche Agronomigue
Unité de Recherche sur les Polysaccharides
leurs Organisations et Interactions
B.P. 71627
44316 Nantes Cedex 3, France
Tel: +33 240675061
Fax: +33 240675066
thibault@nantes.inra.fr

Dr. Jaap Visser
Sec. Mol. Genetics of Industrial Microorganisms, Wageningen
Agricultural University

Dreijenlaan 2
6703 HA Wageningen, The Netherlands
Tel: +31 317484439
Fax: +31 317484011
office@algemeen.mgim.wau.nl

Prof. A.G.J. Voragen
Department of Food Technology and Nutritional Sciences
Food Chemistry Group
Wageningen Agricultural University
Bomenweg 2
6703 HD Wageningen, The Netherlands
Tel: +31 317483209
Fax: +31 317484893
Fons.Voragen@chem.fdsci.wau.nl

Dr. Mike Gidley
Unilever UK Central Resources Ltd
Plant Science Unit, Unilever Colworth
Colworth House
MK44 1LQ Sharnbrook, Bedford, United Kingdom
Tel: +44 1234222566
Fax: +44 1234222401
mike.gidley@unilever.com

Prof. Bauke W. Dijkstra
Groningen University
Laboratory of Biophysical Chemistry
Nijenborgh 4
9747 AG Groningen, The Netherlands
Tel: +31 503634381
Fax: +31 503634800
bauke@chem.rug.nl
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