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Engineering direct fructose production in processed potato tubers by expressing a bifunctional alpha-amylase/glucose isomerase gene complex

Beaujean, A; Ducrocq-Assaf, C; Sangwan, R S; Lilius, G LU ; Bülow, L LU and Sangwan-Norreel, B S (2000) In Biotechnology and Bioengineering 70(1). p.9-16
Abstract

Manipulation of starch biosynthesis/degradation and formation of novel molecules in storage organs of plants through genetic engineering is an attractive but technically challenging goal. We report here, for the first time, that starch was degraded and glucose and fructose were produced directly when crushed potato tubers expressing a starch degrading bifunctional gene were heated for 45 minutes at 65 degrees C. To achieve this, we have constructed a fusion gene encoding the thermostable enzymes: alpha-amylase (Bacillus stearothermophilus) and glucose isomerase (Thermus thermophilus). The chimeric gene was placed under the control of the granule-bound-starch synthase promoter. This enzymatic complex produced in transgenic tubers was... (More)

Manipulation of starch biosynthesis/degradation and formation of novel molecules in storage organs of plants through genetic engineering is an attractive but technically challenging goal. We report here, for the first time, that starch was degraded and glucose and fructose were produced directly when crushed potato tubers expressing a starch degrading bifunctional gene were heated for 45 minutes at 65 degrees C. To achieve this, we have constructed a fusion gene encoding the thermostable enzymes: alpha-amylase (Bacillus stearothermophilus) and glucose isomerase (Thermus thermophilus). The chimeric gene was placed under the control of the granule-bound-starch synthase promoter. This enzymatic complex produced in transgenic tubers was only active at high temperature (65 degrees C). More than 100 independent transgenic potato plants were regenerated. Molecular analyses confirmed the stable integration of the chimeric gene into the potato genome. The biochemical analyses performed on young and old tubers after high-temperature treatment (65 degrees C) revealed an increase in the formation rate of fructose and glucose by a factor of 16.4 and 5. 7, respectively, in the transgenic tubers as compared to untransformed control tubers. No adverse discernible effect on plant development and metabolism including tuber formation and starch accumulation was observed in the transgenic plants before heat treatment. Our results demonstrate that it is possible to replace starch degradation using microbial enzymes via a system where the enzymes are produced directly in the plants, but active only at high temperature, thus offering novel and viable strategies for starch-processing industries.

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publication status
published
subject
keywords
Aldose-Ketose Isomerases, Amino Acid Sequence, Biotechnology, Blotting, Southern, Cloning, Molecular, DNA, Plant, Fructose, Geobacillus stearothermophilus, Glucose, Molecular Sequence Data, Plants, Genetically Modified, Plasmids, Promoter Regions, Genetic, Solanum tuberosum, Temperature, Thermus thermophilus, alpha-Amylases
in
Biotechnology and Bioengineering
volume
70
issue
1
pages
8 pages
publisher
John Wiley & Sons
external identifiers
  • scopus:0034610055
ISSN
0006-3592
DOI
10.1002/1097-0290(20001005)70:1<9::AID-BIT2>3.0.CO;2-7
language
English
LU publication?
yes
id
0dbccd33-c98f-48f7-9061-7f8d16b17030
date added to LUP
2016-04-18 15:58:47
date last changed
2017-05-02 17:53:26
@article{0dbccd33-c98f-48f7-9061-7f8d16b17030,
  abstract     = {<p>Manipulation of starch biosynthesis/degradation and formation of novel molecules in storage organs of plants through genetic engineering is an attractive but technically challenging goal. We report here, for the first time, that starch was degraded and glucose and fructose were produced directly when crushed potato tubers expressing a starch degrading bifunctional gene were heated for 45 minutes at 65 degrees C. To achieve this, we have constructed a fusion gene encoding the thermostable enzymes: alpha-amylase (Bacillus stearothermophilus) and glucose isomerase (Thermus thermophilus). The chimeric gene was placed under the control of the granule-bound-starch synthase promoter. This enzymatic complex produced in transgenic tubers was only active at high temperature (65 degrees C). More than 100 independent transgenic potato plants were regenerated. Molecular analyses confirmed the stable integration of the chimeric gene into the potato genome. The biochemical analyses performed on young and old tubers after high-temperature treatment (65 degrees C) revealed an increase in the formation rate of fructose and glucose by a factor of 16.4 and 5. 7, respectively, in the transgenic tubers as compared to untransformed control tubers. No adverse discernible effect on plant development and metabolism including tuber formation and starch accumulation was observed in the transgenic plants before heat treatment. Our results demonstrate that it is possible to replace starch degradation using microbial enzymes via a system where the enzymes are produced directly in the plants, but active only at high temperature, thus offering novel and viable strategies for starch-processing industries.</p>},
  author       = {Beaujean, A and Ducrocq-Assaf, C and Sangwan, R S and Lilius, G and Bülow, L and Sangwan-Norreel, B S},
  issn         = {0006-3592},
  keyword      = {Aldose-Ketose Isomerases,Amino Acid Sequence,Biotechnology,Blotting, Southern,Cloning, Molecular,DNA, Plant,Fructose,Geobacillus stearothermophilus,Glucose,Molecular Sequence Data,Plants, Genetically Modified,Plasmids,Promoter Regions, Genetic,Solanum tuberosum,Temperature,Thermus thermophilus,alpha-Amylases},
  language     = {eng},
  month        = {10},
  number       = {1},
  pages        = {9--16},
  publisher    = {John Wiley & Sons},
  series       = {Biotechnology and Bioengineering},
  title        = {Engineering direct fructose production in processed potato tubers by expressing a bifunctional alpha-amylase/glucose isomerase gene complex},
  url          = {http://dx.doi.org/10.1002/1097-0290(20001005)70:1<9::AID-BIT2>3.0.CO;2-7},
  volume       = {70},
  year         = {2000},
}