BioMatNet Item: FAIR-CT96-1896 - Production of novel fructans through genetic engineering of crops and their applications

FAIR-CT96-1896
Production of novel fructans through genetic engineering of crops and their applications

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Detergents : FAIR Area 1.2 - Green Chemicals and Polymers Chain : Fine Chemicals : Paints/Coatings/Plastics : Pharmaceuticals/Cosmetics : Plant Genetics

	Proposal No:	FAIR-CT96-1896
	Date Prepared:	July 2001, May 1999
	Source:	Final Report Abstract and Executive Summary Second Annual Progress Report

Final Report Abstract

Source: Progress Report January 2001

Consortium: The project is co-ordinated by Advanta Seeds, Rilland (The Netherlands) in partnership with Botanisches Institut der Universitaet Basel, (Switzerland), Deptartment of Molecular Cell Biology of the University of Utrecht (The Netherlands), Cooperatie Cosun U.A., Sensus, Roosendaal (The Netherlands), Instituto Nazionale per L'analisi e Protezione Degli Agroecosistemi, Firenze (Italy) and SES Europe NV/SA, Tienen, Belgium.

Summary

Objectives: The overall objective of this RTD project is to achieve through genetic engineering of crops, the cost-effective production of tailor-made fructans varying in degree of polymerisation, branching etc. with a wide range of food and non-food applications. Production of fructans with different qualities at relative low costs will be achieved by the controlled expression of specific fructosyltransferase genes from microbial and plant origin. The novel fructans obtained in this way can be extracted and used as functional foods or food ingredients such as low-calorie sweeteners, dietary fibre, bulking agent, Bifidus stimulants etc. Moreover, such fructans and their derivatives can be used as raw material in a wide range of industrial products such as biodegradable plastics (plasticiser), detergents, adhesives, cosmetics and sequestrants.

Activities: The attainment of the major goals of the project is pursued through realisation of several research objectives. One of the first goals is to extend the studies on identification and characterisation of ftuctosyltransferases of different origin and the cloning of the corresponding genes. The fructosyltransferases will be characterised by their different reaction products (fructan molecules with different lengths, branching patterns and chemical bonding) which determine the application of the corresponding gene. Subsequently, chimeric genes are developed comprising the structural part of the fructosyltransferase gene either or not linked to an intra-cellular targeting signal as well as regulatory sequences. These regulatory sequences will be promoter sequences which drive gene expression in plants either constitutively, developmentally regulated or in a tissue specific way. The different chimeric genes are functionally tested in model plant species. The production of transgenic plants which possess different fructan profiles is an important work program objective. Transformation of chicory and potato is carried out with the objective to extrapolate the results achieved within these model crops to the crop of choice, which is sugar beet. Transgenic plants containing different fructan profiles are multiplied to carry out thorough biochemical analyses as well as agronomic studies of this plant material under laboratory, greenhouse and field conditions. Protocols for the extraction of different types of fructans from different crops are developed. Yield and purity will be important criteria in order to carry out further research on applications. Samples of different extracted fructans are used extensively to explore various applications in food (bakery industry, drinks, yoghurts etc.) and non-food (plasticiser in biodegradable plastics etc.).

Progress: The progress made so far within most of the tasks of the project has been largely according to the planned activities described in the Technical Annex. The activities addressing the isolation and functional analysis of genes encoding putative fructosyltransferases (Fr) have made steady progress. An isolated FT promoter hooked to a reporter gene shows the same expression profiles as the native FT gene. In addition, the analysis of transformants as well as their offspring expressing different (combinations of) fructosyltransferase genes has been quite successful. Fructo-oligosaccharide levels in sugar beet transformants were promising. Field trials planned with transgenic chicory were decided to be converted into greenhouse experiments because of better environmental control.

Achievements: The fourth reporting period is marked by better prospects for commercial levels of Fructo-oligosaccharide (FOS) in transgenic sugar beet expressing one of our FT genes. Nevertheless we have observed that interfering with the sugar beet carbohydrate metabolism is not as straight forward as one may have anticipated, since the introduction of other FTgenes was less successful. It has been shown that sugar beet can be converted into a new crop, delivering a new, economically important product. Also the extraction of the compound from the root tissue does not seem to be a problem.

Future actions: The main activity during the final reporting period will be to further characterise the collection of transgenic material obtained thus far. It is anticipated that the currently available transgenic plant material is of sufficient quality and quantity to reach a conclusion with respect to the technical feasibility of the main objective of this project, which is the production of tailor-made fructans in transgenic crops. In addition, the role of fructans in stress protection in plants will be further elaborated.

Source: Progress Report january 2001

Final Report Executive Summary

EXECUTIVE SUMMARY

Introduction

The overall objective of this RTD project is to achieve through genetic engineering of crops, the cost-effective production of tailor-made fructans varying in degree of polymerisation, branching etc. with a wide range of food and non-food applications. Production of fructans with different qualities at relative low costs will be achieved by the controlled expression of specific fructosyltransferase genes from microbial and plant origin. The novel fructans obtained in this way can be extracted and used as functional foods or food ingredients such as low-calorie sweeteners, dietary fibre, bulking agent, Bifidus stimulants etc. Moreover, such fructans and their derivatives can be used as raw material in a wide range of industrial products such as biodegradable plastics (plasticiser), detergents, adhesives, cosmetics and sequestrants.

Activities

During this reporting period the following tasks were addressed.

Task 1: Gene isolation; Isolation and characterisation of enzymes involved in fructan metabolism and cloning of the corresponding genes which will be used to obtain different fructan profiles (degree of polymerisation, type of chemical bonding between the fructose moieties and branching) in transgenic plants.

Task 2: Construct development and transformation of model species; Development of chimeric genes and transfer into model species tobacco, potato and chicory to achieve constitutive or developmentally regulated accumulation of different fructan profiles within different cellular compartments

Task 3: Sugar beet transformation; Transformation of the crop sugar beet with optimised chimeric genes for fructan production

Task 4: Evaluation of transformants; Evaluation of transgenic crop and model plants with respect to their fiuctan profile and accumulation level as well as the (bio)chemical evaluation under laboratory and greenhouse conditions

Task 5: Field trials; Evaluation of transgenic plants with respect to their performance under different field conditions

Task 6: Fructan extraction; Extraction and characterisation of fructans from transgenic plants

Task 7: Applications; Exploration of food and non-food applications of fructans extracted from transgenic plants

Results

Task 1: Gene isolation The novel fructosyltransferase purified from Festuca has been further characterised in assessing the natural expression profiles in fescue. The pattern of mRNA levels in the segments matched the levels of I -SST activity investigated earlier with high levels in the growth zone of the leaves and decreasing levels towards the leaf tip, indicating transcriptional regulation of I -SST. Overexpression in Pichia pastoris was further studied for 4 genes; 6-SFT from barley, 1-SST from fescue, 1-SST from onion and an FFT cDNA clone from barley. The first 2 constructs showed activity in Pichia, the latter two did not. Five new FFT cDNA clones from barley are under study. Purification of the barley 6-SFT protein proved possible after the addition of a His-tag, which had no effect on enzyme activity.

Task 2: Construct development and transformation of model species Fructan synthesis enzymes are likely to be regulated at the transcriptional level. This triggers interest in studying the promoters from the genes involved. To this end the promoter from the 6-SFT gene was isolated from barley. Expression of the GUS reporter gene driven by the isolated 1.6 kb promoter fragment, showed good correlation with fructan synthesis patterns, indicating that the complete promoter was isolated, including all cis-acting elements.

Task 3: Sugar beet transformation Sugar beet plants containing the LevanSucrase gene (plastid targeted; fd-LS) showed a severe yellowing phenotype that seemed to be lethal. Roots were therefore analysed at an early stage, but showed no fructans. With the other constructs, 38 transformants (out of 598) containing pUBI:: I-SST were identified that produced fructan, as well as 8 (out of 65) with pUTBI::I-SST + pUBI::6-SFT and 15 (out of 149) with PUBI::1-SST + pUBI::6- GFFT. The experiments to analyse the transfortnants further are still in progress.

Task 4: Evaluation of transformants In order to understand the function of the I-SST fructosyltransferase from fescue in planta, a stable transformation of the cDNA in tobacco (Nicotiana tabacum) was obtained under the control of the CAMV 35S RNA promoter. The sugar content of young leaves of 46 independent primary transformed lines was tested by HPLC, but the production of iso-kestose was not detected. F1 progeny plants from two primary sugar beet transformants, containing the fd-LS gene and 6-SFT respectively, were analysed. Germination was good. Unexpectedly neither of the F1 plants proved to contain intact T-DNA inserts. Transgenes were either corrupted or absent (as judged from PCR studies).

Vacuolar targeting could not be tested in tobacco harbouring a bacterial fructosyltransferases from Acetobacter diazotrophicus fused to the vacuolar- targeting signal of sporamine, because no fructan or enzymatic activity could be detected. Transgenic tobacco lines harbouring different combinations of fructosyltransferases were found negative for enzymatic activity and fructan as well. Transgenic sugar beet lines expression the onion 1-SST showed 1-kestose production after incubation with sucrose.

Task 5: Field trials Field trials planned with transgenic chicory were decided to be converted into greenhouse experiments because of better environmental control. The chicory seed proved very poor in germination. Moreover the plants that did germinate proved vulnerable to diseases. Most of them died. A new round of experiments has been started in new and better facilities.

Task 6: Fructan extraction Leaf samples from more than 800 sugar beet transformants containing either SST, SFT or 6-GFFT were analysed for DP-3 content. Forty-five events were selected with DP-3 levels above those of the non-transformed control. These plants were allowed to grow further and develop tap roots, DP-3 levels in the roots corresponded well with those in leaves. Fructo-oligosaccharide (FOS) levels in the transformants ranged from 35-61% of the carbohydrate fraction. The best event was SST406 with 61% FOS. These are levels that are commercially interesting.

Task 7: Applications From the literature it is known that fructans may stabilise biological membranes in various systems. It was investigated whether this property could be used to improve the shelf life of various probiotic bacteria, especially in yoghurt-type food systems. Various strains were tested with different fructans (FOS, normal inulin, long chain inulin, and bacterial levans) but so far no improvements in shelf life (tested in pasteurised yoghurt) were found.

Discussion

The fourth reporting period is marked by a very important break through. We have achieved commercial levels of Fructo-oligosaccharide (FOS) in transgenic sugar beet expressing the 1-SST gene from onion. This is even more remarkable given the finding that interfering with the sugar beet carbohydrate metabolism is not as straightforward as one may have anticipated, given the fact that the fd-LS construct and the 6-SFT construct were not only hard to introduce into sugar beet, but also proved not to be transferred to the next (sexual) generation. Whether the particular event SST406 (with 61 % FOS) itself is a commercial event remains to be seen, given the fact that it contains the GUS gene, the NPT-II gene and possibly the amp gene, which are all controversial with respect to public acceptance and official registration and deregulation. In any case, it has been shown that sugar beet can be converted into a new crop, delivering a new, economically important product. Also the extraction of the compound from the root tissue does not seem to be a problem.

Future actions

A structure-function analysis has been started with barley 6-SFT and fescue 1-SST using the Pichia pastoris expressions system. The aim is to determine structural elements responsible for the specificities of these enzymes. The activities of the new barley cDNA clones are still to be investigated, hoping to identify a new cDNA encoding an FFT. Furthermore, the investigation of the transgenic tobacco, harbouring the 1-SST from tall fescue needs to be completed with the second generation. T'he molecular and biochemical analysis of the available transgenic sugar beet material will continue, including the transformants obtained with double constructs. The resulting material will show whether combined activities of different enzymes will lead to the accumulation of fructan molecules of different complexity and functionality when compared to the fructans obtained by a single enzyme. The transgenic TPS-chicory material currently available will be used to study the effect of this gene on the inulin composition under different environmental conditions. The experiments will be conducted in a contained environment.

Second Annual Progress Report

Objectives
The overall objective of this RTD project is to achieve, through genetic engineering of crops, the cost-effective production of tailor-made fructans varying in degree of polymerization, branching etc. with a wide range of food and non-food applications. Production of fructans with different qualities at relative low costs will be achieved by the controlled expression of specific fructosyltransferase genes from microbial and plant origin. The novel fructans obtained in this way can be extracted and used as functional foods or food ingredients such as low-calorie sweeteners, dietary fiber, bulking agent, Bifidus stimulants etc. Moreover, such fructans and their derivatives can be used as raw material in a wide range of industrial products such as biodegradable plastics (plasticiser), detergents, adhesives, cosmetics and sequestrants.

Technical Approach
The major objectives of the project depends on the realisation of several research goals. One of the first goals is to extend the studies on identification and characterization of fructosyltransferases of different origin and the cloning of the corresponding genes. The fructosyltransferases will be characterized by their different reaction products (fructan molecules with different lengths, branching patterns and chemical bonding) which determine the application of the corresponding gene. Subsequently, chimeric genes will be developed comprising the structural part of the fructosyltransferase gene either linked or not linked to an intra-cellular targeting signal as well as regulatory sequences. These regulatory sequences will be promoters that drive gene expression in plants either constitutively, developmentally regulated or in a tissue specific way. The different chimeric genes will be functionally tested in model plant species.

The production of transgenic plants which possess different fructan profiles is an important objective of the work program . Transformation of chicory and potato will be carried out with the objective of extrapolating the results achieved with these model crops to the crop of choice, which is sugar beet.

Transgenic plants containing different fructan profiles are to be multiplied to provide sufficient material to carry out thorough biochemical analyses as well as agronomic studies of this plant material under laboratory, greenhouse and field conditions.

Protocols for the extraction of different types of fructans from different crops will be developed. Yield and purity will be important criteria in selecting material to use in carrying out further research on applications, using samples of different extracted fructans in food (bakery industry, drinks, yogurts etc.) and non-food (plasticiser in biodegradable plastics etc.).

Results
The progress made so far within the different tasks of the project has, so far, been largely according to that planned and described in the Technical Annex. The isolation of genes encoding fructosyltransferase, which is fundamental to the progress of the project, has been very successful. A substantial amount of transgenic material has been generated which will now be assessed for its value in the downstream tasks required for exploitation of the fructan technology.

A number of key enzymes involved in fructan metabolism of barley, onion and tall fescue have been cloned. Microbial systems have been set up successfully to assess functionality of the proteins encoded by the expressed cDNAs. A number of different cDNAs obtained were inserted in expression vectors suitable for transformation of model species such as tobacco or potato. For each construct a representative set of primary transgenic events was obtained which resulted in accumulation of variable levels of fructan. Newly isolated genes, as well as combinations of genes which were already available at an earlier stage of the project, have been introduced into model plants to assess their functionality. Thorough analysis of the transgenic plant material obtained will reveal the key factors at the molecular, biochemical and physiological levels that need to be addressed experimentally in order to attain the objectives of the project.

A large number of independent transgenic sugar beet plants have been produced using different plant-derived fructosyltransferase genes. This material will be analyzed both molecularly and biochemically. It is anticipated that this will result in the proof of concept for the efficient production of fructans through this target crop. Preliminary results have indicated that fructan accumulating plants have acquired enhanced tolerance towards the adverse effects of abiotic stress.

Extraction and characterisation studies have been initiated using transgenic chicory material expressing plant-derived fructosyltransferase genes and shown to accumulate modified inulin. These novel types of fructan have been extracted in sufficient quantities to carry out initial physico-chemical characterisation and application studies.

Future activities
The main activities during the next reporting period will be to characterise the collection of transgenic material obtained thus far. In addition new (combinations) of genes will be introduced into model species to assess the possibility to synthesize novel types of structurally diverse fructan in sufficient quantity for application studies. In addition, the role of fructans in stress protection in plants will be further investigated.