Altering Carbohydrate Composition Fructan

A goal oriented to improving food quality is the production of fructans in transgenic plants. Fructans are regarded as "prebiotic" because of antitumoral effects that seem to be associated with their stimulative effects on Bifidobacteria in the human intestine. This stimulation causes an increased propionate and butyrate fermentation and lowers the concentration of tumor promoting substances like ammonia and p-glucuronidase. In a mutant mouse strain which spontaneously develops colon tumors because of a genetic defect in the APC gene, a fructo-oligosaccharide-containing diet significantly reduces the number of colon tumors, whereas starch and wheat bran have no effect on total tumor number, as reported by Pierre and his coworkers.13

Besides, fructans are interesting low calorie fibers because the p-linkage of the fructose moieties cannot be cleaved by human enzymes. Bacterial fermentation and resorption of

Table 15.4. Mean fresh weight per tuber in g and total tuber yield per plant in kg ± standard deviation of potato expressing apoplastic yeast invertase

Table 15.4. Mean fresh weight per tuber in g and total tuber yield per plant in kg ± standard deviation of potato expressing apoplastic yeast invertase

Mean fresh weight/ tuber (g)

Total yield per plant

(kg)

Control

143

1.21 ± 0.08

U-In1-3

192

1.30 ± 0.10

U-In1-41

179

0.91 ± 0.07

U-In1-33

209

1.23 ± 0.09

Control: untransformed wild type (Var. Désirée). U-In1-3, -41, -33: different transgenic plant lines expressing yeast invertase targeted to the apoplast.

Table 15.5. Maximal photosynthetic rate of potato plants in mmol O2/m2*h ± standard deviation

Max

Photosynthetic

Rate

Control

74 ± 7.2

aSp13

71 ± 7

aSp43

54 ± 7.5

aSp5

46 ± 7.1

aSp34

38 ± 12.7

Control: untransformed wild type (Var. Désirée). aSp13, 43, 5, 34: different transgenic plant lines expressing an antisense RNA to the sucrose transporter transcript.

fermentation products yields an energy value of 1 kcal/g, which is about 30% of that for the free monomers. The texture of the fiber gives a fat-like feeling in the mouth, and therefore fructans are excellent bulking agents for low calorie foods. Fructans are fructose polymers that are synthesized from sucrose as substrate by transfer of the fructose moiety from sucrose to a growing chain. The glycosidic C-2 hydroxyl group can be transferred to the C-6 position; in this case, a levan type fructan is synthesized. Alternatively, it can be transferred to the C-1 position, leading to an inulin type fructan. In both cases the fructan chain contains a terminal glucose and is therefore a non-reducing sugar.

Fructan synthesis is widespread in evolutionary terms: It occurs among bacteria and plants and there are also some reports of fungal fructan production. Nevertheless, bacterial and plant metabolic pathways of fructan production have not much in common. The responsible enzymes are different and so are the synthesized carbohydrates. Bacteria need only one enzyme for the synthesis of fructans, and this enzyme is capable of synthesizing a fairly huge polymer having a molecular mass of several million. In most cases bacterial fructans are of the levan type; only one high molecular weight inulin has been described.

Plant fructans are of low molecular weight and their linkage type depends on their origin. Monocotyledonous plants usually synthesize levan type fructans, whereas the typical dicot fructan is inulin. All studies on beneficial effects of fructans on human health rely on low molecular weight fructans that are either isolated from plants like chicory or Jerusalem artichoke, which are both inulin producers, or can be produced with the help of fungal invertases. This method is very important in Japan. Fructan synthesis in plants is dependent on at least two enzymes, one of them producing the trisaccharide kestose, the other being a transfructosylase that uses fructans as donor and acceptor of fructosyl residues.

We chose artichoke as the source for the fructosyl transferase genes. Artichoke produces the largest inulin known among the plant kingdom. We believe this can possibly influ

' 1 1 1 I ' 1 1 1 I 1 1 1 1 —11,1 I 1 1 1 ' I 1 1 1 1 I 1 I - ■ ■ ■ I

Minutes

' 1 1 1 I ' 1 1 1 I 1 1 1 1 —11,1 I 1 1 1 ' I 1 1 1 1 I 1 I - ■ ■ ■ I

Minutes

Fig. 15.3. Inulin isolated from artichoke and transgenic potato plants expressing artichoke SST and FFT. The inulin preparation was analyzed by high pressure anion exchange chromatography (HPAEC) with pulsed amperometric detection.

ence yield, because longer chains would cause lower osmotic load on storage organs than short ones, considering that fructans are—in contrast to strach—water soluble carbohydrates. We have cloned both genes needed for inulin synthesis in artichoke and expressed them in potato plants. u' 12

Transformation of potato with the SST and FFT genes was performed in two steps. At first, we transformed potato with an SST construct under control of the CaMV (cauliflower mosaic virus) 35S promoter. The plants produce the trisaccharide 1-kestose and also nystose, which is the next higher homolog, in substansial amounts. Oligofructans are located in the vacuole and would be subject to degradation by invertases. Fortunately, invertase activity is low during loading of tubers with photosynthates. Under conditions of cold storage, only longer chains would be resistant to invertase activity. Transformation of the SST-expressing potato with an FFT-construct led to the accumulation of inulin in tubers. The inulin resembles the artichoke inulin in size (Fig. 15.3; see previous page), and the yield reaches up to 1% of the fresh weight, which is high considering the low concentration of sucrose in potato. A closer look at the carbohydrate composition of the potato tubers reveals that fructan synthesis might take place at the expense of starch, but as starch content is about 20-fold higher, the reduction is not significant, at least in greenhouse experiments (Table 15.6). We are now performing field tests to better assess biomass production.

Coming to a conclusion, we can summarize that it is possible to modify carbohydrate composition by manipulating activities of endogenous enzymes. This allows the production, for example, of starches with new properties that are normally not found in nature and gives access to a wide array of renewable resources for industrial production and also of food substances with improved quality. Introducing new synthetic pathways by sequential transformation with genes encoding heterologous enzymes allows the production of carbohydrates that are uncommon to a given plant species and substantially alters its nutritional value.

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