Engineering of Primary Metabolism Carbohydrate Metabolism

The majority of metabolic fluxes inside a plant cell center on the formation and utilization of sugars, the primary products of photosynthesis and their conversion into storage and structural carbohydrates, such as starch and cellulose. Starch is the principle constituent of many of harvestable organs, such as tubers or grain. Besides its importance as a staple in human and animal diets, it is also used as a renewable raw material for a wide range of industrial applications (Jobling 2004). Starch is a relatively simple polymer composed of glucose molecules that are linked in two different forms. Amylose is an essentially linear polymer in which the glucose moieties are joined end-to-end by a(l!4) linkages. Amylopectin is a much larger branched molecule, in which about 5% of the glucose units are joined by a(l!6) linkages. The ratio between amylose and amylopectin is dependent on the plant species or variety, respectively, and is one determinant of the physico-chemical properties of plant derived starches which are important for technical uses. The biochemical pathways leading to starch formation are well documented and the key enzymatic steps have been identified (Fernie et al. 2002; Geigenberger 2003). Starch metabolism in potato tubers is particularly well characterized and attempts to both increase the accumulation of starch and to modify its structural properties by metabolic engineering have received considerable attention (see also Chap. 20). For starch synthesis in growing potato tubers, sucrose delivered from the phloem is cleaved by sucrose-synthase into uridine-diphosphoryl-glucose (UDP-glucose) and fructose, which are converted to hexose phosphates by UDP-glucose pyropho-sphorylase and fructokinase, respectively. Glucose-6-phosphate is then imported into the amyloplast via a glucose-6-phosphate transporter (GPT; Kammerer et al. 1998) and is converted via plastidial phosphoglucomutase and ADP-glucose pyrophosphorylase (AGPase) to ADP-glucose (Fig. 11.1). This process requires

Starch Synthesis Pathway
  1. 11.1 Principal pathway leading to the formation of starch in storage organs. The alternative route of ADP-glc via generation within the cytosol and subsequent uptake into the amyloplast, as it occurs in the endosperm cells of graminaceous species, is shown by dotted arrows. Susy sucrose synthase, UDP-glc UDP-glucose, F6P fructose-6-phosphate, G1P glucose-6-phosphate, AGPase ADP-glucose pyrophosphorylase, ADP-glc ADP-glucose
  2. 11.1 Principal pathway leading to the formation of starch in storage organs. The alternative route of ADP-glc via generation within the cytosol and subsequent uptake into the amyloplast, as it occurs in the endosperm cells of graminaceous species, is shown by dotted arrows. Susy sucrose synthase, UDP-glc UDP-glucose, F6P fructose-6-phosphate, G1P glucose-6-phosphate, AGPase ADP-glucose pyrophosphorylase, ADP-glc ADP-glucose

ATP, which is imported into the amyloplast via an ATP transporter (Tjaden et al. 1998). The glycosyl moiety of ADP-glucose is the substrate for the synthesis of starch via various isoforms of starch synthase.

Starch synthesis in the endosperm of cereals differs from that in other organs in that the synthesis of ADP-glucose occurs in the cytosol, via a cytoplasmic isoform of AGPase. ADP-glucose is imported into the plastid via a specific nucleotide transporter (Tomlinson and Denyer 2003).

To increase the efficiency of the pathway and thus to increase starch accumulation in crop plants, molecular strategies have initially concentrated on AGPase, the enzyme assumed to catalyze the rate-limiting step of starch synthesis. In an early attempt to increase the activity of the starch biosynthetic pathway in potato tubers, Stark et al. (1992) overexpressed a deregulated bacterial AGPase in the potato variety Russet Burbank. Overall, the transformed lines were reported to have an average of 35% more tuber starch than the controls. However, this effect was lost upon transformation of a different potato cultivar (Sweetlove et al. 1996). In the latter case, starch degradation was up-regulated in addition to starch synthesis, resulting in no net change in starch accumulation.

Attempts to increase starch contents through manipulation of AGPase in cereal seeds have made use of a variant of the maize AGPase gene (shrunken2) whose gene product is less sensitive to inhibition by phosphate when compared to the wild-type protein.

Smidansky and colleagues (2002, 2003, 2007) showed that maize, rice and wheat plants expressing this AGPase allele in the endosperm and grown under controlled conditions display an increase in individual seed weight as well as in seed yield per plant. However, in field trials transgenic wheat plants only showed a yield enhancement under conditions of minimal inter-plant competition and optimal water supply (Meyer et al. 2007).

Starch content in potato tubers is very sensitive to manipulation of the plastidial adenylate transporter providing the ATP necessary for the AGPase reaction. Overexpression of an adenylate transporter from Arabidopsis in potato tubers resulted in 16-36% more starch per gram fresh weight, indicating that ATP supply to the plastid limits starch synthesis (Tjaden et al. 1998; Geigenberger et al. 2001). Recently, a further increase in potato tuber starch content was achieved by the simultaneous overexpression of a GPT from pea and an Arabidopsis adenylate translocator. Double transformants exhibited an increase in tuber yield of up to 19% in addition to an increase in starch content of 28%, when compared to control plants (Zhang et al. 2008). Both effects taken together led to a calculated increase in potato tuber starch of up to 44%. The authors concluded that starch synthesis in potato tubers is co-limited by the ATP supply as well as by the import of carbon skeletons into the amyloplast (Zhang et al. 2008). Further evidence for an energy limitation of starch synthesis in potato tubers comes from transgenic plants with reduced expression of plastidial adenylate kinase (ADK; Regierer et al. 2002). In this study a strong negative influence of ADK activity on starch accumulation was found, suggesting that ADK normally competes with starch synthesis for plastidial ATP.

Taken together, successful attempts to increase starch content through metabolic engineering are scarce. The analyses so far suggest that in potato tubers considerable control of starch synthesis lies outside of the linear pathway as both the adenylate transporter as well as the plastidial ADK appear to exert higher control over the pathway than AGPase, the enzyme widely believed to be rate-limiting (Geigenberger et al. 2004).

To provide improved raw material for the starch processing industry considerable effort has been aimed at altering starch quality which is mainly defined by the amylopectin to amylose ratio (Jobling 2004). Most of the work in this direction has been done on potato tubers as these are one of the major sources for industrial starches.

The synthesis of amylose is accomplished through the activity of a particular isoform of starch synthase, GBSS, and antisense inhibition of this gene leads to amylose-free potato starch (Visser et al. 1991). Amylose-free potato starch can be expected to find application in both the food industry and in paper manufacture. Large-scale field trials with transgenic amylose-free potato varieties have been conducted in Europe and this crop is currently going through the regulatory approval process.

High-amylose starches are also of great interest, e.g. for improved frying or for industrial use as gelling agents or thickeners. Recently, an innovative approach to increase the amylose content in potato tubers involved the inhibition of starch-branching enzyme A (SBE A) activity, the enzyme responsible for the introduction of a1!6 linkages into amylopectin (Jobling et al. 2003). The authors of this study expressed a single-chain antibody targeted against the active site of SBE A in transgenic potato tubers, thereby neutralizing the enzymatic activity. They found that immunomodulation of SBE A increased the amylose content of starch granules from about 20% in wild-type tubers to 74% in the best transgenic line, exceeding the concentration of amylose achieved be conventional antisense strategies (Jobling et al. 2003). Production of Novel Carbohydrates in Transgenic Plants

In addition to attempts aiming at manipulating the contents and properties of endogenous carbohydrates, there have been several successful approaches for the production of novel carbohydrates in transgenic plants.

Fructans, or polyfructosylsucroses, are an alternative storage carbohydrate that are highly soluble and are stored within the vacuole. Fructans are present in approximately 15% of all flowering plants (Hellwege et al. 2000). Fructan synthesis is initiated by sucrose:sucrose 1-fructosyltransferase (SST) which catalyzes the fructosyl transfer from one sucrose molecule to another, resulting in the trisaccha-ride 1-kestose. In subsequent steps, fructosyltransferase (FFT) catalyzes the reversible transfer of fructosyl residues from one fructan to another, producing a mixture of fructans with different chain length (Ritsema and Smeekens 2003). One of the simplest fructans is inulin, which consists of p(1^2)-linked fructose residues while fructans of the levan type are p(2^6)-linked fructose polymers.

From a biotechnological viewpoint, interest in fructans has continued to increase, as they have been recognized as beneficial food ingredients. As part of the human diet, they are considered to be prebiotics as they selectively promote the growth of beneficial intestinal bacteria. Furthermore, fructans are assumed to have anti-cancer activity, promote mineral absorption, decrease cholesterol levels and decrease insulin levels. Fructans are normally isolated from plants with low agronomic value, such as the Jerusalem artichoke (Helianthus tuberosus) and chicory (vegetables are also covered in Chap. 25). Thus, attempts have been made to produce transgenic plants with higher fructan yield or making fructans with specific properties. Transformation of sugar beet with an SST gene from Jerusalem artichoke resulted in the conversion of 90% of the vacuolar sucrose into fructan (Sevenier et al. 1998), since the sugar beet accumulates to concentrations approaching 600 mM sucrose, this represents a massive fructan yield. Other researchers have introduced an SST along with an FFT from onion into sugar beet which resulted in an efficient conversion of sucrose into complex, onion-type fructans, without the loss of storage carbohydrate content (Weyens et al. 2004). Potato, as another crop naturally not accumulating fructans, was used to express plant fructo-syltransferases. The SST and FFT enzymes from globe artichoke were engineered into potato and led to the accumulation of the full range of fructans found in globe artichoke itself (Hellwege et al. 2000).

Another recent example of the use of potato tubers as bioreactors is the production of isomaltulose (IM), a sucrose isomer that is an excellent sucrose substitute in foods as it shares many physico-chemical properties with sucrose but is non-metabolizable and non-cariogenic. A gene encoding a sucrose isomerase (pall) which catalyzes the conversion of sucrose into IM has been isolated from the bacterium Erwinia rhapontici (Bornke et al. 2001). Expression of the pall gene within the apoplast of transgenic potato tubers led to a nearly quantitative conversion of sucrose into IM. Despite the soluble carbohydrates having been altered within the tubers, growth of PalI expressing transgenic potato plants was indistinguishable from wild-type plants. Therefore, expression of a bacterial sucrose isomerase provides a valid tool for high level IM production in storage tissues of transgenic crop plants (Bornke et al. 2002). Towards this direction, Wu and Birch (2007) introduced a sucrose isomerase gene tailored for vacuolar compart-mentation into sugar cane. Transgenic lines accumulated substantial amounts of IM in their culm. Remarkably, this was not at the expense of sucrose levels, resulting in up to doubled total sugar concentration in juice harvested from sucrose isomerase expressing transgenic sugar cane lines. The reason for this boost in sugar concentration is not understood but it has been hypothesized that IM accumulation in the culm leads to enhanced sink strength that fosters import of additional carbon from source tissues (Wu and Birch 2007). It remains to be shown whether this strategy allows increasing total sugar content in other sucrose storing crops such as sugar beet.

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