Bio-Farming of Carbohydrate | Genetics

In this article we will discuss about the bio-farming of carbohydrates.

Plants synthesize wide array of carbohydrate. Several of these are commercially impor­tant. Plant produces some of the most commercially available carbohydrates are cellulose and the starch. Cellulose is widely used for manufacture paper from trees, fibres from cotton in the manufacture of industrially important polymers.

Starch is extensively used in food industry and also animal feed as well as industrial use. Apart from these two, several other carbohydrates like cyclodextrins, polyfructan and sugar alcohol like mannitol and sorbitol are widely employed in various fields. Biotechnology has done great endevour towards modifying and improvement of carbohydrate for the bulk produc­tion using transgenic technology.

Synthesis of carbohydrate is confined to different compart­ment in the cell for example, starch is synthesised in the plastid. Sugar and sugar alcohols are synthesised into cytosol and accumulate throughout the cell. Several of the sugar alcohol are synthesised in response to abiotic stress.

Some of the storage carbohydrate like fructans are synthesised and stored in vacuole. Among cereals, carbohydrate is synthesised and stored transiently in the leaves and their reserves accumulate in the endosperm.

Starch:

This is generally found in the form of starch grains whose morphology varies according to plant species. Major starch producing crops are cereals and potatoes, commonly known as chemical feed stocks. Biosynthesis of starch to takes place utilising triose-phosphate in the Calvin cycle. Triosephosphate can be trafficking between cytoplasm and plastids.

In plastids this is converted into hexose phosphate and ADP glucose. This acts as major susbtrate for starch biosynthesis. Starch is generally consisting of amylose, amylopectin. Hexose molecules are directly transported into amyloplast, which is generally specialized for starch accumulation. However, in cereal endosperm ADP-glucose is synthesised in the cytoplasm and transported di­rectly into the plastids.

Enzymes starch synthase (SS) and starch branching enzymes (SBE) are the two major classes of enzymes participate in the synthesis of starch. Starch synthase facilitate the addition of glucose residue from ADP-glucose to growing chain of D-glucose, joined by α-1, 4- glucosidic linkages. Amylase is one of the form of unbranched starch consist of unbranched chains of glucose of about 1000 residue long. Amylopectin is another form of branched starch.

The branch­ing nature is inflicted by the enzyme SBE, which creates side chains by α-1, 6 glucosidic linkages, which have same structure as the main chains and which are 20 to 25 glucose residue long. Some starch contains only amylopectin. Those containing amylose and amylopectin, the respective pro­portion of the two constituents and the length of chains vary according to the type of starch.

Genetic engineering strategy can be enforced in the improvement of carbohydrate syn­thesis based on their biosynthetic principles. One of the main primary metabolite pools, hexose phosphate sugar is converted to starch. ADP-glucose is the immediate precusor of starch cata­lysed by the enzyme ADP glucose pyrophosphorylase.

By altering ADP glucose production, pos­sible to manipulate over all level of starch biosynthesis. Thus, optimising the expression of ADP glucose pyrophosphorylase can result in enhanced amount of starch production. As starch con­tains particular proportions of amylose and amylopectin (20-30% amylose to 70-80% amylopectin), the ratio can be engineered by manipulating one of the branches on the path­ways.

The overall properties of starch are influenced by the presence of proportion of amylase to amylopectin. It is highly desirable to have starch with complete or high proportions of either amylose or amylopectin depending upon their usage in industries. For example, utilization of starch in food industries involves boiling and cooling process.

But presence of amylose tends to produce undesirable product. Thus, limits its usage in industries. Another example, unbranched amylose starch is indispensable as valuable starch as feed stock. Therefore, it is indispensable to produce amylose free starch for food industries and amylopectin free starch for feed stock.

These kinds of manipulations have been achieved in potato tuber using transgenic technology. As granule bound starch synthase (GBSS₁) is responsible for amylose chain, down regulation of GBSS₁ by antigense gene technology produce amylose free starch for food industry. On the other hand, antisense down regulation of starch branch enzyme (SBE) results in amylopectin free starch but having high amylose, which is useful as valuable feedstock.

Production of Cyclodextrins:

Cyclodextrins are commercially important carbohydrate derivative. They are produced from starch using conventional bacterial fermentation. These are particularly employed in medical field as it facilitates solubilization of pharmaceuticals such as steroids. Cyclodextrins are made up of seven membered rings of glucopyranose subunits. These subunits are attached by α-1, 4 linkages.

In view of their commercial significance, biofarming of cyclodextrins seems to be indispensable. Presently, cyclodextrins are produced using fermentation industry because of the presence of bacterial enzyme glucosyltransferase. In one of the published report, which claims to have produced, cyclodextrins from transgenic potato?

Due to the high content of starch in potato tuber, cyclodextrin production has been attempted, by transferring cyclodextrin glycosyltransferase gene from Klebsiella pneumoniae into potato plant by fusing gene to a plastid targetting sequence driven by patatin gene promoter to express specifically in potato tuber. Due to the immobilization of enzyme in the starch reduced its conversion to cyclodextrin. The conversion efficacy hardly reaches 0.01%.

Polyfructans:

Production of polyfructans by the modification of carbohydrate is another example of transgenic modification of carbohydrate. Polymers of fructose synthesised and stored in the vacuole. These accumulate in approximately 15% of the flowering plants as reserve carbohy­drate. They are currently used in several food products hold promise as a feedstock for indus­trial application.

Fructans have a typical structure of glucose-fructose (fructulose) and glycoside linkage is found between fructose residue resulting in straight and branched polymers. Biosynthesis of fructon takes place by involvement of an enzyme sucrose fructosyl transferase (SSF), which transfer fructose from donor sucrose to ketose. These are then donating fructose to the growing fructan chain with the help of fructose fructosyl transferase (FFT).

Fructose can be used as a low calorie food ingredients and small low molecular weight. Fructoses have a sugar taste and could be used as a natural low calorie sweetner. Similarly, high molecular weight fructans is used as food ingredients in industries because of the fat like struc­ture.

Fructose can be a suitable alternative to high caloric value sucrose. Fructose cannot be used as a carbon source as cannot be degraded by human digestive enzyme and moreover consumption of the fructose can stimulate beneficial bacterial resident in the colon of the human body.

In Western Europe, fructans are commercially extracted from chicory roots, and in most of the coun­tries they are extracted from Jerusalem artichoke tuber. Due to its poor agronomic value of the crop, and unfavourable distribution of chain length, additional processing steps are required dur­ing fructan production. One of the novel options is to switch over to transgenic method.

Several strategies have been adapted to improve commercial production of fructans in fructan accumulating crops. One such fruitful approach in which expressing chichory fructosyl fructosyl transferase (FT) gene derived from onion. Among plants only a inulin type of fructan is found.

In another strategy sugar beet have been chosen to produce low-calorie fructans. One of the main reasons for the selection of sugar beet is due to its high agronomic value. Sugar beet stores sucrose in the vacuole upto 0.6 M. Therefore, it is easy to convert vacuole stored sucrose into fructose by enzymatic process.

Thus, in transgenic method, the gene encoding sucrose sucrose fructosyl transferase, which is derived from Helianthus tuberoses, was introduced into sugar beet, which is able to convert sucrose into mixture of low molecular weight fructan. In the tap root of sugar beet transformed with t-sst gene, the stored sucrose in the vacuole is almost totally converted into low molecular weight fructose.

When same t-sst gene expressed in leaves, other than tap root, resulted in only low levels of, fructose due to the reduced availability of sucrose in the leaves. Transformation of sugar beet was achieved with stomatal guard cell protoplast. The cDNA encoding 1-sucrose-sucrose fructosyl transferase was isolated from Jerusalem artichoke and introduced into sugar beet.

Number of bacterial gene have been tried to produce polyfructose in transgenic plants and have now been developed using genes. SacB genes of Bacillus subtilis encodes levan sucrose and ftf gene from streptomyces. Polyfructan have been produced in transgenic potato using the same vacuolar targeted sac gene driven under the control of potato tuber specific patatin promoter.

In potato, sucrose is diverted from starch accumulation in the tuber and towards fructan in the vacuole has generated a novel modal system not only for efficient production but also for regulation of sucrose metabolism. The same sac B gene of Bacillus subtilis have also been expressed in endosperm of maize under the control of Zein promoter.

Different vacuolar specific sequences were fused to target the enzyme specific to the vacuoles of endosperm cells. For example, the two vacuole sequences such as potato sporamin signal peptide and the barley lectin signal peptide was fused to N-terminal end of the enzyme. Accumulation of fructan reached up to 8-10 mg per gram of seed.