Modification of potato starch composition by introduction and expression of bacterial branching enzyme genes

Starch consists of two major components; amylose and amylopectin. Amylose is synthesized by the enzyme Granule-Bound Starch Syntase (GBSS) and consists of essentially linear chains of α-1,4 linked glucose residues. Amylopectin is synthesized by the combined activity of the enzymes Soluble Starch Synthase (SSS) and Branching enzyme (BE) and consists of linear α-1,4 linked glucosidic chains with α-1,6 linked branchpoints. The amount and fine structure of each of the components determine the starch physico-chemical properties and, therefore, the possibilities for industrial applications. Modification of starch composition by genetic engineering can be a way to improve starch quality or design starches with characteristics for new industrial purposes. In order to obtain potato starch with a higher degree of branching, prokaryotic genes encoding branching enzyme were introduced into the potato. These genes are involved with glycogen biosynthesis in bacteria which is analogous to amylopectin biosynthesis in higher plants. The differences between arnylopectin and glycogen (both α-1,4 α-1,6 branched glucan) are found in the ordered structure of amylopectin with clusters of branches as opposed to the random branching in glycogen. Furthermore, glycogen is much more branched compared to amylopectin. The branching enzyme encoding genes (glgB) from Escherichia coli and Anacystis nidulans were placed under the control of the tuberspecific GBSS promoter of potato in the binary plasmid pBIN 19 . Targeting was ensured by fusing the gIgB genes to the transitpeptide sequence of either GBSS or to that of the potato small subunit of Ribulose bisphosphate carboxylase (Rubisco) including a N-terminal addition of variable size, thus creating chimeric proteins. The plasmids were transformed by Agrobacterium tumefaciens to a diploid amylose-free potato mutant done, lacking GBSS activity, as well as to diploids with normal amylose containing starch. Transgenic plants were obtained which expressed the heterologous branching enzyme as was shown by the presence of mRNA and protein in the tubers, although expression levels were relatively low. All four constructs used for transformation were found to change the starch of transgenic tubers of amylose-free and/or wildtype plants in the same way and to a similar degree. Analysis of the starch structure showed an increase in the branching degree (DE), representing up to 25% more branchpoints in the amylose-free mutant background. An increase of up to 35% more branchpoints was observed in the amylose-containing background. The increase in the number of branchpoints was partly caused by the presence of more short chains, the so called A chains, with a degree of polymerization of ≤16 glucose- residues. Changes in other characteristics of the starch, such as average chainlength and λ max , indicated a more branched structure for starch from transgenic plants as well. Among all the transgenic plants, the starch branching degree was never found to be higher than approximately DE 5. Values for the untransformed control starches were 3.6 for amylose-containing and 3.9 for amylose- free starch. Apart from low expression levels or activity of the introduced branching enzyme, this restricted increase in DE might be due to the structure of the starch granule itself, allowing little space for changes in amylopectin structure. Especially if the activity of the introduced branching enzyme is lagging behind normal starch biosynthesis as was proposed in a model to explain the possible mechanism of branching of starch by the introduced bacterial branching enzymes. The introduced bacterial branching enzymes were not only capable of branching arnylopectin, but amylose in wildtype starch as well. It was assumed that the bacterial branching enzymes are located in the amorphous region of the growing starch granule to branch amylose and simultaneously transfer short A chains to the outerside of the amylopectin clusters in the region where all the other branchpoints are located. In some of the transgenic plants with an increased starch branching degree, the amylose content had decreased to a level comparable to that of amylose-free starch (Chapter 3). Starch granules of those transformants showed after iodine staining a blue core surrounded by redstaining starch. Gel permeation chromatography showed that the essentially unbranched amylose was replaced by a so-called intermediate fraction, with a higher degree of branching. For a number of transgenic plants with an increased starch branching degree, in the arnylose-free as well as in an amylose-containing background, enough starch was available to determine some physico-chemical characteristics (Chapters 4 and 5). No change in granule size or morphology could be observed for the altered starches of these transgenic plants compared to their (un)transformed controls. Regardless of the presence or absence of amylose, starches with an increased branching degree showed similar results: a shift towards more short chains in the arnylopectin and a lowered peak viscosity of starch suspensions during heating. For the arnylose-free starch gels it was also found that the starches with an increased branching degree showed a tendency to form weaker gels. The presence of amylose was shown to be the most important characteristic for determining the physico-chemical properties of the starch, as was expected. amylose had a negative effect on the swelling power of starch in water and it lowered the temperature of the onset of gelatinization. Our results in potato confirmed the structural-functional relationships described in literature for starch from maize mutants. More research is needed to establish the extend of changes in rheological properties of the starches from the transgenic plants and to investigate the possible new applications of the altered types of starches.