Expression of different glucansucrases in potato tubers: Implications for starch biosynthesis Géraldine Kok-Jacon Promotor : Prof. Dr. R.G.F. Visser Hoogleraar in de Plantenveredeling Co-promotor : Dr. Ir. J.-P. Vincken Universitair docent Laboratorium voor Plantenveredeling Promotiecommissie : Prof. Dr. Ir. E. Jacobsen, Wageningen Universiteit Prof. Dr. L.H.W. van der Plas, Wageningen Universiteit Prof. Dr. L. Dijkhuizen, Rijks Universiteit Groningen Dr. M. Quanz, Bayer Bioscience, Potsdam, Duitsland Dit onderzoek is uitgevoerd binnen de onderzoekschool Experimentele Planten Wetenschappen Expression of different glucansucrases in potato tubers: Implications for starch biosynthesis G.raldine Kok-Jacon Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof. dr. M. J. Kropff, in het openbaar te verdedigen op maandag 19 september 2005 des namiddags te vier uur in de Aula. Expression of different glucansucrases in potato tubers: Implications for starch biosynthesis. Géraldine Kok-Jacon. Thesis Wageningen University, The Netherlands, 2005. With references - with summaries in English, Dutch and French. ISBN 90-8504-262-3 à Sjaak, Contents Chapter 1 General introduction 9 Chapter 2 Production of dextran in transgenic potato plants 33 Chapter 3 Mutan produced in potato amyloplasts adheres to starch granules 53 Chapter 4 Production of alternan in transgenic potato plants 73 Chapter 5 Granule-bound mutansucrase alters melting temperature of starch granules 87 Chapter 6 General discussion 107 Summary, Samenvatting, Résumé 117 Nawoord 129 Curriculum Vitae and Publications 131 9 : C hapter 1/ General introduction Towards a more versatile α-glucan biosynthesis in plants Géraldine A. Kok-Jacon Jean-Paul Vincken Qin Ji Richard G.F. Visser Graduate School Experimental Plant Sciences, Laboratory of Plant Breeding, Wageningen University, P.O. Box 386, 6700 AJ Wageningen, The Netherlands. Abstract : Starch is an important storage polysaccharide in many plants. It is composed of densely packed α-glucans, consisting of 1,4- and 1,4,6-linked glucose residues. The starch polymers are used in many industrial applications. The biosynthetic machinery for assembling the granule has been modified in many different ways in order to gain insight in the starch biosynthesis process, and to engineer starches with improved functionalities. With respect to the latter, two generic technologies with great potential are developed: (i) introduction of new linkage types in starch polymers (1,3- and 1,6-linkages), and (ii) engineering granule- boundness. The toolbox to engineer this new generation of starch polymers is discussed. Part of this Chapter was published in: Journal of Plant Physiology 160; 765-777 (2003). Reprinted with permission of Blackwell Publishing Key words: starch modification, glucansucrase, starch-binding domain, granule-boundness, transgenic potato 10 : Chapter 1/ General introduction Introduction During the last 10 years, the increased need for starches with novel properties has occupied the research community, and many efforts have concentrated on unraveling the starch biosynthesis pathways. The knowledge generated in these investigations was subsequently used to produce tailor-made starches in higher plants using recombinant DNA technology. Examples of starches with new functionalities are those with a modified degree of branching (Schwall et al. 2000; Shewmaker et al. 1994; Kortstee et al. 1996) and the amylose-free starch (Visser et al. 1991a; Kuipers et al. 1994), some of which hold potential for applications in the paper-, textile-, plastics-, food and pharmaceutical industry. The accumulation of more starch has also been an objective, but this will not be discussed further here (Slattery et al. 2000). In our laboratory, we have embarked on two generic technologies with a very wide range of applicability: (i) introduction of new linkage types and structural elements using glucansucrases, and (ii) engineering granule-boundness by using microbial starch- binding domains (SBDs). It is expected that these technologies will contribute significantly to the biosynthesis of more versatile α-glucans in the near future, leading to starches with improved functionalities that cannot be obtained by conventional breeding. In this paper, these recent developments in starch modification using heterologous expression of microbial genes will be reviewed, with emphasis on the potato crop. Starch structure and biosynthesis Starch is an important reserve carbohydrate found in many plant species, and is deposited as granules in the chloroplasts of green leaves (transitory starch) and in amyloplasts of tubers, roots and seeds (storage starch) (Kossmann and Lloyd 2000). Each starch granule has a highly organized structure defined by the succession of crystalline and amorphous lamellae (Fig. 1). The two main components of starch, amylopectin and amylose, are polymers of glucose. The major polysaccharide of the two, amylopectin (70-80%) is a highly branched molecule, mainly composed of α-1,4- linked glucosyl residues and 4-6% of α-1,4,6-linked branch points. The distribution of the branch points is not at random , enabling the unique, cluster-based structure of amylopectin (Thompson 2000). Clustering of the branch points in the amorphous lamellae enables the polymer chains to line up in a parallel fashion. The chains can 11 : Chapter 1/ General introduction Figure 1. Overview of the various l evels of polymer organization within the starch granule, which is composed of amylopectin and amylose molecules associate with each other to form double helices (Smith et al . 1997; Ball et al . 1998). This chain organization forms the basis of the semi-crystalline structure of the starch granule (Myers et al . 2000). In contrast to amylopectin, amylose (about 20-30%) is an essentially linear polysaccharide with less than 1% of branch points. In plant storage organs, starch biosynthesis takes place within the amyloplast (Fig. 2) and is the result of different reactions such as synthesis (polymerization of glucosyl residues), rearrangement and degradation, in which various starch synthases (E.C.2.4.1.21), transferases (branching (E.C.2.4.1.18) and disproportionating enzymes (E.C.2.4.1.25)), and hydrolytic enzymes (debranching enzyme (E.C.3.2.1.41)), respectively, play key roles. Sucrose is the starting point of starch biosynthesis, which is converted into hexose-phosphate sugars in the cytoplasm. In potato, glucose-6- phosphate (Glc-6P) is transported into the amyloplast (Kammerer et al. 1998). It is first converted to glucose-1-phosphate (Glc-1P) by plastidial phosphoglucomutase (PGM) (E.C.5.4.2.2) and, subsequently, to ADP-glucose (ADP-Glc) by ADP-glucose pyrophosphorylase (AGPase, E.C.2.7.7.27) (Tauberger et al . 2000; Müller-Röber et al . 1992; Stark et al . 1992). ADP-Glc serves as a substrate for the different starch synthase isoforms, some of which are mainly present in the soluble phase or stroma (SS-isoforms), while others are associated with the granule. The exclusively granule- 12 : Chapter 1/ General introduction Figure 2. Schematic representation of starch biosynthesis in a potato tuber cell. A: sucrose synthase (E.C.2.4.1.13); B: UDP-Glc pyrophosphorylase (E.C.2.7.7.9); C: phosphoglucomutase; D: Glc-6P isomerase (E.C.5.3.1.9); E: hexose kinase (E.C.2.7.1.1); F: plastidial phosphoglucomutase; G: ADP-Glc pyrophosphorylase; H: many enzymes such as starch synthase, branching enzymes, debranching enzymes, etc. ; I: non-native glucansucrases. Suc = sucrose, Glc = glucose; Frc = fructose. : translocators; 1: putative sucrose transporter; 2: G6PT (Glc-6P/Pi antiporter) (Kammerer et al. 1998); 3: putative hexose transporter related to the GLUT family (Weber et al . 2000); 4: putative sucrose transporter. : transport of solutes; : reaction catalyzed by enzymes. Dotted arrows indicate putative reactions in transformed tuber cells after introduction of a bacterial glucansucrase. bound starch synthase (GBSSI) catalyzes the formation of amylose (Kuipers et al. 1994). Furthermore, it was shown that GBSSI can also contribute to amylopectin synthesis in potato, pea and the unicellular alga, Chlamydomonas reinhardtii (Baba et al. 1987; Denyer et al. 1996; van de Wal et al . 1998). All starch synthases elongate glucan chains by transferring the glucose moiety of ADP-Glc to the non-reducing end of α-1,4 linked glucans. The branching enzymes (BE) cleave α-1,4 linkages and form α- 1,6 linkages. Schwall et al. (2000) showed that antisensing these enzymes in potato led to less branching of the forth-coming starch. Additional enzymes such as the disproportionating enzymes (D-enzyme) cleave and rejoin α-1,4 linkages in starch 13 : Chapter 1/ General introduction polymers and the debranching enzymes (DBE) hydrolyze the α-1,6 linkage at branch points. Figure 3. Schematic representation of the starch biosynthesis pathway. The timing of the biosynthesis process is as follows: the darker the background colour, the later the event takes place. The role of the various enzymes is described in detai l in the text. The starch biosynthesis process is summarized schematically in Fig. 3. The model shows clearly that amylopectin is synthesized first, and that amylose is formed later. Two possible mechanisms for amylose biosynthesis have been proposed: (i) the amylopectin-primed pathway, and (ii) the malto-oligosaccharides (MOS)-primed pathway (Mouille et al . 1996; Ball et al . 1998; van de Wal et al. 1998, 2000; Denyer et al. 1999;