Starch Meets Biotechnology in Planta Modification of Starch Composition and Functionalities

Starch Meets Biotechnology In Planta Modification of Starch Composition and Functionalities Xuan Xu Thesis committee Promotor Prof. Dr R.G.F. Visser Professor of Plant Breeding Wageningen University & Research Co-promotor Dr L.M. Trindade Associate professor, Wageningen UR Plant Breeding Wageningen University & Research Other members Prof. Dr H.A. Schols, Wageningen University Prof. Dr M.J.E.C. van der Maarel, University of Groningen Dr J.C.P. Hopman, Averis Seeds B.V., Valthermond Dr C.G. Boeriu, Wageningen University & Research This research was conducted under the auspices of the Graduate School of Experimental Plant Sciences Starch Meets Biotechnology In Planta Modification of Starch Composition and Functionalities Xuan Xu Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr A.P.J. Mol, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 14 October 2016 at 11 a.m. in the Aula. Xuan Xu Starch Meets Biotechnology - In Planta Modification of Starch Composition and Functionalities 170 pages. PhD thesis, Wageningen University, Wageningen, NL (2016) With references, with summary in English ISBN 978-94-6257-920-0 DOI 10.18174/389540 Table of Contents Chapter 1 General introduction 7 Chapter 2 Starch modification by biotechnology: 21 state of art and perspectives Chapter 3 Engineering Potato Starch with a Higher Phosphate Content 39 Chapter 4 Starch phosphorylation plays an important role in starch 63 biosynthesis Chapter 5 Introduction and Expression of Arabidopsis Starch Dikinases in 87 Potato Chapter 6 Expression of an (engineered) 4,6-α-glucanotransferase in 107 potato results in changes in starch characteristics Chapter 7 General Discussion 129 References 143 Summary 157 Acknowledgements 159 About the author 165 Overview of training activities 167 Chapter 1 General Introduction 8 Chapter 1 Starch, an attractive bio-resource Starch is the most widespread and predominant storage carbohydrate in higher plants, which can provide energy for plant development. It is synthesized from assimilated sugars and assembled into a granular form in specific tissues. In the leaves of plants, starch serves as short-term energy reserve to ensure continued supply of sugars in day- 1 to-day carbohydrate metabolism. This type of starch is accumulated during the daytime photosynthesis and remobilized subsequently during the night and is thus named “transitory starch”. On the other hand, starch accumulated in heterotrophic organs (e.g. seed, roots and tubers) is termed “storage starch”, as this type of starch serves as a long- term energy source to fuel regrowth upon sprouting or germination. Additionally, and importantly, storage starch plays a multifunctional role in human consumption, serving as a primary source of dietary calories in human diet and also as an inexpensive, renewable and biodegradable material in a broad range of industrial applications. Due to the wide uses of starch, the global production of starch containing crops outweighs other industrial and food crops. At present, the main sources of storage starch are from maize, cassava, wheat and potatoes, which are mainly produced by China, North America, Europe, Southeast Asia and South America (Food and Agriculture Organization of the United Nations, http://faostat3.fao.org/home/E). The most important outlet by far of all starch-derived products is relevant to food; however, the consumption of starch has greatly increased with starch becoming an important component for bio-fuels, paper and glue production, textile weaving and finishing and the fermentation industry. To illustrate, in Europe, the total production of starch in 2014 was 10.5 million tons with 47.5%, 39.2% and 13.3% for maize, wheat and potatoes, respectively, which was increased by 21% compared to 2004 (European Starch Industry Association, www.aaf-eu.org). These starches were produced from in total 23 million tons of agricultural materials, which consist of 16 million tons of cereals and 7 million tons of starch potatoes (Fig. 1a and b). Of this amount, the largest single outlet was (has always been) for sweeteners production (Fig. 1c) and was mainly consumed in confectionary and drinks (Fig. 1d). The global starch market has continuously expanded over the years. It is expected to exceed 150 million metric tons by 2020 driven by the increasing population, cost benefits and sustainable consumption patterns. Correspondingly, the global sales of starches and derivatives are projected to reach $77.4 billion by 2018, up from $51.2 billion in 2012 (BCC research, http://www.bccresearch.com/market-research/food-and-beverage/starch- glucose-fod037b.html). Nonetheless, a big challenge ahead is that the rapid market expansion conflicts with a shortening availability of suitable raw materials at tolerable prices and increasing energy costs. General introduction 9 To overcome this challenge and to meet this rapid growing demand from a growing human population in a changing climate, producing starch with high quality in large quantities through a more innovative, cost-effective, and sustainable manner is inevitable and warranted. At present, while much of the researches are focusing on productive output, an increasing number of studies has emerged to enhance functional characteristics of starches, aiming at producing starches that are more convenient to distribute and easier to process and have specific physico-chemical properties required by the target markets. 1 These value-added starches can provide society with high-value raw materials to satisfy market needs, thereby broadening the path towards a robust starch industry and a truly bio-based and circular economy. FIGURE 1. Overview of European starch production and consumption in 2014. Source: European Starch Industry Association, www.aaf-eu.org. 10 Chapter 1 Green technology, genetic modification, is needed for starch modification Genetic modification of starch, an inexorable trend The competitive advantage and commercial success of starches is mainly determined by 1 their functional characteristics. Surprisingly, most of the native starches are inherently not suitable for industrial applications, notwithstanding the large variations in their functional properties. This is mainly due to the high and unstable viscosity of the cooked starch, retrogradation and gelling tendencies of starch pastes. Therefore, starches are often modified to eliminate deficiencies and achieve the more appropriate properties for different industrial applications, which in turn enhances the starch’s versatility and satisfies the consumers’ demands. Several methods of starch modification are used in industry: chemical, physical and enzymatic modification. Chemical modification is one common method to modify starches for industry. Using various chemical reagents, chemical moieties can be added on the a-D-glucopyranosyl units of starches by molecular scission, oxidation, esterification, etherification or molecular arrangements (Chiu and Solarek, 2009; Shrestha and Halley, 2014), thereby enhancing the functionality of starch. Additionally, physical modification is also widely used in industries to generate pregelatinized, milled, and cold-water-soluble starches through drum drying, spray drying, extrusion cooking, annealing and heat- moisture treatment (Haghayegh and Schoenlechner, 2011). Enzymatic modification is another strategy that evolved not long ago and hydrolyzing enzymes are mainly used to modify starch for industrial applications. The products are very suitable for coating food items with seasonings, flavors and colorants, as they retain a good adhesion property during hydrolysis, which can be applied at high solids to minimize the energy required to remove moisture after application (Chiu and Solarek, 2009). All in all, these methods have largely extended the end uses of starch; however, they are expensive, time-consuming and frequently generate hazardous wastes. Our society is becoming more and more aware that producing modified starch through these conventional methods could not fully meet the requirements for environmental and economic sustainability. Thus, large-scale research has been executed to explore green technologies, and one of them is genetic modification of starch in planta. This approach allows us to generate tailor-made starches in plants that are directly suitable for a particular industrial use, thereby circumventing expensive and environment-unfriendly post-harvest processes and thus providing tremendous economic and environmental advantages. In other words, genetic modification of starches provides opportunities to produce novel starches with enhanced properties in a sustainable and economical manner, which is an inexorable trend for realizing a robust starch industry. General introduction 11 Strategies for genetic modification of starch Our understanding of starch biosynthesis was greatly improved with studies on transitory starch in Arabidopsis thaliana, which provides a bulk of information for targeted design of novel starches. However, the main challenge is to apply the acquired knowledge in economically important starch crops for improving starch quality. In the past decades, continuous efforts have been made to realize this purpose. There are two main strategies 1 to genetically modify starch in planta: one is to modulate endogenous genes involved in starch biosynthesis; another is to express heterologous genes

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