Theory of starch biosynthesis and their structures for the rational design of starches: a mathematical modelling approach

Abstract

Starch is stored transiently in leaves during the day and degraded during the night to fuel respiration and continual growth. In seeds, starch is reserved for seed germination. We rely on starch as the main source of food energy and, in addition, it has a wide range of industrial applications. Rational design of starches for improved quality and quantity has significant global implications. This thesis describes an approach to study starch biosynthesis-structure relation through mathematical model developments. The structure of interest is the starch chain-length distribution (CLD). First, this thesis optimized procedures for obtaining accurate starch CLDs using size-exclusion chromatography (SEC) and fluorophore-assisted carbohydrate electrophoresis (FACE) and found that the commonly used alkali dissolution method causes degradation in the long amylopectin chains and amylose chains. A milder dissolution procedure using dimethyl sulfoxide at 80°C was found to be effective at minimizing artifactual results. Mathematical models for both transient and reserve starch biosynthesis are presented in this thesis which involve linear algebra and differential equations. Analytical solutions and considerable insight is obtained by expressing the solutions in terms of eigenanalysis. Numerical solutions of the models were computed by FORTRAN programs developed in this thesis. These models provide a means by which a small number of key parameters defining the core enzymatic activities can be used to parameterize starch CLDs, providing the basis for focusing studies on the rational design of starch structure. The reserve-starch biosynthesis model predicts defined restrictions on particular ratios of enzymatic activities apply. The model independently proved the absolute requirement of debranching enzymes for the synthesis of starch previously inferred by genetic and biochemistry studies. The model provides a mechanistic basis for understanding how successive arrays of crystalline lamellae are formed, based on the identification of two independent types of long amylopectin chains, one type remaining in the amorphous lamella, while the other propagates into, and is integral to the formation of, an adjacent crystalline lamella. The defined restrictions on ratios of enzymatic activities predicted by the reserve-starch biosynthesis model imply that starch CLD cannot be dramatically altered if the plant is to be viable. However, the model suggests that altering the specificity of branching enzymes so that different chain lengths are transferred during branching is a workable option for producing starch with altered CLD. This prediction is tested in a collaborative work by mutating the conserved amino acids in the catalytic domain of maize branching enzyme IIa (mSBEIIa). One of the so produced mSBEIIa mutant (R436K) was capable of transferring chains with a degree of polymerization (DP) of 7 like the wild-type mSBEIIa. The reserve-starch biosynthesis model predicts that a higher branching enzyme activities causes a lower amount of long amylose chains (degree of polymerization (DP) 700–40,000); branching enzyme activities have no correlation with the short amylose chains (DP 100–700). The transient-starch biosynthesis model predicts the effect of different enzyme combinations on the rate of transient starch biosynthesis. It indicates that α-amylase, in addition to the core starch biosynthetic enzymes, is also involved in the modification of glucans for the synthesis of insoluble starch granules. The model predicts the involvement of β-amylase, in the absence of α-amylase in mutants, slows the rate of attaining a crystalline-competent CLD for crystallization of glucans to form insoluble starch. This is consistent with the minor role of β-amylase in shaping normal starch synthesis. The model predicts that debranching of glucans is an efficient mechanism for the attainment of crystalline-competent CLD; however, attaining this is still possible, albeit significantly slower, through combinations of α- and β-amylase in the absence of isoamylase-type debranching enzyme. This thesis is a step forward on the fundamental research on processes that are involved in starch the regulation of starch synthesis and granular formation for the rational design of starches.