1 Starch: Structure, Function, and Biosynthesis Outline Introduction to starch Starch biosynthesis and genetic modifications Starch analysis Functional properties of food starch Modification of starches Starch digestibility 2 Introduction to Starch Starch Carbon and energy storage material for plants A major component of food and feed, providing source of energy and carbon Used for texturizing and to provide specific functionalities in processed foods Starting material for ethanol production Broad applications in the paper and textile industries 3 Introduction to Starch Industrial Starch Sources Corn starch Common corn Waxy maize (amylose ~ 0%) High-amylose (amylose 55 and 70%) Other mutants Root/Tuber starch Potato Cassava (Tapioca) Other Cereals Wheat Rice Barley 4 Introduction to Starch Partial list of documented starch mutants of maize Single mutants Waxy (wx) Amylose-extender (ae) sbe1 sbe2a Sugary-1 (su1) Zpu1 Sugary-2 (su2) Dull-1 (du1) Brittle-1 (bt1) Brittle-2 (bt2) Shrunken-1 (sh1) Shrunken-2 (sh2) double mutants ae wx sbe1 wx ae su1 ae su2 ae du1 du1 su1 dul su2 du1 wx su1 wx su2 wx su1 su2 Triple/quadruple mutants ae du1 su1 ae du1 su2 ae du1 wx ae su1 su2 ae su1 wx ae su2 wx sbe1 ae wx du1 su1 su2 du1 su1 wx du1 su2 wx su1 su2 wx ae du1 su1 wx 5 Introduction to Starch World starch production and utilizations Starch Production Corn Potato Wheat Tapioca Others 83% 6% 6% 4% 1% Refinery Products Dextrose and maltose High fructose corn syrup Maltodextrin Ethanol Starch Products Paper (ca. 60%) Food (ca. 20%) Textile (ca. 20%) 6 Introduction to Starch End-use demand for starch in major markets Approx. % US Paper and paperboard High-fructose sweetener Other sweeteners Fuel ethanol Food Other industrial a Corn EU 22 5 28 2 18 26 Japan 11 27 27 5 21 10 10 31 12 42 3 2 Annual 2001, Corn Refiners association 7 Introduction to Starch Starch granules 8 Introduction to Starch Starch granules 9 Introduction to Starch Starch granules Normal light Polarized light Birefringence indicates that the granules are semi-crystalline 10 Introduction to Starch Starch granules 11 Introduction to Starch Starch granules Two populations of wheat starch: large size A granules, and small size B granules 12 Introduction to Starch Starch granule size distributions 13 1,000 - 100,000 nm Overview of Starch Granule C rysta lline H ard S he ll (120-400 nm thick) S em i-crysta lline S oft S hell H ilum 20-500 nm C rysta lline hard she ll W h ole G ra nule P o re s (100-300 nm in d iam eter) G ra nule S urfac e S upe rhe lix o f A P T ilte d C lus te rs S ide P la n C rysta lline Lam ellae A m orpho us Lam ellae A xis of A P helices S em i-crysta lline ha rd she ll A P double he lice s Am orph ous c ha n ne ls P itc h of lam ella r he lice s (9 nm ) b locklet b locklet 10-50 nm & 1- 5 c lusters B lo cklet (S ize 50 -500 nm depend ing o n bota nica l so urce a nd locatio n in gra nule ) 9 -1 0n m O ute r rad ius (9 nm ) T op P lan P ie-shaped Lam ellar M otif Inne r R ad ius (4 nm ) C entra l ho le w ith the sam e density as am orp ho us lam e llae 1.07nm S ide V ie w 2.12nm T op V ie w 123.5 0 1.17nm ~1 nm Am y lope c tin C lus te r A M Lipid Lipid 1nm 6nm T y pe A 3nm T y pe B AM 1.04nm C olum n of w ater 120 0 1.85nm 1.85nm 10-15 nm in d iam eter 14 Amylose and Amylopectin Amylose A linear molecule comprising of 1,4 linked alpha-D-glucopyranosyl units. There is a small degree of branching by 1,6 alpha linkages The smaller of the two polysaccharides making up starch Amylopectin A highly branched molecule comprising both 1,4 linked and 1,6 linked alpha-D-glucopyranosyl units Branches are non-randomly distributed in clusters The larger of the two polysaccharides making up starch 15 Amylose and Amylopectin Diagrams of amylopectin branches & clusters 16 Amylose and Amylopectin Reducing end Open ring Aldehyde oxidized to carboxylic acid 17 Amylose and Amylopectin Different type of chains for amylopectin Three chain types A, B and C A chains are not branched B chains are branched C chain has the reducing end R One reducing end per amylopectin molecule Wang et. al., J. Exp. Botany (1998) 49, 481 18 Amylose and Amylopectin Structural parameters of amylopectin clusters ICL of B3 (53.5 DP) ae wx Cluster repeat distance (26.5 DP) Average number of branches per cluster (ANBPC) ICL of B2 (26.5 DP) wx wx β-limit dextrin B chain stub 1 or 2 DP A chain stub 2 or 3 DP ICL of B1-a (3.2 DP) B2 chain, chains connecting two clusters B3 chain, chains connecting three clusters B1a chain, short intra-cluster chains B1b chain, long intra-cluster chains A chains Yao et al. 2004, Plant Physiology, 136: 3515–3523 19 Amylose and Amylopectin Starch-lipid complex Polar (hydrophilic) head outside Nonpolar (hydrophobic) tail inside Buleon et al., Int. J. Biol. Macromol. (1998) 23, 85 20 Starch Crystallinity Crystalline forms A: Cereal starch B: Tuber starch, high amylose starch V: amylose crystallized with lipids, iodine, etc Vh: hydrated V form Buleon et al., Int. J. Biol. Macromol (1998) 23, 85 21 Starch Crystallinity Crystalline A structure Crystalline A structure for starch. An (a,b) plane projection of the unit showing helix packing, water molecules, and hydrogen bonding Imberty et al. J. Mol. Biol. (1988) 201, 365 22 Starch Crystallinity Crystalline B structure Crystalline B structure for starch. An (a,b) plane projection of the unit showing nearby helices and the center channel of organized water molecules Imberty et al. Biopolymers (1988) 27,1205 23 Amylose and Amylopectin Estimation of Crystallinity α Amorphous Crystalline area = 435 α = 435 / 1460 = 30% Total area = 1460 Diagram from Dr. Rengaswami Chandrasekaran 24 Starch Biosynthesis and Genetic Starch Modifications of Maize 25 Outline An introduction from maize kernel to amylopectin clusters Functional behaviors of enzymes synthesizing starch Mutant starches of maize, single mutants Mutant starches of maize, multiple mutants Mutant starches of maize, patents associated with foods Genetic starch modifications, challenges Genetic starch modifications, our approaches 26 From Maize Kernel to Amylopectin Clusters Starch formation in plastids Starch granules are classified as transitory or reserve Transitory starch granules accumulate for only a short period of time before they are degraded, e.g. Starch forms in leaf chloroplasts during the day And hydrolyzed and transported to other plant parts at night as simple sugar Reserve starch, an energy storage for germination, a major component of food and feed, and an industrial starting material, is formed in amyloplasts We will be discussing starch biosynthesis in maize endosperm 27 From Maize Kernel to Amylopectin Clusters A kernel of yellow dent corn Diagram adapted from Wheat Flours Institute, Chicago, Illinois, 1964 28 From Maize Kernel to Amylopectin Clusters Comparison of kernels and starch granules of two wx mutants wx ae wx Genetic modifications provide diverse kernel phenotypes, starch granule sizes, internal granular structures, and starch molecular structures 29 From Maize Kernel to Amylopectin Clusters Let’s look at the big picture before “zooming in”…… We are going here! Gallant et al, 1997, Carbohydrate Polymer, 32:177-191 30 From Maize Kernel to Amylopectin Clusters Amylopectin branches are non-randomly clustered Thompson, 2000, Carbohydrate Polymers, 43: 223-239 31 From Maize Kernel to Amylopectin Clusters Structural parameters of amylopectin clusters Keywords: amylopectin, clusters, branches, A chains, B chains, branching pattern ICL of B3 (53.5 DP) ae wx Cluster repeat distance (26.5 DP) Average number of branches per cluster (ANBPC) ICL of B2 (26.5 DP) wx wx β-limit dextrin B chain stub 1 or 2 DP A chain stub 2 or 3 DP ICL of B1-a (3.2 DP) Yao et al. 2004, Plant Physiology, 136: 3515–3523 32 From Maize Kernel to Amylopectin Clusters Chain length profiles are used to calculate AP parameters Debranched amylopectin DEGREE of POLYMERIZATION, DP 1000 100 50 20 10 3 Debranched β-limit dextrin DEGREE of POLYMERIZATION, DP 60 NORMALIZED D.R.I 1000 100 50 B3 20 10 3 NORMALIZED D.R.I 60 40 20 0 10 12 14 16 18 20 B3 ICL + 2.5 B2 B1-b B1-a A B2 ICL+ 2.5 B1-b ICL+ 2.5 40 20 B1-a ICL+ 2.5 0 10 12 14 16 18 20 DEGREE of POLYMERIZATION, DP ae wx wx RELATIVE MOLAR AMOUNT DEGREE of POLYMERIZATION, DP RELATIVE MOLAR AMOUNT 1000 100 50 20 10 3 5 4 3 2 1 0 10 12 14 16 18 20 RETENTION TIME, min 15 1000 100 50 B3 B2 20 10 B1-b B1-a 3 A Average CL 10 A chain amount Cluster amount 5 B1-a ICL+ 2.5 0 10 12 14 16 18 20 RETENTION TIME, min 33 From Maize Kernel to Amylopectin Clusters What can genetic starch modifications do for us? Improve the starch yield of major crops Acquire starches with desirable functionalities and high value Suitable starting materials for chemical and enzymatic modifications Modified digestibility or degradability as food, feed, and industrial raw materials Retarded or enhanced retrogradation after cooking, leading to extended shelf life or unique functionalities Unique granular and nano structures for high-end uses, e.g. as carriers for controlled release 34 From Maize Kernel to Amylopectin Clusters Successful genetic starch modification is based on the knowledge of starch biosynthesis Where are starch granules synthesized? What enzymes are involved in starch biosynthesis? What are the functional behaviors of these enzymes? Can we tailor starch structure for desirable functions? 35 Enzymes Synthesizing Starch Starch biosynthesis in a cell of maize endosperm Starch debranching enzyme Starch branching enzyme starch granules amylopectin + amylose ADP AMYLOPLAST D-enzyme? Soluble starch synthase Granule-bond starch synthase Glucosyl primer (UDP-Glc:protein transglucosylase?) ADP-Glc transporter ADP-Glc ADP-Glc ATP Phosphoglucomutase ATP PPi ADP-Glc pyrophosphorylase UTP UDP-Glc pyrophosphorylase ADP Glc-1-P + PPi Fructose UDP-Glc Sucrose synthase Sucrose UDP CYTOSOL 36 Enzymes Synthesizing Starch ADP-glucose pyrophosphorylase (AGPase) ATP PPi O P AGPase ADP-glucose ADP 37 Enzymes Synthesizing Starch Starch synthase (SS) Isoforms identified: GBSSI, SSI, SSIIa, SSIIb, & SSIII + ADP O O O O SS ADP O O O O O Soluble starch synthase (SSS) responsible for amylopectin synthesis Granule-bond starch synthase (GBSS) responsible for amylose synthesis 38 Enzymes Synthesizing Starch Starch branching enzyme (SBE) Isoforms identified: SBEI, SBEIIa, & SBEIIb O O O O O O Reaction I O O SBE O O O O O O O + O O O O O O O O Reaction II O O SBE O O O O O O + O O O 39 Enzymes Synthesizing Starch Starch debranching enzyme (DBE) Isoforms identified: Isoamylase-like (SU1) & Pullulanase-like (ZPU1) O O O O O O DBE O O O O O 40 Enzymes Synthesizing Starch Specific substrates may detect enzyme specificity Plant tissue Protein extraction Native PAGE Electroblotting Incubation Iodine staining Photo recording SBEIa SS-4 SS-5 SS-6 ae wx sbe1a ae wx wx sbe1a wx ae wx × 1/2 Glucan Glucan dispersion Glucan gel Potato starch sbe1a ae wx × 1/2 Waxy maize starch Liver glycogen sbe1a ae wx × 1/2 wx sbe1a wx ae wx sbe1a ae wx wx × 1/2 sbe1a wx × 1/2 ae wx × 1/2 sbe1a ae wx × 1/2 SS-1 SS-2 SS-3 ae wx sbe1a ae wx wx sbe1a wx ae wx × 1/2 SBEIIa SBEIIb DBE 41 Enzymes Synthesizing Starch Starch branching enzymes change gel from blue to purple or yellow sbe1a ae wx × 1/2 sbe1a ae wx × 1/2 wx sbe1a wx ae wx sbe1a ae wx wx × 1/2 sbe1a wx × 1/2 ae wx × 1/2 sbe1a ae wx × 1/2 SS-1 SS-2 SS-3 SBEIa SS-4 SS-5 SS-6 ae wx sbe1a ae wx wx sbe1a wx ae wx sbe1a ae wx wx sbe1a wx ae wx × 1/2 SBEIIa SBEIIb DBE Potato starch ae wx × 1/2 Waxy maize starch Liver glycogen All SBEs may branch both amylose & amylopectin SBEI preferentially branches amylose SBEIIa & SBEIIb preferentially branches amylopectin 42 Enzymes Synthesizing Starch Starch debranching enzymes change gel from purple to blue sbe1a ae wx × 1/2 sbe1a ae wx × 1/2 wx sbe1a wx ae wx sbe1a ae wx wx × 1/2 sbe1a wx × 1/2 ae wx × 1/2 sbe1a ae wx × 1/2 SS-1 SS-2 SS-3 SBEIa SS-4 SS-5 SS-6 ae wx sbe1a ae wx wx sbe1a wx ae wx × 1/2 SBEIIa SBEIIb DBE Potato starch Waxy maize starch ae wx sbe1a ae wx wx sbe1a wx ae wx × 1/2 Liver glycogen Shown here is a pullulanase-like DBE (ZPU1) An isoamylase-like DBE (SU1) is not shown 43 Enzymes Synthesizing Starch Starch synthases change gel from yellow to dark blue sbe1a ae wx × 1/2 sbe1a ae wx × 1/2 wx sbe1a wx ae wx sbe1a ae wx wx × 1/2 sbe1a wx × 1/2 ae wx × 1/2 sbe1a ae wx × 1/2 SS-1 SS-2 SS-3 SBEIa SS-4 SS-5 SS-6 ae wx sbe1a ae wx wx sbe1a wx ae wx × 1/2 SBEIIa SBEIIb DBE Potato starch Waxy maize starch ae wx sbe1a ae wx wx sbe1a wx ae wx × 1/2 Liver glycogen At least 5 bands are shown here, with different activities We will assign these bands to known SS isoforms 44 Mutant Starches from Maize A list of documented starch mutants of maize Single mutants Waxy (wx) Amylose-extender (ae) sbe1 sbe2a Sugary-1 (su1) Zpu1 Sugary-2 (su2) Dull-1 (du1) Brittle-1 (bt1) Brittle-2 (bt2) Shrunken-1 (sh1) Shrunken-2 (sh2) double mutants ae wx sbe1 wx ae su1 ae su2 ae du1 du1 su1 dul su2 du1 wx su1 wx su2 wx su1 su2 Triple/quadruple mutants ae du1 su1 ae du1 su2 ae du1 wx ae su1 su2 ae su1 wx ae su2 wx sbe1 ae wx du1 su1 su2 du1 su1 wx du1 su2 wx su1 su2 wx ae du1 su1 wx 45 Mutant Starches, Single Mutants Waxy (wx) Deficiency of granule-bond starch synthase Identified in maize, sorghum, rice, barley, wheat, & potato Kernels of wx are full Starch and dry weight are equal to normal Mutant wx is epistatic to other known mutants, e.g. multiple mutants containing wx has NO amylose Dosage effect shown for Wx wx wx Broad applications in the food and non-food industries 46 Mutant Starches, Single Mutants Amylose-Extender (ae) Deficiency of starch branching enzyme IIb (SBEIIb) for maize Identified in maize, rice, peas, & barley Kernels of ae have a smaller size than normal Dosage effect exists for ae gene Amylose concentration usually ranges 50-75% by blue value tests. Chromatographic analysis shows lower amylose value and the presence of intermediate materials Granules are smaller than normal, and some may be nonbirefringent B-type x-ray pattern is shown for ae starch Broad applications 47 Mutant Starches, Single Mutants Sugary-1 (su1) Deficiency of starch debranching enzyme SU1 Identified in maize, sorghum, & rice Standard sweet corn is su1 homozygous Kernels of su1 accumulates phytoglycogen >25% of kernel dry weight. Small starch granules may be isolated with amylose content possibly higher than normal Kernel development shows a pattern of: Starch granules formed → granules partially degraded and replaced by phytoglycogen → granules mostly degraded and replaced by phytoglycogen Kernels have intermediate particles ranging from amylopectin to phytoglycogen 48 Mutant Starches, Single Mutants Sugary-2 (su2) Possible deficiency of a starch synthase SSIIa Identified in maize, possible in sorghum & rice Kernel dry weight is often reduced Starch granules have extensive internal fractures Amylose content is 10-15% higher than normal. Amylopectin has increased proportion of short chains Thermal properties by DSC are related to su2 dosage 49 Mutant Starches, Single Mutants Dull-1 (du1) Deficiency of a starch synthase SSIII Kernel ranging from full size to semi-collapsed Average granule size smaller than normal Amylose content 5-10% higher than normal. Amylopectin has increased proportion of short chains Mutant du1 in sweet corn background may accumulate phytoglycogen 50 Mutant Starches, Multiple Mutants Amylose-Extender Waxy (ae wx) Deficiency of both SBEIIb and GBSSI Reduced in size, dry weight, and starch content (~50%) Blue value test indicates 15-26% amylose, but chromatographic separation indicates solely amylopectin Both ae and wx functions independently Amylopectin has increased proportion of long chains Dosage effect exists for both ae and wx genes 51 Mutant Starches, Multiple Mutants Amylose-Extender Sugary-1 (ae su1) Deficiency of both SBEIIb and SU1 Kernels not as full as ae, but fuller than su1. Weight and starch concentration are lower than normal Increased doses of ae results in reduced phytoglycogen Blue value test indicates 51-60% amylose content In su1, the initially formed starch granules are broken down and may be utilized to produce phytoglycogen. In ae su1, ae interferes with excessive branching and starch granules are formed along with small amount of phytoglycogen 52 Mutant Starches, Multiple Mutants Dull-1 Waxy (du1 wx) Deficiency of both SSIII and GBSSI Kernels weight similar to du1 and wx, slightly less than normal Sugar concentrations are higher, and starch concentration is lower than in either normal, du1, or wx Gene wx is epistatic to du1, so du1 wx has 100% of amylopectin Amylopectin has increased portion of shorter chains 53 Mutant Starches, Multiple Mutants Amylose-Extender Dull-1 Waxy (ae du1 wx) Deficiency of SBEIIb, SSIII, and GBSSI Starch concentration is low compared with the component single and double mutants. Sugar contents are several fold higher. Sweetness is between standard sweet corn (su1) and sh2 mutation. Amylopectin branching is different from either ae wx or du1 wx, and somewhat intermediate between wx and du1 wx 54 Mutant Starches, Multiple Mutants Amylopectin of mutants containing ae, wx, and du1 Chain length profile of amylopectin DEGREE of POLYMERIZATION Chain length profile of β-limit dextrin DEGREE of POLYMERIZATION 1000 100 50 20 10 3 70 NORMALIZED D.R.I. 60 NORMALIZED D.R.I. 1000 100 50 20 10 3 60 50 40 30 20 10 0 10 12 14 16 18 20 RETENTION TIME, min 50 40 30 20 10 0 10 12 14 16 18 20 RETENTION TIME, min wx du1 wx ae wx du1 ae wx 55 Mutant Starches, the Potential Potential of mutant combinations Single mutants Waxy (wx) Amylose-extender (ae) sbe1 sbe2a Sugary-1 (su1) Zpu1 Sugary-2 (su2) Dull-1 (du1) Brittle-1 (bt1) Brittle-2 (bt2) Shrunken-1 (sh1) Shrunken-2 (sh2) double mutants ae wx sbe1 wx ae su1 ae su2 ae du1 du1 su1 dul su2 du1 wx su1 wx su2 wx su1 su2 Triple/quadruple mutants ae du1 su1 ae du1 su2 ae du1 wx ae su1 su2 ae su1 wx ae su2 wx sbe1 ae wx du1 su1 su2 du1 su1 wx du1 su2 wx su1 su2 wx ae du1 su1 wx 56 Mutant Starches, Patents A list of mutant starch patents in last 20 years 2004, Method of grain production for heterozygous waxy sugary-2 maize 1996, Foodstuffs containing a waxy waxy amylose extender starch 1992, ae du1 batter starch for deep fat fried food 1989, Foodstuffs containing starch of a waxy shrunken-2 genotype 1989, Foodstuffs containing starch of an amylose extender sugary-2 genotype 1988, Food stuffs containing starch of a dull sugary-2 genotype 1988, Food stuffs containing starch of an amylose extender dull genotype 1988, Food stuffs containing starch of a dull waxy genotype 1988, Starch of the duh genotype and products produced therefrom 1988, Starch of wx fl1 genotype and products produced therefrom 1988, Starch of the wx sh1 genotype and products produced therefrom 1986, Bread containing wx su2 genotype starch as an anti-stalent 57 Mutant Starches, Patents An example: Double Mutant Homozygous wx Heterozygous su2 Freeze-thaw stability of different starches (US patent # 6,828,474) Starch A B waxy 2 doses Opacity 3 2