Chapter 20 Engineering Plant Quality and Proteins

1. Chapter 20Engineering Plant Quality and Proteins 呂維茗 (Wei-Ming Leu) wmleu@nchu.edu.tw 22840328, ext. 767 食品暨生物科技大樓, 704室

2. Modification of plant nutritional content Amino acids Lipids Vitamins Iron Phosphorus • Modification of food plant taste and appearance Preventing discoloration Sweetness Starch • (Genetic manipulation of flower pigmentation) • Plants as bioreactors Antibodies Polymers Edible vaccines • Plant yield Increasing iron content Altering lignin content (Erect leaves) Increasing oxygen content • phytoremediation

3. Modification of plant nutritional content Amino Acids • Want increase amount of essential amino acids • Eight amino acids are generally regarded as essential amino acid for humans: phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, leucine, and lysine. Additionally, cysteine (or sulphur-containing amino acids), tyrosine (or aromatic amino acids), histidine and arginine are required by infants and growing children. • cereal seeds: deficient in lysine and tryptophan • legume seeds: deficient in methionine and cysteine (S-amino acids)

4. Lupin • 1. By expressing storage protein from other plants • Lupine in Australia (>800,000 tons)- deficient in methionine and cycteine, engineered to produce sunflower seed albumin, which not only rich in sulfur-containing aa but also can escape microbial breakdown in the rumen.

5. Figure 20.2 • 2. by deregulating lysine biosynthesis pathway • Take Lysine feedback-insensitive gene for AK and DHDPS from bacteria • Add transit peptide • Transform maize • Free lysine: ~100 X • Total lysine in seed: 2~5X

6. Lipids • Oil body in formed from ER in plants • Oil body: not an organelle, are constituted by a lot of TAG covered by oleosin proteins Oil body TAG oleosin

7. 12 1 13 • TAG (triacylglycerol)= glycerol + 3 fatty acid * * * * All in “cis” form

8. Four major oil crops account for ~80% of world wide vegetable-oil production : soybean, sunflower, oil palm and rapeseed (canola). • Canola (油菜) is closely related to Arabidopsis (阿拉伯芥，擬南芥) and can be transformed easily, therefore form “Canola Technology” • Different from essential aa, essential FAs are not the target for oil engineering. (essential FAs: linoleic acid, C18:2; linolenic acid, C18:3, Arachidonic acid, C20:4, as mammals lack the enzymes to insert double bonds at carbon atoms beyond C-9 in the fatty acid chain ) • Instead, novel oil composition, higher oil content, more stable oil, oil healthier for human, etc.,are the targets

9. Healthier edible oil-1 • 1. low in saturated fats • 2. high oleic acid (as oleic acid lowers LDLs without affecting HDLs) • 3. high omega-3 fatty acid (include two types, one is plant ALA, alpha-linolenic acid, the other is marine EPA and DHA) • Canola (Canada oil low erucic acid): been bred for 40 years, erucic acid decreased from 40% to <1%, receive award on 1989.(table 20.2)

10. Healthier edible oil-2

11. More omega-3 and omega-6 fatty acid-1 • Mainly from marine and fish oils currently • Linoleic and a-linolenic acid can be the C-18 precursors of the long chain omega-3 and omega-6 fatty acids • Test metabolic engineering in Arabidopsis (fig. 20.4), find that plant growth is fine. Need to try soybean or canola with seed-specific expressions.

12. Figure 20.4 • More omega-3 and omega-6 fatty acid-2 omega-6 fatty acid omega-3 fatty acid ~7% ~3%

13. Different modified oil • Increase stearic acid content by antisense of the desaturase gene • Generate FA with conjugated double bond (so the oxidation rate is increased, suitable for use as drying agents in paints and inks)

14. Vitamins • Vitamin E – human require 400 IU or 250 mg daily • Seeds extract contain high level of total tocopherols but not -tocopherol, which require –tocopherol methyltransferase (-TMT) to convert. (Fig. 20.5) Figure 20.5

15. A long story of looking for -tocopherol methyltransferase gene • Finally obtain clone from Arabidopsis. (Fig. 20.6.) • Express in corn embryo or soybean seeds. Figure 20.6

16. Figure 20.7 • Vitamin A • Engineer rice to produce provitamin A (-carotene), normally found in plant photosynthetic membranes. • Require three genes, all fused with transit peptide (Fig. 20.4) • Golden rice 1 (2000): only 1.6 ug -carotene per gram of rice • Golden rice 2 (2005): 23-fold higher (replace daffodil phytoene synthease with the corn version). • 150 g rice daily is enough • Mistrust of GMO • Need to introduce to Indica rice by breeding (mid-2008, IRRI start the first field trial of Indica variety) Daffodil psy gene Bacterial crt gene Daffodil lcy gene Endogenous human gene

17. Figure 20.8 • Folate (tetrahydrofolate, or vitamin B9)- 400 g daily • Rice is poor in folate providing • Fig. 20.8 (* indicate rate-limiting steps) • A tripartite from: two parts from chloroplast and cytosol, respectively, and assemble in mitochondria. • Two genes from Arabidopsis, transform rice or tomato. • ~100X increase

18. Metallothionein (cys-rich) phytase Figure 20.10 Iron • 30% of the world’s population, especially vegetable-based, have iron deficiency • Phytate often prevent the absorption of iron from plant • Ferritin • 24 monomeric ferritin contain 4500 iron atoms in its central cavity • Take ferritin gene from soybean, transform rice, increase iron content to 2.5X in seeds, therefore 150 g rice can provide 30~50% of the daily necessity. • However, to improve the bioavailability of iron, two more genes are introduced. (Fig. 20.10, all controlled by endoseperm-specific promoter)

19. Figure 20.11 Phosphorus • Phytate (phytic acid,植酸，or inositol hexaphosphate), deposit in the protein storage vacuoles, can’t be used by nonruminant animals. • Express phytase at low level

20. tuber-specific tuber-specific Figure 20.12 • Modification of food plant taste and appearance Preventing discoloration • Polyphenol oxidase- oxidize monophenols and o-diphenols to o-quinones, 59 kDa, localized in chloroplast and mitochondrial membranes.

21. Sweetness • Fructans (果聚醣) • Polymers of fructose not degraded by human digestive tract but can feed beneficial bacteria. • Small fructans (<6 mer) have a sweet taste. • Expensive: usually produced by fungal invertase from sucrose or extracted from the root of chicory or Jerusalem antichoke tubers. • Transform sugar beet with 1-sucrose:sucrose fructosyl transferase gene from Jerusalem antichoke, accumulate fructan to ~40% dry wt.

22. Monellin: 3000 X sweeter than sucrose in wt basis • A dimer contain A (45 aa) and B (50 aa) which is easily dissociated by heat or acid. • Chemically synthesize a gene contain both parts, transform tomato and lettuce under controlled by fruit-specific promoter E8. Figure 20.13

23. Other sweet proteins

24. Starch • Ratio of amylose(A) and amylopectin(B) have profound effect on the physical and chemical property of the starch (A) (B) Figure 20.14

25. Starch synthesis pathway Starch synthase Starch branching enzyme Figure 20.15

26. Generate high amylose potato to produce fructose • Antisense the starch branching enzyme • amylose increase from 28% to ~80%) • 2. Overexpress a bifunctional enzyme- -amylase/glucose isomerase (Fig. 20.16) • Only effective at high temperature (65C), glucose and fructose were increased to 3.9 and 14.7-fold, respectively. Figure 20.16

27. Increase the total amount of starch Figure 20.17 • Increase supply of ATP by decrease adenylate kinase expressions in chloroplast (Fig. 20.17) • Both the yield of potato tubers and amount of starch were increased significantly. (table 20.5) * * *

28. Genetic manipulation of flower pigmentation • Major cut flower: • - ~70% are roses, carnations, tulips, chrysanthemums • - transformation protocols established • Species such as rose, tulip, and carnation areNOT naturally blue as they lack the "enzymatic machinery" to synthesize blue colored pigments. • Blue chrysanthemum?? • Floral pigments – anthocyanins (花青素，water-soluble) in vacuole and carotenoids (類胡蘿蔔素，water-insoluble) in chloroplast or chromoplast.

29. Biosynthesis of anthocyanin (花青素) 1. CHS 1. CHI • All are colorless before DFR!!! • The more hydroxyl group, the more blue. 2. F3H (DHK) 2. F3'5'H 2. F3'H • Two genes are responsible for producing the blue pigment, delphinidin, in the vacuole of petunia cells. 3. DFR, DFGT 3. DFR, DFGT delphinidin (blue) pelargonidin (brick red) cyanidin (red)

30. Engineering of blue rose 3. Iris DFR gene from Suntory 2 1 1 3 • Turn off the rose DFR gene • Insert pansy F3’5’hydroxylase gene to open the blue door • Insert iris DFR gene to make blue pigment 2. Pansy F3'5'H gene

31. Product of blue carnation FLORIGENE MOONSERIES Florigene Moonshadow™ Florigene Moondust™ Florigene Moonvista™ Florigene Moonshade™ Florigene Moonlite™ Florigene Moonaqua™

32. Why not blue, but purple? • The delphinidin pigment (blue pigment) acts very much like litmus paper(石蕊試紙)—in the alkaline vacuolar environment of the petunia, delphinidin is blue, but in the acidic environment of the rose vacuole, it is pink. • The Florigene company had better luck with carnations, because their petals run to a higher pH (~5.5). • Blue shades should be achievable if researchers can make the rose's petals (pH ~4.5) less acidic.

33. Plants as bioreactors • advantages • No mammalian virus contamination • Produced in seeds for storage concerns • disadvantages • Long processing time: ~2 years from construct to small scale production • Very low expression level (0.0001%~0.001%) • High in purification cost

34. Potential therapeutic proteins

35. Potential vaccine antigens

36. Edible vaccines (oral vaccine) • Test in potato first but may choose banana (although its tree require several years to mature), tomato (although spoil readily), lettuce, carrot, peanut, corn (for vaccinating animals), etc. to develop. • Use cholera toxin B subunit as adjuvant to stimulate immune response. • Some antigen can reach GALT M cell intestine epidermis GALT (Gut-Associated Lymphoid Tissue) Figure 20.22

37. Two fusion antigen • Prevent three pathogen infections- rotaviurs, E. coli, and Vitrio cholera Figure 20.23 Figure 20.24 • Shiga Toxin (志賀毒素) • AB5

38. Antibodies (plantibodies) (for use on mucosal surface)

39. Plantibodies • Produced by viral vectors • No viral coat protein, so no virus particle • High expression level: 0.5 mg/g of fresh leaf biomass Figure 20.21

40. Biodegradable Polymers (PHA, poly (3-hydroxybutyric acid)) • Bacterial fermentation production of biodegradable polymer is expensive • Three genes are required for PHA synthersis. • Send into chloroplast as there are abundant acetyl-CoA • Reach 1 mg/g of PHA. However, plants are stunt. Figure 13.36

41. Plant yield Altering lignin content • Lignin- the second most abundant organic compound on earth (15~35% dry wt. of wood) • Impede pulp and paper industry, decrease efficiency for producing renewable biofuels (Fig. 20.27) • Decrease nutritional value of forage crops • Lginin biosynthesis is highly conserved across plant kingdom • Antisense construct for 4-coumarate: coenzyme A ligase (Fig. 20.26) • The transgenic tree have larger leaves and thicker stems • Transformed aspen has ~45% decrease in lignin content and 15% increase in cellulose.

42. Figure 20.27

43. Figure 20.26

44. Figure 20.25 Increasing iron content • In alkaline soil, iron is largely present as insoluble ferric hydroxides. • Siderophores: small iron-binding molecules (three bidentate bind one trivalent ferric iron). • Plant siderophores (e.g. mugineic acid) is synthesized from L-methionine (Fig. 20.25) • Overexpresion of Nicotianamine aminotransferase (have feed back inhibition control as its own promoter was used)

45. Siderophores

46. Increasing oxygen content • Oxygen is an essential substrate for plant respiratory metabolism. • A dimeric hemoglobin from G(-) Vitreoscilla can increase bacterium cell density or 80~100% plant dry wt when overexpressed..

47. Phytoremediation • Def: Use of plants to remove, destroy, or sequester hazardous substrates from the environment. • Phytoextraction-absorption and concentration of metals from the soil into the plant • Rhizofiltration- use plant root to remove metals from effluents • Phytostabilization- use of plants to reduce the spread of metals in the environment • Phytovolatilization- uptake and release into the atmosphere of volatile materials, such as mercury- or arsenic-containing compound. • Hyperaccumulators- not useful yet

48. Figure 20.31 • Remove lead and cadmium from soil • Accumulate in either vacuoles or cell walls (less harmful to plant) • Require two transporters • e.g. use yeast YCF1 (an ABC transporter), although expressed at very low level, can effectively sequester lead and cadmium to 2~4 fold.

49. organomercural lyase Figure 20.32 mercuric ion reductase • Remove organic mercury from soil • Organic forms of mercury, especially methyl mercury, are highly toxic to both plants and animals. • Obtain genes from mercury-resistant bacteria (Fig. 20.32) • Transform chloroplast of tobacco (Fig. 20.33) Figure 20.33