1. Biosynthesis of Plant-derived flavor compounds By Dudsadee Uttapap

2. References 1. “Flavor Chemistry and Technology”, H.B. Heath, G. Reineccius, 1986. 2. “Flavor Chemistry”, D.B. Min, http://class.fst.ohio-state.edu/fst820/default.htm 3.“Biosynthesis of plant-derived flavor compounds”, The Plant Journal (2008) 54, 712–732 4.“Plant Biochemistry” http://www.uky.edu/~dhild/biochem/lecture.html Biosynthesis of plant-derived flavor compounds

3. Flavor compounds Flavor molecules constitute a heterogeneous group of compounds, with straight-chain, branched-chain, aromatic and heteroaromatic backbones bearing diverse chemical groups such as hydroxyl, carbonyl, carboxyl, ester, lactone, amine, and thiol functions. More than 700 flavor chemicals have been identified and catalogued

4. Chemical synthesis VS Biosynthesis Most commercial flavorants are ‘nature identical’, which means that they are the chemical equivalent of natural flavors but are chemically synthesized, mostly from petroleum-derived precursors Bioproduction, including the extraction from natural sources, de novo microbial processes (fermentation), and bioconversion of natural precursors using micro-organisms or isolated enzymes

5. Biological functions of plant volatiles Compounds emitted by flowers most probably serve to attract and guide pollinators volatiles might also protect the carbohydrate-rich nectar by inhibiting microbial growth. vegetative plant tissue release volatiles following herbivore damage. Some of these substances attract arthropods that prey upon or parasitize the herbivores. Volatiles also act as direct repellents or toxicants for herbivores and pathogens. In fruits, volatile emission and accumulation facilitate seed dispersal by animals and insects. vegetative tissues often produce and release many of the volatiles after their cells are disrupted. These volatile flavor compounds may exhibit anti-microbial activity. “associated with defensive and attractive roles”

6. Aromatic compounds responsible for odor and flavor of fruits comprise; Alcohols Carbonyls Acids Esters Lactones Phenols R-OH R- CHO R-CO-R’ R-COOH R-COO-R’ R OCOR OCO

7. Estimated world consumption of selected aroma chemicals in flavor and fragrance compositions

10. Calvin cycle

11. N enters roots as NO3- or NH4+. The NH4+ is incorporated into amino acids in roots and leaves and the amino acids accumulate in proteins. The main if not sole function of some proteins is to provide a store of amino acids Amino acid synthesis

16. Glycolysis

17. isoprenoid biosynthesis proceeds either via the "classical" or most well studied, mevalonate pathway (cytosolic) (for the synthesis of sterols, sesquiterpenes, triterpenoids) or via the non-mevalonate (1-deoxy-D-xylulose-5- phosphate, DXP) pathway for plastidic isoprenoids (carotenoids, phytol [side-chain of chlorophylls], plastoquinone, isoprene, monoterpenes and diterpenes).

20. Biosynthesis of flavors in vegetables and fruits develop when tissue damage occurs (Intact vegetable generally contains few volatiles) Vegetable flavors are formed during brief ripening period Fruit flavors

21. Minute quantities of lipids, CHO, protein (amino acids) are enzymatically converted to volatile flavors. BIOGENESIS OF FRUIT AROMA develops entirely during ripening period of plant

22. FRUIT FLAVOR COMPOUNDS Apple n-hexanal, ethyl butyrate, 1-propyl propionate, 1-butyl acetate, trans-2-hexenal, ethyl 2- methylbutyrate, 2-methylbutyl acetate, 1- hexanol, hexen-1-ol, trans-2-hexen-1-ol, hexyl acetate, Esters; alcohols; aldehydes; ketone; acids; including hexanal; ethyl 2-methyl butyrate Banana alcohols; esters, including amyl acetate, isoamyl acetate, butyl butyrate, amyl butyrate Peach Ethyl acetate, dimethyl disulfide, cis-3-hexenyl acetate, methyl octanoate, ethyl octanoate, 6- pentyl alpha pyrone, gamma decalactone

23. Biosynthesis of fruit volatiles Carbohyd rate Amino acid Cinnamic acid Terpe ne Fatty acid Acetyl- CoA Malonyl CoA Acetyl CoA Pyruva te Mevalonyl CoA Shikimic acid

24. Flavorants from carbohydrate metabolism Furanones and pyrones “fruit constituents” Only a limited number of natural volatiles originate directly from carbohydrates without prior degradation of the carbon skeleton.

25. Furanones and pyrones Carbohydrate-derived flavor molecules, including 4-hydroxy-2,5-dimethyl-3(2H)- furanone (furaneol), 2,5-dimethyl-4-methoxy-3(2H)-furanone (methoxyfuraneol), 4- hydroxy-5-methyl-3(2H)-furanone (norfuraneol), 2-ethyl-4-hydroxy-5-methyl-3(2H)- furanone (homofuraneol), 4-hydroxy-2-methylene-5-methyl-3(2H)- furanone (HMMF) and 3-hydroxy-2-methyl-4H-pyran-4-on (maltol).

26. Glycolysis Glucose ( 6 C) 2 Pyruvate ( 3 C) EthanolLactate TCA Cycle CO 2 +O 2 -O 2 Flavorants from carbohydrate metabolism

28. “the most interesting is terpene biosynthesis” Terpenoids are enzymatically synthesized from acetyl CoA and pyruvate provided by the carbohydrate pools in plastids and the cytoplasm. Terpenoids constitute one of the most diverse families of natural products, with over 40 000 different structures of terpenoids Many of the terpenoids produced are non-volatile and are involved in important plant processes such as membrane structure (sterols), photosynthesis (chlorophyll side chains, carotenoids), redox chemistry (quinones) and growth regulation (gibberellins, abscisic acid, brassinosteroids) Flavorants from carbohydrate metabolism

29. Important plant-derived volatile terpenoids.

30. Biosynthesis of Terpenes “isoprene is derived from acetyl-CoA”

31. Classification of Terpenes

32. Apocarotenoid formation Carotenoid substrates are oxidatively cleaved to yield the apocarotenoid derivatives (right).

33. Some of the volatile organic compounds in wine come from the grape's skin, or exocarp, while others come from the grape's flesh, or mesocarp. Organic acids give wine its tartness, and sugars give it sweetness. Terpenes provide floral or fruity flavors. Norisoprenoids impart a honeylike character. Thiols are the sulfur-based compounds behind complex wine aromas such as guava, passionfruit or grapefruit — but when thiols go wrong, they can make a wine taste "funky."

34. products; acids, alcohols, diketones, ketones, esters of these compounds. Lipids metabolic pathway for lipid biosynthesis plays a significant role in flavor formation. Alpha-, Beta-oxidation Oxidation via lipoxygenase

35. Lipoxygenase activity is believed to be the major source of volatiles in plants. Oxidation via Lipoxygenase Major products: volatile C6 and C9 aldehydes and alcohols Substrate: unsaturated fatty acid (linoleic and linolenic acids). Lipoxygenase enzymes (dioxygenase) catalyze reactions between O 2 and polyunsaturated fatty acids

36. Linolenic acid-derived flavor molecules. AAT, alcohol acyl CoA transferase; ADH, alcohol dehydrogenase; AER, alkenal oxidoreductase; AOC, allene oxide cyclase; AOS, allene oxide synthase; HPL, hydroperoxide lyase; JMT, jasmonate methyltransferase; LOX, lipoxygenase; OPR, 12- oxo-phytodienoic acid reductase; 3Z,2E-EI, 3Z,2E-enal isomerase.

37. Fatty acid precursors (Tomato)

38.  - and  -oxidation of fatty acids Palmitoyl-CoA (16:0) Myristoyl-CoA (14:0) + Acetyl-CoA the specific pathways in plants are not well understood

39. Formation of pear flavors via beta-oxidation

40. Lactones

41. Amino Acid Metabolism Amino acid metabolism yields short chain aliphatic and aromatic alcohols, acids, carbonyls and esters They are the primary source of branched chain aliphatic flavor compounds their pathways have been barely analyzed in plants.

43. amino acid precursors (Tomato)

44. (a) Catabolism of branched-chain amino acids leading to methyl branched flavor compounds, and (b) postulated biosynthesis of sotolon. Formation of aldehyde (a) from amino acids requires the removal of both carboxyl and amino groups. The sequence of these removals is not fully known and could be the opposite to that shown or aldehyde could be formed in one step by aldehyde synthase Biosynthesis of amino acid-derived flavor compounds

45. Starting amino acids: Tyrosine and phenylalanine products: phenolic/spicy in character

46. Shikimic acid formation

47. Vegetable Flavors

48. Vegetable flavors flavor again arises from major metabolic processes - e.g. Lipids, CHO & amino acids. The role or importance of S compounds to vegetable flavor is quite significant. the precursors, enzymes and end flavors are quite different from fruits.

49. Carbohydrate Fatty acidAmino acid Formation of flavor in vegetables

50. Vegetable Flavor Categories Genus Allium Enzymes produce volatiles from derivatives of cysteine (sulfoxides) Genus Brassica Enzymes produce volatiles from glucosinolates

51. Alliaceous vegetables garlic (Allium sativum L.) onion (Allium cepa L.) chive (Allium schoenoprasum L.) leek (Allium porrum L.)

52. Characteristic flavors not exist in the bulb before processing are produced when the cellular tissues are ruptured by cutting or chewing flavor is produced very rapidly by the action of an enzyme on the odorless precursors which coexist in the cells

53. Onion and Garlic Flavor Enzymatic reaction of cysteine derivative

56. Glucosinolate precursors are important to the flavor of both the Brassica and Cruciferae family Cruciferae family includes radish, horseradish, mustard. GLUCOSINOLATES

57. thiocyanate, nitrile, or isothiocyanate & glucose Hydrolysis of the glucosinolate glucosinolate thioglucosidase

60. Natural carbon pools for the production of flavor compounds, and the pathways

67. “the most interesting is terpene biosynthesis” most of essential oils get flavor from terpenoids (10 carbon) Limonene - a monoterpene hydrocarbon - is the major terpene in many or most citrus products. Orange > 95% of the essential oil is limonene, lemon ~ 65% limonene, yet is of little flavor significance. Citral - oxygenated monoterpene - seldom comprises > 2% of the essential oil of lemon - largely carries the lemon flavor. Flavorants from carbohydrate metabolism

68. (n = total number of carbon atoms) 1 Methane 11 Undecane 21 Henicosane 31 Hentriacontane 2 Ethane 12 Dodecane 22 Docosane 32 Dotriacontane 3 Propane 13 Tridecane 23 Tricosane 33 Tritriacontane 4 Butane 14 Tetradecane 24 Tetracosane 40 Tetracontane 5 Pentane 15 Pentadecane 25 Pentacosane 50 Pentacontane 6 Hexane 16 Hexadecane 26 Hexacosane 60 Hexacontane 7 Heptane 17 Heptadecane 27 Heptacosane 70 Heptacontane 8 Octane 18 Octadecane 28 Octacosane 80 Octacontane 9 Nonane 19 Nonadecane 29 Nonacosane 90 Nonacontane 10 Decane 20 Icosane 20 Icosane 30 Triacontane 100 Hectane

69. Isoamyl acetate, a strong fruity odor described as similar to banana or pear 2-Methyl-butyl acetate has a strong apple scent