Secondary_plant_products.pptx

Secondary Plant Products and
Metabolic Engineering
PB522

PB522 - Secondary Plant Products and
Metabolic Engineering
● Unit 1: Introduction to secondary plant products
● Unit 2: Sub-classification, structures and biosynthesis of secondary
plant products:
● Unit 3: Evolution of secondary pathways,
● Unit 4: Plant poisons
● Unit 5: Polyamines and non-protein aminoacids
● Unit 6: Biotechnological application of secondary plant products
● Unit 7: Pathway Engineering: Principles and case studies

Unit 3: Evolution of secondary pathways, occurrence,
biological functions of:
● Flavor and fragrance substances
● Volatiles
● Colourants
● Medicinal compounds
● Herbal products
● Insecticidal compounds (biopesticides)
● Non-sacchariferous sweeteners
Industrial applications of plant secondary metabolites
and herbal products.

https://foodadditives.net/colors/caramel-color/
E338, phosphoric acid

Red Dye 40 is a synthetic color additive or food dye made from petroleum. It's one of the nine certified color additives
approved by the Food and Drug Administration (FDA) for use in foods and beverages
Yellow 6 is made synthetically from petroleum.
Yellow 5: Tartrazine, also referred to as FD&C yellow #5, is an artificial (synthetic) food dye. It is one of several azo
food dyes that are made from petroleum products.
Blue No. 1 is called "brilliant blue" and, as is typical of modern dyes, was originally derived from coal tar, although
most manufacturers now make it from an oil base.

https://slidetodoc.com/unit-10-chemistry-of-flavor-odour-and-taste/
Methyl salicylate · Gaultheria procumbens · Gaultheria

Flavour and fragrance substances
What is the difference between fragrance
and flavour ?
The main difference between flavour and
fragrance is that flavour is felt with the
tongue whereas fragrance is felt with
the nose.
However, flavour and fragrance cannot be
considered as separate entities since the
flavour is often a result of both taste
and smell.
Thus, fragrance has a major influence on
flavour
https://pediaa.com/difference-between-flavour-and-fragrance/

Flavour
Food Flavours are classified into three major
categories:-
1. Natural Flavours
2. Processed Flavour
3. Added Flavour
https://hmhub.in/classification-food-flavours-uses/
https://www.foodprocessing.com.au/content/training-education/article/flavour-definitions-and-
classifications-1166826690

Natural Flavours
● Herbs- Basil, mint
● Spices- Cardamom, clove, turmeric
● Aromatic Seeds- Aniseed, Cumin
● Fruits- Orange, Lemon
● Vegetables- Peas, Onions, Garlic

Processed Flavour
● Caramelized
● Roasted
● Fermented
● Toasted
● Baked

Added Flavour
Natural Extracted Flavour
● Essential Oil
● Essence
● Extracts
Synthetic Flavour
● Fruit Flavour
● Savoury Flavour

Fragrance
Fragrance wheel -
invented in 1983 by
Michael Edward,
British fragrance
expert.
http://www.historyofperfume.net/perfume-facts/perfume-classification-and-fragrance-notes/
https://scentbeauty.com/blogs/scent-101/scent-101-fragrance-families-explained

Flavours set up a great platform in food
industry. Without flavours, consumer's
avidity on food will be gone.
Foodstuffs containing artificial and
synthetic flavours are mostly avoided,
because the consumers doubt that these
flavour producing components are toxic to
their health

Most of the food flavouring compounds are
produced by chemical synthesis or extracted
from natural resources. However, recent
market surveys have analyzed that consumers
also prefer foodstuff that should be labelled
''natural".
Natural aromas produced by microorganisms
are finally recognized as natural and are safe

Furthermore, chemically synthesized flavours
will often result in environmentally unfriendly
production processes will also cut the
substrate selectivity, which may also result in
formation of undesirable by-products, thus
reducing process yield
and increasing the downstream costs.
On the other hand, the producing flavours by
direct extraction from plants is also subjected
to various problems

These raw materials often has low
concentrations of the desired compounds,
making the extraction even more expensive.
These disadvantages of both methods and
the establishing interest in natural products
have to manage many investigations
towards the search for other strategies to
produce natural flavours.

Nowadays, many researchers and industries
have switched to bio-catalytic flavour
synthesis due to consumer's inclination
towards natural flavours over chemical ones.
These reactions use very mild operating
techniques, have high specificity with
reducing the side reactions and produces
good purity of flavoured compounds by
avoiding the more expensive separation
techniques

In view of the emerging concept of
biological production of natural flavours, the
term 'natural' is explained in the USA as well
as in Europe.

In USA, a distinct difference is made between natural and
artificial flavour compounds and according to the 'Code of
Federal Regulations' (1990), the 'natural flavour' means the
● essential oil
● essence or extractive
● oleoresin
● protein hydrolyzate
● distillate of any product of roasting, heating
which has flavouring constituents derived from a,
● A species fruit juices
● edible yeast
● herb
● vegetable or
● vegetable juice, parts of plants like (bud, bark, root, leaf) or
similar plant material

● meat,
● poultry,
● seafood,
● eggs,
● dairy products or
● fermentation products thereof,
whose noteworthy functions of food is imparting flavours and
not nutrition.

Although the biocatalytic approaches to
these compounds are often expensive,
different applications have been described
Eco-friendly conditions and high chemical
selectivity make biocatalytic approaches
attractive.

In using biocatalytic approaches, two major
scenarios emerge:
1. Industrial production of flavouring
compounds
1. Academic synthesis of selected flavours
(synthesis not used for industrial
production but mainly for scientific
interest).

Few applications are related to the first case in which
isolated enzymes, fermentation products, bio-
transformations are mainly used.
Lipases is the most liked catalyst because they show
remarkable chemo-selectivity, regio-selectivity and
enantio-selectivity.
Moreover, they are easily available on a large scale
and remain active in organic solvent

Yeast alcohol
dehydrogenase
ol
cherry and almond-tasting
benzaldehyde
Cyanogenic Glycoside
L-Menthol
Menthylesters Menthyl acetate

Flavour
Flavour is defined as the combination of
taste and odor. It is, however, influenced by
other sensations such as pain, heat, cold
and tactile sensations, often referred to as
the ‘texture’ of foods.
Flavour compounds that primarily impart
smell, although several of them might also
interact with taste receptors.

Odorants are volatile chemical compounds that are
carried by inhaled air to the olfactory epithelium
located in the nasal cavities of the human nose. The
odorant must possess certain molecular properties
in order to produce a sensory impression.
It must have a certain degree of lipophilicity and
sufficiently high vapor pressure so it can be
transported to the olfactory system,
some water solubility to permeate the thin layer of
mucus, and must occur at a sufficiently high
concentration to be able to interact with one
or more of the olfactory receptors

The knowledge and use of plants as flavoring and
seasoning to enhance the quality of foods, beverages
and drugs is as old as the history of mankind.
Plants used as spices and condiments are usually
aromatic and pungent owing to the presence of
varying types of essential oils. In addition, people
have also used perfume oils and unguents made
from plants on their bodies for thousands of years in
lesser or greater amounts dependent on fashion
whims.
The first perfumes were all natural.

In the 19th century, the commercialization of
flavors and fragrances on an industrial scale
started with the isolation of single chemicals
responsible for the characteristic aroma of natural
products
E.g. cinnamaldehyde isolated from cinnamon oil
and benzaldehyde from bitter almond oil at a time
that is characterized by significant technological
breakthroughs, largely in chemistry.

Today (2008), the total market for flavors and
fragrances is estimated at USD 18 billion, with
market shares between the flavor and fragrance
businesses being almost equal.
The global flavors and fragrances market size
was valued at USD 25.89 billion in 2021. The
market is projected to grow from USD 26.54
billion in 2022 to USD 36.49 billion by 2029,
exhibiting a CAGR of 4.7% during the forecast
period.

The largest markets are in Europe
(36%) and North America (32%),
followed by the Asian Pacific
region (26%).
Eight major global companies
share 60% of the world market.
The flavor and fragrance industry
is a composite of four closely
interrelated and overlapping
business sectors
(Figure 1).

Essential oils and other natural extracts are
usually defined as aromatic materials obtained from
botanical or animal sources by distillation, cold pressing,
solvent extraction or maceration.
Essential oils represent complex aroma mixtures of
potentially hundreds of chemical constituents. Aroma
chemicals are organic compounds with a defined chemical
structure.
They are produced by organic or biocatalytic synthesis or
isolated from microbial fermentations, plants or animal
sources, and are used to compound flavours and fragrances.

Formulated flavors are used by the food and
beverage, tobacco and pharmaceutical industries.
Formulated fragrances are used to give pleasant
scents to fine fragrances, personal care and
household products

Selected volatile substances even reach
annual consumption rates of more than
5000 tonnes (Table 2).
About 40% of the fragrance chemicals are
also used in making flavors, but 80% of the
global consumption of vanillin, menthol,
eugenol, limonene, and esters of lower
alcohols and lower fatty acids is used in
making flavors.

Today, due to the high cost or lack of availability of
natural flavor extracts, 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

Because chemical synthesis often uses environmentally
unfriendly production processes such as heavy metal
catalysts, and crude oil represents a limited source, it is
desirable to switch to bioproduction, including the
extraction from natural sources, de novo microbial
processes (fermentation), and bioconversion of natural
precursors using microorganisms or isolated enzymes.

Why do plants produce volatile
compounds ?

Biological functions of plant volatiles ?
● Essentially all plant parts such as leaves, flowers, fruits
and roots emit volatiles, which have multiple functions
that are not always solely related to their volatility
(Pichersky and Gershenzon, 2002).
● Because plant volatiles are involved in species-specific
ecological interactions and are often restricted to
specific lineages, they have been considered to be
associated with defensive and attractive roles (Pichersky
et al., 2006).
● It is believed that they are not essential for plant survival
but provide adaptive characteristics under strong
environmental selection.

● Compounds emitted by flowers most probably serve to
attract and guide pollinators, but only a few studies have
demonstrated the ability of individual substances to
attract specific pollinators (Dudareva et al., 2004).
● However, volatiles might also protect the carbohydrate-
rich nectar by inhibiting microbial growth.
● Similar to humans, it is probably the qualitative and
quantitative composition of the flavor molecules that
imparts the specific sensory impression for the pollinators
rather than the presence of a certain individual compound.
Volatiles may be a better signal at night than floral color or
shape to draw insect pollinators.

● Because volatiles show anti-microbial and anti-
herbivore activity, it is believed that they serve to
protect valuable reproductive parts of plants from
enemies.
● For example, one monoterpenol (S-linalool) and its
derivatives significantly repelled an agricultural
pest – the aphid Myzus persicae– in dual-choice
assays (Aharoni et al., 2003).
R-linalool S-linalool green peach aphid - Myzus persicae (Sulzer)

https://www.parasite-
journal.org/articles/parasite/full_html/2013/01/parasite130015/parasite130015.html

Isoprene, a ubiquitous volatile hydrocarbon, acts
to increase the tolerance of photosynthesis to
high temperature by stabilizing the thylakoid
membranes or quenching reactive oxygen species
(Dudareva et al., 2004).

A general property of vegetative plant tissue is the release of
volatiles following herbivore damage
Some of these substances have been demonstrated to serve
as indirect plant defenses through multi-trophic interactions
because they attract arthropods that prey upon or parasitize
the herbivores, thus minimizing further damage to plant
tissue.
However, volatiles also act as direct repellents or toxicants for
herbivores and pathogens, and some have the potential to
eliminate reactive oxygen species.

This also includes root-emitted volatiles, which may
function as anti-microbial or anti-herbivore
substances or
exhibit allelopathic activities that increase the
ecological competitiveness of the plant (Steeghs et
al., 2004).

Accordingly, plant volatiles can minimize the growth
suppression of epiphytic bacteria by the
phytopathogenic fungus Botrytis cinerea and thus
affect population dynamics on leaf surfaces
(Abanda-Nkpwatt et al., 2006a),
while simple alcohols emitted by leaves may provide
a carbon and energy source for epiphytic
methylotrophs (Abanda-Nkpwatt et al., 2006b).
Volatiles also attract female insects to lay eggs on
flower buds and berries (Tasin et al., 2007).

In fruits, volatile emission and accumulation have
probably evolved to facilitate seed dispersal by
animals and insects.
For humans, volatiles in fruits have a considerable
economic impact, as parameters of food quality
and consumer preference.

The function of fruit volatiles as a signal of ripeness
and as an attractant for seed-dispersing organisms
is supported by the fact that some substances are
specifically formed by ripe fruits but are absent in
vegetative tissues and non-ripe fruit.

Unlike ripe fruits and flowers, vegetative tissues
often produce and release many of the volatiles
sensed as flavors only after their cells are
disrupted.
These volatile flavor compounds may exhibit anti-
microbial activity and have anti-cancer activities but
can be toxic at high doses (Goff and Klee, 2006).

Chemical nature of flavour molecules
From the chemical perspective, 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.

Chemical nature of flavour molecules
More than 700 flavor chemicals have been identified and
catalogued (Surburg and Panten, 2005)
http://www.flavornet.org/flavornet.html 👆
Most are from various plant sources of diverse plant
families and are major constituents of essential oils. The
biosynthetic pathways of important plant volatiles have
been traced back up to intermediates of primary
metabolism (Croteau and Karp, 1991).
It has been shown that carbohydrates, fatty acids and
amino acids represent the natural carbon pools for flavor
compounds, which can also be liberated from their
polymers (Figure 2).

Biosynthesis of plant‐derived flavor compounds

Biosynthetic pathways
As many plant flavor compounds are
accumulated and biosynthesized in specialized
anatomical structures (Figure 3)

Glandular and non-glandular trichomes in Origanum dayi Post
(Lamiaceae) (a–c) and Pelargonium graveolens (Geraniaceae) (d–f).
Leaves (a, d) and light microscopy of the leaf surface (b,e) and isolated
glandular trichomes (c, f) isolated according to the procedure first
developed by Gershenzon et al. (1992).

Biosynthetic pathways
The development of techniques for isolation of the
secretory cells in such structures has proven to be of
crucial importance in our understanding of the key
biosynthetic pathways and their regulation.
Moreover, as these tissues contain many of the enzymes and
significantly express many of the genes involved in the
production of such metabolites, the isolation of secretory
cells has greatly contributed to characterization of many of
the enzymes and genes involved in the formation of many
plant natural products
E.g. peppermint, sweet and lemon basil, as well as tomato
and other crops.

Carbohydrate-derived flavor compounds
Furanones and pyrones:
Furanones and pyrones are important fruit constituents or
have been isolated from the bark and leaves of several tree
species (Schwab and Roscher, 1997).
Although hexoses and pentoses are the primary
photosynthetic products and serve as excellent flavor
precursors in the Maillard reaction,
only a limited number of natural volatiles originate directly
from carbohydrates without prior degradation of the carbon
skeleton. Eg. Furanones and pyrones.
Furan Furanone Pyrone

https://www.compoundchem.com/2015/01/27/maillardreaction/

Carbohydrate-derived flavor molecules:
● 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)
● 3-hydroxy-2-methyl-4H-pyran-4-on (maltol).

Substituted 4-hydroxy-3(2H)-furanones and the pyrone maltol
constitute an uncommon group of flavor molecules with
exceptional low odor thresholds. Furanones have been detected in
a few plant species in which they are emitted only by the fruits.
Maltol has been isolated from the bark and leaves of
Larix deciduas, Evodiopanax innovans, Cercidiphyllum japonicum
and four kinds of Pinaceae plants (Tiefel and Berger, 1993).
Katsura (Cercidiphyllaceae)
European Larch Ginseng

Incorporation experiments using labeled precursors
revealed that d-fructose-1,6-diphosphate is an efficient
biogenetic precursor of furaneol.
In strawberry (Fragaria × ananassa) and tomato
(Solanum lycopersicum), the hexose diphosphate is
converted by an as yet unknown enzyme to 4-hydroxy-
5-methyl-2-methylene-3(2H)-furanone, which serves as
the substrate for an enone oxidoreductase recently
isolated from ripe fruit.

A highly similar sequence was identified in
an EST collection for pineapple (Ananas
comosus), another species which produces
furaneol in its fruits.

In strawberry, furaneol is further metabolized by an
O-methyltransferase (FaOMT) to methoxyfuraneol.
An ortho-diphenolic structure was identified as a
common structural feature of the accepted
substrates, and is also present in the dienolic
tautomer of furaneol.
methoxyfuraneol
Genetic transformation of strawberry with the
FaOMT sequence in the antisense orientation,
under the control of a constitutive promoter,
resulted in a near total loss of methoxyfuraneol,
demonstrating the in vivo methylation of furaneol
by FaOMT.
However, the reduced level of methoxyfuraneol
was only perceived by one third of the volunteer
panelists, consistent with results obtained by
aroma extract dilution assays.

https://www.chemicalsafetyfacts.org/chemistry-context/the-sweet-chemistry-of-five-summer-fruits/

Norfuraneol and homofuraneol have been
identified in tomato and melon fruits,
respectively, but their biogenetic pathways and
that of maltol remain unknown.
However, studies in tomato and yeast have
identified phosphorylated carbohydrates as
potential precursors of the furanones.

The furanones are mutagenic to bacteria and
cause DNA damage in laboratory tests.
However, they are also very effective anti-
carcinogenic agents in the diets of animals, and
their antioxidant activity is comparable to that of
ascorbic acid .
Norfuraneol has been identified as a male
pheromone in the cockroach Eurycolis florionda
(Walker), and furaneol deters fungal growth.

Furaneol is also one of the key flavor compounds in the
attractive aroma of fruits (Farine et al., 1994). It has been
proposed that the evolved biological function of the furanones is
to act as inter-organism signal molecules in various systems.
The 4-hydroxy-3(2H)-furanones associated with fruit aromas act
to attract animals to the fruit, which ensures seed dispersal.
In the case of humans, the coincidental chemical synthesis of
these compounds in foods during preparation results in these
foods appearing particularly attractive through transferred
operation of the original signaling mechanisms (Slaughter,
1999).

Terpenoids
The terpenoids, also known as isoprenoids, are a large
and diverse class of naturally occurring organic
chemicals derived from the 5-carbon compound
isoprene, and the isoprene polymers called terpenes.
E.g.
Citral, menthol, camphor, salvinorin A in the plant Salvia
divinorum, ginkgolide and bilobalide found in Ginkgo
biloba and the cannabinoids found in cannabis. The
provitamin beta-carotene is a terpene derivative called
a carotenoid.

Terpenoid pathway.
Terpenoids are enzymatically synthesized de
novo from acetyl-CoA and pyruvate provided
by the carbohydrate pools in plastids and
the cytoplasm.
Although fatty acid oxidation is one of the
major pathways producing acetyl CoA, this
process probably does not contribute to the
formation of terpenoids as it takes place in
peroxisomes.

Peroxisomal fatty acid
oxidation
Although most fatty acid oxidation takes place in mitochondria,
some oxidation takes place in cellular organelles called
peroxisomes.
These organelles are characterized by high concentrations of
the enzyme catalase, which catalyzes the dismutation of
hydrogen peroxide into water and molecular oxygen.
Fatty acid oxidation in these organelles, which halts at octanyl
CoA, may serve to shorten long chains to make them better
substrates of β oxidation in mitochondria.

Terpenoids constitute one of the most diverse
families of natural products, with over 40,000
different structures of terpenoids discovered so far.
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)
(Croteau et al., 2000).

The volatile terpenoids – hemiterpenoids (C5),
monoterpenoids (C10), sesquiterpenoids (C15)
and some diterpenoids (C20) – are involved in
interactions between plants and insect
herbivores or pollinators and are also implicated
in general defense or stress responses
Terpenoids, mainly the C10 and C15 members of
this family, were found to affect the flavor
profiles of most fruits and the scent of flowers
at varying levels (Figure 5).
Latin: one and a half
Sesqui = ?

Important plant-derived volatile terpenoids.

Citrus fruit aroma consists mostly of mono- and
sesquiterpenes, which accumulate in specialized oil
glands in the flavedo (external part of the peel) and
oil bodies in the juice sacs.
The monoterpene R-limonene normally accounts
for over 90% of the essential oils of the citrus fruit
(Weiss, 1997).

The sesquiterpenes valencene and α- and β-sinensal, although
present in minor quantities in oranges, play an important role in
the overall flavor and aroma of orange fruit
Nootkatone, a putative derivative of valencene, is a small
fraction of the essential oils, but has a dominant role in the flavor
and aroma of grapefruit

The monoterpene S-linalool was found to be an important
general strawberry aroma compound (Aharoni et al., 2004;
Larsen and Poll, 1992) and is found in many other fruits
including peaches, guavas, nectarines, papayas, mangoes,
passion fruits, tomatoes, litchi, oranges, prickly pears and
koubos.
The combination of the monoterpenes geraniol, citronellol and
rose oxide is a key component of the characteristic aroma of
aromatic muscat grapes as well as the special scent of roses.

Terpenoids
The principal pathway for
monoterpene biosynthesis
in peppermint.
The responsible enzymes
are: geranyl diphosphate
synthase (1), (4S)-(−)-
limonene synthase (2),
cytochrome P450 (−)-
limonene-3-hydroxylase
(3), (−)-trans-isopiperitenol
dehydrogenase (4), (−)-
isopiperitenone reductase
(5), (+)-cis-isopulegone
isomerase (6), (+)-pulegone
reductase (7), and (−)-
menthone reductase (8).
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC59171/
(a) and with two cells (b). C, cuticle; E, epidermis;
CG, glandular cell (GX400).

Terpenoids: primary constituents of the essential oils of
many herbs.
The peltate glandular trichomes of peppermint produce
copious amounts of a commercially valuable, menthol-rich
essential oil, composed primarily of p-menthane
monoterpenes (Turner and Croteau, 2004). The glandular
trichomes of sweet basil (Ocimum basilicum) are rich in
phenylpropenes as well as monoterpenes and
sesquiterpenes (Iijima et al., 2004a).

Lemon-scented herbs of various plant families, such as
lemon basil (Ocimum × citratus, Lamiaceae), lemongrass
(Cymbopogon citratus, Poaceae) and lemon verbena
(Aloysia citriodora, Verbenaceae), accumulate citral,
a mixture of the cis–trans isomeric monoterpene aldehydes
neral and geranial.

Therefore, many terpenoids are commercially important
and are widely used as flavoring agents, perfumes,
insecticides, anti-microbial agents and important raw
material for the manufacture of vitamins and other key
chemicals.
Many terpenoids have medicinal properties;
consequently they are of interest to the pharmaceutical
industry as anti-retroviral agents or anti-malarial
compounds (Modzelewska et al., 2005)..

As a result, modulation of terpenoid biosynthesis in
medicinal and aromatic plants has received much
interest
Synthetic variations and derivatives of natural terpenes
and terpenoids also greatly expand the variety of aromas
used in perfumery and flavors used in food additives.

Despite their diversity, all terpenoids derive from
the common building units
1. isopentenyl diphosphate (IDP) and its isomer
2. dimethylallyl diphosphate (DMADP)

Fates of 3-Hydroxy-3-Methylglutaryl CoA. In the cytosol, HMG-
CoA is converted into mevalonate. In mitochondria, it is converted
into acetyl CoA and acetoacetate
(HMG-CoA reductase)
Ketone body formation

In plants, both IDP and DMADP are synthesized via two
parallel pathways, the mevalonate (MVA) pathway, which is
active in the cytosol, and the methylerythritol 4-phosphate
(MEP) pathway, which is active in the plastids
It is generally recognized that the cytosolic pathway is
responsible for the synthesis of sesquiterpenes, phytosterols
and ubiquinone,
whereas monoterpenes, gibberellins, abscisic acid,
carotenoids and the prenyl moiety of chlorophylls,
plastoquinone and tocopherol are produced in plastids
but indications of cross-talk between the plastidic and
cytosolic pathways have been found in tobacco, Arabidopsis
and snapdragon petals.

Mevalonate is converted into 3-isopentenyl pyrophosphate in three consecutive
reactions requiring ATP. Decarboxylation yields isopentenyl pyrophosphate, an
activated isoprene unit that is a key building block for many important
biomolecules throughout the kingdoms of life.
Synthesis of Isopentenyl Pyrophosphate. This activated intermediate is formed from
mevalonate in three steps, the last of which includes a decarboxylation.
6C
5C

Squalene (C30) Is Synthesized from Six Molecules of
Isopentenyl Pyrophosphate (C5)
This stage in the synthesis of cholesterol starts with the
isomerization of isopentenyl pyrophosphate to dimethylallyl
pyrophosphate.
Squalene is synthesized from isopentenyl pyrophosphate by the
reaction sequence
C5 → C10 → C15 → C30

One molecule of dimethyallyl
pyrophosphate (DMADP) and two
molecules of isopentenyl
pyrophosphate (IDP) condense to form
farnesyl pyrophosphate.
The tail-to-tail coupling of two
molecules of farnesyl pyrophosphate
yields squalene.

The direct precursors of terpenoids, linear geranyl
diphosphate (GDP, C10), farnesyl diphosphate (FDP,
C15) and geranylgeranyl diphosphate (GGDP, C20),
are produced by the activities of three prenyl
transferases.
Terpene synthases are the primary enzymes
responsible for catalyzing the formation of
hemiterpenes (C5), monoterpenes (C10),
sesquiterpenes (C15) or diterpenes (C20) from the
substrates DMADP (dimethylallyl diphosphate),
GDP, FDP or GGDP, respectively.

Prenyl transferases catalyze the addition of
IDP (isopentenyl diphosphate) units to prenyl
diphosphates with allylic double bonds to
the diphosphate moiety.
Most of the prenyl transferases accept
DMADP as the initial substrate, but they also
bind GDP or FDP depending on the particular
prenyltransferase .

The availability of GDP and FDP are often the key factor
in the production of monoterpenes and sesquiterpenes in
plants.
This problem was elegantly overcome in metabolic
engineering experiments by the co-expression of GDP
and FDP synthases with appropriate monoterpene and
sesquiterpene synthases over-expressed in tobacco (Wu
et al., 2006).
This strategy, together with targeting of the over-
expression to the plastid compartment, resulted in
increased synthesis of the sesquiterpenes amorpha-4,11-
diene and patchoulol and the monoterpene S-limonene.

The third phase of terpene volatile biosynthesis involves
conversion of the various prenyl diphosphates DMADP,
GDP, FDP and GGDP to hemiterpenes, monoterpenes,
sesquiterpenes and diterpenes, respectively, by the large
family of the terpene synthases.
Triterpenes (and sterols) and tetraterpenes (such as
carotenoids) are derived from the condensation of two
molecules of FDP or GGDP, respectively.
Terpene synthases

Triterpenes (and sterols)
tetraterpenes (such as
carotenoids)

Plant hemiterpene, monoterpene, sesquiterpene and
diterpene synthases are evolutionarily related to each
other and are structurally distinct from triterpene or
tetraterpene synthases.
Many terpene synthases have been isolated and
characterized from various plant species

While many terpene volatiles are direct products of
terpene synthases, many others are formed through
transformation of the initial products by
oxidation,
dehydrogenation,
acylation and other reactions
For example, (−)-(1R,2S,5R)-menthol, the principal
monoterpene of commercial peppermint essential oil
and the component responsible for the familiar cooling
sensation of peppermint and its products, is formed by
eight enzymatic steps involving monoterpene synthases,
isomerases and reductases

https://en.wikipedia.org/wiki/Menthol

The biosynthesis starts with the formation of 4S-
limonene from GPP and ends with the reduction of (−)-
menthone to (−)-menthol.
Mentha arvensis is the primary species of mint used to
make natural menthol crystals and natural menthol
flakes.
As with many widely used aroma chemicals, the annual
demand for menthol of 6300 tonnes greatly exceeds the
supply from natural sources.

Peppermint (Mentha × piperita, also known as Mentha
balsamea Wild), is a hybrid mint, a cross between
watermint (Mentha aquatica) and spearmint (Mentha
spicata).

Metabolic engineering of the terpenoid pathway is
a constantly improving tool, used for the
fundamental study of terpenoid biosynthesis
In addition, this tool is being used more and more
for the understanding of chemical diversity in crops
as well as improvement of traits in crops such as
disease and pest resistance enhanced and altered
aroma formation and production of medicinal
compounds .

A recent example in which flavor engineering was
detected by non-trained test panelists involved
ectopic expression of the lemon basil geraniol
synthase gene under the control of the fruit
ripening-specific tomato polygalacturonase
promoter.
This caused diversion of the plastidial terpenoid
pathway for production of lycopene to the
accumulation of high levels of geraniol and about
ten novel geraniol derivatives, and had a profound
impact on tomato flavor and aroma, as evaluated
organoleptically.

Apocarotenoids.
Carotenoids are tetraterpenoid pigments that
accumulate in the plastids of leaves, flowers and
fruits, where they contribute to the red, orange and
yellow coloration.
In addition to their roles in plants as photosynthetic
accessory pigments and colorants, carotenoids are
also precursors of apocarotenoids (also called
norisoprenes) such as the phytohormone abscisic
acid, the visual and signaling molecules retinal and
retinoic acid, and aromatic volatiles such as β-ionone.

https://www.aicr.org/resources/blog/carotenoid-foods-may-protect-against-certain-breast-cancers/
women with
higher blood
concentrations of
these carotenoids
are at decreased
risk of the type of
breast cancer
called estrogen
receptor (ER)
negative.

Evidence, based on comparative genetics, has indicated
that carotenoid pigmentation patterns have profound
effects on the apocarotenoid and monoterpene aroma
volatile compositions of tomato and watermelon fruits
(Lewinsohn et al., 2005a,b).
This work indicated that the various flavors and aromas
of otherwise similar fruit of different colors have a real
chemical basis and are not solely due to psychological
preconception.

Indeed, enzymes capable of cleaving carotenoids at
specific sites were found to be involved in the
synthesis of a number of apocarotenoids.
Carotenoid cleavage dioxygenases (CCDs) catalyze
the oxidative cleavage of carotenoids, resulting in
production of apocarotenoids (Schmidt et al., 2006).

Carotenoid cleavage dioxygenases
(CCDs)
CCDs often exhibit substrate promiscuity, which probably
contributes to the diversity of apocarotenoids found in
nature.
Apocarotenoids are commonly found in the flowers, fruits,
and leaves of many plants (Winterhalter and Rouseff, 2002)
and possess flavor aroma properties together with low
aroma thresholds.

Carotenoid cleavage dioxygenases
(CCDs)
They are found among the potent flavor compounds in
wines and contribute to floral and fruity attributes .
Therefore, they have been subject to extensive research in
recent years with regard to their structure and flavor
potential .

The synthesis of β-ionone, geranyl acetone and 6-methyl-5-hepten-2-
one in tomato fruits increases 10–20-fold during fruit ripening, and
these compounds were produced by the activity of the genes
LeCCD1A and LeCCD1B that were isolated from tomato fruits.
In tomato fruit, β-ionone is present at very low concentrations (4 nl
l−1), but due to its low odor threshold (0.007 nl l−1) is the second most
important volatile contributing to fruit flavor ).

Silencing of LeCCD1A and LeCCD1B resulted in a significant decrease
in the β-ionone content of ripe fruits, implying a role for these genes
in C13 norisoprenoid synthesis in vivo.
Reduction of Petunia hybrida CCD1 transcript levels in transgenic
plants led to a 58–76% decrease in β-ionone synthesis in the corollas
of selected petunia lines, indicating a significant role for this enzyme
in volatile synthesis.

Carotenoid cleavage
dioxygenases
Also, a potential CCD gene was identified among a Vitis vinifera L. EST
collection, and recombinant expression of VvCCD1 confirmed that the
gene encodes a functional CCD that cleaves zeaxanthin symmetrically
yielding 3-hydroxy-β-ionone and a C14 dialdehyde. e

Carotenoid cleavage
dioxygenases
CCDs were also found to be involved in the formation of important
aroma compounds in melon (Cucumis melo).
The product of the CmCCD1 gene, whose expression is up-regulated
upon fruit development, was shown to cleave carotenoids, generating
geranylacetone from phytoene, pseudoionone from lycopene, β-ionone
from β-carotene, and α-ionone and pseudoionone from δ-carotene

Carotenoids and their degradation products.
Carotenoid substrates (left) are oxidatively cleaved to yield the
apocarotenoid derivatives (right).

Fatty acid-derived and other
lipophylic flavor compounds
The majority of plant volatiles on a quantitative and
qualitative basis originate from saturated and unsaturated
fatty acids.
Fatty acid-derived straight-chain alcohols, aldehydes, ketones,
acids, esters and lactones are found ubiquitously in the plant
kingdom at high concentrations, and are basically formed by
three processes, α-oxidation, β-oxidation and the
lipoxygenase pathway.

In plants, fatty acids are stored as triacylglycerides and
therefore enzymatic oxidative degradation of lipids is
preceded by the action of acyl hydrolase, liberating the free
fatty acids from acylglycerols.
However, identification of a number of oxylipin-containing
phosphatidylglycerols, monogalactosyldiacylglycerols and
digalactosyldiacylglycerols demonstrated that direct
oxidation of the fatty acid side chain in acylglycerides is
possible
.

Oxylipin
While plants lack an immune system in the sense that
it exists in animals, they do possess mechanisms
that are functionally equivalent in that they recognize
potential pathogens and stress vectors and then
initiate defence responses.
It has become evident that various types of
oxygenated fatty acids, collectively termed ‘oxylipins’,
are involved in responses to
1. physical damage by animals, insects, or
2. abiotic stress (e.g., freeze-thawing), and
3. attack by pathogens.

Oxylipin
Among these, the jasmonates have a special
importance, and they are present ubiquitously in
land plants.
These lipid mediators are similar in many ways to
the eicosanoids derived from arachidonate in
animals, which have so many varied functions but
especially in inflammatory processes.
They are also phytohormones, which are
intimately involved in the growth and development
of plants.

Eicosanoids
All these compounds are extremely potent chemicals that serve as hormonal mediators. They also have
many other medical applications and can cause medical problems. They're also known as eicosanoids-from
the Greek for twenty, which alludes to the presence of 20 carbon atoms (see Figure 8-12).
Arachidonic acid- a 20-carbon, polyunsaturated
fatty acid - serves as the direct or indirect starting
material for the formation of prostaglandins,
thromboxanes, and leukotrienes. Cells synthesize
both leukotrienes and prostaglandins from
arachidonic acid. Additional prostaglandins and
thromboxanes come from the prostaglandin
derived from arachidonic acid.
All three classes of compounds are local hormones.
Unlike other hormones, they're not transported via
the bloodstream. They're short-lived molecules that
alter the activity of the cell that produces them as
well as neighboring cells.

Oxylipin
Related oxylipins function in fungi, yeasts and
mosses.
Some plant oxylipins act directly by being
distasteful to insect predators,
some are sufficiently volatile that they can alert
neighbouring plants, while others can
communicate the information on cell damage
over long distances within a plant to coordinate
a comprehensive response.

Lipoxygenase pathway (in-chain
oxidation).
Saturated and unsaturated volatile C6 and C9
aldehydes and alcohols are important contributors
to the characteristic flavors of fruits, vegetables and
green leaves.
They are widely used as food additives because of
their ‘fresh green’ odor.
The short-chain aldehydes and alcohols are
produced by plants in response to wounding and
play an important role in the plants defense
strategies and pest resistance

Lipoxygenase pathway (in-chain
oxidation).
At least four enzymes are involved in the
biosynthetic pathway leading to their
formation:
1. lipoxygenase (LOX),
2. hydroperoxide lyase (HPL),
3. 3Z,2E-enal isomerase and
4. alcohol dehydrogenase (ADH) .

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.
Linolenic acid-derived flavor molecules.

https://en.wikipedia.org/wiki/E%E2%80%93Z_not
ation
E–Z configuration, or the E–Z convention, is the
IUPAC preferred method of describing the
absolute stereochemistry of double bonds in
organic chemistry.
It is an extension of cis–trans isomer notation
(which only describes relative stereochemistry)
that can be used to describe double bonds having
two, three or four substituents.

LOX is a non-heme, iron-containing
dioxygenase that catalyzes the regio- and
enantio-selective dioxygenation of unsaturated
fatty acids (e.g. linoleic and α-linolenic acid)
containing one or more 1Z,4Z-pentadienoic
moieties.

Numerous plant LOXs have been characterized
because they are essential components of the
oxylipin pathway, converting fatty acids into
hydroperoxides and finally flavors such as 3Z-
hexenol, 2E-hexenal and 2E,6Z-nonadienal.
Products of the LOX pathway are involved in
wound healing, pest resistance and signaling, or
have anti-microbial and anti-fungal activity.

LOX enzymes have been classified with respect to
their positional specificity with regard to fatty acid
oxygenation.
Oxygenation at C9 (9-LOX) or at C13 (13-LOX) of
the hydrocarbon backbone leads to the (9S)- and
(13S)-hydroperoxy derivatives, respectively.

Classification of LOXes
Plant LOX can also be grouped into two gene sub-
families according to their overall sequence similarity.
Type 1 LOX:
Enzymes carrying no plastidic transit peptide show a
high sequence similarity (>75%) to one another.
Type 2 LOX:
harbour an N-terminal extension and have only a
moderate overall sequence similarity (approximately
35%).
The three-dimensional protein structures of soybean
LOX-1 and -3 have been elucidated and essential amino
acids identified (Liavonchanka and Feussner, 2006).

In vegetative tissues, LOX provides Z,E-
configured hydroperoxides that can be
metabolized to compounds that are crucial
elements of plant defense.
It is less clear why seeds and tubers have
large amounts of LOX.
Genetic removal of specific LOX isoforms
appears not to compromise plant health
(Baysal and Demirdöven, 2007).

In tomato, five LOX genes (TomLoxA, B, C, D
and E) are expressed during ripening.
Antisense suppression of TomLoxA and B in
tomato fruit resulted in no significant
changes in the fruit flavor, but co-
suppression of TomLoxC strongly affected
the production of fatty acid-derived volatiles
(Chen et al., 2004).

HPL (Hydroperoxide lyase) cleaves the LOX products,
resulting in the formation of ω-oxo acids and volatile C6
and C9 aldehydes.
Similar to LOX, HPL can be classified into two groups
according to substrate specificity. HPL is a member of
the cytochrome P450 family CYP74B/C, and acts on a
hydroperoxy functionality in a lipid peroxide without any
co-factor.
Recently, a hemi-acetal has been identified as primary
product of HPL (Matsui, 2006). Down-regulation of HPL
has been performed in potato plants (Salas et al., 2005).
Such silencing of HPL induced an increase in LOX
activity but a decrease of most of the C6 volatiles.

3Z,2E-enal isomerase
The β, γ-unsaturated carbonyl functionality in
the HPL products is prone to isomerization,
either enzymatically catalyzed by a 3Z,2E-enal
isomerase or non-enzymatically.
Although 3Z,2E-enal isomerase activity has
been described in soybeans (Glycine max L.)
and alfalfa (Medicago sativa L.), neither a
protein nor a corresponding gene has been
cloned yet.

Alcohol dehydrogenase
C6 and C9 aldehydes can be further metabolized by
ADH to form the corresponding alcohols.
ADH genes that are suspected to participate in the
production of aromas are expressed in a
developmentally regulated manner, particularly during
fruit ripening (Manriquez et al., 2006).

Over-expression of the tomato ADH2 gene
has led to improved flavor of the fruit by
increasing the levels of alcohols, particularly
3Z-hexenol (Speirs et al., 1998).
An acyltransferase catalyzes the formation
of 3Z-hexenyl acetate from 3Z-hexenol and
acetyl CoA, and 2-alkenal reductase can
reduce 2E-hexenal to hexanal (D’Auria et al.,
2003; Mano et al., 2002).

Jasmonic acid pathway
The reaction sequence leading from α-
linolenic acid to the signaling molecule
jasmonic acid involves the enzymes 13-LOX,
allene oxide synthase, allene oxide cyclase
and 12-oxo-phytodienoic acid reductase,
followed by three successive β-oxidation
steps (Figure 7).

Jasmonic acid and its volatile methyl ester act as
phytohormones and are involved in plant
responses to stress and developmental
processes.
In addition, methyl jasmonate is the main
component of the scent of jasmine flowers and
contributes to the precious flavors of Rosmarinus,
Gardenia, Artemisia and lemon oil.
cis-jasmone, which acts as either an attractant or
a repellent for various insects, is a decarboxylated
derivative of jasmonic acid generated by oxidative
degradation of jasmonate (Schaller et al., 2005).

α- and β-oxidation.
α- and β-oxidation. Although the degradation
of straight-chain fatty acids by α- and β-
oxidation is a major process for the
formation of flavor molecules in all
organisms, the specific pathways in plants
are not well understood.
The fatty acid α-oxidation mechanism in
plants involves free fatty acids (C12–C18)
that are enzymatically degraded via one or
two intermediates to C(n−1) long-chain fatty
aldehydes and CO2.

A dual-function α-dioxygenase/peroxidase
and NAD+ oxidoreductase catalyze the α-
oxidation of fatty acids in plants (Saffert et
al., 2000).
β-Oxidation results in successive removal of
C2 units (acetylCoA) from the parent fatty
acid.

Forward and reverse genetic screens have
revealed the importance of β-oxidation
during plant development and in response to
stress (Baker et al., 2006).
Combinations of mutations show much
stronger phenotypes, but it is unclear
whether the necessity for β-oxidation is to
provide an energy source or a lipid-derived
signal molecule.

Short- and medium-chain linear carboxylic
acids that are formed by repeated β-
oxidative cycles followed by the action of an
acylCoA hydrolase have been found in many
essential oils isolated from different plant
sources (Figure 8a).
As a second pathway, de novo synthesis and
hydrolysis of acyl acyl carrier protein (acylACP) can
also provide volatile acids.

Figure 8
Biosynthesis of (a) short-chain acids, aldehydes, alcohols, esters and lactones, and (b) methylketones.
AAT, alcohol acyl CoA transferase; MKS, methylketone synthase; ACP, acyl carrier protein.
acylCoA hydrolase

Aliphatic acids up to C10 play a significant role in
flavors due to their sharp, buttery and cheese-like
odors, not only on their own, but particularly as
substrates in the form of their acylCoAs for
biosynthesis of other flavors.
Aliphatic short- and medium-chain aldehydes and
alcohols are emitted by various plant parts and are
probably formed by enzymatic reduction of the
parent acylCoAs (Flamini et al., 2007).

Alternatively, alcohols can also be formed by
ADH-mediated hydrogenation of aldehydes,
and medium-chain aldehydes are
intermediates of the α-oxidation cycle
starting with common fatty acids (Hamberg
et al., 1999).
However, alcohols are less important as
flavor molecules due to their high odor
thresholds in comparison with their
aldehyde homologues.

Most plant ADHs are Zn-dependent medium-chain
dehydrogenases that are thought to be involved in
the response to a wide range of stresses, including
anaerobiosis and elicitors (Chase, 2000).
An ADH with specific substrate preference has
been isolated from melons (Manriquez et al., 2006).

Specifically, flavor ester production relies upon the
supply of acylCoAs formed during β-oxidation and
alcohols.
Alcohol acyl transferases (AAT) are capable of
combining various alcohols and acylCoAs, resulting
in the synthesis of a wide range of esters, thus
accounting for the diversity of esters.

Figure 8
Biosynthesis of (a) short-chain acids, aldehydes, alcohols, esters and lactones,
AAT, alcohol acyl CoA transferase;

Numerous AAT (Alcohol acyl transferases)
genes have been isolated and characterized
in fruit and vegetables
Aliphatic esters contribute to the aroma of
nearly all fruits and are emitted by vegetative
tissues.
Some are responsible for a particular fruit
aroma or for the smell of a flower.
However, many of these esters possess a
non-specific fruity odor.

Lactones are cyclic esters of organic acids. It is a
condensation product of an alcohol group and a
carboxylic acid group in the same molecule of
hydroxycarbonic acid.
The most stable structures are the five-membered
(gamma-lactone) and six-membered lactones
(delta-lactone).
Lactones

Another major group of fatty acid-derived flavor
molecules are alkanolides, which have γ-(4-) or δ-
(5-)-lactone structures (Figure 8a).
Sensory important lactones usually possess 8–
12 carbon atoms and some are very potent flavor
components for a variety of fruits (Basear and
Demirci, 2007).
The fact that both the optical purity and the
absolute configuration vary for identical lactones
isolated from different sources supports the idea
of different biosynthetic pathways.

Figure 8

However, all lactones originate from their
corresponding 4- or 5-hydroxy carboxylic
acids, which are formed by either
(i) reduction of oxo acids by NAD-linked
reductase,
(ii) hydration of unsaturated fatty acids,
(iii) epoxidation and hydrolysis of unsaturated
fatty acids, or
(iv) reduction of hydroperoxides
Enzymes and genes specifically involved in
the formation have not yet been reported.

In contrast to 4- and 5-hydroxy fatty acids,
3-hydroxy acids, the normal intermediates of
the β-oxidation, do not form lactones.
However, they are converted to methyl- or
ethyl-3-hydroxyesters in plants and
contribute to the aroma of fruits.

Short-length methylketones (C5–C11) are
highly potent flavor molecules that have
been found in numerous plants, while
medium-length methylketones (C7–C15) are
highly effective in protecting plants from
numerous pests.).

Recently, the first methylketone synthase
was isolated from tomato, which catalyzes
the hydrolysis and subsequent
decarboxylation of C12, C14 and C16 β-
ketoacyl ACPs (acyl carrier protein) to give
C11, C13 and C15 methylketones,
respectively (Fridman et al., 2005).

In contrast, in fungi, methylketones are
derived from β-oxidative degradation of fatty
acids through β-ketoacyl CoA intermediates
(Schwab and Schreier, 2002).
Methylketones are assumed to be
precursors of aroma-active secondary
alcohols such as 2-pentanol and 2-heptanol,
which are important flavor molecules
produced by passion fruits (Figure 8b)
(Strohalm et al., 2007).

Figure 8
methylketone
synthase;

Amino acid-derived flavor
compounds
Although aldehydes and alcohols derived from the
degradation of branched-chain and aromatic amino
acids or methionine constitute a class of highly
abundant plant volatiles, their pathways have been
barely analyzed in plants. Especially important are
branched-chain volatiles derived from branched-chain
amino acids.

Isoamyl acetate, an ester with a strong fruity odor
described as similar to banana or pear, is one of the key
constituents of banana flavor.
2-Methyl-butyl acetate has a strong apple scent and is
associated with apple varieties that are rich in aroma
such as ‘Fuji’, ‘Gala’ and ‘Golden Delicious’.
Amino acid-derived flavor compounds
https://www.chemistryworld.com/podcasts/isoamyl-acetate/7609.article

Amino acid-derived flavor
compounds
Methyl 2-methyl butanoate determines the characteristic
aroma of prickly pear, while a combination of several
volatile esters imparts the unique aroma of melons, with
isoamyl acetate and 2-methyl-butyl acetate being
prominent in many varieties

Transaminases (Aminotransferases)
Aminotransferases catalyze the transfer of an α-amino group from an α-amino acid to an
α-ketoacid.
These enzymes, also called transaminases, generally funnel α-amino groups from a
variety of amino acids to α-keto-glutarate for conversion into NH4 +
Alpha amino acid Keto acid

Acids, alcohols, aldehydes, esters, lactones
and N- and S-containing flavor molecules.
In micro-organisms, the catabolism of amino
acids has been analyzed in detail and is initiated
by aminotransferases forming 2-ketoacids that
serve as substrates for three biochemical
reactions:
(i) oxidative decarboxylation to carboxylic acids,
(ii) decarboxylation to aldehydes, and
(iii) reduction to 2-hydroxyacids (Figure 9a)

Biosynthesis of amino acid-derived flavor compounds.
(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 (Kaminaga et al., 2006; Tieman et al., 2006).

Compounds derived from leucine such as 3-
methylbutanal, 3-methylbutanol and 3-methylbutanoic
acid, as well as phenylacetaldehyde and 2-
phenylethanol formed from phenylalanine, are
abundant in various fruits such as strawberry, tomato
and grape varieties (Aubert et al., 2005).
In addition, alcohols and acids derived from amino
acids can be esterified to compounds with a large
impact on fruit odor, such as 3-methylbutyl acetate
and 3-methylbutyl butanoate in banana (Nogueira et
al., 2003).

Genes encoding enzymes responsible for
the direct decarboxylation of phenylalanine
have been isolated from tomato, petunia and
rose, showing that alternative catabolic
pathways exist in plants (Figure 9a)
(Kaminaga et al., 2006; Tieman et al., 2006).
Although the enzymes display subtle
differences in sequences and enzymatic
properties, their down-regulation led to
reduced emission of 2-phenylacetaldehyde
and 2-phenylethanol.

Over-expression of the amino acid decarboxylase in
tomato resulted in fruits with up to 10-fold
increased levels of 2-phenylacetaldehyde, 2-
phenylethanol and 1-nitro-2-phenylethane (Tieman
et al., 2006).
The modulation of the emission of 2-phenylethanol
and 2-phenylacetaldehyde is important because
these substances exert a dual effect.
At low concentrations, both compounds are
associated with pleasant sweet flowery notes, while
at high levels, the pungent aroma of 2-
phenylacetaldehyde is nauseating and unpleasant
(Tadmor et al., 2002).

4,5-Dimethyl-3-hydroxy-2(5H)-furanone
(sotolon) is the major flavor-impact
compound of dried fenugreek seeds
Trigonella foenum-graecum L., and is
probably formed from 4-hydroxy-l-isoleucine
(Slaughter, 1999).

Sulfur-containing flavor compounds
Sulfur-containing flavor compounds originating
from methionine and cysteine are responsible for :
1. odor of garlic (methanethiol, dimethyl disulfide,
S-methyl thioacetate),
2. onions (propanthial S-oxide),
3. boiled potato (methional) and
4. cooked cabbage (methanethiol)
In Arabidopsis, the cleavage of methionine is
catalyzed by methionine γ-lyase, resulting in the
production of methanethiol, 2-ketobutyrate and
ammonia (Rébeilléet al., 2006).

In onion (Allium cepa) and garlic (A. sativum), a
series of volatile sulfur compounds is generated by
cleavage of odorless S-alk(en)yl cysteine sulfoxide
(ASCOs) flavor precursors catalyzed by the
enzymes allinase and lachrymatory-factor synthase
(Jones et al., 2004; Lanzotti, 2006).
The biosynthetic pathway involves alk(en)ylation of
the cysteine in glutathione, followed by cleavage
and oxidation to form the sulfoxides or
(thio)alk(en)ylation of cysteine or O-acetyl serine.

Catabolism of S-alk(en)yl-L-cysteine sulfoxides (ASCOs) by alliinase enzyme generates a spectrum
https://www.researchgate.net/figure/Catabolism-of-S-alkenyl-L-cysteine-sulfoxides-ASCOs-by-alliinase-enzyme-generates-a_fig1_337078335

Once the plant tissue is damaged, the flavor
precursors are enzymatically cleaved by allinase to
give a series of volatile sulfur compounds that
undergo further vapor-phase chemical
transformations (Figure 10a).

Because the levels of the flavor precursors amount
to 1–5% dry weight in certain Allium species, it is
supposed that they play a major role for the plant.
Two roles that have been ascribed are
1. defense against pests and predation, particularly
in the over-wintering bulb, and
1. carbon, nitrogen and sulfur storage and transport
(Jones et al., 2004).

Figure 10
Biosynthetic pathways for (a) thiosulfinates and their degradation products, (b) formation of indole
in maize, (c) volatiles produced from glucosinolates, and (d) cyanogenic glucosides.

Volatile biogentic amines are another group
of flavor molecules that are synthesized
from amino acids or their precursors.
The volatile indol is formed in maize by the
cleavage of indole-3-glycerol phosphate, an
intermediate in tryptophan biosynthesis
(Figure 10b) (Frey et al., 2000).

Figure 10
(b) formation of indole in maize,
indol

Glucosinolates
Cruciferous vegetables such as mustard,
broccoli, cauliflower, kale, turnips, collards,
Brussels sprouts, cabbage, radish and
watercress contain glucosinolates, which
are natural precursors of flavor molecules.

Glucosinolates, which are synthesized from certain
amino acids, are sulfur-rich, nitrogen-containing
thioglycosides that, upon hydrolysis by endogenous
thioglucosidases, produce volatile products such as
isothiocyanates, thiocyanates and nitriles (Figure
10c).
These are the active substances that serve as
defense compounds or attractants for the plant.
For humans, they function as cancer-preventing
agents,
biopesticides and flavor compounds.

Figure 10
(c) volatiles produced from glucosinolates

Cyanogenic glycosides, which are β-
glycosides of α-hydroxynitriles, are another
group of amino acid-derived flavor
precursors

Cyanogenic glycosides, which are β-
glycosides of α-hydroxynitriles, are another
group of amino acid-derived flavor
precursors (Figure 10d) (Bak et al., 2006).
Cyanogenesis is the process by which
hydrogen cyanide and volatile ketones or
aldehydes are released from cyanogenic
glycosides and is dependent on glycosidase
activities (Vetter, 2000).

Enzymatic hydrolysis yields an unstable
hydroxynitrile intermediate that
spontaneously decomposes under certain
conditions to hydrogen cyanide and a
carbonyl compound. Alternatively, the
intermediate can be broken down
enzymatically by α-hydroxynitrile lyase.
Ecological studies have shown that
cyanogenic glycosides can act as either
feeding deterrents or phagostimulants,
depending on the insect species (Vetter,
2000).

Phenylpropenes and other aromatic
derivatives.
Benzenoid and phenylpropanoid volatile compounds,
primarily derived from phenylalanine, contribute to the
aromas and scents of many plant species and play
important roles in plant communication with the
environment (Dudareva and Pichersky, 2006; Knudsen and
Gershenzon, 2006; Pichersky et al., 2006).
Several enzymes that catalyze the final steps in the
biosynthesis of these compounds have been isolated and
characterized.
However, the early steps leading to the formation of the
benzenoid backbone remain unclear (Beuerle and
Pichersky, 2002; Schnepp and Dudareva, 2006; Wildermuth,
2006).

In general, biosynthesis of benzenoids from phenylalanine
requires shortening of the carbon skeleton side chain by a C2
unit, which can potentially occur via either the β-oxidative
pathway or non-oxidatively (Boatright et al., 2004).
Experiments with stable isotope-labeled precursors in
tobacco (Nicotiana tabacum) leaves (Ribnicky et al., 1998)
suggested that benzoic acid is produced from phenylalanine-
derived cinnamic acid via the β-oxidative pathway, first
yielding benzoyl CoA, which can then be hydrolyzed by a
thioesterase to free benzoic acid. In contrast, labeling
experiments, together with initial enzyme characterization, in
Hypericum androsaemum cell cultures (Ahmed et al., 2002)
supported the existence of a pathway for non-oxidative
conversion of cinnamic acid to benzaldehyde with
subsequent formation of benzoic acid, which can be further
converted to benzoyl CoA (Beuerle and Pichersky, 2002).

In vivo isotope labeling and metabolic flux analysis of the
benzenoid network in petunia (Petunia hybrida) flowers revealed
that both pathways yield benzenoid compounds, and that benzyl
benzoate is an intermediate between l-phenylalanine and
benzoic acid (Boatright et al., 2004). Transgenic petunia plants
were generated in which expression of benzoyl-CoA:
phenylethanol/benzyl alcohol benzoyltransferase (BPBT), the
gene encoding the enzyme that uses benzoyl CoA and benzyl
alcohol to make benzyl benzoate, was reduced or eliminated.

Elimination of benzyl benzoate formation decreased the
endogenous pool of benzyl acid and methyl benzoate emission
but increased emission of benzyl alcohol and benzylaldehyde,
confirming the contribution of benzyl benzoate to benzoic acid
formation (Orlova et al., 2006). Labeling experiments with 2H5-
phenylalanine revealed a dilution of isotopic abundance in most
measured compounds in the dark, suggesting an alternative
pathway from a precursor other than phenylalanine, possibly
phenylpyruvate.

Methyl salicylate and methyl benzoate
Methyl salicylate and methyl benzoate are common
components of floral scent and are believed to be
important attractants of insect pollinators (Dobson,
1994; Dudareva and Pichersky, 2000; Dudareva et al.,
1998, 2000).
Methyl salicylate Methyl benzoate

Methyl salicylate and methyl benzoate
Enzymes that catalyze the formation of methyl
salicylate and methyl benzoate from salicylic acid (SA)
and benzoic acid (BA), respectively, have been
characterized from flowers of Clarkia breweri,
snapdragon (Antirrhinum majus), petunia (Petunia
hybrida), Arabidopsis thaliana and Stephanotis
floribunda (Chen et al., 2003; Murfitt et al., 2000; Negre
et al., 2002; Pott et al., 2002; Ross et al., 1999).
Clarkia breweri - Onagraceae Stephanotis floribunda - Apocynaceae
Antirrhinum majus Plantaginaceae

While these enzymes use S-adenosyl-l-methionine as the methyl
donor as do many previously characterized methyltransferases
that act on a variety of substrates (e.g. DNA, protein,
phenylpropanoids),
these SA and BA carboxyl methyltransferases have primary amino
acid sequences that show no significant sequence identity to other
methyltransferases.
Interestingly, a group of N-methyltransferases involved in
biosynthesis of the alkaloid caffeine, including theobromine
synthase, share sequence similarity with the SA and BA carboxyl
methyltransferases (D’Auria et al., 2003).
SAM

These enzymes were therefore grouped into a new class of
methyltransferases designated the SABATH methyltransferases,
and this family now also includes jasmonic acid
methyltransferase (Seo et al., 2001), indole-acetic acid
methyltransferase (Zubieta et al., 2003) and cinnamic/p-
coumaric acid methyltransferase (Kapteyn et al., 2007).
The recently obtained three-dimensional structure of C. breweri
SA carboxyl methyltransferase (Zubieta et al., 2003), combined
with in silico modeling of the active site pocket in the Nicotiana
suaveolens and S. floribunda enzymes (Pott et al., 2004), also
indicates that these enzymes have a unique structure that is
distinct from those of unrelated methyltransferases found in
plants (Noel et al., 2003).

Vanillin
Vanillin (4-hydroxy-3-methoxybenzaldehyde) is the most
widely used flavor compound in the world. It is the
principal flavor component of the vanilla extract obtained
from cured pods (beans) of the orchid Vanilla planifolia
Andrews.

Vanillin
Vanillin accumulates in the secretion around the seeds in
the mature fruits. A unique secretory tissue composed of
closely packed unicellular hairs is located in three gaps
between the placentas along the central fruit cavity.
These cells seem to be responsible for vanillin secretion
(Joel et al., 2003).
Vanilla extract is valued as a natural flavor, but, because
of its cost and limited availability, less than 1% of the
annual world demand for vanillin is isolated from its
natural source (Walton et al., 2003).

Vanillin
Most of the vanillin used by the flavor industry originates
from chemical methods that use guaiacol, eugenol or
lignin as starting materials (Rao and Ravishankar, 2000).
Vanillin is believed to be synthesized from
phenylpropanoid precursors, and various biosynthetic
pathways have been proposed.
Guaiacol eugenol lignin
Lignin is a polymeric
material that consists of
the cross-linked
component of three
monolignols: coumaryl
alcohol (H), coniferyl
alcohol (G), and
sinapyl alcohol (S)

Vanillin
A three-step pathway for vanillin biosynthesis from 4-
coumaric acid has been proposed based on precursor
accumulation and on feeding cell cultures of V. planifolia
with the proposed precursors (Havkin-Frenkel et al.,
1999).

Vanillin
In this pathway, 4-coumaric acid is first converted to 4-
hydroxybenzaldehyde by 4-hydroxybenzaldehyde synthase
through a chain-shortening step (Podstolski et al., 2002).
Then, hydroxylation at position 3 on the ring is performed by
4-hydroxybenzaldehyde synthase, converting p-hydroxybenzyl
alcohol to 3,4-dihydroxybenzyl alcohol or aldehyde.
The final enzymatic step was shown to be catalyzed by a
multifunctional O-methyltransferase from V. planifolia that
has a broad substrate range, including 3,4-
dihydroxybenzaldehyde (Pak et al., 2004).
4-hydroxybenzaldehyde

References
https://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2008.03446.x
https://asianjournalofchemistry.co.in/user/journal/viewarticle.aspx?ArticleID=29_11_2
https://www.tandfonline.com/doi/full/10.1080/10408398.2020.1769547

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