Biotechnology for the production of essential

oils, avours and volatile isolates. A review.

,
Y. Gounaris*
ABSTRACT: Various applications of biotechnological methods for the production of volatile compounds useful to the foodand
pharmaceutical industries are discussed. The yields obtained fromintact or genetically modied plants are compared to those
achieved by microbial methods. Plant yields are too low for the products to compete commercially to those synthesized
chemically. Still lower yields are obtained with in vitro-cultured plant tissues. Trangenic plants with altered methylerythritol
path gave 50% more essential oil in the best case. The 100-fold increases in shikimate-derived volatiles, obtained with over-
expressed alcohol dehydrogenase and ve-fold more C6 volatle aldehydes and 2-phenylethanol, were produced with overex-
pressed lipoxygenase and 2-phenylethanol dehydrogenase, respectively. However, the most spectacular yields were observed
with biotransformations catalysed by microorganisms. Kluyveromyces marxianus, produces over 26 g/l 2-phenylethanol from
phenyalanine, whereas Candida sorbophila, Mucor circillenoides or Yarrowia lipolytica can produce 540 g/l g-decalactone from
ricinoleic acid. Vanillin production from ferulic acid is in the range 1260 g/l with Amycolatopsis and Streptomyces species.
Vanillin can be produced at 5 g/l by Escherichia coli and amorphadiene yields of 37 g/l have been observed with Saccharomyces
cerevisiae, both with the genetically overexpressed methylerythritol path. Genetically engineered b-oxidation genes result in
yields of 10 g/l g-decalactone byYarrowia lipolytica andupto 80 g/l dicaboxylic acids by various yeasts. These results far exceed
the theoretical limit of about 1 g/l required for consideration of a procedure as a commercially interesting process, alternative
to chemical sythesis. Copyright 2010 John Wiley & Sons, Ltd.
Keywords: bioreactor; biotechnology; essential oil; terpenoids; volatiles
Introduction
There are hundreds of thousands of dierent secondary metabo-
lites produced by plants, four times more than the number pro-
duced by microorganisms. This number is estimated to represent
only 10%of the secondary metabolites existing in plants and still
waiting to be isolated and identied. Of these, terpenoids com-
prise the largest and structurally most varied class, numbering
over 40 000 dierent molecules. Members of the 10-carbon ter-
penoids, the monoterpenoids, are constituents of essential oils
produced by plants. The essential oil monoterpenoids are vola-
tile, which means that they pass in to the air in sucient concen-
trations to be detected by, and to act on, other organisms.
Essential oils can also contain sesquiterpenoids, phenypro-
panoids and benzenoids. In addition, plant tissues can produce
volatile aldehydes and their corresponding alcohols, and acids as
well as volatile ketones. These compounds are occasionally found
in essential oils, but are usually formed in specic plant tissues
and under specic physiological conditions that favour catabolic
reactions. They can be considered as belonging to the primary
metabolism, although they can have useful fragrance, avour or
medicinal qualities.
The commercial interest on volatiles stems from their aro-
matic and avour qualities. Several of them, have signicant
antimicrobial and antineoplastic activity. Others act as messen-
gers in communication between plants themselves or with
other organisms. Volatiles are obtained from plants by distilla-
tion at or by extraction with ethanol, diethyl ether, chloroform,
pentane, hexane, benzene or other organic solvents. Unfortu-
nately, volatiles, like most secondary metabolites, are present in
plant tissues in limited quantities. Plant seeds, owers, stems
and roots usually contain 0.110% v/w fresh weight essential oil
and often <0.1%. Cases of up to 20% v/w or even higher essen-
tial oil concentrations in plant tissues are also known. Even so, a
single compound can constitute 4090% of the oil and usually
is not the most useful one. The most desirable volatiles are often
present in the essential oil at concentrations <10% and even
less than 1%. Considering that in most cases the essential oil is
produced in only specic plant tissues, such as seeds or owers,
whose total mass in a single harvesting season is a small weight
percentage of the whole plant, it is obvious why obtaining
useful volatiles from cultivated plants can be an expensive
operation. Chemical manufacture of small organics, such as the
majority of plant volatiles, is often cheaper, so that the natural
product occupies a small percentage of the market. An example
is vanillin, nine-tenths of whose market involves the synthetic
product.
[1]
A factor determining the share market of the natural
* Correspondence to: Y. Gounaris, 10 Stoimenidou Street, 62045 Alistrati,
Greece. E-mail: igouna@otenet.gr

This article is part of the Special Issue of Flavour and Fragrance Journal
entitled, Aromatic Plants, Spices and Volatiles in Food and Beverages, edited
by Ana Cristina Figueiredo and M. Graa Miguel
University of Thessaly, Department of Agriculture, Fytokou Street, 38446 New
Ionia, Magnesia, Greece

This article was published online on 12 May 2010. The funding information in
this footnote has been removed. This notice is included in the online and print
versions to indicate that both have been corrected 28 May 2010
Review
Received: 27 March 2010; Accepted: 27 March 2010; Published online in Wiley Online Library: 12 May 2010
(wileyonlinelibrary.com) DOI 10.1002/j.1996
3
6
7
Flavour Fragr. J. 2010, 25, 367386 Copyright 2010 John Wiley & Sons, Ltd.
product compared to that of the synthetic is the recent
preference of consumers for it, partially stemming from the
belief that it is free from traces of harmful manufacturing arte-
facts and left-overs. Also, the chemical synthesis often results in
racemic mixtures of the product, giving an approximation only
of the natural avour qualities. There is a need to reduce the
cost of the natural product, so it becomes available to a wider
range of consumers. From an environmental aspect, the produc-
tion of useful volatiles by non-chemical environmentally
friendly methods is always a preferred and often necessary
alternative.
Biotechnology attempts to facilitate the production, and
therefore to reduce the market cost, of natural volatiles by
employing a variety of non-polluting methods. The initial
attempts with plant materials consisted of eorts to produce
volatiles in plant cell or tissue cultures, either by de novo synthesis
or by biotransformation of cheap precursors into high-value
products. Before that, biotransformation eorts involved fungi,
yeast and bacterial cultures. Semi-synthetic methods, in which a
precursor is transformed into a useful product by isolated
enzyme preparations, crude or puried, have also been
attempted. Recently, most eorts have involved metabolic engi-
neering of the biosynthetic pathways leading to the synthesis of
the desired volatile. The various methods of biotechnology for
producing useful volatiles are discussed in this paper.
Types of Volatile Metabolites with Flavour
and Fragrance Qualities or with Biological
Action and Their Biosynthesis
Of the terpenoids, only members of the mono- (C10) and ses-
quiterpenoid (C15) classes are suciently volatile. Volatility is
determined not only by the size of the molecule and its stere-
ochemistry, but mainly by its ability to form hydrogen bonds.
Monoterpenoids whose molecule contains only carbons and
hydrogens are very volatile. Those with one hydroxyl, keto,
peroxy, or epoxy group are still volatile, but those with more
hydroxyls are only slightly or not at all volatile. The same holds
for the sesquiterpenoids, where one hydroxyl group seems to
be the maximum tolerated hydrogen bond-forming function for
sucient volatility to be preserved in the molecule. Triterpe-
noids and higher-order terpenoids are not volatile. Phenylpro-
panoids and benzenoids, bearing up to one hydroxyl and no
carboxyl, are volatile. The simultaneous presence of a keto
group does not abolish the volatility. However, more hydroxyls
drastically reduce or completely abolish the volatile properties.
(Hydroxy)cinnamic acids are not suciently volatile, due to the
presence of the carboxyl group, unless it is esteried with a
volatile alcohol. Aliphatic and olenic aldehydes, monoalcohols
and monoketones are volatile for at least up to 12-carbon sizes.
As the molecule becomes smaller than ve carbons, even the
acids are volatile. Organic monocarboxylic acids, esteried with
volatile alcohols, are also volatile. This type of compounds can
be divided in two categories. One has linear carbon chains and
the other has a methyl side-chain.
Genetic engineering is a powerful method used to alter the
rate of volatile production by acting on the biosynthetic path-
ways leading volatile synthesis. A short discussion of these path-
ways seems pertinent at this point. The monoterpenoids are
produced by the plastidic methylerythritolphosphate (MEP)
path, whose sequence and enzymatic properties have been
elucidated almost to completion.
[29]
Its rate-limiting step is the
one catalysed by 1-deoxy-D-xylulose 5-phosphate synthase
(DXS). NADPH, CTP and ATP are required for its operation. Con-
densation of dimethylallyl diphosphate (DMADP) with isopente-
nyl diphosphate (IDP) by the action of geranyl diphosphate
(GDP) synthase leads to formation of the monoterpenoid pre-
cursor GDP. Like all prenyltransferases, the GDP synthase is a
rather slow catalyst. The cis isomer neryl diphosphate (NDP) is
also formed. Cyclization of GDP is catalysed by the also slow
cyclases, membrane-bound enzymes in the plastids and endo-
plasmic reticulum. The hydroxylations of linear or cyclic monot-
erpenes are catalysed by NADPH-consuming, cyt450-dependent
monoxygenases, utilizing molecular oxygen, but also able to use
hydrogen peroxide produced from any source. These hydroxy-
lases are inducible by a variety of biotic or abiotic stress factors.
Sesquiterpenoids are considered to be synthesized in the
cytosol from farnesyl diphosphate (FDP), derived from the
mevalonic acid pathway. Two NADPH and three ATP molecules
are consumed for FDP synthesis and the rate-limiting step is
catalysed by 3-hydroxymethylglutaryl coenzyme A reductase
(HMGR). Sesquiterpene cyclases act on FDP to produce at least
200 types of cyclic sesquiterpenoids.
The phenylpropanoids are produced from phenylalanine and
tyrosine, both derived from the shikimic acid pathway. A multi-
tude of feedback-inhibited steps and a need for NADPH and ATP
in the shikimic acid path ensure a tight regulation of Tyr and Phe
synthesis. The requirement for NADPH is even greater in the
transformation of Phe and Tyr into phenylpropanoids. It is
required for the removal of the carboxyl group of the propenyl
side-chain by successive reductions that form the corresponding
volatile aldehydes, alcohols and phenylpropenes. The carboxyl
group is rst esteried to coenzyme A, by specic ligases. Then it
is transformed to an aldehyde group by an oxidoreductase and
the aldehyde is reduced into an alcohol by an alcohol dehydro-
genase (ADH). Phenylpropenes are then produced from the
alcohols. NADPH is also required for the hydroxylations of the
aromatic ring. Benzoic and phenolic acids come from the corre-
sponding hydroxycinnamic acids by b-oxidation of the propenyl
chain, followed by oxidative decarboxylation. This process is
tightly regulated by feedback inhibition. Volatile derivatives
are then formed by the reduction of the carboxyl group, as in
phenylpropanoids.
Non-branched volatile aldehydes and their corresponding
alcohols can be derived by degradation of unsaturated fatty
acids, mainly linoleic and linolenic acid,
[1012]
by the sequential
action of lipoxygenases (LOX), hydroperoxide lyases (HPL) and
ADH. The initial introduction of molecular oxygen into the
carboncarbon double bonds requires NADPH. These are cata-
bolic reactions of the primary metabolism. Methyl-branched
compounds, such as isovaleric and isobutyric acid, are derived
from leucine and valine catabolism. Isovaleric acid could poten-
tially be produced from DMADP of the terpenoid synthesis
pathways. According to Hschle et al.,
[13]
branched volatiles can
be produced by catabolism of lineal terpenoids and of leucine-
derived 3-methyl-crotonyl-CoA, the precursor of isovaleric acid,
via degradation of the produced 3-methyl glutaryl-CoA. In bac-
teria at least, they could be produced from linear monoterpe-
noid or even sesquiterpenoid degradation.
[14,15]
Unlike the LOX
path, the degradations of leucine, valine, monoterpenoids and
DMADP, leading to volatile aldehyde, alcohol and acid produc-
tion, do not need reductive equivalents but rather produce
them.
3
6
8
Y. Gounaris
Flavour Fragr. J. 2010, 25, 367386 View this article online at wileyonlinelibrary.com Copyright 2010 John Wiley & Sons, Ltd.
De novo Production of Volatiles by Tissue
and Cell Cultures
Plant Tissue and Cell Cultures
Volatiles in callus and cell cultures. Producing compounds in
plant cell, callus or tissue cultures has been attempted to ensure
a stable supply and quality of the product. Some of the plants
used as sources for the desired substance are rare, slow-growing
and found in not easily approachable regions of the world and
dicult to cultivate. The in vitro cultures were expected to speed
up the biomass propagation rate and to have it under controlled
conditions and immediately available. These attempts encoun-
tered serious diculties. The rate of secondary metabolite pro-
duction by in vitro-cultured plant cells is orders of magnitude
lower than that in the intact plant, usually in the range 0.1
0.01 g/l day.
[16]
Volatile compound yields are still lower and vola-
tile secondary metabolites are present often in trace amounts
detected in cultures of various plant species, examples of which
are given in Table 1. Although cases of cultures showing higher
yields of secondary metabolites than the intact plant are
known,
[41]
they are not involving volatiles. In many cases, the
volatiles found in the intact plant are not present at all in the in
vitro cultures. They are often dierent than those present in the
intact plant. Volatile aldehydes, alcohols, ketones and acid esters
appear more frequently or for the rst time in in vitro cultures. The
terpenoids produced are in most cases glycosylated.
The reasons for the reduced ability of the in vitro cultures
to produce volatiles, and secondary metabolites in general,
are not known with certainty. The cultured cells and callus seem
to have some enzymatic activity for terpenoid production.
[4244]
Geranyl diphosphate synthase activity has been detected in
plastids
[45]
but sesquiterpene cyclase has not.
[4648]
According
to Falk et al.,
[49]
the inability of cultured plant cells and callus
to accumulate signicant amounts of monoterpenes, could be
due to the combined eect of lower enzymatic activity and
their higher catabolic rate. Concerning the phenylpropanoid
synthesis potential, enzymatic activities of phenylalanine
ammonia lyase, shikimate dehydrogenase, cinnamic acid-4-
hydroxylase, p-coumaric acid-3-hydroxylase, cinnamoyl-CoA
reductase, 4-coumarate:CoA ligase, 4-hydroxycinnamate:CoA
ligase, cinnamyl alcohol dehydrogenase and caeic acid-O-
methyltransferase have been detected in callus or cell suspen-
sions and are often equal to those in the intact plant.
[5054]
Of the
enzymes of the volatile aldehyde and alcohol synthesis path,
discussed below, lipoxygenase and hydroperoxide lyase activity
has been found to be present in in vitro-cultured plant tis-
sues.
[29,55,56]
In cell suspension cultures of alfalfa, the hydroper-
oxide lyase activity was rate-limiting.
[57]
The ability of cultured
plant tissue and cells to produce volatiles is inducible by a
variety of chemical and physical factors, as is also the ability for
secondary metabolite synthesis in general. The induction treat-
ments increase the essential oil yield by up to ve-fold
[58,59]
in
some oils containing novel terpenes or in oils of altered relative
percentage.
[6062]
Even under the optimum induction conditions,
the yield of essential oil by in vitro-cultured plant tissues and
cells is usually less than that achieved by the intact untreated
plant. Therefore, using cultured plant cells and calli for volatile
production, even with the inclusion of elicitors and other
inducers, does not seem to be a particularly promising
undertaking.
Volatiles in hairy roots. It is a general observation that sec-
ondary metabolite yield by cell and callus cultures increases if
some degree of cell dierentiation is induced. Genetic transfor-
mation of plant tissue by insertion of the T-DNA regions of the Ri
plasmid of Agrobacterium rhizogenes results in the formation of
small, ne, hair-like root structures, known as hairy roots. Four
of the 18 ORFs in theT
L
-DNAare essential for hairy root formation,
of which ORF11 (rolB) is absolutely necessary. The T
R
-DNA carries
two auxin synthesis genes, but by itself does not provoke hairy
roots formation. Hairy roots lack geotropism, are highly branched
and can be cultured in bioreactor facilities needing no plant
growth regulators, since the inserted T-DNA carries genes for
auxin synthesis. They grow as fast, or faster, than normal roots,
with meristem cell cycles averaging 10 h. They produce second-
ary metabolites at levels and patterns similar to those of normal
roots, but also metabolites produced in aerial parts of the plant.
Often novel compounds are also produced. Unlike cell or callus
cultures, hairy roots are biochemically stable and the T-DNA is
stably integrated.
Excellent reviews on the culture methodologies and morpho-
logical and biochemical characteristics of hairy root cultures,
including their potential for secondary metabolite production,
have been published.
[42,6366]
Most of these reviews cover the wide
spectrum of secondary metabolites and the preponderance of
the cited cases concerns the production of non-volatile com-
pounds, primarily alkaloids and secondarily some phenolics and
non-volatile higher terpenoids, with a few cases involving vola-
tiles. However, Figueiredo et al.
[63]
focused on essential oils only;
they listed 11 plant species whose hairy root cultures can synthe-
size essential oil constituents. Among these, Pimpinella anisum
and Achillea millefolium hairy roots are capable of essential oil
yields similar to, or even higher than, those obtained with the
roots of the parent plants. It is clear that hairy roots have the
potential for synthesizing both volatile and non-volatile
terpenoids.
Table 2 presents a list of additional examples, specically for
volatile compounds produced by hairy roots, mostly drawn from
Figueiredo et al.
[63]
In these, the essential oil was analysed to some
extent, although the authors also cite the cases of Daucus carota
and Leontopodium alpinum hairy roots, whose main component
was not identied. The yields are greatly elevated in comparison
to those of cell or callus cultures and can be further increased by
the inclusion of abiotic or biotic elicitors in the culture medium. A
prospect for further increasing the volatile production fromhairy
roots is to genetically engineer their volatile production paths
using transgenes inserted into the T-DNA region.
Volatile Synthesis by Cultured Microorganisms
Although a great deal of research on the biotechnology of vola-
tiles still involves plants, especially eorts to increase terpenoid
and phenolics production in transgenic plants, most of the recent
eort is directed to using microorganisms. Volatile aldehydes and
alcohols are far more easily produced by cultured microorgan-
isms, and eorts to genetically alter microbes for producing or
biotransforming terpenoids or phenolics were met with reward-
ing success. Therefore, in most cases microorganisms are used for
their production, instead of plant cell cultures. Also, microorgan-
isms (bacteria, algae and fungi, including yeasts) are sturdier than
plant cells under bioreactor conditions. They are better suited to
withstand the frictional stress imposed by the shaking proce-
dures as well as various temporary extremes of pH, temperature
3
6
9
Biotechnology for essential oils, avours and volatile isolates
Flavour Fragr. J. 2010, 25, 367386 View this article online at wileyonlinelibrary.com Copyright 2010 John Wiley & Sons, Ltd.
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3
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Y. Gounaris
Flavour Fragr. J. 2010, 25, 367386 View this article online at wileyonlinelibrary.com Copyright 2010 John Wiley & Sons, Ltd.
and salt or deleterious metabolite concentrations created during
the culture process. All classes of volatiles can be produced by
microorganisms (Table 3), but aldehydes, alcohols and organic
acid esters are in far greater preponderance, indicating a strong
participation of catabolic processes. Microorganisms producing
volatiles have also been previously cited in several review
articles.
[93,118124]
In most cases the yield of the main compound is
<100 mg/l. Far better yields are attained when a substrate bio-
chemically more immediate to the volatile product is used, or
when an immediate precursor is included in the culture medium
together with the crude substate, a case closer to the biotrans-
formation procedures discussed in the next section. For example,
2-phenylethanol production by Kluyveromyces and other yeasts
can exceed 400 mg/l.
[91,112]
2-Phenylethanol is a rose-like aroma
with an annual market of 7000 tons. Chemically produced, is 250
times cheaper than its natural counterpart.
[91]
High yields of
benzaldehyde (almost 10 g/l) were also achieved by cultured
Goetrichum candidum.
[88]
Production of the peach-like aroma
g-decalactone by Yarrowia lipolytica and various Candida species
can exceed 10 g/l if castor oil is included in the culture.
[81,116]
Biotransformations
Biotransformations by Plant and Microorganism Cultures
Not all steps leading to the synthesis of a volatile are blocked to
the same extent in cultured plant cells and calli. Included in the
culture media, an intermediate compound of the path can often
be (bio)transformed or (bio)converted into volatile products by
residual enzymatic activity downstream of the blocked step(s).
Such biotransformation procedures are most successfully used
with microorganisms cultured in supplemented media. The
yields are far better than those in media not supplemented with
a close precursor molecule. Ideally, to keep the cost low and the
process economically worthwhile, cheap abundant precursors
are sought, able to biotransform into high-value products. The
issue of secondary metabolite bioconversions has been devel-
oped in several recent reviews
[41,78,93,118127]
and the discussion
here is limited to volatiles only. Some examples are given in
Table 4.
Acommon problemin biotransformations involving volatiles is
that asubstratemight not bedissolvedeasily inaqueous medium.
Agents facilitatingits dissolutioncanbeused, but it must beborne
in mindthat above a thresholdconcentration the substrate might
hinder cell growth. The same holds for the volatile product, which
above a threshold concentration could feedback-inhibit its own
synthesis or could be poisonous to the cells. These practical prob-
lems limit productivity. Methods for continuous addition of
substrateat non-harmful concentrations andremoval of theprod-
uct(s) have evolved. Among these, the pervaporation method
[230]
and the inclusion of volatile-binding materials in the culture
media are commonly used. The issue of sequestering the product
has been discussed by Ramachandra and Ravishankar
[41]
and
Georgiev et al.
[65]
Addition to the culture media of a non-soluble
solid or non-miscible hydrophobic liquid phase creates two-
phase systems, consisting of two immiscible liquids or of a solid
and a liquid phase, in which systemthe desired volatile is seques-
teredbybindingnon-covalentlytothesolidor tothehydrophobic
liquid phase. For example, including the Amberlite resin XAD-4 in
the cell cultures of Vanilla fragrans increased the yield of vanilla
avour. Alternatively, theproduct canbeboundnon-covalently to
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Biotechnology for essential oils, avours and volatile isolates
Flavour Fragr. J. 2010, 25, 367386 View this article online at wileyonlinelibrary.com Copyright 2010 John Wiley & Sons, Ltd.
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Flavour Fragr. J. 2010, 25, 367386 View this article online at wileyonlinelibrary.com Copyright 2010 John Wiley & Sons, Ltd.
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Biotechnology for essential oils, avours and volatile isolates
Flavour Fragr. J. 2010, 25, 367386 View this article online at wileyonlinelibrary.com Copyright 2010 John Wiley & Sons, Ltd.
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3
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4
Y. Gounaris
Flavour Fragr. J. 2010, 25, 367386 View this article online at wileyonlinelibrary.com Copyright 2010 John Wiley & Sons, Ltd.
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3
7
5
Biotechnology for essential oils, avours and volatile isolates
Flavour Fragr. J. 2010, 25, 367386 View this article online at wileyonlinelibrary.com Copyright 2010 John Wiley & Sons, Ltd.
soluble additives. b-Cyclodextrins are water-soluble molecular
cages that, properly modied, could sequester the desired
product into their cavities. In addition, this connement of the
product into the clathrate cage protects it from exposure to
degrading conditions in the culture medium.
Of the volatiles listed in Table 4, vanillin is of the highest
importance, followed by benzaldehyde, capsaicin, g-decalactone
and 2-phenylethanol. The commercial aspects, the organoleptic
and bioactivity qualities, the uses and the production method-
ologies of these volatiles have been discussed by Cheetham,
[202]
Dignum et al.,
[1]
Garavaglia et al.,
[91]
Krings and Berger,
[118]
Ramachandra and Ravishankar,
[41]
Schade et al.
[231]
and
Vandamme and Soetaert.
[78]
Vanillin is produced at about 12 000
tonnes annually, primarily from guaiacol. Only 20 tonnes are
produced by extraction from vanilla beans. It is used as a food
additive, but also possesses antimicrobial, antioxidant and anti-
mutagenic qualities. The biotransformation of ferulic acid to van-
illin using Amycolatopsis can produce up to 12 g/l culture
medium,
[207]
whereas yields in the range 13>60 g/l have been
achieved with Streptomyces species.
[227229]
Capsaicin, the major
pungent compound in chilli peppers, is produced by the same
biosynthetic path as vanillin. It is used as a food additive and a
counter-irritant in rheumatisms and neuralgias. Of commercial
interest could be the method of producing it using immobilized
placenta cultures of Capsicum frutescens, biotransforming cou-
maric acid.
[232]
The avour compound benzaldehyde is the
second most important volatile. Currently, it is obtained form
amygdalin. Biotransformation of phenylalanine to benzaldehyde
by Ischnoderma benzoinum seems to be a possible alternative,
since yields of 1 g/l can be achieved by in situ recovery meth-
ods.
[118,213]
The peach-smelling g-decalactone, product of lipid
catabolism, is also easily made using chemical synthesis. The
biotransformation of ricinoleic acid by Candida sorbophila can
produce up to 40 g/l g-decalactone;
[174]
Mucor circillenoides
achieves over 10 g/l from fatty acid esters;
[186,187]
and Yarrowia
lipolytica produces 5 g/l of a mixure of g-lactones, mainly
g-decalactone, from methyl ricinoleic acid.
[204,205]
These values are
much higher than the theoretical limit of about 1 g/l required for
consideration of a procedure as an alternative commercially
interesting process. The same holds for the rose avour com-
pound 2-phenylethanol, product of the phenylpropanoid path,
obtained by biotransformation of phenylalanine. Kluyveromyces
marxianus, cultured in the presence of phenylalanine, produces
over 26 g/l 2-phenylethanol
[123,185]
and Sacharomyces cerevisiae
yields 12.6 g/l from oleic acid and 7 g/l from phenylalanine.
[198]
An inspection of Tables 3 and 4 shows the superiority of micro-
orgamisms compared to cultured plant cells, as far as yields of
volatile compound production are concerned.
Biotransformations by Isolated Enzymes
Isolated enzyme systems often have a higher biotransformation
rate than cell-based procedures, but the cost of isolating the
enzyme is a basic factor determining their employment.
[233]
Enzymes exhibit high stereo-selectivity in the conversion
process, minimizing the production of undesirable isomer and
enantiomer by-products often accompanying cell-mediated
biotransformations.
[234]
However, only about 7% of the studies
on biotransformation involve isolated enzymes, the rest being
concerned with whole-cell systems.
[125]
Although they have
been applied to a signicant extent,
[126]
only a few cases of
biotransformations involve volatiles. Some examples are given
in Table 5. The yields are improved by immobilizing the enzyme
on polymer, glass or other supports, or by encapsulating it inside
membranes. The resulting easy recoverability and re-use of the
enzyme lowers the cost of operation. Overall, and with the
exceptions of glycosidases and esterases, most of the enzymes
used in bioconversions catalyse the introduction of some
oxygen function in the substrate molecule. This reduces its vola-
tility, unless it splits the molecule into smaller volatile parts.
The majority of the isolated enzyme-catalysed bioconversions
involve lipid-derived volatile products. In the phenolic and ter-
penoid category, the most promising use is in liberating volatiles
from their glycosylated forms. Of particular importance, as far as
yields are concerned, is the formation of green note avours,
hexenols and hexenals, from fatty acids by the action of hetero-
geneous crude enzyme preparations
[262]
and the liberation of
vanillin from glycovanillin by the action of commercial pectinase
and cellulase.
Metabolic Engineering
Genetic engineering of metabolic paths is the newest and most
promising eort for improvement of volatile production. There
are several excellent reviews on metabolic engineering of the
secondary metabolite pathways in general
[263266]
and also on
terpenoid paths specically.
[267269]
This review concentrates
on cases concerning the production of volatile compounds.
Genetically Engineered Microorganisms
Bacteria and yeasts have been genetically altered, either to
improve their synthesis of volatiles or to produce new ones the
microorganism is normally unable to synthesize. A list of promis-
ing examples is given on Table 6. Microorganisms are particularly
well suited for fermentation and general culturing procedures.
Genetically manipulating them to produce volatiles from raw
substrates, in commercially worthwhile quantities, would be a
major breakthrough in biotechnology. Although most of the
achieved yields are still in the mg/l scale, vanillin can be produced
at 5 g/l by E. coli carrying the 3-deoxyarabino-heptulosonic acid
7-phosphate synthase gene
[271]
and amorphadiene yields of
37 g/l have been observed with S. cerevisiae transformed with
operons of the methylerythritol and mevalonate path genes.
[283]
Yarrowia lipolytica with genetically engineered genes of the
b-oxidation genes is able to reach 10 g/l g-decalactone produc-
tion from ricinoleic acid esters.
[285289]
Volatile dicarboxylic acids
can accumulate at amounts up to 80 g/l, when various yeasts
having genetically interrupted b-oxidation and amplied
w-hydroxylation were fed with fatty acids and alkanes.
Genetically Engineered Plants
Terpenoids. A compilation of some of the metabolic engi-
neering eorts concerning the monoterpene- and
sesquiterpene-synthesizing paths is presented in Table 7. The
CaMV35S promoter was commonly used in such studies.
However, expression of the transgene often proved tissue-
specic. Co-suppression instead of overexpression was also a
common occurrence. Most successful was the overexpression of
the DXR of the MEP path, where a 50% increase in total essential
oil yield in peppermint was attained.
[293]
Monoterpene cyclases
are slow enzymes and higher yields of cyclic monoterpenes
were achieved by overexpressed limonene synthase, especially
3
7
6
Y. Gounaris
Flavour Fragr. J. 2010, 25, 367386 View this article online at wileyonlinelibrary.com Copyright 2010 John Wiley & Sons, Ltd.
T
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Biotechnology for essential oils, avours and volatile isolates
Flavour Fragr. J. 2010, 25, 367386 View this article online at wileyonlinelibrary.com Copyright 2010 John Wiley & Sons, Ltd.
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Flavour Fragr. J. 2010, 25, 367386 View this article online at wileyonlinelibrary.com Copyright 2010 John Wiley & Sons, Ltd.
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9
Biotechnology for essential oils, avours and volatile isolates
Flavour Fragr. J. 2010, 25, 367386 View this article online at wileyonlinelibrary.com Copyright 2010 John Wiley & Sons, Ltd.
when directed to plastids. Although overexpression of rate-
limiting steps (DXR, cyclases) increased the total or specic yield
of monoterpenes, altering the expression of enzymes for non-
rate-limiting steps, or for more downstream steps, usually
altered their relative percentages. This might be advantageous if
the yield of the desired compound increases. Boosting the pro-
duction of monoterpene alcohols, as by overexpressed linalool
synthase, also increases the rate of their glycosidation, so that
the yield of the glycosidated forms can far exceed that of the
free form. Overexpressing transgene prenyltransferases of ses-
quiterpene synthesis, such as the farnesene, nerolidol and TSP10
synthases, increases the linear and some cyclic sesquiterpenes
appreciably, and these can even be detected in plants not
normally producing them. More limited was the success with
non-regulated sesquiterpene cyclases, where only small
increases in the specic sesquiterpene yield were observed.
Shikimic acid-derived volatiles. Eects of both, overexpres-
sion and silencing of enzymes for synthesis of shikimate-derived
phenolic in transgenic plants are known (Table 8). Manipulating
the pre-tyrosine and pre-phenylalanine part of the shikimic acid
path does not seem very promising, due to the presence of mul-
tiple feedback-inhibited regulatory steps, which would nullify the
overexpression results. The same seems to be true for the pro-
duction of benzenoids from phenylpropanoids, although paths
not involving b-oxidation have been reported, whose regulation
characteristics, however, are still not dened.
[315]
At downstream
sites, overexpressions of aromatic amino acid decarboxylases,
alcohol dehydrogenase and alcohol acetyltransferase all resulted
in higher volatile phenolic yield. Reports on the eect of overex-
pression of transcription factor genes on volatile yield, as
reported by Memelink et al.
[263]
for avonoid and indole alkaloids,
are not yet known for the shikimic acid path. This approach
seems, however, to be most promising and RNAi silencing of a
myb-type trans-factor in Petunia strongly reduced the levels of
volatile benzenoids.
Lipid and amino acid-derived volatiles. Metabolic engineer-
ing of volatile production achieved its most spectacular results
with the levels of aldehydes, alcohols and organic acid esters.
Examples are given in Table 9. For compounds produced by deg-
radation of unsaturated fatty acids, the largest increases (even
ve-fold) were observed by overexpression of desaturases and
lipoxygenases. Only moderate increases were achieved by over-
expressed hydroperoxide lyase. With alcohol dehydrogenase the
results were not unambiguous. In some cases, its upregulation
only increased the amount of alcohols, leaving the aldehydes
unchanged, and in other cases no signicant changes were
observed for either type of compound. Since ethylene is involved
in the signal transduction path for stimulation of lipid-derived
aldehyde and alcohol synthesis, attempts were made to change
the activity of 1-amino- cyclopropane-1-carboxylate synthase
(ACS), the enzyme catalysing the synthesis of the ethylene pre-
cursor 1-amino-cyclopropane-1-carboxylic acid. Up to now, only
downregulation of ACS has been reported, accompanied, as
expected, by drastic decreases in the levels of volatile aldehydes
and alcohols.
Acknowledgements
The author would like to thank his colleagues for their recom-
mendations on improving this article.
T
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Y. Gounaris
Flavour Fragr. J. 2010, 25, 367386 View this article online at wileyonlinelibrary.com Copyright 2010 John Wiley & Sons, Ltd.
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Biotechnology for essential oils, avours and volatile isolates
Flavour Fragr. J. 2010, 25, 367386 View this article online at wileyonlinelibrary.com Copyright 2010 John Wiley & Sons, Ltd.
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