-
Biotechnology for the production of essentialoils, avours and
volatile isolates. A review.,
Y. Gounaris*
ABSTRACT: Various applications of biotechnologicalmethods for
the production of volatile compounds useful to the food
andpharmaceutical industries are discussed. The yields obtained
from intact or genetically modied plants are compared to
thoseachieved by microbial methods. Plant yields are too low for
the products to compete commercially to those
synthesizedchemically. Still lower yields are obtained with in
vitro-cultured plant tissues. Trangenic plants with altered
methylerythritolpath 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, themost spectacular yields were observedwith
biotransformations catalysed by microorganisms. Kluyveromyces
marxianus, produces over 26 g/l 2-phenylethanol fromphenyalanine,
whereas Candida sorbophila, Mucor circillenoides or Yarrowia
lipolytica can produce 540 g/l g-decalactone fromricinoleic 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 observedwith Saccharomycescerevisiae, bothwith the genetically
overexpressedmethylerythritol path. Genetically engineered
b-oxidation genes result inyields of 10 g/l g-decalactone
byYarrowia lipolytica and up to 80 g/l dicaboxylic acids by various
yeasts. These results far exceedthe theoretical limit of about 1
g/l required for consideration of a procedure as a commercially
interesting process, alternativeto chemical sythesis. Copyright
2010 JohnWiley & Sons, Ltd.
Keywords: bioreactor; biotechnology; essential oil; terpenoids;
volatiles
IntroductionThere 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
representonly 10% of the secondary metabolites existing in plants
and stillwaiting to be isolated and identied. Of these, terpenoids
com-prise the largest and structurally most varied class,
numberingover 40 000 dierent molecules. Members of the 10-carbon
ter-penoids, the monoterpenoids, are constituents of essential
oilsproduced 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
producevolatile aldehydes and their corresponding alcohols, and
acids aswell as volatile ketones. These compounds are occasionally
foundin essential oils, but are usually formed in specic plant
tissuesand under specic physiological conditions that favour
catabolicreactions. They can be considered as belonging to the
primarymetabolism, although they can have useful fragrance, avour
ormedicinal qualities.
The commercial interest on volatiles stems from their aro-matic
and avour qualities. Several of them, have signicantantimicrobial
and antineoplastic activity. Others act as messen-gers in
communication between plants themselves or withother 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,
stemsand roots usually contain 0.110% v/w fresh weight essential
oiland often
-
product compared to that of the synthetic is the
recentpreference of consumers for it, partially stemming from
thebelief that it is free from traces of harmful manufacturing
arte-facts and left-overs. Also, the chemical synthesis often
results inracemic mixtures of the product, giving an approximation
onlyof the natural avour qualities. There is a need to reduce
thecost of the natural product, so it becomes available to a
widerrange of consumers. From an environmental aspect, the
produc-tion of useful volatiles by non-chemical
environmentallyfriendly methods is always a preferred and often
necessaryalternative.
Biotechnology attempts to facilitate the production,
andtherefore to reduce the market cost, of natural volatiles
byemploying a variety of non-polluting methods. The initialattempts
with plant materials consisted of eorts to producevolatiles in
plant cell or tissue cultures, either by de novo synthesisor by
biotransformation of cheap precursors into high-valueproducts.
Before that, biotransformation eorts involved fungi,yeast and
bacterial cultures. Semi-synthetic methods, in which aprecursor is
transformed into a useful product by isolatedenzyme preparations,
crude or puried, have also beenattempted. Recently, most eorts have
involved metabolic engi-neering of the biosynthetic pathways
leading to the synthesis ofthe desired volatile. The various
methods of biotechnology forproducing useful volatiles are
discussed in this paper.
Types of Volatile Metabolites with Flavourand Fragrance
Qualities or with BiologicalAction and Their BiosynthesisOf the
terpenoids, only members of the mono- (C10) and ses-quiterpenoid
(C15) classes are suciently volatile. Volatility isdetermined 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 andhydrogens are very volatile.
Those with one hydroxyl, keto,peroxy, or epoxy group are still
volatile, but those with morehydroxyls are only slightly or not at
all volatile. The same holdsfor the sesquiterpenoids, where one
hydroxyl group seems tobe the maximum tolerated hydrogen
bond-forming function forsucient 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 nocarboxyl, are volatile. The simultaneous presence of
a ketogroup does not abolish the volatility. However, more
hydroxylsdrastically reduce or completely abolish the volatile
properties.(Hydroxy)cinnamic acids are not suciently volatile, due
to thepresence of the carboxyl group, unless it is esteried with
avolatile alcohol. Aliphatic and olenic aldehydes, monoalcoholsand
monoketones are volatile for at least up to 12-carbon sizes.As the
molecule becomes smaller than ve carbons, even theacids are
volatile. Organic monocarboxylic acids, esteried withvolatile
alcohols, are also volatile. This type of compounds canbe divided
in two categories. One has linear carbon chains andthe other has a
methyl side-chain.
Genetic engineering is a powerful method used to alter therate
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 areproduced by
the plastidic methylerythritolphosphate (MEP)path, whose sequence
and enzymatic properties have been
elucidated almost to completion.[29] Its rate-limiting step is
theone 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 arather slow catalyst. The
cis isomer neryl diphosphate (NDP) isalso formed. Cyclization of
GDP is catalysed by the also slowcyclases, membrane-bound enzymes
in the plastids and endo-plasmic reticulum. The hydroxylations of
linear or cyclic monot-erpenes are catalysed by NADPH-consuming,
cyt450-dependentmonoxygenases, utilizing molecular oxygen, but also
able to usehydrogen 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
thecytosol from farnesyl diphosphate (FDP), derived from
themevalonic acid pathway. Two NADPH and three ATP moleculesare
consumed for FDP synthesis and the rate-limiting step iscatalysed
by 3-hydroxymethylglutaryl coenzyme A reductase(HMGR).
Sesquiterpene cyclases act on FDP to produce at least200 types of
cyclic sesquiterpenoids.
The phenylpropanoids are produced from phenylalanine
andtyrosine, both derived from the shikimic acid pathway. A
multi-tude of feedback-inhibited steps and a need for NADPH and
ATPin the shikimic acid path ensure a tight regulation of Tyr and
Phesynthesis. The requirement for NADPH is even greater in
thetransformation of Phe and Tyr into phenylpropanoids. It
isrequired for the removal of the carboxyl group of the
propenylside-chain by successive reductions that form the
correspondingvolatile aldehydes, alcohols and phenylpropenes. The
carboxylgroup is rst esteried to coenzyme A, by specic ligases.
Then itis transformed to an aldehyde group by an oxidoreductase
andthe aldehyde is reduced into an alcohol by an alcohol
dehydro-genase (ADH). Phenylpropenes are then produced from
thealcohols. NADPH is also required for the hydroxylations of
thearomatic ring. Benzoic and phenolic acids come from the
corre-sponding hydroxycinnamic acids by b-oxidation of the
propenylchain, followed by oxidative decarboxylation. This process
istightly regulated by feedback inhibition. Volatile derivativesare
then formed by the reduction of the carboxyl group, as
inphenylpropanoids.
Non-branched volatile aldehydes and their correspondingalcohols
can be derived by degradation of unsaturated fattyacids, mainly
linoleic and linolenic acid,[1012] by the sequentialaction of
lipoxygenases (LOX), hydroperoxide lyases (HPL) andADH. The initial
introduction of molecular oxygen into thecarboncarbon double bonds
requires NADPH. These are cata-bolic reactions of the primary
metabolism. Methyl-branchedcompounds, such as isovaleric and
isobutyric acid, are derivedfrom leucine and valine catabolism.
Isovaleric acid could poten-tially be produced from DMADP of the
terpenoid synthesispathways. According to Hschle et al.,[13]
branched volatiles canbe 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 LOXpath, the degradations of
leucine, valine, monoterpenoids andDMADP, leading to volatile
aldehyde, alcohol and acid produc-tion, do not need reductive
equivalents but rather producethem.
368
Y. Gounaris
Flavour Fragr. J. 2010, 25, 367386View this article online at
wileyonlinelibrary.com Copyright 2010 John Wiley & Sons,
Ltd.
-
De novo Production of Volatiles by Tissueand Cell Cultures
Plant Tissue and Cell Cultures
Volatiles in callus and cell cultures. Producing compounds
inplant cell, callus or tissue cultures has been attempted to
ensurea stable supply and quality of the product. Some of the
plantsused as sources for the desired substance are rare,
slow-growingand found in not easily approachable regions of the
world anddicult to cultivate. The in vitro cultures were expected
to speedup the biomass propagation rate and to have it under
controlledconditions 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
magnitudelower than that in the intact plant, usually in the range
0.10.01 g/l day.[16] Volatile compound yields are still lower and
vola-tile secondary metabolites are present often in trace
amountsdetected in cultures of various plant species, examples of
whichare given in Table 1. Although cases of cultures showing
higheryields of secondary metabolites than the intact plant
areknown,[41] they are not involving volatiles. In many cases,
thevolatiles found in the intact plant are not present at all in
the invitro cultures. They are often dierent than those present in
theintact plant. Volatile aldehydes, alcohols, ketones and acid
estersappearmore frequently or for the rst time in in vitro
cultures. Theterpenoids produced are in most cases
glycosylated.
The reasons for the reduced ability of the in vitro culturesto
produce volatiles, and secondary metabolites in general,are not
known with certainty. The cultured cells and callus seemto have
some enzymatic activity for terpenoid production.[4244]
Geranyl diphosphate synthase activity has been detected
inplastids[45] but sesquiterpene cyclase has not.[4648] Accordingto
Falk et al.,[49] the inability of cultured plant cells and callusto
accumulate signicant amounts of monoterpenes, could bedue to the
combined eect of lower enzymatic activity andtheir higher catabolic
rate. Concerning the phenylpropanoidsynthesis potential, enzymatic
activities of phenylalanineammonia lyase, shikimate dehydrogenase,
cinnamic acid-4-hydroxylase, p-coumaric acid-3-hydroxylase,
cinnamoyl-CoAreductase, 4-coumarate:CoA ligase,
4-hydroxycinnamate:CoAligase, 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 theenzymes of the volatile aldehyde and alcohol
synthesis path,discussed below, lipoxygenase and hydroperoxide
lyase activityhas 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 culturedplant tissue and cells to produce volatiles is
inducible by avariety of chemical and physical factors, as is also
the ability forsecondary metabolite synthesis in general. The
induction treat-ments increase the essential oil yield by up to
ve-fold[58,59] insome oils containing novel terpenes or in oils of
altered relativepercentage.[6062] Even under the optimum induction
conditions,the yield of essential oil by in vitro-cultured plant
tissues andcells is usually less than that achieved by the intact
untreatedplant. Therefore, using cultured plant cells and calli for
volatileproduction, even with the inclusion of elicitors and
otherinducers, does not seem to be a particularly
promisingundertaking.
Volatiles in hairy roots. It is a general observation that
sec-ondary metabolite yield by cell and callus cultures increases
ifsome degree of cell dierentiation is induced. Genetic
transfor-mation of plant tissue by insertion of the T-DNA regions
of the Riplasmid of Agrobacterium rhizogenes results in the
formation ofsmall, ne, hair-like root structures, known as hairy
roots. Fourof the 18ORFs in theTL-DNA are essential for hairy root
formation,of which ORF11 (rolB) is absolutely necessary. The TR-DNA
carriestwo auxin synthesis genes, but by itself does not provoke
hairyroots formation. Hairy roots lack geotropism, are highly
branchedand can be cultured in bioreactor facilities needing no
plantgrowth regulators, since the inserted T-DNA carries genes
forauxin 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
normalroots, but also metabolites produced in aerial parts of the
plant.Often novel compounds are also produced. Unlike cell or
calluscultures, hairy roots are biochemically stable and the T-DNA
isstably 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 thewidespectrum of secondary metabolites and the
preponderance ofthe cited cases concerns the production of
non-volatile com-pounds, primarily alkaloids and secondarily some
phenolics andnon-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 anisumand Achillea millefolium hairy roots are capable
of essential oilyields similar to, or even higher than, those
obtained with theroots of the parent plants. It is clear that hairy
roots have thepotential for synthesizing both volatile and
non-volatileterpenoids.
Table 2 presents a list of additional examples, specically
forvolatile compounds produced by hairy roots, mostly drawn
fromFigueiredo et al.[63] In these, the essential oil was analysed
to someextent, although the authors also cite the cases of Daucus
carotaand Leontopodium alpinum hairy roots, whose main componentwas
not identied. The yields are greatly elevated in comparisonto those
of cell or callus cultures and can be further increased bythe
inclusion of abiotic or biotic elicitors in the culturemedium.
Aprospect for further increasing the volatile production from
hairyroots is to genetically engineer their volatile production
pathsusing 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
terpenoidand phenolics production in transgenic plants, most of the
recenteort is directed to usingmicroorganisms. Volatile aldehydes
andalcohols are far more easily produced by cultured
microorgan-isms, and eorts to genetically alter microbes for
producing orbiotransforming terpenoids or phenolics were met with
reward-ing success. Therefore, in most cases microorganisms are
used fortheir production, instead of plant cell cultures. Also,
microorgan-isms (bacteria, algae and fungi, including yeasts) are
sturdier thanplant cells under bioreactor conditions. They are
better suited towithstand the frictional stress imposed by the
shaking proce-dures as well as various temporary extremes of pH,
temperature
369
Biotechnology for essential oils, avours and volatile
isolates
Flavour Fragr. J. 2010, 25, 367386 View this article online at
wileyonlinelibrary.comCopyright 2010 John Wiley & Sons,
Ltd.
-
Table
1.Vo
latiles
detected
inplant
cellor
callu
scu
ltures
Plan
tProd
ucts/rem
arks
Referenc
e
Agastacherogo
sa(Koreanmint)
VolatileC9-alde
hyde
san
dalcoho
ls,b
utan
edione
.Dieren
tfrom
thosein
intact
plants
[17]
Artem
isia
dracun
culus(tarrago
n)Prod
uctio
nof
phe
nylpropen
esof
theessentialo
il[18]
Citrus
sp.
C.pa
radisicallu
sprodu
ced40
volatiles
(mon
o-,sesqu
iterpen
es,ade
hyde
san
dhy
droc
arbon
s),186
mg/kg
FW.Thisis5%
ofpee
loilyield.
C.lim
onprodu
ced11
mon
oterpen
esan
dn-no
nana
l,40
mg/kg
FW.C
.auran
tifolia
gave
onlylim
onen
e,4.4mg/kg
FW
[19]
Citrus
sine
nsis
Novo
latilecompon
entswerede
tected
,but
embryog
eniccallu
sprodu
ced10
ingred
ientsof
oran
geoil
[20]
Citrus
aurantifo
liaProd
uced
citrals,terpen
ylacetate,do
decana
l[21]
Coleon
emaalbu
mMon
oterpen
es.M
oreifcu
ltures
unde
rlig
ht[22]
Eucalyptus
camaldu
lensis
Alkan
es,alken
es,alcoh
olsin
callide
rived
from
stam
ens
[23]
Eucalyptus
citriodo
raMon
oterpen
esin
callide
rived
from
immatureo
wers
[24]
Melissa
ocina
lis(balm)
Lowam
ountsof
2-phe
nylethan
ol,d-octalactone
[25]
Very
lowam
ountsof
C6-alde
hyde
s,-alcoh
olsan
d-acetate
asters.Large
conc
entrations
ofglycosides
ofne
rol,citron
ellol,
geraniol,1-octen
-3-ol
[26]
Men
thapiperita(pep
permint)
Mintoilcom
pon
ents
[27]
Ocimum
basilicum
Essentialo
ilingred
ients
[28]
Oleaeuropa
ea(olivetree
)Prod
ucemostof
thevo
latileC6-alde
hyde
s,-alcoh
olsan
d-acetylester
foun
dalso
inoliveoil
[29]
Orig
anum
acutiden
tsOrig
anum
oiling
redien
ts(38)
[30]
Orig
anum
vulgare
Form
ationof
n-alkane
s.Lack
ofterpen
oids
even
ingree
ncalli
[31]
Oryza
sativa
Volatilehy
droc
arbon
s,alcoho
ls,keton
esalde
hyde
s,esters.M
ostwerepresent
intheintact
plant
also
[32]
Petroselinum
crispu
m(parsley)
Both
types
ofcu
ltures
produ
cedno
nana
land
decana
l.Cellculturesprodu
cedalso
limon
ene,acetop
heno
ne,n
otfoun
din
callu
sor
inintact
plants.Nophe
lland
rene
,apiole,m
enthatrie
ne,tha
tas
foun
din
intact
plants
[33]
Salvia
ocina
lis(sag
e)Lo
wam
ountsof
essentialo
il[34]
Smyrnium
perfoliatum
a-Pine
ne[35]
Frag
aria
sp.(strawberry)
Lowam
ountsof
ethy
lbutyrate,butylbutyrate
[36]
1,2-Prop
aned
iol(a
vour
precu
rsor)
[37]
Tacomasambu
cofoliu
mAccum
ulationof
phe
nylpropan
oidglycosides.
[38]
Taraxacum
ocina
le(dan
delio
n)Acetate
butylester,2-methy
l-1-propan
ol,n
-butan
ol,4-phe
nyl-1
-butan
ol,terpineo
ls,4-hyd
roxy-4-m
ethy
l-2-pen
tano
ne,
acetate
[39]
Vanilla
plan
ifolia
Vanillin
[40]
370
Y. Gounaris
Flavour Fragr. J. 2010, 25, 367386View this article online at
wileyonlinelibrary.com Copyright 2010 John Wiley & Sons,
Ltd.
- and salt or deleterious metabolite concentrations created
duringthe culture process. All classes of volatiles can be produced
bymicroorganisms (Table 3), but aldehydes, alcohols and organicacid
esters are in far greater preponderance, indicating a
strongparticipation of catabolic processes. Microorganisms
producingvolatiles have also been previously cited in several
reviewarticles.[93,118124] In most cases the yield of the main
compound is
-
Table
3.Vo
latilecompou
ndsprodu
cedby
cultured
microorga
nism
s
Microorga
nism
Substrate/culture
type
Prod
uct/remarks
Referenc
e
Acetob
actersp.
Fuselo
ilMethy
lbutyricacid
(precu
rsor
toarom
as)
[78]
Aspergillu
sniger
Rice
branoil(4g/lferulicacid)
2.8g/lVan
illin
[79]
Cocon
utfat
2-Und
ecan
one,2-no
nano
ne,2-hep
tano
ne.40%
yield
[80]
Aspergillu
soryzae
castor
oil
g-Decalactone
,0.86g/l
[81]
Botryodiplod
iatheobrom
aeJasm
onicacid,110
0mg/l(10
0mg/gdrycells)
[82]
Cand
idagu
illierm
ondiiand
othe
rCa
ndidaspecies
Castoroil,de
cano
icacid
g-Decalactone
,upto
10g/lw
ithcastor
oilh
ydrolysate
[81]
Ceratocystism
briata
Co
eehu
sks/So
lid-state
12Vo
latiles,inc
luding
etha
nol,acetalde
hyde
,ethylacetate(m
aincompon
ent,25
0mg/lp
erkg
drysubstrate),ethy
lpropiona
te,isoam
ylacetate
[83]
Pervap
orationbioreactor
Estersan
dalcoho
ls.B
anan
a-likea
vour
[84]
Ceratocystismon
iliform
isPe
rvap
orationbioreactor
Ethy
l-,propy
l-,isob
utyl-a
ndisoa
myl-acetates,citron
ellol,ge
raniol.A
ll
