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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.
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