Banana flavor: insights into isoamyl acetate production

Sebastian Torres1, Ashok Pandey2 and Guillermo R. Castro3,4*

Planta Piloto de Procesos Industriales Microbiolgicos (PROIMI).

Av. Belgrano y Pasaje Caseros, T4001 MVB Tucumn, Argentina.

Biotechnology Division, Regional Research Laboratory.

Trivandrum-695 019, India.

CINDEFI - Universidad Nacional de La Plata (CONICET, CCT La Plata).

Calle 50 y 115 (B1900AJL) La Plata, Buenos Aires, Argentina.

Department of Biomedical Engineering, School of Engineering, Tufts University.

4 Colby Street, Medford, MA 02155, USA.

*Corresponding author: Guillermo R. Castro

CINDEFI. Dept of Chemistry, School of Sciences,
Universidad Nacional de La Plata.
Calle 50 y 115. CP B1900AJL. La Plata, Argentina
E-mail: grcastro@gmail.com
Phone/fax: ++54.221.483.37.94 ext 132/103
Cell: ++549.221.155.778.776

Index
Title
1.2.-

3.-

4.5.-

Abstract
Introduction
Enzyme and whole-cell mediated synthesis of isoamyl acetate
a.Enzymatic synthesis of isoamyl acetate: lipases and carboxylesterases
b.- Whole-cell catalyzis for isoamyl acetate synthesis
Isoamyl acetate production by microbial cultures
a.Microbial fermentation processes
b.- Metabolic engineering of cells to improve fermentation processes
c.Bioconversion using wild-type and genetically engineered microorganism
Conclusions
References

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Abstract
Isoamyl acetate is one of the most important flavor compounds used in food industries
because of its characteristic banana flavor. The ester is used as a flavoring compound in many
foods and drinks, such as honey, butterscotch, artificial coffee, beverages and perfumes. Also
it is one of the major flavor components of fermented alcoholic beverages, such as sake, beer
and wines. The isoamyl acetate production is traditionally carried out via chemical synthesis
by Fischer esterification mechanism. However, because consumers are moving to foods
containing natural flavors due to environmental and health issues, biotechnology is emerging
as a competitive alternative to traditional chemical synthesis for the production of isoamyl
acetate. Enzyme and whole-cell biocatalysis and fermentation were recently proposed for
isoamyl acetate production that can be considered close to natural. These new bioprocesses
are in development stages, and most of them limited only to laboratory scale, however, they
have high potential to be industrially useful and could offer an alternative way to obtain
natural banana flavor. The current review discusses the myriad of reaction systems developed
for isoamyl acetate production.

1.- Introduction
Flavor esters are compounds of a great commercial importance due their application
mainly in food and cosmetic industries, but also as -environmentally benign- solvents and
intermediates in chemical and pharmaceutical processes (Torres et al., 2009a). Traditionally,
flavor compounds have been produced by chemical synthesis or extracted from plant
materials. Although high costs plus low yields of natural compounds procedure made this
technique inadequate at large scale.
Raising awareness among consumers about the safety of the products they use,
especially food and beverages they choose, preference for natural products, like flavor esters
has been increased. Additionally, legislation in the EU regarding to inform consumers about
natural or artificially flavoring substances contained in supplemented foods is mandatory
(Regulation EC n 1334). Also, in some cases artificial flavoring substances in supplemented
food were banned. So, high demand for natural flavors has led to extensive biotechnology
research to develop alternative production methods. Products derived from bioprocesses
starting with natural substrates can be considered as natural if they have already been
identified in natural sources. However, natural flavors are still limited to premium products
because they are expensive compared to chemically synthesized ones (Schrader et al., 2004).
An example is one of the most common flavor chemical, vanillin. Artificial vanillin is a cheap
flavor compound about US$ 11 - 20 kg1, meanwhile bio-vanillin derived from microbial
processes currently costs up to US$ 1400 kg1 and yields an estimated market volume of
5,000 tons annually (Schrader et al., 2004, Feron and Wach, 2006).
Although biotechnological processes are generally more expensive than chemical
ones, inherent advantages of biocatalysis have driven the research activities in this field.
Biocatalysis bring environmental and health advantages since inorganic catalysts in chemical
synthesis are classified as toxic and their products are labeled in foods as artificial. Enzymes
can be reused minimizing the catalyst inactivation and reaction residues (Romero et al.,
2007). In addition, enzymatic syntheses are typically very selective and highly specific (e.g.
production of optically-active compounds) and they are performed under mild reaction
conditions (moderate temperatures and pressures) compared with chemical syntheses
(Krishna et al., 2001). Despite supercritical media (Romero et al., 2005), and even aqueous
and free-solvent systems, have been studied as potential reaction media for enzymatic
synthesis (Guvenc et al., 2002, Macedo et al., 2003, Romero et al., 2007), the most used
media in biocatalysis are still organic solvents (Torres and Castro, 2004; Torres et al., 2009b).
Among flavor esters, low molecular weight ones from C1 (methyl) to C5 (amyl and
isoamyl) alkyl esters are the most wanted in food industries. Particularly, isoamyl acetate due
its strong banana flavor is the number one ester required in food industries (Torres et al.,
2009a). Isoamyl acetate is widely used as a flavoring compound in a variety of foodstuffs,
such as honey, butterscotch, artificial coffee and beverages. It is also one of the major flavor

components of fermented alcoholic beverages, such as sake, beer and wines. Annual demand
for isoamyl acetate in USA alone amounts to about 74,000 Kg (Welsh et al., 1990). In nature,
esters can be formed by several means. A well studied process is the esterification of alcohols
(which usually derived from amino acid degradation) catalyzed by lipases or esterases. Due
the advantages of these reactions, the synthesis of short chain esters catalyzed by these
hydrolytic enzymes has been extensively investigated for years (Macedo et al., 2003; Akoh et
al. 2004; Adachi and Kobayashi 2005; Joseph et al. 2008). Another well known process for
ester synthesis involves alcohol acyltransferases (EC 2.3.1.84) that convert alcohols and acylCoAs to their corresponding esters. Both processes have been studied as environmentally
friendly and health benign alternatives for the production of isoamyl acetate. The current
production of isoamyl acetate is traditionally carried out via chemical synthesis by Fischer
esterification mechanism using concentrated sulfuric acid as catalyst (Figure 1) (Welsh et al.,
1990). On the other side, extraction of this ester from plants in order to obtain this in a natural
way does not satisfy the global market needs, and is too expensive for commercial
exploitation. However, as it was previously mentioned biotechnology is emerging to compete
with chemical synthesis for the green production of isoamyl acetate. Enzyme, whole-cell
biocatalysis and fermentation were proposed for isoamyl acetate production that can be
accepted as natural. These new bioprocesses are in early stages, most of them limited to
laboratory scale only. However, might have high potential to be industrially useful and could
offer an alternative way to obtain natural banana flavour. The current article is reviewing the
state of the art of all the reaction systems studied for isoamyl acetate production.
2.- Enzyme and whole-cell mediated synthesis of isoamyl acetate
2.a.- Enzymatic synthesis of isoamyl acetate: lipases and carboxylesterases.
Carboxylesterases (E.C. 3.1.1.1, carboxyl ester hydrolases) and lipases (triacylglycerol
hydrolases, E.C. 3.1.1.3) are enzymes widely distributed among all forms of life; their
physiological functions have been implicated in many activities such as carbon source
reutilization, pathogenicity, and detoxification (Ewis et al., 2004). These biocatalysts have a
number of unique enzyme characteristics such as substrate specificity, regio-specificity, and
chiral selectivity (Jung et al., 2003). Useful reactions performed by these hydrolases that can
be highlighted are the resolution of racemic mixtures by transesterification, the
enantioselective hydrolysis of esters for obtaining optically pure compounds, and the
synthesis of flavor esters, among which include isoamyl acetate (Bornscheuer, 2002; Macedo
et al., 2003; Torres et al., 2008).
Many reaction systems were tested for carboxylesterase and lipase catalyzed the
esterification of isoamyl acetate. Some of them were summarized in Table 1. Generally, most
of these reactions occur in non-aqueous environments, achieving in many cases yields above
90% (Krishna et al., 2001). Also, the vast majority of esterifications were performed in

immobilized enzyme reactors to increase the stability and reuse of the biocatalyst in presence
of the organic solvent, which also enables their application in a continuous mode or over
several production batches. The best yield was obtained in n-hexane using acetic anhydride
and commercial immobilized Candida antarctica lipase B (CAL-B) as acyl donor and
catalyst, respectively (Romero et al., 2005b and 2007). Krishna et al. were able to synthesize
isoamyl acetate in n-heptane solvent, using an immobilized lipase from Rhizomucor miehei
obtaining yields above 95% (Krishna et al., 2000 and 2001). Employing acetic anhydride as
acyl donor instead of acetic acid the authors achieved an efficient synthesis of isoamyl acetate
with high yields, even at high concentrations of the acyl donor substrate. Direct esterification
using acetic acid as the acyl donor usually leads to low yields due to the inhibition of the
enzyme activity by the acid (Krishna et al., 2001).
Despite isoamyl acetate is a short-chain ester, the enzymatic synthesis reactions
studied up to now were mainly carried out using lipases. This concept is based on the popular
knowledge about lipases as very strong enzymes, like work horses, and also because they
are able to act efficiently at organic-aqueous interfaces and in water-restricted environments.
However, recently some researchers described the use of esterases for the successful synthesis
of isoamyl acetate. Immobilized type II esterase from Bacillus licheniformis S-86 was used to
synthesize isoamyl acetate from isoamyl alcohol and p-nitrophenyl acetate (acyl donor) in nhexane (Torres et al., 2009a). It is noteworthy that in contrast with other lipase-catalyzed
reactions, the resulting ester yield (42.8%) was obtained at a low temperature (28C) and with
a very low amount of enzyme (4.6 x 10-5 mg ml-1) (Torres et al. 2009a). These two
parameters, enzyme concentration and low reaction temperature are very important from the
economic viewpoint, making this enzyme attractive for a possible practical application.
The use of organic solvents in these reactions result of special interest since they
thermodynamically favors the occurrence of esterification reaction (Torres and Castro, 2004).
However, since isoamyl acetate is used above all as an ingredient in foods and drinks would
be ideal to reduce or eliminate the use of toxic organic solvents for enzymatic synthesis.
Elimination of organic solvents can also simplify downstream processing and make the
process economically exploitable by reducing production cost and safety. With this premise
Gvenc et al. (2002) carried out the synthesis of isoamyl acetate in a solvent-free system
using acetic acid and isoamyl alcohol as substrates, and two commercial immobilized lipases
(Lipozyme RM IM and Novozym 435) as biocatalysts. Novozym 435 resulted more efficient
than Lipozyme RM IM for isoamyl acetate production. After 6 hours of reaction at 30 C an
isoamyl acetate yield of 80% was obtained using alcohol:acid molar ratio of 2:1 (Gvenc et
al., 2002). Macedo et al. were able to synthesize isoamyl acetate with 80% yield in a solventfree system using a free lipase from Rhizopus sp. using the same substrates. This conversion
was achieved using an alcohol:acid molar ratio (2:1) after 48 hours at 40 C and in the same
conditions used in previous work (Gvenc et al. 2002; Macedo et al., 2003). The

transesterification of isoamyl alcohol with vinyl acetate using immobilized Rhizopus oryzae
NRRL 3562 lipase in a solvent-free system was also developed (Kumari et al., 2009). The
substrates had no inhibitory effect on immobilized lipase and maximum ester conversion of
95 % isoamyl acetate was reached after 8 hours of reaction at 40 C and 200 rpm.
Other alternatives include the use of green solvents (e.g. one of the substrates) or nonconventional solvents like supercritical fluids or ionic liquids (Wolfson et al., 2009; Varma
and Madras, 2008). In this procedure, triacetin (glycerol triacetate), a non-toxic and
biodegradable solvent, was successfully used as both green solvent and acyl donor in the
transesterification of isoamyl alcohol to produce isoamyl acetate using free and immobilized
lipase B from Candida antarctica (Wolfson et al., 2009). The use of triacetin also simplifies
the separation of product by simple extraction with petroleum ether or by distillation. On the
other hand, immobilized enzyme could be easily recovery by filtration.
Supercritical fluids are defined as fluids above their critical pressure and critical
temperature that represent a unique class of non-aqueous media for biocatalysis (Kamat et al.,
1995). Several reviews have described in detail enzymatic reactions in supercritical fluids
(Kamat, et al., 1995; Beckman, 2004; Hobbs and Thomas, 2007; Wimmer and Zarevcka,
2010). Strictly speaking, supercritical fluids do not play a chemical role in the reaction;
however, their special physical properties are generally used to enhance the reaction rates.
These fluids have gas-like low viscosities and high diffusivities that increase the mass transfer
rates of substrates to enzyme and product to the reaction medium. Contrariwise, supercritical
fluids posses liquid-like densities that result in higher solubilizing power than those observed
for gases. Nevertheless, unlike gases and liquids, the physical properties of a supercritical
fluid can be adjusted over a wide range by relatively small changes in pressure or temperature
(Kamat et al., 1995). Among the supercritical fluids that can be used as a solvent,
supercritical carbon dioxide (ScCO2) is extensively used because of its advantages: non-toxic,
less expensive, near ambient critical temperature (31.1 C), and moderate critical pressure
(72.8 atm). In ScCO2, lipozyme TM catalyzed the transesterification between ethyl acetate
and isoamyl alcohol with a yield of 120 g of isoamyl acetate per kilogram of enzyme per hour
(van Eijs et al., 1988). Romero et al. successfully synthesized isoamyl acetate from isoamyl
alcohol and acetic anhydride in ScCO2 (Romero et al., 2005). The authors used two different
immobilized lipases (Novozym 435 from Candica antarctica and Lipozyme RM-IM from
Rhizopus miehei) to synthesize isoamyl acetate in continuous operation. An esterification
extent of 100% was obtained with Novozym 435 and 77 % isoamyl acetate yield could be
reached in only 15 minutes. Besides, the advantages of supercritical fluids in biocatalysis, it is
important to realize that the equipment is still at laboratory scale only. Production, like in
pilot plants or higher scale, will require further equipment developments and intensive
process engineering research.

The other non-conventional media that have recently (since 2000) appeared as clean
alternative to classical organic solvents for a wide variety of enzymatic transformations are
ionic liquids (de los Ros et al., 2008; Pohar et al., 2009). Some ionic liquids have been
considered to be the best non-aqueous media for biocatalytic processes. The main advantage
of these media is their low vapor pressure, which besides its environmental benefit, also
facilitates recycling by means of evaporation. Ionic liquids are also non-flammable, non-toxic
and present high thermal, chemical and electrochemical stability; possess very good solubility
properties for a wide range of both inorganic and organic materials. Furthermore, the
physicochemical properties of ionic liquids can be modified by replacing the cation or anion
or by simple derivatization procedure, and the optimal ionic liquid can be designed for each
specific reaction system (de los Ros et al., 2008). In addition to all these advantages, ionic
liquids also exert a positive effect on enzymes, increasing their stability and activity and also
could modify their enantioselectivity. Lipases are part of these assertions, showing increased
activity and stability in ionic liquids. Candida antarctica lipase B (CaLB) has already shown
to be more thermally and operationally stable in imidazolium type ionic liquids and its
activity and solubility in ionic liquids were proved to be anion dependent and mutually
exclusive (Pohar et al., 2009). CaLB lipase was used in ionic liquids for the production of
isoamyl acetate from acetic acid or acetic anhydride (as acyl donor) and isoamyl alcohol.
Fehr et al. carried out the synthesis of this ester in biphasic mixture of isoamyl alcohol (in
excess) and 1-butyl-3-methylimidazolium-hexafluorophosphate ionic liquid. The optimized
reaction conditions gives near 100% isoamyl acetate yield (Fehr et al., 2008). Furthermore,
the ionic liquid and the enzyme were successfully reused together for 10 cycles, which make
the system very attractive from the practical application viewpoint and also environmental
aspect. CaLB lipase was also used in a continuously operated microreactor in the 1-butyl-3methylpyridinium dicyanamide/n-heptane two-phase system that enables the simultaneous
esterification and product removal with highly efficiency (Pohar et al., 2009). This solvent
system dissolved the lipase, which was attached to the ionic liquid/n-heptane interfacial area
due to its amphiphilic properties. The microchannel system used allowed the formation of a
flow pattern responsible of intense emulsification, which provided a large interfacial area for
the reaction and simultaneous product extraction. By using an excess of alcohol, a maximum
of about 150 % isoamyl acetate yield was achieved in this two-phase system (Pohar et al.,
2009). However, the main issue for ionic liquids application in biocatalysis is the absence of
systematic knowledge about the solvent properties. So, an efficient enzymatic reaction in
ionic solvents means the development of an extensively screening procedure of solvents
combined with the choice of all parameters of the system (i.e. enzyme, substrates, products,
biphasic or monophasic).
2.b.- Whole-cell catalyzis for isoamyl acetate synthesis.

The use of whole-cells as catalysts avoids some drawback in costs and labor:
production and purification of enzymes for their use as biocatalysts in industrial
biotransformation processes (Len et al., 1998). Furthermore, enzymes are often used in
immobilized forms and the use of a whole-cell may, in principle, protect the enzymes just as
an immobilization matrix does. Whole-cell systems are also especially advantageous where
enzyme cofactors or more than one enzyme participate in the biotransformation reaction.
Nevertheless, few studies on the production of isoamyl acetate using whole-cell catalysts
were performed. Hansenula mrakii cells cultured up to stationary phase under aerobic
conditions were used as biocatalysts for the production of isoamyl acetate ester from isoamyl
alcohol and acetic acid in a solvent-free system (Inoue et al., 1997). Esterification reaction
was carried out using 100 mg of intact cells in phosphate buffer at 25 C during 1 hour. Under
these conditions 33.9 g.l-1 of isoamyl acetate/10 g of cells/ hour were synthesized. The same
experience was made incubating H. mrakii cells in presence of isoamyl alcohol and acetylCoA for alcohol acyltransferase (AATFase) biocatalysis, isoamyl acetate was synthesized, but
in reduced amount compared with the synthesizes performed by esterase (Inoue et al., 1997).
With the discovery of extremophile microorganisms able to grow in presence of
organic solvents the use of cellular biotransformation systems has been extended (Torres et
al., 2005 and 2009b). This finding allowed the development of whole-cell biocatalytic process
that profits the well-known advantages of organic solvents catalysis. The main criterion is the
choice of the solvent. An adequate solvent selection is essential to exploit the full potential
that whole-cell catalysis offer (Nikolova and Ward, 1993; Len et al., 1998). The most
relevant criteria for solvent selection are high product recovery capacity and biocompatibility,
as well as a non-hazardous nature and low price. Whole-cell biocatalysis in organic solvents
was studied for the synthesis of isoamyl acetate; however, its application to the production of
this ester has not been investigated in depth. Lyophilized whole-cells of Rhizopus oryzae CBS
112-07 were used to promote the synthesis of isoamyl acetate in n-heptane (Molinari et al.,
1995). The direct esterification of isoamyl alcohol with acetic acid was performed using 20
g.l-1 lyophilized Rhizopus oryzae cells suspended in the organic solvent at 47 C during 24
hours. Although only a 12 % yield of the ester was achieved. The use of cells as catalysts may
be an attractive alternative to enzymatic synthesis for the production of isoamyl acetate and
should be studied in more detail.
3.- Isoamyl acetate production by microbial cultures
3.a.- Microbial fermentation processes
Microorganisms historically have played an essential role in the production of the
flavor components of many foods (Longo and Sanromn, 2006). They can be used to produce
flavor compounds, either specifically for application as food additives or in situ
accompanying the food fermentation processes. The aroma profile of alcoholic beverages

such as beer, wine or sake is dominated by those components that are formed during
microbial fermentation. In these fermented drinks, one of the most popular esters found is
isoamyl acetate, which is recognized as an important factor for determining the flavor quality
of beer, wine and especially sake. Isoamyl acetate is particularly important to confer the so
much appreciated fruit-like flavor to the latter.
In most cases synthesis of isoamyl acetate during microbial fermentation is carried out
intracellularly by enzyme alcohol acetyltransferase (AATase), which catalyzes the reaction
between isoamyl alcohol and acetyl-CoA coenzyme (Furukawa et al., 2003; Verstrepen et al.,
2003). In yeast, higher alcohols such as isoamyl alcohol are formed during fermentation via
catabolic (Erlich) and anabolic pathways (Ehrlich, 1904; Chen, 1978; Oshita et al., 1995). The
Ehrlich pathway implies the catabolism of branched-chain amino acids such as leucine and
valine, which begin with amino acids transamination by aminotransferases encoded by BAT1
and BAT2 to form -keto acid, a precursor of isoamyl alcohol (Eden et al., 1996). The
anabolic pathway catalyzes glucose (during amino acid biosynthesis) mainly through the
intermediate -keto acid, to form isoamyl alcohol. Isoamyl acetate ester is then synthesized in
the AATase catalyzed reaction between isoamyl alcohol and acetyl-CoA. In Saccharomyces
species three distinct AATases (AATase I, its closely related homologue Lg-AATase I, and
AATase II) were identified which are encoded by ATF1, Lg-ATF1, and ATF2 genes
(Verstrepen et al., 2003). These enzymes are strongly repressed when the yeasts are cultured
under aerobic conditions. Yeasts cells also produce esterases that hydrolyze esters, including
isoamyl acetate (Fukuda et al., 1998a). The major isoamyl acetate-hydrolyzing esterase was
encoded by IAH1 gene. Therefore, an appropriate balance of this esterase and AATases
activities is critical for an efficient production of isoamyl acetate (Fukuda et al., 1998a).
In order to improve the production of isoamyl acetate in fermented alcoholic
beverages, and hence the sensorial quality of the final product, several microbiological studies
were carried out. Wild-type microorganisms able to produce isoamyl acetate are summarized
in Table 2. Furukawa et al. discovered that inositol limitation in sake mash increases isoamyl
acetate content in sake by increasing Saccharomyces cerevisiae AATase activity. Inositol
addition increased phosphatidylinositol content in yeast cells, which strongly inhibited
AATase activity possibly due to its high adsorptive capacity for the AATase protein
(Furukawa et al., 2003). The production of isoamyl acetate, together with ethyl acetate and
amyl alcohol, by S. cerevisiae cultured using wort and molasses was improved when EDTA
and zinc ions were added to this media (Quilter et al., 2003). EDTA binds metal ions present
in the fermentation medium and minimizes their toxicity that could favor yeast growth. With
regard to zinc, it was reported that its addition to the brewing of lager fermentation increased
synthesis of higher alcohols by enhancing decomposition of -keto acids to their
corresponding aldehydes and their subsequent conversion to alcohols (Quilter et al. 2003).

10

The ability to produce isoamyl acetate was also studied in several so-called nonSaccharomyces yeasts. Plata et al. examined the performance of wine yeast strains in the
formation of isoamyl acetate and ethyl acetate during grape juice fermentation. The authors
studied seven yeast species that prevail at different times during fermentation process: low
activity fermentative yeasts (Picchia, Candida, Hansenula, and Kluyveromyces spp.) are the
first to grow, continued by intermediate yeasts (Kloeckera, Torulaspora spp.), and finally by
Saccharomyces cerevisiae which has high capacity of fermentation (Plata et al., 2003).
Kloeckera apiculata exhibited the highest ability for isoamyl acetate formation, which was
more strongly influenced by the availability of isoamyl alcohol than by AATase activity.
Whereas, Hansenula subpelliculosa, Kluyveromyces marxianus, Torulaspora delbrueckii and
Saccharomyces cerevisiae were able to produce intermediate levels of isoamyl ester (Plata et
al., 2003). Other non-Saccharomyces yeast, Williopsis saturnus (formerly Hansenula
saturnus) was employed for the production of natural isoamyl acetate using sugar beet
molasses as carbon source under anaerobic conditions. Williopsis saturnus was capable to
produce about 25 mg.l-1 isoamyl alcohol from 10 Brix sugar beet molasses, and this alcohol
was used for the ATTase catalyzed biosynthesis of 20.7 mg.l-1 isoamyl (Yilmaztekin et al.,
2008). Isoamyl acetate production by this strain could be improve when fusel oil
(approximately 4555% amyl alcohols content), a by-product obtained from the distillation of
alcohol made by fermentation of molasses, was added to fermentation medium (Yilmaztekin
et al., 2009). Bioconversion of isoamyl alcohol present in fusel oil led to an increase in 3-fold
isoamyl acetate concentration after the addition of 1% fusel oil to molasses based
fermentation medium.
Unlike the vast majority of yeasts, which have esterases that hydrolyze isoamyl acetate
ester, some yeasts, such as Hansenula mrakii, can use the reverse reaction of esterases for
synthesizing isoamyl acetate in the absence of acetyl-CoA (Inoue et al., 1997). This allows
Han. mrakii produce isoamyl acetate ester, also when the cells are cultured under aerobic
conditions. Similar to Han. mrakii, the Pichia anomala wine yeast was also proved to be an
efficient producer of isoamyl acetate under aerobic conditions (Rojas et al., 2001). This
property allows its use in mixed starters for wine production, in order to increase the
production of aroma compounds during the early stages of wine fermentation (when
respiration may be important). Indeed, mixed cultures of Saccharomyces cerevisiae T73 and P.
anomala 10590 produce in grape must 1.3-fold higher isoamyl acetate regarding S. cerevisiae
T73 pure culture (Rojas et al. 2003). An improved production of isoamyl acetate was also
observed using mixed cultures of Saccharomyces cerevisiae and Kloeckera apiculata (its
telemorph Hanseniaspora uvarum) or Williopsis saturnus under aerobic and anaerobic
conditions, respectively (Moreira et al., 2008; Erten et al., 2010).
Besides yeast, some fungi species are also able to produce isoamyl acetate during
fermentation processes, such as the fungus Ceratocystis moniliformis, which was able to

11

produce this ester among several aromas (Bluemke and Schrader, 2001). Soares et al. reported
the production of various flavor compounds, including isoamyl acetate, by Ceratocystis
fimbriata when solid-state fermentation was carried out using coffee husk as a substrate. The
addition of leucine to this medium increased ethyl acetate and isoamyl acetate production, and
generated a strong banana odour (Soares et al., 2000).
3.b.- Metabolic engineering of cells to improve fermentation processes
The major efforts to improve the microbial production of isoamyl acetate involve genetic
modification of cells (Table 3). In general, these modifications are aimed to increase the
production of isoamyl alcohol (a major rate-limiting factor for isoamyl acetate production),
the overproduction of AATases or disruption of esterases that hydrolyze isoamyl acetate.
As the substrate of AATases, enhancement of isoamyl alcohol levels in the culture
medium may increase isoamyl acetate synthesis. In Saccharomyces cerevisiae the enzyme isopropyl malate synthase, encoded by the LEU4 gene, catalyzes the production of isoamyl
alcohol. And the formation of -keto acid (an intermediate in the synthesis of isoamyl
alcohol) is prevented by the leucine-mediated feedback inhibition. In early attempts, Ashida et
al. isolated from sake, wine and beer Saccharomyces cerevisiae mutants lacking the feedback
inhibition of isoamyl alcohol synthesis caused by accumulated L-leucine. Using these
mutants, the concentration of isoamyl alcohol increased about three to four times and higher
amounts of isoamyl acetate were obtained consequently (Ashida et al., 1987). Later, Hirata et
al. achieved the overexpression of the LEU4 gene using a delta sequence in recombinant
Saccharomyces cerevisiae, which resulted in a strain yielding 1.75 times higher concentration
of isoamyl alcohol than the wild strain. In another attempt to overcome the feedback
inhibition, several mutants of Saccharomyces cerevisiae and Saccharomyces servazzi from
fermenting Japanese radish pickles were selected for resistance to a leucine analog (5,5,5trifluoroleucine). Some of these mutant strains were able to produce twice the concentration
of isoamyl alcohol (1,700 g.l-1) and isoamyl acetate (162 g.l-1) of the parental strains when
cultivated in wort and koji medium (Quilter et al. 2003; Tominaga et al. 2003).
An increase in the production of isoamyl alcohol, and thus in isoamyl acetate was also
obtained by overexpression of BAT2 gene in Saccharomyces cerevisiae, which encodes
cytosolic branched-chain amino acid aminotransferase (Yoshimoto et al., 2002). This enzyme
catalyzed amino acids transamination to form the corresponding alcohol intermediate -keto
acid. Overexpression of BAT2 gene resulted in 1.3-fold increase in production of isoamyl
alcohol, and a 1.5-fold increase in isoamyl acetate yield. In a similar way, HPG1 mutants of
Saccharomyces cerevisiae with a defect in their Rsp5 ubiquitin ligase, were able to produce
high amounts isoamyl alcohol (and its acetate ester) due to enhanced leucine uptake (Abe et
al., 2005). HPG1 mutation enhances the uptake of leucine probably by stabilizing leucine
permease Bap2 and/or Bap3.

12

As was previously described, accumulation of isoamyl acetate is dependent on the ratio

of activities between AATases that synthesize it, and esterase(s) that simultaneously
hydrolyze it. In order to produce large amounts of isoamyl acetate one possible approach is
the disruption of esterase(s) that hydrolyze this ester. Yanagiuchi et al. isolated sake yeast
mutants exhibiting low esterase activity. These mutants showed high productivity of isoamyl
acetate compared with the parent strain (Yanagiuchi et al., 1989). In subsequent studies, this
research group demonstrated that isoamyl acetate-hydrolyzing esterase(s) play a crucial role
in the accumulation of isoamyl acetate during fermentation (Fukuda et al., 1996 and 1998a).
Different mutant strains of S. cerevisiae deficient in EST2 esterase, involved in hydrolysis of
isoamyl acetate, were capable of producing in laboratory scale sake brewing, between 2- and
19-times higher amounts of this ester compared to the wild-type strains.
Other methods designed to optimize the production of isoamyl acetate were aimed at
increasing the levels of AATases. A recombinant strain of S. cerevisiae was engineered to
overproduce AATase I (Fujii et al. 1994). Transformants harboring the multicopy plasmid
carrying the ATF1 gene produced 27-times higher amounts of isoamyl acetate compared to
cultures of the parental yeast strain. Fukuda et al. further studied how balance of both enzyme
activities (ATTases/esterase) in yeasts affected the accumulation of isoamyl acetate Fukuda et
al. 1998b). Using yeast strains with different numbers of copies of the AATase gene (ATF1)
and the isoamyl acetate-hydrolyzing esterase gene (IAH1), the authors observed that the
higher the ratio of AATase/esterase activities, the greater the production of isoamyl acetate. In
another study, overexpression of Kluyveromyces lactis ATF gene in Saccharomyces cerevisiae
showed 2-fold increases in isoamyl acetate concentrations compared to the control strain (Van
Laere et al. 2008).
Previously was mentioned the influence, in Saccharomyces cerevisiae, of cytosolic
branched-chain amino acid aminotransferase (encoded by BAT2 gene) in the production of
isoamyl alcohol and therefore isoamyl acetate (Yoshimoto et al., 2002). However, null mutant
strain of the BAT2 gene that overexpressed the ATF1 gene exhibited a decrease in the
production of isoamyl alcohol of 52%, and 4.7-fold increase in isoamyl acetate yield. This
modification could enhance the diversity of flavors in the fermented products, leading to the
development of new types of yeast-fermented alcoholic beverages (Yoshimoto et al., 2002).
In other study, isoamyl acetate production could be enhanced depending on itself AATase
activity, but not on the concentration of isoamyl alcohol. 1-Farnesylpridinium (FPy), an
isoprenoid farnesol analog, strongly inhibited sake yeast growth and isoamyl acetate
production, was used to select FPy-resistant mutant cells (Hirooka et al., 2005). FPy-resistant
strain A1 showed major AATase activity and produced 1.4-fold higher amounts of isoamyl
acetate than wild strain, in spite of a limited production of alcohol.
Saccharomyces cerevisiae AATase II and Lg-AATase I were characterized to have a
lesser role than AATase I in isoamyl acetate formation (Verstrepen et al. 2003). The over

13

expression of the genes that encodes these AATases caused smaller increases in isoamyl
acetate formation than overexpression of ATF1 gene (Verstrepen et al., 2003). Additionally, a
double atf1 atf2 mutant could not produce isoamyl acetate (Verstrepen et al. 2003). However,
despite the smaller effect of ATF2 and Lg-ATF1 overexpression on ester production,
sometimes could be desirable in industrial applications rather than the dramatic effects of
ATF1 overexpression (Verstrepen et al., 2003). Similarly, Lilly et al. noted that yeast strains
with overexpression of ATF1 gene significantly increased isoamyl acetate concentration,
while overexpression of ATF2 had only a minor effect on isoamyl acetate production (Lilly et
al., 2006). Even, some authors reported that ATF2 did not show any effects upon isoamyl
acetate production (Ano et al., 2009). However, despite these observations, overexpression of
ATF2 gene was successfully carried out in a Kyokai No 9 yeast strain, as host cell, for an
improved production of isoamyl acetate (Sahara et al., 2009). Sahara et al. constructed, from
a heterozygous integrant, a homozygous diploid that overexpresses ATF2 gene, applying the
high-efficiency loss of heterozygosity (HELOH) method for disrupting genes in diploid sake
yeast. During sake brewing, the homozygous integrant was capable to produce 1.4 times more
isoamyl acetate than the parental, heterozygous strain, but preserving those characteristics
required for industrial applications (Sahara et al., 2009).
3.c.- Bioconversion using wild-type and genetically engineered microorganisms
Bioconversion of isoamyl alcohol by wild-type and metabolic engineered
microorganisms could represent an alternative way to produce natural isoamyl acetate. Most
studies of bioconversion have covered the genetic manipulation of microbial cells in order to
improve the production of isoamyl acetate. However, there is scarce information about wild
cells used with this purpose. One of these investigations was carried out with organic solventtolerant wild-type Bacillus licheniformis S-86 (Torres et al., 2009a). This strain produces
esterases that are active and stable in organic solvents, and which were the responsible for the
conversion of isoamyl alcohol into isoamyl acetate. The yield of isoamyl acetate obtained
using B. licheniformis S-86 was similar to those obtained with other wild-type bacterium and
yeast strains (Kashima et al., 2000; Abe and Horikoshi, 2005; Hirooka et al., 2005). The B.
licheniformis S-86 esterases could represent one of the tolerance mechanisms to decrease
organic solvents toxicity. When B. licheniformis S-86 was cultured in presence of isoamyl
alcohol, an increase in the esterases production was observed (Torres et al., 2009c). The
conversion of hydrophilic isoamyl alcohol into more hydrophobic ester molecules, leads to a
reduction of cellular toxicity of the organic solvent.
The use of esterases for the bioconversion of isoamyl acetate is another alternative that
has been little considered. In a similar fashion to B. licheniformis S-86, the synthesis of
isoamyl acetate with Acetobacter sp. also occurred via esterification of the alcohol. Kashima
et al. demonstrated that flavor esters production by Acetobacter sp. is mostly catalyzed by an

14

intracellular esterase (esterase-1). The authors also observed that the overexpression of
esterase-1 is effective for increasing the production of isoamyl acetate (Kashima et al., 2000).
Esterase-1 overproducing strains of A. pasfeurianus, N-23(pME122P) and DElK(pME122P),
were capable of producing a 1.5-fold higher amount of isoamyl acetate than the wild-type
strain in culture media supplemented with isoamyl alcohol.
Most applications of metabolic engineering have been carried out in order to express
yeast AATases genes in bacteria, mainly in E. coli strains. Due to extensive knowledge of
their genetics and the simplicity of culture cultivation, E. coli strains are, with few exceptions,
the preferred host cells for exploitation of AATases. These studies revealed that although
ATF2 appears to be redundant in yeast, could be useful in isoamyl acetate biosynthesis when
was expressed in bacteria (Horton et al., 2003). E. coli strain carrying the ATF2 gene was able
to produce the ester isoamyl acetate from intracellular acetyl-CoA when isoamyl alcohol was
added externally to the cell culture medium (Vadali et al., 2004a). To produce more isoamyl
acetate, expression (and overexpression) of ATF2 gene in combination with increased
intracellular levels of CoA and acetyl-CoA was studied. Different strategies were successfully
applied (even all together) to achieve acetyl-CoA accumulation, such as overexpression of
pantothenate kinase, and the deletion of the two major acetate-producing pathways, pyruvate
oxidase (poxB) and acetate kinase/phosphotransacetylase (ackA-pta), plus the overexpression
of pyruvate dehydrogenase (PDH) (Vadali et al., 2004a; 2004b; Dittricht et al., 2005).
4.- Conclusions
The characteristic banana aroma of isoamyl acetate makes it one of the most widely used
flavor compounds in the food industries. It is one of the major flavor components of
fermented alcoholic beverages, such as beer and wines and the major in sake. With the rise of
natural products, new ways to produce this ester are necessary to replace the unhealthy and
environmental unfriendly chemical synthesis. Since non-synthetically extraction of isoamyl
acetate from plant materials is often in short supply, enzymatic synthesis and microbial
biosynthesis emerge as potential alternatives to carry out the synthesis of this flavor
compound in a natural Green Chemistry fashion.
Most of these innovative strategies for isoamyl acetate production are in development
stages; limited only to laboratory scale, however, have high potential to be industrially useful.
Particularly, protein and metabolic engineering arise as extremely useful tools for achieving
natural isoamyl acetate produced in quantities that meet market needs. The improved
genomic and proteomic methodologies provide access to new enzymes including those
playing key roles in microbial ester biosynthesis. Nevertheless, the goal of all these researches
should be to develop production systems safe for health and the environment. An example of
this is the use of triacetin, a biodegradable, non-toxic solvent, and GRAS human food

15

ingredient, which was successfully used as solvent and acyl donor in the lipase-catalized
transesterification of isoamyl alcohol into isoamyl acetate.
Acknowledgments
The authors want to thank DST (India) and MinCyT (Argentina) for support under
Indo-Argentina Bilateral Collaborative Scientific Program. Also, the financial support from
ANPCyT (PICT 14-32491, Argentina) to GRC is gratefully acknowledged.

16

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22

Figure 1. Synthesis of isoamyl acetate by Fisher esterification mechanism.

H2SO4

H2O

23

Table 1. Examples of reaction systems used in esterase and lipase-catalyzed

synthesis of isoamyl acetate.
Enzymea

Reaction conditionsb

Yield (%)

Reference

Type II esterase
B. licheniformis S-86

n-hexane, pNP-acetate, 28C, enzyme

immobilized en DEAE sepharose CL-6B

42.8 d

Torres et al.
(2009a)

Novozym 435
Candida antarctica,

n-hexane, acetic anhidride, 40C, enzyme

immobilized in acrylic resin

> 100.0 c

Romero et al.
(2007)

Novozym 435
Candida antarctica

n-hexane, acetic anhidride, 40C, enzyme

immobilized in acrylic resin

> 100.0 c

Romero et al.
(2005a)

Novozym 435
Candida antarctica

n-hexane, ammonium acetate, 40C, enzyme

immobilized in acrylic resin

< 10.0 c

Novozym 435
Candida antarctica

n-hexane, acetic acid, 40C, enzyme

immobilized in acrylic resin

< 25.0 c

Novozym 435
Candida antarctica

n-hexane, ethyl acetate, 40C, enzyme

immobilized in acrylic resin

~ 50.0 c

Novozym 435
Candida antarctica

ScCO2, acetic acid, 40C, enzyme

immobilized in acrylic resin

10.0 d

Novozym 435
Candida antarctica

ScCO2, acetic anhidride, 40C, enzyme

immobilized in acrylic resin

95.0 d

Lipozyme RM-IM
Rhizomucor miehei

ScCO2, acetic anhidride, 40C, enzyme

immobilized in acrylic resin

30.0 d

Lipase
Geotrichum sp.

Water, acetic acid, 60C, free enzyme

24.0 d

Lipase
Rhizopus sp.

Water, acetic acid, 60C, free enzyme

55.0 d

Lipozyme IM-20
Rhizomucor miehei

n-heptane, acetic acid, 40 C, enzyme

immobilized in anionic exchange resin

95.0 c

Lipozyme IM-20
Rhizomucor miehei

n-hexane, acetic acid, 40C, enzyme

immobilized in anionic exchange resin

70.2 c

Lipozyme IM-20
Rhizomucor miehei

n-heptane, acetic acid, 39 C, enzyme

immobilized in anionic exchange resin
Duolite

40.0 c

Romero et al.
(2005b)

Macedo et al.
(2003)

Krishna et al.
(2001)

Krishna et al.
(2000)

a, Enzyme used for the ester synthesis, except type II esterase all the others enzymes were lipases; b,
Reaction conditions: solvent, acyl donor, temperature, reaction time, free or immobilized enzyme; c, Yield
from acyl donor; d, Yield from isoamyl alcohol.

24

Table 2. Comparative production of isoamyl acetate in wild-type microorganisms.

Straina

Enzymeb / Culture Medium

Yield (%)

AATase C / grape must

5.1 e

Saccharomyces
cerevisiae

Reference

Erten et al. 2010

S. cerevisiae + Williopsis
saturnus NCYC22
B. licheniformis S-86

Sake yeast strain 2NF

Acetobacter sp. N23

AATase

/ grape must

5.7

Type II esterase / Synthetic

medium + 0.6% isoamyl alcohol

1.1 d

AATase C / YPD medium

1.1 e

AATase C / Sake brewing

1.5 e

Esterase / YPGE medium +

0.05% isoamyl alcohol

3.7 d

Torres et al.
(2009c)
Hirooka et al.
(2005)
Kashima et al.
(2000)

a, Wild-type strains; b, Enzyme which catalyses the ester synthesis; c, Alcohol acetyltranferase; d,
Highest yield from isoamyl alcohol supplemented to the medium; e, Highest yield from isoamyl alcohol
synthesized by the strain. Abbreviations: YPD: yeast extract, Polypepton, dextrose; YPGE: yeast
extract, Polypepton, glucose, ethanol.

25

Table 2. Metabolic engineered microorganisms for an improved production of isoamyl

acetate.

Enzyme a

Strain

Genetic Modification

Escherichia coli
YBS121

Overexpression of ATF1 and ATF2

genes. Deletion of ackA-pta gene

Singh et al.
(2008)

Saccharomyces
cerevisiae
FAY171E

High-pressure growth mutant /

defect in RSP5 ubiquitin ligase

Abe and
Horikoshi (2005)

Sake yeast strain

2NF

1-Farnesylpyridinium resistant
mutant

Hirooka et al.
(2005)

E. coli CD61
(pATCA)

Overexpression of E. coli panK and

yeast ATF2 genes. Deletion of
ackA-pta gene

E. coli CD6158
(pATCA)

Overexpression of E. coli panK and

yeast ATF2 genes. Deletion of
ackA-pta and poxB genes

Reference

Dittrich et al.
(2005)

AAT
Clostridium
acetobutylicum
ATCC824

Overexpression of ATF2 gene

Horton et al.
(2003)

E. coli TOPO

Overexpression of ATF1 and ATF2

genes

Yeast strain
KY1060

Overexpression of BAT2 gene

Yeast strain
KY1061

Overexpression of ATF1 gene

Acetobacter sp.
N23 (pME122E)

Overexpression of est1 gene

Yoshimoto et al.
(2002)
Esterase

Kashima et al.
(2000)

a, Enzyme which catalyses the ester synthesis.

26