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Banana flavor: insights into isoamyl acetate production

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

1Planta Piloto de Procesos Industriales Microbiológicos (PROIMI).

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

2Biotechnology Division, Regional Research Laboratory.

Trivandrum-695 019, India.

3CINDEFI - Universidad Nacional de La Plata (CONICET, CCT La Plata).

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

4Department 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

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Index

Title Page Abstract 3 1.- Introduction 4 2.- Enzyme and whole-cell mediated synthesis of isoamyl acetate 6 a.- Enzymatic synthesis of isoamyl acetate: lipases and carboxylesterases 6 b.- Whole-cell catalyzis for isoamyl acetate synthesis 11 3.- Isoamyl acetate production by microbial cultures 12 a.- Microbial fermentation processes 12 b.- Metabolic engineering of cells to improve fermentation processes 16 c.- Bioconversion using wild-type and genetically engineered microorganism 19 4.- Conclusions 21 5.- References 23

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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.

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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 nº 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 kg−1, meanwhile bio-vanillin derived from microbial processes currently costs up to US$ 1400 kg−1 and yields an estimated market volume of 5,000 tons annually (Schrader et al., 2004, Feron and Waché, 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

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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 acyl-CoAs 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

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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 n-hexane (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 (28°C) 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 Güvenc 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 (Güvenc et al., 2002). Macedo et al. were able to synthesize isoamyl acetate with 80% yield in a solvent-free 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 (Güvenc et al. 2002; Macedo et al., 2003). The

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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 non-conventional 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 Zarevúcka, 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.

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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 Ríos 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 Ríos 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. Fehér 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 (Fehér 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-3-methylpyridinium 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.

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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 (León 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 µg.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 acetyl-CoA 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; León 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 Sanromán, 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

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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 α-keto acid, a precursor of isoamyl alcohol (Eden et al., 1996). The anabolic pathway catalyzes glucose (during amino acid biosynthesis) mainly through the intermediate α-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 α-keto acids to their corresponding aldehydes and their subsequent conversion to alcohols (Quilter et al. 2003).

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The ability to produce isoamyl acetate was also studied in several so-called non-Saccharomyces 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 45–55% 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

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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 α-isopropyl malate synthase, encoded by the LEU4 gene, catalyzes the production of isoamyl alcohol. And the formation of α-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 (5,5,5-trifluoroleucine). Some of these mutant strains were able to produce twice the concentration of isoamyl alcohol (1,700 µg.l-1) and isoamyl acetate (162 µg.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 α-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.

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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

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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 solvent-tolerant 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

  15

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

  16

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.

  17

5.- References Abe F. & Horikoshi K. (2005) Enhanced production of isoamylalcohol and isoamyl acetate by

ubiquitination-deficient Saccharomyces cerevisiae Mutants. Cellular & Molecular Biology Letters, 10, 383 – 388.

Adachi S. & Kobayashi T. (2005) Synthesis of esters by immobilized lipase-catalyzed condensation reaction of sugars and fatty acids in water-miscible organic solvent. Journal of Bioscience and Bioengineering, 99, 87–94.

Akoh C.C., Lee G.C. & Shaw J.F. (2004) Protein engineering and applications of Candida rugosa lipase isoforms. Lipids, 39, 513–526.

Ano A., Suehiro D., Cha-Aim K., Aritomi K., Phonimdaeng P., Nontaso N., Hoshida H., Mizunuma M., Miyakawa T. & Akada R. (2009) Combinatorial gene overexpression and recessive mutant gene introduction in sake yeast, Bioscience Biotechnology and Biochemistry, 73, 633–640.

Aono R. & Kobayashi H. (1997) Cell surface properties of organic solvent-tolerant mutants of Escherichia coli K-12. Applied Environmental Microbiology, 63, 3637-3642.

Ashida S., Ichdcawa E., Suginami K. & Imayasu S. (1987) Isolation and application of mutants producing sufficient isoamyl acetate, a sake flavor component. Agricultural and Biological Chemistry, 51, 2061-2065.

Beckman E.J. (2004) Supercritical and near-critical CO2 in green chemical synthesis and processing. Journal of Supercritical Fluids, 28, 121–191.

Bluemke W. & Schrader J. (2001) Integrated bioprocess for enhanced production of natural flavors and fragrances by Ceratocystis moniliformis, Biomolecular Engineering, 17, 137–142.

Bornscheuer U.T. (2002). Microbial carboxyl esterases: classification, properties and application in biocatalysis. FEMS Microbiology Reviews, 26, 73-81.

Chen E.C.H. (1978) The relative contribution of Ehrlich and biosynt- hetic pathways to the formation of fusel alcohols. Journal of American Society of Brewing Chemistry, 36, 39-43.

de los Ríos A.P., Hernández-Fernández F.J., Tomás-Alonso F., Gómez D. & Víllora G. Synthesis of flavour esters using free Candida antarctica lipase B in ionic liquids. Flavour and Fragrance Journal, 23, 319–322.

Dittricht C.R., Bennet G.N. & San K.Y. (2009) Metabolic engineering of the anaerobic central metabolic pathway in Escherichia coli for the simultaneous anaerobic production of isoamyl acetate and succinic acid. Biotechnology Progress, 25, 1304-1309.

Eden A., Simchen G. & Benvenisty N. (1996) Two yeast homologs of ECA39, a target for c-Myc regulation, code for cytosolic and mitochondrial branched chain amino acid aminotransferases. Journal of Biologycal Chemistry, 271, 20242- 20425.

Ehrlich F. (1904) Uber das natürliche isomere des leucins. Berichte der Deutschen Chemisten Gesellschaft, 37, 1809-1840.

  18

Erten H. & Tanguler H. (2010) Influence of Williopsis saturnus yeasts in combination with Saccharomyces cerevisiae on wine fermentation. Letters in Applied Microbiology, 50, 474–479.

Ewis H.E., Abdelal A.T. & Lu C.-D. (2004) Molecular cloning and characterization of two thermostable carboxylesterases from Geobacillus stearothermophilus. Gene, 329, 187–195.

Fehér E., Illeova V., Kelemen-Horvath I., Belafi-Bako K., Polakovic M. & Gubicza L. (2008) Enzymatic production of isoamyl acetate in an ionic liquid–alcohol biphasic system. Journal of Molecular Catalysis B: Enzymatic, 50, 28–32.

Feron G. & Waché Y. (2006) Microbial biotechnology of food flavor production, In: Food Biotechnology, Second Edition, Kalidas S., Paliyath G., Pometto A. & Levin R.E. (Eds). CRC Press Taylor and Francis Group, Florida, 407-442.

Fujii T., Nagasawa N., Iwamatsu A., Bogaki T., Tamai Y. & Hamachi M. (1994) Molecular cloning, sequence analysis, and expression of the yeast alcohol acetyltransferase gene. Applied and Environmental Microbiology, 60, 2786-2792.

Fukuda K., Yamamoto N., Kiyokawa Y., Yanagiuchi T., Wakai Y, Kitamoto K., InoueY. & Kimura A. (1998a) Balance of activities of alcohol acetyltransferase and esterase in Saccharomyces cerevisiae is important for production of isoamyl acetate. Applied and Environmental Microbiology, 64, 4076–4078.

Fukuda K., Yamamoto N., Kiyokawa Y., Yanagiuchi T., Wakai Y, Kitamoto K., InoueY. & Kimura A. (1998b) Brewing properties of sake yeast whose EST2 gene encoding isoamyl acetate-hydrolyzing esterase was disrupted. Journal of Fermentation and Bioengineering, 85,101–106.

Furukawa K., Yamada T., Mizoguchi H. & Hara S. (2003) Increased alcohol acetyltransferase activity by inositol limitation in Saccharomyces cerevisiae in sake mash. Journal of Bioscience and Bioenginering, 96, 3809-386.

Güvenç A., Kapucu N., Mehmetoglu Ü. (2002) The production of isoamyl acetate using immobilized lipases in a solvent-free system. Process Biochemistry, 38, 379-386.

Hirata D., Aoki S., Watanabe K.I., Tsukooka M. & Suzuki T. (1992) Stable overexpression of isoamyl alcohol by Saccharomyces cerevisiae with chromosome-integrated multicopy LEU4 genes. Bioscience Biotechnology and Biochemistry, 56, 1682–2683.

Hirooka K, Yamamoto Y, Tsutsui N & Tanaka T, 2005, Improved production of isoamyl acetate by a sake yeast mutant resistant to an isoprenoid analog and its dependence on alcohol acetyltransferase activity, but not on isoamyl alcohol production, Journal of Bioscience and Bioengineering, 99, 125-129

Hobbs H.R. & Thomas N.R. (2007) Biocatalysis in supercritical fluids, in fluorous solvents, and under solvent-free conditions. Chemical Reviews, 107, 2786–2820.

  19

Horton C.E., Huang K.X., Bennett G.N. & Rudolph F.B. (2003) Heterologous expression of the Saccharomyces cerevisiae alcohol acetyltransferase genes in Clostridium acetobutylicum and Escherichia coli for the production of isoamyl acetate. Journal of Industrial Microbiology and Biotechnology, 30, 427–432.

Inoue Y., Trevanichi S., Fukuda K., Izawa S., Wakai Y. & Kimura A. (1997) Roles of esterase and alcohol acetyltransferase on production of isoamyl acetate in Hansenula mrakii. Journal of Agricultural and Food Chemistry, 45, 644-649.

Joseph B., Ramteke P.W. & Thomas G. (2008) Cold active microbial lipases: some hot issues and recent developments. Biotechnology Advances, 26, 457–470.

Jung Y.-J., Lee J.-K., Sung C.-G., Oh T.K. & Kima H.K. (2003) Nonionic detergent-induced activation of an esterase from Bacillus megaterium 20-1. Journal of Molecular Catalysis B Enzymatic, 26, 223-229.

Kamat S.V., Beckman E.J. & Russell A.J. (1995) Enzyme activity in supercritical fluids. Critical Reviews in Biotechnology, 15, 41-71.

Kashima Y., Iijima M., Nakano T., Tayama K., Koizumi Y., Udaka S. & Yanagida F. (2000) Role of intracellular esterases in the production of esters by Acetobacter pasteurianus, Journal of Bioscience and Bioengineering, 89, 81-83.

Krishna S.H., Divakar S., Prapulla S.G. & Karanth N.G. (2001). Enzymatic synthesis of isoamyl acetate using immobilized lipase from Rhizomucor miehei. Journal of Biotechnology, 87, 193–201.

Krishna S.H., Manohar B., Divakar S., Prapulla S.G., & Karanth N.G. (2000). Optimization of isoamyl acetate production by using immobilized lipase from Mucor miehei by response surface methodology. Enzyme and Microbial Technology, 26, 131–136.

Kumari A., Mahapatra P., Kumar Garlapati V., Banerjee R. & Dasgupta S. (2009) lLpase mediated isoamyl acetate synthesis in solvent-free system using vinyl acetate as acyl donor. Food Technology and Biotechnology, 47, 13–18.

León R., Fernandes P., Pinheiro H.M. & Cabral J.M.S. (1998) Whole-cell biocatalysis in organic media. Enzyme and Microbial Technology, 23, 483–500.

Lilly M., Bauer F.F., Lambrechts M.G., Swiegers J.H., Cozzolino D. & Pretorius I.S. (2006) The effect of increased yeast alcohol acetyltransferase and esterase activity on the flavour profiles of wine and distillates. Yeast, 23, 641–659.

Longo M.A. & Sanromán M.A. (2006) Production of food aroma compounds: microbial and enzymatic methodologies. Food Technology and Biotechnology, 44, 335–353.

Macedo G.A., Pastore G.M. & Rodrigues M.I. (2003). Lipase-catalyzed synthesis of isoamyl acetate in free-solvent media optimization using a central composite design. Journal of Food, Agriculture and Environment, 1, 59-63.

Molinari F., Marianelli G. and Aragozzini F. (1995) Production of flavour esters by Rhizopus oryzae. Applied and Microbial Biotechnology, 43, 967-973.

  20

Moreira N., Mendes F., Guedes de Pino P., Hoog T., Vasconcelos I. (2008) Heavy sulphur compounds, higher alcohols and esters production profile of Hanseniaspora uvarum and Hanseniaspora guilliermondii grown as pure and mixed cultures in grape must. International Journal of Food Microbiology, 124, 231–238.

Nikolova P. & Ward O.P. (1993) Whole cell biocatalysis in nonconventional media. Journal of Industrial Microbiology, 12, 76-86.

Oshita K., Kubota M., Uchida M., Ono M. (1995) Clarification of the relationship between fusel alcohol formation and amino acid assimilation by brewing yeast using 13C-labeled amino- acid. Proceedings of the European Brewery Convention Congress; IRL Press: Oxford, U.K., 387−394.

Park Y.C.& Horton Shaffer C.E. & Bennett G.N. (2009) Microbial formation of esters. Applied Microbiologyand Biotechnology, 85, 13–25.

Plata C., Mill!an C., Mauricio J.C., Ortega J.M. (2003) Formation of ethyl acetate and isoamyl acetate by various species of wine yeasts. Food Microbiology, 20, 217–224.

Pohar A., Plazl I. & Znidarsic–Plazl. (2009) Lipase-catalyzed synthesis of isoamyl acetate in an ionic liquid/n–heptanes two-phase system at the microreactor scale. Lab on a Chip, 9, 3385–3390.

Quilter M.G., Hurley J.C., Lynch F.J. & Murphy M.G. (2003) The production of isoamyl acetate from amyl alcohol by Saccharomyces cerevisiae. Journal of the Institute of Brewing, 109, 34–40.

Rojas V., Gil J.V., Piñaga F., Manzanares P. (2001) Studies on acetate ester production by non-Saccharomyces wine yeasts. International Journal of Food Microbiology, 70, 283–289.

Rojas V., Gil J.V., Piñaga F., Manzanares P. (2003) Acetate ester formation in wine by mixed cultures in laboratory fermentations. International Journal of Food Microbiology, 86, 181–188.

Romero M.D., Calvo L., Alba C. & Daneshfar A. (2007). A kinetic study of isoamyl acetate synthesis by immobilized lipase-catalyzed acetylation in n-hexane. Journal of Biotechnology, 127, 269–277.

Romero M.D., Calvo L., Alba C., Daneshfar A. & Ghaziaskar H.S. (2005b) Enzymatic synthesis of isoamyl acetate with immobilized Candida antarctica lipase in n-hexane. Enzyme and Microbial Technology, 37, 42–48.

Romero M.D., Calvo L., Alba C., Habulin M., Primozic M. & Knez Z. (2005a). Enzymatic synthesis of isoamyl acetate with immobilized Candida antarctica lipase in supercritical carbon dioxide. Journal of Supercritical Fluids, 33, 77–84.

Sahara H., Kotaka A., Kondo A., Ueda M. & Hata Y. (2009). Using promoter replacement and selection for loss of heterozygosity to generate an industrially applicable sake yeast

  21

strain that homozygously overproduces isoamyl acetate. Journal of Bioscience and Bioengineering, 108, 359–364.

Schrader J., Etschmann1 M.M.W., Sell D., Hilmer J.-M. & Rabenhorst J. (2004) Applied biocatalysis for the synthesis of natural flavour compounds – current industrial processes and future prospects. Biotechnology Letters 26, 463–472.

Singh R., Vadlani V., Harrison M.L., Bennett G.N. & San K.-Y. (2008) Aerobic production of isoamyl acetate by overexpression of the yeast alcohol acetyl-transferases AFT1 and AFT2 in Escherichia coli and using low-cost fermentation ingredients. Bioprocess and Biosystems Engineering, 31, 299–306.

Soares M., Christen P., Pandey A. & Soccol C.R. (2000) Fruity flavour production by Ceratocystis fimbriata grown on coffee husk in solid-state fermentation. Process Biochemistry, 35, 857–861.

Tominaga T., Okuzawa Y., Kato S. & Suzuki M. (2003) The first isolation of two types of trifluoroleucine resistant mutants of Saccharomyces servazzii. Biotechnology Letters, 25, 1735–1738.

Torres S. & Castro G.R. (2004) Non-aqueous biocatalysis in homogeneous solvent systems. Food Technology and Biotechnology, 42, 271–277.

Torres S., Baigorí M.D. & Castro G.R. (2005) Effect of hydroxylic solvents on cell growth, sporulation, and esterase production of Bacillus licheniformis S-86, Process Biochemistry, 40, 2333-2338.

Torres S., Baigorí M.D., Pandey A. & Castro G.R. (2008). Production and Purification of a solvent-resistant esterase from Bacillus licheniformis S-86. Applied Biochemistry and Biotechnology, 151, 221-232.

Torres S., Baigorí M.D., Swathy S.L, Pandey A. & Castro G.R. (2009a) Enzymatic synthesis of banana flavour (isoamyl acetate) by Bacillus licheniformis S-86 esterase. Food Research International, 42, 454-460.

Torres S., Martinez M.A., Pandey A. & Castro G.R. (2009b) An organic-solvent-tolerant esterase from thermophilic Bacillus licheniformis S-86. Bioresource Technology, 100, 896-902.

Torres S., Pera L.M., Pandey A. and Castro G.R. (2009c) Study on the effects of organic solvent stress on Bacillus licheniformis S-86. Chapter 1. In: Current Topics on Bioprocesses in Food Industry, Vol III. Asiatech Publishers, Inc., New Delhi. Pp. 1-13.

Vadali R.V., Bennett G.N. & San K.Y. (2004a) Applicability of CoA/acetyl-CoA manipulation system to enhance isoamyl acetate production in Escherichia coli. Metabolic Engineering, 6, 294–299.

Vadali R.V., Horton C.E., Rudolph F.B., Bennett G.N. & San K.Y. (2004b) Production of isoamyl acetate in ackA-pta and/or ldh mutants of Escherichia coli with overexpression of yeast ATF2. Applied Microbiology and Biotechnology, 63, 698–704.

  22

van Eijs A.M.M., de Jong J.P.L., Doddema H.J. & Lindeboom D.R. (1988) Enzymatic transesterification in supercritical carbon dioxide. Proceedings of International Symposium in Supercritical Fluids, 2, 933.

Van Laere S., Saerens S., Verstrepen K., Van Dijck P., Thevelein J. & Delvaux F. (2008) Flavour formation in fungi: characterisation of KlAtf, the Kluyveromyces lactis orthologue of the Saccharomyces cerevisiae alcohol acetyltransferases Atf1 and Atf2. Applied Microbiology and Biotechnology, 78, 783–792.

Varma M.N. & Madras G. (2008) Kinetics of synthesis of butyl butyrate by esterification and transesterification in supercritical carbon dioxide. Journal of Chemical Technology and Biotechnology, 83, 1135–1144.

Verstrepen K.J., Van Laere S.D.M., Vanderhaegen B.M.P., Derdelinckx G., Dufour J.-P., Pretorius I.S., Winderickx J., Thevelein J.M. & Delvaux F.R. (2003) Expression levels of the yeast alcohol acetyltransferase genes ATF1, Lg-ATF1, and ATF2 control the formation of a broad range of volatile esters. Applied and Environmental Microbiology, 69, 5228–5237.

Welsh F.W., Williams R.E.and Dawson K.H. (1990) Lipase mediated synthesis of low molecular weight flavor esters. Journal of Food Science, 55, 1679-1682.

Wimmer Z. & Zarevúcka M. (2010) A review on the effects of supercritical carbon dioxide on enzyme activity. International Journal of Molecular Science, 11, 233–253.

Wolfson A., Atyya A., Dlugy C. & Tavor D. (2009) Glycerol triacetate as solvent and acyl donor in the production of isoamyl acetate with Candida antarctica lipase B. Bioprocess Biosystem Engineering, 33: 363-366.

Yanagiuchi T., Kiyokawa Y. & Wakai Y. (1989) Isoamyl acetate accumulation in sake mash and isoamyl acetate hydrolysis activity of sake-yeast strains. Hakkokogaku, 67, 419-425.

Yilmaztekin M., Erten H. & Cabaroglu T. (2008). Production of isoamyl acetate from sugar beet molasses by Williopsis saturnus var. saturnus. Journal of the Institute of Brewing, 114, 34–38.

Yilmaztekin M., Erten H. & Cabaroglu T. (2009). Enhanced production of isoamyl acetate from beet molasses with addition of fusel oil by Williopsis saturnus var. saturnus. Food Chemistry, 112, 290–294.

Yoshimoto H., Fukushige T., Yonezawa T., Sone H. Genetic and physiological analysis of branched-chain alcohols and isoamyl acetate production in Saccharomyces cerevisiae. (2002) Applied Microbiology and Biotechnology, 59, 501–508.

Yoshioka K. & Hashimoto N. (1987) Ester formation by alcohol acetyltransferase from brewers' yeast. Agricultural and Biological Chemistry, 45, 2183-2190.

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+ +     Δ

H2SO4H2O

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

  24

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, 28°C, enzyme immobilized en DEAE sepharose CL-6B

42.8 d Torres et al. (2009a)

Novozym 435 Candida antarctica,

n-hexane, acetic anhidride, 40°C, enzyme immobilized in acrylic resin

> 100.0 c Romero et al. (2007)

Novozym 435 Candida antarctica

n-hexane, acetic anhidride, 40°C, enzyme immobilized in acrylic resin

> 100.0 c

Novozym 435 Candida antarctica

n-hexane, ammonium acetate, 40°C, enzyme immobilized in acrylic resin

< 10.0 c

Novozym 435 Candida antarctica

n-hexane, acetic acid, 40°C, enzyme immobilized in acrylic resin

< 25.0 c

Novozym 435 Candida antarctica

n-hexane, ethyl acetate, 40°C, enzyme immobilized in acrylic resin

~ 50.0 c

Romero et al. (2005a)

Novozym 435 Candida antarctica

ScCO2, acetic acid, 40°C, enzyme immobilized in acrylic resin

10.0 d

Novozym 435 Candida antarctica

ScCO2, acetic anhidride, 40°C, enzyme immobilized in acrylic resin

95.0 d

Lipozyme RM-IM Rhizomucor miehei

ScCO2, acetic anhidride, 40°C, enzyme immobilized in acrylic resin

30.0 d

Romero et al. (2005b)

Lipase Geotrichum sp.

Water, acetic acid, 60°C, free enzyme 24.0 d Macedo et al. (2003)

Lipase Rhizopus sp.

Water, acetic acid, 60°C, free enzyme 55.0 d

Lipozyme IM-20 Rhizomucor miehei

n-heptane, acetic acid, 40 ºC, enzyme immobilized in anionic exchange resin

95.0 c Krishna et al. (2001)

Lipozyme IM-20 Rhizomucor miehei

n-hexane, acetic acid, 40°C, enzyme immobilized in anionic exchange resin

70.2 c

Lipozyme IM-20 Rhizomucor miehei

n-heptane, acetic acid, 39 ºC, enzyme immobilized in anionic exchange resin

Duolite

40.0 c 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.

 

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

Straina Enzymeb / Culture Medium Yield (%) Reference

Saccharomyces cerevisiae

AATase C / grape must 5.1 e

S. cerevisiae + Williopsis saturnus NCYC22 AATase C / grape must 5.7 e

Erten et al. 2010

B. licheniformis S-86 Type II esterase / Synthetic medium + 0.6% isoamyl alcohol 1.1 d Torres et al.

(2009c)

Sake yeast strain 2NF AATase C / YPD medium

AATase C / Sake brewing

1.1 e

1.5 e Hirooka et al.

(2005)

Acetobacter sp. N23 Esterase / YPGE medium + 0.05% isoamyl alcohol

3.7 d 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.

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

acetate.

Strain Genetic Modification Enzyme a Reference

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

Dittrich et al. (2005)

Clostridium acetobutylicum ATCC824

Overexpression of ATF2 gene

E. coli TOPO Overexpression of ATF1 and ATF2 genes

Horton et al. (2003)

Yeast strain KY1060

Overexpression of BAT2 gene

Yeast strain KY1061

Overexpression of ATF1 gene

AAT

Yoshimoto et al. (2002)

Acetobacter sp. N23 (pME122E)

Overexpression of est1 gene Esterase Kashima et al. (2000)

a, Enzyme which catalyses the ester synthesis.