Production of flavours and fragrances via bioreduction of (4R)-(-)-carvone and (1R)-(-)-myrtenal by non-conventional yeast whole-cells

Article abstract of DOI:10.3390/molecules18055736

As part of a program aiming at the selection of yeast strains which might be of interest as sources of natural flavours and fragrances, the bioreduction of (4R)-(-)-carvone and (1R)-(-)-myrtenal by whole-cells of non-conventional yeasts (NCYs) belonging to the genera Candida, Cryptococcus, Debaryomyces, Hanseniaspora, Kazachstania, Kluyveromyces, Lindnera, Nakaseomyces, Vanderwaltozyma and Wickerhamomyces was studied. Volatiles produced were sampled by means of headspace solid-phase microextraction (SPME) and the compounds were analysed and identified by gas chromatography-mass spectroscopy (GC-MS). Yields (expressed as % of biotransformation) varied in dependence of the strain. The reduction of both (4R)-(-)-carvone and (1R)-(-)-myrtenal were catalyzed by some ene-reductases (ERs) and/or carbonyl reductases (CRs), which determined the formation of (1R,4R)-dihydrocarvone and (1R)-myrtenol respectively, as main flavouring products. The potential of NCYs as novel whole-cell biocatalysts for selective biotransformation of electron-poor alkenes for producing flavours and fragrances of industrial interest is discussed.

Full text of DOI:10.3390/molecules18055736

Molecules 2013, 18, 5736-5748; doi:10.3390/molecules18055736
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Production of Flavours and Fragrances via Bioreduction of
(4R)-(-)-Carvone and (1R)-(-)-Myrtenal by Non-Conventional
Yeast Whole-Cells
Marta Goretti 1, Benedetta Turchetti 1, Maria Rita Cramarossa 2, Luca Forti 2,*
and Pietro Buzzini 1,*
1
Department of Agricultural, Environmental and Food Sciences & Industrial Yeasts Collection DBVPG,
University of Perugia, Borgo XX Giugno 74, Perugia 06121, Italy;
E-Mails: martago22@hotmail.it (M.G.); bturchetti@unipg.it (B.T.)
2
Department of Life Sciences, University of Modena & Reggio Emilia, via G. Campi 183,
Modena 41125, Italy; E-Mail: mariarita.cramarossa@unimore.it
* Authors to whom correspondence should be addressed: E-Mails: luca.forti@unimore.it (L.F.);
pbuzzini@unipg.it (P.B.); Tel.: +39-059-2055110 (L.F.); Fax: +39-059-373543 (L.F.);
Tel.: +39-075-5856455 (P.B.); Fax: +39-075-5856470 (P.B.).
Received: 29 March 2013; in revised form: 29 April 2013 / Accepted: 10 May 2013 /
Published: 16 May 2013
Abstract: As part of a program aiming at the selection of yeast strains which might be of
interest as sources of natural flavours and fragrances, the bioreduction of (4R)-(−)-carvone
and (1R)-(−)-myrtenal by whole-cells of non-conventional yeasts (NCYs) belonging to the
genera Candida, Cryptococcus, Debaryomyces, Hanseniaspora, Kazachstania, Kluyveromyces,
Lindnera, Nakaseomyces, Vanderwaltozyma and Wickerhamomyces was studied. Volatiles
produced were sampled by means of headspace solid-phase microextraction (SPME) and the
compounds were analysed and identified by gas chromatography–mass spectroscopy
(GC-MS). Yields (expressed as % of biotransformation) varied in dependence of the strain.
The reduction of both (4R)-(−)-carvone and (1R)-(−)-myrtenal were catalyzed by some
ene-reductases (ERs) and/or carbonyl reductases (CRs), which determined the formation of
(1R,4R)-dihydrocarvone and (1R)-myrtenol respectively, as main flavouring products. The
potential of NCYs as novel whole-cell biocatalysts for selective biotransformation of electron-poor
alkenes for producing flavours and fragrances of industrial interest is discussed.
Keywords: biocatalysis; non-conventional yeasts (NCYs); ene-reductases; carbonyl reductases;
monoterpenoids; (4R)-(−)-carvone; (1R)-(−)-myrtenal

Molecules 2013, 18
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1. Introduction
Biocatalysis represents an effective and sometimes preferable alternative to the standard synthesis
of fine and/or optically active chemicals [1–7]. Overall, reactions catalysed by biological systems
frequently exhibit high selectivity (chemo-, regio-, and stereo-selectivity) and can be considered
environmentally acceptable because they typically occur under mild conditions. Both isolated enzymes
and whole-cells can be used of as biocatalysts, but whole-cell biocatalysts are often preferable to the
former because they are more convenient and stable sources of enzymes, with no need for costly
enzyme purification and coenzyme addition. Moreover, because of the enzymes are kept within the
natural environment of living cells, usually less enzyme inactivation occurs.
Flavours play a very important role in the quality perception of food and beverages, whereas
fragrances represent an important part of soap and perfume industry [8–10]. Consumers have a strong
preference for natural food additives over chemically synthesized ones. Both United States (US) [11]
and European (EU) [12] laws have already labelled as “natural flavour” all those obtained from living
cells, including Generally Regarded As Safe (GRAS) microorganisms [8]. Thus, products obtained by
microorganisms and enzymes can be considered natural as long as natural raw materials are used. As a
result, the “natural” label, allocated by EU and US food legislation, represents a strong marketing
advantage [9,10].
Monoterpenes are one of the largest classes of flavouring compounds (over 400 different naturally
occurring structures), and represent a valuable resource for the flavour and fragrance industry. The
consumer requests for natural flavours and fragrances have encouraged a growing part of scientific
community to study and develop novel biocatalysts for producing this class of molecules. Thus, the
microbial and enzymatic biotransformation of some monoterpenoids, in particular a few ketones and
aldehydes (e.g., carvone, menthol, citronellol, myrtenal and geraniol) into highly valuable flavouring
derivatives is becoming of increasing interest because of their economic potential for the perfume,
soap, food, and beverage industry [13–22]. Carvone is produced by over 70 different plants. It is found
basically in two distinct stereoisomeric forms, which differ between them for their flavouring
attributes: (i) (4R)-(−)-carvone, which is the principal constituent in spearmint (Mentha spicata) oil,
and (ii) S-(+)-enantiomer, which is present in oils extracted from caraway (Carum carvi) seeds and
from dill (Anethum graveolens) seeds. Biocatalytic transformation of carvone has recently been the
focus of several studies, reporting that some enzymes may catalyze the reduction of C=C and C=O
double bonds competitively, affording a mixture of saturated ketones, saturated alcohol and, more
rarely, the allylic alcohol [14,20,23–32]. From an industrial point of view, carvone and related
compounds are important flavours and fragrances of industrial interest [33]. In particular, due their
high volatility, dihydrocarvones are potent inhibitors of bacteria and filamentous fungi, as well as
prospective insect repellents [34], and have been used as chiral starting compounds in the synthesis of
natural products (e.g., striatenic acid, pechueloic acid) [35–37], antimalarial drugs [38] and valuable chiral
synthons [39,40]. Dihydrocarveols are valuable fragrance ingredients currently used in decorative
cosmetics, fine fragrances, shampoos, soaps and other toiletries as well as in household products such
as cleaners and detergents [41].
Although the use of yeast whole-cells as biocatalysts is a well-established practice, and a few
yeast-catalysed processes have been even successfully scaled up from laboratory to the industrial

Molecules 2013, 18
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level [5,42–45], if compared with other microbial domains (e.g., bacteria, filamentous fungi), the
number of studies reporting the use of yeast whole-cells to catalyse the biotransformation of
monoterpenes represents only a little percentage of the literature published so far [46]. To most people,
yeasts are exemplified by the common baker’s yeast (taxonomically defined as belonging to the
ascomycetous species Saccharomyces cerevisiae). This is in spite of the fact that this species
represents only a small fragment of the huge taxonomic and metabolic diversity occurring in the
yeast world. In fact, in recent decades, biotech-oriented research had paid its attention to the so-called
non-conventional yeasts (NCYs), which demonstrated sometimes a superior biocatalytic aptitude than
S. cerevisiae [43,44,47].
As a part of a program aiming at the selection of yeast strains as novel sources of natural flavouring
molecules, the production of flavours and fragrances via bioreduction of (4R)-(−)-carvone and
(1R)-(−)-myrtenal by whole-cells of non-conventional yeasts (NCYs), belonging to the genera
Candida, Cryptococcus, Debaryomyces, Hanseniaspora, Kazachstania, Kluyveromyces, Lindnera,
Nakaseomyces, Vanderwaltozyma, and Wickerhamomyces was studied.
2. Results and Discussion
The biotransformations of the α,β-unsaturated ketone (4R)-(−)-carvone (1) catalyzed by whole-cells
of NCYs in aqueous media were investigated. The possible reaction pathway is illustrated in Scheme 1.
According to the proposed scheme, the biotransformation resulted in the reduction of the
α,β-unsaturated C=C bond of the cyclic ketone, catalyzed by ene-reductases (ERs) associated to the
yeast cells, to give two dihydrocarvones 2a,b. The ER-catalysed reduction was thus followed by the
subsequent reduction of the carbonyl group of both dihydrocarvone isomers, catalyzed by carbonyl
reductases (CRs), which determined the formation of a mixture of four dihydrocarveols 3a–d (Scheme 1).
Scheme 1. Bioconversion pathway of (4R)-(-)-carvone by whole-cells of NCYs.
O
O
O
2a
1
2b
(1S, 4R)-dihydrocarvone
(4R)-(-)-carvone
(1R, 4R)-dihydrocarvone
OH
OH
OH
OH
3a
3b
3c
(1R, 2S, 4R)-
dihydrocarveol
3d
(1S, 2S, 4R)-
dihydrocarveol
(1S, 2R, 4R)-
dihydrocarveol
(1R, 2R, 4R)-
dihydrocarveol

Molecules 2013, 18
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Although the use of purified ERs needs an accompanying regeneration system for the nicotinamide
cofactor [NAD(P)H] to close the catalytic cycle and improve the bioreduction efficiency, we assumed
that lyophilized cells contain the needed recycling system except for the co-substrate. Accordingly,
glucose was added because we found that this compound was the best co-substrate for co-factor
recycling system [23]. In fact, from a quantitative point of view, the presence of glucose in the reaction
mixture [acting as auxiliary substrate for NAD(P)H regeneration] resulted critical for enhance the % of
conversion of precursor, in close agreement with current literature [23,48]. With no glucose addition,
whole-cells of NCYs showed only a little ability to reduce (4R)-(−)-carvone. Only three strains gave
acceptable results: Hanseniaspora guilliermondii DBVPG 6790 (conversion about 14%), Lindnera
amylophila DBVPG 6346 (about 10%) and Vanderwaltozyma polyspora DBVPG 6243 (about 8%).
The prevalent catalytic activity of whole-cells of NCYs was the ER-catalysed reduction of
(4R)-(−)-carvone into a mixture of (1R,4R)- and (1S,4R)-dihydrocarvone, with a clear-cut preference
towards the production of (1R,4R)-diastereomer. Only traces of dihydrocarveols 3a-d, derived from the
subsequent CR-catalysed reduction of the carbonyl group of (4R)-(−)-carvone were found. As
expected, the addition of glucose to the reaction mixture, visibly increased the aptitude of some strains
to reduce (4R)-(−)-carvone 1 (Table 1).
Table 1. Bioconversion of (4R)-(−)-carvone 1 into derivative products after 120 h by
whole-cells of NCYs in the presence of glucose.
Products (mol %)
3b
Conversion
(mol %)
Species and strain
2b
2a
3a
0
3d
3c
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
C. maltosa DBVPG 6021
12.93 ± 3.87
4.71 ± 0.15
14.81 ± 4.46
0.05 ± 0.05
1.09 ± 0.26
2.05 ± 0.24
0.74 ± 1.08
7.38 ± 12.43
13.45 ± 17.77
0.17 ± 0.04
7.79 ± 4.91
4.29 ± 0.83
0.94 ± 0.42
11.38 ± 16.72
4.43 ± 0.28
63.59 ± 15.44
6.26 ± 1.45
16.93 ± 3.20
0.24 ± 0.04
1.44 ± 0.12
0.33 ± 0.01
1.27 ± 0.80
0.05 ± 0.01
0.020± 0.04
12.25 ± 10.56
9.76 ± 4.49
3.73 ± 0.25
6.69 ± 1.22
0.05 ± 0.04
1.00 ± 0.23
0.36 ± 0.12
0.43 ± 0.56
7.24 ± 12.21
13.35 ± 17.65
0.17 ± 0.04
7.37 ± 4.64
2.87 ± 0.29
0.90 ± 0.40
10.85 ± 16.23
3.98 ± 0.25
62.11 ± 14.99
4.90 ± 1.05
7.59 ± 0.24
0.24 ± 0.04
1.28 ± 0.11
0.30 ± 0.02
1.16 ± 0.70
0.05 ± 0.01
0.02 ± 0.04
11.68 ± 10.26
0.55 ± 0.04
0.33 ± 0.03
0.57 ± 0.08
0.01 ± 0.01
0.01 ± 0.01
0
2.63 ± 0.93
0.66 ± 0.09
7.40 ± 3.19
0
0
Cr. gastricus DBVPG 6057
C. oregonensis DBVPG 6149
C. sake DBVPG 6162
0
0
0
0.15 ± 0.13
0
0
C. freyschussii DBVPG 6208
W. canadensis DBVPG 6211
Cr. albidus DBVPG 6237
Cr. terreus DBVPG 6242
0.07 ± 0.02
0
0
1.32 ± 0.15
0.13 ± 0.22
0.24 ± 0.22
0.30 ± 0.53
0.14 ± 0.22
0.10 ± 0.13
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
V. polyspora DBVPG 6243
K. lodderae DBVPG 6308
L. amylophila DBVPG 6346
K. exigua DBVPG 6469
0
0
0.42 ± 0.28
0.22 ± 0.04
0.05 ± 0.02
0.46 ± 0.54
0.46 ± 0.03
1.49 ± 0.45
0.41 ± 0.09
0.63 ± 0.02
0
0
1.20 ± 0.53
Cr.s terreus DBVPG 6685
Kaz. spencerorum DBVPG 6746
Kaz. spencerorum DBVPG 6748
H. guilliermondii DBVPG 6790
H. occidentalis DBVPG 6798
C. shehatae DBVPG 6850
K. marxianus DBVPG 6854
Kaz. africana DBVPG 6934
N. bacillisporus DBVPG 6945
N. bacillisporus DBVPG 6962
D. nepalensis DBVPG 7123
D. coudertii DBVPG 7124
Kaz. naganishii DBVPG 7133
0
0.06 ± 0.10
0
0
0.95 ± 0.33
8.71 ± 2.99
0
0
0
0
0
0
0
0.16 ± 0.01
0.03 ± 0.00
0.10 ± 0.11
0
0
0.57 ± 0.30

Molecules 2013, 18
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In particular, whole-cells of H. guilliermondii DBVPG 6790 exhibited a superior level of
conversion (about 63%). Interestingly, the ERs associated with whole-cells of this strain exhibited a
high chemoselectivity, thus resulting in the preferential reduction of the α,β-unsaturated C=C bond of
(4R)-(−)-carvone to give almost exclusively (1R,4R)-dihydrocarvone 2b (about 62%). No formation of
dihydrocarveols was observed (Table 2). The high diasteroisomeric excess observed for this reaction
(d.e. = 95%) suggested that the ERs of H. guilliermondii may attack hydrogen at the conjugated C=C
double bond in a stereoselective way from the si-face at C-1 and re-face at C-6 by anti-addition.
Table 2. Bioconversion of (1R)-(−)-myrtenal 4 into derivative products after 120 h by
whole-cells of NCYs
Main products (mol %)
Strain
Conversion (mol %)
5
6
7
C. maltosa DBVPG 6021
97.86 ± 0.47
78.22 ± 20.3
98.82 ± 0.29
91.36 ± 1.24
100 ± 0.00
95.56 ± 1.34
65.64 ± 4.77
97.93 ± 0.33
0
0
2.29 ± 0.98
2.41 ± 0.15
0.89
Cr. gastricus DBVPG 6057
C. oregonensis DBVPG 6149
C. sake DBVPG 6162
0
0
74.80 ± 5.25
16.56 ± 4.81
4.15 ± 0.65
23.07 ± 8.65
67.48 ± 38.82
1.63 ± 1.49
1.80 ± 0.78
0.33 ± 0.19
82.65 ± 5.30
25.67 ± 44.15
0
C. freyschussii DBVPG 6208
W. canadensis DBVPG 6211
Cr. albidus DBVPG 6237
Cr. terreus DBVPG 6242
96.58 ± 1.87
27.62 ± 47. 84
2.26 ± 1.96
36.27 ± 55.37
79.06 ± 7.20
84.20 ± 4.05
0
0
52.58 ± 38.44
99.52 ± 0.82
38.49 ± 53.49
83.34 ± 4.43
87.13 ± 1.79
99.19 ± 0.17
43.46 ± 37.32
0
1.89 ± 1.68
0
0.58 ± 0.51
V. polyspora DBVPG 6243
K. lodderae DBVPG 6308
L. amylophila DBVPG 6346
K. exigua DBVPG 6469
0
0
0
17.65 ± 28.62
0
0.14 ± 0.24
Cr.s terreus DBVPG 6685
Kaz. spencerorum DBVPG 6746
Kaz. spencerorum DBVPG 6748
H. guilliermondii DBVPG 6790
H. occidentalis DBVPG 6798
C. shehatae DBVPG 6850
K. marxianus DBVPG 6854
Kaz. africana DBVPG 6934
N. bacillisporus DBVPG 6945
N. bacillisporus DBVPG 6962
D. nepalensis DBVPG 7123
D. coudertii DBVPG 7124
Kaz. naganishii DBVPG 7133
0
0
0
0
0
0
0
0
0
0
0
0
0
100 ± 0.00
98.68 ± 0.44
70.23 ± 1.62
64.20 ± 55.60
71.59 ± 12.25
57.08 ± 32.76
0
1.67 ± 0.39
0.70 ± 0.81
27.13 ± 28.72
6.10 ± 1.36
1.46 ± 0.35
4.61 ± 5.32
5.96 ± 2.66
1.15 ± 0.18
11.16 ± 7.85
0
70.81 ± 0.92
99.33 ± 1.16
78.53 ± 13.46
58.50 ± 33.41
32.65 ± 56.55
40.87 ± 13.58
51.64 ± 2.84
97.02 ± 3.27
53.97 ± 51.15
54.41 ± 51.27
0
35.85 ± 53.03
50.75 ± 2.58
49.78 ± 57.48
53.97 ± 51.15
54.41 ± 51.27
0
0
0
Similarly to the biotransformation yields observed when H. guilliermondii was used, the addition of
glucose to the reaction mixture increased the aptitude of some strains (otherwise practically incapable
of catalysing the reaction) to reduce (4R)-(−)-carvone to levels of bioconversion > 10%: Candida
shehatae DBVPG 6850 (17%), Candida oregonensis DBVPG 6149 (15%), Candida maltosa DBVPG
6021 and V. polyspora DBVPG 6243 (13%), Kazachstania naganishii DBVPG 7133 (12%) and
Kazachstania spencerorum DBVPG 6746 (11%) (Table 1).
The increase of the bioconversion yield caused by glucose addition described above is not
surprising. ERs are NAD(P)H-dependent flavoproteins and need an accompanying co-factor acting as

Molecules 2013, 18
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regeneration system for the nicotinamide cofactor [NAD(P)H]. ERs often show a marked specificities
for NADH or NADPH as co-factor, which allows to choose the best co-substrate for cofactor-recycling
system on a case-to-case basis. A few authors [48] reported that yeast whole-cells contain only
catalytic amounts of NAD(P)H, so its regeneration must take place by means of metabolism of an
electron donor. In this framework, glucose could act as co-substrate for cofactor-recycling systems,
thus exhibiting a significant efficacy in increasing the ER-catalysed reduction of (4S)-(−)-carvone.
This result is consistent with current literature [44] that postulate that the presence of auxiliary
substrates (e.g., glucose, formate, isopropanol, etc.) for cofactor-recycling system could reduce the
problem of co-factor consumption and, therefore, greatly enhance the bioconversion yield.
Surprisingly, C. shehatae reduced the α,β-unsaturated C=C bond of (4R)-(−)-carvone in an
chemoselective way divergent from that discussed above for H. guilliermondii, to give a slightly
excess of (1S,4R)-dihydrocarvone (d.e. = 10%) (Table 2). This evidence could apparently support the
hypothesis that the ERs associated with whole-cells of C. shehatae may attack hydrogen at the
conjugated C=C double bond from both the si- and re-face at C-1. This could be due to the possibility
of (4R)-(−)-carvone to flip into the enzymatic pocket of ER, or, alternatively, to the presence of a
second ER (associated to whole-cells too) showing an opposite stereo-preference.
The biotransformations of the α,β-unsaturated aldehyde (1R)-(−)-myrtenal (4) catalyzed by
whole-cells of NCYs were investigated. The possible reaction pathway is reported in Scheme 2. Based
on the proposed scheme, ERs associated to whole-cells of NCYs catalysed the reduction of the
α,β-unsaturated C=C bond of (1R)-(−)-myrtenal to give dihydromyrtenals 6. The carbonyl group of the
aldehyde was further reduced by CRs to give dihydromyrtenols 7. A concurrent reaction, giving the
production of (1R)-(-)-myrtenol 5 was also observed in some strains (Scheme 2).
Scheme 2. Bioconversion pathway of (1R)-(-)-myrtenal by whole-cells of NCYs
OH
O
O
OH
5
4
6
7
(1R)-myrtenol
(1R)-(-)-myrtenal
dihydro-myrtenals
dihydro-myrtenols
Overall, NCYs used in this study showed good (in some cases excellent) aptitudes to biotransform
(1R)-(−)-myrtenal (4) into derivative compounds, even in the absence of co-substrates for NAD(P)H
regeneration (i.e., glucose): about one third of strains gave percentage of conversion ≥ 95%. Among
them, Candida freyschussii DBVPG 6208 and Kazachstania spencerorum DBVPG 6746 converted
100% of the precursor (Table 3). Interestingly, in almost all cases, biocatalytic ability was prevalently
related to the activity of CRs associated to whole-cells, affording myrtenol 5 as the main product of the
bioconversions. On the contrary, the results obtained apparently suggest that (1R)-(−)-myrtenal (4) is
not a good substrate for the ER-activity. In fact, only a few strains showed prevalent ER-catalysed
asymmetric C=C bioreduction of the α,β-unsaturated aldehyde: they produced dihydromyrtenals 6,
which was then further reduced by CRs to give dihydromyrtenols 7, that resulted the main products
when Cryptococcus albidus DBVPG 6237 and Lindnera amylophila DBVPG 6346 were used as
biocatalysts (Table 2).

Molecules 2013, 18
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Surprisingly, in some cases, (in particular when the biotransformation was promoted by
Cryptococcus gastricus DBVPG 6057, L. amylophila DBVPG 6346, Kluyveromyces marxianus
DBVPG 6854 and Nakaseomyces bacillisporus DBVPG 6962), the sum of (1R)-(−)-myrtenol 5 +
dihydromyrtenals 6 + dihydromyrtenols 7 was apparently lower than % of conversion (Table 2). This
could be due to the fact that GC-MS analysis did not reveal additional volatile by-products. This
suggests that the actually lower biotransformation yield might be the result of the possible presence of side
reactions. Among them, a special mention could be dedicated to myrtenoic acid, which was detected in
little amounts (< 10%) in some strains and was revealed after extraction of samples with ethyl acetate.
The production of this compound (not indicated in the bioconversion pathway reported in Scheme 2
may be due to a dismutation side reaction of aldehyde into the corresponding alcohol and carboxylic
acid, promoted by an alcohol dehydrogenase (ADH), which was probably associated to whole-cells [49].
Among naturally occurring oxygen-containing monoterpene derivatives, (1R)-(−)-myrtenal is one
of the most widespread flavouring compounds distributed in the Asteraceae (Compositae) family. It is
a constituent of the cumin seed, juniper berry, pepper, peppermint, scotch spearmint and it is
ubiquitous in the essential oils of flowers, stems, and leaves which possess a sweet and spicy
cinnamon-like odour highly demanded as a key constituent of some fragrances [50]. Although only
one study describing the biotransformation of myrtenal by algae has been so far published [51], the
industrial importance of its structurally-related monoterpene alcohol derivative myrtenol is becoming
greater and greater: this compound is a fragrance ingredient used in decorative cosmetics, fine
fragrances, shampoos, toilet soaps and other toiletries as well as in non-cosmetic products such as
household cleaners and detergents [52].
3. Experimental
3.1. Chemicals and Culture Media
(4R)-(−)-Carvone (5-isopropenyl-2-methyl-2-cyclohexenone) and (1R)-(−)-myrtenal [(5R)-6,6-
dimethylbicyclo[3.1.1]hept-3-ene-4-carbaldehyde] were from Fluka (Sigma-Aldrich Co, St. Louis,
MO, USA).
The following microbiological culture media were used: i) growth phase: YEPG: yeast extract 10 g L−1,
peptone 10 g L−1, glucose 20 g L−1, agar 15 g L−1); ii) biotransformation phase: Yeast medium (YM):
yeast extract 3 g L−1, malt extract 3 g L−1, peptone 5 g L−1, glucose 10 g L−1, pH 6.5. Ingredients of
culture media were from Difco (BD, Franklin Lakes, NJ, USA).
3.2. Yeast Strains
Twenty-five NCY strains belonging to the genera Candida, Cryptococcus, Debaryomyces,
Hanseniaspora, Kazachstania, Kluyveromyces, Lindnera, Nakaseomyces, Vanderwaltozyma, and
Wickerhamomyces, were used (Table 3). They were preliminarily selected from amongst over 200 strains of
environmental origin (representative of about 80 ascomycetous and basidiomycetous yeast species) for
their ability to catalyse the biotransformation of (4S)-(+)-carvone (which was preliminarily used as
model target substrate) into reduced products (dihydrocarvones and dihydrocarveols) [23]. All strains
are conserved at the Industrial Yeasts Collection DBVPG of the University of Perugia, Italy [53] and

Molecules 2013, 18
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are publicly available upon request. NCY strains were routinely maintained in frozen form (−80 °C).
Working cultures were grown on YEPG agar slants at 25 °C.
Table 3. Salient information on NCY strains used in the present study.
Pecies and Strain
Origin
Location
Japan
Candida maltosa DBVPG 6021
Soil
Cryptococcus gastricus DBVPG 6057
Candida oregonensis DBVPG 6149
Candida sake DBVPG 6162
Soil
New Zealand
USA
Frass of Tsuga heterophylla
Soil
Sweden
Candida freyschussii DBVPG 6208
Wickerhamomyces canadensis DBVPG 6211
Cryptococcus albidus DBVPG 6237
Cryptococcus terreus DBVPG 6242
Vanderwaltozyma polyspora DBVPG 6243
Kazachstania lodderae DBVPG 6308
Lindnera amylophila DBVPG 6346
Kazachstania exigua DBVPG 6469
Cryptococcus terreus DBVPG 6685
Kazachstania spencerorum DBVPG 6746
Kazachstania spencerorum DBVPG 6748
Hanseniaspora guilliermondii DBVPG 6790
Hanseniaspora occidentalis DBVPG 6798
Candida shehatae DBVPG 6850
Wood pulp
Sweden
Wood pulp
Sweden
Soil
Hungary
Buthan
Soil
Soil
South Africa
South Africa
USA
Soil
Frass of Pinus taeda
Soil
South Africa
ex USSR
South Africa
South Africa
France
Soil
Soil
Gut of Psychidae larva
Trachea of bee
Soil
West Indies
Brazil
Rain forest drosophilids
Rain forest drosophilids
Soil
Kluyveromyces marxianus DBVPG 6854
Kazachstania africana DBVPG 6934
Nakaseomyces bacillisporus DBVPG 6945
Nakaseomyces bacillisporus DBVPG 6962
Debaryomyces nepalensis DBVPG 7123
Debaryomyces coudertii DBVPG 7124
Kazachstania naganishii DBVPG 7133
Brazil
Zimbabwe
USA
Exudate of Quercus emoryi
Exudate of Quercus emoryi
Soil
USA
Nepal
Dropping of Aptenodytes patgonica France
Decaying leaves Japan
3.3. Preparation of Lyophilized Whole-Cells Biocatalysts
Lyophilized NCY cells were obtained as previously described [23], with some modifications.
Aliquots (200 μL) of 24 h cell suspensions, calibrated to A580 = 0.5 (average cell concentration =
106 cells mL−1), were used to inoculate 5 mL of YM. Once the NCY biomass was reached following
48 h of incubation, cells and supernatants were separately harvested. NCY cells were washed three
times by using 50 mM phosphate buffer (pH 6.5), centrifuged each time for 15 min at 4,000 rpm, snap
frozen (−80 °C) and lyophilized for 48 h in a Lyophilizer Modulyo (Edwards, Sanborn, NY, USA).
3.4. Bioconversion Reactions
Thirty mg of lyophilized NCY cells were resuspended in 25 mL sterile vials containing 5 mL of
50 mM phosphate buffer (pH 6.5) + 50 mM glucose [acting as cofactor-recycling system for
NAD(P)H]. Each substrate (final concentration 10 mM) was added and the vials were incubated on an
orbital shaker (120 rpm) at 25 °C for 120 h. After incubation vials were sealed and frozen (−30 °C)

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until SPME + GC-MS analysis. All the reactions were carried out in triplicate. In order to determine
whether substrates were spontaneously reduced in the absence of NCY cells, blank (cell-free) vials
containing 50 mM phosphate buffer + 50 mM glucose and each substrate were analyzed at 120 h.
As with previous samples, following incubation vials were sealed and frozen (−30 °C) until
SPME + GC-MS analysis.
3.5. SPME and GC-MS Analyses
(4R)-(−)-Carvone and (1R)-(−)-myrtenal derivatives occurring in vial headspace after bioreduction
reactions were detected by GC-MS using the solid-phase microextraction (SPME) sampling technique.
Sealed vials containing the yeast suspensions were thawed by immersion in a silicon oil bath at 35 °C
for 15 minutes. Headspace was analyzed using a 1-cm needle containing a fiber coated with 75 μM
Carboxen/polydimethylsiloxane bonded to a flexible fused silica core (Supelco, Sigma-Aldrich Co,
St. Louis, MO, USA). The needle was inserted into the vial through the septum and the fiber was
exposed to headspace volatiles for 5 min at 30 °C. After direct desorption into the injector port at
280 °C for 5 min, the products were analyzed using a Hewlett Packard (Palo Alto, CA, USA) G1800C
Series II gas chromatograph–mass spectrometer equipped with a HP-5 column (25 m × 0.2 mm, 0.5 μM
film thickness) coated with (5%)-diphenyl-(95%)-dimethylpolysiloxane copolymer.
Compounds derived from biotransformation of (4R)-(−)-carvone and (1R)-(−)-myrtenal were
identified by comparing their respective mass fragmentation patterns (EI, 70 eV) with the database
library NIST05 (Varian MS Library Software, Palo Alto, CA, USA). A concentration of monoterpenes
was measured in the vial headspace by an internal standard method in which thawing vial contents
were spiked with 50 μL of a freshly prepared chlorobenzene solution (0.05 mg/mL in deionized water).
Myrtenoic acid obtained after (1R)-(−)-myrtenal bioconversion were detected via GC-MS after
extraction with a solution of octanol (internal standard) in ethyl acetate 0.1% V/V (5 mL).
All the results were expressed as biotransformation yield, i.e., % of substrate converted to a given
derivative. The total ene-reductase (ER) or carbonyl reductase (CR) activities of NCY whole-cells
were calculated as the sum of products obtained by reduction of a given substrate (catalyzed by ERs or
CRs, respectively). All the results represented the average of three independent experiments, and the
statistical significance of these average data was assessed via ANOVA.
When (4R)-(−)-carvone was used as substrate, the % of diasteroisomeric excess (d.e.) of
1R,4R-isomers (or alternatively, 1S,4R-isomers) was calculated as follow:
d.e. = [(1R,4R-isomers − 1S,4R-isomers − or vice versa)/(1R,4R-isomers + 1S,4R-isomers)] × 100
4. Conclusions
Although prokaryotic and eukaryotic microorganisms (including yeasts) are recognized to produce
flavouring compounds potentially attractive for industry, the number of processes that can be
considered truly competitive with chemical synthesis is at present limited, primarily because of their
low yields [43]. Accordingly, as underlined by previous authors [46,53], the potential offered by yeast
diversity, still far from being fully explored, could represent a noteworthy source of novel biocatalysts
for the synthesis of flavours and fragrances. Moreover, the use of whole-cells is nowadays adequately

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accepted, not only as laboratory curiosity, but also in the industrial scale, because they are considered
much more economical and easy-to-handle than purified enzymes [23,54,55].
In this paper, we selected some active NCYs for the stereoselective reduction of α,β-unsaturated
C=C bond of cyclic ketones and aldehydes [namely (4R)-(−)-carvone and (1R)-(−)-myrtenal] to
produce valuable flavouring derivatives. Overall, just one strain (H. guilliermondii DBVPG 6790)
exhibited a good bioconversion yield of (4R)-(−)-carvone (about 63%), coupled with an excellent
chemoselectivity yet (d.e. = 98%), compatible with its application as potential source of
dihydrocarvone. Alternatively, some strain showed an excellent aptitude to totally transform
(1R)-(−)-myrtenal to its flavoured derivative myrtenol (% of conversion ≥ 95%). Their further study
for industrial scale-up is in progress.
Acknowledgments
Authors are deeply grateful to Elisa Caselli for her valuable technical assistance in the course of the
SPME and GC-MS analyses.
Conflict of Interest
The authors declare no conflict of interest.
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© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).