Selective enzymatic synthesis of the grapefruit flavor (+)-nootkatone

Article abstract of DOI:10.1002/cctc.201402952

(+)-Nootkatone is a high-value sesquiterpenoid known for its grapefruit-odor impression. Its isolation from natural plant sources suffers from low yields, and chemical syntheses involve carcinogenic or hazardous compounds. Herein, a biocatalytic route for the synthesis of (+)-nootkatone that combines two enzymes in one pot is presented. In the first step, a cytochrome P450 monooxygenase catalyzes the selective allylic hydroxylation of the sesquiterpene (+)-valencene to the intermediate alcohol nootkatol. In the second step, nootkatol is further oxidized to (+)-nootkatone by an alcohol dehydrogenase (ADH). The challenging task of finding a suitable cofactor regeneration system was solved by careful selection of an appropriate cosubstrate for the ADH, which works in a dual-functional mode. After reaction optimization, involving cosolvent and cosubstrate screening, (+)-nootkatone concentrations of up to 360mg L^-1 and a space-time yield of 18mg L^-1 h^-1 were achieved. Fruitful synthesis: A P450 monooxygenase and an alcohol dehydrogenase are combined in a one-pot cascade to afford the high-value grapefruit flavor (+)-nootkatone by selective oxidation of (+)-valencene.

Full text of DOI:10.1002/cctc.201402952

DOI: 10.1002/cctc.201402952
Communications
Selective Enzymatic Synthesis of the Grapefruit Flavor
+)-Nootkatone
(
[a]
[a]
[a]
[a]
[b]
Sebastian Schulz, Marco Girhard, Sarah K. Gaßmeyer, Vera D. Jꢀger, Daniel Schwarze,
[b]
[a]
Andreas Vogel, and Vlada B. Urlacher*
[4]
(
+)-Nootkatone is a high-value sesquiterpenoid known for its
water as byproduct. The existing biocatalytic approaches for
grapefruit-odor impression. Its isolation from natural plant
sources suffers from low yields, and chemical syntheses involve
carcinogenic or hazardous compounds. Herein, a biocatalytic
route for the synthesis of (+)-nootkatone that combines two
enzymes in one pot is presented. In the first step, a cytochrome
P450 monooxygenase catalyzes the selective allylic hydroxyl-
ation of the sesquiterpene (+)-valencene to the intermediate
alcohol nootkatol. In the second step, nootkatol is further oxi-
dized to (+)-nootkatone by an alcohol dehydrogenase (ADH).
The challenging task of finding a suitable cofactor regeneration
system was solved by careful selection of an appropriate co-
substrate for the ADH, which works in a dual-functional mode.
After reaction optimization, involving cosolvent and cosub-
strate screening, (+)-nootkatone concentrations of up to
(+)-valencene oxidation based on P450s are hampered by in-
[5]
complete conversion of nootkatol to (+)-nootkatone. The
odor threshold of (+)-nootkatone is approximately 50 times
À1
[2,6]
lower (ꢀ1 mgL ) compared to that of nootkatol,
therefore,
complete conversion to (+)-nootkatone is desired.
We have developed a two-enzyme reaction sequence for
the synthesis of (+)-nootkatone, operating in a one-pot mode
in aqueous solution. The reaction includes P450-catalyzed re-
gioselective allylic C2-hydroxylation of (+)-valencene (1) to
yield the intermediate alcohols cis- (2a) and trans-nootkatol
(2b), which are both further oxidized to (+)-nootkatone (3) by
an unselective alcohol dehydrogenase (ADH) (Scheme 1). The
À1
À1 À1
360 mgL
and a space-time yield of 18 mgL
h
were
achieved.
Selective (enzymatic) oxyfunctionalizations of readily available
terpene molecules have attracted attention of chemists and
biotechnologists because the resulting products are often
[
1]
sought-after compounds with high market values. One exam-
ple is the sesquiterpenoid (+)-nootkatone, a high-price constit-
uent of grapefruit with applications in the flavor, fragrance,
and pharmaceutical industries. Isolation of (+)-nootkatone
[
2]
from natural sources suffers from low yields. Its chemical syn-
thesis by means of (+)-valencene oxidation involves carcino-
genic tert-butyl chromate and sodium dichromate, or hazard-
ous compounds, such as tert-butyl peracetate and tert-butyl
Scheme 1. Cosubstrate-supported two-enzyme cascade for selective allylic
oxidation of (+)-valencene (1). P450 catalyzes the hydroxylation step to cis-
[
2–3]
hydroperoxide.
Hence, biotechnological routes for (+)-noot-
(
2a) and trans-nootkatol (2b). An unselective ADH oxidizes both nootkatol
[
2]
katone synthesis have become important. Cytochrome P450
monooxygenases (EC 1.14.–.–; P450) represent an attractive en-
zymatic alternative for the allylic oxidation of (+)-valencene.
P450s catalyze the direct insertion of one atom of molecular
oxygen into (non-)activated CÀH bonds upon formation of
(2) isomers to (+)-nootkatone (3) and simultaneously converts cosubstrates
to regenerate the cofactor NADH.
same ADH converts an appropriate cosubstrate to ensure ef-
fective regeneration of the cofactor NADH.
Two previously developed P450BM3 (CYP102A1) mutants,
[5d]
F87A/A328I (BM3-AI) and F87V/A328V (BM3-VV),
were ap-
[a] S. Schulz, Dr. M. Girhard, S. K. Gaßmeyer, V. D. Jꢀger, Prof. Dr. V. B. Urlacher
plied for the initial hydroxylation of 1. BM3-AI produced a mix-
ture of 2a and 3 (and minor amounts of 2b), whereas BM3-VV
generated almost exclusively 2b and minor amounts of 3 (Sup-
Institute of Biochemistry
Heinrich-Heine-Universitꢀt Dꢁsseldorf
Universitꢀtsstraße 1, Bldg. 26.42.U1, 40225 Dꢁsseldorf (Germany)
Fax: (+49)211 81 13117
E-mail: vlada.urlacher@uni-duesseldorf.de
[5d]
porting Information, section 2.7), as described previously.
Thus, in contrast to the common demand for stereoselective
ADHs, we were interested in an enzyme with no pronounced
stereoselectivity for either of the two isomers, 2a and 2b.
To find a nootkatol-oxidizing ADH, we screened the propriet-
ary enzyme collection at c-LEcta (c-LEcta GmbH, Leipzig, Ger-
[b] D. Schwarze, Dr. A. Vogel
c-LEcta GmbH
Perlickstraße 5, 04103 Leipzig (Germany)
Supporting Information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201402952.
ChemCatChem 2015, 7, 601 – 604
601
ꢁ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communications
many), which included wild-type enzymes from biodiversity
[
7]
screenings and well-studied ADHs, as well as mutants there-
of. Remarkably, only two active evolved ADH variants could be
identified for the desired reaction, whereas none of the
screened wild-type ADHs showed detectable activity. Based on
conversion experiments and kinetic data with 2 (Supporting In-
formation, section 2.1), ADH-21 was chosen for the P450–ADH
cascade.
+
ADH-21 showed a clear preference for the cofactor NAD ;
+
+
activity with NADP was <1% of the activity with NAD . Al-
though P450BM3 prefers NADPH, it has been reported to also
[
8]
accept NADH. Similar levels of conversion of 1 with P450BM3
mutants, supported by a glucose dehydrogenase (GDH) for co-
factor regeneration, could be achieved regardless of whether
NADPH or NADH was applied (Supporting Information, sec-
tion 2.5). Based on this finding we combined the P450BM3
mutants and ADH-21 in one pot with NADH as the cofactor.
Additionally, we intended to further optimize BM3-AI for NADH
acceptance by introduction of two previously described amino
Figure 1. Cosubstrate-supported BM3-AI–ADH-21 cascade for (+)-nootka-
tone synthesis. A) Formation of C2-oxidized products (sum of 2 and 3)
versus time. Conditions: 500 mL, 258C, Tris-HCl buffer (50 mm, pH 7.5 with
2
mm MgCl
2
), DMSO (2% v/v), substrate 1 (200 mm), cosubstrate (20 mm),
À1
NADH (400 mm), BM3-AI (1 mm), ADH-21 (100 mUmL ), catalase
(1200 UmL ). Reaction without cosubstrate (w/o CS) served as control.
À1
B) Influence of cosubstrate concentration on formation of 3. Conditions:
[8b]
1 mL, 258C, 20 h, Tris-HCl buffer (50 mm, pH 7.5 with 2 mm MgCl
2
), DMSO
acid substitutions in the reductase domain (R966D/W1046S).
(
2% v/v), substrate 1 (10 mm), cosubstrate (as indicated), NADH (400 mm),
However, the resulting mutant was approximately 50% less
productive than BM3-AI when using NADH (Supporting Infor-
mation, section 2.3).
À1
À1
BM3-AI (5 mm), ADH-21 (500 mUmL ), catalase (600 UmL ). Where error
bars are not recognizable, they are smaller than symbols or bar lines.
Initial attempts to develop the two-enzyme cascade led, as
expected, to the formation of 3, however, the reaction stopped
after 30 min. Uncoupling reactions in which NAD(P)H is con-
sumed by the P450, but the target substrate is not oxidized,
pletely oxidized to 3 in the 2-butanol system, and was detecta-
ble only in very low amounts in the 2-pentanol system (Sup-
porting Information, section 2.7).
[
9]
often occur in reactions with non-natural substrates. This was
also observed for the oxidation of 1 by BM3-AI (coupling effi-
ciency of 33%). We concluded that the cascade reaction
stopped as the NADH was depleted. Indeed, when doses of
NADH were added during the reaction course, conversion of
As the C2 selectivity of BM3-AI (up to 97%) was higher than
that of BM3-VV (up to 86%) in the systems with cosubstrates
(Supporting Information, section 2.7), BM3-AI was selected for
reaction scale-up and optimization experiments at higher sub-
strate concentrations (up to second phase formation at 10 mm
1).
1
continued (Supporting Information, section 2.2).
The addition of stoichiometric amounts of costly NADH is
Monitoring of the NADH concentration during the reactions
revealed that the presence of a cosubstrate clearly induced co-
factor regeneration compared to the reactions without cosub-
strate (Supporting Information, section 2.6). As expected from
the determined activities of ADH-21, the cofactor regeneration
was higher with 2-pentanol than with 2-butanol.
not economically feasible. Addition of GDH for cofactor regen-
eration along with BM3-AI and ADH-21 resulted in incomplete
conversion of 2 to 3 (data not shown), which can be explained
+
by the reduced availability of NAD for ADH-21 owing to GDH.
To address this issue we screened for an ADH-21 cosubstrate
to effectively regenerate NADH while ensuring simultaneous
complete oxidation of 2 to 3. Consequently, ADH-21 should
serve as a dual-functional enzyme, which performs both the
oxidation of 2 and cofactor regeneration by cosubstrate con-
version (Scheme 1). Several alcohols were identified as poten-
tial cosubstrates for ADH-21, which displayed an apparent pref-
erence for secondary alcohols (Supporting Information, sec-
tion 2.4). All measured volumetric activities were lower com-
pared with that of 2; an important factor to achieve complete
conversion of 2. 2-Butanol and 2-pentanol, for which ADH-21
displayed 9 and 42% activity relative to the activity for 2, re-
spectively, were chosen to establish a cosubstrate-supported
P450–ADH cascade on an analytical scale. Indeed, addition of
either of the cosubstrates to the reaction resulted in double
the concentration of C2-oxidized products (sum of 2 and 3) in
comparison with the control reaction without cosubstrate (Fig-
ure 1A and Supporting Information, section 2.5). In reactions
with either BM3-AI or BM3-VV intermediate alcohol 2 was com-
Unexpectedly, during optimization of the individual cosub-
strate concentrations the highest concentration of 3 amount-
ing to 1.2 mm was achieved with 100 mm 2-butanol, whereas
at an optimized 2-pentanol concentration (20 mm) only
0.7 mm of 3 was produced (Figure 1B). Further increase of 2-
butanol or 2-pentanol concentrations led to reduced amounts
of 3. Although 2-pentanol initially seemed to be the better co-
substrate for ADH-21 (Supporting Information, sections 2.4 and
2.6), conversions with 2-butanol yielded higher concentrations
of 3 (Figure 1B). This could be explained by a negative effect
of 2-pentanol on either protein stability or activity, demonstrat-
ing the importance of careful cosubstrate choice in the devel-
opment of P450–ADH cascades.
The developed P450–ADH cascade with 2-butanol as cosub-
strate was scaled up linearly (Table 1). Similarly to the results
À1
from using a reaction volume of 1 mL, 1.0 mm (221 mgL ) of
3 was obtained in 20 mL (Table 1, entry 3). Strikingly, in any of
the reactions, 2a and 2b were detected in traces only, again
ChemCatChem 2015, 7, 601 – 604
www.chemcatchem.org
602
ꢁ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communications
1
, which was obtained in technical grade (>70%) from Sigma–Al-
Table 1. (+)-Nootkatone titers achieved with the BM3-AI–ADH-21
enzyme cascade under optimized reaction conditions.
drich. Catalase (from bovine liver) was from Sigma–Aldrich. Lyophi-
lized enzyme preparations of ADH-21 are commercially available
from c-LEcta GmbH (Germany). Enzyme production in E. coli BL21
(DE3), measurements of enzyme concentrations and activity assays
are described in the Supporting Information. All enzymes were ap-
plied as cleared cell lysates without any further purification steps
[
a]
[b]
Entry Scale
mL]
Cosolvent/additive (+)-Nootkatone (3) Nootkatol (2)
À1
[
[mM]
[mgL
]
[%]
[
[
c]
1
2
3
4
control DMSO (2%)
0.22
1.03
1.01
48
225
221
336
28
<2
<1
17
d]
1
20
1
DMSO (2%)
DMSO (2%)
(except for coupling measurements). Chemical synthesis and analy-
[5c]
[
e]
[f]
sis of 2 was performed as described previously. Reaction condi-
tions are specified in Figure and Table captions. Analytical proce-
dures are described in the Supporting Information. Data are given
as means with errors representing absolute deviations (nꢁ2).
methyl-b-CD (4%) 1.54
[a] Conditions: 258C, 20–21 h, Tris-HCl buffer (50 mm, pH 7.5 with 2 mm
MgCl
2
), DMSO (2% v/v) or methyl-b-CD (4% w/v), substrate 1 (10 mm), 2-
À1
butanol (100 mm), NADH (400 mm), BM3-AI (5 mm), ADH-21 (500 mUmL ),
catalase (600 UmL ). [b] Percentage of 2 of formed C2-oxidized products
À1
(
[
estimated from GC peak areas). [c] Control (1 mL) without ADH-21.
d] Reactions with either NADH or NAD showed similar results. [e] NAD
+
+
Acknowledgements
was applied as cofactor. [f] Value represents the average of three inde-
pendent experiments. The maximal value observed in a single experiment
was 1.65 mm (360 mgL ). Errors are <10%.
À1
We thank the Ministry of Innovation, Science, and Research of
North Rhine-Westphalia (Germany) and Heinrich-Heine University
Dꢁsseldorf for financial support (scholarship within the CLIB
Graduate Cluster Industrial Biotechnology to S.S.). We are grateful
to Melanie Wachtmeister for technical assistance.
demonstrating a very efficient applicability of ADH-21 for the
oxidation of both nootkatol (2) isomers.
To increase the solubility of 1, a cyclodextrin (CD) was added
instead of DMSO. CDs enhance the solubility of hydrophobic
compounds in aqueous solution and have been applied for
Keywords: biocatalysis · cascade reactions · enzyme catalysis ·
oxidation · synthetic methods
[
10]
[11]
biocatalytic conversions of steroids,
lipophilic ketones,
[
12]
fatty acids, and others. Indeed, in the presence of methyl-b-
[
1] S. Schulz, M. Girhard, V. B. Urlacher, ChemCatChem 2012, 4, 1889–1895.
[2] M. A. Fraatz, R. G. Berger, H. Zorn, Appl. Microbiol. Biotechnol. 2009, 83,
5–41.
CD, the concentration of 3 could be increased up to 1.65 mm
À1
(360 mgL ; Table 1, entry 4). It should be noted, however, that
3
reactions containing methyl-b-CD displayed a residual 17% of
the intermediate 2, which is presumably because of complexa-
[
3] a) G. L. K. Hunter, W. B. Brogden, J. Food Sci. 1965, 30, 876–878;
b) J. A. R. Salvador, J. H. Clark, Green Chem. 2002, 4, 352–356; c) G. W.
Shaffer, E. H. Eschinasi, K. L. Purzycki, A. B. Doerr, J. Org. Chem. 1975, 40,
2181–2185; d) C. W. Wilson, P. E. Shaw, J. Agric. Food Chem. 1978, 26,
tion of 2 by CD.
The space-time yields of 17–18 mgL
À1 À1
h
achieved with the
1430–1432.
optimized cascade were higher than those previously reported
[
4] V. B. Urlacher, M. Girhard, Trends Biotechnol. 2012, 30, 26–36.
À1 À1 [13]
for oxygenase systems (13 mgL h ), whereas the achieved
[5] a) C. Gavira, R. Hçfer, A. Lesot, F. Lambert, J. Zucca, D. Werck-Reichhart,
Metab. Eng. 2013, 18, 25–35; b) K. Cankar, A. van Houwelingen, D.
Bosch, T. Sonke, H. Bouwmeester, J. Beekwilder, FEBS Lett. 2011, 585,
(
+)-nootkatone concentrations were comparable (317–
À1
3
20 mgL for Pleurotus sapidus cells or recombinant Pichia
178–182; c) M. Girhard, K. Machida, M. Itoh, R. D. Schmid, A. Arisawa,
[
13]
pastoris cells in 24 h). The presented cascade offers the clear
advantage of rapid production of the biocatalysts in E. coli (1–
V. B. Urlacher, Microb. Cell Fact. 2009, 8, 36; d) A. Seifert, S. Vomund, K.
Grohmann, S. Kriening, V. B. Urlacher, S. Laschat, J. Pleiss, ChemBioChem
2
009, 10, 853–861; e) S. Takahashi, Y.-S. Yeo, Y. Zhao, P. E. O’Maille, B. T.
Greenhagen, J. P. Noel, R. M. Coates, J. Chappell, J. Biol. Chem. 2007,
82, 31744–31754; f) R. J. Sowden, S. Yasmin, N. H. Rees, S. G. Bell, L.-L.
2
days), and their direct application as cleared cell lysates with-
out further purification. Future work will be focused on optimi-
zation of P450BM3 by protein engineering with the goal to
obtain higher product concentrations in shorter reaction times.
In this regard, preliminary data indicate that the coupling effi-
ciency seems to be a more important factor than high enzyme
activity alone for the successful establishment of P450–ADH
cascades.
2
Wong, Org. Biomol. Chem. 2005, 3, 57–64.
[6] a) H. G. Haring, F. Rijkens, H. Boelens, A. van der Gen, J. Agric. Food
Chem. 1972, 20, 1018–1021; b) R. Kaspera, U. Krings, T. Nanzad, R. G.
Berger, Appl. Microbiol. Biotechnol. 2005, 67, 477–483.
[
7] a) K. Abokitse, W. Hummel, Appl. Microbiol. Biotechnol. 2003, 62, 380–
86; b) T. Greiner-Stoeffele, A. Vogel, R. Schmiedel, S. Kçpke, (c-LEcta
3
GmbH), Patent Appl. WO2013102619A3, 2013; c) K. Inoue, Y. Makino, T.
Dairi, N. Itoh, Biosci. Biotechnol. Biochem. 2006, 70, 418–426; d) K. Ito, Y.
Nakajima, E. Ichihara, K. Ogawa, N. Katayama, K. Nakashima, T. Yoshimo-
to, J. Mol. Biol. 2006, 355, 722–733.
8] a) H. M. Girvan, A. J. Dunford, R. Neeli, I. S. Ekanem, T. N. Waltham, M. G.
Joyce, D. Leys, R. A. Curtis, P. Williams, K. Fisher, M. W. Voice, A. W.
Munro, Arch. Biochem. Biophys. 2011, 507, 75–85; b) S. C. Maurer, K.
Kꢂhnel, L. A. Kaysser, S. Eiben, R. D. Schmid, V. B. Urlacher, Adv. Synth.
Catal. 2005, 347, 1090–1098.
The described dual-functional mode of an ADH that cata-
lyzes the second oxidation step and simultaneously utilizes
a cosubstrate for cofactor regeneration represents a valuable
concept that generally could extend the applicability of P450–
[
[
[
14]
ADH cascades. Thereby, the careful choice of a “low activity”
cosubstrate was demonstrated to be key for the successful de-
velopment of such a system.
9] a) I. G. Denisov, T. M. Makris, S. G. Sligar, I. Schlichting, Chem. Rev. 2005,
1
05, 2253–2277; b) P. J. Loida, S. G. Sligar, Biochemistry 1993, 32,
1530–11538.
[10] P. Bracco, D. B. Janssen, A. Schallmey, Microb. Cell Fact. 2013, 12, 95.
11] T. Zelinski, M.-R. Kula, Biocatal. Biotransform. 1997, 15, 57–74.
1
Experimental Section
[
Chemicals were purchased from Sigma–Aldrich, AppliChem,
[12] M. Theurer, Y. El Baz, K. Koschorreck, V. B. Urlacher, G. Rauhut, A. Baro, S.
GERBU, and VWR (all Germany) with a purity of ꢁ98%, except for
Laschat, Eur. J. Org. Chem. 2011, 4241–4249.
ChemCatChem 2015, 7, 601 – 604
www.chemcatchem.org
603
ꢁ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communications
[
[
13] a) M. A. Fraatz, S. J. L. Riemer, R. Stçber, R. Kaspera, M. Nimtz, R. G.
Berger, H. Zorn, J. Mol. Catal. B: Enzym. 2009, 61, 202–207; b) T. Wriess-
negger, P. Augustin, M. Engleder, E. Leitner, M. Mꢂller, I. Kaluzna, M.
Schꢂrmann, D. Mink, G. Zellnig, H. Schwab, H. Pichler, Metab. Eng. 2014,
Staudt, E. Burda, C. Giese, C. A. Mꢂller, J. Marienhagen, U. Schwaneberg,
W. Hummel, K. Drauz, H. Grçger, Angew. Chem. Int. Ed. 2013, 52, 2359–
2363; Angew. Chem. 2013, 125, 2415–2419.
24, 18–29.
14] a) C. A. Mꢂller, B. Akkapurathu, T. Winkler, S. Staudt, W. Hummel, H.
Received: November 26, 2014
Grçger, U. Schwaneberg, Adv. Synth. Catal. 2013, 355, 1787–1798; b) S.
Published online on February 2, 2015
ChemCatChem 2015, 7, 601 – 604
www.chemcatchem.org
604
ꢁ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim