Wickerhamomyces subpelliculosus as whole-cell biocatalyst for stereoselective bioreduction of ketones

Article abstract of DOI:10.1016/j.molcatb.2016.11.003

Newly isolated strains of Wickerhamomyces subpelliculosus were recognized as excellent whole-cell biocatalyst for bioreduction of various ketones. The biocatalytic properties of the new strains were demonstrated in this study by stereoselective bioreduction of acetophenone 1a, 2-heptanone 1b, phenylacetone 1c, 3,4-dimethoxyphenylacetone 1d and 1-cyclopropyl-2-(2-methoxy-4-nitrophenoxy)ethanone 1e. Our study is the first report on application of W. subpelliculosus as whole-cell biocatalyst for stereoselective bioreduction of prochiral ketones. In these processes, both the freshly harvested cell paste and the lyophilized cell powder were tested as biocatalyst using glucose or 2-propanol at various concentrations as cosubstrates for cofactor regeneration. The newly isolated strains of W. subpelliculosus showed diverse characteristics, including optimal pH, temperature and organic solvent tolerance. Bioreductions of phenylacetone 1c applying glucose as cosubstrate under various mild conditions resulted (S)-1-phenylpropanol [(S)-2c] in good to excellent conversion (c = 63.4%–99.9%) with excellent enantiomeric excess [ee_(S)-2c = 98.7%–99.8%].

Full text of DOI:10.1016/j.molcatb.2016.11.003

Accepted Manuscript
Title: Wickerhamomyces subpelliculosus as whole-cell
biocatalyst for stereoselective bioreduction of ketones
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Author: Viktoria Bodai Laszlo Nagy-Gyor Robert Orkenyi
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Zsofia Molnar Szabolcs Kohari Balazs Erdelyi Zsuzsanna
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Nagymate Csaba Romsics Csaba Paizs Laszlo Poppe Gabor
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Hornyanszky
PII:
S1381-1177(16)30216-8
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2016.11.003
MOLCAB 3468
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
4-5-2016
4-11-2016
6-11-2016
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Please cite this article as: Viktoria Bodai, Laszlo Nagy-Gyor, Robert Orkenyi, Zsofia
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Molnar, Szabolcs Kohari, Balazs Erdelyi, Zsuzsanna Nagymate, Csaba Romsics, Csaba
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Paizs, LaszloPoppe, GaborHornyanszky, Wickerhamomycessubpelliculosusaswhole-
cell biocatalyst for stereoselective bioreduction of ketones, Journal of Molecular
Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2016.11.003
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Wickerhamomyces subpelliculosus as whole-cell biocatalyst for stereoselective
bioreduction of ketones
Viktória Bódai,a,b* László Nagy-Győr,c Róbert Örkényi,c Zsófia Molnár,a Szabolcs Kohári,a
Balázs Erdélyi,a Zsuzsanna Nagymáté,d Csaba Romsics,d Csaba Paizs,e László Poppe,c,e,f
Gábor Hornyánszkyc,f
a Fermentia Ltd, Berlini u 47-49., H-1045 Budapest, Hungary
b Witaria Ltd, Csata u. 24-26., H-1135 Budapest, Hungary
c Department of Organic Chemistry and Technology, Budapest University of Technology and
Economics, Műegyetem rkp. 3., H-1111 Budapest, Hungary
d Department of Microbiology, Eötvös Loránd University, Pázmány Péter stny 1/C, H-1117,
Budapest, Hungary
e Biocatalysis and Biotransformation Research Group, Babeş-Bolyai University, Arany János
11., Ro-400028, Cluj-Napoca, Romania
f SynBiocat Ltd, Lázár deák u. 4/1., H-1173 Budapest, Hungary
* Corresponding author. Tel.: +36-1-369-6024; fax: +36-1-920-2024; E-mail address:
viktoria.bodai@fermentia.hu
1

Graphical abstract
Highlights

Wickerhamomyces subpelliculosus was recognized as excellent whole-cell biocatalyst for
bioreduction of ketones.


Glucose and 2-propanol were useful as cosubstrates in the bioreductions.
Strains of W. subpelliculosus showed diverse characteristics, including optimal pH,
temperature and organic solvent tolerance.
Abstract
Newly isolated strains of Wickerhamomyces subpelliculosus were recognized as excellent
whole-cell biocatalyst for bioreduction of various ketones. The biocatalytic properties of the
new strains were demonstrated in this study by stereoselective bioreduction of acetophenone
1a, 2-heptanone 1b, phenylacetone 1c, 3,4-dimethoxyphenylacetone 1d and 1-cyclopropyl-2-
(2-methoxy-4-nitrophenoxy)ethanone 1e. Our study is the first report on application of W.
subpelliculosus as whole-cell biocatalyst for stereoselective bioreduction of prochiral ketones.
In these processes, both the freshly harvested cell paste and the lyophilized cell powder were
tested as biocatalyst using glucose or 2-propanol at various concentrations as cosubstrates for
cofactor regeneration. The newly isolated strains of W. subpelliculosus showed diverse
characteristics, including optimal pH, temperature and organic solvent tolerance. Bioreductions
of phenylacetone 1c applying glucose as cosubstrate under various mild conditions resulted (S)-
2

1-phenylpropanol [(S)-2c] in good to excellent conversion (c = 63.4% – 99.9%) with excellent
enantiomeric excess [ee(S)-2c= 98.7% – 99.8%].
Keywords: stereoselective bioreduction, Wickerhamomyces subpelliculosus, whole-cell
biotransformation, ketone, ketoreductase, yeast
1.
Introduction
Nature is rife with ketoreductases. Researchers successfully isolated and characterized
members of this enzyme group from microbes as well as human samples [1]. In the past two
decades the development of ketoreductases has been sharply increasing, mainly due to the fact
that yeast-mediated reduction of prochiral ketones results in high stereoselectivity and still
acceptable yield[2,3]. The number of syntheses carried out with stereoselective reduction of
prochiral ketones by ketoreductases increased significantly. A number of chiral alcohols
produced by yeasts from prochiral ketones are already well known as chiral building blocks in
the synthesis of various pharmaceuticals[4-,5,6,7,8].
The use of isolated enzymes in industrial synthesis to produce chiral synthons and
enantiomerically pure products is, however, often hindered by the low stability of enzymes
under process conditions[9]. A convenient way to stabilize enzymes is the use of the whole
microbial cell as a biocatalyst[10,11]. Production of recombinant cells containing
ketoreductases with a proper cofactor regeneration system takes months for a team of skilled
scientists [12]. Applications with native hosts allows deferral of recombinant biocatalyst
production [13] or may compete with recombinant whole-cell biocatalysts.
Ketoreductase activity of various yeast strains aiming industrial applications was studied
earlier (Scheme 1) [5,15]. Ketones 1a, 1c and 1d were successfully reduced to the
3

corresponding secondary chiral alcohols (S)-2a,c,d using lyophilized whole cells of
Zygosaccharomyces rouxii and Debaryomyces hansenii [5]. Reductions of 2c with yeast-cell-
based biocatalysts of Debaryomyces carsonii, Candida guilliermondii, Pichia kijperi and
Saccharomyces bayanus resulted in modest yields of (S)-2c. The produced alcohols (S)-2a-c
were useful in the syntheses of various enantiomerically pure chemicals [16-,17,18], (S)-2d was
a key intermediate in the synthesis of benzodiazepines [19]. Furthermore, 1e and both
enantiomers of 2e were important building blocks of antagonists for melanin concentrating
hormone receptor-1 (MCHR-1)[20,21].
The yeast Wickerhamomyces subpelliculosus was first described and classified by
Bedford[22] as Hansenula subpelliculosus, which was revised and reclassified by Kurtzman
[23]. As a result of morphological, physiological and genetic tests, the genus Hansenula
including Hansenula subpelliculosus was transferred to the genus Pichia [24]. Recently, genera
Issatchenkia, Starmera, Williopsis and Pichia were reclassified and species Pichia
subpelliculosus proposed to belong genus Wickerhamomyces based on the phylogenetic
analysis of the genes coding the large-subunit (LSU) and the small-subunit (SSU) rRNA and
the elongation factor 1α (EF-1α)[25].
Herein we report the taxonomic identification of several novel yeast isolates as
Wickerhamomyces subpelliculosus and their application as whole-cell biocatalysts in highly
stereoselective bioreduction of three simple prochiral ketones 1a-c and two further ketones 1d,e
leading to secondary alcohols of pharmacological importance.
4

2.
Materials and methods
2.1. Reagents, solvents
All chemicals and starting materials were purchased from Sigma-Aldrich (St. Louis, MO,
USA), Fluka (Milwaukee, WI, USA) and Alfa Aesar Europe (Karlsruhe, Germany) and were
purified when it was necessary. Prior to use, solvents from Merck KGDA (Darmstadt,
Germany) were dried and/or freshly distilled.
2.2. Analytical methods
The NMR spectra of compounds 1a-d and 2a-d were recorded in CDCl3 on a Bruker DRX-
1
13
300 spectrometer operating at 300 MHz for H and 75 MHz for C. The NMR spectra of
compounds 1e and 2e were recorded in CDCl3 on Varian spectrometer operating at 400 MHz,
1
13
20 °C for H and 100.5 MHz for C nuclei. NMR signals are given in ppm on the δ scale.
Infrared spectra were recorded on a Bruker ALPHA FT-IR spectrometer and wavenumbers of
bands are listed in cm-1. Optical rotation was measured on PerkineElmer 241 polarimeter at the
D-line of sodium. The polarimeter was calibrated with measurements of both enantiomers of
menthol.
Bioreductions of 1a-d were analyzed by GC on Agilent 4890 or Agilent 5890 equipment with
FID detector and Hydrodex β-6TBDM column (25 m × 0.25 mm × 0.25 µm film with heptakis-
(2,3-di-O-methyl-6-O-t-butyldimethylsilyl)-β-cyclodextrine; Macherey &Nagel) or Hydrodex
β-TBDAc column (25 m × 0.25 mm × 0.25 µm film with heptakis-(2,3-di-O-acetyl-6-O-t-
butyldimethylsilyl)-β-cyclodextrine; Macherey&Nagel), using H2 carrier gas (injector: 250°C,
FID detector: 250 °C, head pressure: 12 psi, 50:1 split ratio). GC data [(oven program); tr (min)]:
for 1a and 2a [(8 min at 120 °C); 1a: 2.7; (R)-2a: 4.8; (S)-2a: 5.0], for 1b and 2b [(50 min at
40°C); 1b: 15.7; (S)-2b: 39.5; (R)-2b: 39.7] for 1c and 2c [(5 min at 80 °C, 80-120°C, 2 °C min-
1); 1c: 15.7; (S)-2c: 20.2; (R)-2c: 20.7] on Agilent GC 4890. For 1d and 2d [(20 min at 60 °C,
5

60-180 °C, 4 °C min-1); 1d: 46.9; (S)-2d: 47.1; (R)-2d: 47.4] on Agilent GC 5890. Conversion
and the enantiomeric excess was determined by GC.
Enantiomeric excess and conversion of 2e was analyzed by HPLC on a Agilent 1100
equipment using Lux Amylose-1 [4.6 mm × 150 mm × 5 µm,]; eluents: A (H2O), B
(acetonitrile/2-propanol 80/20); flow rate: 0.5 mL min-1; method: A= 50% (0-10 min), A= 50–
65 % (10-13 min); A: 50 %, 13-16 min; column temperature: 20 °C, λ = 240 nm. Retention
times, tr (min): (R)-2e: 10.73; 1e: 11.41; (S)-2e: 12.15. A comparative measurement to assign
the elution order of the enantiomers of 2e was carried out on CHIRALPAK® IC, [4.6 mm x
250 mm x 5 um] column; eluents: A (0.05% TFA in water/methanol 80/20), B (0.05% TFA in
acetonitrile/methanol 80/20); flow rate: 0.5 mL min-1; method: B= 30% (start), B= 55% (0-25
min), B= 100% (25-30 min), B= 100% (30-40 min), at room temperature, UV detection 240
and 340 nm. Retention times, tr (min): (R)-2e: 24.9; (S)-2e: 26.7; 1e: 32.7 [20].
2.3. Biocatalysts
Wickerhamomyces subpelliculosus CBS 5767 was purchased from Centraalbureau voor
Schimmelcultures, Utrecht, The Netherlands. Wickerhamomyces subpelliculosus WY3 and
Wickerhamomyces subpelliculosus WY13 were isolated, identified and deposited in Witaria
Yeast collection (Witaria LLC, Budapest, Hungary).
2.3.1. Production of biocatalysts
Strains are stored in lyophilized ampules in Witaria’s strain culture collection. Yeasts are
maintained and cultivated on Yeast Malt Agar (HiMedia M424) and Yeast Malt Broth
(HiMedia M425). Cultivation was carried out as described earlier [5] in 10 liter fermenter.
2.3.2. Lyophilization of the yeast cells
6

Following the cultivation, the cells were sedimented by centrifugation at 4000 g (Sigma 6K,
rotor 12500H, 6000 rpm) at 8 °C for 15 min. The freeze-dried cell paste (as 150 g aliquots)
supplemented with skim milk (10 w/w%, Scharlau 06-019) was placed onto sterilized and pre-
cooled (-35 °C) shelves of a freeze-dryer (Christ Epsilon 2-6D LSCplus Pilot Freeze). Freeze-
drying was carried out applying a constant vacuum of 0.850 mbar and the temperature was
gradually increased until the temperature reached 15 °C (1.66 °C h-1, 33 h). The final drying
was performed at 25 °C and 100 mbar for 1 h. Batches of the lyophilized powder (moisture
content between 5 and 8 m/m%) were stored in hermetically closed plastic bags at RT.
2.4. DNA isolation, PCR amplification and phylogenetic analysis
In order to extract the genomic DNA, the strains were suspended in 25 µL of 0.5 M NaOH
solutions. Following an incubation for 15 min at room temperature, 25 µL of 1M TRIS-buffer
(pH 8) and 300 µL of diethyl pyrocarbonate (DEPC)-treated water was added to the suspension
and the slurry was centrifuged at 10,000 g for 3 min. The 18S-25-28S ribosomal DNA internal
transcribed spacer (ITS) region was amplified by PCR using ITS1F (5’- TCC GTA GGT GAA
CCT GCG G-3’) and ITS4R (5’-TCC TCC GCT TAT TGA TAT -3’) primers [25] in a 2720
Thermal Cycler (Applied Biosystems, Foster City, CA, USA) with the following temperature
profile: initial denaturation at 98 °C for 5 min, followed by 32 amplification cycles of 30 s at
94 °C, 45 s at 55 °C and 1 min at 70 °C, followed by a final extension at 70 °C for 10 min. The
PCR reaction mixture contained 1U of LC Taq DNA Polymerase (Fermentas, Vilnius,
Lithuania), 200 mM of each deoxynucleoside triphosphate, 1× Taq buffer with (NH4)2SO4
(Fermentas, Vilnius, Lithuania), 2 mM of MgCl2, 0.65 mM of each primer, 1 µL of DNA
template in a final volume of 50 µL. Amplification was performed by GeneAmp PCR System
(Model 2400, Applied Biosystems, Foster City, USA). PCR products were visualized by UV
light in 1% agarose gel stained with ethidium-bromide. To determine the nucleotide sequences
of the PCR products purified by EZ-10 Spin Column PCR Products Purification Kit (Bio Basic,
7

Amherst, USA), Sanger sequencing reaction was applied. Sequences were aligned by Basic
Local Alignment Search Tool (BLAST; http://www.ncbi.nih.gov/BLAST) using the NCBI
GenBank nucleotide database.
2.5. Synthetic procedures
2.5.1. Synthesis of 1-cyclopropyl-2-(2-methoxy-4-nitrophenoxy)ethanone 1e
1-Cyclopropyl-2-(2-methoxy-4-nitrophenoxy)ethanone 1e was prepared according to Zhao
et al. [20] with slight modifications. Briefly, 1-cyclopropylethanone was brominated and the
resulted 2-bromo-l-cyclopropylethanone was coupled with potassium 2-methoxy-
nitrophenolate in DMF.
1e: yellow powder; mp: 117-118 °C {lit. mp: 112-113 °C [20]}; IR (KBr): 3096, 2958, 1717,
1590, 1516, 1498, 1334, 1285, 1088, 1013, 863 and 747 cm-1; 1H NMR (400 MHz, CDCl3) δ
ppm: 1.0 (m, 2H, CH2), 1.2 (m, 2H, CH2), 2.2 (m, 1H, CH), 4.0 (s, 3H, OCH3), 4.9 (s, 2H,
OCH2), 6.8 (d, J = 8.9 Hz, 1H, Ar-H), 7.8 (d, J = 2.6 Hz, 1H, Ar-H), 7.9 (dd, J = 8.9 Hz, J =
2.6 Hz, 1H, Ar-H); 13C NMR (100.5 MHz, CDCl3) δ ppm: 12.2 (CH2), 17.2 (CH), 56.4 (OCH3),
73.6 (OCH2), 107.1 (Ph), 111.8 (Ph), 117.5 (Ph), 142.3 (Ph), 149.2 (Ph), 152.8 (Ph), 205.4
(OC). 1H NMR and 13C NMR spectra were in accordance with literature data [20].
2.5.2. Synthesis of racemic 1-cyclopropyl-2-(2-methoxy-4-nitrophenoxy)ethanol 2e
Reduction of 1-cyclopropyl-2(2-methoxy-4-nitrophenoxy)ethanone 1e (3.50 g 13.9 mmol),
according to the published method [20], provided racemic alcohol (2e, 3.22g).
rac-2e: yield: 92.8%; yellow powder; mp: 78-79 °C; IR (KBr): 3566, 3444, 3010, 1589,
1525, 1336, 1280, 1093, 1019, 865 and 747 cm-1; 1H NMR (400 MHz, CDCl3) δ ppm: 0.3 – 0.4
(m, 1H, CH2), 0.4 – 0.5 (m, 2H, CH2), 0.5 – 0.7 (m, 2H, CH2), 0.9 – 1.7 (m, 1H, CH), 2.4 – 2.5
(br.s, 1H, OH), 3.4 (ddd, J = 8.1 Hz, J = 8.1 Hz, J = 2.9 Hz, 1H, CH), 3.9 (s, 3H, OCH3), 4.1
(dd, J = 9.6 Hz, J = 7.9 Hz, 1H, OCH2), 4.3 (dd, J = 9.6 Hz, J = 2.9 Hz, 1H, OCH2), 6.9 (d, J =
8

8.9 Hz, 1H, Ar-H), 7.7 (d, J = 2.6 Hz, 1H, Ar-H), 7.9 (dd, J = 8.9 Hz, J = 2.6 Hz, 1H, Ar-H);
13C NMR (100.5 MHz, CDCl3) δ ppm: 2.1 (CH2), 2.8 (CH2), 13.5 (CH), 56.2 (OCH3), 73.6
(OCH2), 74.4 (OCH2), 106.7 (Ph), 111.6 (Ph), 117.7 (Ph), 141.8 (Ph), 149.2 (Ph), 153.8 (Ph).
1H NMR and 13C NMR spectra were in accordance with literature data [20].
2.6. Bioreduction of ketones 1a-d
2.6.1. Comparison of bioreductions of 1c with freshly harvested and lyophilized yeast cells
Freshly harvested and sedimented cell paste (100 mg) or lyophilized cells (300 mg) as
biocatalyst was added to a mixture of ketone 1c (20 mg), glucose (6 w/v%) in phosphate buffer
(5 mL, 100 mM, pH 7.5) in 20 ml Erlenmeyer-flasks. The mixtures were shaken on an orbital
shaker (150 rpm) at 30 °C for 24 h. Then 0.5 mL from the mixture was extracted with ethyl
acetate (1 mL) dried over Na2SO4 and analyzed by GC.
2.6.2. Bioreduction of ketones 1a-d with lyophilized cells in the presence of various
cosubstrates
Lyophilized cell powder (300 mg) was suspended in phosphate buffer (5 mL, 100 mM, pH
7.5) containing one of the ketones 1a, 1b, 1c or 1d (20 mg) and glucose (1, 2, 4, 8 or 16 w/v%)
or 2-propanol (1, 2, 4, 8 or16 v/v%) or without cosubstrate in 20 mL Erlenmeyer-flasks. The
mixture was shaken on an orbital shaker (150 rpm) at 30 °C for 24 h. Then 0.5 ml from the
mixture was extracted with ethyl acetate (1 mL) dried over Na2SO4 and analyzed by GC.
2.6.3. Bioreduction of ketones 1a-d on preparative scale
Lyophilized cell powder (1500 mg) was suspended in phosphate buffer (25 mL, 100 mM,
pH 7.5) containing one of the ketones 1a, 1b, 1c or 1d (100 mg) and glucose (4 w/v%) in 20
mL Erlenmeyer-flasks. The mixture was shaken on an orbital shaker (150 rpm) at 30 °C for 24
h. Then the mixture was extracted with ethyl acetate (50 mL) dried over Na2SO4 and
9

concentrated under reduced pressure. The residue was purified by plate chromatography (silica
gel, eluent: hexane – ethyl acetate 10:4) to yield the corresponding secondary alcohol (S)-2a-d.
[ ] 25
(S)-2a: yield: 20.3%; ee: 99.1% (by GC); colorless oil; Rf: 0.65; 훼 = -64.2 (c 1.0, CHCl3);
D
[ ] 22
[ ] 22
D
{lit.: 훼 = -62.8 (c 1.0, CHCl3), for ee= 98.5%[26], 훼 = -57.7 (c 1.0, CHCl3), for ee=
D
89%[27]}; IR (film): 3331, 2972, 1449, 1075, 1009, 897, 759 and 697 cm-1; 1H-NMR (CDCl3,
300 MHz) δ ppm: 1.5 (d, J= 6.4 Hz, 3H, CH3), 2.0 (s, H, OH), 4.9 (m, H, CH) 7.3-7.4 (m, 5H,
13
Ar-H); C-NMR (CDCl3, 75 MHz) δ ppm: 25.4 (CH3), 70.6 (CH), 125.6 (Ph), 127.6 (Ph),
128.7 (Ph), 146.6 (Ph). 1H and 13C NMR data agreed with the reported spectra [27,28].
[ ] 25
(S)-2b: yield: 76.8%; ee: 99.9% (by GC); colorless liquid; Rf: 0.71; 훼 D : +11.6 (c 1.0,
[ ] 25
[ ] 25
CHCl3); {lit.: 훼 D = +12.1 (c 0.66, ethanol)[29], 훼 = +10.4 (c 0.52, CHCl3)[30]}; IR (film):
D
3332, 2959, 2927, 2859, 1459, 1375, 1112 and 950 cm-1; 1H-NMR (CDCl3, 300 MHz) δ ppm:
0.9 (t, J= 6.4 Hz, CH3); 1.2 (d, J= 6.4 Hz, CH3); 1.3-1.5 (m, 6H, C3H6); 1.6 (s, 1H, OH); 3.8
13
(m, 1H, CH); C-NMR (CDCl3, 75 MHz) δ ppm: 14.2 (CH3), 22.8 (CH2), 23.6 (CH3), 25.6
1
(CH2), 32.0 (CH2), 39.5 (CH2), 69.4 (CH). IR and H-NMR data agreed with the reported
spectra[31].
[ ] 25
(S)-2c: yield: 58.4%; ee: 99.9% (by GC); colorless oil; Rf: 0.53; 훼 퐷 = +38.2 (c 1.0,
[ ] 22
[ ] 25
CHCl3); {lit.: 훼 = +4.9 (c 1.0, CHCl3), for ee= 56.4% [26] 훼 퐷 = +48.1 (c 1.0,
퐷
benzene)[32]}; IR (film): 3356, 2986, 1453, 1116, 1078, 936, 740, 697 and 504 cm-1; 1H-NMR
(CDCl3, 300 MHz) δ ppm: 1.3 (d, J= 6.1 Hz, 3H CH3), 1.8 (s, 1H, OH), 2.7-2.8 (m, 2H, CH2),
4.1 (m, 1H, CH), 7.3-7.4 (m, 5H, Ar-H); 13C-NMR (CDCl3, 75 MHz) δ ppm: 22.8 (CH3), 45.8
(CH), 98.9 (CH), 126.1 (Ph), 128.59 (Ph), 129.5 (Ph), 130.6 (Ph). 1H and 13C NMR data agreed
with the reported spectra [26].
[ ] 25
(S)-2d: yield: 63.9% ee: >99.9% (by GC); colorless oil; Rf: 0.20; 훼 D : +25.4 (c 1.0,
[ ] 25
CHCl3); {lit.: 훼 = +26.4 (c 1.85, CHCl3) [19]}; IR (film): 3319, 2962, 2837, 1519, 1540,
D
10

1263, 1238, 1159, 1028 and 806 cm-1; 1H-NMR (CDCl3, 300 MHz) δ ppm: 1.3 (d, J= 5.7 Hz,
3H, CH3), 1.6 (s, 1H, OH), 2.6-2.8 (m, 2H, CH2), 3.6 (s, 3H, O-CH3), 3.9 (s, 3H, O-CH3), 4.0
13
(m, 1H, CH), 6.7-6.8 (m, 3H, Ar-H); C-NMR (CDCl3, 75 MHz) δ ppm: 22.9 (CH3), 45.5
(CH2), 56.1 (CH3), 56.2 (CH3), 69.1 (CH), 111.6 (Ph), 112.7 (Ph), 121.5 (Ph), 131.2 (Ph), 147.9
(Ph), 149.2 (Ph). IR, 1H and 13C NMR data agreed with the reported spectra [19].
2.6.4. Temperature effects on bioreductions with Wickerhamomyces subpelliculosus strains
Lyophilized cell powder (300 mg) was suspended in phosphate buffer (5 mL, 100 mM, pH
7.5) containing one of the ketones 1a, 1b, 1c or 1d (20 mg) and glucose (4 w/v%) in 25 mL
Erlenmeyer-flasks. The mixture was shaken on an orbital shaker (150 rpm) at a given
temperature (30 °C; 35 °C; 40 °C; 45 °C; 50 °C; 55 °C or 60 °C) for 24 h. Then a 0.5 mL portion
of the mixture was extracted with ethyl acetate (1 mL), and the extract was dried over Na2SO4
and analyzed by GC.
2.6.5. pH effects on bioreductions with Wickerhamomyces subpelliculosus strains
The effect of pH on ketoreductase activity was determined in various pH ranges using
appropriate buffers (pH 3.0 – 5.0: 100 mM sodium citrate buffer, pH 6.0 – 8.0: 100 mM sodium
phosphate buffer, pH 9.0 – 11.0: 100 mM sodium carbonate – sodium bicarbonate buffer).
Lyophilized cell powder (300 mg) was suspended in the buffer (5 mL) containing the ketone
(20 mg of 1a, 1b, 1c or 1d) and glucose (4 w/v%) in 25 mL Erlenmeyer-flasks. The mixture
was shaken on an orbital shaker (150 rpm) at 30 °C for 24 h. Then a 0.5 mL portion of the
mixture was extracted with ethyl acetate (1 mL), and the extract was dried over Na2SO4 and
analyzed by GC.
2.6.6. Effect of organic solvents on ketoreductase activity of Wickerhamomyces
subpelliculosus strains
11

Lyophilized cell powder (300 mg) was suspended in phosphate buffer (5 mL, 100 mM, pH
7.5) containing ketone 1c (20 mg), glucose (4 w/v%) and various organic solvents (2.5 or 5
v/v%) in 25 mL Erlenmeyer-flasks. The mixture was shaken on an orbital shaker (150 rpm) at
30 °C for 6 h. Then a 0.5 mL portion of the mixture was extracted with ethyl acetate (1 mL),
and the extract was dried over Na2SO4 and analyzed by GC.
2.6.7. Bioreduction of 1-cyclopropyl-2-(2-methoxy-4-nitrophenoxy)ethanone 1e
Lyophilized cell powder (300 mg) was suspended in phosphate buffer (5 mL, 100 mM, pH
7.5) containing of ketone 1e (10 mg), glucose (4 w/v%) and DMSO (2.5 or 5 v/v%) in 20 mL
Erlenmeyer-flasks. The mixture was shaken on an orbital shaker (150 rpm) at 30°C for 24 h.
Then a 0.5 mL portion of the mixture was extracted with ethyl acetate (1 mL), and the extract
was dried over Na2SO4 and analyzed by HPLC. The residue was purified by plate
chromatography (silica gel, eluent: hexane – ethyl acetate 1:1) to yield the corresponding
secondary alcohol (S)-2e.
[ ] 25
(S)-2e: yield: 51.5%; ee: 94.2 % (by HPLC); yellow powder; Rf: 0.25; 훼 퐷 : +13.6 (c 1.0,
CHCl3); The IR, 1H and 13C-NMR spectra of (S)-2e were indistinguishable from that of rac-2e.
3.
Results and discussion
3.1. Strain selection and identification of Wickerhamomyces subpelliculosus strains as
potential biocatalysts for bioreductions
In preliminary experiments, more than 300 different bacterium and yeast strains of Witaria
Strain Collection (WY) were isolated and screened for ketoreductase activity. After preliminary
activity tests 10 candidates were selected for more detailed investigations and for selectivity
screenings. Two yeast strains with high ketoreductase activity were taxonomically identified as
Wickerhamomyces subpelliculosus (Table 1). To our best knowledge, there were only three
12

examples published on bioreductions of various ketones with strain Hansenula subpelliculosa
MY 1552 [33,34] and Pichia subpelliculosa CBS 5767 [35] (which are considered as synonyms
of Wickerhamomyces subpelliculosus) but Wickerhamomyces subpelliculosus has never been
mentioned as biocatalyst in any stereoselective bioreduction.
3.2. Bioreduction of prochiral ketones 1a-e
This study describes the application of W. subpelliculosus strains (WY3, WY13) in
stereoselective bioreduction of ketones 1a-e (Scheme 1). Five structurally different ketones,
acetophenone 1a (aryl methyl), 2-heptanone 1b (alkyl methyl), phenylacetone 1c, 3,4-
dimethoxy-phenylacetone 1d (aralkyl methyl) and 1-cyclopropyl-2-(2-methoxy-4-
nitrophenoxy)-ethanon 1e (aryl cyclopropyl) were chosen to characterize the ketoreductase
activity. The W. subpelliculosus strains were used first for screening the bioreduction on
analytical scale and later on preparative scale as well.
Ample information is available about the bioreduction of these ketones, hence we can easily
compare W. subpelliculosus with other strains published earlier [16-18]
First, the newly isolated strains W. subpelliculosus WY3 and WY13 were compared with the
W. subpelliculosus CBS 5767 in the bioreduction of phenylacetone 1c (Table 2). In the
preliminary tests freshly harvested and lyophilized cells were compared under the reactions
conditions which were applied for bioreductions of benzofuranyl ketones by freshly harvested
and lyophilized Saccharomyces cerevisiae cells [36]. Bioreduction of phenylacetone 1c either
by the freshly harvested or the lyophilized cells of the control yeast CBS 5767 resulted in low
yield (conversion under 10%). Contrarily, the bioreduction of phenylacetone 1c either by the
fresh or the lyophilized WY3 and WY13 cells resulted in (S)-2c in good conversion and in
excellent selectivity (ee >99%). Based on this result W. subpelliculosus WY3 and WY13 strains
13

were further investigated. In the subsequent experiments we used only lyophilized cells, since
they are easy to store, use and transport [5].
3.3. Effect of cosubstrates on bioreductions of ketones 1a-d
Next, the efficacy of W. subpelliculosus WY3 and WY13 were examined in the bioreductions
of 1a-d in the presence of glucose and 2-propanol as proper cosubstrates for cofactor
regeneration in yeast-mediated bioreductions [5]. It was found that the nature of cosubstrates
significantly influenced the conversion. Reductions of the ketones 1a-d in the presence of
glucose (Table 3) gave significantly higher conversions than their counterparts supplemented
with 2-propanol (Table 4).
The moderate conversion observed in bioreductions of ketones 1a-d in the presence of 1 and
2 w/v% glucose almost doubled in most cases in the presence of 4 w/v% glucose. The 8 w/v%
glucose concentration proved to be the optimal cosubstrate amount for the reduction of 1c
resulting in (S)-2c in excellent conversions and enantiomeric excess (c(S)-2c = 99.8% and ee(S)-
2c= 99.6% with strain WY3; and c(S)- = 99.9%, ee(S)-2c= 99.8% with strain WY13). Worth
2c
mentioning that the reductions of 1b and 1d by the newly isolated strains WY3 and WY13
yielded the products in extremely high enantiomeric excess (ee(S)-
>99.9%) but were only
2b,d
moderately productive. The lowest conversion and enantiomeric excess were observed in
reductions of 1a (c(S)-2a = 8.2–40.6 %, ee(S)-2a = 92.4–97.8 %, respectively).
In the next series of experiments 2-propanol was tested as cosubstrate at different
concentrations in the yeast-mediated reductions of 1a-d. Unfortunately, only low cosolvent
tolerance of the strains W. subpelliculosus WY3 and WY13 was observed as moderate to
negligible ketoreductase activities were found at 2-propanol concentrations over 4 v/v% (Table
4). The high content of this cosolvent might be harmful for these strains. The optimal 2-
propanol concentrations were in WY3 mediated bioreductions 2 v/v% for 1a and 1d and 4 v/v%
14

for 1b and 1c. In case of WY13 the highest productivity and enantiomer selectivity were
observed in reductions of 1a,b,d with 2 v/v% 2-propanol and 1c was used with 4 v/v% 2-
propanol. The highest productivity was observed in the bioreduction of 1a by the strain W.
subpelliculosus WY13 in presence of 2 v/v% 2-propanol (c(S)-2a = 72.4% and ee(S)-2a= 94.9%).
The fact, that optima of the bioreductions of 1a-d were found at 2 or 4% amounts of 2-propanol
(Table 4) indicated that lower efficiency of 2-propanol as cosubstrate compared to glucose
(Table 3) is only partially due to the deleterious effect of 2-propanol as organic solvent (see
Section 3.6).
The results of bioreductions of ketone 1c with the newly isolated strains W. subpelliculosus
WY3 (c2c= 98.9%, ee(S)-2c= 98.7%) and WY13 (c2c= 99.3%, ee(S)-2c= 99.8%) at 2 w/v% glucose
concentration (Table 3) surpassed the outcome of bioreductions of 1c with lyophilized cells of
Zygosaccharomyces rouxii; (c2c= 99%, ee(S)-2c= 82%) or Debaryomyces hansenii (c2c= 78%,
ee(S)-2c= 77%)[5]. The enantiotope selectivity of W. subpelliculosus WY3 and WY13 was
excellent in bioreductions of ketone 1d at 2 w/v% glucose concentration (c2d= 21.4%, ee(S)-
2d
>99.9% with WY3 and c2d= 48.5%, ee(S)-2d >99.9% with WY13) and higher than with Z. rouxii
(c2d= 17%, ee(S)-2d= 80%) or D. hansenii (c2d= 58%, ee(S)-2d= 99%)[5].
3.4. Effect of temperature on bioreductions of ketones 1a-d
Ketoreductase activity of the two isolates (WY3 and WY13) was investigated in
bioreductions of four ketones 1a-d at various temperatures (Figure 1). Expectedly, both isolates
showed modest thermal tolerance in bioreductions being the most active at around 30 °C.
Temperature over 40 °C proved to be detrimental for the ketoreductase activity of the strains.
Interestingly, 35 °C was the most suitable temperature for the bioreduction of 1c with WY13
strain (c(S)-2c = 73.1%, ee(S)-2c >99.9%).
3.5. Optimal pH in bioreductions of ketones 1a-d
15

The effect of pH on ketoreductase activity and selectivity of the WY3 and WY13 strains was
studied using appropriate buffers with pH values between 3.0 and 11.0 (Figure 2). The optimal
pH was determined in bioreductions of four ketones 1a-d with both yeast strains separately.
Overall, it could be concluded that strain WY13 was active in a wider range of pH than strain
WY3 (mostly due to the higher activity in the alkaline pH range between 8–10) but the
enantiotope selectivity of the reduction was usually higher with strain WY3 than with WY13.
In addition, the pH optima for activity with strains WY3 and WY13 were different towards the
various ketones 1a-d. As the optima for activity and selectivity were often different in the
bioreductions of the ketones 1a-d the optimal operating pH was determined by taking the
selectivity seriously into account. In the bioreduction of 1a the optimal operating pH was 6 with
strain WY13 (c(S)-2a =49.0%, ee(S)-2a = 99.5%) but 7 with strain WY3 (c(S)-2a =27.6%, ee(S)-
=
2a
99.5%). In biotransformations of 1b the optimal operating pH shifted one pH unit lower for
both strains to 5 with strain WY13 (c(S)-2b =56.1%, ee(S)-2b = 99.9%) and to 6 with strain WY3
(c(S)- =35.6%, ee(S)- = 99.9%). With both strains the most robust bioreductions could be
2b
2b
performed from ketone 1c in wide operating pH range 3–10 for WY13 (c(S)- >99%, ee(S)-
2c
2c
>98%) or 5–8 for WY3 (c(S)-2c >99%, ee(S)-2c >99%). Although with lower conversions (c(S)-
2d
21–62% with WY13 and 9–49% with WY3), ketone 1c could be reduced by excellent
enantiotope selectivity (ee(S)-2d >99.5%) in a wide pH range of 3–11.
Because these isolates (WY3 and WY13) of W. subpelliculosus are whole-cell biocatalysts,
it is obvious that they contain a number of different ketoreductases with various properties.
Thus, the variations in optimal pH/operation condition for bioreductions of the various ketones
1a-d are understandable by assuming slightly different expression levels/concentrations of
these enzymes within the two different strains.
3.6. Effect of organic solvents on ketoreductase activity in bioreductions of phenylacetone 1c
16

The significant influence of organic solvents on ketoreductase activity and selectivity is
well-known in case of many ketoreductase-producing strains [37,38]. Strains from
Chryseobacterium sp. could tolerate a wide selection of organic solvents [39], furthermore in
bioreductions with baker’s yeasts several organic solvents had a positive impact on activity
and/or selectivity [29,40]. This prompted us to study the effect of several organic solvents on
ketoreductase activity and selectivity of the strains WY3 and WY13 in the bioreduction of 1c
as an appropriate substrate (Table 5). Because in the absence of organic solvents the
bioreductions of 1c resulted in 99% conversion after 24 h, the reaction time in the organic
solvent tolerance tests has been shortened to 6 h to enable observation of the positive effects.
Although with some variations, the organic solvent tolerance of the strains WY3 and WY13
were similar. The best tolerated solvents by the two W. subpelliculosus isolates were dimethyl
sulfoxide and i-octane with >50% residual conversions and ee >94% up to 5 v/v% concentration
level compared to the bioreduction without cosolvent. n-Hexane was somewhat less tolerated
up to 5 v/v% level (>37% residual conversions and ee >94%). Methanol could be tolerated only
up to 5 v/v% level (>52% residual conversions and ee >96%). The worst tolerated solvents by
the strains WY3 and WY13 were acetonitrile and toluene casing significantly decreased
ketoreductase activity and selectivity even at 2.5 v/v% level (<10% residual conversions and
ee <80%). Thus, apparently no correlation was observed between solvent inactivation and
solvent logP values [-1.3 (dimethyl sulfoxide), -0.85 (methanol), 3.1 (toluene), 3.5 (n-hexane)
and 4.5 (iso-octane)]. Interestingly, when 2-propanol was added to the system in the presence
of 4% glucose, the conversions were significantly lower (e.g c(S)-2c = 7.6% and 3.9% at 2.5% 2-
propanol content with WY3 and WY13, respectively in Table 5) than found in the presence of
2-propanol as sole cosubstrate (e.g c(S)-2c = 45.6% and 46.0% at 4% 2-propanol content with
WY3 and WY13, respectively in Table 4). The solvent tolerance results can be best explained
by assuming at least two ketoreductases acting on substrate 1c, the (S)-selective one(s) with
17

lower organic substrate tolerance as major biocatalyst and another (R)-selective one(s) with
better organic solvent tolerance as minor contributor to the bioreduction.
In addition to the role organic solvents as modifiers of activity and/or selectivity, they can
act as co-solvents improving the solubility of not-well-soluble substrates. This advantageous
effect was clearly demonstrated by this study in the biotransformations of 1e (Table 6). When
bioreduction of ketone 1e was tried under the “standard” conditions (4 w/v% of glucose as a
cosubstrate in phosphate buffer at pH 7.5 and 30 °C) with either of the strains WY3 or WY13,
apparently no product formation was observed in 24 h. Successful bioreductions of 1e with
modest conversions and good enantiotope selectivities could be performed in presence of
2.5 v/v% DMSO as co-solvent (c(S)-2e = 51.5%, ee(S)-2e = 94.2% with WY3 and c(S)-2e = 47.0%,
ee(S)- = 92.8% with WY13). The bioreductions performed at presence of 5 v/v% DMSO
2e
indicated that larger amounts of DMSO are harmful for the whole-cell bioreduction (c(S)-
=
2e
11.3% with WY3 and c(S)-2e = 1.5% with WY13). The fact that DMSO tolerance profiles were
different for bioreductions of ketones 1c (Table 5) and 1e (Table 6) further supported the
hypothesis that in bioreductions of ketones 1a-e with these W. subpelliculosus isolates at least
two ketoreductases were involved with different solvent tolerance.
4.
Conclusion
Our study – related to our program aiming discovery of novel strains as whole-cell
biocatalysts with significant ketoreductase activity – revealed two novel strains of
Wickerhamomyces subpelliculosus WY3 and WY13 applicable as efficient biocatalyst for
bioreduction of ketones. To our best knowledge, this is the first study exploring the excellent
ketoreductase activity and selectivity of W. subpelliculosus strains. Not only the freshly
harvested cells of the two newly isolated W. subpelliculosus WY3 and WY13 strains but also
their lyophilized cells – even after long-term storage – proved to be excellent biocatalysts in
18

bioreduction of various ketones 1a-e. Both 2-propanol and glucose were investigated as
cosubstrate in the bioreductions. As expected, glucose was tolerated at higher concentration (up
to 16 w/v%) than 2-propanol (up to 4 v/v%). The excellent results achieved in bioreduction of
phenylacetone 1c even at high concentration of glucose (8 w/v %) as cosubstrate [c= 99.8%,
ee(S)-2c= 99.6% with WY3; and c= 99.9%, ee(S)-2c= 99.8%] indicated the usefulness of these
novel whole-cell biocatalysts. Both strains showed moderate tolerance of temperature (up to
40 °C) stability in wide pH range (3–10) and variable tolerance of a few organic solvents up to
5 w/v%. Based on our results, W. subpelliculosus can be recommended as an excellent
extension to the toolbox of whole-cell biocatalyst with ketoreductase activity for production of
chiral alcohols in high enantiopurity.
Acknowledgments
This work was supported by a grant of the “Közép-Magyarország Operatív Program”
(KMOP-1.1.4-11/a-2012-0007). LP and GH thank for the funding from the New Hungary
Development Plan (TÁMOP-4.2.1/B-09/1/KMR-2010-0002). LP and PC thank COST Action
CM1303 “Systems Biocatalysis” and project P_37-273 “NEMSyB” for financial support. ÖR
thanks the Gedeon Richter Talentum Foundation and the Pro Progressio Foundation for their
financial support. Thanks are due to Ferenc Faigl (BME, Budapest, Hungary) and to János Éles
and István Greiner (Richter Gedeon PLC, Budapest, Hungary) for scientific support and
valuable discussions.
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25

Figure 1. Effect of temperature on bioreduction of 1a (A), 1b (B), 1c (C) and 1d (D) with
strains WY3 (conversion: ■, enantiomeric excess: ●) and WY13 (conversion: □, enantiomeric
excess: ○); substrate to dry cell ratio: 1:15; glucose: 4 w/v%; pH 7.5; 24 h. Due to the large
uncertainty of enantiomeric excess for reactions of low conversion, no ee values are shown
when conversion remained under 2%.
B
A
100
80
60
40
20
0
100
95
90
85
80
75
70
100
80
60
40
20
0
100
95
90
85
80
75
70
30 35 40 45 50 55 60
Temperature (°C)
30 35 40 45 50 55 60
Temperature (°C)
D
C
100
80
60
40
20
0
100
100
80
100
95
90
85
80
75
70
95
90
85
80
75
70
60
40
20
0
30 35 40 45 50 55 60
Temperature (°C)
30 35 40 45 50 55 60
Temperature (°C)
26

Figure 2. Effect of pH on bioreduction of 1a (A), 1b (B), 1c (C) and 1d (D) with strains WY3
(conversion: ■, enantiomeric excess: ●) and WY13 (conversion: □, enantiomeric excess: ○).
Buffers: 100 mM sodium citrate (pH 3.0 – 5.0), 100 mM sodium phosphate (pH 6.0 – 8.0) and
100 mM sodium carbonate – sodium bicarbonate (pH 9.0 – 11.0); substrate to dry cell ratio:
1:15; glucose: 4 w/v%; 30 °C; 24 h. Due to the large uncertainty of enantiomeric excess for
reactions of low conversion, no ee values are shown when conversion remained under 2%.
A
B
100
80
60
40
20
0
100
95
90
85
80
75
70
100
100
95
90
85
80
75
70
80
60
40
20
0
3
4
5
6
7
8
9 10 11
3
4
5
6
7
8
9 10 11
pH
pH
C
D
100
80
60
40
20
0
100
95
90
85
80
75
70
100
80
60
40
20
0
100
95
90
85
80
75
70
3
4
5
6
7
8
9 10 11
3
4
5
6
7
8
9 10 11
pH
pH
27

Scheme 1. Bioreduction of prochiral ketones 1a-e by Wickerhamomyces subpelliculosus cells
28

Table 1. The phylogenetic affiliation of the fungal strains based on their 18S-28S ribosomal
DNA internal transcribed spacer (ITS) region sequences compared to that of Wickerhamomyces
subpelliculosus NR 111336 as closest phylogenetic relative
Strain ID
Similarity
(sequenced/target DNA)
99.8% (494/495)
WY3
WY13
99.5% (559/561)
CBS5767
100.0% (574/574)
29