Yarrowia lipolytica dehydrogenase/reductase: An enzyme tolerant for lipophilic compounds and carbohydrate substrates

Article abstract of DOI:10.1016/j.bmcl.2013.03.064

Yarrowia lipolytica short chain dehydrogenase/reductase (YlSDR) was expressed in Escherichia coli, purified and characterized in vitro. The substrate scope for YlSDR mediated oxidation was investigated with alcohols and unprotected carbohydrates spectrophotometrically, revealing a preference for secondary compared to primary alcohols. In reduction direction, YlSDR was highly active on ribulose and fructose, suggesting that the enzyme is a mannitol-2-dehydrogenase. In order to explore substrate tolerance especially for space-demanding, lipophilic protecting groups, 5-O-trityl-d-ribitol and 5-O-trityl-α,β-d-ribose were investigated as substrates: YlSDR oxidized 5-O-trityl-d-ribitol and 5-O-trityl-α,β-d-ribose and reduced the latter at the expense of NADP(H).

Full text of DOI:10.1016/j.bmcl.2013.03.064

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3393–3395
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Yarrowia lipolytica dehydrogenase/reductase: An enzyme tolerant
for lipophilic compounds and carbohydrate substrates
Kamila Napora a, Tanja M. Wrodnigg b, Patrick Kosmus b, Martin Thonhofer b, Karen Robins c,
Margit Winkler a,
⇑
a Acib GmbH, Petersgasse 14, Graz 8010, Austria
b Glycogroup, Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse 9, Graz 8010, Austria
c Lonza AG, Rottenstrasse 6, Visp 3930, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 6 April 2013
Yarrowia lipolytica short chain dehydrogenase/reductase (YlSDR) was expressed in Escherichia coli, puri-
fied and characterized in vitro. The substrate scope for YlSDR mediated oxidation was investigated with
alcohols and unprotected carbohydrates spectrophotometrically, revealing a preference for secondary
compared to primary alcohols. In reduction direction, YlSDR was highly active on ribulose and fructose,
suggesting that the enzyme is a mannitol-2-dehydrogenase. In order to explore substrate tolerance espe-
Keywords:
Short chain dehydrogenase/reductase (SDR)
Carbohydrates
Yarrowia lipolytica
Biooxidation
cially for space-demanding, lipophilic protecting groups, 5-O-trityl-
D
-ribitol and 5-O-trityl- ,b-
a
D-ribose
were investigated as substrates: YlSDR oxidized 5-O-trityl-
D
-ribitol and 5-O-trityl- ,b-D-ribose and
a
reduced the latter at the expense of NADP(H).
Bioreduction
Ó 2013 Elsevier Ltd. All rights reserved.
Oxidation reactions in general and selective oxidation reactions
on carbohydrates in particular are very important chemical trans-
formations and are frequently carried out using toxic (chromate
based or sulfide releasing), expensive (Dess–Martin) and often
unselective reagents.1 To circumvent these typically harsh and
uneconomic reaction conditions of chemical oxidation we were
interested to find an enzyme with the ability to oxidize polar as
well as non-polar carbohydrate compounds. Yarrowia lipolytica is
a non-conventional yeast that is often found in lipid-rich media2
and its machinery of enzymes seems to be well developed to
metabolize both polar and non-polar substrates.3 This prompted
us to investigate enzymes from this organism.
at OD600 0.6–0.8 and the expression phase carried out for 16–
18 h at 28 °C. Collected cells were frozen at À20 °C for whole cell
biotransformations or sonicated and the YlSDR protein purified
by Ni-affinity chromatography over a GE Healthcare HisTrap FF
column according to the manufacturers protocol. The eluate was
re-buffered into 100 mM Tris–HCl, pH 10.5 containing 50 mM
NaCl, 1 mM tris(2-carboxyethyl)phosphine (TCEP) and 1% v/v glyc-
erol, the protein concentrated by spin column centrifugation and
aliquots were stored at À80 °C.
First, in order to explore the substrate scope of YlSDR, several
commercially available compounds such as simple primary and
secondary alcohols of different chain length, selected aldoses, ke-
toses and polyols as well as two aldehydes were tested. Whereas
for SalM the preferred cofactor is NAD+,4 it was NADP+ for YlSDR.6
Its closest homologue for which a crystal structure is known (Can-
dida parapsilosis carbonyl reductase, pdb: 3ctm) was also reported
to be NADP(H) dependent.7 To explore enzyme characteristics and
the substrate scope of YlSDR, the consumption of NADP+ was mon-
itored at 340 nm.6 Substrate oxidation was tested between 22 and
38 °C and YlSDR showed the highest activity between 25 and 28 °C.
The optimal pH for the reaction appears to be 10.0 (Fig. 1), how-
ever, further increase resulted in a drastic enzyme deactivation.
In a recent publication, 5-chloro-5-deoxy-a,b-D-ribose was oxi-
dized to the respective 5-chloro-5-deoxy- -ribonolactone by the
D
short chain dehydrogenase/reductase (SDR) SalM from the marine
organism Salinispora tropica.4 We amplified the gene of a homolo-
gous short chain dehydrogenase (NCBI Accession No.
XM_500963.1, 30% identity to SalM) from genomic DNA of the
Yarrowia lipolytica CLIB122 strain and cloned the gene into the
pK470 vector,5 introducing also an N-terminal His-tag to facilitate
protein purification. The construct was transformed into Esche-
richia coli BL21 (DE3) Gold. The strain was cultivated in Luria Broth
medium, YlSDR expression induced with 0.1 mM isopropyl b-
thiogalactopyranoside (IPTG) and supplemented with 1% glucose
D
-
Whereas polyols such as xylitol or
dized according to the photometric measurements, sugars such
as -xylose, -glucose or -mannose were not. In fact, secondary
D-mannitol were readily oxi-
D
D
D
alcohols were preferred: for the tested compounds, YlSDR showed
the highest activity for racemic 2-heptanol (1.34 0.44 U/mg),
⇑
Corresponding author. Tel.: +43 316 873 9333; fax: +43 316 873 9308.
E-mail address: margit.winkler@acib.at (M. Winkler).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.064

3394
K. Napora et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3393–3395
Table 2
Apparent kinetic parameters for YlSDR
Oxidation
rac-2-heptanol
NADP+
4.91 0.31
3.17 0.15
0.65 0.08
Reduction
ribulose
Km [mM]
kcat [sÀ1
kcat/Km [sÀ1 mMÀ1
0.793 0.026
6.15 0.28
7.75 0.06
8.80 2.10
5.42 0.14
0.62 0.24
]
]
The determination of kinetic parameters for oxidation and
reduction corroborated the different specific activities for oxidation
and reduction, showing that the kcat value for reduction of ribulose
is significantly higher than for oxidation of racemic 2-heptanol (Ta-
ble 2). Whereas oxidations appeared most efficient at pH 10.0,
reductions may be carried out between pH 4.5 and 9.0 (Fig. 1). This
remarkably broad operational stability for a wild type enzyme ren-
ders it a good candidate for industrial applicability.
Figure 1. pH Optima of YlSDR in 50 mM buffer. pH 4.5–6.0: citrate, pH 6.5–8.0 and
11.5–12.0: potassium phosphate, pH 7.0–9.0: Tris–HCl, 8.5–10.0: glycine, 9.5–11.0:
carbonate and 10.5–12: piperidine; Ç oxidation of rac-2-heptanol; s reduction of
fructose.
In parallel, we investigated partly protected carbohydrates as
substrates for this enzyme. The results for SalM driven oxidation
of 5-chloro-5-deoxy-
Table 1
a,b-D
-ribose4 prompted us to investigate 5-
Exploration of substrate spectrum of YlSDR
O-trityl- -ribitol (5) and 5-O-trityl-a,b-D-ribose (2) as model sub-
D
Entry Substrate
Relative
Substrate
Relative
strates. We reasoned that trityl ethers should exhibit better com-
patibility with biocatalytic processes than, e.g., typical ester or
amide protective groups which would be cleaved by omnipresent
esterase or amidase/peptidase activities in whole cell biocatalysts.
The trityl group is, however, space demanding (Scheme 2) and
lipophilic, and therefore literature reports on the use of tritylated
substrates in biocatalytic steps are scarce and limited to lipase cat-
alyzed esterifications.10
For the synthesis of substrates 2 and 5 conventional carbohy-
drate chemistry was applied (Scheme 1). Starting from D-ribose
(1), the primary hydroxy group at position C-5 underwent regiose-
lective ether formation employing triphenylmethyl chloride in
pyridine at 50 °C.11 Instead of straight forward reduction of 5-O-tri-
oxidation
activity (%)
reduction
activity (%)
1
2
rac-
28
51
Phenylacet-
aldehydeb
Acetophenone
16
0
Phenylethanol
rac-1-Phenyl-
1,2-Ethanediol
2-Propanol
(2R,3R)-
Butanediol
rac-4-Methyl-
2-pentanol
Cyclohexanol
rac-2-
3
4
3
3
Acetone
0
5
6
6
7
8
Cyclohexanone
2-Heptanone
0
19
100a
Heptanol
8
9
1-Octanol
rac-2-Octanol
(R)-2-Octanol
(S)-2-Octanol
Octanal
1-Nonanol
2-Nonanol
1-Decanol
1-Dodecanolc
Ribitol
0
70
97
36
7
5
27
3
4
0
18
9
24
55
Octanal
2-Octanone
0
16
tyl-
the anomeric position employing bromine and BaCO3 in water12 to
the corresponding 5-O-trityl- -ribono-1,4-lactone (3), as this com-
a,b-D-ribose (2) to ribitol derivative 5, aldose 2 was oxidized at
10
11
12
13
14
15
16
17
18
19
20
21
D
pound was needed for investigating biotransformation reactions of
YlSDR. The 1,4-lactone 3 was reduced employing NaBH4 in metha-
nol and a per-O-acetylation step appeared to be necessary for puri-
fication reasons. Finally, 5-O-trityl-
deacetylation under Zemplen conditions in very pure form.13
In the following, 5-O-trityl- ,b- -ribose (2) was used as a sub-
D-ribitol 5 was obtained by
Ribulosed
Arabinose
Xylose
100
0
0
0
33
Arabitol
Xylitol
Sorbitol
Mannitol
a
D
strate for oxidative whole cell biotransformation reactions.14
According to HPLC/MS, YlSDR mediated oxidation occurred NADP+
dependent at the C-1 position and gave 97% conversion of 2 to 3.
The product peak showed identical retention time and mass spec-
trum as the authentic standard prepared by chemical means and
was in pH-dependent equilibrium with the respective open chain
acid. The whole cell biotransformation result was confirmed by
reaction with purified YlSDR,15 yielding 96% 3. Subsequently, 5-
Glucose
Fructosed
The specific activity for the oxidation of rac-2-heptanol was 1.4 0.4 U mgÀ1
TritonX 100 was used as solubilizer in 0.15% end concentration.
DMSO was used as co-solvent in 5% end concentration. 5% DMSO reduced the
.
a
b
c
activity towards rac-2-octanol oxidation by 25%.
d
Similar to other aldoses, ribose and mannose were not reduced.
O-trityl-D-ribitol (5) was subjected to oxidation with whole cells
and purified enzyme, which resulted in equally low conversion to
mixtures of <5% 2 and approximately 10% of 3. Whereas YlSDR was
unable to reduce 3, compound 2 was partly reduced to 5. However,
in course of the reduction, NADP+ is produced, which is consumed
for the formation of 3. Due to a reversible step (interconversion of 5
and 2) in combination with an irreversible step (oxidation to 3), the
resultant mixture of products typically ends in approximately 10%
5, 20% 2 and 70% 3 in reactions with purified enzyme, after over-
night incubation.14
These findings seem to be in contradiction to the spectrophoto-
metric results discussed above, in which neither D-ribitol nor D-ri-
bose gave significant activities. However, it needs to be considered
that typical photometric assays are restricted to initial rate
followed by racemic 2-octanol. Although the enzyme had higher
activity for (R)-2-octanol, also its (S) enantiomer was oxidized
(Table 1).
Furthermore, YlSDR turned out to be an efficient reductase for
selected substrates: whereas 2-heptanone and 2-octanone were
reduced with similar efficiency to the oxidation of the correspond-
ing alcohols (0.94 0.24 and 0.81 0.38 U/mg), specific activities
for fructose and ribulose reduction were 1.65 0.47 and
4.96 0.57 U/mg.8 Since aldopyranoses were not reduced within
the observed timeframe, YlSDR may be a member of the NADP(H)
dependent mannitol-2-dehydrogenase family (EC1.1.1.138), as
also suggested by its sequence similarity to other proteins of this
family.7,9

K. Napora et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3393–3395
3395
OH
HO
OTrit
HO
OTrit
aldoses may be explored in the presence of for example NADPH
oxidases to prevent shunt oxidation to the corresponding lactones.
O
O
O
O
OH
OH
a
b
Acknowledgments
HO
OH
OH
OH
1
2
3
Thomas Prossliner, Manoj N. Sonavane, Marcelina Bilicka, and
Gerlinde Offenmüller are kindly acknowledged for technical sup-
port. We are grateful to Petra Köfinger and Zalina Magomedova
for vector pK470. This work has been supported by the Austrian
BMWFJ, BMVIT, SFG, Standortagentur Tirol and ZIT through the
Austrian FFG-COMET-Funding Program.
OTrit
OTrit
OAc
OH
OAc
OH
c
d
Trit =
AcO
OAc
HO
OH
4
5
Supplementary data
Scheme 1. Synthesis of 5-O-trityl-D-ribitol 5: (a) Pyr, TritylCl, 50 °C, 50%; (b) Br2,
BaCO3, H2O, 45%; (c) (1) NaBH4, MeOH, (2) Ac2O, Pyr, rt, 54%; (d) NaOMe 1 M,
MeOH, rt, 86%.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.03.
064.
OTrit
OTrit
HO
References and notes
OH
O
OH
OH
1. Madsen, R. In Oxidation and Reduction in Glycoscience; Fraser-Reid, B. O.,
Tatsuta, K., Thiem, J., Eds.; Springer: Berlin, Heidelberg, New York, 2001; pp
195–229.
2. Barth, G.; Gaillardin, C. In Yarrowia lipolytica in Nonconventional Yeasts in
Biotechnology; Wolf, K., Ed.; Springer: Berlin, Heidelberg, New York, 1996; pp
313–388.
HO
OH
OH
2
5
3. Fickers, P.; Benetti, P.-H.; Waché, Y.; Marty, A.; Mauersberger, S.; Smit, M. S.;
Nicaud, J.-M. FEMS Yeast Res. 2005, 5, 527.
4. Kale, A. J.; McGlinchey, R. P.; Moore, B. S. J. Biol. Chem. 2010, 285, 33710.
5. Balzer, D.; Ziegelin, G.; Pansegrau, W.; Kruft, V.; Lanka, E. Nucleic Acid Res. 1992,
20, 1851.
OTrit
OTrit
O
O
O
OH
6. Unless otherwise stated, purified tagged YlSDR (1–10 lM) was assayed in
50 mM Tris–HCl containing 2 mM MgCl2, pH 8.0, 10 mM substrate (for
lipophilic substrates, additional 0.15% Tween 20) and 1 mM NADP+ or NAD+.
The increase of absorbance at 340 nm was monitored at 28 °C for 10 min (or
12 h for ribose). For NAD+, no activity was observed under these conditions. The
reported values represent the average of at least four measurements with
appropriate blanks substracted. One activity unit is defined as the amount of
HO
OH
HO
OH
2
3
enzyme catalyzing the reduction of
1 lM
of NADP+ per minute. Kinetic
Scheme 2. Oxidation of 5-O-trityl-D-ribitol (5) and 5-O-trityl-a,b-D-ribose (2).
parameters for oxidation were determined at pH 10.0 from unweighted non-
linear least-square fits of experimental data using the program Sigmaplot
(version 12.3).
7. Zhang, R.; Zhu, G.; Zhang, W.; Cao, S.; Ou, X.; Li, X.; Bartlam, M.; Xu, Y.; Zhang,
X. C.; Rao, Z. Protein Sci. 2008, 17, 1412.
8. Unless otherwise stated, purified tagged YlSDR (1–10 lM) was assayed in
measurements. Due to short monitoring times, interesting activi-
ties may easily be overlooked. We therefore monitored the oxida-
tion of ribose for 12 h and found a specific activity of 10 mU/mg,
admittedly much lower than those for other substrates (see Ta-
ble 2) but certainly a useful starting point for protein engineering.
Summarizing, we have shown that YlSDR is a versatile enzyme
that catalyzes oxidation and reduction of polar as well as non-polar
substrates at a very broad pH range. Secondary alcohols are pre-
ferred in the oxidation reaction compared to primary alcohols
and aldehydes. Substrate selectivity was found for the reduction
of medium chain length ketones with the carbonyl function at po-
sition C-2. In case of the carbohydrate substrates, alditols are pre-
ferred over aldoses as substrates in the oxidizing mode and ketoses
(2-uloses) are accepted for the reduction step whereas aldoses are
not. Most interestingly, a slightly different picture was obtained
50 mM Tris–HCl containing 2 mM MgCl2, pH 8.0, 10 mM substrate (for
lipophilic substrates, additional 0.15% Tween 20) and 0.75 mM NADPH. The
decrease of absorbance at 340 nm was monitored at 28 °C for 10 min. The
reported values represent the average of at least four measurements with
appropriate blanks substracted. One activity unit is defined as the amount of
enzyme catalyzing the oxidation of
1 lM of NADPH per minute. Kinetic
parameters for reduction were determined at pH 5.0.
9. (a) Hörer, S.; Stoop, J.; Mooibroek, H.; Baumnn, U.; Sassoon, J. J. Biol. Chem. 2001,
276, 27555; (b) Nüss, D.; Goettig, P.; Magler, I.; Denk, U.; Breitenbach, M.;
Schneider, P. B.; Brandstetter, H.; Simon-Nobbe, B. Biochimie 2010, 92, 985.
10. (a) Henly, R.; Elie, C. J. J.; Buser, H. P.; Ramos, G.; Moser, H. E. Tetrahedron Lett.
1993, 34, 2923; (b) MacManus, D. A.; Vulfson, E. N. Enzyme Microb. Technol.
1997, 20, 225; (c) Faigl, F.; Thurner, A.; Farkas, F.; Battancs, M.; Poppe, L.
Chirality 2007, 19, 197; (d) Palocci, C.; Falconi, M.; Chronopoulou, L.; Cernia, E. J.
Supercrit. Fluids 2008, 45, 88.
11. Kam, B. L.; Oppenheimer, N. J. Carbohydr. Res. 1979, 69, 308.
12. Andersen, S. M.; Ebner, M.; Ekhart, C. W.; Gradnig, G.; Legler, G.; Stütz, A. E.;
Withers, S. G.; Wrodnigg, T. Carbohydr. Res. 1997, 301, 155.
13. Jung, M. E.; Xu, Y. Tetrahedron Lett. 1997, 38, 4199.
14. Typically, 100 mg of thawed whole cells were dispersed in 50 mM Tris–HCl
buffer (pH 8.0 for oxidation, pH 6.2 for reduction) containing 20 mM MgCl2 and
with 5-O-trityl-D-ribitol, -ribose and -ribonolactone. In the oxida-
tion step 5-O-trityl-
a
,b- -ribose (3) turned out to be a far better
D
substrate than the corresponding ribitol 5. In the reduction the
same compound is a substrate whereas the lactone is not. In the
light of these results, we are planning to explore the substrate
scope of YlSDR biotransformations in a structure activity relation-
ship study by investigating further tritylated sugar substrates such
8 mM NADP(H). The reaction was started by addition of 10 lL of 2, 3 or 5
(200 mM in DMSO) to give final concentrations of 4 mM substrate and 2 % v/v
DMSO. The reaction proceeded at 30 °C in an Eppendorf Thermomixer at
1000 rpm for 16–18 h and was then stopped by addition of 500 lL of
acetonitrile. After centrifugation, the supernatant was analyzed by HPLC
using a Chromolith Performance RP-18 column with 1.2 mL minÀ1 0.1% formic
acid and acetonitrile as the mobile phase. The respective HPLC traces are
shown in the Supplementary data.
as 5-O-trityl-D-mannitol and 6-O-trityl-D-fructose. Furthermore,
different aromatic substituents at the terminal position of the car-
bohydrate moieties such as various nitro phenyl groups will be in-
cluded to this study. In addition, YlSDR mediated reductions of
15. Bioconversions with purified enzymes contained 2.7 mg of purified YlSDR at
pH 10.5. All other ingredients, workup and analysis see Ref. 14.