Simple Preparation of Rhodococcus erythropolis DSM 44534 as Biocatalyst to Oxidize Diols into the Optically Active Lactones

Article abstract of DOI:10.1002/chir.22623

In the current study, we present a green toolbox to produce ecological compounds like lactone moiety. Rhodococcus erythropolis DSM 44534 cells have been used to oxidize both decane-1,4-diol (2a) and decane-1,5-diol (3a) into the corresponding γ- (2b) and δ-decalactones (3b) with yield of 80% and enantiomeric excess (ee)?=?75% and ee?=?90%, respectively. Among oxidation of meso diols, (?)-(1S,5R)-cis-3-oxabicyclo[4.3.0]non-7-en-2-one (5a) with 56% yield and ee?=?76% as well as (?)-(2R,3S)-cis-endo-3-oxabicyclo[2.2.1]dec-7-en-2-one (6a) with 100% yield and ee?=?90% were formed. It is worth mentioning that R. erythropolis DSM 44534 grew in a mineral medium containing ethanol as the sole source of energy and carbon Chirality 28:623–627, 2016.

Full text of DOI:10.1002/chir.22623

CHIRALITY (2016)
Short Communication
Simple Preparation of Rhodococcus erythropolis DSM 44534 as
Biocatalyst to Oxidize Diols into the Optically Active Lactones
2
ENRIQUETA MARTINEZ-ROJAS,1 TERESA OLEJNICZAK,2 KONRAD NEUMANN,3 LEIF-ALEXANDER GARBE1,3 AND FILIP BORATYÑSKI
*
1Neubrandenburg University of Applied Sciences, Neubrandenburg, Germany
2Department of Chemistry, University of Environmental and Life Sciences, Wrocław, Poland
3Department of Biotechnology, TU Berlin, Bioanalytics GG6, Berlin, Germany
ABSTRACT
In the current study, we present a green toolbox to produce ecological com-
pounds like lactone moiety. Rhodococcus erythropolis DSM 44534 cells have been used to oxidize
both decane-1,4-diol (2a) and decane-1,5-diol (3a) into the corresponding γ- (2b) and δ-
decalactones (3b) with yield of 80% and enantiomeric excess (ee) = 75% and ee = 90%, respec-
tively. Among oxidation of meso diols, (À)-(1S,5R)-cis-3-oxabicyclo[4.3.0]non-7-en-2-one (5a)
with 56% yield and ee = 76% as well as (À)-(2R,3S)-cis-endo-3-oxabicyclo[2.2.1]dec-7-en-2-one
(6a) with 100% yield and ee = 90% were formed. It is worth mentioning that R. erythropolis
DSM 44534 grew in a mineral medium containing ethanol as the sole source of energy and
carbon Chirality 00:000–000, 2016. © 2016 Wiley Periodicals, Inc.
KEY WORDS: Bacteria; diols; lactones; oxidation; Rhodococcus erythropolis DSM 44534;
stereoselectivity
The biological properties of chiral compounds are often
strongly related to the absolute configuration. Therefore,
looking for new stereoselective methods to synthesize opti-
cally pure molecules with various biological activities is one
of the most studied areas in current chemistry. Lactones are
widespread natural products, which exhibit a wide spectrum
of biological activities such as antimicrobial, antioxidant, and
anticancer.1–3 Furthermore, lactone moieties have been
found as natural ingredients in different enantiomeric compo-
sitions used in the essences and fragrances industry.4
Although the organocatalytic methods of lactonization of
diols with application of toxic metal complexes under extreme
experimental conditions are still applied,5–8 the use of
biocatalysts has considerably increased in the preparation of
enantiomerically pure or enriched lactones under mild and non-
toxic conditions. Biotransformations are carried out under mild
temperature, pH, and pressure conditions and their most signif-
icant advantages are high chemo-, regio-, and stereoselectivity.
In the literature, different microbial/enzymatic methods of
lactone biosynthesis were presented.9–14 A chemoenzymatic
route with application of modified lipase from Pseudomonas
sp. in order to catalyze a stereoselective acylation of 5-
hydroxy acid esters and subsequent lactonization into opti-
cally active lactones has been optimized.13 Reduction of car-
bonyl groups in γ- and δ-ketoacids or ketoesters as well as
lactonization process was catalyzed by different species of
yeast.15 Hydrolytic dehalogenation of bis(4-chloro-n-butyl)
ether with Rhodococcus erythropolis DSM 44354 afforded γ-
butyrolactone as a major metabolite and 1,4-butanediol as
well as γ-butyrolactol as intermediate products.16
whole cells of Acetobacter aceti contributed to drospirenon
production.18 A native as well as a recombinant alcohol dehy-
drogenase from horse liver (HLADH) has been broadly used
in the oxidation of racemic 1,4-diols,19 1,5- and 1,6-diols,12 as
well as meso diols20 into the enantiomerically enriched lac-
tones. Recently, commercially available HLADH has also
been used for the desymmetrization oxidation of diols by
adding organic cosolvents to prepare lactones with enantio-
meric excess (ee) and improved yield.21
In our previous work, it was observed that decane-1,4-diol
as well as nonane-1,4-diol were converted by R. erythropolis
DSM 44354 into optically active γ-R-decalactone and γ-R-
nonalactone, respectively.22 Additionally, the oxidation pro-
cess of meso diols possessing monocyclic and bicyclic struc-
tures into corresponding chiral lactones by R. erythropolis
DSM 44354 was established during the microbial alcohol de-
hydrogenase screening.23 Herein we report an optimized pro-
tocol for the oxidation of racemic aliphatic diols as well as
meso cyclic diols into the corresponding lactone products
using whole cells of R. erythropolis DSM 44354 as biocatalyst.
MATERIALS AND METHODS
Strain and Cultivation Conditions
R. erythropolis DSM 44534 was obtained from the German Collection of
Microorganisms (DSMZ, Braunschweig, Germany). The biomass pro-
duction was performed as follows: R. erythropolis cells were grown for
5 days at 28 °C in shake flasks containing mineral medium supplemented
Contract grant sponsor: National Science Centre; Contract grant number:
2011/03/B/NZ9/05005.
*Correspondence to: Filip Boratyñski, Department of Chemistry, University of
Environmental and Life Sciences, Norwida 25, 50-375, Wrocław, Poland. E-
mail: filip.boratynski@up.wroc.pl
Received for publication 31 December 2015; Accepted 24 June 2016
DOI: 10.1002/chir.22623
Published online in Wiley Online Library
(wileyonlinelibrary.com).
Also worth mentioning is the biooxidative methods in lac-
tone synthesis. Microbial Baeyer-Villiger oxidation with appli-
cation of whole cell cultures in the synthesis of lactones from
α- and β-thujone was successfully applied.17 Biooxidation of 1-
alkylbutane-1,4-diols into the corresponding γ-lactones by
© 2016 Wiley Periodicals, Inc.

MARTINEZ-ROJAS ET AL.
with 1.8 g LÀ1 (50 mM) of ethanol as carbon and energy source. Biomass
Determination of Enantiomeric Excess of Lactones 1b-6b
growth was followed by measurements of optical density at OD620. Cells
from late-exponential phase were harvested by centrifugation, washed
twice with 50 mM potassium phosphate buffer, pH 7.5 (KPi), and frozen
at À20 °C.
Gas chromatography analyses (GC, FID, carrier gas H2) were carried
out on Agilent Technologies 7890 N (Palo Alto, CA; GC System) with
HP-5 column (cross-linked methyl silicone, 30 m x 0.32 mm x 0.25 μm).
Enantiomeric excesses of the lactone products was determined on chiral
columns: Lipodex E (50 m x 0.31 mm x 0.25 μm) for lactones 1b-3b and
Cyclosil-B (30 m x 0.25 mm x 0.25 μm) for lactones 4b-6b under the fol-
lowing temperature program: initial temperature of 120 °C, then in-
creased to 220 °C with a rate of 10 °C minÀ1, finally to 250 °C at a rate
Biotransformation Experiments
Biotransformation assays with washed cells were performed in 24-
deepwell polypropylene microtiter plates (MTP) containing 2 mL of cells
dissolved in KPi buffer until a final concentration of 20 mg mLÀ1. The sub-
strate 1a-6a was previously dissolved in KPi buffer supplemented with
0.1% Triton X-100 and added to the cells solution to reach a concentration
of 2 mM. Subsequently, the MTP was incubated at 30 °C on a rotary
shaker at 180 rpm. For the time-course analysis at appropriate intervals,
200 μL of the reaction mixture was transferred to corresponding wells
in empty MTP. Then each well was acidified with 10 μL of 100 mM HCl
and extracted with diethyl acetate (500 μL). Finally, the organic phase
from each well of MTP was transferred to a gas chromatography (GC)
vial and analyzed on GC-FID-MS. The biotransformation experiments
were set up in triplicate. Heat-killed cells (100 °C for 15 min) were used
as a control.
of 20 °C minÀ1
.
RESULTS AND DISCUSSION
Chemical Synthesis of Diols 1a-6a
The substrates for microbial oxidation, racemic aliphatic di-
ols 1a-3a as well as meso diols 4a-6a, were obtained by the
reduction method of the corresponding lactones 1b-3b and
anhydrides 4c-6c, respectively (Fig. 1).
The synthesized diols 1a-6a were divided into two
groups: aliphatic and cyclic diols. The first group were com-
prised of nonane-1,4-diol (1a), decane-1,4-diol (2a), and
decane-1,5-diol (3a). The second group was represented
by monocyclic meso diols with cyclohexane ring cis-1,2-
bis-(hydroxymethyl)-cyclohexane (4a), its unsaturated ana-
log cis-4,5-bis-(hydroxymethyl) cyclohexene (5a), and un-
saturated bicyclic diol with the structure of [2.2.1] cis-endo-
2,3-bis (hydroxymethyl) bicyclo[2.2.1]hept-5-ene (6a). The
structures of isolated compounds were confirmed by spec-
tral data (1H NMR,13C NMR, and IR). Furthermore, GC–
MS analyses were carried out in order to compare the ob-
tained spectra with the compounds described in the
literature.23
Chemicals
γ-Nonalactone (5-pentyl-dihydrofuran-2-one (1b)), γ-decanolactone (5-
hexyl-dihydrofuran-2-one
(2b)),
δ-decanolactone
(6-pentyl-
tetrahydropyran-2-one (3b)), cis-4-cyclohexene-1,2-dicarboxylic anhy-
dride (5c), cis-5-norbornene-endo-2,3-dicarboxylic anhydride (6c), and
LiAlH4 were purchased from Sigma-Aldrich Chemical (St. Louis, MO),
while cis-cyclohexane-1,2-dicarboxylic anhydride (4c) was purchased
from Fluka BioChemika (Buchs, Switzerland). All solvents were of analyt-
ical grade or distilled before use.
Synthesis of Diols 1a-6a
Biotransformation of Diols
A standard procedure was performed for the synthesis of racemic 1,4-
diols 1a-3a from the corresponding lactones 1b-3b. In brief, racemic lac-
tones 1b-3b were reduced with lithium aluminum hydride in dried tetra-
hydrofuran.22 Standard workup and silica gel chromatography afforded
the diols 1a-3a in 65–75% yield and more than 95% GC purity.
Meso diols 4a-6a were synthesized by reduction of the corresponding
anhydrides 4c-6c mixed in diethyl ether: tetrahydrofuran (1:1) with
LiAlH4 dissolved in diethyl ether. The reactions were carried out accord-
ing to the procedure described before.23 The yields of diols 4a-6a ob-
tained were in the range 53–73%.
R. erythropolis grew in a mineral medium containing etha-
nol as the sole source of energy and carbon. After 5 days of
growth, the cells were collected and then living cells were
prepared in a concentration of 20 mg of dry weight per
1 mL KPi buffer. The cell suspension was used to perform
the transformation of diols 1a-6a and to detect metabolites
within the degradation pathway. Heat-killed cells were used
GC-FID/GC–MS Analysis
The metabolites formed during the biotransformation were identified
by their GC retention times and their electron-impact mass spectrometry
fragmentations (EI-MS), which were identical to those of commercially
available compounds or reference standards from chemical synthesis.
In addition, EI-MS fragments were in accordance with the NIST database.
Qualitative and Quantitative Analyses of Lactones 1b-6b
Analyses were performed on a DB-WAX capillary column (60 m x
0.32 mm x 0.25 μm J&W Scientific, Folsom, CA) installed in a GC-FID
(Fision Instruments Model 8165). Nitrogen was used as a carrier gas with
a flow rate of 2 mL minÀ1. The detector temperature was set to 240 °C.
The initial oven temperature was held at 160 °C for 4 min, then increased
to 230 °C with a rate of 10 °C minÀ1 and finally held for 3 min. GC–MS
was performed on a Hewlett Packard HP 5890 GC equipped with a DB-
WAX capillary column (30 m x 0.25 mm x 0.25 μm) coupled to a
TSQ7000 mass spectrometer (EI-MS ionization energy =70 eV). Carrier
gas was He (5.6) at 120 kPa. The initial oven temperature was set to
70 °C for 2 min, raised to 120 °C at a rate of 5 °C minÀ1, then raised to
260 °C at a rate of 40 °C minÀ1, and finally held for 8 min. Data were proc-
essed with HP ChemStation (v. A.01.03, 1988) software.
Fig. 1. Chemical synthesis of aliphatic (1a-3a) and meso diols (4a-6a) by
reduction of the corresponding lactones (1b-3b) and anhydrides (4c-6c).
Chirality DOI 10.1002/chir

BIOOXIDATION OF DIOLS BY RHODOCOCCUS ERYTHROPOLIS DSM 44534
as a control. Initially, the experiments were set up to establish
the optimal reaction time.
appeared in less than 65%. Contrary to Figure 2, the results
obtained with the disappearance of the corresponding meso
diols as well as with heat-killed cells (as control) were not
shown due to the same tendency as in the oxidation of ali-
phatic diols 1a-3a.
Biotransformation of Racemic Aliphatic Diols 1a-3a
Within 2 h more than 50% of the initial concentration
(2 mM) of diol 1a was oxidized and 0.9 mM of γ-nonalactone
(1b) was formed, which was 56% of 1b entirely produced
(1.6 mM). After 3 h of the incubation, 20% of the initial amount
of diol 1a concentration was detected and 1b concentration
had increased slightly (1.4 mM). The highest 1b concentra-
tion (1.6 mM) was detected after 4 h incubation. The level de-
creased gradually and after 8 h 1b was not detected at all
(Fig. 2).
Opposite to our previous findings,23 we report the
highest lactone 4b-6b concentration after
7
days
incubation during the screening tests in the microtiter
plate. Apparently, such disagreement could be an effect of
the different composition of the growth medium or the
amount of biocatalyst used in the experiments. During this
assay the oxidation time of meso diols 4a-6a was reduced
from
7 to 4 days with nearly the same yield and
In the oxidation of diol 2a after 6 h more than 80% of γ-
decalactone (2b) was detected. In comparison with the oxida-
tion of 1a, the concentration of the product did not decrease
drastically and after 8 h 50% of lactone 2b was present.
When diol 3a was oxidized into the corresponding δ-
decalactone (3b) the highest conversion was observed after
4 h of incubation. Finally, after 17 h of biotransformation,
the concentration of 3b was still detected (40%) (data not
shown).
stereoselectivity, which shows good potential to scale-up
the process with high concentrations of cells.
The biotransformation reaction was not 100% equimolar,
because from the initial concentration of diol (2 mM), in the
case of aliphatic diols as substrates, only 1.6 mM of product
was detected. The rest (0.4 mM) was converted into minor
compounds, which were not identified.
The decreasing concentration of synthesized lactones is
also observed in yeast cultures, which are able to assemble
de novo aroma compounds and use these molecules as a car-
bon source. Among a number of reports describing lactone
degradation, reconsumption of γ-decalactone (1b) in
Yarrowia lipolytica culture was presented by Waché et al.24
Although the metabolic pathway of flavoring lactone degrada-
tion in R. erythropolis cells is not fully explored, N-
acylhomoserine lactonase activity has been described in
Rhodococcus genus. This discovery opens the possibility to
find out the enzyme or group of enzymes responsible for
the lactone degradation.25
Biotransformation of Meso Diols 4a-6a
Microbial oxidation of meso monocyclic diols 4a and 5a,
whose chemical structures differ in the presence of the unsat-
urated bond, and the bicyclic diol 6a were performed in the
same reaction conditions as done in the oxidation of the race-
mic aliphatic diols 1a-3a. The only exception was the bio-
transformation time, which was prolonged to 10 days with
respect to earlier experiments. In Figure 3 the results ob-
tained from oxidation of meso diols 4a-6a are summarized.
Under these experimental conditions after 2 days 60%, 40%,
and 30% conversion was achieved, which corresponds to mo-
lar concentrations of 1.2 mM for cis-3-oxabicyclo[4.3.0]nonan-
2-one (4b), 0.6 mM for cis-endo-3-oxabicyclo[2.2.1]dec-7-en-2-
one (6b), and 0.8 mM for cis-3-oxabicyclo[4.3.0]non-7-en-2-
one (5b), respectively. The highest concentration of products
4b and 6b was reached after 4 days of the incubation. Con-
trary to this, meso diol 5a was not completely oxidized and af-
ter 8 days of the incubation the corresponding lactone 5b had
Determination of the Process Enantioselectivity
The enantioselectivity of the biooxidation process was de-
termined by chiral GC analysis when the highest concentra-
tion of the lactone product was reached. The results of the
microbial oxidation of diols 1a-6a are summarized in
Table 1. R. erythropolis preferentially oxidized the R enantio-
mer of all aliphatic diols 1a-3a. The observed ee of lactones
enhanced with increasing alkyl chain length (ee = 43% for
Fig. 2. Product yield (%) after biotransformation of racemic aliphatic diols (1a-3a) into the corresponding optically pure lactones (1b-3b) with whole cells of R.
erythropolis. Disappearance of 1,4-nonadiol is represented with living cells (ÀÀ + À-), with heat-killed cells as a control.
Chirality DOI 10.1002/chir

MARTINEZ-ROJAS ET AL.
Fig. 3. Time courses for the biotransformation of meso diols 4a-6a by R. erythropolis cells.
TABLE 1. The conversion (according to chiral GC) of diols 1a-
6a in the biotransformation process
CONCLUSION
The biotransformation yields obtained with whole cells of R.
erythropolis are quite significant and are interesting due to the
simplicity of preparation of the biocatalyst. Among the biocon-
version processes carried out, oxidation of decan-1,5-diol (3a)
and cis-endo-2,3-bis(hydroxymethyl)bicyclo[2.2.1]hept-5-ene
(6a) showed the best conversion yield as well the highest en-
antiomeric excess. The biotransformation time was signifi-
cantly reduced due to application of an increased amount of
biocatalyst. In this sense, a potential interesting application as-
sumes the oxidation of decan-1,5-diol (3a) to optically active δ-
decanolactone (3b), which has a creamy-coconut and peach-
like aroma and is an important flavor constituent of many
types of fruits, cheese, and other dairy products. In the case
of δ-decanolactone (3b), both isomers as well as racemic mix-
tures indicate different aromas. The (+)-R-isomer, preferen-
tially found in nature, is the favored one for practical
applications.
Lactone
No. Diol
Time
Conversion of diol
[%]
[hours]
ee [%] Enantiomer
1
2
3
4
5
6
1a
2a
3a
4a
5a
6a
4
6
6
96
96
96
80
85
95
100
56
100
43
75
90
20
76
90
(+)-4R
(+)-4R
(+)-5R
(+)-(1S,5R)
(À)-(1S,5R)
(À)-(2R,3S)
1b and ee = 75% for 2b). Comparing the lactone ring size, lac-
tones with pyran ring were characterized by higher ee
(ee = 90% for 3b) than lactones with furan ring (ee = 43% for
1b and ee = 75% for 2b).
Among monocyclic meso diols 4a and 5a, R. erythropolis
preferred the oxidation of 4a (conv. = 100%) over its unsatu-
rated analog 5a (conv. = 56%). However, the process was
characterized by significantly lower enantioselectivity
(ee = 20% for 4b and ee = 76% for 5b). As it turned out, the
more bulky substrate bicyclic diol 6a was completely oxi-
dized into the corresponding lactone 6b with the highest ee
(ee = 90%). Oxidation of all meso diols 4a-6a took a longer
time (96 h) in comparison to aliphatic racemic diols 1a-3a
(4–6 h).
Organocatalytic reaction based on the oxidation of diol 3a
into the corresponding lactone 3b in occurrence of co-
oxidants was proposed by Jhulki et al.26 The reaction yield
afforded 55% in 15 h. This result is opposite to our findings,
where the highest yield of lactone 3b was reached after 6 h
of biotransformation. It is worth pointing out that the corre-
sponding R-isomer of lactone 3b was formed with high ee
(ee = 90%).
Our experiments assumed oxidation of a number of diols,
among them aliphatic racemic diols 1a-3a and cyclic meso di-
ols 4a-6a. Due to the manifoldness of enzyme and metabolic
versatility (ability to grow in an unconventional carbon source
such as ethanol), R. erythropolis is a promising candidate for a
wide range of biotransformation processes. Our future work
will focus on the optimization of the biooxidation conditions,
by implementing a downstream process on a large scale to
minimize undesirable side reactions. Furthermore, the en-
zyme or group of enzymes responsible for diols oxidation will
be purified and characterized in order to offer another tool-
box for green chemistry.
ACKNOWLEDGMENTS
Publication supported by Wroclaw Centre of Biotechnol-
ogy, programme The Leading National Research Centre
(KNOW) for years 2014–2018 (http://know.wroc.pl).
Chirality DOI 10.1002/chir

BIOOXIDATION OF DIOLS BY RHODOCOCCUS ERYTHROPOLIS DSM 44534
15. Forzato C, Gandolfi R, Molinari F, Nitti P, Pitacco G, Valentin E. Microbial
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Chirality DOI 10.1002/chir