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CAQ31055.1) were ordered at DNA2.0 (California, USA) in pJex-
press-404 and 401 expression vectors, respectively. Plasmid DNA
was retrieved according the protocol delivered with the genes and
the sequence was verified by sequencing (LGC genomics). Electro-
competent E. coli BL21 (DE3) Gold cells were cotransformed with
plasmid DNA of both constructs (1.13 mg of pJexpress-404-CAR
and 0.11 mg of pJexpress-401-EcPPTase). Positives were selected on
LB-plates supplemented with 100 mgmLꢁ1 Amp, and 50 mgmLꢁ1
kanamycin after overnight incubation at 378C.
Conclusions
We have presented the first reductive enzyme-catalyzed
method to the natural antioxidant 3-hydroxytyrosol (3-HT).[32]
The applied whole-cell biocatalyst combines the reactivity of
a carboxylate reductase with a number of endogenous reac-
tions, including those for cofactor regeneration, to reduce the
precursor 3,4-dihydroxyphenylacetic acid in two steps to the
corresponding alcohol. Product loss by quinone formation was
avoided by performing the reaction at pH 6.0 and by tailoring
the reaction medium. The “design of experiments” methodolo-
gy was used for the simultaneous optimization of six critical
parameters with a manageable number of experiments. On an-
alytical level, we detected full substrate conversion at 10 mm
concentration under several conditions and up to 27.9 mm of
3-HT within 21 h using 30 mm of DOPAC. First preparative-
scale reactions allowed isolation and purification of 3-HT in
19% overall yield and >99% purity. An extension of the
“design of experiments” methodology including further param-
eters, for example, from the biocatalyst preparation and the
scale-up is envisaged and will certainly lead to further
improvements.
Cell cultivation
A glycerol stock sample of E. coli BL21 (DE3) Gold:pJexpress-404-
NiCAR/pJexpress-401-EcPPTase was used for inoculation of LB
medium containing 100 mgmLꢁ1 Amp and 50 mgmLꢁ1 kanamycin.
Standard cultivation was performed and the main culture was in-
duced with 1 mm IPTG at an OD600 between 0.4 and 0.6. The pro-
tein expression phase proceeded for 4 h at 378C and 120 rpm. The
cells were collected by centrifugation and washed by resuspending
them in 50 mL of cold buffer (50 mm MES, 10 mm MgCl2, 1 mm
EDTA, 1 mm DTT, pH 6.0) prior to another centrifugation step for
10 min at 48C and 2831ꢂg. Washing was repeated and all superna-
tants were discarded. Washed cells were dispersed in 50% glycerol,
shock-frozen in liquid nitrogen and stored at ꢁ808C. Typically, the
cells were used within a few days because the overall activity
decreased significantly upon prolonged storage.
Experimental Section
Typical procedure for resting cell biotransformation
Materials and equipment
Frozen cell suspensions were thawed and washed twice in reaction
buffer, before finally suspending the pellet in reaction buffer (Ap-
proximately 80 mg of wet cell pellet per sample). Unless otherwise
stated, cofactor (typically 2 equiv.) or cofactor recycling compo-
nents were added. The reactions were started by addition of
DOPAC solution in 10–50 mm final concentration with adjusted pH
(6.0) and proceeded in a mixer incubator (Eppendorf Thermomixer)
with open lid at 288C and 1000 rpm. The final OD600 of the sample
was approximately 120. To compensate for evaporation, the 1.5 mL
reaction tubes were refilled to the 500 mL mark with water. Subse-
quently, the biomass was removed by centrifugation at 16100ꢂg
at RT for 5 min, and the supernatants were filtered before HPLC
analysis under conditions described above.
Ampicillin (Amp), CoA, and 3,4-dihydroxyphenylacetic acid were
purchased from Sigma Aldrich, and acetonitrile (ACN) from J. T.
Baker. ATP and NAD(P)(H) cofactors were purchased from Roche Di-
agnostics, isopropyl b-d-1-thiogalactopyranoside (IPTG) from Bio-
synth and all other chemicals and buffer components were ob-
tained from Carl Roth. GDH was obtained from DSM Innovative
Synthesis BV and 1 mg corresponds to 2.7 U for NADP+ and 3.4 U
for NAD+ reduction. Formate dehydrogenase (FDH) was used as
cell-free extract of an E. coli Rosetta (DE3) containing the plasmid
pRSF:FDH from Candida boidinii as described in [31] and 1.08 mg
corresponds to 0.375 U for NAD+ reduction. NMR spectra were re-
corded on a Bruker AVANCE III 300 spectrometer (1H: 300.36 MHz;
13C: 75.53 MHz) and chemical shifts were referenced to residual
protonated solvent signals as internal standard. For HPLC measure-
ments, an Agilent Technologies 1200 Series equipped with G1379B
degasser, G1312B binary pump SL, G1367C HiP-ALS SL autosam-
pler, a G1314C VWD SL UV detector, G1316B TCC SL column oven,
and a G1956B mass-selective detector (MSD) were used. Samples
were filtered through AcroPrep 96 Filter Plate (Pall Life Science)
with 0.2 mm polypropylene membrane prior to analysis. 3,4-Dihy-
droxyphenylacetic acid, 3,4-dihydroxyphenylacetaldehyde, and 3-
HT were separated on a Chromolith Performance RP-18 endcapped
100–4.6 column at 308C by using a 95% aqueous eluent (0.1%
formic acid) and 5% ACN at a flow of 1.2 mLminꢁ1, which was split
to 0.8 mLminꢁ1 before the mass-selective detection. Vanillic acid,
vanillin, and vanillyl alcohol were separated with 30% ACN content
under otherwise identical conditions.
Design of experiments (DoE)
The reactions were performed as described above with the follow-
ing modifications: All cells used for these experiments were frozen
for 16 h. The thawed cells were preincubated with citrate and glu-
cose for 1 h to revitalize their metabolic activity. The reaction time
was 21 h. Planning of the experiments and analysis of the experi-
mental data was performed by using commercially available soft-
ware (such as Design-Expert from Stat-Ease, Inc).
Preparative-scale 3-HT production
An aliquot of frozen resting cells was thawed on ice, diluted with
50 mm MES buffer pH 6.0 (supplemented with 10 mm MgCl2, 1 mm
DTT and 1 mm EDTA), and centrifuged for 20 min at 48C and
2831ꢂg. The cell pellet (3 g) was washed two more times at the
same conditions except the centrifugation time, which was 10 min.
The cells were finally dispersed in 50 mm MES buffer pH 6.0. Stock
solutions of sodium citrate and glucose were added to give con-
Strain generation
The genes coding for N. iowensis NRRL 5646 CAR (EMBL accession
no. Q6RKB1, gene sequence optimized for expression in E. coli) and
PPTase entD from E. coli BL21 (DE3) (NCBI accession no.
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemCatChem 2014, 6, 1089 – 1095 1094