Advanced Synthesis & Catalysis
10.1002/adsc.201900155
coli RARE. Specifically, 3 wells were used for negative
controls (e.g. pETDuet:EcPPTaseNiCARA, an A-domain
deletion variant which lacks a stretch of the A-domain, 3
wells sterile controls and 6 wells for positive controls
Mecklenburg-Vorpommern for financial support. We would like to
thank K.L.J. Prather for providing the RARE strain.
(
pETDuet:EcPPTaseNiCAR). 18 additional wells were
References
inoculated with pETDuet:EcPPTaseNiCARA for on-plate
calibration. These master plates were incubated over night
at 37 °C and 1000 rpm on a Heidolph Titramax 1000 until
cells were in the exponential growth phase. Freshly
[1]
M. Breuer, K. Ditrich, T. Habicher, B. Hauer, M.
Keßeler, R. Stürmer, T. Zelinski, Angew. Chem. Int. Ed.
2
004, 43, 788-824.
[
2]
3]
A. L. Carroll, S. H. Desai, S. Atsumi, Y.-S. Jin, J.-H.
Seo, Curr. Opin. Biotechnol. 2016, 37, 8-15.
J. X. Zheng, S. Chevance, C. Darcel, J. B. Sortais,
Chem. Commun. 2013, 49, 10010-10012.
[
17]
prepared 96-DWPs filled with 800 µL LB-5052
containing 50 µg/mL ampicillin were inoculated from the
master plates by a microplate replicator. The copy step
from the master plate to the expression plate ensured the
comparable starting point for expression as described by
Dörr et al.[ Expression plates were incubated for 4 h at
[
[4]
a) A. M. Kunjapur, K. L. J. Prather, App. Environ.
Microbiol. 2015, 81, 1892-1901; b) F. Hollmann, I. W.
C. E. Arends, D. Holtmann, Green Chem. 2011, 13,
18]
2
285-2314.
3
7 °C and 1000 rpm on a Heidolph Titramax 1000,
[
[
5]
6]
M. Winkler, Curr. Opin. Chem. Biol. 2018, 43, 23-29.
D. Gahloth, M. S. Dunstan, D. Quaglia, E. Klumbys, M.
P. Lockhart-Cairns, A. M. Hill, S. R. Derrington, N. S.
Scrutton, N. J. Turner, D. Leys, Nat. Chem. Biol. 2017,
subsequently the temperature was lowered to 20 °C for
expression with the same shaking speed. After 24 h total
time for growth and expression, cells were harvested by
centrifugation at 3220 x g for 20 min and the supernatant
was removed by decantation. The remaining cell pellets
were resuspended in 400 µL of conversion buffer (M9
medium without ammonium chloride, containing 0.8%
glucose and 2.5 mM of 1f, Supporting Information, chapter
1
3, 975-981.
[7]
P. Venkitasubramanian, L. Daniels, J. P. N. Rosazza, J.
Biol. Chem. 2007, 282, 478-485.
[8]
A. He, T. Li, L. Daniels, I. Fotheringham, J. P. N.
Rosazza, App. Environ. Microbiol. 2004, 70, 1874-
1
881.
2
). Cells in ‘on-plate calibration wells’ were resuspended in
buffer with 2f in a concentration range between 0.1 and
.5 mM). The plates were incubated at 25°C and 1000 rpm
[9]
a) D. Schwendenwein, G. Fiume, H. Weber, F. Rudroff,
M. Winkler, Adv. Synth. Catal. 2016, 358, 3414-3421;
b) M. Winkler, C. K. Winkler, Chem. Monthly 2016,
1
for 5 h. Next, 400 µL of ABAO-solution (sodium acetate
buffer 100 mM, pH 4.5 containing 5% DMSO and 10 mM
of ABAO) were added and the plates were centrifuged at
1
47, 575-578; c) S. P. France, S. Hussain, A. M. Hill, L.
J. Hepworth, R. M. Howard, K. R. Mulholland, S. L.
Flitsch, N. J. Turner, ACS Catal. 2016, 6, 3753-3759; d)
Y. Duan, P. Yao, X. Chen, X. Liu, R. Zhang, J. Feng, Q.
Wu, D. Zhu, J. Mol. Catal. B 2015, 115, 1-7; e) M. K.
Akhtar, N. J. Turner, P. R. Jones, Proc. Natl. Acad. Sci.
USA 2013, 110, 87-92.
3
220 x g for 30 min. The supernatants (150 µL) were
transferred to a fresh plate via multichannel pipette and
analyzed at 380 nm. On-plate calibration curves were used
to calculate the amount of 2f produced by each clone.
[
10]
11]
a) N. Ladkau, A. Schmid, B. Buehler, Curr. Opin.
Biotechnol. 2014, 30, 178-189; b) T. Bayer, S. Milker,
T. Wiesinger, M. Winkler, M. D. Mihovilovic, F.
Rudroff, ChemCatChem 2017, 9, 2919-2923; c) L. J.
Hepworth, S. P. France, S. Hussain, P. Both, N. J.
Turner, S. L. Flitsch, ACS Catal. 2017, 7, 2920-2925.
E. H. Hansen, B. L. Moller, G. R. Kock, C. M. Bunner,
C. Kristensen, O. R. Jensen, F. T. Okkels, C. E. Olsen,
M. S. Motawia, J. Hansen, App. Environ. Microbiol.
Whole cell biotransformation
Biotransformations were conducted in 100 ml flasks in a
volume of 10 ml. Resting cells were prepared as described
in Supporting information chapter 2.3.1. The
biotransformation was performed at an OD600 of 10 in M9
medium (no nitrogen, 0.8% glucose) with a carboxylic acid
concentration (1f) of 2.5 mM. The flaks were shaken at 100
rpm at 25 °C in a Multitron shaker and samples were taken
after 0.5, 1, 2 and 3 h and analyzed by HPLC-UV.
Reactions were performed in triplicates.
[
2
009, 75, 2765-2774.
[
12]
13]
H. Stolterfoht, D. Schwendenwein, C. W. Sensen, F.
Rudroff, M. Winkler, J. Biotechnol. 2017, 257, 222-
2
32.
[
A.
K.
Ressmann,
D.
Schwendenwein,
S.
Leonhartsberger, M. D. Mihovilovic, U. T.
Bornscheuer, M. Winkler, F. Rudroff, Adv. Synth.
Catal. 2019, 361, DOI: 10.1002/adsc.201900154.
A. M. Kunjapur, Y. Tarasova, K. L. J. Prather, J. Am.
Chem. Soc. 2014, 136, 11644-11654.
W. Finnigan, A. Thomas, H. Cromar, B. Gough, R.
Snajdrova, J. P. Adams, J. A. Littlechild, N. J. Harmer,
ChemCatChem 2017, 9, 1005-1017.
Acknowledgements
[
14]
15]
Financial support by FWF (grant: P28477-B21) and TU Wien
ABC-Top Anschubfinanzierung are gratefully acknowledged. This
work has been supported by the Austrian BMWD, BMVIT, SFG,
Standortagentur Tirol, Government of Lower Austria and
Business Agency Vienna through the Austrian FFG-COMET-
Funding Program. A.K.R. & D.S. thank the COST-Action
CM1303 (Systems Biocatalysis) for financing a short-term
scientific mission stay at University Greifswald. M.D. and U.T.B.
thank the DFG (INST 292/118-1 FUGG) and the federal state
[
[16]
[17]
E. Y. Jeong, K. S. Cho, H. S. Lee, J. Food Prot, 2012,
75, 118-122.
a) F. W. Studier, Prot. Expr. Purification 2005, 41, 207-
2
34; b) F. W. Studier, in Structural Genomics: General
6
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