Cascade Biotransformation to Access 3-Methylpiperidine in Whole Cells

Article abstract of DOI:10.1002/cctc.201900702

Synthesis of 3-methylpiperidine from 1,5-diamino-2-methylpentane in preparative scale is reported by using recombinant Escherichia coli cells expressing a variant of the diamine oxidase from Rhodococcus erythroprolis and an imine reductase from Streptosporangium roseum. Optimization of process parameters for cultivation and bioconversion led to substantial improvements in the initial laboratory procedure. The transformation of the methyl-substituted diamine substrate to the N-heterocyclic product was successfully scaled-up from shake-flask to a 20 L bioreactor with increased substrate concentrations. Remarkably, we obtained 67 % of 3-methylpiperidine product from 140 g substrate within 52 h.

Full text of DOI:10.1002/cctc.201900702

Accepted Article
Title: Cascade biotransformation to access 3-methylpiperidine in whole
cells
Authors: Niels Borlinghaus, Leonie Weinmann, Florian Krimpzer,
Philipp Scheller, Ammar Al-Shameri, Lars Lauterbach, Anne-
Sophie Coquel, Claus Lattemann, Bernhard Hauer, and
Bettina Nestl
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10.1002/cctc.201900702
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Cascade biotransformation to access 3-methylpiperidine in whole
cells
Niels Borlinghaus,ꝉ[a] Leonie Weinmann,ꝉ[a] Florian Krimpzer,[b] Philipp N. Scheller,[a] Ammar Al-
Shameri,[c] Lars Lauterbach,[c] Anne-Sophie Coquel,[b] Claus Lattemann,[b] Bernhard Hauer,[a] and
Bettina M. Nestl*[a]
Abstract: Synthesis of 3-methylpiperidine from 1,5-diamino-2-
methylpentane in preparative scale is reported by using recombinant
Escherichia coli cells expressing a variant of the diamine oxidase from
Rhodococcus erythroprolis and an imine reductase from
Streptosporangium roseum. Optimization of process parameters for
cultivation and bioconversion led to substantial improvements in the
initial laboratory procedure. The transformation of the methyl-
substituted diamine substrate to the N-heterocyclic product was
successfully scaled-up from shake-flask to a 20 L bioreactor with
increased substrate concentrations. Remarkably, we obtained 67% of
3-methylpiperidine product from 140 g substrate within 52 h.
have begun to provide alternative strategies for the synthesis of
substituted pyrrolidines and piperidines from diketones,
ketoaldehydes, diamines, and ketoacids by combining
transaminases[17–24]
reductases[29,30]
,
amine oxidases[25–28]
with imine
reductases[31–38]
,
carboxylic acid
The
.
diastereoselective synthesis of disubstituted pyrrolidines and
piperidines has also been reported with transaminases and
chemical reducing agents.[39–41] Although several methods are
presented for the asymmetric synthesis of 2- as well as 2,6-
disubstituted piperidines, the synthesis of 3-substituted has been
subject to little scientific research. 3-Methylpiperidine is an
intermediate in the industrial production of nicotinic amide and
nicotinic acid, which is an essential vitamin (vitamin B3).[42] In this
regard, the synthesis of 3-substituted piperidines is of
considerable importance.
Recently, we reported an enzymatic cascade for the
preparation of N-heterocyclic pyrrolidines and piperidines from
the corresponding diamine substrate. Substituted pyrrolidines and
piperidines were obtained with up to 97% product formation in a
one-pot reaction directly from the corresponding diamine
substrates.[43] Our approach relies on the application of the
engineered putrescine oxidase (PuO) from Rhodococcus
erythropolis for the oxidation of diamines to the amino aldehyde,
which undergoes spontaneous cyclization to the corresponding
imine. Subsequent reduction mediated by the imine reductase
from Streptosporangium roseum (IRED_Sr) provides access to
enantioenriched N-heterocycles (Scheme 1).
The development of cascade reactions for industrially relevant
compound classes has recently gained increasing interest since
intermediate purifications and isolations can be avoided.[1–5]
Biocatalytic reactions are generally performed under mild reaction
conditions, like ambient temperature and physiological pH. For
that reason, enzymes are usually compatible with each other,
opening the possibility of combining several reactions in the same
reaction vessel. In the last years, enzymatic cascades have
reached a remarkable level of complexity allowing the one-pot
preparation of valuable organic molecules. Nitrogen-containing
heterocycles are privileged structures that are ubiquitously found
in nature and in various drug candidates.[6] Consequently, the
development of synthetic methods for the preparation of these
compounds constitutes an area of current interest.[7] Piperidines,
in particular, are among the most common heterocyclic building
blocks in approved pharmaceuticals.[8] Conventionally such
compounds have been prepared by reduction of the
corresponding pyridines[9], dinitriles[10], isoxazolinones[11] and
heteroaromatic N-oxides[12], alkylation and reduction of bicyclic
lactams, hydroaminations[13,14]
,
ring-closing metathesis[15] or
cyclization of linear diamines.[16] Biocatalytic cascade reactions
[a]
Niels Borlinghaus, Dr. Leonie Weinmann, Dr. Philipp N. Scheller,
Prof. Dr. Bernhard Hauer, Dr. Bettina M. Nestl
Institute of Biochemistry and Technical Biochemistry, Chair of
Technical Biochemistry, Universitaet Stuttgart
Allmandring 31, 70569 Stuttgart (Germany)
E-mail: bettina.nestl@itb.uni-stuttgart.de
ꝉ Both authors contributed equally.
[b]
[c]
Dr. Anne-Sophie Coquel, Florian Krimpzer, Dr. Claus Lattemann
Sanofi Chimie, Pharmaceutics Development Platform, Impasse des
Ateliers 1, 94400 Vitry sur Seine (France)
Scheme 1. Enzyme cascade for the synthesis of 3-methylpiperidine 2
Ammar Al-Shameri, Dr. Lars Lauterbach
Institute of Chemistry, Technical University of Berlin, Strasse des
17. Juni 135, 10623 Berlin (Germany)
In continuation with our recent studies on the synthesis of -
heterocycles, we became interested in optimization of our
enzymatic cascade for the production of 3-methylpiperidine 2 as
Supporting information for this article is given via a link at the end of
the document.
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10.1002/cctc.201900702
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well as transferring this cascade to an Escherichia coli (E. coli)
whole cell system with facilitated up-scaling.
generation of a single plasmid pBAD18_PuOM1_IRED (Figures
S3-S6). An alternative approach is the mixing of whole cells
producing PuOM1 with whole cells producing IRED.
We began by examining the combination of IRED_Sr and triple
variant PuOM1 (L200I/E203S/I206L). In our previous study, the
directed evolution of PuO has been conducted. From the random
mutant library created by error-prone PCR, we obtained a single
variant E203G, which exhibited increased oxidation activity of
non-natural diamines compared with the PuO wild-type.[43] This
result suggests that glutamic acid at position 203 may play an
important role in terms of substrate specificity and prompted us to
consider whether PuO activity in the oxidation of 1,5-diamino-2-
methylpentane 1 can be further improved by semi-rational
mutagenesis. Amino acid residues located on a loop adjacent to
residue E203 were modified to improve the substrate acceptance.
Four variants PuOM1 – PuOM4 were constructed with the variant
PuOM1 indeed being more active than variant E203G in the
oxidation of C5 diamine substrates cadaverine and 1 (Table 1 and
Table S1 in the Supporting Information).Using PuOM1 a 2-fold
increased activity for the oxidation of 1 (Table 1) and a 3-fold
increased kcat (Table S2, Figures S1 and S2) compared to the
PuO wild-type was observed.
SDS-PAGE analysis confirmed the synthesis of both
enzymes. It further suggested that the whole cells with the single
plasmid showed reduced production level of imine reductase
compared to the cells with two compatible plasmids (Figure S4).
The mixing of cells producing either PuOM1 or IRED displayed the
lowest activity, even if the amount of cells was varied at different
ratios (data not shown). Methylpiperidine formation of the
recombinant whole cells was further evaluated and optimized.
Also, to assure efficient production of 2 and to abolish the need
for exogenous NADPH cofactor supplementation, the cascade
was performed at different buffer and glucose concentrations.
Highest product formation was obtained within 48 h with 10 mM
glucose in a 50 mM Tris-HCl buffer pH 7.5 (82.5%, Figure 1,
shake flask). This result corresponds with recent observations on
the formation of N-heterocycles combining a 6-hydroxy-D-nicotine
oxidase with an IRED, which revealed that glucose concentration
could have a significant impact on the product formation.[25] When
more than 10 mM glucose has been applied the formation of 2
was decreased.
Table 1. Comparison of activity of putrescine oxidase wild-type (WT) with single
a
variant E203G and triple variant PuOM1
Table 2. Design of Experiment (DoE) for evaluating the influence of main
process parameters during bioconversion.
Batch
process #
Substrate
[mM]
Product
formation [%]a
pH
T [°C]
pO2 [%]
TON [min-1
]
6 h
12
23
67
64
37
21
21
11
15
40
21
48 h
64
82
98
89
93
70
67
49
66
78
57
1
2
7.2
7.2
7.8
7.8
7.8
7.2
7.2
7.2
7.8
7.8
7.8
20
30
30
30
20
30
30
20
20
30
20
10
50
50
10
50
50
10
50
50
10
10
15
15
15
15
15
35
35
35
35
35
35
WT
1128 ± 47
940 ± 42
509 ± 8
98 ± 9
105 ± 2
121 ± 11
47 ± 8
43 ± 1
82 ± 3
E203G
PuOM1
3
4
[a]
Formation of oxidized 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid
(ABTS) was followed spectrophotometrically for 15 min at 405 nm wavelength.
5
6
7
The cascade coupling of IRED_Sr and PuOM1 was used to
transform 1 to 3-methylpiperidine (2) in one-pot (Scheme 1). At
10 mM substrate concentration, 1 was converted with excellent
product formation of 90.4 ± 4.7 % within 5 h. Buoyed by the
compatibility of IRED_Sr and PuOM1, we intended to transfer the
in vitro enzymatic cascade into whole cells to achieve product
formation in multi-gram scale. The bioconversion system which
involves a three-reaction, two-enzyme, one-pot process was
established by using recombinant E. coli expressing genes of
engineered PuOM1 and imine reductase. The optimization of
fermentation and bioconversion of the cascade was investigated
to achieve considerable product formation. The design and
construction of the cascade started with evaluating suitable gene
expression systems to produce the chosen biocatalysts. For the
generation of PuOM1 and IRED protein production in the
arabinose-deficient strain E. coli JW5510, two different
approaches were applied: (i) the combination of two compatible
plasmids pBAD18_PuOM1 and pBAD33_IRED and (ii) the
8
9
10
11
[a]
A significant part of the positive effect on final product formation is the
increase of the initial substrate concentration, pH and temperature.
To demonstrate the synthetic applicability of the one-pot
cascade, the protein production and conversion of 1 to 2 was
scaled-up to a 20 L bioreactor (Figure 1). Initial fermentation and
bioconversion studies using 10 mM 1 illustrated that the activities
declined for the scaled-up process with product formations of less
than 30% (Figure S19, red bars). As a consequence, the process
parameters of the individual process phases such as biomass
production, enzyme production and bioconversion (Table S3 and
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10.1002/cctc.201900702
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Figure S20) have been investigated to optimize the
biotransformation. Specifically, the cultivation medium has been
optimized to allow significantly higher cell mass as well as key
process parameters concerning both protein production and
reaction conditions. The highest conversion of 1 has been
obtained applying the following reaction conditions: 14 h protein
production at 30°C after induction with 0.04% (w/v) arabinose at
OD600 10-15 in mineral salt medium supplemented with casamino
acids and glycerol (high cell density medium), followed by whole
cell bioconversion (48 h, 30°C) adding 15 mM substrate 1 and 10
mM glucose directly to the fermentation broth (see also Figures 1
as well as 2, and Figure S19, highlighted in purple).
To gain further insights into the process parameters
affecting the extent of 2 productions, several process parameters,
including temperature, pH, dissolved oxygen concentration and
initial substrate concentration, were varied applying Design of
Experiment (DoE) (Table 2, for more details see SI Tables S4-S5
and Figures S8-S13).The effects of these modifications on the
whole cell bioconversion were investigated after 6 h, 24 h and 48
h reaction time. Parallel runs with the same process conditions
were performed prior to DoE in 20 L and 7 L fermenters to
investigate whether the scale of the bioreactor used for the
bioconversion could affect the performance of production (data
not shown).
conducted using 15 mM as well as 35 mM of 1 to generate 2 (see
SI Figures S14-S17). For the major part of the experiments
performed with an initial substrate concentration of 15 mM, no
residual substrate could be detected at 48 h bioconversion.
We observed that after 24 h reaction, the maximum titer was
obtained and all substrate was converted. Batch 3 highlights the
excellent conversion of 1 with 98% of formed product 2 (Table 1
and Figure 2). We thus assume that the combination of higher pH
(from pH 7.2 to 7.8), as well as temperature (from 20°C to 30°C),
enhanced the overall enzymatic cascade. On the basis of these
promising results for the conversion of 15 mM, we investigated
the whole cell cascade by using 35 mM of 1. Highest product
formation was observed in batch 8 with 78% of formed product
within 48 h applying an initial substrate concentration of 35mM
(Table 2 and Figure 1). The statistical analysis of DoE data further
revealed that the increase of pH and temperature to 7.8 and 30°C,
respectively, had a positive impact on the bioconversion reaction.
Moreover, within these conditions highest product titer was
observed through the increase of initial substrate concentration
from 15 mM to 35 mM, which is accompanied by a lower
conversion rate at high substrate concentration. Finally, optimized
reaction conditions were used in the conversion of 100 mM 1 (140
g, batch 12 in Figure 1, Table S5, Figure S18) combined with
additional glucose intake to improve NADPH recycling. Substrate
1 along with 100 mM glucose were directly added to the
fermentation medium. The
F E R M E N TAT I O N
O P T I M I Z AT I O N
D E S I G N O F E X P E R I M E N T S
bioconversion was performed
at pH 7.8 and 30°C with a cell
density of 8 g L-1 (cell dry
weight).
Substrate
and
6
5
4
3
2
1
product concentrations were
measured after 52 h, resulting
in 66.7 mM 2 (see also Figure
S18). Batch #12 was further
applied
downstream
including
separation by centrifugation
or membrane separation,
distillation and liquid-liquid
extraction to transfer and
enrich 3-methylpiperidine 2 in
organic solvent. Due to the
to
study
the
processing
solid-liquid
0
fact
that
solid-liquid
SHAKE FLASK
# 1 # 2 # 3 # 4 # 5
# 6 # 7 # 8
# 9 # 10 # 11
# 12
separation and distillation
resulted in unsatisfactory
10 mM
15 mM
35 mM
100 mM
Substrate conc.:
product recovery, we focused our efforts on liquid-liquid extraction.
The following candidate solvents were selected for extraction of 2
at alkaline conditions (pH 12), namely 1-heptanol, 1-octanol and
2-ethyl-1-hexanol. These solvents are considered as ‘green
solvents’ contributing to a sustainable process development.
They have proved suitable for the application in liquid-liquid
extraction of 500 mL samples with isolated yields of up to 96%
(data not shown). That allows us to conclude that liquid-liquid
extraction can be applied for product purification of N-
heterocycles in future processes.
Figure 2. Evolution of process performance in the preparative-scale synthesis
of 2. Shake flask experiments of the substrate (shown in orange) were optimized
through improvement of the fermentation process and use of Design of
Experiments (DoE). In the course of process optimization, substrate
concentrations (substrate conc.) were gradually increased from 10 mM to 100
mM. Detected product concentrations after 48 h reaction time are shown.
Product formation using 100 mM of the substrate was performed for 52 h.
No differences were observed between the two applied
bioconversion scales allowing the use of both 7 L and 20 L
bioreactors. To demonstrate the potential for industrialization of
the developed process, various 20 L scale operations were
In summary, a one-pot cascade using engineered diamine
oxidase and imine reductase has been successfully implemented
for the synthesis of 3-methylpiperidine from the corresponding
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Production of 3-methylpiperidine at a larger scale
1,5-diamino-2-methylpentane. The compatibility of both enzymes
allowed the development of a whole cell system at bioreactor
scale under controlled reaction conditions eliminating the
necessity of costly enzyme, coenzyme as well as cosubstrates
and intermediate purification steps. Operational improvements of
the vital fermentation and bioconversion parameters enabled the
production of 3-methylpiperidine in high amounts. Our results
demonstrate that this enzymatic cascade established as whole
cell biotransformation with living cells is promising and can be
applied in a large-scale format opening up the perspective
towards the production of high-value piperidine compounds that
might be difficult to obtain by organic chemistry.
The conversion experiments were carried out in a 20 L bioreactor with 12
L working volume. The substrate 1,5-diamino-2-methylpentane (15 mM to
100 mM) was added into the system by one-time without prior dissolution
in DMSO or other organic solvents. Glucose was added with
a
concentration of 20 mM followed by glucose fed-batch feeding 48 h of the
bioconversion. 72 mM of glucose was converted over 48 h. NH4OH (25%)
and H3PO4 (30%) were used to control and maintain the pH at 7.8. The
concentration of 1,5-diamino-2-methylpentane and 3-methylpiperidine
was determined using the GC methods described in the SI.
Acknowledgements
Experimental Section
Research leading to these results received funding from the
Innovative Medicines Initiative Joint Undertaking under grant
agreement no 115360 (FP7/2007-2013; and EFPIA companies
in-kind contribution) and the German Research Foundation (DFG,
project number 284111627). Lars Lauterbach was supported by
the Deutsche Forschungsgemeinschaft under Germany´s
Excellence Strategy – EXC2008/1 UniSysCat (DFG, project
number 390540038). We thank Youcef Athmani and Jeremy
Parize, technicians at Sanofi who performed all the scale-up
experiments.
Production of PuOM1 and IRED
The plasmids containing the putrescine oxidase variant PuOM1 (Leu192Ile,
Glu203Ser, Ile206Leu, pITB1381) and the imine reductase
(pBAD33_IRED-Sr, pITB1236) were transformed into arabinose deficient
chemical competent E. coli JW5510 and plated onto selection plates
containing 100 µg mL-1 ampicillin and 34 µg mL-1 chloramphenicol. The
production was performed in 400 mL TB-media containing 100 µg mL-1
ampicillin and 34 µg mL-1 chloramphenicol in 2 L shake flasks, which were
inoculated with an overnight culture to an OD600 of 0.02. The cells were
grown at 37°C and 180 rpm to an OD600 of about 0.6 in 2 to 3h and then
induced with 0.04% of L-arabinose. The production was performed at 25°C
overnight. The cells were harvested by centrifugation (centrifuge Avanti J
26S XP, Beckman Coulter, Krefeld, Germany; rotor JLA 8.1) for 25 min at
4°C with 9000 g and a 400 mL culture resulted in average in a cell pellet
of 5.5 g. The production of the enzymes was visualized by SDS-PAGE
analysis.
Keywords: whole cell enzyme cascade • imine reductase •
upscaling • methylpiperidine • amine oxidase
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10.1002/cctc.201900702
ChemCatChem
COMMUNICATION
COMMUNICATION
Scaling Up! The coupling of
engineered putrescine oxidase and
imine reductase realizes the
preparation of piperidine
N. Borlinghaus, L. Weinmann, F.
Krimpzer, P. N. Scheller, A. Al-
Shameri, L. Lauterbach, A.-S. Coquel,
C. Lattemann, B. Hauer, B. M. Nestl*
heterocycles. Particulary, the
production of 3-methylpiperidine at
20 L scale was demonstrated using
an E. coli whole cells system.
Page No. – Page No.
Title
This article is protected by copyright. All rights reserved.