Stereoselective Biotransformations of Cyclic Imines in Recombinant Cells of Synechocystis sp. PCC 6803

Article abstract of DOI:10.1002/cctc.201901592

Light-driven biotransformations in recombinant cyanobacteria allow to employ photosynthetic water-splitting for cofactor-regeneration and thus, to save the use of organic electron donors. The genes of three recombinant imine reductases (IREDs) were expressed in the cyanobacterium Synechocystis sp. PCC 6803 and eight cyclic imine substrates were screened in whole-cell biotransformations. While initial reactions showed low to moderate rates, optimization of the reaction conditions in combination with promoter engineering allowed to alleviate toxicity effects and achieve full conversion of prochiral imines with initial rates of up to 6.3 mM h^?1. The high specific activity of up to 22 U g_CDW ^?1 demonstrates that recombinant cyanobacteria can provide large amounts of NADPH during whole cell reactions. The excellent optical purity of the products with up to >99 %ee underlines the usefulness of cyanobacteria for the stereoselective synthesis of amines.

Full text of DOI:10.1002/cctc.201901592

Accepted Article
Title: Stereoselective biotransformation of cyclic imines in recombinant
cells of Synechocystis sp. PCC 6803
Authors: Hanna Büchsenschütz, Viktorija Vidimce-Risteski, Bettina
Eggbauer, Sandy Schmidt, Christoph Winkler, Jörg H
Schrittwieser, Wolfgang Kroutil, and Robert Kourist
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10.1002/cctc.201901592
ChemCatChem
COMMUNICATION
Stereoselective biotransformations of cyclic imines in
recombinant cells of Synechocystis sp. PCC 6803
Hanna C. Büchsenschütz,#[b] Viktorija Vidimce-Risteski#,[a] Bettina Eggbauer,[b] Sandy Schmidt,[b]
Christoph K. Winkler,[a,c] Joerg H. Schrittwieser,[c] Wolfgang Kroutil*[a,c] and Robert Kourist*[a,b]
Abstract:
Light-driven
biotransformations
in
recombinant
corresponding heterotrophic Pseudomonas taiwanensis VLB120
with the same enzymes.[12,15] Cyanobacteria offer additional
advantages such as the ease to extract the product from reaction
solution[10] and the possibility of an in situ production of oxygen as
cyanobacteria allow to employ photosynthetic water-splitting for
cofactor-regeneration and thus, to save the use of organic electron
donors. The genes of three recombinant imine reductases (IREDs)
were expressed in the cyanobacterium Synechocystis sp. PCC 6803
and eight cyclic imine substrates were screened in whole-cell
biotransformations. While initial reactions showed low to moderate
rates, optimization of the reaction conditions in combination with
promoter engineering allowed to alleviate toxicity effects and achieve
full conversion of prochiral imines with initial rates of up to 6.3 mM h-1.
cosubstrate for oxyfunctionalizations.[11,16] Despite
a
few
encouraging examples, however, photoautotrophic organisms yet
need to fulfill their promise. Besides efficiency, selectivity and
robustness, a broad scope toward different substrates is crucial
for the wide application of a biocatalyst.[1] Potential toxicity of
substrates and products may have an immense influence on the
system. Furthermore, the transport of substrate and product
across cell membranes can restrict the application of whole-cell
biocatalysts.[17] We were interested to which extent light-driven
whole-biotransformations could be applied for the cofactor
regeneration of imine reductases (IREDs) for the production of
secondary amines. IREDs were discovered by Mitsukura et al. in
Streptomyces sp. Since then, IREDs have rapidly assumed an
important role as highly stereoselective catalysts for amine
snythesis.[18–24] IREDs strictly require NADPH and are
preferentially used in cellular systems, which makes them very
suitable for application in photosynthesis-driven whole-cell
biotransformations.
-1
The high specific activity of up to 22 U gCDW demonstrates that
recombinant cyanobacteria can provide large amounts of NADPH
during whole cell reactions. The excellent optical purity of the products
with up to >99 %ee underlines the usefulness of cyanobacteria for the
stereoselective synthesis of amines.
Biocatalysis has emerged as important catalytic technology with
significant contributions for the development of clean reactions.[1]
A large number of biocatalytic processes employ oxidoreductases,
which often require a stoichiometric supply of sacrificial organic
cosubstrates for cofactor regeneration, which in turn results in a
very poor atom economy. In this context, cyanobacteria have
been
receiving
increasing
attention
as
hosts
for
biotransformations to exploit photosynthetic water splitting for the
supply of reductive enzymatic reactions with electrons. Beside the
application of wildtype strains,[2–5] heterologous enzyme
production is of main interest and has been greatly facilitated by
the impressive progress in molecular biology of photoautotrophic
organisms.[6–8]
So far, ene-reductases,
Baeyer-Villiger
monooxygenases, carboxylic acid reductases, the AlkBGT-
system and CYP450 monooxygenases have successfully been
used in light driven biotransformations.[9–14] In particular, cells
expressing the genes encoding for recombinant oxidoreductases
-1 [10]
showed reaction rates as high as 123 U gDCW
.
In case of
CYP450 from Acidovorax sp. CHX100, recombinant
cyanobacteria exhibit higher specific activity than the
[a]
[b]
[c]
Prof. Dr. Robert Kourist, Viktorija Vidimce-Risteski, Dr. Christoph
Winkler, Prof. Dr. Wolfgang Kroutil
Austrian Centre of Industrial Biotechnology (ACIB GmbH),
Petersgasse 14/ Mozartgasse 12/II, 8010 Graz, Austria
E-mail: kourist@tugraz.at
Prof. Dr. Robert Kourist, Dr. Sandy Schmidt, MSc. Hanna
Büchsenschütz
Institute of Molecular Biotechnology
Graz University of Technology, NAWI Graz, BioTechMed,
Petersgasse 14, 8010 Graz, Austria
Dr. Jörg H. Schrittwieser
Institute of Chemistry, Organic & Bioorganic Chemistry,
NAWI Graz, BioTechMed Graz, University of Graz
Heinrichstraße 28/II, 8010 Graz, Austria
Scheme 1. Biotransformation of imines 1a-h by recombinant IREDs in
Synechocystis sp. PCC 6803. The required cofactor NADPH is regenerated by
natural photosynthesis.
# both authors contributed equally to this work.
Supporting information for this article is given via a link at the end of
the document.
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10.1002/cctc.201901592
ChemCatChem
COMMUNICATION
The simplicity of an imine reduction in cyanobacteria is shown in
equation (1). Water is used as electron donor and molecular
oxygen is released as side-product.
(Supplementary Figure S1). While the enzymes showed activity
in whole-cell biotransformations, protein bands for these enzymes
could not be detected in SDS-PAGE (Supplementary Figure S2).
This is not unusual for Synechocystis.[10,27] For an initial substrate
screening, cells were concentrated to densities of 3.6 gCDW L-1
(correspond to OD750 = 15) and substrate conversion was
monitored up to 25 h.
R-CH=NH-R‘ + H2O  R-CH2-NH2R‘ + ½ O2
[1]
To investigate strengths and limitations of cyanobacterial imine
reduction, we chose a set of eight substrates and three enzymes
as representatives, namely IRED-A from Streptomyces sp.
GF3587, IRED-J from Kribbella flavida DSM 17836 and IRED-M
from Saccharothrix espanaensis ATCC 51144.[25] In a recent
study comparing 15 IREDs, these enzymes proved to be
particularly suitable for our approach.[25] IRED-A was most active
on a variety of smaller imine substrates and exceled with stability
toward high temperature, high substrate concentrations and its
selectivity. IRED-J on the other hand was the most active (S)-
selective IRED characterized by Velikogne et al. but also IRED-M
showed high conversions and selectivites especially toward bulky
imine substrates.[25]
We were pleased to detect product formation for substrate 1a-e
in most cases (Table 1). No or very low product formation was
detected for reactions with larger substrate 1f-h. The low activity
of IREDs in Synechocystis toward bulkier substrates
demonstrates a clear difference to the activity of the same
[25]
enzyme in E. coli
and might be attributed to the capacity of
transport of substrates and products across the cell membrane.
The measured conversions were low (≤ 36%) in all cases except
for 1b and IRED-A. Furthermore, cells changed their visible
appearance which was not observed for analogous reactions with
the ene-reductase YqjM from B. subtilis[10] or cyclohexanone
monooxygenase from A. calcoaceticus[9] and is an indication for
toxicity effects. The toxicity of some substrates such as 1g was
recognized by a color change to blue or brown of the cells during
the reaction (Supplementary Figure S3). To investigate the extent
of toxicity, cells used in biotransformations were streaked out on
BG11-agar plates after different reaction time points. The vitality
of cells treated with 5 mM 1b or 2b was reduced considerably
within one hour while cells treated with 0.5 mM grew back after
24 h (Figure 1). Furthermore, toxicity experiments with all other
imine substrates showed high toxicity of 1g-h, moderate toxicity
of 1b-e and lowest toxicity of 1a and 1f (Supplementary Figure
S4).
Table 1. Specific activities (Aspec.) and conversions (C) for the light-driven
imine reduction of substrates (S) 1a-1h using three different recombinant
Synechocystis strains. All heterologous IREDs are under the control of
psbA2 promoter.
PpsbA2 IRED-A
PpsbA2 IRED-J
PpsbA2 IRED-M
[a]
[a]
[a]
S
Aspec.
C[b]
[%]
Aspec.
C[b]
[%]
Aspec.
C[b]
[%]
-1
-1
-1
[U gCDW
]
[U gCDW
]
[U gCDW
]
1a[c]
1b[c]
1c[c]
1d
0.2 ± 0.1
2.7 ± 0.3
18
0.6 ± 0.1
0.9 ± 0.2
n.c.
32
0.4 ± 0.3
n.c.
19
83
21
n.c.
n.c.
12
0.6 ± 0.02
0.2 ± 0.04
0.2 ± 0.1
n.c.
36
n.c.
6
n.c.
15
0.1 ± 0.03
0.1 ± 0.04
n.c.
0.2 ± 0.1
n.c.
1e
9
9
n.c.
6
1f
n.c.
n.c.
n.c.
n.c.
n.c.
n.c.
0.1 ± 0.04
n.c.
1g
n.c.
n.c.
n.c.
n.c.
1h
n.c.
n.c.
n.c.
Figure 1. Growth of Synechocystis wild-type and a mutant expressing the gene
of IRED-A in presence of different concentrations of 1b and 2b. Reaction
conditions: 150 µmolphotons m-2 s-1, 30 °C, Vtotal = 1 mL, 3.6 gCDW L-1, in BG11
(pH = 8)
[a] determined from the substrate decay in the first 12 h using GC-FID
analysis;
[b] determined from the substrate decay after 18-25 h using GC-FID
analysis; exact time points are given in the supplements
The substrate toxicity is an explanation why recombinant
Synechocystis strains with the imine reductases show very
different substrate spectra than observed for the same enzymes
in E. coli. Although Synechocystis cells are viable in the presence
low substrate or product concentrations, the toxicity reveals an
intrinsic limitation for achieving high space-time-yields.
[c] samples containing these substrates were derivatized before analysis
n.c = not converted.
Reaction conditions: 5 mL scale, 5 mM substrate, 150 µmolphotons m-2 s-1,
30 °C, 3.6 gCDW L-1, in BG11 (pH = 8)
Genes for all recombinant IREDs were integrated into the gene
locus slr0168 of the cyanobacterium Synechocystis sp. PCC 6803
(in the following Synechocystis) under the control of the light-
inducible promoter PpsbA2. This native promoter is of moderate
strength[10,26] and seemed to be very suitable to obtain a
reasonable amount of soluble enzyme without overloading the
folding machinery. Integration of the expression cassette into all
genome copies of Synechocystis was verified by PCR
In the initial screen, the highest activities were observed with
IRED-A and hence, this enzyme was chosen for further
optimization. We expected an increase of expression levels of the
recombinant IRED-A by changing the promoter psbA2 to the
stronger promoter cpc.[28,29] Indeed, we were pleased to find that
the effect of the promoter exchange was very pronounced and the
recombinant Synechocystis strain with Pcpc_IRED-A converted
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10.1002/cctc.201901592
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COMMUNICATION
83 % 1b already 3 h (Supplementary Figure S6). The effect of a
stronger promoter is a clear indication that the intracellular
concentration of the imine reductase is a limiting factor.
The control reaction with 1b in darkness led to a specific activity
in the same order of magnitude than the light-illuminated
counterparts (Figure 2). This is not surprising as in case of the
recombinant ene-reductase YqjM, cells could convert 5-7 mM in
absence of light, while the conversion of a higher amount of
substrate required illumination.[10] Accordingly, reactions under
In subsequent experiments, we compared the specific rates of our
Pcpc-IRED-A strain using different cell densities at a light intensity
of µmolphotons m-2 s-1 (Figure 2). For light-driven biotransformations, darkness with 8 mM 1a showed some conversion, but with clearly
we expected constant specific activities when increasing cell
densities until a point where self-shading of the cells reduces light-
availability as we observed this trend for other oxidoreductases
and cell densities above 2.4 gCDW L-1 per litre.[9] Very surprisingly,
the activity of Pcpc-IRED-A towards 1b was lowest when using
1.2 gCDW L-1 cells, constant between 2.4 and 4.8 gCDW L-1 cells and
highest when 6 gCDW L-1 cells were used. In a reaction vessel with
a diameter of 1 cm and at these high densities, self-shading is
expected to reduce the reaction rate (Figure 2). This indicates that
a higher cell density remediates toxicity effects on the cells. The
increase of the cell density allowed a significant improvement of
the productivity of up to 11.6 ± 1 U gCDW-1, which is comparable to
activities obtained for ene-reductases,[10] Baeyer-Villiger
lower activity and conversion compared to reactions under light
(Supplementary Table S6). This can be attributed to the presence
of storage carbohydrates that were produced during the
cultivation under photoautotrophic conditions. As the toxicity of
the imines prohibits increasing the substrate concentration above
a few mM, it is difficult to differentiate whether NADPH-
regeneration is fueled by photocatalytic water-splitting or the
degradation of (photosynthetically produced) storage sugars. In
both cases, however, the reaction is driven directly or indirectly by
the photosynthetic system of Synechocystis.
Encouraged by the promising results for 1b we were interested if
the promoter exchange also increased activity of recombinant
Synechocystis toward the other substrates, particularly for 1a as
this imine showed lowest toxicity in the plate assay. Indeed, 8 mM
1a was completely converted within 3 h and the achieved specific
monoogygenases,[9]
oxygenases.[12]
AlkBGT[11,13]
and
CYP450
mono-
-1
activity of around 22 U gCDW was two magnitudes higher than
90
80
70
60
50
40
30
20
10
0
that observed for PpsbA2-IRED-A (Table 2). Furthermore, activities
were greatly improved for substrates 1c-f with full conversion for
1c and nearly full conversion of 1d within 24 h. The results
indicate that the amount of enzyme in the cell was the limiting
factor in the initial screening. All biotransformations were
performed in small scale batch modus, with shaking or stirring. In
order to provide the cells with enough CO2, the head space inside
the biotransformation tubes was even higher than 50 % of the total
volume. Taking in consideration all these cultivation parameters,
low product formations were attributed to the volatility of the
products during biotransformations or a possible degradation of
the product. The substrate scope of the whole-cell biocatalysts
producing IRED-A with preference toward smaller imines is
analogously obtained with purified enzyme or in E. coli as
reported by Velikogne et al. [25]
1.2 gCDW L-1
2.4 gCDW L-1
3.6 gCDW L-1
4.8 gCDW L-1
6.0 gCDW L-1
-10
0
2
4
6
time [h]
20
18
16
14
12
10
8
80
70
60
50
40
30
20
10
0
We were pleased to find a high enantiomeric excess of up to 99%
under optimized reaction conditions for compounds 2a-c and 2f
as well as moderate ee for 2e. Derivatization and volatility
complicated the detection of 2a and 2b, therefore, ee% cannot be
given with higher accuracy than >94%. The results nicely
correspond to the selectivity of the isolated enzymes and in case
of 2f achieved purity was even higher (Supplementary Table
S5).[25] The stereoselectivity of the recombinant cells underlines
the usefulness of photosynthesis-driven biocatalysts for the
synthesis of optical pure amines. The recombinant cyanobacteria
fully reduced 8 mM 1a within 3 h showing that our system is
comparable to other systems which avoid organic
cosubstrates,[17,30,31] and to biotransformations using hydrogen as
electron donor such as the recently reported imine reduction
coupled to a hydrogenase.[32] The main limitation of the reported
system is the toxicity of substrates and products for
6
4
2
1.2
2.4
3.6
4.8
6.0
47.82dark
cell density [gCDW L-1]
Figure 2. Conversion using recombinant Synechocystis harbouring
Pcpc_IRED-A as biocatalyst at different cell densities. Top: Time course of the
conversions; bottom: Specific activity from the same experiment (black dots)
and total conversion after 6 h (bars). Standard deviation for three biological
replicates is represented by error bars. Reaction conditions: 2 mL scale, 4 mM
1b, 30 °C, 150 µmolphotons m-2 s-1, in BG11 (pH = 8)
Synechocystis. In principle, biphasic systems are applicable to
[13]
cyanobacteria,
and it remains to be studied to which extent
approaches such the one in micro-aqueous environment reported
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10.1002/cctc.201901592
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COMMUNICATION
by Maugeri et al. will be applicable to the imine reductases in
cyanobacteria.[33] As the number of genetically amenable
cyanobacterial strains is rapidly expanding, future research will
focus on identifying strains less sensitive towards the toxicity of
the reactants.
Experimental Section
Detailed description to the used methods can be found in the supplements.
Cultivation of Synechocystis sp. PCC 6803
Synechocystis sp. PCC 6803 wildtype and mutant strains were cultivated
under constant light intensity between 20-60 µmolphotons m-2 s-1, at 30 °C
and 50 % humidity in BG11 or on BG11 agar plates. The medium for the
mutants was supplemented with 50 µg mL-1 kanamycin. For
biotransformations, cells were harvested at OD750 between 1 and 3 and
concentrated to the respective density of the reaction.
Table 2. Imine reduction of substrates 1a-1f using Pcpc-IRED-A in recombinant
Synechocystis The specific activity (Aspec.), the intial rate and the conversion
(C) were calculated from the substrate depletion.
[a]
Biotransformations using recombinant cells
S
c(S)
[mM]
Aspec.
T [h] C[b]
ee
[%] Product[c] Rate[a]
-1
[U gCDW
]
[%]
[%]
[mM h-1]
All biotransformations (5 mL or 2 mL) with Synechocystis sp. PCC 6803
strains expressing heterologous IREDs harboring strains were performed
in a photo-bioreactor at 30 °C, 140 rpm and with a light intensity of
150 µmolphotons m-2 s-1. The reaction was started by mixing BG11 medium
(2.25 mL or 900 µL), substrate stock solution (100 mM in isopropanol or
N,N-DMF, 250 µL or 100 µL) and cell suspension (OD750 = 30, 2.5 mL or
1 mL) in a glass vial (20 mL or 4 mL). The amount of concentrated cells
was varied to obtain different ODs for biotransformations. Eight to ten
sampling time points were spread over 40 h of biotransformation to monitor
the reaction curve. Samples of the cell suspension (300 µl) were taken and
the reaction was quenched by the addition of NaCl (1 spatula tip) and
saturated Na2CO3 solution (200 µL). Till analysis, samples were frozen at
-20 °C.
1a
1a
4
8
19.5 ± 2.2
21.8 ± 3.0
1 h
84
> 94 (R)
82
65
4.8 ± 0.6
24 h > 99 > 94 (R)
1 h
3 h
65
> 99
> 94 (R)
63
88
66
6.3 ± 0.8
24 h > 99 > 94 (R)
1b
1c
1d
1e
1f
4
5
4
5
5
8.9 ± 1.2
5.5 ± 1.9
10.2 ± 0.9
3.1 ± 1
1 h
24 h 83
61
> 94 (R)
> 94 (R)
49
81
2.6 ± 0.3
1.4 ± 0.6
2.9 ± 0.3
0.9 ± 0.3
0.5 ± 0.1
1 h
47
>99 (R)
N/A
24 h > 99 >99 (R)
Biotransformations using wild-type cells
Control reactions with the wild type strain Synechocystis sp. PCC 6803
were performed in 1 mL total volume. Cell suspension (OD750 = 20, 500 µl),
BG11 medium (450 µL) and substrate stock solution (100 mM in
isopropanol or N,N-DMF, 50 µL) were mixed in glass vials (1.5 mL) and
the biotransformations were performed at 30 °C in a photo-bioreactor with
a light intensity of 150 µmolphotons m-2 s-1. Magnetic stirrers were used for
stirring. After specific time points (0 h, 1 h, 6 h, 24 h) samples of the cell
suspension (100 µL) were taken and immediately frozen at -20 °C.
1 h
24 h 99
64
N/A
49
88
1 h
24 h 94
18
71 (R)
63 (R)
13
67
1.6 ± 0.2
1 h
24 h 26
10
88 (R)
94 (R)
N/A
Sample derivatization and extraction
Samples were extracted with equal volumes ethyl acetate (300 µL)
containing acetophenone (2 mM) as internal standard and directly
measured on GC FID and HPLC. Monocyclic amines 2a, 2b and 2c were
acetylated prior chiral GC analysis. Therefore, acetic anhydride (20 µL),
saturated Na2CO3 solution (200 µL) and NaCl (1 spatula tip) were added
to the organic phase and the suspension was mixed by inverting. After
centrifugation (2 min, 13 000 g), the organic phase was dried with
anhydrous K2CO3, vortexed briefly and centrifuged (2 min, 13 000 g). The
organic phase of all other samples was dried on MgSO4. Samples were
analyzed on chiral GC FID.
[a] determined from the substrate decay after 0.5 h or 1 h
[b] determined from the substrate depletion
[c] product formation is based on product quantification by achiral GC-FID
Reaction conditions: 2 mL scale, 150 µmolphotons m-2 s-1, 30 °C, 3.6 gCDW L-1, in
BG11 (pH = 8)
N/A = not analysed
In conclusion, we showed that recombinant cyanobacteria are
promising hosts for imine reduction. Yet, high susceptibility of
Synechocystis toward imine substrates and amine products is
considered as main bottleneck for any synthetic application of the
cyanobacterial imine reduction. Furthermore, dark control
reactions indicated that the cofactor recycling might be largely
fueled from storage compounds. The observed biotransformation
is therefore not a light-driven reaction, but nevertheless does not
need the addition of organic cosubstrates for cofactor
regeneration. Our approach represents a simple system for highly
selective imine reduction which we optimized by promoter
exchange and increasing cell densities. Beside the excellent
enantioselectivity, the highest specific activities of around
22 U gCDW-1 and product formation rates of up to 6.3 mM h-1 for 1a
with full conversion within 3 h puts the use of photoautotrophs for
cofactor recycling in line with other presently used systems and
hosts and is a promising basis for further optimization.
Acknowledgements
This work has been supported by the Federal Ministry of Science,
Research and Economy (BMWFW), the Federal Ministry of Traffic,
Innovation and Technology (bmvit), the Styrian Business
Promotion Agency SFG, the Standortagentur Tirol, the
Government of Lower Austria and Business Agency Vienna
through the COMET-Funding Program managed by the Austrian
Research Promotion Agency FFG. H.B. and R.K. are indebted to
the Deutsche Bundesstiftung Umwelt (DBU) for financial support
(AZ 33741/01-32).
Keywords: biotechnology · enzyme catalysis · photobiocatalysis
· photosynthesis · imine reductase
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10.1002/cctc.201901592
ChemCatChem
COMMUNICATION
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COMMUNICATION
Synechocystis sp. PCC 6803
harbouring recombinant IREDs
can selectively reduce imines.
With activities of up to 22
U gCDW-1 and corresponding
product formations of up to
6.3 mM h-1, the system is
comparable to other
Hanna C. Büchsenschütz,
Viktorija Vidimce-Risteski,
Bettina Eggbauer, Sandy
Schmidt, Christoph K. Winkler,
Jörg H. Schrittwieser, Wolfgang
Kroutil* and Robert Kourist*
Page No. – Page No.
approaches avoiding the
addition of organic
Title
cosubstrates for NADPH
regeneration.
This article is protected by copyright. All rights reserved.