Effect of Photocaged Isopropyl β-d-1-thiogalactopyranoside Solubility on the Light Responsiveness of LacI-controlled Expression Systems in Different Bacteria

Article abstract of DOI:10.1002/cbic.202000377

Photolabile protecting groups play a significant role in controlling biological functions and cellular processes in living cells and tissues, as light offers high spatiotemporal control, is non-invasive as well as easily tuneable. In the recent past, photo-responsive inducer molecules such as 6-nitropiperonyl-caged IPTG (NP-cIPTG) have been used as optochemical tools for Lac repressor-controlled microbial expression systems. To further expand the applicability of the versatile optochemical on-switch, we have investigated whether the modulation of cIPTG water solubility can improve the light responsiveness of appropriate expression systems in bacteria. To this end, we developed two new cIPTG derivatives with different hydrophobicity and demonstrated both an easy applicability for the light-mediated control of gene expression and a simple transferability of this optochemical toolbox to the biotechnologically relevant bacteria Pseudomonas putida and Bacillus subtilis. Notably, the more water-soluble cIPTG derivative proved to be particularly suitable for light-mediated gene expression in these alternative expression hosts.

Full text of DOI:10.1002/cbic.202000377

Combining Chemistry and Biology
Accepted Article
Title: Effect of Photocaged Isopropyl β-d-1-Thiogalactopyranoside
Solubility on Light-Responsiveness of LacI-controlled Expression
Systems in Different Bacteria
Authors: Fabian Hogenkamp, Fabienne Hilgers, Andreas Knapp, Oliver
Klaus, Claus Bier, Dennis Binder, Karl-Erich Jaeger, Thomas
Drepper, and Joerg Pietruszka
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.202000377
Link to VoR: https://doi.org/10.1002/cbic.202000377
01/2020

10.1002/cbic.202000377
ChemBioChem
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Effect of Photocaged Isopropyl β-D-1-thiogalactopyranoside
Solubility on Light-Responsiveness of LacI-controlled Expression
Systems in Different Bacteria
Fabian Hogenkamp+[a], Fabienne Hilgers+[b], Andreas Knapp[b], Oliver Klaus[b], Claus Bier[a], Dennis
Binder[b], Karl-Erich Jaeger[b, c], Thomas Drepper*[b] and Jörg Pietruszka*[a, c]
Abstract: Photolabile protecting groups play a significant role for
controlling biological functions and cellular processes in living cells
and tissues, since light offers a high spatiotemporal control, is non-
invasive as well as easily tuneable. In the recent past, photo-
responsive inducer molecules such as 6-nitropiperonyl-caged IPTG
(NP-cIPTG) have been used as optochemical tools for Lac repressor
controlled microbial expression systems. To further expand the
applicability of the versatile optochemical on-switch, we now
investigated if the modulation of cIPTG water solubility can improve
the light-responsiveness of appropriate expression systems in
bacteria. To this end, we developed two new cIPTG derivatives with
varying hydrophobicity and demonstrated both an easy applicability
for the light-mediated control of gene expression and a simple
transferability of this optochemical toolbox to the biotechnologically
relevant bacteria Pseudomonas putida and Bacillus subtilis. Notably,
the more water-soluble cIPTG derivative has proven to be particularly
suitable for light-mediated gene expression in these alternative
expression hosts.
on light-responsive two- or one-component systems, are
extensively studied and have been successfully employed as
reversible photoswitches for light-mediated in vivo signal
transduction in various biological applications.[2]
Besides the use of photoreceptors photolabile protecting groups
were established as optochemical tools for a variety of diverse
applications.[3] In recent years, many approaches were published,
in which photocaged compounds have been used for controlling
different cellular processes, ranging from cell signalling[3b, 4], over
drug delivery[5] to gene expression.[6] In this context, especially 2-
nitrobenzyl-photocaging groups (NB) and their derivatives such
as 6-nitropiperonyl (NP) were commonly used to mediate an
adequate and well-characterised UV-A light triggered release of
7]
bioactive molecules.[3d, To implement caged compounds as
versatile optochemical switches,
a variety of photolabile
protecting groups has been developed focusing on the (i)
redshifted absorption,[3b, 8] (ii) higher quantum yields[9] and (iii) an
improved solubility.[10] Especially for in vivo approaches an
excellent stability towards enzymatic hydrolysis, good
biocompatibility, and low overall toxicity of caged compounds
(also including the photolysis products) are indispensable.[11] In
addition, the extend of the caged compound’s solubility could
further modulate their ability to pass bacterial cell membranes
either via passive processes including free diffusion and porin-
based uptake or by active, membrane transporter-mediated
processes.[12]
Introduction
In general, optogenetics combines genetic and optical methods to
allow fast control of cellular functions with high spatiotemporal
resolution and in a non-invasive fashion.[1] The control over gene
expression by light can basically be realised by employing
genetically encoded photoreceptors or chemically photocaged
(bio)molecules. Recombinant photoreceptors are typically based
In the recent past, photoresponsive inducer molecules such as
caged
thiogalactopyranoside
carbohydrates[6c,
derivatives
of
doxycycline,[13]
isopropyl
several
β-D-
other
6b]
(IPTG)[6a,
or
6d]
have been used as non-reversible
optochemical switches for appropriate microbial expression
systems. Especially the applicability of 6-nitropiperonyl
photocaged IPTG (NP-cIPTG, 1) for bioengineering approaches
using Escherichia coli[14] and Corynebacterium glutamicum[15] as
production hosts could be demonstrated. However, a further
expansion of the applicability in different expression hosts was for
instance hindered by the low water-solubility of NP-cIPTG (1)
(0.7 mM), as appropriately high inducer concentrations were not
soluble in the cultivation medium.
[a]
[b]
F. Hogenkamp, Dr. C. Bier, Prof. Dr. J. Pietruszka
Institute of Bioorganic Chemistry, Heinrich Heine University
Düsseldorf at Forschungszentrum Jülich
Stetternicher Forst, 52426 Jülich (Germany)
E-mail: J.Pietruszka@fz-juelich.de
F. Hilgers, Dr. A. Knapp, O. Klaus, Dr. D. Binder, Prof. Dr. K.-E.
Jaeger, Dr. T. Drepper
Institute of Molecular Enzyme Technology Heinrich Heine University
Düsseldorf at Forschungszentrum Jülich
Stetternicher Forst, 52426 Jülich (Germany)
E-mail: T.Drepper@fz-juelich.de
Prof. Dr. K.-E. Jaeger, Prof. Dr. J. Pietruszka Institute of Bio- and
Geosciences (IBG-1: Biotechnology), Forschungszentrum Jülich
Stetternicher Forst, 52426 Jülich (Germany)
These authors contributed equally to this work.
Derivatives of the 2-nitrobenzyl group with improved solubility in
aqueous media have been applied before (Figure 1 A). Tsien and
[c]
[+]
co-worker as well as Ni et al. conceived
a
4,5-
bis(carboxymethoxy)-2-nitrobenzyl protecting group (BC, 2),
which they stated to be highly water-soluble.
Supporting information for this article is given via a link at the end of
the document
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Figure 1. Photolabile protection groups and their application in this work. A) A variety of previously published photolabile protection groups with improved
aqueous solubility or membrane-permeability based on the NB photocaging group. B) Three photolabile protection groups were used in this work to construct
the photocaged IPTG variants NP-cIPTG (1), BEC-cIPTG (10a) and BC-cIPTG (10b), strongly differing in their water-solubility. These caged inducer
molecules (red dot with blue frame) are biologically inactive, however, upon illumination with UV-A light their activity can be restored by a two-step cleavage
process. Subsequently, the IPTG binds its respective repressor protein LacI leading to a release of LacI from the PT7, Ptac or Pgrac promoter and thus to the
induction of gene expression. This principle was applied to analyse the effect of cIPTG solubility on the inducibility of LacI repressor-controlled target gene
expression in E. coli, P. putida and B. subtilis.
However, they masked the carboxylate 2 as acetoxymethyl ester
3 to facilitate diffusion across cell membranes.[12a, 12b] Russell et
al. published a similar derivative 4, but bearing an additional third
carboxy group in the benzylic position, for the synthesis of
photolabile tyrosine, whereby a solubility of at least 30 mM was
reached.[10b] As the formation of a dioxolane is required for the
protection of IPTG, previously reported α-carboxy-2-nitrobenzyl
(α-CNB, 5–8) photocages[10a, 10c, 16] were not considered, because
the α-carboxy-group increases solubility, but concurrently blocks
the position where the dioxolane is later formed.
Based on these results the BC protecting group 2 was chosen in
this work as a candidate for the synthesis of a charged, highly
water-soluble photocaged IPTG-derivative (Figure 1 B) and was
further applied to determine the influence of the solubility and the
charge on the inducer uptake through the cell membrane and the
resulting expression response. In addition, the 4,5-
photophysical properties as well as photolysis in aqueous media
were characterised. Subsequently, the in vivo applicability of the
newly synthesised compounds for light-inducible gene expression
was analysed in comparison to the well-established NP-cIPTG (1)
in E. coli in a time-resolved manner. Finally, we investigated
whether optochemical control of gene expression can also be
implemented in the alternative expression hosts Pseudomonas
putida and Bacillus subtilis, which exhibit individual morphological
and physiological properties. Therefore, we used the photocaged
IPTG derivatives 1, 10a, and 10b together with appropriate LacI
repressor-controlled expression systems and comparatively
evaluated their light-responsiveness.
Results
bis(ethoxycarbonylmethoxy)-2-nitrobenzyl
protecting
group
Synthesis and photochemical properties of cIPTGs
(BEC, 9) harbouring lipophilic ester moieties, was selected as an
alternative caging group, which might facilitate its passive
diffusion across cell membranes. Afterwards, enzymatic
hydrolysis of the ester moiety could lead to intracellular
accumulation.[12c] To comparatively analyse the effect of caged
inducer solubility on light dependent control of gene expression in
bacteria, the two new cIPTG derivatives BEC-cIPTG (10a,
derived from 9) and BC-cIPTG (10b, derived from 2) were
synthesised and the maximum solubility was quantified. The
The BC-cIPTG (10b) was synthesised in a three-step reaction
(Scheme 1; yield over three steps: 24%) from 4,5-
bis(ethoxycarbonylmethoxy)-2-nitrobenzaldehyde (11), which
was obtained following the previously reported procedure by Ni et
al. (see Supporting methods).[12b] The 2-nitrobenzaldehyde
derivative 11 was reacted with triethyl orthoformate to form the
corresponding acetal 12 in 89% yield, which then was converted
to BEC-cIPTG (10a) (45%) via a transacetalisation, as the direct
acetalization was not feasible. In this step the triethyl orthoformate
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was preferred to the trimethyl orthoformate due to the occurrence
of transesterification during the acid-catalysed reaction, which
was leading towards a mixture of products. After deprotection
under basic conditions the BC-cIPTG (10b) could be obtained in
59% yield as the corresponding lithium-salt, which promised
advantageous solubility properties compared to the free-acid. NP-
cIPTG (1) was synthesised from 6-nitropiperonal (13) according
to literature procedures.[6a, 6b] The purity of BEC-cIPTG (10a), BC-
cIPTG (10b) and NP-cIPTG (1) was determined by qNMR (Table
S3).
derivative 10b about ~5% of the starting material remained after
irradiation for 30 min (Figure S15).
Scheme 2. Two-step release sequence after photolysis of BEC- and BC-
photocaged IPTG 10a and 10b by irradiation with UV-A light and a subsequent
enzymatic hydrolysis by a microbial esterase as previously described.[6a, 6b]
The BC-cIPTG (10b) showed a maximum solubility of 147 mM in
deionised and degassed water, which is over 200 times higher
than the maximum solubility of NP-cIPTG (1),[6b] but only ~8% of
the maximum solubility of IPTG (14) itself (Table 1). Other
previously reported photocaged carbohydrates were in the range
of 4–58 mM.[6d] In contrast, the BEC-cIPTG (10a) displayed a
more than 7-times lower solubility of <0.1 mM, as expected due to
the ester-protected carboxylic acids. Since the possible higher
membrane permeability of BEC-cIPTG might result in an
improved in vivo applicability, this cIPTG derivative was
additionally used for further investigations.
Scheme 1. Synthesis of BEC-, BC- and NP-photocaged IPTG 10a, 10b and 1:
a) Triethyl orthoformate, pyridinium p-toluenesulfonate, ethanol, reflux, 19 h
(89%); b) IPTG, p-toluenesulfonic acid, CH2Cl2, RT, 20 h (45%); c) 0.2 M LiOH
(aq.), MeOH, 0 °C - RT, 1 h (59%); d) IPTG, sulfuric acid, DMSO, 0 °C - RT,
24 h (21%).
Due to the structural similarity of the newly synthesised caged
compounds 10a and 10b to the NP-cIPTG (1), IPTG (14) should
be released upon UV-A light exposure in
a
two-step
photocleavage reaction as previously described.[6a, 6b] In the first
step the irradiation with UV-A light leads to the formation of ester
intermediates 15 and 16, which might subsequently be cleaved
by a microbial esterase. The corresponding nitroso compounds
17 are formed as the photo by-product (Scheme 2).
Table 1. Spectral and (photo-)chemical properties of caged IPTG
1, 10a, 10b and 14.
[b]
[d]
[a]
Compound
λmax
ε[a]
t0.5
s[c]
Φu
εΦu
-1
-1
[nm]
[M cm-1]
[min]
[mM]
[M cm-1]
The in vitro characterisation (Table 1, Table S2, Figure S1-3) of
the new photocaged compounds 10a and 10b showed uncaging
1[e]
241
336
1690
3.4
0.7
0.50
845
quantum yields (Φu) and molar extinction coefficients (ε) in the
10a[e]
10b[f]
298
1810
3543
2.2
3.5
<0.1
147
0.68
0.46
1230
1630
17]
range of previously reported caged compounds.[6d,
The
resulting photolytic efficiencies (εΦu) are all in the same order of
magnitude. However, more importantly the uncaging half-life time
of the photolytic cleavage amounts to 2.2 min for BEC-cIPTG
(10a), 3.5 min for BC-cIPTG (10b), and 3.4 min for NP-cIPTG (1).
This underlines the fast formation of the ester intermediates 15
and 16 (Figure S4, Table S2). Full photoconversion of the cIPTG
variants (1 mM) by irradiation with UV-A light (375 nm,
6.4 mW cm-2) was achieved in less than 30 min for 10a and 1. For
242
340
14
204
—
—
1941
—
—
[a] ε = molar extinction coefficient at λ = 375 nm, [b] t0.5 = uncaging half-life time,
[c] s = solubility in deionised and degassed water, [d] Φu = uncaging quantum
yield upon 375 nm irradiation, [e] measured in MeOH, [f] measured in sodium
phosphate buffer (0.1 mM, pH 7.5).
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1 mW cm-2) did not lead to considerable growth impairments in
the presence (Figure S6) or absence (Figure S7) of IPTG (14) and
its photocaged derivatives 1, 10a and 10b. Furthermore, the
stability of 1, 10a and 10b were analysed by measuring the
fluorescence intensity of cultures in the dark (Figure S6 A). The
data clearly reveals a pronounced in vivo stability of the new
cIPTG derivatives 10a and 10b over 20 h in LB medium at 30 °C.
Applicability of cIPTGs for light-controlled gene expression
in bacteria
After the successful synthesis of BEC- and BC-cIPTG (10a and
10b), we next analysed whether the different solubility of the
cIPTG derivatives (solubility in aqueous solvents: 10b>>1>10a,
see Table 1) affect the inducibility of LacI repressor-controlled
expression systems. The regulatory system, which originally
controls the lactose consumption in E. coli, is one of the most
often used regulation mechanisms for triggering heterologous
gene expression in this host.[18] The development of different
recombinant promoters (e.g. Ptac, Ptrc, PT7), whose activities can
be tightly and gradually controlled by the concentration of the
added inducer (e.g. the non-hydrolysable lactose analogue IPTG)
led to its broad applicability in basic research and biotechnological
production processes. Furthermore, the development of light-
responsive NP-cIPTG (1) allowed for non-invasive light-mediated
control of gene expression in E. coli.[6a-c] To further optimise light
responsiveness of this promising optochemical on-switch in
E. coli and to facilitate its transferability to other industrially
relevant microbes, we used the following Gram-negative and -
positive bacteria as appropriate model hosts offering individual
morphological and physiological properties: (i) E. coli Tuner(DE3)
is a lactose permease-deficient strain and was shown to be well
suited for NP-cIPTG-based light control of gene expression,
because the uptake of appropriate inducers is solely dependent
on passive diffusion processes. Previous studies using E. coli
Expression studies in E. coli: To further evaluate the applicability
of the new cIPTG derivatives 10a and 10b in comparison to 1 in
E. coli, we used the well-established strain E. coli Tuner(DE3)
carrying the eYFP expression vector pRhotHi-2-lacI-EYFP[6b, 14]
.
Initially, we could observe that, in contrast to the variants 1 and
10a which form an emulsion-like structure at relevant
concentrations in LB medium without considerable amounts of
ethanol or DMSO, variant 10b can be completely dissolved in the
cultivation medium, superseding the use of additional solvents. To
compare the UV-A light-induced gene expression mediated by
differently soluble photocaged IPTG variants during E. coli
cultivation, light exposure was carried out for 30 min in order to
ensure sufficient photoconversion of 1, 10a and 10b (see Figure
S4). First, the general applicability of cIPTG variants was
evaluated by analysing eYFP expression in cultures that reached
the stationary growth phase. As shown in Figure 2A, illumination
of the already established NP-cIPTG resulted in comparable
eYFP expression levels as in the control experiment, where
conventional IPTG (14) was added. In contrast, the new water-
soluble BC-cIPTG (10b) and the more hydrophobic BEC-cIPTG
(10a) led to a slight decrease of reporter gene expression in this
experimental setup.
To analyse the properties of the cIPTG variants in more detail,
eYFP expression was subsequently online monitored during
batch cultivation of E. coli. Illumination of BC-cIPTG (10b)
resulted in the fastest induction response in the early logarithmic
growth phase (4-7 h after inoculation) as also indicated by a
lower half-maximal responsiveness with t0.5 final = 4.16 h when
compared to NP-cIPTG (1) and BEC-cIPTG (10a) (t0.5 final = 4.41
and 4.51 h, respectively, Table S4 and Figure S8). Thus, these
results give a first indication that NB caging group derivatives with
improved water-solubility such as BC might slightly facilitate the
overall uptake of cIPTG in E. coli. However, the lower final eYFP
expression levels in the respective cultures point to a less efficient
enzymatic release of IPTG from ester intermediates 15 and 16,
which is eventually caused by the increasing size of these
photolabile protecting groups. All in all the differential solubility of
tested cIPTG variants in aqueous solvents seems to play a minor
role for optochemical in vivo applications in E. coli, since only
marginal differences of light-controlled gene expression could be
observed.
Tuner(DE3) revealed
a
very stringently controlled and
homogeneous gene expression that gradually responded to
changes of illumination time or light intensity.[6b, 6c, 14] (ii) P. putida
KT2440 is a rod-shaped, Gram-negative soil bacterium, which
offers a pronounced tolerance towards xenobiotics[19] as well as
redox stress.[20] Besides its genetic accessibility and its FDA
certification as a host-vector biosafety system,[21] P. putida
exhibits an extraordinary versatile metabolism that makes it
especially suited for a variety of biotechnological applications
including the production of various high-value natural products
and their derivatives.[22] (iii) Bacillus subtilis DB430 is a Gram-
positive bacterium commonly used as a ‘microbial cell factory’ for
high-level production and secretion of proteins for industrial
applications.[23] In contrast to the Gram-negative bacteria used in
this study, B. subtilis possesses a more rigid and thick cell wall
which might act as an additional diffusion barrier for the
photocaged IPTG molecules, but lacks an outer membrane. For
all the here tested bacterial hosts, expression systems
encompassing LacI-controlled, IPTG-inducible promoters have
been successfully established in recent studies (Table S1).[6b, 18,
22c, 24]
To exclude detrimental effects of the new caged inducers or UV-
A light exposure on cell viability, we first analysed the growth of
E. coli, P. putida and B. subtilis cells in the presence of the cIPTG
derivatives 10a and 10b as well as their corresponding
photoproducts in comparison to conventional IPTG (14). For
these studies, we used inducer concentrations that were sufficient
to fully induce reporter gene expression in the respective
expression hosts (Figure S5). Comparative growth of all strains
clearly demonstrated that UV-A light exposure (30 min, 365 nm,
Expression studies in P. putida: Next, we analysed, if the
optochemical cIPTG/LacI system can be transferred to the Gram-
negative bacterium P. putida KT2440 and if the solubility of the
caged inducer has an effect on its in vivo applicability. In the
following experiments, we used P. putida KT2440 carrying the
expression vector pVLT33 harbouring a GFPmut3 gene, which is
under control of the Ptac promoter (Table S1), and the same
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experimental setup as established for reference strain E. coli
Tuner(DE3).
fluorescence in P. putida cultures that reached the stationary
growth phase demonstrates an induction of reporter gene
expression of about 70% for BC-cIPTG (10b) when compared to
conventional IPTG (14). In contrast, the use of NP- and BEC-
cIPTG (1 and 10a) led to a lower induction response of 50% or
less. For BC-cIPTG (10b) the maximal responsiveness value
t0.5 final of 2.62 h is significantly slower than IPTG (14) (t0.5 final
=
1.41 h) (Figure S8 and Table S4). In summary, cIPTG constitutes
an optochemical tool that can be used as an optogenetic switch
for LacI-controlled expression systems in P. putida, but
comparative expression studies revealed that modified IPTG
variants 10a, 10b and 1 work less efficient than in E. coli.
Remarkably, only the variant BC-cIPTG (10b) that offers an
increased solubility in aqueous solution showed a satisfactory
applicability for controlling gene expression by light. Similar to the
E. coli Tuner(DE3), P. putida lacks
a
specific lactose
permease.[30] Therefore, IPTG can only pass the cytoplasmic
membrane via passive diffusion processes. Furthermore, in
pseudomonads including P. putida, the outer membrane exhibits
a reduced permeability as compared to E. coli. The uptake of
small water-soluble molecules is mainly mediated by a defined set
of specific porins such as OprF, which is characterised by a
significantly slower diffusion rate compared to the more unspecific
E. coli porins OpmF and OmpC.[25-26] As a consequence, the
water-soluble compound 10b could be transported over the outer
membrane in a slower process.
Expression studies in B. subtilis: The Gram-positive bacterium
B. subtilis was used as an expression host to determine the effect
of inducer solubility on the uptake process, which is here solely
influenced by the permeability of the cytoplasmic membrane and
the surrounding cell wall. As this bacterium is not able to utilise
lactose as a carbon source, and a lactose permease-encoding
gene could not be identified in the genome,[27] the uptake of
inducer molecules is most probably restricted to passive diffusion.
To evaluate the cIPTG applicability, we used the B. subtilis
DB430/pHT01-sfGFP strain, where fluorescence reporter
expression is driven by the LacI-controlled Pgrac promoter.[24b]
Similar to P. putida, we added the respective inducer at a
concentration of 1 mM to ensure full induction of recombinant
gene expression (Figure S5). Remarkably, illumination of BC-
cIPTG (10b) led to a strong and fast induction response
comparable to the results obtained with IPTG (14) (Figure 2C,
Figure S8, Table S4). In contrast, the induction with BEC-cIPTG
(10a) led to a sfGFP expression level of around 75% in
comparison to IPTG (14), while addition of NP-cIPTG (1) resulted
in only 50% sfGFP fluorescence. Based on this observation, we
cannot exclude that the cell wall of B. subtilis, which is much
thicker (20–80 nm) than in Gram-negative organisms (5–
10 nm),[28] is less permeable for the more hydrophobic cIPTG
variants. In addition, the extremely fast responsiveness of BC-
cIPTG (10b) in B. subtilis (t0.5 final  2.3 h), which also outperforms
the respective induction response in E. coli (t0.5 final  4.3 h), might
indicate an efficient catalytic cleavage of the ester intermediate
after photoconversion. It should be noted that addition of BC- and
BEC-cIPTG resulted in an increased basal target gene
expression in non-illuminated cultures, which might be due to a
Figure 2. Light-controlled gene expression in E. coli Tuner(DE3)/pRhotHi-2-
lacI-EYFP (A), P. putida KT2440/pVLT33-GFPmut3 (B), B. subtilis
DB430/pHT01-sfGFP (C) using NP-, BC-, and BEC-cIPTG. A: In vivo eYFP
fluorescence (ex = 508 nm, em = 532 nm) of E. coli cultures supplemented with
50 µM of each cIPTG variant is shown in relation to a 50 µM IPTG (14) after 20 h
(stationary growth phase). Induction was performed after 2.5 h via UV-A light
exposure at 365 nm (1 mW cm-2) for 30 min or the addition of 50 µM 14. B: In
vivo GFPmut3 fluorescence (ex= 508 nm, em = 532 nm) of P. putida cultures
supplemented with 1 mM of each cIPTG variant is shown in relation to a 1 mM
IPTG (14) control after 20 h (stationary growth phase). Induction was performed
after 3 h via UV-A light exposure at 365 nm (1 mW cm-2) for 30 min or the
addition of 1 mM 14. C: In vivo sfGFP fluorescence (ex = 488 nm, em = 520 nm)
of cultures supplemented with 1 mM of each cIPTG variant is shown in relation
to a 1 mM IPTG (14) control after 20 h. Induction was performed after 5 h via
UV-A light exposure at 365 nm (1 mW cm-2) for 30 min or the addition of 1 mM
14. In vivo fluorescence intensities were normalized to cell densities and values
are means of triplicate measurements. Error bars indicate the respective
standard deviations.
Since we could observe only basal induction of gene expression,
when 50 µM IPTG (14) was added to P. putida expression
cultures (Figure S5), 1 mM of each IPTG derivative was utilised.
As depicted in Figure 2B, the comparison of GFPmut3
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slightly reduced stability of these cIPTG derivatives probably
caused by a minimal catalytic release of the respective caging
groups.
esterases, and (iii) the individual permeability of cell membranes
for cIPTG, the ester intermediates or released inducer. Thus, the
individual physiological and morphological properties of the
chosen microbial expression host might exhibit relevant
differences such as the respective membrane composition or the
ability for active inducer uptake via appropriate transporters. In
Gram-negative bacteria, for example, the inducer has to pass two
membranes, a process that occurs via (i) free diffusion (both
membranes), (ii) passive transport processes involving unspecific
or specific porins (outer membrane), and (iii) active transport
mechanisms that are facilitated by suitable permeases
(cytoplasmic membrane). In Gram-positive bacteria, even though
only one membrane needs to be passed, the surrounding cell wall
is much thicker than in Gram-negative hosts and thus a distinct
interaction with the differently soluble cIPTG variants might
additionally influence their uptake. However, to unravel the role of
individual properties of respective bacterial strains for cIPTG
uptake and IPTG release, further experiments have to be
performed in future studies.
Analysis of expression heterogeneity: Finally, we elucidated, if the
differential solubility of the applied cIPTG derivatives has an effect
on the expression heterogeneity. For E. coli strain Tuner(DE3),
we have previously proven a homogeneous induction response
for both IPTG (14) and NP-cIPTG (1), which is primarily due to the
absence of the permease and the resulting inducer uptake
via diffusion.[6b] In contrast, for Bacillus species considerable
expression heterogeneities are frequently described.[29] For the
direct comparison of expression heterogeneity, fluorescence of
the reporter proteins was determined at the single-cell level in
light-exposed and non-illuminated cell cultures of E. coli and
B. subtilis using flow cytometry. The results indicate that reporter
gene expression was induced homogenously in E. coli cells
irrespective of the added cIPTG variant (Figure S9 A) thereby
corroborating observations from microfluidic investigations with
NP-cIPTG (1).[6b] Similarly, the differential solubility of cIPTG
variants did not affect the rate of expression heterogeneity in B.
subtilis although it is generally more pronounced than in E. coli
(Figure S9 B). Thus, expression heterogeneity is not provoked by
a varying efficiency of inducer uptake.
In conclusion, we constructed two new caged IPTG variants,
characterised their (photo-)chemical properties and demonstrated
an easy applicability for the light-mediated control of gene
expression in Gram-negative and Gram-positive bacteria.
Because of their differential solubility, BC-, NP- and BEC-cIPTG
constitute a valuable “starter set” which enables an easy access
to a robust, light-responsive expression system in a broad variety
of different hosts. Due to the non-invasive nature, the here
presented optochemical on-switches additionally allow the
external triggering of gene expression in closed biological
systems thereby making e.g. anaerobic expression hosts more
accessible in the near future.
Discussion
We developed the two new cIPTG derivatives 10a and 10b with
varying hydrophobicity and aimed to analyse whether the change
of cIPTG solubility affects the inducibility of LacI repressor-
controlled target gene expression in E. coli, P. putida and
B. subtilis. In the here presented in vivo studies, the derivatives
are stable against spontaneous hydrolysis and did not induce
elevated basal expression of target genes in the dark. In E. coli,
only marginal differences of light-controlled gene expression
could be observed for the new cIPTG variants in comparison to
the well-established NP-cIPTG (1). Nevertheless, the increased
water-solubility of derivative 10b and its homogeneous dispersion
without addition of an organic cosolvent, noticeably improves the
applicability of this cIPTG derivative. The transfer to P. putida and
B. subtilis clearly demonstrated that the solubility of photocaged
inducer molecules is an important aspect that has to be
considered for the establishment of a light-controlled expression
system. Here, BC-cIPTG (10b), the variant that offers an
increased solubility in aqueous solution, resulted in high
expression levels together with a comparable or even increased
induction factor in comparison to IPTG (for direct comparison of
cIPTG derivatives’ induction factors see Tab. S5). In this context
it should be noted that, besides the improved solubility in microbial
cultivation media, the diverging hydrophobicity of the cIPTG
variants as well as the negative charge in case of BC-IPTG might
additionally affect the complex processes that are involved in
light-induced gene expression. These processes include (i) the
efficiency of photoconversion under the applied cultivation and
illumination conditions, (ii) the enzymatic hydrolysis of cIPTG
ester intermediates via cytoplasmic, periplasmic or extracellular
Experimental Section
General remarks: All chemicals for synthesis were obtained from
commercial suppliers and used without further purification unless stated
otherwise. Solvents were reagent grade and were dried as well as purified
by common methods. Thin-layer chromatography (TLC) was performed
using pre-coated silica gel plates (Polygram® SIL G/UV, Macherey-Nagel)
and components were visualised via oxidative staining or UV-light. Flash
chromatography was performed on silica gel (Merck silica gel 60
(0.063−0.200 µm) and solvents for flash chromatography (petroleum
ether/ethyl acetate) were distilled prior to use. Optical rotation was
determined at 20 °C on a Perkin Elmer Polarimeter 241 MC against
sodium D-line and melting points were recorded using a Büchi melting
point B-545 apparatus. The NMR spectra (1H and 13C) were measured at
20 °C on a Bruker Avance/DRX 600 spectrometer in deuterated solvents
(CDCl3, DMSO-d6, D2O). The chemical shifts are given in ppm relative to
the solvent (1H: CDCl3 = 7.26 ppm, 1H: DMSO-d6 = 3.31 ppm or 1H: D2O =
4.79 ppm / 13C: CDCl3 = 77.16 ppm or 13C: DMSO-d6 = 39.52 ppm).
Signals were assigned by means of H-COSY-, HSQC- and HMBC-
experiments and splitting patterns are reported as singlet (s), doublet (d),
triplet (t), multiplet (m), and broad singlet (brs). The IR spectra were
recorded with
a Perkin Elmer SpectrumOne IR-spectrometer ATR
(Waltham, USA). HRMS (ESI) spectra were recorded by the centrum of
analytics of the Heinrich Heine University. UV-Vis absorption spectra were
recorded on
a Genesys 10S UV/VIS Spectrophotometer (Thermo
Scientific) and uncaging experiments were performed in a quartz cuvette
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10.1002/cbic.202000377
ChemBioChem
FULL PAPER
with the LUMOS 43® from Atlas Photonics at 375 nm. Light intensity was
quantified using a Thermal Power Sensor (S302C, Thorlabs Inc, USA) and
the decay was detected by a Jasco HPLC system [column: Hyperclone 5
µ ODS (C18) 120 (Phenomenex)] combined with an UV/Vis-detector.
Synthesis of BC-cIPTG (10b)
A solution of BEC-cIPTG (10a) (200 mg, 0.35 mmol) in MeOH (3.5 mL)
was cooled to 0 °C and a 0.2 M solution of LiOH (3.5 mL) was added. The
reaction mixture was stirred for 1 h at room temperature. After the reaction
was completed as indicated by TLC, the MeOH was evaporated under
reduced pressure and the remaining solution was lyophilised overnight.
The residue was suspended in THF, sonicated for 15 min and filtrated.
After washing with small amounts of cold THF a white solid (107 mg,
0.21 mmol, 59%) was obtained. m.p. 190 °C (decay); [α] = -92 (c = 1.0 in
Synthesis of 4,5-Bis(ethoxycarbonylmethoxy)-2-nitrobenzylaldehyde
diethyl acetal (12)
To a solution of 4,5-bis(ethoxycarbonylmethoxy)-2-nitrobenzaldehyde (11)
(3.00 g, 8.44 mmol) in ethanol (50 mL) triethyl orthoformate (1.88 g,
12.6 mmol, 1.50 equiv.) and pyridinium p-toluenesulfonate (424 mg,
1.69 mmol, 0.20 equiv.) were added and heated under reflux for 19 h. A
dean-stark trap filled with molecular sieve (3Å) was utilised for the constant
removal of water. After the reaction was completed as indicated by TLC, it
was washed with saturated NaHCO3 solution. The aqueous phase was
then extracted with CH2Cl2 and the combined organic phase was dried with
anhydrous Na2SO4 and concentrated under reduced pressure. The
residue was purified by flash-column chromatography on SiO2 (petroleum
ether/ethyl acetate 85:15) to yield a yellow solid (3.22 g, 7.51 mmol, 89%).
H2O); 1H-NMR (600 MHz, D2O): δ = 1.29 (d, JCH3-a,SCH = 6.8 Hz, 3 H,
CH3-a), 1.31 (d, 3JCH3-b,SCH = 6.8 Hz, 3 H, CH3-b), 3.26 (septet, 3JSCH,CH3-a/b
= 6.8 Hz, 1 H, SCH), 3.66 (t, 3J5″,6″ = 9.8 Hz, 1 H, 2″-H), 3.71−3.82 (m, 2 H,
3
3
3″-H, 5″-H), 4.18 (m, 2 H, 6″-H), 4.37 (d, J4″,3″ = 3.6 Hz, 1 H, 4″-H), 4.60
(s, 2 H, 8′-H), 4.62 (d, 3J1″,2″ = 9.8 Hz, 1 H, 1″-H), 4.67 (d, J = 2.6 Hz, 2 H,
8-H), 6.20 (s, 1 H, 7-H), 7.32 (s, 1 H, 6-H), 7.55 ppm (s, 1 H, 3-H); 13C-
NMR (151 MHz, D2O): δ = 22.9 (CH3-a), 23.3 (CH3-b), 35.0 (SCH),
67.3(C-8′), 67.4 (C-8), 69.1 (C-2″), 69.3 (C-6″), 69.6 (C-5″), 72.8 (C-3″),
76.5 (C-4″), 84.8 (C-1″), 96.4 (C-7), 109.2 (C-3), 110.8 (C-6), 126.3 (C-1),
140.0 (C-2), 147.2 (C-4), 151.5 (C-5), 175.1 (C-9), 175.4 ppm (C-9′); IR
(ATR-film): ṽ = 3124, 3043, 1605, 1522, 1398, 1335, 1277, 1077, 1047,
1
Rf = 0.25 (petroleum ether/ethyl acetate 80:20) m.p. 62.1 °C; H-NMR
(600 MHz, [D6]DMSO): δ = 1.12 (t, 3J2′,1′ = 7.1 Hz, 6 H, 2′-H), 1.22 (t, 3J11,10
1024, 824 cm-1; UV-Vis (H2O): λmax (ε)
(3191 dm3 mol-1 cm-1); HRMS (ESI): m/z calcd for C20H29N2O13S+:
537.1385 [M + NH4]+; found: 537.1382.
= 245 (5008), 342 nm
and 11′,10′ = 7.1 Hz, 6 H, 11-H and 11′-H), 3.50 (dq, 2J1′a,1′b = 9.3 Hz, 3J1′a,2′
=
7.1 Hz, 2 H, 1′a-H), 3.62 (dq, 2J1′b,1′a = 9.3 Hz, 3J1′b,2′ = 7.1 Hz, 2 H, 1′b-H),
4.18 (q, 3J10,11 or 10′,11′ = 7.1 Hz, 2 H, 10-H or 10′-H), 4.19 (q, 3J10,11 or 10′,11′
=
7.1 Hz, 2 H, 10-H or 10′-H), 4.96 (s, 2 H, 8′-H), 4.99 (s, 2 H, 8-H), 5.88 (s,
1 H, 7-H), 7.09 (s, 1 H, 6-H), 7.57 ppm (s, 1 H, 3-H); 13C-NMR (151 MHz,
[D6]DMSO): δ = 14.0 (C-11 and C-11′), 14.9 (C-2′), 60.8 (C-10 or C-10′),
60.9 (C-10 or C-10′), 65.5 (C-8 or C-8′), 65.6 (C-8 or C-8′), 97.7 (C-7),
110.6 (C-3), 111.5 (C-6), 127.9 (C-1), 141.4 (C-2), 146.5 (C-4), 150.2
(C-5), 168.1 (C-9 or C-9′), 168.1 ppm (C-9 or C-9′); IR (ATR-film): ṽ = 2981,
1755, 1692, 1581, 1526, 1446, 1346, 1291, 1196, 1176, 1080, 878,
796 cm-1; HRMS (ESI): m/z calcd for C19H27NO10+: 447.1973 [M+NH4]+;
found: 447.1972.
Determination of purity by qNMR: The purity of the photocaged IPTG
derivatives 10a, 10b and 1 was determined via quantitative NMR. 3,5-
bis(trifluoromethyl)bromobenzene was utilised as internal standard for 10a
as well as 1 and (methanesulfonyl)methane for 10b. The spectra were
measured at 20 °C on a Bruker Avance/DRX 600 spectrometer with 64
scans each and 30 µs relaxation time between each scan. The results in
Table S3 are means of triplicate measurements.
Solubility analysis: The solubility of 10a, 10b and 14 was determined
photometrically at 25 °C using a spectrophotometer Shimadzu UV-1800
(CPS-240A). The absorbance of a serial dilution in degassed and
deionised water was measured at the absorption maximum of the
respective compound. A saturated solution was measured under the same
conditions. The solubility was calculated using the Lambert-Beer law.[13b]
Synthesis of BEC-cIPTG (10a)
To a solution of 4,5-bis(ethoxycarbonylmethoxy)-2-nitrobenzylaldehyde
diethyl acetal (12) (1.00 g, 2.33 mmol, 1.50 equiv.) in dry CH2Cl2 (6 mL)
IPTG (370 mg, 1.55 mmol) was added. After 5 min p-TSA (11.8 mg,
0.06 mmol, 4 mol%) was added to the suspension and it was stirred at
room temperature for 20 h. After the reaction was completed as indicated
by TLC, a small amount of triethylamine was added and the reaction was
concentrated under reduced pressure. The residue was purified by flash-
column chromatography on SiO2 (petroleum ether/ethyl acetate 50:50 to
20:80) to yield a white solid (403 mg, 0.70 mmol, 45%). Rf = 0.35
(petroleum ether/ethyl acetate 20:80); m.p. 104.5 °C; [α] = -68 (c = 1.0 in
CHCl3); 1H-NMR (600 MHz, CDCl3): δ = 1.31 (t, 3J11,10 or 11′,10′ = 7.2 Hz, 6 H,
11-H and 11′-H), 1.35 (d, 3JCH3-a/b,SCH = 6.8 Hz, 3 H, CH3-a or CH3-b), 1.36
(d, 3JCH3-a/b,SCH = 6.8 Hz, 3 H, CH3-a or CH3-b), 2.56 (brs, 2 H, 2″-OH and
Hydrolytic stability: For the determination of the hydrolytic stability, a
1 mM solution of the respective compound in methanol or sodium
phosphate buffer (0.1 M, pH 7.5) was stored in the dark at room
temperature. Samples were removed after 0 and 24 h and analysed by
reversed-phase HPLC.
Quantification of uncaging half-life times: A 1 mM solution of each
photocaged compound in methanol or sodium phosphate buffer (0.1 M, pH
7.5) was prepared. In a cuvette 1 mL of this solution was irradiated at room
temperature using the LUMOS 43 (375 nm) for a certain time period. The
sample was then analysed by reverse phase HPLC Jasco HPLC system
[column: Hyperclone 5 µ ODS (C18) 120 (Phenomenex)]. For each
photocaged compound, the procedure was repeated for different
irradiation times. The decrease of concentration was measured by an UV
detector.[6d]
3
3
3″-OH), 3.25 (septet, JSCH,CH3-a/b = 6.8 Hz, 1 H, SCH), 3.52 (dt, J5″,6″
3
=1.7 Hz, J5″,4″ = 1.2 Hz, 1 H, 5″-H), 3.64 – 3.70 (m, 2 H, 2″-H and 3″-H),
2
3
4.08 (dd, J6″b,6″a = 12.5 Hz, J6″b,5″ = 1.7 Hz, 1 H, 6″-Hb), 4.24 – 4.31 (m,
3
6 H, 10-H / 10′-H / 4″-H / 6″-Ha), 4.41 (d, J1″,2″ = 8.7 Hz, 1 H, 1″-H), 4.77
(s, 2 H, 8-H), 4.82 (s, 2 H, 8′-H), 6.21 (s, 1 H, 7-H), 7.35 (s, 1 H, 6-H),
7.54 ppm (s, 1 H, 3-H); 13C-NMR (151 MHz, CDCl3): δ = 14.3 (C-11 or
C-11′), 14.3 (C-11 or C-11′), 24.1 (CH3-a or CH3-b), 24.3 (CH3-a or CH3-b),
35.5 (SCH), 61.8 (C-10 or C-10′), 61.9 (C-10 or C-10′), 66.4 (C-8 or C-8′),
66.6 (C-8 or C-8′), 69.8 (C-6″), 70.1 (C-5″), 70.3 (C-3″), 73.9 (C-2″), 76.2
(C-4″), 85.7 (C-1″), 96.6 (C-7), 111.5 (C-3), 112.8 (C-6), 127.7 (C-1), 141.3
(C-2), 147.6 (C-4), 151.7 (C-5), 167.9 (C-9 or C-9′), 167.9 ppm (C-9 or
C-9′); IR (ATR-film): ṽ = 3478, 2967, 2916, 2866, 1747, 1520, 1287, 1176,
Determination of uncaging quantum yields: The quantum yields of 1,
10a and 10b were determined by a relative method in comparison to the
quantum yield of 2-nitropiperonylacetate (NPA-Ac), as this substrate
shows a sufficient similarity to 1, 10a and 10b. The procedure was followed
as previously described in literature (Supporting Information: Figure S4
and Table S2).[6c, 30]
1097, 1077, 1027, 989 cm-1; UV-Vis (MeOH): λmax (ε)
(8006 dm3 mol-1 cm-1); HRMS (ESI): m/z calcd for C24H37N2O13S:
593.2011 [M + NH4]+; found: 593.2011.
= 298 nm
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10.1002/cbic.202000377
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FULL PAPER
Bacterial strains and plasmids: The E. coli strain DH5α[31] was used for
all cloning procedures, while the E. coli strain S17-1[32] and Tuner(DE3)
(Novagen) were applied for conjugation and expression studies,
respectively. All E. coli strains, the P. putida strain KT2440[33] and the
B. subtilis strain DB430[34] were grown on LB agar plates or in liquid LB
medium (Luria/Miller, Carl Roth®), at 37 °C (E. coli) or 30 °C (P. putida,
B. subtilis). Media were supplemented either with kanamycin (50 µg mL-1),
gentamicin (25 µg mL-1), irgasan (25 µg mL-1) or chloramphenicol
(5 µg mL-1), when appropriate.
cell aggregates, the cells were also analysed regarding their size (forward
scatter, FSC) and granularity (side scatter, SSC). FSC was measured
using an FSC laser (nm) with 80% of the laser power for E. coli and 50%
for B. subtilis and
a 456/51 nm bandpass filter for detection. For
determination of SSC a nm-light laser with 80% of the laser power for
E. coli and 50% for B. subtilis (773/56 nm bandpass filter) was used.
Based on the scatter plots, bacterial cells were gated from irrelevant
counts for fluorescence analysis. Flow cytometric data were evaluated with
the CellStreamTM Analysis Software (Merck, now Luminex Corporation,
Austin, TX, USA).
All bacterial strains and plasmids used in this study are listed in Table S1,
Supporting Information.
Acknowledgements
Plasmid construction: All recombinant DNA techniques were carried out
as described by Sambrook et al..[35] For the construction of the B. subtilis
expression vector pHT01-sfGFP, the sfGFP-encoding gene was
synthesised with flanking NdeI and HindIII restriction sites (Eurofins
Genomics, Germany) and subsequently cloned into pET-22(b) (Novagen,
Merck, Germany). The resulting vector pET-22(b)-sfGFP was used as
template for SLIC cloning[36] of a DNA fragment encompassing the sfgfp
gene into the B. subtilis expression vector pHT01 (MoBiTec, Germany)
using oligos 3-6 (Table S1, Supporting Information). The construction of
the P. putida expression vector pVLT33-GFPmut3 was performed via
restriction and ligation. To this end, the gfpmut3 gene was amplified with
flanking EcoRI and XbaI restriction sites via PCR using oligos 1-2 (Table
S1, Supporting Information). Afterwards, the EcoRI/XbaI hydrolysed
fragment was ligated into the likewise hydrolysed vector backbone
pVLT33, resulting in the final expression vector pVLT33-GFPmut3. Correct
nucleotide sequences of all constructs were confirmed by Sanger
sequencing (Eurofins Genomics, Germany).
The work was supported by grants from German Federal Ministry
of Education and Research (BMBF, FKZ: 031A167A), the
Bioeconomy Science Center, and the European Regional
Development
Fund
(ERDF:
34.EFRE-0300096
and
34.EFRE-0300097) within the project CLIB-Kompetenzzentrum
Biotechnologie CKB). The scientific activities of the Bioeconomy
Science Center were financially supported by the Ministry of
Innovation, Science and Research of the German federal state of
North Rhine-Westphalia MIWF within the framework of the NRW
Strategieprojekt BioSC (No. 313/323-400-00213). The authors
thank Dr. Anita Loeschcke for the helpful discussion, Sonja
Kubicki for the construction of the plasmid pVLT33-GFPmut3,
Vera Ophoven for synthetic, and Birgit Henßen for analytical
support.
Cultivation conditions: All E. coli, P. putida and B. subtilis expression
Keywords: caged compounds, gene expression, optogenetics,
cultures were grown in 48-well Flowerplates® in
a
BioLector
photochemistry, synthetic biology
microbioreactor system (m2p labs, Germany) (800 µL LB medium, 1200
rpm, 30 °C), inoculated with an optical density at 580 nm of 0.05. During
cultivation, the cell density was measured online via scattered light
intensity at 620 nm. In addition, fluorescence of eYFP and GFP variants
(GFPmut3 and sfGFP) were continuously determined using a 508/532 nm
and 488/520 nm filter, respectively. cIPTG variants 10a, 10b or NP-cIPTG
(1) were added prior inoculation (final concentration: 50 µM for E. coli,
1 mM for P. putida and B. subtilis; purities of cIPTG variant after synthesis
were taken into account accordingly) and expression of reporter genes
was induced during the early logarithmic growth phase (after approx. 2.5 h
for E. coli, 3 h for P. putida and 5 h for B. subtilis) via UV-A light-exposure
(VL-315.BL lamp, Vilber Lourmat, France; 1 mW cm-2, 30 min exposure)
or by addition of equal amounts of conventional IPTG (14) after
illumination.
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FULL PAPER
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10.1002/cbic.202000377
ChemBioChem
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Extended toolbox: New photocaged
Fabian Hogenkamp+, Fabienne Hilgers+,
Andreas Knapp, Oliver Klaus, Claus
Bier, Dennis Binder, Karl-Erich Jaeger,
Thomas Drepper* and Jörg Pietruszka*
IPTG
derivatives
with
varying
hydrophobicity for light-induced gene
expression were synthesised and
tested with three different expression
host strains. The most water-soluble
Page No. – Page No.
compound
showed
superior
performance, especially in Bacillus
subtilis.
Effect of Photocaged Isopropyl β-D-1-
thiogalactopyranoside Solubility on
Light-Responsiveness of LacI-
controlled Expression Systems in
Different Bacteria
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.