New Prodigiosin Derivatives Obtained by Mutasynthesis in Pseudomonas putida

Article abstract of DOI:10.1021/acssynbio.7b00099

The deeply red-colored natural compound prodigiosin is a representative of the prodiginine alkaloid family, which possesses bioactivities as antimicrobial, antitumor, and antimalarial agents. Various bacteria including the opportunistic human pathogen Serratia marcescens and different members of the Streptomycetaceae and Pseudoalteromonadaceae produce prodiginines. In addition, these microbes generally accumulate many structurally related alkaloids making efficient prodiginine synthesis and purification difficult and expensive. Furthermore, it is known that structurally different natural prodiginine variants display differential bioactivities. In the herein described mutasynthesis approach, 13 different derivatives of prodigiosin were obtained utilizing the GRAS (generally recognized as safe) classified strain Pseudomonas putida KT2440. Genetic engineering of the prodigiosin pathway together with incorporation of synthetic intermediates thus resulted in the formation of a so far unprecedented structural diversity of new prodiginine derivatives in P. putida. Furthermore, the formed products allow reliable conclusions regarding the substrate specificity of PigC, the final condensing enzyme in the prodigiosin biosynthesis pathway of S. marcescens. The biological activity of prodigiosin toward modulation of autophagy was preserved in prodiginine derivatives. One prodiginine derivative displayed more potent autophagy inhibitory activity than the parent compound or the synthetic clinical candidate obatoclax.

Full text of DOI:10.1021/acssynbio.7b00099

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Article
New Prodigiosin Derivatives obtained by
Mutasynthesis in Pseudomonas putida
Andreas Sebastian Klein, Andreas Domröse, Patrick Bongen, Hannah U. C. Brass, Thomas Classen,
Anita Loeschcke, Thomas Drepper, Luca Laraia, Sonja Sievers, Karl-Erich Jaeger, and Jörg Pietruszka
ACS Synth. Biol., Just Accepted Manuscript • Publication Date (Web): 15 May 2017
Downloaded from http://pubs.acs.org on May 17, 2017
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New Prodigiosin Derivatives obtained by Mutasynthesis in Pseudomonas putida
Andreas S. Klein,a Andreas Domröse,b Patrick Bongen,a Hannah U. C. Brass,a Thomas Classen,c Anita
Loeschcke,b Thomas Drepper,b Luca Laraia,d Sonja Sievers,e Karl-Erich Jaegerb,c and Jörg Pietruszka*,a,c
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a Institute of Bioorganic Chemistry, Heinrich Heine University Düsseldorf located at
Forschungszentrum Jülich, Jülich (Germany)
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b Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf located at
Forschungszentrum Jülich, Jülich (Germany)
c Insitute of Bio- and Geosciences (IBG-1), Forschungszentrum Jülich, Jülich (Germany)
d Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Dortmund
(Germany)
e Compound Management and Screening Center (COMAS), Max Planck Institute of Molecular
Physiology, Dortmund (Germany)
Supporting information for this article is given via a link at the end of the document.
ABSTRACT
The deeply red-colored natural compound prodigiosin is a representative of the prodiginine alkaloid
family, which possesses bioactivities as antimicrobial, antitumor and antimalarial agents. Various
bacteria including the opportunistic human pathogen Serratia marcescens and different members of
the Streptomycetaceae and Pseudoalteromonadaceae produce prodiginines. In addition, these
microbes generally accumulate many structurally related alkaloids making efficient prodiginine
synthesis and purification difficult and expensive. Furthermore, it is known that structurally different
natural prodiginine variants display differential bioactivities. In the herein described mutasynthesis
approach, 13 different derivatives of prodigiosin were obtained utilizing the GRAS (generally
recognized as safe) classified strain Pseudomonas putida KT2440. Genetic engineering of the
prodigiosin pathway together with incorporation of synthetic intermediates thus resulted in the
formation of a so far unprecedented structural diversity of new prodiginine derivatives in P. putida.
Furthermore, the formed products allow reliable conclusions regarding the substrate specificity of
PigC, the final condensing enzyme in the prodigiosin biosynthesis pathway of S. marcescens. The
biological activity of prodigiosin towards modulation of autophagy was preserved in prodiginine
derivatives. One prodiginine derivative displayed more potent autophagy inhibitory activity than the
parent compound or the synthetic clinical candidate obatoclax.
Table of Contents Graphic
Keywords
Antibiotics, antitumor agents, metabolic engineering, mutasynthesis, natural product production,
autophagy activator, prodiginine
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INTRODUCTION
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Natural products are a precious source for the development of new drugs. Prodiginines are a class of
red-colored microbial pigments, which exhibit several important pharmacological properties
including antitumor, antimalarial and antimicrobial activities against pathogens such as
Salmonella typhi, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus.1-3
Furthermore, immunosuppressive and antifungal activities have been demonstrated for
prodiginines.4 Prodigiosin, as well as the synthetic derivative obatoclax mesylate (GX15-070), have
been shown to modulate autophagy.5 Autophagy is a cellular recycling process which is used by cells
to dispose of cellular toxic waste such as aggregated proteins or damaged organelles as well as to
survive during starvation periods.6 Modulation of autophagy is discussed as a promising therapeutic
approach for many diseases like neurodegenerative disorders, metabolic disease or cancer.
Autophagy is a highly dynamic process. The hallmark of autophagy is the generation of
autophagosomes, small vesicles that receive input from the endocytic pathway. In the last step of
autophagy, autophagosomes fuse with lysosomes thus enabling the degradation of cargo and
recycling of nutrients. Weakly basic amine-containing drugs like prodigiosin have been shown to be
sequestrated into lysosomes and induce their alkalization.7 Thereby, the compounds interfere with
autophagy by blocking the last step in the recycling process.
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In the main, prodiginines are naturally produced from amino acids and acetate building blocks by
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Serratia, Hahella, Vibrio, Pseudoalteromonas, and Streptomyces species.8, Contrary to the large
number of natural producers, there is only a minor chemical diversity in the presently discovered
prodiginines. They can be generally classified into two groups, namely compounds with n-alkyl
residues like prodigiosin (1a) or undecylprodigiosin (2) and cyclized derivatives like
cycloprodigiosin (3) and butyl-meta-cycloheptylprodigiosin (4) (Scheme 1).1 Total chemical syntheses
of various prodiginines and derivatives thereof have been reported, but they are typically associated
with high synthetic cost and low overall yields of multistep synthetic routes.10 Therefore, attention
has been drawn to exploitation of prodiginine biosynthesis. All prodiginines are biologically
synthesized in
a bifurcated pathway producing the bipyrrole 4-methoxy-2,2'-bipyrrole-5-
carbaldehyde (MBC, 5; A+B-ring) and a monopyrrole (e.g. 2-methyl-3-amylpyrrole, MAP, 6a; C-ring).8,
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These two intermediates are finally condensed to the tripyrrolic natural compound (compare
Scheme 1). Cyclic variants are then generated from alkyl residue prodiginines by a subsequent
oxidative cyclization catalyzed by non heme iron oxidases.12
Scheme 1. Examples of natural prodiginine derivatives biosynthetically produced in Serratia, Hahella, Vibrio,
Pseudoalteromonas and Streptomyces species.
The MBC biosynthesis is highly conserved in all producing species, whereas the pathways of the
monopyrrole intermediates are different in terms of both, substitution pattern and functional
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moiety. Hence, the monopyrroles are responsible for the natural diversity of prodiginines.
Accordingly, previous studies reported flexible substrate recognition of the condensing enzyme PigC
derived from the prodigiosin biosynthetic pathway within Serratia marcescens towards analogs of
MAP (6a) as well as MBC (5). For example, MAP could successfully be replaced by
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2,4-dimethylpyrrole (7) and 2,4-dimethyl-3-ethylpyrrole (8), whereas also derivatives of MBC altered
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in the aromatic A-ring were widely accepted by PigC
These results highlight the possibility of
using PigC for mutasynthesis approaches to gain access to new prodigiosin (1a) derivatives.
Therefore, we envisioned that a combination of chemical synthesis of MAP (6a) derivatives and their
subsequent biocatalytic conversion would constitute a highly appealing strategy to create diversity in
the C-ring of prodiginines and to determine the substrate specificity of PigC.
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Mutasynthesis comprises the feeding of a precursor analog to a mutant strain, which carries a block
in the biosynthesis towards an early key intermediate.15, 16 The artificial precursor (mutasynthon)
thus can be fed into the recombinant pathway thereby generating (non-natural) derivatives. The use
of a mutasynthesis approach, commonly in homologous hosts, has been successfully reported for
instance for the generation of ansamitocin, rapamycin, vancomycin, and platensimycin analogs.17-22
Previously, we have demonstrated an effective heterologous prodigiosin production in the GRAS-
certified strain P. putida KT2440 by expression of the S. marcescens pig gene cluster (P. putida pig-r2)
and the straightforward product recovery from culture broth using polyurethane (PU) foam cubes as
an adsorbent for prodiginines.23 Consequently, a mutasynthesis approach encompassing a MAP (6a)
deficient, but still MBC (5) producing mutant of this safe recombinant production strain presents a
fast and cost efficient alternative to chemical synthesis of prodigiosin (1a) derivatives. Furthermore,
the approach offers a potential model system to characterize the substrate spectra of condensing
enzyme PigC and the production of new and more complex prodiginines with altered bioactivities.
RESULTS AND DISCUSSION
Precursor acceptance of prodigiosin production strain
As a pretest for our mutasynthetic endeavors, the import into the cells and the acceptance of
precursors by PigC was first proven in a precursor-directed biosynthesis. Therefore, both, the natural
precursor MAP (6a) and derived pyrroles 6e as well as 6f were synthesized via a Trofimov reaction
(for details see Supporting Information S6) and fed to the prodigiosin (1a) producing strain P. putida
pig-r2.23-25 Supplementation of MAP (6a) in ethanol resulted in a 52% higher production of
prodigiosin, as measured by means of photometric absorption (Figure S2), whereas the MAP
derivatives lead to a mixture of the natural (1a) and unnatural product (1e or 1f), respectively (for
LC-MS traces see Figure S27 and S28). The results indicate that pyrrole precursors are imported and
the condensing enzyme PigC accepts minor changes of the precursor structure.
Construction of knockout strain and development of mutasynthesis
Next, we aimed at establishing an effective mutasynthesis setup. To this end, we constructed a
mutant strain (P. putida pig-r2 ΔpigD) blocked in the very first step of the monopyrrole biosynthesis
(Figure 1A and B). Therefore, the mutant strain is unable to produce the monopyrrole MAP (6a),
while the pathway of MBC (5) and the condensing enzyme PigC remain unaffected. In contrast to
P. putida pig-r2, which formed deeply red cultures, the phenotype of P. putida pig-r2 ΔpigD remained
colorless (Figure 1C) and and cells accumulated the intermediate MBC (5) as demonstrated by HPLC
analysis (Figure S1). The biosynthesis of prodigiosin (1a) could be restored by feeding of exogenous
MAP (6a) to this MAP-deficient mutant (Figure 1C). Using the natural precursor 6a, the optimal
reaction conditions for the incorporation of supplemented precursors into the prodiginine were
investigated concerning the co-solvent (DMSO) and the effective precursor concentration. A final
DMSO concentration of 2% (v/v), which proved to be nontoxic, and a precursor concentration of
1.25 mM (analytical scale) and 0.5 mM (preparative scale) were utilized for further mutasynthesis
approaches, respectively (Figure S29 and S30).
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Figure 1. Strategy for the mutasynthesis approach. A) Prodigiosin pig gene cluster of S. marcescens (blue: genes
for MBC (5) biosynthesis; green: genes for MAP (6a) biosynthesis; red: condensing enzyme). B) P. putida
pig-r2 ΔpigD strain is blocked in the very first step of the MAP (6a) biosynthesis by knockout of pigD. Different
monopyrrole derivatives (6a-u) were supplemented to this knockout strain. C) Phenotypes of the prodigiosin
(1a) producing strain P. putida pig-r2 (I) and the MAP (6a) deficient strain P. putida pig-r2 ΔpigD (II). The red
phenotype could be re-established by feeding of exogenous MAP (6a) (III). The produced prodigiosin (1a) is
adsorbed from culture broth by polyurethane (PU) foam cubes (IV).
In vivo exploration of substrate acceptance in mutasynthesis
The substrate acceptance range of P. putida pig-r2 ΔpigD and the condensing enzyme PigC was
exploited by mutasynthesis in vivo with different synthetic precursor derivatives (Table 1) (for
synthesis details see Supporting Information S6).
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Table 1. Mutasynthesis with the MAP (6a)-deficient mutant P. putida pig-r2 ΔpigD.
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entry substrate
R1
R2
product
1b
1c
1d
1e
1a
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
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methyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
H
H
methyl
n-propyl
n-butyl
n-pentyl
n-hexyl
n-octyl
n-decyl
n-dodecyl
ethyl
n-propyl
n-pentyl
n-hexyl
n-octyl
n-undecyl
n-pentyl
n-propyl
n-butyl
n-pentyl
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6l
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6o
6p
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6r
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H
H
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ethyl
n-butyl
n-pentyl
n-hexyl
methyl
methyl
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6t
6u
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2-propenyl 1t
4-pentenyl 1u
In the first series of mutasynthesis approaches, the influence of the alkyl chain length in 3-position of
the pyrrole was investigated by feeding monopyrroles 6a–i to the mutant strain
P. putida pig-r2 ΔpigD. By analogy to the natural precursor MAP (6a), the methyl group in 2-position
remained untouched. As shown in Figure 2, the mutasynthons could be incorporated by P. putida
and were widely accepted by PigC resulting in the formation of seven red-colored prodiginines (1a,
1c–h, for LC-MS traces see Supporting Information S11) including the natural prodigiosin. Among
these, prodiginines 1c and 1h, where monopyrrole moieties differed most from the natural precursor
MAP, could only be detected in traces by LC-MS and the extracts showed only minor coloration.
Clearly, the results demonstrate that the substrate spectrum of PigC in mutasynthesis is limited by
the alkyl chain length in 3-position of the monopyrrole precursor (R2 in Table 1); however, the
presence of an alkyl chain in this position is indispensable because addition of precursor 6b did not
result in prodiginine formation. Based on these results, a second series of mutasynthesis was
intended to investigate the importance of the methyl group in 2-position (R1 in Table 1) by using
demethylated precursors 6j–o. As shown in Figure 2, the acceptance of these monopyrroles by P.
putida and PigC decreased drastically in comparison to 6a–i. Prodiginine formation could only be
detected after supplementation with mutasynthons 6l–n (Figure 2, for LC-MS traces see Supporting
Information S11), which are closely related to the naturally occurring precursor MAP (6a) in terms of
2-position chain length. These results clearly indicate the importance of a methyl group present at
this position of the precursor. The combination of the results obtained from experimental series one
and two led us to devise series three altering both, the 2- and 3-position. Here, elongation of the
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essential methyl group, together with an alteration of the alkyl chain in 3-position, was investigated
by feeding pyrroles 6p–s.26 Remarkably, in contrast to the flexibility in 3-position, an elongation of
the alkyl chain length exceeding methyl in 2-position was almost impossible as only precursor 6p was
tolerated in mutasynthesis. The product of pyrrole 6q, prodiginine 1q, could again only be detected
in traces by LC-MS. Finally, series 4 of experiments was conducted to further enhance the
functionalization of prodiginines. Here, the terminal alkene-pyrroles 6t and 6u were fed as
mutasynthons as a proof of concept for functional diversification and could successfully be
incorporated in analogy to pyrroles 6d and 6a.
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Figure 2. Mutasynthesis in vivo and in vitro of prodignines using differently substituted monopyrrole
precursors. Ethanolic extracts are shown obtained from in vivo mutasynthesis experiments and in vitro
biotransformations catalyzed by isolated PigC with monopyrroles 6a-u. The structure of natural precursor MAP
(6a) is highlighted in red. Four experimental series were carried out using monopyrrole precursors with
different alkylation patterns in positions 2 and/or 3. [n.d.: not determined; *: quantification relies on predicted
extinction coefficient (see Methods)]
In vitro substrate acceptance of condensation enzyme PigC
The mutasynthesis data shown in Figure 2 indicate that the length of the alkyl chains of the
mutasynthons is important for their recognition by the condensing enzyme PigC. Additionally, we
decided to develop a cell-free assay with isolated PigC where direct substrate access is granted to
exclude putative physiological effects, e.g. caused by hampered cellular incorporation of particular
precursors, and to further verify the substrate spectrum of the condensing enzyme. To this end,
protein extracts were subjected to an appropriate in vitro assay using MBC with the substrates 6a-u.
As indicated by an overall matching pattern of in vivo and in vitro coloration (i.e. product formation)
in Figure 2, the substrate spectrum of PigC is in accordance with the spectrum observed in
mutasynthesis (for LC-MS traces see Supporting Information S13). Thus, the here observed
boundaries in prodigiosin diversification during mutasynthesis are not due to a limitation of substrate
access, but can be clearly correlated to the substrate acceptance of the condensing enzyme.
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Preparative scale production of prodiginines in mutasynthesis
The mutasynthetic strategy was proven successful for most fed precursors in analytical scale.
Furthermore, preparative scale (500 mL) reactions feeding the natural precursor MAP (6a) and the
structurally related alkene-pyrrole precursor 6u were performed to assess feasibility. In these
approaches, the previously reported product recovery with PU foam was applied. The respective
yields were 17.2 mgL-1 (53.2 µmol, 11%) of prodigiosin (1a) and 19.8 mgL-1 (61.6 µmol, 12%) of
prodiginine 1u. In comparison to these product titers, the strain P. putida pig-r2 is able to produce
48 mgL-1 (148.4 µmol) prodigiosin at the same cultivation conditions. The lower yields might be a
consequence of decomposition of the fed precursors in aerated aqueous medium or reduced
accumulation of the aldehyde intermediate MBC (5) in the knockout strain. In addition to these well-
accepted precursors, mutasynthons 6d and 6g were also applied in preparative scale reactions to
investigate the limitation in production regarding a shorter and a longer alkyl moiety in 3-position.
The respective yields were 10.5 mgL-1 (35.5 µmol, 7%) of prodiginine 1d and 3.8 mgL-1 (10.4 µmol,
2%) of prodiginine 1g. Nevertheless, in contrast to a total synthesis, the mutasynthesis is able to
provide an effective alternative method for the rapid production of prodiginines based on easily
synthesizable precursors without the need of expensive catalysts or starting materials which are
obligatory in the time consuming synthesis of MBC (5).
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Biological evaluation of prodiginines
Several bioactivities, including the modulation of autophagy, are known for prodigiosin (1a) as well as
obatoclax mesylate (GX15-070, 9, Figure 3D), a synthetic derivative investigated in several phase II
clinical trials.27-30 To investigate the influence of the alkyl chain length in 3-position (C-ring) towards
autophagy, the four prodiginines (1a, 1d, 1g and 1u), produced in the preparative scale reactions,
were tested for their ability to modulate eGFP-LC3 puncta, an autophagosomal marker. After 3 h
incubation of eGFP-LC3-MCF7 cells with prodiginines, a clear accumulation of eGFP positive
autophagosomes was detected (Figure 3A and B). Compound 1d showed the most potent
autophagosome accumulation with an EC50 of approximately 150 nM, which was about two-fold
more potent than prodigiosin and obatoclax mesylate (for EC50 values of 1a, 1d, 1g, 1u and obatoclax
mesylate see Table S3). On protein level, derivative 1d caused the accumulation of lipidated LC3
(LC3-II) in relation to the Actin control, as expected for a compound that inhibits the autophagosome
lysosome fusion (Figure 3C). This occurred to a much greater extent than with the known fusion
inhibitor chloroquine (CQ) as verified under both fed and starved conditions. Inhibition of autophagic
flux was also confirmed, as 1d inhibited autophagic degradation of protein p62, a known autophagy
substrate, after induction of autophagy through starvation.31 Compound 1d also caused growth
inhibition in a dose-dependent manner, which resulted in cell death through induction of apoptosis
(Figure 3E and F). Both were assessed using live-cell imaging of MCF7 cells incubated with a caspase
3/7 selective probe. It is interesting to note that while 1d inhibited autophagy more potently than
Obatoclax, the effect on the induction of apoptosis was less pronounced at equivalent
concentrations (Figure 3F, 0.11 µM). This is presumably because Obatoclax has been optimized for
inhibition of Bcl-2, an anti-apoptotic protein, and thus the pro-apoptotic activity is likely to have
multiple origins.
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Figure 3. Accumulation of autophagosomes in eGFP-LC3-MCF7 cells upon treatment with prodiginine 1d.
Whereas DMSO treated cells (A) show a diffuse eGFP-LC3 staining, (B) 0.3 µ
accumulation of autophagosomes (green: GFP fluorescence; blue: nuclei detection with Hoechst; scale
bar = 150 µ ). C) Treatment of eGFP-LC3-MCF7 cells with 1d for 3 h resulted in increased LC3-II levels and
M 1d treatment for 3 h leads to
M
decreased p62 degradation after starvation-induced autophagy. Using DMSO as a negative and obatoclax
mesylate (9, D, Obato.) as a positive control, the influence of treatment with different doses of compound 1d
on cell growth (measured as % confluence over 72 h) (E), and on occurrence of apoptosis (after 24 h of
treatment) (F) were determined by live-cell imaging and automated image analysis using the Incucyte Zoom.
Shown data represent mean values and respective standard deviations (n = 3).
In summary, we have successfully explored the potential of a mutasynthesis approach to create
diverse prodiginines. Beside the natural product prodigiosin (1a), four known and nine new
derivatives of this pharmacologically promising class of compounds could be obtained by feeding
pyrrole precursors to an accordingly engineered P. putida KT2440 mutant strain. By alteration of the
monopyrrole alkylation pattern, the precursor substrate spectrum of the condensing enzyme PigC
was characterized for the first time and was proven in an in vitro biotransformation in addition to the
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mutasynthesis. Although the here presented results indicate a restricted acceptance of pyrrole
precursors by PigC, this study offers a potential model system for the application and investigation of
other condensing enzymes in P. putida. The potential of the mutasynthesis approach was further
demonstrated by applying preparative scale reactions in combination with product recovery during
cell cultivation using a cost efficient PU foam-based procedure. This provided the possibility to access
four prodiginines for testing their efficacy against autophagy in eGFP-LC3-MCF7 cells. All prodiginines
caused accumulation of autophagosomes. Prodiginine 1d was the most potent of the series and
inhibited autophagic flux as well as cell growth, leading to cell death via apoptosis. Indicating
promising perspectives, the acceptance of alkene-pyrrole precursors establishes the access to
prodiginine building blocks for semisynthesis purposes and therefore offers high potential for the
production of new biologically active compounds.
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METHODS
Oligonucleotides used in this study are listed in Table S1.
Bacterial strains and culture conditions: Escherichia coli strains DH5α, S17-1 and BL21(DE3) (applied
for cloning, conjugation and gene expression) were cultivated on LB-agar plates or under constant
shaking (120 rpm) at 25–37 °C in LB (lysogeny broth) liquid medium (LB medium (Luria/Miller), Carl
Roth®, Karlsruhe, Germany: 10 g/L tryptone, 5 g/L yeast extract, 10 g/L sodium chloride) or TB
(terrific broth) liquid medium (Carl Roth®, Karlsruhe, Germany: 12 g/L casein, 24 g/L yeast extract,
12.54 g/L K2HPO4, 2,31 g/L KH2PO4, 4 mL/L glycerol).32-34 Standard conditions for Pseudomonas putida
prodigiosin production strains (P. putida pig-r2) and P. putida pig-r2 ΔpigD mutant strains were
cultivation on LB-agar plates or in liquid LB or TB medium under constant shaking (120 rpm) at 28 °C.
Cultivation media were prepared using distilled water and sterilized by autoclaving. Antibiotics were
added to the culture medium to the following final concentrations [µg/mL]: E. coli: 50 (kanamycin,
Kan); P. putida: 25 (gentamicin, Gm) 80 (streptomycin, Sm).
DNA manipulation equipment: Enzymes for DNA manipulation and PCR-reagents were obtained
from ThermoFisher Scientific, Walkham, USA, and oligonucleotides from Sigma-Aldrich, Steinheim,
Germany, and Eurofins Genomics GmbH, Ebersberg, Germany. PCR reactions were performed in
100 μL thin-wall tubes using a TProfessional Basic thermocycler (Biometra, Göttingen, Germany).
DNA amounts in plasmid samples were determined via a NanoDrop 2000c (Thermo Fischer Scientific)
spectrophotometer. Commercially available kits were applied for DNA isolation and purification. DNA
sequencing was conducted at GATC Biotech.
Construction of P. putida pig-r2 ΔpigD mutant strain
The MAP biosynthesis deficient pigD deletion mutant strain was constructed based on the
constitutive prodigiosin production strain P. putida pig-r2 described in Domröse, Klein et al.23 A part
of pigD was exchanged for the streptomycin resistance gene aadA via homologous recombination
using the vector pSUP202-pigCaadApigD.
The vector was generated in two steps: First, a 4.6 kb Eco72I fragment of vector pUC19-pig
containing parts of pigC and pigD was ligated with ScaI hydrolyzed vector pSUP202 (6.5 kb), resulting
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in pSUP202-pigCD (11.1 kb).23, In the second step, a partial deletion in pigD was realized by
MauBI/AsiSI restriction hydrolysis, removing a 294 bp part of pigD, and subsequent ligation with a
0.9 kb MauBI/AsiSI fragment of the promoterless aadA gene. The aadA gene was amplified by PCR
from pHP45omega vector as DNA template using oligonucleotide primers #3 and #4. The PCR
reaction contained 1 U Phusion® High-Fidelity DNA Polymerase, 100 ng plasmid, 200 μ
M of each
dNTP and 200 n forward and reverse primer in 50 µL High-Fidelity buffer. The applied temperature
M
protocol was 30 s 98 °C, 35 cycles of 10 s 98 °C and 20 s 72 °C, a final step of 10 min at 72 °C and
cooling to 10 °C. Correct vector assembly was verified by sequencing using primers #1, #2, #5 and #6.
The resulting mutagenesis vector pSUP202-pigCaadApigD was transformed into E. coli S17-1 and
further transferred to P. putida pig-r2 via conjugation. The pigC-aadA-pigE cassette was designed to
incorporate the promoterless aadA gene in the same orientation as pig genes instead of pigD in the
pig gene cluster. As in P. putida pig-r2 expression of pig genes is constitutively driven by an intrinsic
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promoter,23 integration of the aadA gene at this locus should drive its expression likewise and confer
corresponding resistance to streptomycin. Since the pSUP202 derivative represents a suicide vector
in P. putida, clones with integrated aadA gene could be directly selected on LBSm agar plates.
Potential streptomycin resistant P. putida pig-r2 ΔpigD mutant strains were first identified by lack of
the red phenotype of pig-r2 due to disruption of prodigiosin biosynthesis, and secondly confirmed by
colony PCR using different combinations of oligonucleotides. Primers #7 and #8 are located in the
aadA gene while #9 binds upstream in pigC and #10 downstream in pigE, both outside the
homologous regions. Thus PCR products of the pairs #7/#9 (1.4 kb) and #8/#10 (3 kb) confirm both
the presence of aadA and its correct orientation.
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Mutasynthesis experiments
Analytical scale: Reactions were performed in 96 x 2000 μL deep-well-plates (conical bottom, solvent
resistant) using a final volume of 750 µL containing TB liquid medium, 1.25 mM pyrrole precursor and
2% (v/v) DMSO. In detail: The TBSm liquid medium was inoculated with an overnight culture of
P. putida pig-r2 ΔpigD to an OD of 0.02. The pyrrole precursor was initially dissolved in DMSO and
added to the culture medium to achieve the final concentration. The plates were sealed with
AeraSeal™ breathable sealing films (Excel Scientific, Victorville, USA). The mutasynthesis proceeded
at 25 °C and 1000 rpm for 24 h. The cells containing the prodiginines were harvested by
centrifugation, the supernatant discarded and the cells extracted with 300 µL of acidified ethanol
(4% (v/v) 1N HCl in ethanol). The extraction was supported using an ultrasonic bath for 5 min.
Afterwards the cell debris was removed by centrifugation and extracts of three samples were
combined. For further LC-MS analysis, ethanol was removed under reduced pressure, 200 µL water
was added and the prodiginines extracted with dichloromethane (2 x 200 µL). Evaporation of
dichloromethane and addition of methanol provided the desired crude extracts for LC-MS analysis.
Preparative scale: The reactions were performed either in a baffled 100 mL flask or 3 L Fernbach
flask using a final volume of 20 mL or 500 mL TB liquid medium, 1.25 mM (for 20 mL scale) or 0.5 mM
(for 500 mL scale) pyrrole precursor and 2% (v/v) DMSO. Polyurethane (PU) foam cubes
(Bornewasser, Göllheim, Germany: Softpur, 25 kg/m3density, 4 kPa compression hardness), each
approximately 1 cm3, were added as an adsorbent for the in situ recovery of prodiginines (275 mg or
5 g).23 In detail: The TBSm liquid medium was inoculated with an overnight culture of P. putida
pig-r2 ΔpigD to an OD of 0.02. The pyrrole precursor was initially dissolved in DMSO and added to the
culture medium to achieve the final concentration. After the addition of the PU foam cubes, the tops
of the shaking flasks were covered with aluminum foil and the mutasynthesis proceeded at 25 °C and
125 rpm for 24 h. The PU foam cubes of the 20 mL scale were wrung out and extracted with diethyl
ether (10 mL) by washing in a beaker. The extraction of prodiginines from the PU foam cubes of the
500 mL cultures was performed with a Soxhlet extractor: The foam cubes were recovered from the
cell culture by sieving, wrung out and charged into a 250 mL Soxhlet apparatus fitted with a reflux
condenser and a 500 mL round bottom flask filled with 300 mL of diethyl ether. The sample was
continuously extracted by heating with the chosen solvent. After extraction, the organic layer was
washed with water, dried over MgSO4 and the solvent was removed under reduced pressure
providing the desired crude extracts for analysis.
Plasmid construction for pigC expression
The pigC gene was PCR amplified from pUC19-pig vector as DNA template using Gibson Assembly®
primers #11 and #12. The PCR reaction contained 0.4 µL Phire Hot Start II DNA polymerase, ≈ 30 ng
plasmid, 200 μM of each dNTP and 500 nM forward and reverse primer, 0.6 µL DMSO in 20 μL buffer.
The applied temperature protocol was 30 sec at 98 °C, 35 cycles of 10 sec at 98 °C, 30 sec at 69.5 °C
and 1 min at 72 °C, a final step of 10 min at 72 °C and cooling to 10 °C. Amplification was confirmed
by agarose gel electrophoresis. The final vector was assembled via the Gibson Assembly® protocol by
using the PCR product and a previously linearized pET28a(+) vector (digested with NdeI and XhoI).36
The obtained vector is referred to as pPIGC. The vector was applied for transformation of E. coli
DH5α cells by standard heat-shock procedure using chemically-competent E. coli DH5α cells.37
Colonies of transformants grew overnight on LBKan agar-plates at 37 °C. The presence of the desired
vector was tested by colony PCR and agarose gel electrophoresis which enabled clone selection for
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cultivation in LBKan medium and plasmid isolation. The same conditions as mentioned before were
applied for the pigC gene amplification in the colony PCR.
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Expression of the pigC gene
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For recombinant, heterologous expression of pigC, chemically competent cells of E. coli BL21(DE3)
were transformed with the pPIGC vector and selected on LBKan agar-plates. In a baffled 3 L Fernbach
flask, 500 mL TBKan medium was inoculated with 1% (v/v) of an overnight culture. The culture was
incubated to an OD600 of about 0.6 at 37 °C (120 rpm), induced with 0.1 m
M isopropyl β-D-1-
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thiogalactopyranoside (IPTG), and incubated for additional 18 h at 25 °C (120 rpm). Cells were
harvested by centrifugation at 4 °C and stored at -20 °C.
PigC in vitro assay
Frozen cells (1 g) were thawed by suspension in potassium phosphate buffer (KPi buffer, 50 mM,
pH 7.0) and disrupted using a SONOPULS Ultrasonic homogenizer (Bandelin, Berlin, Germany) for
2 times 5 min and 5 cycles. The cell debris was removed by centrifugation. The assay solution
contained 440 µL cell-free supernatant, 25 µL of a pyrrole-precursor (6a-u) solution in DMSO
(20 m
M
), 25 µL of MBC-precursor (5) solution in DMSO (20 m
M) and 10 µL of an ATP∙Na2 solution in
water (62.5 m
M). The mixture was shaken in a 1.5 mL reaction tube at 300 rpm and 30 °C for 4 h.
Afterwards, the supernatant was discarded after centrifugation and the produced prodiginine
extracted by the addition of 300 µL of ethanol. Ethanolic extracts were separated by centrifugation,
transferred to a new reaction tube and the solvent was removed under reduced pressure. The
production of prodiginines was analyzed by LC-MS. To exclude a self-condensation of MAP (6a) and
MBC (5), the assay was carried out without the addition of cell-free supernatant. Furthermore, a
second negative control was performed without the addition of ATP.
Quantification of prodiginines
Prodiginines were quantified either by absorption in acidified ethanol (4% v/v of 1 N HCl) or by mass
after column chromatography. For prodigiosin (1a), the previously reported molar extinction
coefficient (ε535 = 139,800 ± 5,100 M
-1cm-1) was used.23 The molar extinction coefficients of
prodiginines 1d and 1g were determined from the slope by plotting the absorption of the pigment
versus the concentration at the absorption maximum (535 nm) in acidified ethanol (4% v/v of
1 N HCl): ε535(1d) = 125,776 ± 6,450
M
-1cm-1, ε535(1g) = 189,904 ± 2,950
M
-1cm-1
.
The extinction
coefficients of other 2-methyl-3-alkyl-prodiginines were predicted by a linear fit after plotting the
above mentioned extinction coefficients versus the chain length in 3-position (for details see
Supporting Information S14).
Evaluation of biological activity of prodiginines
Stable (eGFP-LC3)-MCF7 breast cancer cells were kindly provided by Georgios Konstantinidis. Cells
were cultured in MEM containing 10% fetal bovine serum, 1% L-glutamine, 1% sodium pyruvate, 1%
non-essential amino acids, 0.01 mg/ml human insulin and 0.2 mg/ml G418. (eGFP-LC3)-MCF7 cells
(4,000 cells/well) were seeded in 384 well plates (Greiner Bio-One, Kremsmünster, Austria). The next
day, compounds were added using Echo dispenser (Labcyte, Sunnyvale, USA). After three hours
incubation at 37 °C and 5% CO2, cells were fixed by addition of 25 µL formaldehyde in phosphate
buffered saline (PBS; 4.6% final concentration) and simultaneous nuclear staining was performed
with 1:500 Hoechst (Stock 1 mg/mL) for 20 min at RT. Fixed cells were washed three times with
1x PBS using plate washer ELX405 (BioTek, Winooski, USA). For visualization, 4 pictures/well were
acquired using ImageXpress Micro XL (Molecular Devices, Sunnyvale, USA) at 20x magnification and
analyzed with the granularity algorithm of MetaXpress Software (Molecular Devices). Dose-response
analysis was carried out starting from 10 µM using a three-fold dilution curve over eight steps. EC50
calculations were performed using Quattro Workflow software (quattro research GmbH, Planegg,
Germany).
Antibodies
Anti-p62/SQSTM1 was purchased from MBL international (Cat# PM045, Woburn, USA) and used at
1:10,000. Anti-LC3B was obtained from Cell Signaling Technology (Cat# 2775, Cambridge, UK) and
used at 1:1,000. Anti-beta-actin was purchased from abcam (Cat# ab8227, Cambridge, UK) and used
at 1:10,000. Goat anti-rabbit-HRP was purchased from Pierce (ThermoFisher Scientific, Walkham,
USA, cat# 31460) and used at a dilution of 1:10,000.
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Western blotting
200,000 (eGFP-LC3)-MCF7 cells in 2 mL media were seeded in 6-well plates and incubated (37 °C,
5% CO2) overnight. The media was removed and the cells were washed with 1x PBS, before adding
test compounds at the required concentrations in EBSS (starvation medium) or MEM (full medium).
Cells were incubated (37 °C, 5% CO2) for 3 h before removing the media, washing with 1x PBS, and
lysing in SDS loading buffer without bromophenol blue. Protein concentrations were determined
using the DC Assay (Bio-Rad Laboratories, Hercules, USA) according to the manufacturer’s
instructions. SDS-PAGE was carried out using 15% polyacrylamide gels run at a constant voltage of
80 V for 15 min followed by 120 V for approximately 1.5 h. Semi-dry transfer onto a polyvinylidene
difluoride (PVDF) membrane was performed at 25 V for 50 min. Membranes were blocked in 5% milk
in Tris-buffered saline with 0.1% Tween20 (TBST) for 1 h at room temperature. The membrane was
incubated with the primary antibody in blocking buffer overnight at 4 °C. After washing with TBST
(3 x 5 min), the membrane was incubated with the secondary antibody in blocking buffer for one
hour at room temperature. Signals were visualized using the SuperSignal West Pico
Chemiluminescent Substrate or the SuperSignal West Femto Maximum Sensitivity Substrate
(ThermoFisher Scientific, Walkham, USA) on a Li-COR Odyssey Fc.
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Confluence and apoptosis measurement using IncuCyte™ Zoom
4,000 (eGFP-LC3)-MCF7 cells in 100 µL medium were seeded in a clear flat-bottomed 96-well plate
and incubated overnight. The media was removed gently and replaced with 100 µL of MEM
containing 5 µM IncuCyte™ Kinetic Caspase-3/7 Apoptosis Assay Reagent (Cat#4440). To this were
added test compounds at a concentration of 6x the desired final concentration in 20 µL MEM. Cells
were incubated at 37 °C for 72 h in the Incucyte Zoom. Images were acquired at 10x magnification in
the phase and green channel every 2 h. Images were analysed using automated image analysis,
where the key parameters were confluence (%) and Caspase 3/7 positive cells (cells/mm2).
ASSOCIATED CONTENT
Supporting Information
Supplementary methods describe the chemical synthesis and characterization of molecules used in
this study. Supporting Information contains additional tables and figures based on the descriptions
provided in the text. This material is available free of charge on the ACS publication website
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-Mail: j.pietruszka@fz-juelich.de
Website: http://www.iboc.uni-duesseldorf.de
Author Contributions
J.P. and K.-E.J. developed the idea for the mutasynthesis approach. A.S.K., A.D., T.C., T.D., and A.L.
designed the experiments. A.S.K. performed the chemical syntheses, the mutasynthesis (analytical
scale), the in vitro biotransformation and analytics. A.S.K. and H.U.C.B. performed the mutasynthesis
(preparative scale). A.D. and A.L. performed the construction of the knockout strain. P.B. performed
the LC-MS analysis. L.L. and S.S. performed the physiological experiments and analysis. A.S.K., A.L.,
A.D., T.C., P.B., T.D., S.S., K.-E.J. and J.P. wrote the manuscript.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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We thank the Ministry of Innovation, Science and Research of the German federal state of North
Rhine-Westphalia and the Heinrich Heine University Düsseldorf (scholarship within the CLIB Graduate
Cluster Industrial Biotechnology for A.S.K. and A.D.). Scientific activities within the Bioeconomy
Science Center were supported by the Ministry of Innovation, Science and Research within the
framework of the NRW-Strategieprojekt BioSC (No. 313/323-400-002 13). The authors wish to thank
David Klebl, M.Sc. for his support in determining the extinction coefficients of prodiginines.
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ABBREVIATIONS
EBSS, Earle´s balanced salt solution (starvation medium); MAP, 2-methyl-3-amylpyrrole; MBC,
4-methoxy-2,2'-bipyrrole-5-carbaldehyde; MEM, minimum essential medium (full medium); PBS,
phosphate buffered saline; PVDF, polyvinylidene difluoride; PU, polyurethane
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sigmatropic rearrangement does not show any regioselectivity using 5-undecanonoxime.
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TOC-Graphic
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