Mutasynthesis of Physostigmines in Myxococcus xanthus

Article abstract of DOI:10.1021/acs.orglett.1c02374

The alkaloid physostigmine is an approved anticholinergic drug and an important lead structure for the development of novel therapeutics. Using a complementary approach that merged chemical synthesis with pathway refactoring, we produced a series of physostigmine analogues with altered specificity and toxicity profiles in the heterologous host Myxococcus xanthus. The compounds that were generated by applying a simple feeding strategy include the promising drug candidate phenserine, which was previously accessible only by total synthesis.

Full text of DOI:10.1021/acs.orglett.1c02374

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Mutasynthesis of Physostigmines in Myxococcus xanthus
Lea Winand, Pascal Schneider, Sebastian Kruth, Nico-Joel Greven, Wolf Hiller, Marcel Kaiser,
Jörg Pietruszka, and Markus Nett*
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ABSTRACT: The alkaloid physostigmine is an approved anti-
cholinergic drug and an important lead structure for the
development of novel therapeutics. Using a complementary
approach that merged chemical synthesis with pathway refactoring,
we produced a series of physostigmine analogues with altered
specificity and toxicity profiles in the heterologous host Myxococcus
xanthus. The compounds that were generated by applying a simple
feeding strategy include the promising drug candidate phenserine,
which was previously accessible only by total synthesis.
hysostigmine is a potent parasympathomimetic alkaloid
the desired compounds by fermentation. For this, a
Pthat is medically used for the treatment of glaucoma and mutasynthetic strategy was pursued. Mutasynthesis involves
cholinergic poisoning.1 The narrow therapeutic window of this
the genetic disruption of the targeted biosynthesis at an early
step. Afterward, the disrupted pathway can be reactivated by
feeding synthetic analogues of the missing intermediate.7 In
the present case, we decided to use serotonin (1) as an entry
point to reroute the biosynthesis, as derivatives of this achiral
intermediate are easily synthetically prepared.8 However,
because a genome sequence of the native physostigmine
producer, Streptomyces griseofuscus NRRL 5324, has not been
published, it remained unclear whether the serotonin-forming
decarboxylase PsmH could be complemented by a house-
keeping enzyme upon its genetic inactivation. To avoid this
scenario, we decided to reconstitute the subsequent bio-
synthetic reactions in a heterologous host, which is not capable
of producing 1. We selected the myxobacterium Myxococcus
xanthus as a production chassis due to its abundant cellular
pool of S-adenosyl methionine (SAM),9 which was expected to
promote the biosynthesis.
After the required biosynthesis genes psmA-F had been
obtained through gene synthesis, they were cloned into a
plasmid derived from the E. coli-Myxococcus shuttle vector
pZJY156.10,11 For expression, the genes were placed under the
control of a σ54-dependent, constitutive myxobacterial
promoter.12 In comparison with σ70, σ54 allows a very tight
control of gene transcription because the corresponding RNA
polymerase-promoter DNA complex is not prone to
spontaneous isomerization to form the transcriptionally
drug as well as its short duration of action have stimulated the
search for derivatives with better toxicity and pharmacokinetic
profiles that could be administered systemically, for example,
for the treatment of Alzheimer’s dementia (AD) or other
diseases.2 Therefore, the generation of physostigmine ana-
logues has attracted considerable attention over the years.2
From a chemical perspective, the asymmetric synthesis of the
pyrroloindoline moiety of physostigmine is of particular
interest, and it has been achieved, among others, from various
oxindole derivatives featuring a C3-quaternary stereogenic
center.3 The preparation of these chiral oxindole precursors
typically involves multiple steps and requires precious metal
catalysts or toxic reagents.4 Nature generates the pyrroloindo-
line scaffold in a much more sustainable way, although the
assembly strategy is remarkably similar to chemical synthesis.
The biosynthesis of physostigmine starts from L-tryptophan,
which is initially oxidized by a housekeeping enzyme to 5-
hydroxy-L-tryptophan and then further processed by the
pathway-specific decarboxylase PsmH to give the intermediate
serotonin (1).5 A series of concerted enzymatic reactions, of
which many are catalyzed by transferases, ultimately give rise
to physostigmine (7) (Figure 1).5 Particularly noteworthy in
this context is the PsmD-mediated stereospecific methylation
of the indole backbone at C3. This enzymatic conversion is
assumed to lead to a highly reactive iminium species, which is
prone to an intramolecular nucleophilic attack by a side-chain
amine, thereby forming the pyrroloindoline skeleton.5,6
Received: July 16, 2021
The total synthesis of natural product derivatives is, in
general, laborious due to the repeated use of identical linear
transformations and the need to individually optimize reaction
conditions. To produce physostigmine analogues, we thus
planned to redirect the biosynthetic pathway and to generate
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Figure 1. Proposed biosynthesis of physostigmine (7) from serotonin (1).5
competent open complex.13 After the transformation of the
M. xanthus type strain FB with the generated expression
plasmid pMEX04, the bacterium was grown in a medium
supplemented with 1. The analysis of this culture by LC-MS
did not provide any evidence of the production of
physostigmine (7), whereas the mass of the fed substrate
was found in high intensity. To test whether this failure was
due to cellular uptake issues of 1, we repeated the feeding
study with the synthetically prepared intermediates N-
acetylserotonin (2) and 5-O-methylcarbamoyl-N-acetylseroto-
nin (3). Although both 2 and 3 are more lipophilic than 1,
evidence of a biosynthetic conversion was obtained only with 3
(Figure 2).
Despite the successful reconstitution of physostigmine
biosynthesis in M. xanthus, only a very low titer had been
achieved (<1 mg/L; Figure 2B). The expression plasmid was
redesigned to improve the productivity. Because our analyses
had corroborated the necessity to feed the carbamoylated
intermediate 3, psmA, psmE, and psmF became dispensable.
The removal of these genes reduced the size of the expression
plasmid and thereby also its metabolic burden. The remaining
biosynthesis genes were fused to the first 45 bp of the
endogenous pilA gene to improve their expression.14 (See the
Supporting Information.) Furthermore, they were assembled in
a multimonocistronic fashion, in which every gene was placed
under the control of a discrete promoter. Using the modified
expression plasmid, pMEX10, the titer of 7 increased to 4.7
mg/L after the feeding of 3 (Figure 2C). This value could be
further raised when M. xanthus NM was used as the host. The
latter strain is closely related to the original host M. xanthus FB
but lacks swarming capabilities due to mutations in its motility
systems.15 In M. xanthus NM:pMEX10, the average production
titer of 7 was determined to be 28.1 mg/L (Figure 2D).
Because the substrate 3 had been completely consumed in the
initial feeding study of M. xanthus NM:pMEX10, we assumed
that even higher titers of 7 would be possible, contingent upon
the availability of a sufficient precursor. Effectively, a maximal
physostigmine titer of 72.0 mg/L could be achieved by
increasing the concentration of 3 in the fermentation broth
from 40 to 200 mg/L.
Figure 2. (A) Biosynthesis of physostigmine (7: R1 = OCONHCH3,
R2, R3 = CH3) or physostigmine analogues in the generated
M. xanthus strains, depending on the feeding of synthetically prepared
precursor molecules (marked in red, e.g., 3: R1 = OCONHCH3, R =
CH3). (B−D) Total ion chromatograms of raw extracts from
M. xanthus FB:pMEX04 (B), M. xanthus FB:pMEX10 (C), and
M. xanthus NM:pMEX10 (D) after the feeding of 3. Red: Extracted
ion chromatogram (EIC) of 3 (m/z 276.13). Green: EIC of
physostigmine (7) (m/z 276.17). The identities of 3 and 7 were
confirmed by comparison with external standards.
action and the toxicity profile of this cholinesterase inhibitor.16
The synthetic substrate 5-O-ethylcarbamoyl-N-acetylserotonin
(3a), which is closely related to 3, was readily converted by the
reconstituted biosynthetic machinery and gave the derivative
7a with a good titer (9.5 mg/L). Moreover, we isolated the
analogue 8-desmethyl-ethyl-physostigmine (8a) with a titer of
19.0 mg/L. We assume that 8a is a biosynthetic precursor of 7a
and that the sequence of biosynthetic reactions in M. xanthus
deviates from the original proposal displayed in Figure 1.5
However, the possibility that 7a is metabolized to 8a in the
heterologous host cannot be completely ruled out, even though
a feeding experiment with 7a did not support this scenario.
With an M. xanthus strain producing sufficient amounts of 7
for further functionalization at our disposal, the structural
boundaries for the in vivo production of analogues could be
explored. We first probed the replacement of the methyl-
carbamoyl moiety in 7 by more lipophilic carbamoyl residues,
which is a proven strategy to improve both the duration of
B
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Table 1. IC50 Values of Physostigmines for the Inhibitory Effects on AChE, BChE, and L6 Cells
a
b
Selectivity was calculated from the ratio AChE IC50/BChE IC50. Not determined.
Next, we evaluated the production of the phenylcarbamate
According to high-performance liquid chromatography
(HPLC) analysis, the lipophilicity increases from N-acetyl-5-
fluorotryptamine via N-acetyl-5-chlorotryptamine to N-acetyl-
5-bromotryptamine.
derivative of physostigmine, which is known under the name
phenserine (7b) and represents a promising drug candidate for
the treatment of AD.16b,17 Upon the feeding of 5-O-phenyl-
carbamoyl-N-acetylserotonin (3b) to a culture of M. xanthus
NM:pMEX10, a new metabolite with a mass corresponding to
7b was detected. The identity of this compound was verified
by chromatography with a commercial standard of 7b and LC-
MS/MS analysis (Figure S1). Although the titer of the
fermentation-derived 7b (<1 mg/L) was low in comparison
with 7 or 7a, this result was still very encouraging regarding the
plasticity of the physostigmine pathway.
In the following studies, we thus attempted to entirely
replace the carbamate moiety of 7 with halogen atoms. The
substitution of this pharmacophore seemed worthwhile despite
the anticipated loss of anticholinergic properties because it
would provide analogues that would be potentially useful as
analgesics or antiplasmodial agents.18 Furthermore, the
introduction of chlorine or bromine atoms into natural
products facilitates subsequent chemical derivatizations, such
as Pd-catalyzed Suzuki−Miyaura and related cross-coupling
reactions.19 For the halogen incorporation, M. xanthus
NM:pMEX10 was fed with N-acetyltryptamines featuring a
fluoro, chloro, or bromo substituent at C5. To our delight, all
three tested substrates were successfully converted into
correspondingly halogenated physostigmine analogues. The
titer of the fluorinated analogue 7c (2 mg/L) was lower than
the chlorinated 7d (4.9 mg/L) or brominated physostigmine
7e (5.6 mg/L). This might indicate a preference for at least
one Psm enzyme for large substituents at C5 but could also be
due to different cellular uptake rates of the substrates.
Except for C5, the methyl group at N1 of 7 was identified as
another possible target for mutasynthetic modifications. The
N-butanoylated analogue of 5-O-methylcarbamoyl-serotonin
(3f), which had been added to cultures of M. xanthus
NM:pMEX10, was converted to the pyrroloindoline 7f (4.7
mg/L). An inspection of the ion chromatogram from the
corresponding culture extract suggested the presence of a
putatively methylated derivative of 7f (m/z 332.19; Figure
S40), but the identity of this compound could not be clarified
due to its low titer. Because no further physostigmine-related
compounds were observed after the feeding of 3f, we assume
that the PsmD product 7f is not efficiently processed by PsmB
or PsmC.
The produced derivatives 7a, 8a, and 7c−7f were tested
together with 7 for their inhibitory activities against
acetylcholinesterase (AChE) and butyrylcholinesterase
(BChE). Although AChE is the major enzyme for regulating
the acetylcholine level in healthy humans, BChE can
compensate its function.20 In patients suffering from AD, the
activity ratio of both enzymes is strongly shifted to BChE,
which is why the latter represents an important pharmaco-
logical target in the treatment of this disease.21 Many AD drugs
actually show a preference for BChE over AChE inhibition.
This is illustrated by the approved drug rivastigmine,22 which
served as a positive control in our study (Table 1).
A comparison of rivastigmine with physostigmine (7), 7a,
8a, and 7f revealed that the physostigmines are more potent
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inhibitors of AChE and BChE, as documented by their lower
IC50 values. On the contrary, the tested physostigmines were
less selective regarding the inhibition of the two cholines-
terases. Whereas the natural product 7 did not discriminate
between AChE and BChE, the selectivity ratio could be
improved in the mutasynthetic analogues 7a, 8a, and 7f. In the
case of 7a and 8a, the gain of selectivity was at the expense of
the activity. In stark contrast, the new derivative 7f not only
showed a significantly improved selectivity ratio but also
improved bioactivity. Notably, this compound also exhibited
reduced toxicity against the myoblast cell line L6 in
comparison with 7.
The halogenated physostigmine analogues 7c−7e, which
lack a carbamoyl moiety, were found to be inactive toward
AChE and BChE, as expected. Because previous studies had
suggested that 7 and its degradation products eseroline and
rubreserine might inhibit Plasmodium falciparum,23 we also
investigated the antiprotozoal activities of the generated
compounds (Table S1). Although the halogenated derivatives
showed only moderate activity in the respective assays, it
became obvious, once more, that the activity profile of 7 can be
tailored by mutasynthetic modification.
In summary, we refactored and reconstituted the late steps
of the physostigmine pathway in the host bacterium
Myxococcus xanthus. This approach, in combination with the
feeding of precursor analogues, enabled the mutasynthetic
production of various physostigmine derivatives, including the
drug candidate phenserine which, until then, had been
accessible only by total synthesis. Of particular note is the
generation of a previously not described physostigmine
analogue featuring a butanoyl moiety at N1. The correspond-
ing compound possesses an improved selectivity and toxicity
profile in comparison with the parental natural product, which
underlines the usefulness of mutasynthesis in drug develop-
ment.
Nico-Joel Greven − Department of Biochemical and Chemical
Engineering, TU Dortmund University, Dortmund 44227
Nordrhein-Westfalen, Germany
Wolf Hiller − Department of Chemistry and Chemical Biology,
TU Dortmund University, Dortmund 44227 Nordrhein-
Westfalen, Germany
Marcel Kaiser − Parasite Chemotherapy Unit, Swiss Tropical
and Public Health Institute, 4002 Basel, Switzerland;
University of Basel, 4001 Basel, Switzerland
Jörg Pietruszka − Institute of Bioorganic Chemistry, Heinrich-
Heine-University Du
Ju
Bio- und Geowissenschaften: Biotechnologie (IBG-1),
Forschungszentrum Julich, Julich 52428 Nordrhein-
sseldorf at Forschungszentrum Julich,
̈ ̈
̈
lich 44227 Nordrhein-Westfalen, Germany; Institut fur
̈
̈
̈
Westfalen, Germany; orcid.org/0000-0002-9819-889X
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.1c02374
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support from the European Regional Development
Fund (ERDF: grants EFRE-0300096, EFRE-0300097, and
EFRE-0300098 to J.P. and M.N.) is gratefully acknowledged.
We thank Dr. Benoit David and Prof. Holger Gohlke from the
Heinrich-Heine-University Dusseldorf for valuable discussions.
̈
Special thanks go to Angela Sester for her helpful advice and to
Chantale Zammarelli for her support with the LC-MS analyses
(both TU Dortmund University). We thank Tanja Becker (TU
Dortmund University) for her assistance in the laboratory.
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AUTHOR INFORMATION
Corresponding Author
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Lea Winand − Department of Biochemical and Chemical
Engineering, TU Dortmund University, Dortmund 44227
Nordrhein-Westfalen, Germany
Pascal Schneider − Institute of Bioorganic Chemistry,
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