Mutational biosynthesis to generate novel analogs of nosiheptide featuring a fluorinated indolic acid moiety

Article abstract of DOI:10.1039/d0ob00084a

Nosiheptide (NOS) is a member of bicyclic thiopeptides possessing a biologically important indolic acid (IA) moiety appended onto the family-characteristic core system. The IA formation relies primarily on NosL, a radical S-adenosylmethionine (SAM) protein that catalyzes a complex rearrangement of the carbon side chain ofl-tryptophan, leading to the generation of 3-methyl-2-indolic acid (MIA). Here, we establish an efficient mutational biosynthesis strategy for the structural expansion of the side-ring system of NOS. ThenosL-deficient mutantStreptomyces actuosusSL4005 complemented by chemically feeding 6-fluoro-MIA is capable of accumulating two new products. The target product 6′-fluoro-NOS contains an additional fluorine atom at C6 of the IA moiety, in contrast with an unexpected product 6′-fluoro-NOSint that features an open side ring and a bis-dehydroalanine (Dha) tail. The newly obtained 6′-fluoro-NOS displayed equivalent or slightly reduced activities against the tested drug-resistant pathogens compared with NOS, but dramatically decreased water solubility compared with NOS. Our results indicate that the modification of the IA moiety of NOS not only affects its biological activity but also affects its activity which will be key considerations for further modification.

Full text of DOI:10.1039/d0ob00084a

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DOI: 10.1039/D0OB00084A
Journal Name
ARTICLE
Mutational Biosynthesis to Generate Novel Analogs of
Nosiheptide Featuring Fluorinated Indolic Acid Moiety
Received 00th January 20xx,
Accepted 00th January 20xx
E Zhang, a Heng Guo, b Dandan Chen, b Qian Yang, b Yafei Fan, a Yu Yin, e Wengui Wang, a, c Daijie
Chen, e Shoufeng Wang,*a, c Wen Liu*b, d
DOI: 10.1039/x0xx00000x
www.rsc.org/
Nosiheptide (NOS) is a member of bicyclic thiopeptides possessing a biologically important indolic acid (IA) moiety
appended onto the family-characteristic core system. The IA formation relies primarily on NosL,
a radical S-
adenosylmethionine (SAM) protein that catalyzes a complex rearrangement of the carbon side chain of L-tryptophan,
leading to the generation of 3-methyl-2-indolic acid (MIA). Here, we establish an efficient mutational biosynthesis strategy
for the structural expansion of the side-ring system of NOS. The nosL-deficient mutant Streptomyces actuosus SL4005
complemented by chemically feeding 6-fluoro-MIA is capable of accumulating two new products. The target product 6’-
fluoro-NOS contains an additional fluorine atom at C6 of the IA moiety, in contrast with an unexpected product 6’-fluoro-
NOSint that features an open side ring and a bis-dehydroalanine (Dha) tail. The newly obtained 6’-fluoro-NOS displayed
equivalent or slightly reduced activities against the tested drug-resistant pathogens compared with NOS, while
dramatically decreased water solubility compared with NOS. Our results indicate that the modification of IA moiety of NOS
not only affect its biological activity but also affect its activity which will be key considerations for further modification.
Introduction
Natural products with diverse chemical structures and ring systems5-8, and finally obtain the mature products.
biological activities are an important source for drug discovery Specifically, NOS bears an indolic acid (IA) moiety within a 19-
and development. However, most of the natural products are membered side-ring system, whereas in TSR, a quinaldic acid
accompanied by biochemical drawbacks that need to be (QA) moiety is appended to the macrocyclic framework by
improved via directional structural modification. Benefitting forming a 27-membered side-ring system. Both the IA moiety
from the maturing methodology in synthetic chemistry and the of NOS and the QA moiety of TSR are derived from L-
deepening understanding of biosynthetic paradigm, the tryptophan. The IA formation relies primarily on NosL, a radical
combination of biological and chemical approaches has S-adenosylmethionine (SAM) protein that catalyzes a complex
become an effective way to obtain natural product derivatives1. rearrangement of the carbon side chain of L-tryptophan and
Nosiheptide (NOS) and thiostrepton (TSR) are two generates 3-methyl-2-indolic acid (MIA)9, 10. In contrast, the QA
representative bicyclic thiopeptides, whose macrocyclic formation is initiated by TsrT, a radical SAM protein catalyzing
frameworks originate from the C-terminal core sequences of the 2-methylation of L-tryptophan to produce 2-methyl-L-
the ribosomally synthesized precursor peptides2-4. The core tryptophan11, which will further go through the key expansion
sequences then undergo precise and ordered post- of an indole to a quinolone intermediate via the action of the
translational modifications, including the incorporation of side- pyridoxal-5’-phosphate-dependent protein TsrA and the
flavoprotein TsrE12, 13 (Fig. 1). Existence of the side ring can
expand the chemical spaces available for biological activities,
therefore, bicyclic thiopeptides seem to exhibit unique mode
of action against pathogen infection14. It is reasonable to
predict that changes in the IA- or QA-containing side-ring
system may significantly affect the biological activities of the
a. School of Chemistry and Chemical Engineering, University of Jinan, 336 West
Road of Nan Xinzhuang, Jinan, 250022, China; E-mail: chm_wangsf@ujn.edu.cn.
b. State Key Laboratory of Bioorganic and Natural Products Chemistry, Center for
Excellence on Molecular Synthesis, Shanghai Institute of Organic Chemistry,
University of Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032,
China; E-mail: wliu@mail.sioc.ac.cn.
corresponding NOS or TSR15, 16
.
c. Shandong Provincial Key Laboratory of Fluorine Chemistry and Chemical
materials, 336 West Road of Nan Xinzhuang, Jinan, 250022, China
d. Huzhou Center of Bio-Synthetic Innovation, 1366 Hongfeng Road, Huzhou
313000, China.
In our previous work, we have applied the strategy of
precursor-directed biosynthesis to introduce structural
diversity into the side-ring system of NOS. By feeding 5-fluoro-
DL-tryptophan into the NOS-producing wild-type strain
e. School of Pharmacy, Shanghai Jiaotong University, Shanghai 200240, China.
†
Electronic Supplementary Information (ESI) available. See DOI:
10.1039/x0xx00000x
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DOI: 10.1039/D0OB00084A
Journal Name
ARTICLE
O
H
N
O
NH2
COOH
IleAlaSerAlaSerCysThrThrCysIleCysThrCysSerSer
Leader Peptide
NH2
OH
N
N
H
COOH
S
3-methyl-2-indolic acid (MIA)
S
N
N
NosL
O
N
H
N
NH2
O
N
HN
N
S
S
O
O
H
NH2
O
HO
NH
S
O
O
COOH
NH
S
O
HN
S
H
O
N
N
H
H
N
S
N
N
H
N
N
N
NH
O
H
H
L-tryptophan
N
O
O
Indolic acid (IA) moiety
N
O
O
H
NH
HN
O
OH
S
OH
O
H
HO
H
TsrA, TsrE
TsrT
Nosiheptide (NOS)
NH
O
N
O
O
H
HN
S
N
H
O
S
N
N
OH
Quinaldic acid (QA) moiety
O
HO
OH
N
COOH
SerCysThrThrCysGlucCysCysCysSerCysSerSer
Leader Peptide
NH2
Thiostrepton (TSR)
Quinolone
COOH
Figure 1. Biosynthetic paradigms of the bicyclic thiopeptides, NOS and TSR. The macrocyclic frameworks originate from the C-
terminal core sequences of the precursor peptides2-4, and the side-ring systems are constructed via the incorporation of the L-
tryptophan-derived precursors5-13
.
Streptomyces actuosus,
a
new compound 5’-fluoro-NOS tryptophan, which is independent of the precursor peptide9, 10
Focusing on the biologically relevant but tunable MIA moiety,
.
showing improved antibacterial activity was generated10.
However, due to the competition of substrates in the native we have considered to introduce the pharmaceutically
biosynthetic machinery, a mixture of NOS and 5’-fluoro-NOS important fluorine onto the side ring of NOS by taking into
were co-produced, leading to complex compound purification account the electronic effect pertinent to drug design. Given
burden. Based on precursor-directed biosynthesis, mutational the fact that 5-fluoro-L-tryptophan have been successfully
biosynthesis can utilize mutant strains deficient in a key aspect introduced into the NOS biosynthetic pathway via the
of the biosynthetic pathway and directionally produce natural endogenous generation of 5-fluoro-MIA10, it’s reasonable to
product analogs via the supplementation of particular design the directional incorporation of exogenous 6-fluoro-
precursors1. By feeding the quinolone derivatives into a tsrT- MIA into an engineered NOS biosynthetic machinery.
For 6-fluorinated MIA preparation, an efficient two-step
chemical synthesis route was developed. Briefly, 1-(2-bromo-
4-fluorophenyl)ethan-1-one was reacted with ethyl
isocyanoacetate in the presence of Cs2CO3 and CuI, resulting in
6-fluorinated indole-2-carboxylic acid ester in 90% yield. The 6-
fluoro-MIA ester was further hydrolyzed by NaOH followed by
acidification, which furnished 6-fluoro-MIA in 88% yield
(Scheme 1). Our method can synthesize 6-fluoro-MIA in gram-
scale, which is superior to the enzymatic conversion of
fluorinated MIA by NosL10.
deficient mutant strain, we have successfully obtained
numbers of TSR analogs with significant activities against drug-
resistant pathogens17-19. Since the competing native precursor
was eliminated in this mutant strain, the resultant TSR analogs
were prepared in high yields and efficiency. Considering the
significant advantages of mutational biosynthesis, we herein
use this strategy to expand the diversity of the NOS side ring
via the establishment of an efficient operation procedure.
We have previously constructed a nosL-deficient mutant
strain, SL4005, in which the biosynthetic route to MIA was
blocked2. NOS production was abolished in SL4005, but could
Results and discussion
MIA formation in the NOS biosynthetic pathway involves the
NosL-catalyzed carbon side chain rearrangement of L-
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a
O
CuI, Cs 2CO3
DMSO
1) NaOH, C2H5OH, reflux overnight
COOEt
COOH
+
CNCH2COOEt
N
H
F
Br
2) H+
F
N
F
H
6-fluoro-MIA
O
H
O
O
N
N
H
N
O
NH2
N
COOH
H
OH
N
b
S
S
S
N
N
S
N
N
N
N
SL4005 (DnosL)
COOH
HN
HN
O
S
S
S
S
O
O
O
N
F
H
HO
NH
S
HO
NH
S
O
O
HN
NH
6-fluoro-MIA
NH
O
O
HN
S
N
S
H
N
N
H
N
N
O
N
OH
F
O
F
O
O
O
OH
6'-fluoro-NOS
6'-fluoro-NOSint
Scheme 1. Chemical synthesis route of the 6-fluorinated MIA (a) and mutational biosynthesis strategy for generating NOS
analogs (b). The target product 6’-fluoro-NOS and the unexpected product 6’-fluoro-NOSint were co-produced in SL4005, a nosL-
deficient mutant, complemented by chemically feeding 6-fluoro-MIA.
be restored by chemical feeding the synthetic MIA (Fig. incorporated into the IA moiety (Fig. S3). To fully elucidate the
2).Therefore, SL4005 was served as an ideal host for the corresponding chemical structures, 6 mg 6’-fluoro-NOS was
biosynthesis of NOS analogs by eliminating the interference of
native precursor. Comparing to the methods described
previously2, 20, we further developed a procedure preferable in
i
shortened fermentation cycle, improved conversion rate, and
reduced separation difficulty. After sporulation, the
ii
Streptomyces actuosus spores were subjected to primary
liquid fermentation, and 0.2 mM synthetic 6-fluoro-MIA was
iii
exogenously fed to the fermentation broth afterward. Notably,
two new compounds, namely 6’-fluoro-NOS (∼2 mg L-1) and 6’-
fluoro-NOSint (∼4 mg L-1) were efficiently produced by SL4005
iv
complemented by chemically feeding 6-fluoro-MIA (Fig. 2).
The target NOS analog, 6’-fluoro-NOS, showed typical NOS-
min
18
14
16
20
22
24
26
like UV absorption pattern (Fig. S1), suggesting the existence
of the NOS bicyclic molecular skeleton. HR-ESI-MS datum
established the molecular formula as C51H42FN13O12S6 ([M+H]+,
calcd 1240.1457, obsd 1240.1459) (Fig. S2), which confirmed
the introduction of a fluorine atom into NOS. Subsequent LC-
MS/MS analysis further supported that the fluorine atom was
Figure 2. HPLC analysis of the fermentation products from
the wild-type NOS-producing strain Streptomyces actuosus
ATCC25421 (i), the nosL-deficient mutant strain SL4005 (ii),
SL4005 complemented by chemically feeding MIA (iii) or 6-
fluoro-MIA (iv). Solid dot (●) indicates NOS. Solid triangle (▲)
indicates the NOS analog, 6’-fluoro-NOS. Solid square ( ■ )
indicates the NOS analog, 6’-fluoro-NOSint.
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Journal Name
purified from 8 L fermentation broth and went through 1D and leading to the generation of a side-ring-opening iVnietwerAmrticeledOiantlinee.
1
2D NMR (1H, 13C and 19F NMR, H-1H COSY, HSQC, HMBC, and Our results show that the introduction oDfOaI:fl1u0o.1r0i3n9e/Da0tOoBm00a0t8C46A
ROESY) experiments (Fig. S6). The NMR data revealed signals of MIA deduce the electron cloud density of MIA and hence
typical for bicyclic thiopeptides and highly resembled to those affect methylene radical electrophilic substitution of MIA,
of NOS, except for the appearance of an additional quaternary which is consistent with the proposed NosN reaction
carbon (δC 160.5, Ind-C6) that suggested a difference in the IA mechanism24.
1
moiety. In the H NMR spectrum, two typical aromatic proton
The side-ring-opening intermediate will go through
signals [δH 6.99 (H-5), 6.99 (H-7)] were observed. Besides, in sequential enzymatic transformations to forms the pyrinie ring
the 19F spectrum, chemical shift (δF) -114.9 was observed as by NosO; however, two oxidation events catalyzed by two
well. Combining the above information with the HMBC cytochrome P450-like mono-oxygenases, NosB acts on Glu at
correlations from H-5 to C-4, C-6, and C-7 in the IA moiety, the its γ-position whereas NosC acts on the Pyr position, did not
chemical structure of 6’-fluoro-NOS was determined, featuring happen since these two steps occur at the tailoring stage, after
an additional fluorine atom at C6 of the IA moiety (Scheme 1).
the main scaffold is formed during NOS biosynthesis25. And the
Besides 6’-fluoro-NOS, an unexpected compound, named as side-ring-opening intermediate also affect the enzymatic
6’-fluoro-NOSint, was co-produced. The UV absorption pattern activity of NosA, a novel protein enabling the cleavage of the
of 6’-fluoro-NOSint was different from that of NOS (Fig. S1), bis-Dha tail through the process of enamine dealkylation21. The
indicating that changes might take place in the molecular side ring closure, catalyzed by NosN, will not only affect the
skeleton. HR-ESI-MS datum established the molecular formula tailoring stage enzymatic transformations but also affect the
as C53H44FN13O12S6 ([M+H]+, calcd 1266.1613, obsd 1266.1604) formation of mature NOS analog.
(Fig. S4), implying the high correlation between 6’-fluoro-
Table 1. Minimum inhibitory concentrations (MIC) values of 6’-
fluoro-NOS, 6’-fluoro-NOSint and NOS against drug-resistant
pathogens. Vancomycin was chosen as a control drug for the tests
NOSint and 6’-fluoro-NOS. Given the LC-MS/MS datum that
clearly provided a fragmental mass of the fluorinated MIA
segment and a fragmental mass of a bis-dehydroalanine (Dha)
segment (Fig. S5), we could basically verify that 6’-fluoro-
NOSint was a 6-fluoro-MIA-S-conjugated, side-ring-opening
NOS derivative with a bis-Dha tail (Scheme 1). The speculative
chemical structure of 6’-fluoro-NOSint is highly similar to NOS
analogue 4 which is isolated from a nosN-deficient mutant
strain5.
To fully elucidate the corresponding chemical structures, 8
mg 6’-fluoro-NOSint was purified from 4 L fermentation broth
and went through 1D and 2D NMR (1H, 13C and 19F NMR, 1H-1H
COSY, HSQC, HMBC, and ROESY) experiments (Fig. S9). The
NMR data highly resembled to NOS analogue 4 which is
isolated from a nosN-deficient mutant strain, the appearance
of an additional quaternary carbon (δC 160.5, 6-F-Ind-C6) that
suggested a difference in the IA moiety. In the 1H NMR
spectrum, two typical aromatic coupling proton signals [δH
7.73 (H-4), 6.96 (H-5)] were observed. Similar to 6-F-Nos, in
the 19F spectrum, chemical shift (δF) -114.9 was also observed.
Combining the above information with the HMBC correlations
from H-5 to C-4, C-6, and C-7 in the IA moiety, besides with the
1H-1H COSY between H-5 and H-4, the chemical structure of 6’-
fluoro-NOSint was determined, featuring an additional fluorine
atom at C6 of the IA moiety (Fig. S11).
The biosynthesis of the side-ring structure requires the
actions of NosI, -J, -K,-L, and –N26. NosN is a radical SAM
protein that selectively build a C1 unit at the S-conjugated MIA
moiety and form an ester linkage to establish the side ring
system5, 8; besides, NosN was identified to close the side ring
before NosO forms the pyridine ring24. Therefore, the
introduction of a fluorine atom at C6 of MIA will not affect the
enzymatic transformations of NosIJK; however, the
introduction of a fluorine atom at C6 of MIA will interfere the
methylation function of NosN, perhaps affect the electrical
properties of MIA with electronegativity of F (4.0) higher than
H (2.1) while atomic radius of F (0.71 Å) similar to H (0.79 Å),
μg mL-1
MIC (
)
Drug-resistant pathogen
MRSA ATCC43300
Vancomycin
6'-fluoro-NOS
6'-fluoro-NOSint
NOS
1
4
0.032
0.016
0.032
0.032
0.032
>0.128
>0.128
VRE ATCC29212
VRE ATCC51299
0.016
0.004
>0.128
>0.128
64
VRE ATCC51559
0.016
>128
Considering the importance of the IA moiety that can
interact with A1067, nucleobase of 23S rRNA that
a
contributes to mutation-induced bacteria resistance22, 23, 6’-
fluoro-NOS, 6’-fluoro-NOSint and NOS were thus evaluated for
their activities against a panel of Gram-positive drug-resistant
pathogens, including methicillin-resistant Staphylococcus
aureus (MRSA ATCC43300) and vancomycin- resistant
Enterococcus (VRE ATCC29212, VRE ATCC51299, VRE
ATCC51559) (Table 1). The newly obtained 6’-fluoro-NOS
displayed equivalent or slightly reduced activities against the
tested pathogens, which was in agreement with the
dramatically decreased water solubility of 6’-fluoro-NOS (0.15
± 0.02 μg mL-1) compared with NOS (4.90 ± 0.11 μg mL-1). The
above results indicate that the modification of IA moiety of
NOS not only affect the biological activity of NOS to drug-
resistant pathogens but also affect the solubility of NOS, which
may be an important bottleneck limiting the acquisition of
bioactive new products. As our expected, the antibacterial
activity of 6’-fluoro-NOSint became very poor, shed light on
the closure of side ring on the biological activity of NOS. Side-
ring-opening increases the degree of freedom of 6’-fluoro-
NOSint, while affects the binding of 6’-fluoro-NOSint to the
target at the same time.
4 | J. Name., 2012, 00, 1-3
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(2019GSF108223), National Key Research and Development
Conclusions
View Article Online
Program of China (grant No. 2018YFA0901903), Chinese
DOI: 10.1039/D0OB00084A
We have successfully applied the strategy of mutational
biosynthesis to generate the novel fluorinated analogs of NOS.
Through the operation procedure we developed, the synthetic
precursor analog, 6-fluoro-MIA, was efficiently fed into SL4005
that deficient in the endogenous MIA preparation, leading to
the accumulation of the target product, 6’-fluoro-NOS. It is
worth mentioning that 6’-fluoro-NOSint, an unexpected
product featuring an open side ring and a bis-Dha tail, was
produced simultaneously. Structure elucidation of 6’-fluoro-
NOSint gives insight into the substrate selectivity of the
enzymes responsible for MIA incorporation and NOS
maturation. The bicyclic NOS analog 6’-fluoro-NOS displayed
equivalent or slightly reduced activities against the tested
drug-resistant pathogens, which might assign to the
unforeseen reduction in water solubility. The closure of side
ring not only affects physicochemical property of NOS but also
interferes with substrate recognition and enzymatic
transformation of the tailor enzymes. This work will deepen
our understanding of natural products biosynthesis machinery
and lay a foundation for the mutational biosynthesis of NOS
analogs.
Academy of Sciences (QYZDJ-SSW-SLH037 and XDB20020200),
Science and Technology Commission of Shanghai Municipality
(17JC1405100), Youth Innovation Promotion Association of the
Chinese Academy of Sciences (2017303), and K. C. Wong
Education Foundation.
Notes and references
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Experimental
Mutational biosynthesis of nosiheptide analogs: For
sporulation, the Streptomyces actuosus strains were cultured
on MS agar plates at 30°C for 3 days. The S. actuosus spores
10 Q. Zhang, Y. Li, D. Chen, Y. Yu, L. Duan, B. Shen and W. Liu,
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were inoculated into a 500-mL flask containing 100 mL of 11 A. J. Blaszczyk, A. Silakov, B. Zhang, S. J. Maiocco, N. D. Lanz,
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primary fermentation medium and incubated at 30℃ and 220
rpm for 30 hrs.
0.2 mL synthetic 6-fluoro-MIA (1mM dissolved in DMSO)
was fed to 100 mL fermentation broth of S. actuosus SL4005,
which would be incubated at 30℃ and 220 rpm for 48 hrs.
The fermentation broth was centrifuged, and the supernatant
was discarded. The mycelia cake was soaked with acetone and
sonicated for 30 min. The acetone sample was then
centrifuged for HPLC analysis on an Agilent Zorbax column.
A total of 8 L fermentation broth was treated by the above
method. The crude product was dissolved in tap water, and
then extracted twice with an equal volume of n-butanol, and
the organic phase was concentrated and purified by silica gel
chromatography eluting with 100% CH2Cl2 followed by CH2Cl2-
MeOH (100:1 to 100:10). The component 6’-fluoro-NOS was
then collected and the organic solvent was evaporated to
obtain a crude extract. The crude extract was further purified
by semi-preparative HPLC performed on an Agilent 1100 with a
Zorbax SB-C18 column (9.4 mm × 25 cm)
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Conflicts of interest
There are no conflicts to declare.
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Acknowledgements
This work was supported in part by grants from the National
Natural Science Foundation of China (31972850, 21750004 and
21520102004), the Shandong Key Research Program
This journal is © The Royal Society of Chemistry 20xx
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