science.abq7333
science.abq7333
science.abq7333
T
he antimicrobial resistance (AMR) crisis ments, a fundamental requirement for nu- The principle and design of ACTIMOT
has been identified by the World Health merous potential applications. For instance, In contrast to conventional molecular cloning,
Organization (WHO) as one of the great- biosynthetic gene clusters (BGCs)—composed ACTIMOT eliminates the need for isolating ge-
est global threats (1). Although AMR un- of multiple genes (varying from several kb to nomic DNA from the source species and rep-
doubtedly poses a major health threat, >100 kb) forming a pathway to produce small licating recombinant DNA in the host species.
its inherent evolutionary mechanism in gene molecules and related congeners—hold im- Instead, ACTIMOT enables the leap of TDRs
transmission appears to be a successful strat- mense value for chemical entities exhibiting from the bacterial chromosome to plasmids
egy in nature. Recent studies have indicated extremely diverse biological activities. Such within the same bacterial cell. We developed
that the spread of antibiotic-resistance genes natural products (NPs) have contributed to the prototype of ACTIMOT as comprising two
(ARGs) occurs through the transfer of AMR 66.8% of all US Food and Drug Administra- distinct Streptomyces–Escherichia coli shuttle
genes from the chromosome to mobile genetic tion (FDA)–approved small-molecule drugs in plasmids, facilitating TDR mobilization and
elements (MGEs) such as multicopy plasmids the last four decades, making them vital for relocation. The release plasmid (pRel) used in
(2, 3). This process generally includes two suc- the treatment of various diseases (9). How- the TDR mobilization step consists of a codon-
cessive events: the mobilization of ARGs typ- ever, technical challenges and high rediscovery optimized Cas9 gene, a single-guide RNA (sgRNA)
ically achieved by either insertion sequences rates have led to a decline in interest from the cassette, and a Streptomyces replicon [from pSG5
(4, 5) or integrons (6, 7) and the subsequent pharmaceutical industry in NP research (10). (29)]. The capture plasmid (pCap) for BGC relo-
relocation of the mobilized ARGs to MGEs (8). Nevertheless, recent genomic investigations cation features a bacterial artificial chromosome
Subsequently, horizontal gene transfer (HGT) have unveiled the heavily underestimated po- (BAC) backbone and a Streptomyces-specific rep-
facilitates the transmission of ARGs among tential of untapped secondary metabolites in licon [either from pSG5 or pIJ101 (30)] (Fig. 2A,
bacteria (2, 3) (Fig. 1A). bacteria, even in extensively studied taxa such figs. S1 and S2, and table S2). The option of the
Inspired by the “mobilization-relocation- as Actinobacteria (11). Although prior efforts in pIJ101 replicon in pCap ensures compatibility
transfer” process naturally occurring during the field developed different strategies to un- with pRel when used together in Streptomyces.
AMR spread, we contemplated the possibility cover NPs from BGCs (12–21), the discon- To mobilize a specific TDR from the bacterial
of artificially simulating the molecular mecha- nection between the enormous biosynthetic chromosome, a functional pRel carrying two
nisms to mobilize and transmit large DNA frag- potential of BGCs and our limited knowledge sgRNA spacers targeting the two ends of TDR is
of encoded chemical entities highlights the required to ensure accurate cleavage on the chro-
1
need for alternative approaches to accelerate mosome (Fig. 1B). Simultaneously, a functional
Helmholtz Institute for Pharmaceutical Research Saarland
(HIPS), Helmholtz Centre for Infection Research (HZI),
the discovery of what are now known as “cryptic” pCap harboring a specific spacer–protospacer
Saarbrücken, Germany. 2Helmholtz International Lab for Anti- NPs (22). adjacent motif (PAM) cassette between the
Infectives, Helmholtz Centre for Infection Research, To accomplish this objective, we have de- pair of homologous arms (HAs) is cleaved by
Braunschweig, Germany. 3PharmaScienceHub, Saarbrücken,
Germany. 4Faculty of Medicine, Saarland University,
vised an approach inspired by ARG transmis- sgRNA-Cas9, exposing the HAs, CapL, and CapR.
Homburg, Germany. 5Key Laboratory of South China sion called ACTIMOT (Advanced Cas9-mediaTed The linearized pCap, the destination for BGC
Agricultural Plant Molecular Analysis and Genetic In vivo MObilization and mulTiplication of relocation, captures the mobilized TDR frag-
Improvement, South China Botanical Garden, Chinese
BGCs). To simulate the mechanisms of gene ment through homologous recombination.
Academy of Sciences, Guangzhou, China. 6Department of
Pharmacy, Saarland University, Saarbrücken, Germany. mobilization, we have leveraged the power Meanwhile, the Cas9-triggered double-strand
7
German Center for Infection Research (DZIF), of clustered regularly interspaced short pal- break on the chromosome is repaired through
Braunschweig, Germany. indromic repeats (CRISPR) and the CRISPR- homology-directed repair by HAs (DelL and DelR)
*Corresponding author. Email: rolf.mueller@helmholtz-hips.de
(R.M.); chengzhang.fu@helmholtz-hips.de (C.F.) associated protein 9 (Cas9) (23–27), a cutting-edge cloned in the functional pRel. The relocated BGC
†These authors contributed equally to this work. genome-editing technology, to artificially lib- on the multicopy plasmid undergoes replication
in vivo, resulting in residual empty pCap in the level production of dozens of compounds with- residue present in 1, as indicated by their
mutants. To further improve the efficiency of out any genetic modifications (Fig. 3, B, D, MS/MS data (fig. S18). In avidistatins 5 to 7,
ACTIMOT, we postulated that a CRISPR-Cas9– and E). We isolated the main compounds and the 2,3-Dab residues were substituted with Dhb
mediated self-cleavage could enhance the ef- structurally elucidated two types of peptides by residues (Fig. 3C and fig. S18), which presum-
ficiency of cutting multicopy plasmids within nuclear magnetic resonance (NMR) and ultra- ably undergo conversion through a C domain–
cells. Therefore, we fused pCap-101-Apr-SAV11-LR high-performance liquid chromatography–high- mediated online dehydration from a threonyl
and pRel-Hyg-SAV11-dsp (materials and methods), resolution mass spectrometry (UHPLC-HRMS) residue (39). This observation suggests the po-
generating the single-plasmid pRelCap-SAV11- (figs. S10 to S13, S50 to S104, and tables S14 to tential promiscuity of the adenylation (A) do-
dsp for the mobilization of Sav11. Similarly to S17). The stereochemistry of amino acid residues main within the second module (fig. S21). We
the dual-plasmid approach, we easily obtained in these compounds was assigned by advanced confirmed the presence of (2S,3R)-Dab using
and verified the correct exconjugants harbor- Marfey’s method (38) (figs. S14 to S17). Addi- the advanced Marfey’s method (fig. S14). The
ing the pCap-Sav11 (fig. S8C). All E. coli colonies tionally, the structures of unisolated derivatives genes avsC, avsD, and avsE are implicated in
were confirmed to carry the correct pCap-Sav11 were elucidated through tandem MS (MS/MS) the formation of (2S,3R)-Dab because they show
after transformation with the plasmids ex- analysis (figs. S18 and S19). similarities to the previously reported (2S,3R)-
tracted from Streptomyces exconjugants (fig. We named the peptide families avidistatins Dab biosynthesis cassette (40). Recent studies
S8D), indicating improved efficiency of the single- (1 to 7) and avidilipopeptins (8 to 22) (Fig. 3, C have corroborated that analogous genes are re-
plasmid ACTIMOT versus the dual-plasmid and F, and fig. S19). Their corresponding BGCs sponsible for the synthesis of (2S,3R)-Dab from
system for BGC mobilization (fig. S8E). were confirmed through gene deletion on the threonine (41, 42), supporting their roles in pro-
These two NRPS BGCs remained silent after pCap-Sav11 (fig. S20). Avidistatin A1 (1) is a lin- viding free substrate available for avidistatin
the successful mobilization in S. avidinii be- ear acetylated pentapeptide composed of unusual assembly. Furthermore, the alignment of the
cause we did not detect prominent new com- nonproteogenic amino acid residues, includ- majority of amino acid building blocks in
pounds in chromatographic analysis in the ing (2S,3R)-2,3-diamino-butyric acid [(2S,3R)- avidistatin A with the A domain–specificity pre-
correct mutants. We hypothesized that the ex- Dab] and (Z)-dehydrobutyrine (Dhb) (Fig. 3C). diction in the avs pathway (table S7) strength-
pression of Sav11 might still be repressed in Avidistatin A2 (2) was identified as an isomer ens the proposed biosynthetic pathway of
the native host (36). Therefore, we transferred of 1, on the basis of their identical molecular avidistatins (fig. S21).
pCap-Sav11 into the heterologous host S. albus formulas and MS/MS data (fig. S18), whereas Avidilipopeptins are linear lipopeptides, in-
Del14 (37) to escape the potential repression avidistatin B1 (3) and B2 (4) were charac- cluding acylated pentapeptides 8 to 12, 17 to
effect in the native strain, which led to the high- terized by the absence of the terminal serine 22, and acylated dipeptides 13 to 16 (Fig. 3F).
All characterized avidilipopeptins share the first covery of armeniaspirols and streptopyrroles cinnamoyl-containing compounds, which is
two residues (D-arginine and L-serine), suggest- (34, 35), underscoring the untapped biosyn- corroborated by the linear polyene moieties
ing a potential pretermination step after the thetic potential to be explored by using different present in ishigamide (43) and colibrimycins
second NRPS module in the avl pathway (fig. strategies. Among these, the 67-kb TDR Sar13 (44). These BGCs were categorized into various
S22). The diverse structures of avidistatins and emerged as a promising target, containing a groups: 6 mop BGCs, 12 ishigamide BGCs, 4
avidilipopeptins highlight the strategies used by cryptic “ladderane”-NRPS BGC mop (Fig. 4A, colibrimycin BGCs, and 12 other unknown BGCs
NRPS pathways to diversify the final products, fig. S23, and table S8). The “ladderane”-NRPS (table S10, and data S5 and S6), suggesting
including variable building blocks introduced BGCs are known to produce polyene-peptide structural diversity encoded within this family.
by the promiscuous A domains and different hybrid compounds, such as ishigamide (43) and Exploring the products and biosynthesis of mop
chain lengths resulting from the optional colibrimycins (44), or cinnamoyl-containing will deepen our knowledge of the structural
chain release from the assembly line (figs. S21 cyclodepsipeptides, such as kitacinnamycins features concealed within this BGC family.
and S22). (45) and skyllamycins (46, 47). BGC mop shares To mobilize and activate Sar13, we constructed
more similarity with ishigamide BGC and the plasmid pRelCap-SAR13-dsp and introduced
BGC multiplication facilitates the discovery of colibrimycin BGCs than with those produc- it into S. armeniacus. Following the same val-
unusual lipopeptides ing cinnamoyl-containing cyclodepsipeptides. idation procedure described in the Sav11 mo-
Building upon the success of the improved ClusterBlast analysis (48) identified 34 similar bilization, we verified the successful Sar13
single-plasmid ACTIMOT, we expanded its ap- BGCs distributed in various Actinobacteria, mobilization in 10 out of 11 plasmids derived
plication to activate other BGCs from different featuring a set of highly reducing type II poly- from four randomly picked starting Strepto-
Streptomyces strains. Despite many BGCs pres- ketide synthase (PKS), NRPS modules, and 2,3- myces exconjugants (fig. S24), revealing a 90.9%
ent in the genome of S. armeniacus DSM19369, diaminopropionic acid (2,3-Dap) formation success rate for Sar13 mobilization. Short-read
previous screening efforts by the pharma- genes. These BGCs lack genes for benzene ring sequencing of pCap-Sar13 further corroborated
ceutical industry have only resulted in the dis- formation (49), deviating their products from the integrity of the entire plasmid (fig. S9B).
The mobilization and multiplication of Sar13 the unusual type II PKS genes (50–52) present mobilipeptin E) at retention times of 6.7 and
promoted the discovery of a series of metab- in the “ladderane” region of Sar13, whereas 7.5 min, respectively (Fig. 4D and fig. S30). These
olites that we named mobilipeptins. The yield the dipeptide is installed by one of the NRPS “transient” compounds exhibited their highest
of mobilipeptins in the mutants carrying mo- genes with the starter condensation (Cs) do- yield on day 2 when cultured in ISP4 medium
bilized Sar13 increased nearly 40-fold (Fig. 4B main (mopI). The substrates predicted for the but faded quickly in the subsequent fermen-
and fig. S25). Discovering mobilipeptins di- two modules in MopI correspond closely with tation (fig. S30). We next purified the tetracyclic
rectly from the WT of S. armeniacus would the observed residues (table S9). However, peptide 27 from a 2-day fermentation of the
have been challenging because of their pro- the sole TE domain in mop is situated after the Sar13 mobilized mutant in ISP4. However, 27
duction in trace amounts that are only barely glycine-recognizing NRPS module in MopJ exhibits extremely poor solubility in various
detectable with selective ion monitoring after (tables S8 and S9), hinting at the potential pres- solvents, including methanol, water, chloroform,
the target mass is identified by using ACTI- ence of precursor compounds with more resi- and dimethyl sulfoxide, making the NMR mea-
MOT (Fig. 4B and fig. S25A). The absence of dues present in the pathway. We hypothesized surement and the ensuing bioactivity test
selection antibiotics did not affect the produc- that this hybrid BGC initially produces precursor challenging. Unexpectedly, we found that 27
tion of mobilipeptins (Fig. 4B and fig. S25B), molecules with a tetrameric cyclic peptide core. exhibited the desired solubility in acetone;
indicating the replication stability of the plas- This hypothesis is supported by the identifica- however, the MS analysis of the solution showed
mid in the cells. This feature shows the potential tion of an additional mobilipeptin congener 25, 27 to convert to a new derivative 28 (fig. S31).
of using ACTIMOT for increasing large-scale featuring an extra glycine residue coupled to the Subsequently, we solved the structure of 28
economic production of high-value NPs. a-amino group of L-2,3-Dap (fig. S29). through HRMS and NMR (figs. S26, S32, S118
Structural elucidation by NMR, UHPLC-HRMS, To explore the possibility of a structurally to S123, and table S19), which suggested that
and Marfey’s method revealed that mobilipeptin different primary product, we conducted fer- 28 is an acetone adduct of 27, which itself
A (23) and B (24) are lipopeptides containing mentation under varying conditions. Numerous most likely exhibits a structure in line with the
a linear conjugated polyene chain coupled with attempts resulted in the detection of short-lived in silico prediction of substrate specificity of
a L-2,3-Dap and an O-methyl-D-tyrosine residue products from the Sar13-activated mutant with all the four A domains in mop (table S9). The
(Fig. 4C; figs. S26 to S28, S106 to S117; and table corresponding molecular ions of [M+H]+ = deduced structure of 27 is a linear polyene chain
S18). The polyene chain is likely derived from 571.29 (26, mobilipeptin D) and 553.28 (27, coupled with tetracyclic peptide comprising
two L-2,3-Dap residues, an O-methyl-D-tyrosine mop by ACTIMOT significantly enhanced the S. avidinii mutants carrying pCap-Sav17, we
residue, and a glycine residue (Fig. 4E). More- yield of mobilipeptins at different biosynthesis identified a series of highly yield-improved
over, the structure of 26 was determined by MS stages (Fig. 4D), which exemplified the distinc- compounds that were easy to neglect in the
fragmentation to be a degradation product of tive capability of ACTIMOT. WT strain because of their meager production
27, resulting from the hydrolysis of the amide (Fig. 5B). This observation indicated the activa-
bond between O-methyl-D-tyrosine and the third Biosynthetic “dark matter” revealed by ACTIMOT tion of a cryptic BGC within Sav17. Subsequent-
residue (L-2,3-Dap) (fig. S29), further support- In our pursuit to uncover hidden biosynthetic ly, the heterologous expression of pCap-Sav17
ing the structure assignment of 27. Hence, we potential, we embarked on mobilizing the co- in S. albus Del14 showed a much higher pro-
propose that 27 is the direct product released lossal TDR Sav17 from the chromosome of duction of the compounds found in S. avidinii
and cyclized from the NRPS of mop, which can S. avidinii DSM40526. This expansive segment and multiple new compounds (Fig. 5B). Through
be hydrolyzed into 26, followed by stepwise encompasses a massive 149-kb region predicted comprehensive analysis using NMR and UHPLC-
cleavage into 23 to 25 (fig. S34). to harbor a giant NRPS BGC (Fig. 5A). This HRMS/MS, we purified and structurally elu-
Although direct degradation of 27 remains BGC comprises 28 NRPS modules with a Cs cidated nine compounds (29 to 36, and 38),
a plausible scenario, the identification of spe- domain in the initial module, suggesting the unveiling a family of benzoxazole-containing
cific intermediates with different numbers of potential production of large lipopeptides (fig. compounds that we named actimotins (Fig.
amino acid residues suggests that the tetra- S35). A thorough ClusterBlast search on this 5C; figs. S38 to S40, S124 to S176; and tables
cyclic peptide precursor could also undergo BGC revealed a lack of similar BGCs within the S20 to S23). The stereochemical configurations
stepwise degradation by an as-yet-unknown current public database (data S7 and S8). After of 31 and 35 were determined with Mosher
mechanism (fig. S34). This process could in- the mobilization procedure using the plasmid ester analysis (fig. S39 and tables S24 and S25),
volve rare enzyme-mediated cleavage, similar pRelCap-SAV17-dsp targeting the two ends of and the stereochemistry of 29 and 30 was
to that observed in the biosynthetic pathways Sav17 on the chromosome (fig. S36), we eval- assigned through density-functional theory
of other peptides (53–55). The discovery of the uated four exconjugants for the TDR mobili- simulation (figs. S41 to S45). MS/MS analysis
cyclic mobilipeptin and a series of processing zation efficiency. Subsequently, five out of eight facilitated the identification of the structures
products of mobilipeptins has not only iden- random E. coli colonies obtained by transform- of three additional derivatives (37, 39, and
tified the likely authentic products of the mop ing plasmids from the starting four S. avidinii 40) (fig. S46). One notable structural feature
pathway but also shed light on the biosynthe- exconjugants were found to harbor the plas- of actimotins, alongside their rare metasubsti-
sis and potential degradation mechanisms of mid pCap-Sav17 that contains the correct 149-kb tuted benzoxazole pattern, is the (1S,2R,4S)-2-
mobilipeptins. Furthermore, the mobilipeptin DNA region (fig. S37). Further short-read se- aminocyclohexane-1,4-diol (ACHD) moiety in
biosynthetic pathway implies that the biosyn- quencing of the plasmids confirmed the in- several congeners (29 to 31).
thesis of ishigamide might also involve a cryptic tegrity of the mobilized Sav17, suggesting the We did not find the reported benzoxazole
cyclic peptide precursor. The direct activation of high fidelity of ACTIMOT (fig. S9C). In the BGC in sav17 (56). The only plausible candidate
is an upstream 20-kb region enriched with var- which contain two 3,4-AHBA units (Fig. 5C). The the most common variant linked to TTR amy-
ious biosynthetic genes, including discrete A do- incorporation rate was low (fig. S48, A and B), loidosis (66). To evaluate a potential similar
main genes, which we initially presumed to which likely results from the use of aspartic acid activity of actimotins, we developed a thioflavin
be part of the giant NRPS BGC. Through gene in primary metabolism and additional catalytic T-based assay to stabilize the TTR-V30M, on
deletions and heterologous expression of the steps required before incorporation into acti- the basis of a previous study (67). In our assays
modified sav17 (Fig. 5A, fig. S47, and table S11), motin (59). A similarly low incorporation rate with 10 mM TTR-V30M, tafamidis, used as posi-
we confirmed that this cryptic 16-gene region is was observed in actimotin A (29) and B (30) tive control, showed activity with a median ef-
responsible for actimotin biosynthesis, distinct (fig. S48, C and D), suggesting that the ACHD fective concentration (EC50) value of 9.8 mM
from the giant NRPS BGC. Thus, ACTIMOT moiety originates from 3,4-AHBA through step- (fig. S50A), which is consistent with the previous
paved the way for discovering the actimotin wise reductions, although alternative path- report (68). Among the isolated actimotins,
biosynthetic pathway, which was not identi- ways cannot be excluded. Further feeding with actimotin J (38) exhibited activity in a similar
fied by in silico prediction owing to the limited 13 15
L-cysteine- C3, N revealed a 4 Da difference range, with an EC50 value of 67.8 mM (fig. S50B).
understanding of the biosynthesis of metasub- between control and isotope-labeled 29 and 30 Actimotin J shares the same structure with a
stituted benzoxazoles. During our study on (fig. S48, E and F), indicating that L-cysteine previously isolated metasubstituted benzoxa-
actimotin biosynthesis, the first BGC encod- serves as the precursor for their N-acetylcysteinyl zole from Nocardiopsis lucentensis DSM44048,
ing for metasubstituted benzoxazoles was pub- moieties. A previous study on grixazole bio- which was also reported to show no cell-based
lished (57). However, genome mining efforts synthesis showed that N-acetylcysteine (NAC) bioactivity (69). Our finding holds promise for
power of ACTIMOT in boosting NP discovery. 13. L. Huo et al., Heterologous expression of bacterial natural 35. C. Fu et al., Armeniaspirol antibiotic biosynthesis: Chlorination
The achievements of ACTIMOT in Streptomyces product biosynthetic pathways. Nat. Prod. Rep. 36, 1412–1436 and oxidative dechlorination steps affording spiro4.4non-
(2019). doi: 10.1039/C8NP00091C; pmid: 30620035 8-ene. ChemBioChem 20, 764–769 (2019). doi: 10.1002/
fuel optimism about its potential applicability 14. K. Scherlach, C. Hertweck, Mining and unearthing hidden cbic.201800791; pmid: 30556942
in rare actinomycetes, owing to compatible rep- biosynthetic potential. Nat. Commun. 12, 3864 (2021). 36. B. Aigle, C. Corre, Waking up Streptomyces secondary
licons (73). Although the current application doi: 10.1038/s41467-021-24133-5; pmid: 34162873 metabolism by constitutive expression of activators or genetic
15. A. G. Atanasov, S. B. Zotchev, V. M. Dirsch, C. T. Supuran, disruption of repressors. Methods Enzymol. 517, 343–366 (2012).
range of ACTIMOT is limited and depends on International Natural Product Sciences Taskforce, Natural doi: 10.1016/B978-0-12-404634-4.00017-6; pmid: 23084947
the genetic tractability of target strains, it shows products in drug discovery: Advances and opportunities. 37. M. Myronovskyi et al., Generation of a cluster-free
potential in other genetically manipulable and Nat. Rev. Drug Discov. 20, 200–216 (2021). doi: 10.1038/ Streptomyces albus chassis strains for improved heterologous
s41573-020-00114-z; pmid: 33510482 expression of secondary metabolite clusters. Metab. Eng.
BGC-rich bacterial species, such as Proteobacteria 16. F. Panter, C. D. Bader, R. Müller, Synergizing the potential of 49, 316–324 (2018). doi: 10.1016/j.ymben.2018.09.004;
and Firmicutes (11). To broaden its utility, bacterial genomics and metabolomics to find novel antibiotics. pmid: 30196100
further efforts are needed to develop compati- Chem. Sci. 12, 5994–6010 (2021). doi: 10.1039/D0SC06919A; 38. P. Marfey, Determination of D-amino acids. II. Use of a
pmid: 33995996 bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene.
ble genetic elements, thereby expanding the Carlsberg Res. Commun. 49, 591–596 (1984). doi: 10.1007/
17. A. Bauermeister, H. Mannochio-Russo, L. V. Costa-Lotufo,
scope of ACTIMOT’s applicability. These future A. K. Jarmusch, P. C. Dorrestein, Mass spectrometry-based BF02908688
applications will allow for a more comprehen- metabolomics in microbiome investigations. Nat. Rev. 39. S. Wang et al., Discovery and biosynthetic investigation of a
Microbiol. 20, 143–160 (2022). doi: 10.1038/s41579-021- new antibacterial dehydrated non-ribosomal tripeptide.
sive assessment of its broader impacts. In sum- Angew. Chem. Int. Ed. 60, 3229–3237 (2021). doi: 10.1002/
00621-9; pmid: 34552265
mary, this study demonstrates the potential 18. M. H. Medema, T. de Rond, B. S. Moore, Mining genomes anie.202012902; pmid: 33107670
of ACTIMOT as a strategy to discover previously to illuminate the specialized chemistry of life. Nat. Rev. 40. C. Müller et al., Sequencing and analysis of the biosynthetic
55. Y.-M. Shi et al., Global analysis of biosynthetic gene clusters reveals Americans. N. Engl. J. Med. 336, 466–473 (1997). thank A. F. Stewart, A. Keller, C. Beemelmanns, and A. A. Gurevich
conserved and unique natural products in entomopathogenic doi: 10.1056/NEJM199702133360703; pmid: 9017939 for their valuable discussions. Funding: C.F. and R.M. acknowledge
nematode-symbiotic bacteria. Nat. Chem. 14, 701–712 (2022). 66. M. L. Soares et al., Haplotypes and DNA sequence variation support from the Helmholtz International Lab (Helmholtz
doi: 10.1038/s41557-022-00923-2; pmid: 35469007 within and surrounding the transthyretin gene: Genotype- Association, InterLabs0007) and the German Federal Ministry of
56. M. Lv, J. Zhao, Z. Deng, Y. Yu, Characterization of the phenotype correlations in familial amyloid polyneuropathy Education and Research (BMBF, 01DO22003). R.M. acknowledges
biosynthetic gene cluster for benzoxazole antibiotics A33853 (V30M) in Portugal and Sweden. Eur. J. Hum. Genet. 12, funding from the German Research Foundation (DFG) and German
reveals unusual assembly logic. Chem. Biol. 22, 1313–1324 225–237 (2004). doi: 10.1038/sj.ejhg.5201095; Center for Infection Research (DZIF). Author contributions:
(2015). doi: 10.1016/j.chembiol.2015.09.005; pmid: 26496684 pmid: 14673473 Conceptualization: C.F., F.X., and R.M. Methodology: C.F., F.X., H.Z.,
57. T. Horch et al., Alternative benzoxazole assembly discovered in 67. T. Yokoyama, Y. Kosaka, M. Mizuguchi, Crystal structures and X.W. Investigation: C.F., F.X., H.Z., J.L., X.Y., M.N., A.A.A., A.K.,
anaerobic bacteria provides access to privileged heterocyclic of human transthyretin complexed with glabridin. J. Med. J.H., and O.V.K. Visualization: C.F., F.X., H.Z., and X.W. Funding
scaffold. Angew. Chem. Int. Ed. 61, e202205409 (2022). Chem. 57, 1090–1096 (2014). doi: 10.1021/jm401832j; acquisition: C.F. and R.M. Project administration: C.F., R.M., and
doi: 10.1002/anie.202205409; pmid: 35656913 pmid: 24422526 F.X. Supervision: C.F. and R.M. Writing – original draft: C.F., F.X., and
58. H. Suzuki, Y. Ohnishi, Y. Furusho, S. Sakuda, S. Horinouchi, 68. C. E. Bulawa et al., Tafamidis, a potent and selective H.Z. Writing – review and editing: C.F., F.X., R.M., H.Z., and X.W.
Novel benzene ring biosynthesis from C(3) and C(4) primary transthyretin kinetic stabilizer that inhibits the amyloid Competing interests: The authors declare that they have no
metabolites by two enzymes. J. Biol. Chem. 281, 36944–36951 cascade. Proc. Natl. Acad. Sci. U.S.A. 109, 9629–9634 (2012). competing interests. Data and materials availability: All tool
(2006). doi: 10.1074/jbc.M608103200; pmid: 17003031 doi: 10.1073/pnas.1121005109; pmid: 22645360 plasmids developed in this study will be deposited in Addgene
59. R. E. Viola, The central enzymes of the aspartate family of 69. M. Sun, X. Zhang, H. Hao, W. Li, C. Lu, Nocarbenzoxazoles (ID nos. 227507, 227508, and 227579 to 227583). All DNA sequences
amino acid biosynthesis. Acc. Chem. Res. 34, 339–349 (2001). A–G, benzoxazoles produced by halophilic Nocardiopsis of TDRs have been deposited in GenBank under accession nos.
doi: 10.1021/ar000057q; pmid: 11352712 lucentensis DSM 44048. J. Nat. Prod. 78, 2123–2127 (2015). OR339702 (Sav11), OR339701 (Sar13), and OR339703 (Sav17). All
60. H. Suzuki, Y. Furusho, T. Higashi, Y. Ohnishi, S. Horinouchi, doi: 10.1021/acs.jnatprod.5b00031; pmid: 26270803 DNA sequences of ACTIMOT toolkit plasmids have been deposited
A novel o-aminophenol oxidase responsible for formation 70. D. Du et al., Genome engineering and direct cloning of in GenBank under accession nos. OR264833 (pRel-Hyg),
of the phenoxazinone chromophore of grixazone. J. Biol. Chem. antibiotic gene clusters via phage ϕBT1 integrase-mediated OR264834 (pRel), OR264835 (pHelp), OR264836 (pCap-SG5-Hyg),
Note: This service is not intended for secure transactions such as banking, social media, email, or purchasing. Use at your own risk. We assume no liability whatsoever for broken pages.
Alternative Proxies: