Diisonitrile Natural Product SF2768 Functions As a Chalkophore That

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Diisonitrile natural product SF2768 functions as a chalkophore that mediates copper acquisition in Streptomyces thioluteus Lijuan Wang, Mengyi Zhu, Qingbo Zhang, Xu Zhang, Panlei Yang, Zihui Liu, Yun Deng, Yiguang Zhu, Xueshi Huang, Li Han, Shengqing Li, and Jing He ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00897 • Publication Date (Web): 13 Nov 2017 Downloaded from http://pubs.acs.org on November 18, 2017

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ACS Chemical Biology

1

Diisonitrile natural product SF2768 functions as a chalkophore that mediates

2

copper acquisition in Streptomyces thioluteus

3

Lijuan Wang,1# Mengyi Zhu,1# Qingbo Zhang,2 Xu Zhang,1 Panlei Yang,1 Zihui Liu,3 Yun Deng,1

4

Yiguang Zhu,2 Xueshi Huang,4 Li Han,4 Shengqing Li3 and Jing He1*

5

1

6

Huazhong Agricultural University, Wuhan 430070, China

7

2

8

Microbiology, Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute

9

of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, P. R.

National Key Laboratory of Agricultural Microbiology, College of Life Science and Technology,

CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, RNAM Center for Marine

10

China

11

3

12

University, Wuhan 430070, China

13

4

14

University, Shenyang 110819, China

15

#These authors contributed equally to this work.

16

*For Correspondence: E-mail: [email protected]; Tel./Fax: +86-27-87280670

State Key Laboratory of Agricultural Microbiology, College of Science, Huazhong Agricultural

Institute of Microbial Pharmaceuticals, College of Life and Health Sciences, Northeastern

1

ACS Paragon Plus Environment


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1

Abstract

2

A non-ribosomal peptide synthetase (NRPS) gene cluster (sfa) was identified in

3

Streptomyces thioluteus to direct the biosynthesis of the diisonitrile antibiotic SF2768.

4

Its biosynthetic pathway was reasonably proposed based on bioinformatics analysis,

5

metabolic profiles of mutants and the elucidation of the intermediate and shunt

6

product structures. Bioinformatics-based alignment found a putative ATP-binding

7

cassette (ABC) transporter related to iron import within the biosynthetic gene cluster,

8

which implied that the product might be a siderophore. However, characterization of

9

the metal-binding properties by high-resolution electrospray ionization mass

10

spectrometry (HR-ESI-MS), metal-ligand titration, thin-layer chromatography (TLC)

11

and chrome azurol S (CAS) assays revealed that the final product SF2768 and its

12

diisonitrile derivatives specifically bind copper, rather than iron, to form stable

13

complexes. Inductively coupled plasma mass spectrometry (ICP-MS) analysis

14

revealed that the intracellular cupric content of S. thioluteus significantly increased

15

upon incubation with the copper-SF2768 complex, direct evidence for the copper

16

acquisition function of SF2768. Further in vivo functional characterization of the

17

transport elements for the copper-SF2768 complexes not only confirmed the

18

chalkophore identity of the compound but also gave initial clues into the copper

19

uptake mechanism of this non-methanotrophic microorganism.

2


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1

Introduction

2

The essential trace element copper usually serves as a catalytic or structural cofactor

3

in a variety of bacterial cuproproteins, including cytochrome oxidase, NADH

4

dehydrogenase, multicopper oxidase (MCO) and particulate methane monooxygenase

5

(pMMO)1-5. By analogy with iron-binding siderophores, a natural product produced

6

by bacteria that chelates and transports extracellular copper is referred to as a

7

“chalkophore”6-8.

8

corresponding copper carriers are expected to universally acquire essential copper

9

from the environment. However, the peptide-based methanobactins are the only

10

well-characterized chalkophores that mediate environmental copper acquisition, in

11

contrast to the widely discovered siderophores9, 10.

12

Methanobactin (Mbn), isolated from Methylosinus trichosporium OB3b, is the first

13

example of this kind of post-translationally modified peptide6. Mbn is produced to

14

fulfil the copper demand of pMMO, a copper-dependent metalloenzyme responsible

15

for the crucial oxidation of methane to methanol in M. trichosporium OB3b11, 12. Mbn

16

binds both Cu(II) and Cu(I) by the oxazolone and thioamide groups, reducing Cu(II)

17

to Cu(I) once the copper ion is chelated6,

18

comprising a TonB-like receptor, MbnT, and a periplasmic binding protein, MbnE,

19

was recently identified in the import of the intact Cu(I)-Mbn complex into M.

20

trichosporium OB3b11, 16. This result constituted the first insight into Cu(I)-Mbn

21

recognition and internalization in methanotrophic bacteria. Moreover, the biosynthetic

22

pathway of Mbn was preliminarily clarified by the identification of its precursor

Since

cuproproteins

are ubiquitous

in

microorganisms,

10, 13-15

. Efficient transport machinery

3



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1

peptide and functional research on post-modification enzymes17-19. In addition to the

2

characterization of the Mbn biosynthetic pathway, its copper chelating ability and the

3

transport of the Cu-Mbn complex strengthened the evidence for its chalkophore

4

identity. Production of Mbn-like compounds was shown to be widespread in

5

methanotrophic bacteria8, 10, 19-21. Similar molecules are considered to play ancillary

6

physiological roles in oxidative stress defence22 and metal detoxification23,

7

addition to their roles in copper capture. Further analysis of genome sequence

8

databases revealed that the Mbn operon is not rare in non-methanotrophic bacteria, as

9

can be expected considering the ubiquitous role played by copper as a cofactor in

10

many important enzymes25, 26. However, although many valuable insights into copper

11

trafficking in methanotrophs have been acquired in the past decade, the mechanism by

12

which non-methanotrophic bacteria collect copper from environment has barely been

13

addressed27.

14

Genome sequencing of a prolific actinomycetes strain, Streptomyces thioluteus DSM

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40027, a producer of aureothin28 and dithiolopyrrolones29, revealed at least 40 poorly

16

studied orphan biosynthetic gene clusters. These clusters include a putative

17

nonribosomal peptide synthetase (NRPS) biosynthetic gene cluster that we

18

successfully cloned from a S. thioluteus genomic library. We identified the product of

19

this pathway as the diisonitrile compound SF2768, which was once considered a

20

cryptic antibiotic whose biosynthesis was triggered by exogenous polyether

21

compounds in other streptomycetes30. Further functional analysis of the cluster by

22

mutagenesis not only revealed the putative biosynthetic pathway but also resulted in 4


24

in

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the production of several analogues of SF2768. In silico homology sequence analysis

2

of the cluster suggested that the product might act as a siderophore since a putative

3

ATP-binding cassette (ABC) transporter related to iron import was observed in the

4

cluster. However, the results of different in vitro and in vivo experiments such as

5

metal-ligand complex detection through high-resolution electrospray ionization mass

6

spectrometry (HR-ESI-MS), metal-ligand titrations, thin-layer chromatography (TLC)

7

and chrome azurol S (CAS) assays revealed that SF2768 specifically binds

8

extracellular copper rather than iron and that the complex is transported into S.

9

thioluteus, suggesting a chalkophore function of this diisonitrile. Moreover, in vivo

10

functional characterization of a major facilitator superfamily (MFS) exporter and an

11

ABC transporter within the cluster pointed to diisonitrile efflux and copper-SF2768

12

complex internalization functions that constituted a diisonitrile-mediated copper

13

acquisition system.

5



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Results and discussion

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Identification of the biosynthetic gene cluster for SF2768

3

A cosmid genomic library of S. thioluteus was constructed in the integrative vector

4

pJTU2554, and all clones were heterologously expressed in the host Streptomyces

5

lividans ZX131. A transformant of this strain, S. lividans::p13A, which harboured the

6

cosmid p13A (containing orf1-28, Figure 1), showed antimicrobial activity against the

7

gram-positive bacterium Bacillus subtilis 168. Subsequent subcloning experiments

8

into the vector pJTU2554, yielding the new cosmid p13C containing orf12-28,

9

conferred the same antimicrobial activity as p13A. This result indicated that this 24 kb

10

region (sfa, GenBank accession No. KY427327, Figure 1) was sufficient to generate

11

the bioactive product. Large-scale fermentation of S. lividans::p13C led to the

12

isolation of compound 1 (obsd m/z 337.1868 [M+H]+, calcd for m/z 337.1870

13

[M+H]+ Figure S1), a colourless oily substance. Detailed analyses by HR-ESI-MS

14

and nuclear magnetic resonance (NMR) data disclosed that compound 1 had the same

15

chemical structure as a known diisonitrile metabolite, SF276830,

16

reported to be present as a mixture of two anomers, which was consistent with the

17

double peak at m/z 337.1870 in the extracted ion chromatogram (EIC) shown in

18

Figures 1 and 4B.

19

The involvement of each encoded sequence in the sfa cluster in the biosynthesis of

20

compound 1 was subsequently investigated by PCR-targeting gene inactivation

21

according to a reported strategy33. The EIC traces at m/z 337.1870 from HR-ESI-MS

22

analyses showed that the inactivated mutants ∆sfaA, ∆sfaB, ∆sfaC, ∆sfaD and ∆sfaE 6


32

. SF2768 was

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1

were no longer capable of producing compound 1, while the other mutants persisted

2

in production, demonstrating the indispensable roles of these five genes in compound

3

1 biosynthesis. In contrast to the other four disrupted strains, the inactivated mutant

4

∆sfaE still displayed antimicrobial activity against B. subtilis. Large-scale

5

fermentation of the mutant ∆sfaE was therefore conducted, and two bioactive

6

analogues, compounds 3 (obsd m/z 323.2076 [M+H]+, calcd for m/z 323.2078

7

[M+H]+ Figure S2) and 4 (obsd m/z 365.2175 [M+H]+, calcd for m/z 365.2183

8

[M+H]+ Figure S3) were isolated by a bioassay-guided fractionation of ethyl acetate

9

extract. The chemical structures of compounds 3 and 4 were determined from NMR

10

analyses. Compound 3 was the pyran ring-opened derivative of compound 1, and

11

compound 4 was an O-acetylated product of compound 3 (Figure S4).

12

Proposed biosynthetic pathway of the diisonitrile compounds

13

Elucidation of the above structures, combined with a sequence analysis of the

14

involved genes, enabled the proposal of a diisonitrile compound SF2768 biosynthetic

15

pathway (Figure 2). A BLAST search (Table 1) revealed that three crucial

16

NRPS-related enzymes, SfaBCD of sfa, shared high sequence homology with

17

MxcEFG of the well-studied biosynthetic cluster for catecholate siderophore

18

myxochelin A biosynthesis (mxc) in Stigmatella aurantiaca; MxcEFG constituted an

19

assembly line for the construction of the myxochelin peptide chain34, 35. In vitro

20

substrate specificity assays of the core NRPS enzyme SfaD confirmed that lysine was

21

most favoured substrate (Figure S5), which is consistent with results from MxcG and

22

supports the possibility that these two enzymes shared similar assembly logic. The 7



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1

AMP ligase SfaB/MxcE and the peptidyl carrier protein SfaC/MxcF were thus

2

expected to be responsible for the adenylation and transfer of the acyl chain building

3

blocks, and SfaD/MxcG would subsequently mediate skeleton assembly and reductive

4

release.

5

The C-terminal NAD(P)H-dependent RED domain in NRPSs usually catalyses the

6

release of peptide chain by an alternative scheme that involves one or two turns of

7

reduction to produce an aldehyde or alcohol compound35-37. This domain inspired a

8

search for the corresponding aldehyde intermediate of compound 1 in the

9

heterologous expression and mutant strains, resulting in the discovery of another

10

metabolite, 2, in ∆sfaE. Although the isolation of compound 2 was hampered by its

11

low yield, the identification of this expected compound was confirmed by

12

HR-ESI-MS (Figure S6). Based on the structures of compounds 1, 2 and 3, we

13

propose that the post-NRPS δ-hydroxylation modification of the lysine residue was

14

performed by the hydroxylase SfaE, and then the hemiacetal spontaneously formed to

15

afford the final product, 1. When SfaE was absent, the accumulating aldehyde 2 was

16

reduced again by the RED domain to generate the alcohol 3. Compound 4 was found

17

only in the ethyl acetate extract and not in the fermentation broth of ∆sfaE, implying

18

that compound 4 might be an artefact acquired through O-acetylation during

19

extraction.

20

Since the functions of SfaBCDE had been assigned, the last essential ORF, SfaA, was

21

assumed to be involved in the biosynthesis of possible 3-isocyanobutanoic acid

22

building blocks. All prior validated isonitrile synthases belong to the PvcA/IsnA 8


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1

family of proteins that catalyse isonitrile group formation by transferring the C2 of

2

ribulose-5-phosphate to an α-amino group through redox reactions38-41. However,

3

SfaA shared no similarity with any members of the PvcA/IsnA family of proteins,

4

hinting that SfaA might mediate an isonitrile incorporation mechanism that was not

5

yet clear. While this manuscript was in preparation, Zhang and co-workers reported

6

that the heterologous expression of two similar gene clusters, mma and sco, from

7

Mycobacterium marinum and Streptomyces coeruleorubidus, respectively, in

8

Escherichia coli produced different isonitrile lipopeptides, including compounds 3

9

and 442. Although direct experimental evidence was still missing, they also proposed

10

the involvement of MmaE/ScoE in isonitrile biosynthesis. Since SfaA and MmaE are

11

similar, we hypothesized that their catalytic mechanism, which needs to be proven in

12

vitro, might be identical. Another detail that cannot be ignored was that they verified

13

that thioesterases MmaD/ScoD were essential but that disruption of the homologue

14

Orf16 within sfa did not abolish the production of compound 1 (Figure 1). If sfa and

15

mma/sco share an identical biosynthetic route, a possible explanation is that a

16

substitute for Orf16 that is endogenous to the host, Streptomyces lividans ZX1,

17

participates in the biosynthesis of compound 1. However, the suspected substitute

18

cannot be found in S. lividans through genome scanning (data not shown), implying

19

that a thioesterase-like protein may not be essential in our case. This issue needs to be

20

clarified in further in vitro investigation.

21

Even though gene clusters similar to sfa are widespread among different

22

actinomycetes such as Streptomyces, Mycobacterium, Kutzneria, Rhodococcus, 9



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1

Actinomadura and Nocardia, the homologues of SfaE are found only in several

2

streptomycetes

3

Mycobacterium, might therefore only produce compounds 3 and 4. This hypothesis is

4

consistent with the results that Zhang and co-workers obtained.

5

Metal chelation properties of SF2768 and its derivatives

6

The wide distribution of gene clusters homologous to sfa suggests that their

7

corresponding products might play an important role in the physiology of these

8

actinomycetes despite most of them having no assigned functions. A putative iron

9

ABC transporter encoded by genes orf19-21 (Table 1) within the sfa cluster, as well as

10

the known affinity of the isonitrile group towards transition elements43, 44, stimulated a

11

search for metal-SF2768 complexes in the fermentation broth by HR-ESI-MS when

12

sixteen different metals were added (Cu(I), Cu(II), Al(III), Ca(II), Cd(II), Cr(II),

13

Fe(II), Fe(III), Mg(II), Mn(II), Mo(V), Pb(II), Zn(II), Co(II), Hg(II) and Ni(II), Figure

14

4). The EIC traces indicated the formation of a copper-SF2768 (Cu-1) complex after

15

supplementation with either Cu(I) or Cu(II), while no change was observed in other

16

samples. Compound 1 unexpectedly degraded upon incubation with Co(II), Hg(II)

17

and Ni(II), resulting in no complex.

18

HR-ESI-MS-based titration experiments performed in vitro with purified 1, 3 and 4 in

19

the presence of different concentrations of Cu(II) determined the chelation

20

stoichiometry of the complexes. Figure 5C shows that compound 1 was completely

21

exhausted upon the addition of 0.5 equivalents of Cu(II), which indicated that all the

22

apo-1 could be fully loaded with copper and form complexes with a stoichiometry of

in

silico

(Figure

3).

The

other

actinomycetes,

10


including

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1

1:2 (Cu:1). The measured m/z value (735.2898) of the Cu-1 complex was consistent

2

with that of a singly charged cupric complex of the form [2M1+Cu(I)]+, suggesting the

3

reduction of Cu(II) to Cu(I) during the chelation process (Figure 5AB). The presence

4

of the prominent M+2 peak at m/z 737.2892 that was expected from

5

height was approximately one-half the height of the base peak, coincided with the

6

natural isotope abundance of copper (Figure 5B). A new spot, representing the

7

copper-dimerized 1 complex, with a Rf (retention factor) value lower than that of

8

monomeric 1 appeared in the TLC plate (Figure 5D), which corroborated the

9

HR-ESI-MS result in Figure 5B. Analogues 3 and 4 behaved similarly in this

65

Cu, whose

10

chelation-based ligand dimerization and metal reduction.

11

It was surprising that the corresponding diisonitrile metabolites formed complexes

12

with Cu(I) and Cu(II) rather than Fe(II) or Fe(III) in the metal binding experiments

13

because sfa had been predicted to be a potential siderophore biosynthetic gene cluster

14

according

15

ion-sequestering small-molecule compounds, are produced and secreted by almost all

16

aerobic bacteria, fungi and higher plants to satisfy the iron demand of the producers45.

17

Moreover, some siderophores bind non-ferric metal ions with different affinities27 to

18

fulfil additional physiological functions such as tolerance to heavy metals46,

19

resistance to host redox defence48, 49 and other potential functions. Nevertheless, their

20

highest affinity was for iron. To further rule out the possibility of diisonitrile

21

compounds binding ferric iron as siderophores and to further determine their genuine

22

role, we conducted Fe- and Cu-CAS assays50, 51, which constitute canonical tests to

to

bioinformatics

analyses.

Siderophores,

11


as

well-known

ferric

47

,


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1

identify siderophores and chalkophores, respectively. A colour change clearly

2

confirmed the ability of compounds 1, 3 and 4 to displace CAS from copper, but not

3

iron (Figure 5E). The three diisonitrile compounds chelated copper with EC50 values

4

approximately 4-fold higher than that of EDTA but 3-fold lower than that of

5

2,2’-bipyridine (Figure 5F). These results collectively supported the potential role of

6

diisonitriles in S. thioluteus as chalkophores and not as siderophores. Previously

7

reported chalkophores or copper-binding natural products usually possess functional

8

groups such as nitrogen heterocycles (oxazolone, imidazolone, thiazole, etc.) or

9

thioamide moieties10, 49, 52. This study is the first demonstrating that the isonitrile

10

groups of compound 1 and its analogues could act as a potential copper-binding motif.

11

Copper complex uptake assay

12

A compound that is defined as a chalkophore should be capable of not only binding

13

Cu(II) or Cu(I) but also shuttling the copper ion into cells. To verify the chalkophore

14

identity of compound 1, we employed ICP-MS to measure the intracellular cupric

15

concentration of wild-type S. thioluteus upon incubation with 5 µM freshly made

16

copper complexes (Cu-1, Cu-3 and Cu-4) for 2 hours, and cells without treatment

17

were used as the control. Cells treated with Cu-3 and Cu-4 contained similar

18

intracellular cupric ion concentrations (9.44±0.86 and 8.91±0.69 ppb Cu/mg dry cells)

19

as the control (8.60±0.59 ppb Cu/mg dry cells), while cells incubated with Cu-1

20

contained significantly more (13.17±1.13 ppb Cu/mg dry cells), which demonstrated

21

that Cu-1 was effectively imported into S. thioluteus (Figure 6A, Table S4). We

22

hypothesized that the complexes Cu-3 and Cu-4 were not efficiently internalized 12


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1

because their subtle structural differences resulted in the inability to be effectively

2

recognized by the receptor responsible for import. These data provided direct

3

evidence that SF2768 chelated extracellular copper and promoted its intake into S.

4

thioluteus.

5

In vivo functional characterization of the transport elements

6

Bioinformatics predicted that the putative ABC transporter Orf19-21 is a candidate

7

transport element for the complex Cu-1 (Table 1). The function of Orf19-21 was

8

investigated by a feeding the cells

9

possesses two stable isotopes in a 2.2:1 ratio (63Cu:65Cu). This ratio is expected to be

10

changed by the addition of 65Cu in this assay. The intracellular concentrations of 63Cu

11

and

12

gene-inactivated mutant strain ∆orf19-21, with and without incubation with 65Cu-1 at

13

a final concentration of 5 µM, were determined by ICP-MS. Although the intracellular

14

63

15

decrease was smaller in ∆orf19-21 than in S. lividans::p13C. This result indicated a

16

loss of function of the putative ABC transporter Orf19-21 that led to reduced uptake

17

of the isotopic complex (Figure 6B, Table S5). The direct interaction between Cu-1

18

and Orf19 needs to be identified through future biochemical and structural biological

19

studies.

20

We also assessed the concentration of compound 1 in the intracellular and supernatant

21

fractions of the heterologous strain S. lividans::p13C and the mutant strain ∆orf12 (in

22

which the gene encoding the putative MFS transporter that might mediate drug efflux

65

Cu-1 in vivo. Naturally occurring copper

65

Cu in the heterologous expression strain S. lividans::p13C and the

Cu:65Cu ratio of both strains declined upon

65

Cu-1 addition, the extent of the

13



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1

was inactivated). Figure 6C shows that the extracellular/intracellular content ratio of

2

compound 1 was significantly decreased when orf12 was knocked out,

3

unambiguously demonstrating that Orf12 was involved in the export of the proposed

4

chalkophore in the model of the SF2768-mediated copper acquisition system.

5

Model of SF2768-mediated copper acquisition system

6

After the elucidation of the diisonitrile natural product SF2768 biosynthetic pathway,

7

we demonstrated the chalkophore activity of this product in S. thioluteus by several

8

experiments. Further identification of the transport elements for the copper-SF2768

9

complex provided new clues to a copper uptake mechanism in a non-methanotrophic

10

strain. A primary model is proposed based on the above data to showcase this

11

diisonitrile-mediated copper acquisition system of S. thioluteus. The metabolite

12

SF2768 is synthesized by the enzymes encoded by the sfa operon and then exported

13

by the MFS transporter Orf12. SF2768 scavenges environmental copper by chelation

14

of Cu(II) and its reduction to Cu(I) ions. The copper-SF2768 complex is then

15

internalized by the ABC transporter Orf19-21. The chelated copper is released via an

16

unknown mechanism and binds cuproproteins. The copper-free SF2768 is exported

17

again and reused.

18

Copper plays a crucially important role in physiological development and secondary

19

metabolite production in Streptomyces species53, 54. The morphological development

20

of many streptomycetes is completely dependent on the bioavailability of copper

21

ions54. Moreover, many proteins in Streptomyces require copper for function,

22

including laccases (for oxidizing a variety of organic and inorganic substrates)55, 56, 14


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1

lipocyanins (involved in electron-transfer processes)57, and multicopper oxidase58 and

2

tyrosinase-like copper-containing monooxygenases (the production of secondary

3

metabolites)59. The wild-type strain S. thioluteus also exhibits distinct denitrifying

4

activity, and a copper-containing dissimilatory nitrite reductase was identified in its

5

denitrification, which may contribute to the global nitrogen cycle60. Despite copper

6

being known to participate in the life cycle of Streptomyces, a copper uptake system

7

was unnoticed and has not been well characterized in bacteria due to copper’s

8

potential toxicity and the low bacterial intracellular requirement for copper27, 61. This

9

relatively unstudied system stands in contrast to the well-studied efflux systems that

10

respond to high concentrations of copper. In our study, a novel copper chelator used to

11

uptake copper from the environment was discovered. The potential transport systems

12

involved in SF2768 export and Cu-SF2768 complex import were also identified. This

13

study provides a new understanding of molecular mechanisms of copper trafficking

14

and homeostasis in non-methanotrophic bacteria. The regulation mechanism by which

15

the SF2768-mediated copper acquisition system maintains copper homeostasis in S.

16

thioluteus will be addressed in our future research.

15



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1

Methods

2

Detailed experimental procedures are described in Supporting Information.

3

Conflict of interest

4

The authors declare no conflict of interest.

5

Acknowledgements

6

We thank G. Kenney from Northwestern University for the helpful discussion about

7

the oxidation state of the complexed copper. This project was supported by the

8

National Natural Science Foundation of China (31270136) and the Fundamental

9

Research Funds for the Central Universities (2009PY006 and 2662014PY053).

10

Supporting Information

11

Detailed experimental procedures; Supplementary figures and tables; Details of

12

strains, plasmids and primers; HR-ESI-MS and NMR spectra of the compounds. This

13

material is available free of charge via the Internet at http://pubs.acs.org.

14

References

15 16 17 18 19 20 21 22 23 24 25 26 27 28

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

Figure legends: Figure 1. Genetic organization of the SF2768 biosynthetic gene cluster (sfa) in Streptomyces thioluteus and the metabolite profile of the gene inactivated strains in this study. The open reading frames involved in biosynthesis and transport are colored. The EIC traces of compound 1 (m/z 337.1870) in the heterologous expression and mutant strains are shown on the right. Observation of the double peak at m/z 337.1870 is consistent with the presence of two anomers of compound 1 as reported. Figure 2. Proposed biosynthetic pathway of the diisonitrile natural products in this study. Abbreviations: C, condensation domain; A, adenylation domain; PCP, peptidyl carrier protein; RED, reductase; 2-OG, α-ketoglutarate. The numbers 1 to 4 denote the corresponding compounds. Figure 3. Homologous gene clusters of sfa in different actinomycetes. The negative sign represents the product of corresponding gene cluster has not yet been reported. Figure 4. HR-ESI-MS detection of the metal-compound complexes in the fermentation broths when different metals were added. Figure 5. (A) HR-ESI-MS analyses of the copper-compound complexes. After incubation with Cu(II), the EIC traces of the diisonitriles (black lines) disappeared and the putative copper-compound complexes were tracked respectively ([2M1+Cu]+, orange; [2M3+Cu]+, red; [2M4+Cu]+, blue). (B) Mass spectra of the complexes with distinct isotopic distribution for copper. (C) Titration of the diisonitriles and Cu(II). Relative abundance of the copper-compound complex was measured and normalized by integration of ion intensities. (D) RP-TLC for detection of the complexes. (E) Concentration dependent Cu-CAS and Fe-CAS assays. (F) Copper chelating activities (EC50) of 1, 3, 4 and positive controls (2,2’-bipyridine and EDTA). The Cu-CAS solution used in chelating activities assay was ten-fold diluted. Figure 6. (A) In vivo characterization of copper-diisonitriles complexes import in S. thioluteus (p=0.005, n=6). (B) In vivo characterization of 65Cu-1 import in S. lividans::p13C and ∆orf19. The cells without isotopic incubation were used as controls. Uptake of 65Cu-1 led to a decreased 63 Cu:65Cu ratio. Results shown are the mean of six replicates with error bars representing SEM (p=0.0148, n=6). (C) SF2768 level in the extracellular (left) and intracellular (middle) fractions and their ratio (right) of the heterologous strain S. lividans::p13C and ∆orf12. Results shown are the mean of three replicates with error bars representing SEM (p=0.0101, n=3). P-values were calculated by two-tailed unpaired t-test using a 95% confidence interval.

21



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Table 1. Deduced functions of the open reading frames in the sfa cluster (GenBank accession No. KY427327). Size ORF

Homologous protein/Genbank accession Deduced functions

(aa)

(Identity/Positives %) SwissProt hits

3 4 5 6 7

Orf12

484

Cephamycin export protein

CmcT/Q04733.1 (40/55)

Orf13

365

3-oxoacyl-ACP synthase

FabH3/O54151.1 (60/72)

Orf14

78

Acyl carrier protein

AcpP/Q47NG1.1 (37/63)

Mxc

Mma

SfaA

295

Tau, Putative dioxygenase

Mb0100/P67756.1 (42/61)

MmaE (43/60)

Orf16

177

FcoT-like thioesterase

Mb0101/P64686.1 (37/53)

MmaD (39/52)

SfaB

530

AMP ligase

MxcE (26/41)

MmaC (32/47)

SfaC

85

Phosphopantetheine attachment domain

MxcF (51/67)

MmaB (28/38)

SfaD

1426

Non-ribosomal peptide synthetase

MxcG (53/67)

MmaA (30/43)

Orf19

330

Iron ABC transporter substrate-binding protein

WP_057576127.1 (73/83)

Orf20

320

Iron chelate uptake ABC transporter

YvrB/O34451.1 (43/62)

Orf21

267

Iron(III) dicitrate transport ATP-binding protein

FecE/P15031.1 (45/59)

SfaE

253

Asparaginyl beta-hydroxylase

Asph/Q8BSY0.1 (31/49)

Orf23

257

Cyclohexyl-isocyanide hydratase

InhA (32/50) Q8G9F9.1

Orf24

333

ABC transporter substrate-binding protein

WP_026219843.1 (75/83)

Orf25

511

Ribose import ATP-binding protein

RbsA/Q9K6J9.1 (37/57)

Orf26

347

L-arabinose transport system permease protein

AraH/P0AE26.2 (35/52)

Orf27

503

Serine protease

WP_052860930.1 (72/80)

Orf28

230

ECF RNA polymerase sigma factor

SigL/H8EXN1.1 (39/50)

The compared gene clusters include Mxc (Stigmatella aurantiaca, Genbank: AF299336) and Mma (Mycobacterium marinum, MmaE: ACC38728, MmaD: ACC38727, MmaC: ACC38726, MmaB: ACC38725 and MmaA: ACC38724). SfaD, MxcG and MmaA were predicted to load lysine based on conserved amino acid residues for the substrate specificity62, SfaD: DAEDVGTV, MxcG: DAEDIGTV, MmaA: DIEDVGSV.

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Figure 1. Genetic organization of the SF2768 biosynthetic gene cluster (sfa) in Streptomyces thioluteus and the metabolite profile of the gene inactivated strains in this study. The open reading frames involved in biosynthesis and transport are colored. The EIC traces of compound 1 (m/z 337.1870) in the heterologous expression and mutant strains are shown on the right. Observation of the double peak at m/z 337.1870 is consistent with the presence of two anomers of compound 1 as reported. 117x98mm (300 x 300 DPI)



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Figure 2. Proposed biosynthetic pathway of the diisonitrile natural products in this study. Abbreviations: C, condensation domain; A, adenylation domain; PCP, peptidyl carrier protein; RED, reductase; 2-OG, αketoglutarate. The numbers 1 to 4 denote the corresponding compounds. 59x25mm (300 x 300 DPI)


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Figure 3. Homologous gene clusters of sfa in different actinomycetes. The negative sign represents the product of corresponding gene cluster has not yet been reported. 84x51mm (300 x 300 DPI)



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Figure 4. HR-ESI-MS detection of the metal-compound complexes in the fermentation broths when different metals were added. 112x187mm (300 x 300 DPI)


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Figure 5. (A) HR-ESI-MS analyses of the copper-compound complexes. After incubation with Cu(II), the EIC traces of the diisonitriles (black lines) disappeared and the putative copper-compound complexes were tracked respectively ([2M1+Cu]+, orange; [2M3+Cu]+, red; [2M4+Cu]+, blue). (B) Mass spectra of the complexes with distinct isotopic distribution for copper. (C) Titration of the diisonitriles and Cu(II). Relative abundance of the copper-compound complex was measured and normalized by integration of ion intensities. (D) RP-TLC for detection of the complexes. (E) Concentration dependent Cu-CAS and Fe-CAS assays. (F) Copper chelating activities (EC50) of 1, 3, 4 and positive controls (2,2’-bipyridine and EDTA). The Cu-CAS solution used in chelating activities assay was ten-fold diluted. 87x54mm (300 x 300 DPI)



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Figure 6. (A) In vivo characterization of copper-diisonitriles complexes import in S. thioluteus (p=0.005, n=6). (B) In vivo characterization of 65Cu-1 import in S. lividans::p13C and ∆orf19. The cells without isotopic incubation were used as controls. Uptake of 65Cu-1 led to a decreased 63Cu:65Cu ratio. Results shown are the mean of six replicates with error bars representing SEM (p=0.0148, n=6). (C) SF2768 level in the extracellular (left) and intracellular (middle) fractions and their ratio (right) of the heterologous strain S. lividans::p13C and ∆orf12. Results shown are the mean of three replicates with error bars representing SEM (p=0.0101, n=3). P-values were calculated by two-tailed unpaired t-test using a 95% confidence interval. 96x84mm (300 x 300 DPI)


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52x32mm (300 x 300 DPI)


Diisonitrile Natural Product SF2768 Functions As a Chalkophore That

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