biosynthetic pathway connects cryptic ribosomally ... · biosynthetic pathway connects cryptic...

Cell Chemical Biology, Volume 23

Supplemental Information

Biosynthetic Pathway Connects Cryptic Ribosomally

Synthesized Posttranslationally Modified Peptide

Genes with Pyrroloquinoline Alkaloids

Peter A. Jordan and Bradley S. Moore

Figure S1, related to “Time-Course of Ammosamides A-C Production” in the Experimental Procedures section:

LCMS analysis of (A) S. coelicolor M512-pCAP01-amm and (B) Streptomyces sp. CNR-698 culture time-course.

Chromatograms at 254 nm are provided above for each time point. Peaks were verified by retention time in references

to standards (bottom peaks), UV absorbance, and mass spectrometry. Deviations in retention time between (A) S.

coelicolor M512-pCAP01-amm and (B) Streptomyces sp. CNR-698 culture time-courses were attributed to two

different Phenomenex Luna 5 μm C18(2) 100 Å, 100 x 4.6 mm columns used for each analysis. The identity of

ammosamides A-C were verified with standards, UV absorbance and MS analysis.

Figure S2, related to “Chemical Complementation of M512-pCAP01/ammamm3” in the Experimental

Procedures section: LCMS analysis of S. coelicolor M512 pCAP01/ammamm3 mutants chemically complemented

with 4 mM rac-6-chloro-tryptophan, “6-Cl-Trp” (2mM of each enantiomer). Chromatograms at 254 nm are provided

above. Peaks for ammosamides A and B are labeled with 1 and 2, respectively, and 6-chloro-tryptophan is indicated

with an asterisk (*). The top trace corresponds to a media blank and the bottom two traces correspond to cultures

complemented with rac-6-chloro-tryptophan, whereas the remaining samples correspond to cultures treated with L-

tryptophan. The decomposition of 6-chloro-tryptophan is evident in the bottom two traces. The absence of either

ammosamide A or B was verified by retention time with respect to the positive control (pCAP01/amm), UV

absorbance, and mass spectrometry. In particular, the peak corresponding to the same retention time as 1 in the bottom

two traces was not related to ammosamide products based upon UV-Vis profile and MS data.

Figure S3, related to “Secondary Metabolite Profiling of S. coelicolor M512-pCAP01/amm and mutants” in the

Experimental Procedures section: Summary of gene deletions to ‘core’ biosynthetic genes in the amm cluster that

share close homology to lymphostin biosynthesis. The absence of ammosamide A-C was verified by retention time

with respect to a positive control (pCAP01/amm), UV absorbance, and mass spectrometry. Related metabolites were

not observed by UV absorbance or extracting masses.

Figure S4, related to figure 2: A. Alignment of predicted precursor peptides from the from gram-positive and gram-

negative bacteria. MUSCLE alignment performed in Geneious 8.1.5. Abbreviations: LD = lantibiotic dehydratase;

O = oxidoreductase; M = methyl transferase; T = transporter. Entry 2 corresponds to an Amm6 homologue found in

a streptomycete genome carrying the lym biosynthetic gene cluster. Entries 3-5 correspond to Amm6 homologues

found in the lymphostin gen clusters from the genus Salinispora. B. Predicted biosynthetic gene clusters from Bacillus

halodurans C-125 (NC_002570, ORF locus tags ctg1_2032-2062) and Desmospora sp. 8437 (NZ_AFHT00000000,

ORF locus tags HMPREF9374_RS06800-RS06705).

Name Predicted Function Size

(a.a.) Predicted Function of Closest Homologue % ID Notes

xan1 MFS Transporter 412 Salinispora arenicola, MFS Transporter 52%

xan2 HP 44 HP, Salinispora multispecies, Streptomyces

sp. CNR698

64%,

49%

lym,

amm

xan3 TPR Repeat Protein 547 HP, Salinispora multispecies, Streptomyces

sp. CNR698

40%,

41%

lym,

amm

xan4 LD 847 LD, Salinispora multispecies, Streptomyces

sp. CNR698

42%,

41%

lym,

amm


sp. CNR698

34%,

39%

lym,

amm

xan6 HP 525 HP, Cystobacter violaceus, Salinispora

arenicola

36%,

34% lym


sp. CNR698

39%,

38%

lym,

amm


sp. CNR698

31%,

29%

lym,

amm


sp. CNR698

31%,

32%

lym,

amm


sp. CNR698

41%,

42%

lym,

amm

xan11 LD 774 LD, Saccharothrix sp. NRRL B-

16314/Salinispora

29%,

28%

lym,

amm

xan12 Flavin Reductase 188 Flavin Reductase, Haliangium ochraceum

DSM 14365 40%

xan13 P 436 P, Salinispora multispecies, Streptomyces sp.

CNR698

34%,

34%

lym,

amm

xan14 P 440 P, Cystobacter violaceus 41%

xan15 Oxidoreductase

(FAD) 367

Oxidoreductase (FAD), Streptomyces sp.

CNR698 35%

lym,

amm

Table S1, related to figure 2: Predicted function of open reading frames (ORFs) in the xan locus of Streptomyces

xanthophaeus NRRL B-3004. Where appropriate, multiple homologues with their respective amino acid identities

are listed. Biosynthetic gene clusters that contain homologues to xan ORFs are indicated in the “Notes” column.

(Abbreviations: HP = hypothetical protein, LD = lantibiotic dehydratase, P = peptidase; lym = lymphostin BGC; amm

= ammosamide BGC.)

Figure S5, related to the characterization of compounds 7 and 8 in the Experimental Procedures section:

Scheme for the synthesis of compounds 7 and 8.

Primer sequences used in this work (Related to the Experimental Procedures section)

Name Purpose Sequence

F-amm-cap-up upstream capture arm ATGTTCAactagtGTCAGGCGAGCCTGATCGAG

R-amm-cap-up upstream capture arm CGTTGTTggatccCTGGCGTCCTTCCGGGGAAG

F-amm-cap-

dwn downstream capture arm ACGCCAGggatccAACAACGTGGACCGCTCCTG

R-amm-cap-

dwn downstream capture arm TATCGTActcgagGTGAGCGACCGCTTAGTACCTTCAG

F-TARscreen yeast colony PCR screening GACGAGGAGACCGGCTGGGT

R-TARscreen yeast colony PCR screening CCGGATGTGCCGCCGGATAC

F-pKY01Scr pkY01 screening and sequencing CTGACCGACGCGGTCCACAC

R-pKY01Scr pkY01 screening and sequencing TGGAAAGCGGGCAGTGAGCG

F-amm6LR amm6 -red primer CGGCCACCGCACAGCACCCCAGCGAACACAGGGAG

TCGCCATATGGCTCACGGTAACTGATGCCG

R-amm6LR amm6 -red primer CAGAAGGCTGGAAATCCGGGGGTCGGACGGGCGGT

TCACCATATGGGAACTTATGAGCTCAGCCA

F-amm7LR amm7 -red primer GCTCGCCCTCGCCCTCAAGGAATCGCGTTCATGGTG

AACGGTCGGGCTGGGAAGTTCCT

R-amm7LR amm7 -red primer GGCGCCAGTGTCCCTCCCCGCTCGTGGCAGCCATCG

TCAGTAGGCTGGAGCTGCTTCGA

F-amm8LR amm8 -red primer CCGCCCGTTCGACCTGATCAACTTCCAGGAGTGACG

ATGGGTCGGGCTGGGAAGTTCCT

R-amm8LR amm8 -red primer CGACGGTGGGCAGGACGTGCCAGGCGGGGGTGCTC

ATACGTAGGCTGGAGCTGCTTCGA

F-amm9LR amm9 -red primer CGAGTTCCGGCTGGCTCTGCGCTGGGAGACCGTATG

AGCGGTCGGGCTGGGAAGTTCCT

R-amm9LR amm9 -red primer GGACGATCAGCAGCTCGCTCATGCGGCCCGCCGCAG

GTAGTAGGCTGGAGCTGCTTCGA

F-amm11LR amm11 -red primer GCTCACCGCGGCCGGCGCCCTCCGGAGGACCCCATG

ACCGGTCGGGCTGGGAAGTTCCT

R-amm11LR amm11 -red primer CCGCCCTGCGGGCATGTTCGGCGAAGACGCGTATCA

CGGGTAGGCTGGAGCTGCTTCGA

F-amm18LR amm18 -red primer GCCCGCCGCGCCGGTCAGCGCCGGAGGGGTGCGGT

GACCGGTCGGGCTGGGAAGTTCCT

R-amm18LR amm18 -red primer GCTCCACCGCGGCGCGGCAACGGTCCCGGTGGCTCA

TCGGTAGGCTGGAGCTGCTTCGA

F-amm23LR amm23 -red primer ACGGTACGCCAACATCAGGCGAAGGAGTGACACGT

CGTGGGTCGGGCTGGGAAGTTCCT

R-amm23LR amm23 -red primer CAACAGGGAGATGGGCGATGTGGTGGTGCCGGAAA

ATCAGTAGGCTGGAGCTGCTTCGA

F-amm3LR amm3 -red primer GGAACCCTCCCTCGTACCCGTCAGGAAGGAAGCGTG

ACGGGTCGGGCTGGGAAGTTCCT

R-amm3LR amm3 -red primer CCGCCGGGGGCAGGACGGGCGTCCCCGTGCGGGTC

ATCGGTAGGCTGGAGCTGCTTCGA

F-amm4LR amm4 -red primer CGCGGGCGGTGGCGAGCGGGCGGGGGGCGTGCGAT

GACCGGTCGGGCTGGGAAGTTCCT

R-amm4LR amm4 -red primer CCCGGCCCGGCTCGGCTCAGCTCGGCTCAGCTCGGC

TCAGTAGGCTGGAGCTGCTTCGA

F-amm14LR amm14 -red primer TGGCGGAGAAAAGGACGGGTTCCCTTGCGCACTCAT

GACGGTCGGGCTGGGAAGTTCCT

R-amm14LR amm14 -red primer TGGTTCGGGCGCCCAGGATGACGTGGTCGATGTTCA

CCGGTAGGCTGGAGCTGCTTCGA

F-amm16LR amm16 -red primer GACCGACGACGGCCCCCTGCTGCTGGAGGTGCCGTG

ACCGGTCGGGCTGGGAAGTTCCT

R-amm16LR amm16 -red primer CGCTCAGGATCACGATCTCGGGCTCGGTGGTCGTCA

TGCGTAGGCTGGAGCTGCTTCGA

F-amm17LR amm17 -red primer CGCCCCGGACCCCATCCCACAGGAGCAGCCCGCATG

ACGGGTCGGGCTGGGAAGTTCCT

R-amm17LR amm17 -red primer CGGGGTCCGTGGGGTGTGCGGTACGGGTGGAGGTC

ACCGGTAGGCTGGAGCTGCTTCGA

F-amm19LR amm19 -red primer CGGCTGCGTCGTCTGGCCGGCGGAGGAGGGCCGAT

GAGCGGTCGGGCTGGGAAGTTCCT

R-amm19LR amm19 -red primer CAGGCCGGCCGAGGGGAGCGGGGAGCCGTCCCGCC

GTCAGTAGGCTGGAGCTGCTTCGA

F-amm6Scr amm6 KO screening CCGAATGCCGAGGATATGACCTGTGC

R-amm6Scr amm6 KO screening GACGCGGTAGTTCTGCTCCGACTC

F-amm7Scr amm7 KO screening GCGGAGATCGCCGCCGAGTC

R-amm7Scr amm7 KO screening CGACCTCCGGGTCTGTGAGGCC

F-amm8Scr amm8 KO screening CGGCTCAGCACCACCCACCC

R-amm8Scr amm8 KO screening GGCTCTTGCGGTGGCCGAGC

F-amm9Scr amm9 KO screening CGTCTTCGCCCATCTGGCGG

R-amm9Scr amm9 KO screening AGAGGGCGTCCAGTGCCTGT

F-amm11Scr amm11 KO screening CACCTTCGTCGCCGACCTGCG

R-amm11Scr amm11 KO screening CGTCGTGGACGTCGGCGCAG

F-amm18Scr amm18 KO screening CCCGATGGGAGTCGGCCTCT

R-amm18Scr amm18 KO screening CACTCCGGCCACCAGTTCGC

F-amm23Scr amm23 KO screening GTCGTTGCGGAGGCTGCTGT

R-amm23Scr amm23 KO screening TCGCTCGGCCATCACCTGGA

F-amm3Scr amm3 KO screening GGTCCTGTGGTGGTGCCTGC

R-amm3Scr amm3 KO screening GGGTTGGGCTCGGGAAAGCC

F-amm4Scr amm4 KO screening CGCAGGTGCTGGTCTCCCAC

R-amm4Scr amm4 KO screening CGGTGTCGGCGAGGGCTTAC

F-amm14Scr amm14 KO screening GTTCCTGCTGCGCGGCAAAC

R-amm14Scr amm14 KO screening GTCGGAGAGGACGGCCCAGT

F-amm16Scr amm16 KO screening GCCTCCTGGGAGACGCCCTC

R-amm16Scr amm16 KO screening CGGCGAAGACGGTCGGATGG

F-amm17Scr amm17 KO screening CGTTCAGCAACACCGCGCAC

R-amm17Scr amm17 KO screening GGACTTCCCGCACCATCGGC

F-amm19Scr amm19 KO screening CGTCCTGGTCGGCGAGATGC

R-amm19Scr amm19 KO screening CCGCAAGAGCCCCTACCCCT

F-amm6-7-

pKY01 Cloning of amm6-7 into pKY01 ATCGTCACATATGAGTGAGACCCAAGTGACCGAG

R-amm6-7-

pKY01 Cloning of amm6-7 into pKY01

ACGCCAGAAGCTTTCACTCCTGGAAGTTGATCAGGT

CG

F-amm3-

pKY01 Cloning of amm3 into pKY01 ATCGTCACATATGACGCACATGCACGGTACCCACG

R-amm3-

pKY01 Cloning of amm3 into pKY01 ACGCCAGAAGCTTTCATCGCACGCCCCCCG

F-amm6-7-

W56stp

W56stp point mutation of

pKY01/amm6amm7 CTCAAGGAATCGCGTTCATGATGAACCGCCCGTCCG

R-amm6-7-

W56stp

W56stp point mutation of

pKY01/amm6amm7 CGGACGGGCGGTTCATCATGAACGCGATTCCTTGAG

F-amm6-7-

W56C

W56C point mutation of

pKY01/amm6amm7 CTCAAGGAATCGCGTTCATGCTGAACCGCCCGTCCG

R-amm6-7-

W56C

W56C point mutation of

pKY01/amm6amm7 CGGACGGGCGGTTCAGCATGAACGCGATTCCTTGAG

Supplemental Experimental Procedures

General

All chemicals and reagents were acquired from Fisher Scientific, VWR Scientific, Sigma-Aldrich and used without

further purification. Restrictions enzymes, T4 DNA ligase, Gibson Assembly Master Mix were purchased from New

England Biolabs. PrimeSTAR DNA polymerase from Clontech was used for PCR reactions. Antibiotics for bacterial

selection were used at the following concentrations: Kanamycin (50 µg/mL), Apramycin (50 µg/mL),

Chloramphenicol (25 µg/mL) and Nalidixic Acid (25 µg/mL). All solvents used for LC purposes were LCMS grade

and all solvents for preparative and semi-preparative reverse phase purifications were HPLC grade. LC-MS/MS

analyses were performed on an Agilent 6530 Accurate-Mass Q-TOF MS equipped with a dual electrospray ionization

source and an Agilent 1260 LC system with diode array detector and Phenomenex Kinetex 2.6μ XB-C18 100 A, 150

x 4.6 mm column. MS and UV data were analyzed with Agilent MassHunter Qualitative Analysis version B.05.00.

Preparative HPLC was carried out using an Agilent 218 purification system (ChemStation software, Agilent) equipped

with a ProStar 410 automatic injector, Agilent ProStar UV-Vis Dual Wavelength Detector, a 440-LC fraction collector

and preparative HPLC column indicated below. Semi-preparative HPLC purifications were performed on an Agilent

1260 Series Instrument with a multiple wavelength detector and Phenomenex Luna 5µm C8(2) 250x100 mm semi

preparative column. Unless otherwise specified, all HPLC purifications utilized a 0.1% trifluoracetic acid buffer and

all analytical LCMS methods included a 0.1% formic acid buffer. NMR data were acquired at the UCSD Skaggs

School of Pharmacy, using a 600 MHz Bruker Avance III spectrometer with a 1.7 mm cryoprobe and at the UCSD

Chemistry Department NMR facility on a 500 MHz Varian XSens 5 mm cryoprobe. All signals are reported in ppm

with the internal DMSO-d6 signal at 2.50 ppm or 39.52 ppm. The data is being reported as (s=singlet, d=doublet,

t=triplet, q=quadruplet, m=multiplet or unresolved, br=broad signal, coupling constant(s) in Hz, integration).

Design and Construction of the amm Specific Capture Vector

To directly capture the 37.5 kb region encompassing the ammosamide (amm) biosynthetic gene cluster, we followed

the general procedure described by Yamanaka et al.(Yamanaka et al., 2014) Briefly, 1kb capture arms corresponding

to the upstream and downstream regions of the amm pathway were PCR amplified from Streptomyces sp. CNR-698

genomic DNA using the following primers: F-amm-cap-up, R-amm-cap-up, F-amm-cap-dwn, R-amm-cap-dwn (see

Table S2) with the included SpeI (actagt), XhoI (ctcgag), and BamHI (ggatcc) restriction sites. The two 1 kb fragments

were then PCR assembled into a single 2 kb fragment with F-amm-cap-up and R-amm-cap-dwn, and the assemble

fragment was ligated into pCAP01 using the SpeI and XhoI restriction sites. Prior to TAR capture, the pCAP01/amm

vector was linearized with BamHI.

Direct Capture of the amm gene cluster using transformation associated recombination

Streptomyces sp. CNR-698 genomic DNA was digested with EcoRI, NdeI and NheI-HF prior to its use in the TAR-

capture as described by Yamanaka et al.(Yamanaka et al., 2014) CNR-698 gDNA was also randomly fragmented

using a pipette tip, however capture experiments using this gDNA fragmentation method did not yield any positive

yeast clones Out of 100 colonies screened, where restriction digested gDNA was used, 3 positive hits were identified

by colony PCR using primers for a 1 kb region of amm3 (see Table S3). Candidates from PCR positive clones were

introduced into E. coli Top 10 by electroporation and evaluated by NcoI restriction digestion. The captured construct

was designated pCAP01/amm.

Calculation of RNA secondary structure downstream of amm6

The RNA secondary structure beginning 8 bases downstream of amm6 stop codon consisted of a stem-loop structure

comprising 15 bases, followed by a second stem loop comprising 50 bases. The structure was calculated using the

mFold server (http://unafold.rna.albany.edu/?q=mfold/RNA-Folding-Form) (Zuker, 2003), and RNAstructure

(http://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1.html) (Reuter and Mathews, 2010).The

calculated lowest free energy structure, and G of folding were very similar-57.7 kcal/mol (mFold) and -57.4

kcal/mol. (RNAstructure). Minimum free energy calculations were performed at 310.14 K (default) with all other

parameter set at default.

Synthesis of acid 8

The synthesis of compound 9 (Figure S5) was previously reported.(Reddy et al., 2010) Boc protected 9 (29.6 mg,

0.060 mmol) was dissolved in 1.5 mL DCM and 140 µL TFA (1.8 mmol) was added. After stirring for 6 hours at

http://unafold.rna.albany.edu/?q=mfold/RNA-Folding-Form

http://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1.html

room temperature, TFA and solvent were removed with a stream of N2 followed by high vacuum. To the resulting

residue was added 6 mL tetrahydrofuran and 1.2 mL 2M LiOH. The reaction was stirred for 1 hour at 40 degrees then

quenched with 0.37 mL TFA (4.8 mmol). The reaction was then poured into a separatory funnel, acidified with 1N

HCl and extracted three times with ethyl acetate. Then organics were dried with acidified brine, followed by

anhydrous sodium sulfate then concentrated. The resulting residue was then purified by preparative HPLC on an

Agilent Pursuit XRS 5 μm C18 100x21.2 mm preparative HPLC column (0-1 min 5% ACN isocratic, 1-10 min 5-

15% ACN at 15 mL/min with a mobile phase buffered with 12.5 mM NH4OH). One third of the purified material was

frozen and lyophilized to yield 2.3 mg of 8 as the ammonium salt. For the other 2/3 of the pure fractions, they were

pooled and the acetonitrile removed by rotovap. The resulting aqueous phase was acidified with HCl and extracted

three times, dried with acidified brine and anhydrous sodium sulfate before. The dried organics were concentrated to

yield 6.3 mg of the free acid. 1H-NMR (500 MHz, DMSO) δ 10.25 (s, 1H), 8.31 (s, 1H), 6.56 (s, 2H), 6.40 (s, 2H). 13C-NMR (126 MHz, dmso) δ 165.7, 164.7, 140.2, 132.5, 131.9, 131.3, 120.1, 117.5, 104.5, 103.9. LCMS analysis

and 1H- and 13C-NMR matched 8 isolated from M512-pCAP01/ammamm3 + pKY01/amm3.

Synthesis of amide 7

The synthesis of compound 10 (Figure S5) was described previously.(Reddy et al., 2010) 2.4 mg of (0.008 mmol)

10 was added 2 mL THF and 200 µL 28% NH4OH. The reaction was stirred at room temperature for 24 hours where

it was determined to be ~40% complete. An additional 200 µL of 28% NH4OH was added and the reaction was then

heated at 40 degrees for 24 hours where it was determined complete. The reaction was diluted with water in separatory

funnel and extracted 3 times with ethyl acetate. The combined organics were dried with brine then sodium sulfate and

concentrated. The resulting residue was then purified by preparative HPLC on an Agilent Pursuit XRS 5 μm C18

100x21.2 mm preparative HPLC column (0-1 min 10% ACN isocratic, 1-15 min 10-58% ACN at 15 mL/min with a

mobile phase buffered with 0.1% trifluoroacetic acid) to yield 0.8 mg. 1H-NMR (500 MHz, DMSO) δ 10.14 (s, 1H),

8.85 (s, 1H), 8.31 (s, 1H), 7.60 (s, 1H), 6.58 (s, 2H), 6.17 (s, 2H). 13C-NMR (126 MHz, DMSO) δ 166.3, 165.1, 144.5,

140.1, 131.7, 131.4, 131.1, 120.0, 115.5, 104.4, 103.7. LCMS analysis and 1H- and 13C-NMR matched 7 isolated

from M512-pCAP01/ammamm23.

1H-NMR (500 MHz, DMSO) and 13C-NMR (126 MHz, DMSO) of Compound 4

Related to “Production and Isolation of Compounds 4 and 8 from M512-pCAP01/ammamm3 + pKY01/amm3” in

the Experimental Procedures section.


Related to “Production and Isolation of Compounds 6 and 7 from M512-pCAP01/ammamm23” in the

Experimental Procedures section.

LCMS and UV-Vis Characterization of Compounds 4 and 8



LCMS and UV-Vis Characterization of Compounds 6 and 7



Reddy, P.V.N., Banerjee, B., and Cushman, M. (2010). Efficient Total Synthesis of Ammosamide B. Org. Lett. 12, 3112-3114. Reuter, J.S., and Mathews, D.H. (2010). RNAstructure: software for RNA secondary structure prediction and analysis. Bmc Bioinformatics 11, 9. Yamanaka, K., Reynolds, K.A., Kersten, R.D., Ryan, K.S., Gonzalez, D.J., Nizet, V., Dorrestein, P.C., and Moore, B.S. (2014). Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A. Proc. Natl. Acad. Sci. U.S.A. 111, 1957-1962. Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406-3415.

biosynthetic pathway connects cryptic ribosomally ... · biosynthetic pathway connects cryptic...

Documents