1. Biochemistry and Chemical Biology
  2. Genetics and Genomics
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Genome mining unearths a hybrid nonribosomal peptide synthetase-like-pteridine synthase biosynthetic gene cluster

  1. Hyun Bong Park
  2. Corey E Perez
  3. Karl W Barber
  4. Jesse Rinehart
  5. Jason M Crawford  Is a corresponding author
  1. Yale University, United States
  2. Yale School of Medicine, United States
Research Article
Cite this article as: eLife 2017;6:e25229 doi: 10.7554/eLife.25229
8 figures, 2 tables and 1 additional file

Figures

Figure 1 with 1 supplement
The pepteridine biosynthetic locus.

Green, pteridine synthesis genes; Blue, pyruvate dehydrogenase-like genes; Red, NRPS-like genes. T, thiolation domain; C, condensation domain.

https://doi.org/10.7554/eLife.25229.003
Figure 1—figure supplement 1
Genome synteny analysis using MicroScope.

Genome synteny analysis revealed a genomic island in P. luminescens TT01 (note absence of biosynthetic genes in phylogenetically related species, lower panel) harboring a number of enzymes possessing homology to pteridine and NRPS biosynthetic machineries (+1, +2, and +3 reading frames, upper panel). The regulator in the genomic island was not included in our design, as the pathway was placed under the control of a T7 promoter.

https://doi.org/10.7554/eLife.25229.004
Figure 2 with 2 supplements
Production of 7,8-dihydroxanthopterin (3) and pterin (4) by heterologous expression.

(A) HPLC traces (310 nm) of butanol extracts showing compounds 3 and 4 being over-produced in the heterologous expression strain. (B) The edge of a culture flask growing E. coli BAP1 carrying an empty vector pET28a (top), and a culture flask growing E. coli BAP1 carrying the wild-type pepteridine pathway (bottom). (C) HPLC traces (310 nm) from HPLC co-injection with authentic pterin (4), and UV absorption spectral comparison between natural and authentic pterin (inset). (D) UV-vis absorption spectra of compounds 3 and 4. (E) 1H NMR comparison of natural (blue) and standard (black) 3 in DMSO-d6.

https://doi.org/10.7554/eLife.25229.005
Figure 2—figure supplement 1
1H and 13C NMR spectra of compound 3.

(A) 1H NMR spectrum of compound 3 in DMSO-d6. (B) 13C NMR spectrum of compound 3 in DMSO-d6.

https://doi.org/10.7554/eLife.25229.006
Figure 2—figure supplement 2
2D- (gCOSY and gHMBCAD) NMR spectra of compound 3.

(A) gCOSY NMR spectrum of compound 3 in DMSO-d6. (B) gHMBCAD NMR spectrum of compound 3 in DMSO-d6.

https://doi.org/10.7554/eLife.25229.007
Figure 3 with 10 supplements
Relative abundances of wild-type pathway-dependent metabolites in wild-type and mutant pepteridine pathways.

The average ionization intensity is depicted for each molecular feature under a given genetic condition (wild-type and Δplu2793 through Δplu2799). Alterations in abundance are correlated with changes in nodal color intensity among genetic constructs, allowing visual assessment of product distributions for a given mutation. See Figure 3—figure supplement 2 for information on mass of pathway-dependent metabolites. Structural characterization of compounds 1 and 2 is shown in Figure 3—figure supplements 410, Table 2, and the Materials and methods section.

https://doi.org/10.7554/eLife.25229.009
Figure 3—figure supplement 1
Untargeted molecular networking of culture extracts from cells harboring the wild-type pepteridine biosynthetic pathway.

The node with the green hue represents pterin (4) networked into the untargeted wild-type map. This network includes abundant molecules from the pathway in addition to primary metabolites and media components.

https://doi.org/10.7554/eLife.25229.010
Figure 3—figure supplement 2
Pathway-targeted molecular networking of the wild-type pepteridine biosynthetic pathway.

Comparative metabolomic analysis between control and experimental culture samples enabled targeting (MS2) of molecular features dependent on the presence of the biosynthetic pathway.

https://doi.org/10.7554/eLife.25229.011
Figure 3—figure supplement 3
LC/MS Extracted Ion Count (EIC) chromatograms of compounds 1 (A) and 2 (B) from butanol extracts of the pepteridine heterologous expression strain.

LC/MS traces were extracted with m/z 224 corresponding to compound 1 (A) and m/z 210 corresponding to compound 2 (B) from butanol extracts of the E. coli BAP1 culture broths carrying the wild-type pepteridine pathway (green) or the empty vector (pET28a, red).

https://doi.org/10.7554/eLife.25229.012
Figure 3—figure supplement 4
1H and 13C NMR spectra of compound 1.

(A) 1H NMR spectrum of compound 1 in DMSO-d6. (B) 13C NMR spectrum of compound 1 in DMSO-d6.

https://doi.org/10.7554/eLife.25229.013
Figure 3—figure supplement 5
2D- (gCOSY and gHSQCAD) NMR spectra of compound 1.

(A) gCOSY NMR spectrum of compound 1 in DMSO-d6. (B) gHSQCAD NMR spectrum of compound 1 in DMSO-d6.

https://doi.org/10.7554/eLife.25229.014
Figure 3—figure supplement 6
2D- (gHMBCAD and NOESY) NMR spectra of compound 1.

(A) gHMBCAD NMR spectrum of compound 1 in DMSO-d6. (B) NOESY NMR spectrum of compound 1 in DMSO-d6.

https://doi.org/10.7554/eLife.25229.015
Figure 3—figure supplement 7
1H and 13C NMR spectra of compound 2.

(A) 1H NMR spectrum of compound 2 in DMSO-d6. (B) 13C NMR spectrum of compound 2 in DMSO-d6.

https://doi.org/10.7554/eLife.25229.016
Figure 3—figure supplement 8
2D- (gCOSY and gHSQCAD) NMR spectra of compound 2.

(A) gCOSY NMR spectrum of compound 2 in DMSO-d6. (B) gHSQCAD NMR spectrum of compound 2 in DMSO-d6.

https://doi.org/10.7554/eLife.25229.017
Figure 3—figure supplement 9
2D- (gHMBCAD and NOESY) NMR spectra of compound 2.

(A) gHMBCAD NMR spectrum of compound 2 in DMSO-d6. (B) NOESY NMR spectrum of compound 2 in DMSO-d6.

https://doi.org/10.7554/eLife.25229.018
Figure 3—figure supplement 10
Amide conformation of compounds 1 and 2.

(A) Plausible cis- (a) and trans- (b) amide conformations of compound 1 (top) and interpretation of 2D NOESY spectral data of compound 1 (bottom), (B) Plausible cis- (a) and trans- (b) amide conformations of compound 2 (top) and interpretation of 2D NOESY spectral data of compound 2 (bottom).

https://doi.org/10.7554/eLife.25229.019
Proposed pepteridine biosynthesis.

α-Ketobutyrate is processed by the atypical dehydrogenase-NRPS-like complex to generate a propionyl-loaded carrier protein (blue). Pteridine enzymes interact with the metabolism of the host to generate tetrahydropterin (green). The NRPS C domain couples these substrates to form the cis-amide bond (red) as illustrated for 1.

https://doi.org/10.7554/eLife.25229.021
Gene deletion and NRPS inactivation analyses on 1 (A–B) and 2 (C–D).

(A–B) Extracted ion chromatograms of 1 for wild-type and mutants are shown. (C–D) Extracted ion chromatograms of 2 for wild-type and mutants are shown.

https://doi.org/10.7554/eLife.25229.022
Figure 6 with 2 supplements
α-Ketobutyrate feeding studies on 1.

(A–B) Production enhancement of 1 in α-ketobutyrate supplementation studies (Arel, relative integration value). (C) 13C4-α-ketobutyrate supplementation leads to 13C3 incorporation in 1 consistent with the proposed biosynthesis.

https://doi.org/10.7554/eLife.25229.023
Figure 6—figure supplement 1
Production of compounds 1 and 2 in dose-dependent α-ketobutyrate (A) and pyruvate (B) feeding studies.

HPLC/MS traces were extracted with m/z 224 corresponding to compound 1 (A) and m/z 210 corresponding to compound 2 (B) from butanol extracts of the culture broths fed with varying concentrations of α-ketobutyrate (A) and pyruvate (B). Dose-response plots for 1 and 2 were determined using extracted peak integration values.

https://doi.org/10.7554/eLife.25229.024
Figure 6—figure supplement 2
Characterization of 13C4-α-ketobutyrate incorporation in compound 1.

Incorporation of 13C4-α-ketobutyrate into compound 1 (B) led to a clear 3 Da mass shift of native 1 (A) via oxidative decarboxylation of α-ketobutyrate.

https://doi.org/10.7554/eLife.25229.025
Figure 7 with 1 supplement
Extracted ion counts LC/HR-ESI-QTOF-MS analysis of pepteridines A (left panel) and B (right panel) from two genetically engineered P. luminescens strains locked in the phenotypic variants M-form (top) and P-form (bottom).
https://doi.org/10.7554/eLife.25229.026
Figure 7—figure supplement 1
Extracted ion counts chromatograms from LC/HR-ESI-QTOF-MS analysis of pepteridines A (left panel) and B (right panel) from the butanol extracts of P-form culture broth (top), standard compounds (middle), and co-injection (bottom).
https://doi.org/10.7554/eLife.25229.027
Quantitative proteomic analysis of a Δlocus strain in a wild-type background (A) and ΔhexA background (B).
https://doi.org/10.7554/eLife.25229.028
Figure 8—source data 1

Proteins increased in WT vs. WTΔlocus strains by LC-MS/MS.

Intensities from label-free quantification calculated using MaxQuant, averaged for biological triplicate samples, then log2 transformed. Proteins presented exhibited ≥2 fold increased average intensity in WT compared with WTΔlocus. Two-tailed t-test performed using a cutoff of FDR = 0.01 and 2-fold signal difference, or p<0.05.

https://doi.org/10.7554/eLife.25229.029
Figure 8—source data 2

Proteins increased in WTΔlocus vs. WT strains by LC-MS/MS.

Intensities from label-free quantification calculated using MaxQuant, averaged for biological triplicate samples, then log2 transformed. Proteins presented exhibited ≥2 fold increased average intensity in WTΔlocus compared with WT. Two-tailed t-test performed using a cutoff of FDR = 0.01 and 2-fold signal difference, or p<0.05.

https://doi.org/10.7554/eLife.25229.030
Figure 8—source data 3

Proteins increased in ΔhexA vs. ΔhexAΔlocus strains by LC-MS/MS.

Intensities from label-free quantification calculated using MaxQuant, averaged for biological triplicate samples, then log2 transformed. Proteins presented exhibited ≥2 fold increased average intensity in ΔhexA compared with ΔhexAΔlocus. Two-tailed t-test performed using a cutoff of FDR = 0.01 and 2-fold signal difference, or p<0.05 (+ indicates statistical significance).

https://doi.org/10.7554/eLife.25229.031
Figure 8—source data 4

Proteins increased in ΔhexAΔlocus vs. ΔhexA strains by LC-MS/MS.

Intensities from label-free quantification calculated using MaxQuant, averaged for biological triplicate samples, then log2 transformed. Proteins presented exhibited ≥2 fold increased average intensity in ΔhexAΔlocus compared with ΔhexA. Two-tailed t-test performed using a cutoff of FDR = 0.01 and 2-fold intensity difference, or p<0.05 (+ indicates statistical significance).

https://doi.org/10.7554/eLife.25229.032
Figure 8—source data 5

All proteins observed in WT and WTΔlocus strains by LC-MS/MS.

Intensities from label-free quantification calculated using MaxQuant and log2 transformed. Intensities for biological triplicate samples are presented. Average fold change shown for WT compared with WTΔlocus. Two-tailed t-test performed using a cutoff of FDR = 0.01 and 2-fold signal difference, or p<0.05 (+ indicates statistical significance).

https://doi.org/10.7554/eLife.25229.033
Figure 8—source data 6

All proteins observed in ΔhexA and ΔhexAΔlocus strains by LC-MS/MS.

Intensities from label-free quantification calculated using MaxQuant and log2 transformed. Intensities for biological triplicate samples are presented. Average fold change shown for ΔhexA compared with ΔhexAΔlocus. Two-tailed t-test performed using a cutoff of FDR = 0.01 and 2-fold signal difference, or p<0.05 (+ indicates statistical significance).

https://doi.org/10.7554/eLife.25229.034

Tables

Table 1

Genetic dependency of molecular features.

https://doi.org/10.7554/eLife.25229.008
GeneDependent molecular features
plu2793114
plu279431
plu279519
plu279637
plu279710
plu279821
plu279926
plu2793/plu279612
  1. Of the wild-type molecular features, this table denotes how many of them were deleted (i.e., not detected) in each mutant strain.

Table 2

NMR spectral data of pepteridine A (1) and B (2) in DMSO-d6.

https://doi.org/10.7554/eLife.25229.020
Pepteridine A (1)
No.δCatypeδHbMult (J in Hz)HMBC
1N
2154.6C
3NH10.07br s
4157.3C
4a93.1C
5N
638.5CH24.54dd (12.2, 3.6)C-1′, C-4a
2.33m
741.9CH23.30d (12.2)
2.97dt (12.0, 4.2)C-6
8NH6.96br sC-4a, C-6, C-7
8a153.1C
9N
1′174.0C
2′26.5CH22.57dt (15.0, 7.4)C-1′, C-3′
2.15dt (14.8, 7.3)C-1′, C-3′
3′9.7CH30.88t (7.4)C-1′, C-2′
NH26.25br s
Pepteridine B (2)
No.δCatypeδHbMult (J in Hz)HMBC
1N
2155.2C
3NH10.04br s
4157.3C
4a93.5C
5N
638.3CH24.52dd (12.1, 3.5)C-1′, C-4a
2.32dt (11.7, 2.4)
741.8CH23.30d (12.0)
2.98dt (11.9, 4.1)C-6
8NH6.96d (4.0)
8a153.3C
9N
1′170.6C
2′22.5CH31.97sC-1′
NH26.22br s
  1. NMR spectra were recorded at b600 MHz for 1H NMR and a100 MHz for 13C NMR, respectively.

Additional files

Supplementary file 1

Molecular feature list and primers used.

(A) Molecular feature list dependent on the presence of the wild-type pathway. (B-D) Primers used.

https://doi.org/10.7554/eLife.25229.035

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