Discovery of a Diazo-Forming Enzyme in Cremeomycin Biosynthesis

Article abstract of DOI:10.1021/acs.joc.8b00367

The molecular architectures and potent bioactivities of diazo-containing natural products have attracted the interest of synthetic and biological chemists. Despite this attention, the biosynthetic enzymes involved in diazo group construction have not been identified. Here, we show that the ATP-dependent enzyme CreM installs the diazo group in cremeomycin via late-stage N-N bond formation using nitrite. This finding should inspire efforts to use diazo-forming enzymes in biocatalysis and synthetic biology as well as enable genome-based discovery of new diazo-containing metabolites.

Full text of DOI:10.1021/acs.joc.8b00367

Subscriber access provided by University of Winnipeg Library
Note
Discovery of a diazo-forming enzyme in cremeomycin biosynthesis
Abraham J. Waldman, and Emily P Balskus
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b00367 • Publication Date (Web): 17 May 2018
Downloaded from http://pubs.acs.org on May 17, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 23
The Journal of Organic Chemistry
1
2
3
4
5
6
Discovery of a diazo-forming enzyme in cremeomycin biosynthesis
Authors: Abraham J. Waldman and Emily P. Balskus*
Author Affiliations
7
8
9
Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St, Cambridge, MA
02138, United States
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Corresponding Author
*Eꢀmail: balskus@chemistry.harvard.edu
Abstract
The molecular architectures and potent bioactivities of diazoꢀcontaining natural products have attracted
the interest of synthetic and biological chemists. Despite this attention, the biosynthetic enzymes
involved in diazo group construction have not been identified. Here, we show the ATPꢀdependent
enzyme CreM installs the diazo group in cremeomycin via lateꢀstage N–N bond formation using nitrite.
This finding should inspire efforts to use diazoꢀforming enzymes in biocatalysis and synthetic biology
and enable genomeꢀbased discovery of new diazoꢀcontaining metabolites.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 2 of 23
1
2
3
4
5
6
7
8
Diazoꢀcontaining compounds are exceptionally useful reagents and intermediates in synthetic
organic chemistry. The diazo group enables a wide scope of unique transformations including 1,3ꢀ
dipolar cycloadditions, alkylations, and carbene insertions (Figure 1A).1ꢀ3 Recently, diazo compounds
have been used extensively as carbene precursors in biocatalysis applications employing engineered
hemeꢀFe and porphyrinꢀIr based metalloenzymes for a variety of X–H (X = C, N, B, Si and S) insertions
and cyclopropanations (Figure 1A).4ꢀ10 Diazoꢀcontaining natural products are made by soilꢀ and marineꢀ
derived bacteria (Figure 1B),11 were first isolated in the 1950s, and have been evaluated as
chemotherapeutics in clinical trials.12ꢀ16 More recently, the exceptional potency of the polyketide
lomaiviticin A has attracted considerable attention from biological and synthetic chemists.17ꢀ23 Despite
significant interest among the chemical community in diazo compounds, no diazoꢀforming enzyme(s)
has been identified or proposed. Discovering these enzymes could advance biocatalysis and synthetic
biology by providing the tools for accessing both reagents and potential therapeutic candidates.
Furthermore, elucidating the genes required for diazo biosynthesis would enable genome mining efforts
to uncover novel and potentially bioactive diazoꢀcontaining small molecules.
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthetic chemists have developed numerous methods for diazo group installation, including
approaches involving C–N and N–N disconnection strategies.2, 24ꢀ25 Biosynthetic studies suggest diazo
biosynthesis may exhibit some parallels. Early feeding studies and genetic disruption experiments with
the producers of the diazoꢀcontaining kinamycin D and pyridazineꢀcontaining azamerone have
implicated a lateꢀstage N–N bond forming, diazotization reaction with an aryl amine intermediate.26ꢀ29
However, recent in vitro characterization of enzymes from the hydrazideꢀcontaining fosfazinomycin
pathway and identification of homologous enzymes in diazofluorene gene clusters imply an alternative
diazoꢀforming strategy involving formation of a discrete, hydrazide intermediate and lateꢀstage C–N
bond formation.30ꢀ32
ACS Paragon Plus Environment

Page 3 of 23
The Journal of Organic Chemistry
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Transformations of diazo compounds and natural products. A) Synthetic and biocatalytic
transformations involving diazo compounds. Proposed metallocarbenoid intermediate involved in hemeꢀ
based biocatalysts. B) Examples of natural products containing a diazo group and the pyridazine natural
product azamerone with its diazoꢀcontaining precursor A80915D.
Additional insight into diazo biosynthesis was obtained from the discovery and characterization
of the cremeomycin (cre) gene cluster (Figure 2A). Cremeomycin is a lightꢀsensitive, antibacterial oꢀ
diazoquinone natural product isolated in 1967.33ꢀ34 In vitro enzyme characterization, feeding studies with
15Nꢀlabeled intermediates, and genetics experiments have demonstrated the final transformation in the
pathway is the diazotization of 3ꢀaminoꢀ2ꢀhydroxyꢀ4ꢀmethoxybenzoic acid (3,2,4ꢀAHMBA) (Figure
2B).35ꢀ36 However, many questions remain regarding the details of this lateꢀstage N–N bond forming
reaction. Nitrite is the proposed source of the distal diazo group nitrogen atom, as two enzymes from the
cre cluster, the flavinꢀdependent Nꢀmonooxygenase CreE and the aspartase homolog CreD, produce
nitrite from Lꢀaspartate (Figure 2B).36 Feeding studies have also implicated nitrite in the biosynthesis of
the diazoꢀcontaining azamerone intermediate A80915D.37 This biosynthetic logic is analogous to wellꢀ
established synthetic methods for diazotization of aryl amines with nitrite under acidic conditions.29, 38
However, neither CreD nor CreE catalyzed diazotization of 3,2,4ꢀAHBMA in vitro, and genetic
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 4 of 23
1
2
3
4
5
6
7
8
disruption experiments by the Ohnishi group did not reveal any candidate diazotizing enzyme(s).36
Given that nitriteꢀbased diazotization of aryl amines requires acidic conditions and the culture medium
remains at pH ~7 throughout cremeomycin production, it appeared an enzyme would be required to
catalyze diazotization of 3,2,4ꢀAHMBA.
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Here, we report the discovery and preliminary characterization of a diazoꢀforming enzyme from
cremeomycin biosynthesis. Using an Escherichia coliꢀbased heterologous expression system, lysate
experiments, siteꢀdirected mutagenesis, and biochemical reconstitution with partiallyꢀpurified enzyme,
we demonstrate the fatty acidꢀcoenzyme A (CoA) ligase homolog CreM catalyzes diazotization of 3,2,4ꢀ
AHMBA with nitrite to afford cremeomycin in vivo and in vitro. In vitro biochemical reconstitution and
in vivo siteꢀdirected mutagenesis experiments suggest CreM uses ATP to promote diazotization.
Additionally, we provide evidence potentially explaining why previous efforts failed to identify this
enzyme.36
Our search for a diazoꢀforming enzyme(s) was informed by the need to activate nitrite to afford
the electrophilic nitrosonium ion required for diazotization. After eliminating genes within the cre
cluster that encoded enzymes with previously characterized roles in cremeomycin biosynthesis, we
identified three candidate diazoꢀforming enzymes, including CreM, a predicted fatty acidꢀCoA ligase
from the ANL superfamily (Figure S1). These ligases use ATP to activate the fatty acid carboxylate
group and catalyze thioesterification with CoA. We hypothesized the general catalytic capabilities of
this enzyme, the activation of a negatively charged, heteroatomꢀcontaining functional group with ATP,
could be used to promote diazotization.39 Specifically, we predicted ATP could activate nitrite to afford
an electrophilic intermediate that could be attacked directly by 3,2,4ꢀAHMBA or generate a nitrosonium
ion. Activation with ATP could also facilitate the dehydration of subsequent N–N linked intermediates,
which would parallel the role of acid catalysis in synthetic methods. The nitrite and N–N linked
intermediate(s) could be activated as phosphate derivatives (using the γꢀphosphate of ATP) or as
adenosine monophosphate (AMP) mixed anhydrides, both wellꢀestablished mechanisms for ATP
ACS Paragon Plus Environment

Page 5 of 23
1
2
3
4
5
6
7
The Journal of Organic Chemistry
activation (Figure 2C).40 However, previous studies demonstrated that the genetic disruption of creM did
not abolish cremeomycin production in a heterologous expression system, suggesting CreM is not
responsible for diazo formation.36 Therefore, the role of CreM in diazo biosynthesis remained unclear.
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. The predicted ATP-dependent enzyme CreM is a candidate diazo-forming enzyme. A)
The cre biosynthetic gene cluster. Blue = characterized genes; Green = predicted regulatory/resistance
genes; Grey = genes with unknown functions. B) Cremeomycin biosynthesis involves lateꢀstage
diazotization of 3,2,4ꢀAHMBA with nitrite. C) Proposed mechanisms by which CreM may use ATP to
activate nitrite or an Nꢀhydroxydiazenyl intermediate.
We began by reconstituting the final diazotization step in vivo using a Streptomyces lividans
TK64 heterologous host expressing the nitriteꢀgenerating enzymes CreD and CreE as well as the
putative diazoꢀforming enzyme CreM. Feeding 3,2,4ꢀAHMBA to this strain followed by culture
supernatants analysis by liquid chromatographyꢀmass spectrometry (LCꢀMS) demonstrated the
production of cremeomycin and its known, twoꢀelectron reduced degradation product 2ꢀhydroxyꢀ4ꢀ
methoxybenzoic acid (2,4ꢀHMBA) in several growth media (Figure S2).35 However, the production of
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 6 of 23
1
2
3
4
5
6
7
8
these metabolites also occurred in an S. lividans strain that expressed only CreD and CreE. Importantly,
the pH of the buffered media remained near neutral during the fermentation. We reasoned components
in the complex growth media could catalyze nonꢀenzymatic diazotization of 3,2,4ꢀAHMBA with the
nitrite generated by CreD and CreE. Indeed, incubating 500 µM 3,2,4ꢀAHMBA and 500 µM nitrite for
24 h at 30 °C in sterile, pH ~7 buffered R5, Q and FM media resulted in varying levels of cremeomycin
production in all media (Figure S3). No cremeomycin was detected when nitrite or 3,2,4ꢀAHMBA were
removed. The ability of Q media to promote the nonꢀenzymatic diazotization of 3,2,4ꢀAHMBA with
nitrite may explain why cremeomycin was previously detected in a creM mutant of a heterologous
host.36
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Since identifying a medium for S. lividans that did not promote diazotization would likely be
difficult, we moved our in vivo system into E. coli. Importantly, standard E. coli LB growth medium did
not catalyze the nonꢀenzymatic diazotization of 3,2,4ꢀAHMBA with nitrite (Figure S3). Culturing
different E. coli strains (Strains 1–8, Table S3) expressing various combinations of CreD, CreE, and
CreM in LB media supplemented with 3,2,4ꢀAHMBA revealed CreMꢀdependent production of 2,4ꢀ
HMBA (Figure 3A, 3B). The pH of the culture remained at pH 7 over the course of the experiment.
While cremeomycin was not detected, we are unaware of any biological route to 2,4ꢀHMBA in E. coli,
suggesting cremeomycin is being produced but subsequently degraded. When 500 µM of cremeomycin
standard was incubated with E. coli in LB under the expression conditions, 2,4ꢀHMBA was detected
(Figure S4). Together, these results implicate CreM in diazo formation, but suggest low production
levels and chemical instability prevent cremeomycin detection.
We next sought to increase cremeomycin titers and more directly determine the role of CreM in
diazotization. When an E. coli strain expressing a maltose binding fusion construct of CreM (MBPꢀ
CreM) was cultured in LB supplemented with 100 µM 3,2,4ꢀAHMBA and 200 µM nitrite, we observed
CreMꢀdependent production of cremeomycin and 2,4ꢀHMBA (Figure 3C, Figure S5).
ACS Paragon Plus Environment

Page 7 of 23
1
The Journal of Organic Chemistry
Removal of 3,2,4ꢀAHMBA or nitrite abolished production of both products. E. coli strains expressing an
empty pET28a vector or the soluble protein CreB did not generate either metabolite, demonstrating that
MBPꢀCreM expression was required for diazotization. Quantification of cremeomycin and 2,4ꢀHMBA
showed a conversion to both products of approximately 7% from the starting material 3,2,4ꢀAHMBA
(Figure S6).
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. CreM expression reconstitutes diazo formation in vivo in E. coli A) In vivo E. coli diazo
formation heterologous expression system. B) LCꢀMS analysis showing CreMꢀdependent production of
2,4ꢀHMBA in E. coli expressing either CreDE or CreDEM in 3,2,4ꢀAHMBAꢀsupplemented LB. C) LCꢀ
MS analysis demonstrating CreMꢀdependent production of cremeomycin in E. coli expressing MBPꢀ
CreM in nitriteꢀ and 3,2,4ꢀAHMBAꢀsupplemented LB medium. EIC = extracted ion chromatogram.
Additionally, we sought to confirm that the catalytic activity of CreM was responsible for diazo
formation by mutating a key active site residue in CreM. Members of the fatty acidꢀCoA ligase family
contain a highly conserved glutamate required to coordinate an essential Mg2+ ion necessary for ATP
binding.39, 41ꢀ42 Using multiple sequence alignments and structural homology modeling, we identified
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 8 of 23
1
2
3
4
5
6
7
8
E352 as the conserved glutamate in CreM (Figure S7 and S8). Upon mutating E352 to alanine (MBPꢀ
CreM_E352A) neither cremeomycin nor 2,4ꢀHMBA could be detected. Taken together, these results
demonstrate CreM catalyzes diazotization of 3,2,4ꢀAHMBA with nitrite to generate cremeomycin and
suggest ATP is required for this transformation.
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To assess the stability of MBPꢀCreM to purification, we incubated cell lysates of E. coli
expressing MBPꢀCreM with 3,2,4ꢀAHMBA and nitrite in TrisꢀHCl pH 7.0 aqueous buffer. These
reactions produced both cremeomycin and 2,4ꢀHMBA, demonstrating MBPꢀCreM remains active in E.
coli lysates (Figure 4B, Figure S9). In agreement with our previous experiments, lysates from strains
expressing CreB or MBPꢀCreM_E352A did not catalyze the reaction. The substrates nitrite and 3,2,4ꢀ
AHMBA were also required. Using clarified lysate abolished production of both metabolites suggesting
MBPꢀCreM may be improperly folded, aggregated, and/or membraneꢀbound, potentially explaining the
relatively low conversion rate seen in vivo.
We next sought to biochemically reconstitute CreM’s activity in vitro, but despite numerous
attempts, CreM always expressed as insoluble inclusion bodies in E. coli. Expression of Nꢀterminal
His6ꢀtagged CreM in S. lividans TK64 yielded protein that could be partially purified using metal ion
affinity chromatography (Figure S10). Incubation of partiallyꢀpurified CreM with 100 µM 3,2,4ꢀ
AHMBA, 200 µM nitrite, and 200 µM ATP in TrisꢀHCl pH 7.0 aqueous buffer yielded both
cremeomycin and 2,4ꢀHMBA (Figure 4C, Figure S11). This activity required CreM, the substrates
3,2,4ꢀAHMBA and nitrite, ATP, and was abolished when CreM was heated prior to use in the assay.
Furthermore, assays run with the more hydrolytically stable ATP analogue γꢀSꢀATP did not yield
cremeomycin and produced ~40ꢀfold less 2,4ꢀHMBA. These results provide additional evidence that
CreM is a diazoꢀforming enzyme and suggest ATP is involved in the transformation. Unfortunately, the
18
low in vitro activity of the CreM preparation prevented labeling studies (e.g. with Oꢀlabeled nitrite) to
gain deeper insights into the diazotization mechanism. Future efforts are aimed at improving the
purification of CreM to characterize the mechanism and structure of this enzyme.
ACS Paragon Plus Environment

Page 9 of 23
The Journal of Organic Chemistry
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Reconstitution of diazo formation with lysate and partially-purified CreM. A)
Reconstitution conditions for diazo formation using lysate or partiallyꢀpurified CreM. B) LCꢀMS
analysis demonstrating diazotization activity in MBPꢀCreM E. coli lysates. C) LCꢀMS analysis showing
in vitro biochemical reconstitution of diazo formation with partiallyꢀpurified NꢀHis6ꢀCreM. heated =
CreM protein heat denatured before use in assay.
In summary, our identification of the diazoꢀforming enzyme CreM has revealed a strategy for
diazo group construction in natural product biosynthesis, addressing a significant challenge in the field.
This work represents a critical step for future biochemical and structural investigations aimed at
understanding this chemistry in greater mechanistic detail. Additionally, the work described here now
informs investigations of other diazoꢀcontaining and related N–N bondꢀcontaining natural products. This
discovery will also facilitate efforts to use CreM and homologs in synthetic biology and biocatalysis
applications to produce diazoꢀcontaining compounds. Finally, the creDEM genes can now be used to
mine sequenced microbial genomes for gene clusters encoding putative diazoꢀcontaining natural
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 10 of 23
1
2
3
4
5
6
7
8
products. Initial mining of sequencing data from the National Center for Biotechnology Information
revealed greater than 90 gene clusters encoding predicted diazo natural products. Characterizing the
products of these gene clusters will not only provide access to new, potentially bioactive compounds but
may also afford additional CreM homologs that could provide starting points for enzyme engineering.
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EXPERIMENTAL SECTION
General Materials and Methods.
DNA polymerases, restriction enzymes, and T4 ligase used for cloning were purchased from
New England BioLabs. Recombinant plasmid DNA was purified with a QIAprep Kit from Qiagen or
with a E.Z.N.A. Plasmid DNA Mini Kit I from Omega BioꢀTek. Gel extraction of DNA fragments and
restriction endonuclease clean up were performed using an Illustra GFX PCR DNA and Gel Band
Purification kit from GE Healthcare. DNA sequencing was performed by Beckman Coulter Genomics
(Danvers, MA) and Eton Bioscience (Charlestown, MA). Nickelꢀnitrilotriacetic acidꢀagarose (NiꢀNTA)
resin was purchased from Qiagen and Thermo Scientific. SDS–PAGE gels (4 – 15% MiniꢀProtean
TGX) were purchased from BioꢀRad. Streptomyces lividans TK64 was obtained from Prof. Wenjun
Zhang at the University of California, Berkeley. The vector pET28ꢀMBP was obtained from Prof. Doug
Mitchell at the University of Illinois UrbanaꢀChampaign.
Optical densities of Escherichia coli cultures were determined with a DU 730 Life Sciences
(Beckman Coulter) or a Genesys 20 UV/Vis spectrophotometer (Thermo Scientific) by measuring
absorbance at 600 nm. Thermocycling was carried out in a C1000 Gradient Cycler (BioꢀRad). Cell lysis
by disruption was performed using an Avestin EmulsiFlexꢀC3. Cell lysis by sonication was performed
using a Branson Digital Sonifier Model 450. Highꢀresolution mass spectral data were obtained at the
Small Molecule Mass Spectrometry Facility, FAS Division of Science, Harvard University. Detection of
cremeomycin and 2ꢀhydroxyꢀ4ꢀmethoxybenzoic acid (2,4ꢀHMBA) by liquid chromatographyꢀmass
spectrometry (LCꢀMS) were carried out on a Thermo Scientific Dionex UltiMate 3000 UHPLC coupled
ACS Paragon Plus Environment

Page 11 of 23
1
The Journal of Organic Chemistry
to a Thermo Q Exactive Plus mass spectrometer system (Thermo Fisher Scientific Inc, Waltham, MA)
equipped with an HESIꢀII electrospray ionization (ESI) source. LCꢀMS data were analyzed with
Chromeleon Xpress software for UHPLC and Thermo Xcalibur software version 3.0.63 for mass
spectrometry and processed with Thermo Xcalibur Qual Browser software version 4.0.27.19. 8 µL of
each sample was injected onto the UHPLC including a HPGꢀ3400RS binary pump with a builtꢀin
vacuum degasser and a thermostatted WPSꢀ3000TRS high performance autosampler. An XTerra MS
C18 analytical column (2.1x100 mm, 3.5 µm) from Waters Corporation (Milford, MA) was used at a
flow rate of 0.3 mL/min with 0.2% acetic acid in water as mobile phase A and 0.2% acetic acid in
acetonitrile as mobile phase B. The column temperature was maintained at room temperature. The
following gradient was applied: 0ꢀ3 min: 5% B isocratic, 3ꢀ12 min: 5ꢀ100% B, 12ꢀ14 min: 100% B
isocratic, 14ꢀ14.5 min: 100ꢀ5%B, 14.5ꢀ20.5 min: 5% B isocratic. The MS conditions were as follows:
positive ionization mode for cremeomycin and negative ionization mode for 2,4ꢀHMBA; full scan mass
range, m/z 50 to 750; resolution, 70.00; AGC target, 1e6; maximum IT, 220 ms; spray voltage, 3500 V;
capillary temperature, 280 °C; sheath gas, 47.5; Aux gas, 11.25; probe heater temperature, 412.5 °C; Sꢀ
Lens RF level, 50.00. A mass window of ± 5 ppm was used to extract the ion of [M+H]+ for
cremeomycin and [MꢀH]ꢀ for 2,4ꢀHMBA. Targets were considered detected when the mass accuracy was
less than 5 ppm, there was a match of isotopic pattern between the observed and theoretical ions, and
there was a match of retention time with the standards.
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Proton nuclear magnetic resonance (1H NMR) spectra and carbon nuclear magnetic resonance
(13C NMR) spectra were recorded on Varian Inovaꢀ500 (500 MHz, 125 MHz) NMR spectrometers.
Chemical shifts are reported in parts per million downfield from tetramethylsilane using the solvent
1
resonance as internal standard for H (CDCl3 = 7.26 ppm) and 13C (CDCl3 = 77.25 ppm). Data are
reported as follows: chemical shift, integration multiplicity (s = singlet, d = doublet, t = triplet, m =
multiplet, q=quartet, qt=quintet), coupling constant, integration, and assignment. NMR spectra were
processed using iNMR Reader, version 5.3.4.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 12 of 23
1
2
3
4
5
6
7
8
Synthetically prepared cremeomycin38 and commercially available 2,4ꢀHMBA from Sigmaꢀ
Aldrich were used as standards in all LCꢀMS analyses. The 3,2,4ꢀAHMBA used in all in vivo
heterologous expression experiments and in vitro assays was synthetically prepared following the
previous report by Varley et al.38 Our synthesis and characterization of 3,2,4ꢀAHMBA following
Varley’s protocol was reported previously by our lab.35
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
As cremeomycin is light sensitive, all procedures undertaken in which cremeomycin was
potentially present (i.e. in vivo heterologous expression experiments in S. lividans and E. coli, in vitro
experiments, nonꢀenzymatic diazotization experiments, and cremeomycin degradation experiments)
were carried out with exclusion of as much light as possible. Lights were turned off while working,
culture flasks and tubes were wrapped in tinfoil during incubations, incubator windows were covered in
tinfoil, Eppendorf tubes were wrapped in tinfoil, centrifuges were covered in tinfoil, and amber, lightꢀ
excluding LCꢀMS sample vials were used for analysis.
Cremeomycin
1H NMR (500 MHz, CDCl3) δ 8.36 (d, J = 8.55 Hz, 1H, aromatic CH), δ 5.97 (d, J = 8.57 Hz, 1H,
aromatic CH), δ 4.05 (s, 3H, OCH3); 13C NMR (125 MHz, CDCl3) δ 177.16 (C2), 165.79 (COOH),
161.87 (C6), 146.47 (C4), 114.67 (C1), 94.87 (C5), 57.55 (CH3). HRMS (ESI): calc’d m/z for
C8H7N2O4 [M+H]+ = 195.0400; found, 195.0401. H and 13C NMR data matched those previously
reported by Moody.38 No paramagnetic reagent was added; therefore the diazo carbon was silent in the
13C NMR.38
+
1
Generation of S. lividans TK64 strains expressing CreD, CreE, and CreM.
The genes creD, creE, and creM were each cloned using the ‘compatible cohesive ends’ method
(using AvrII and NheI restriction sites) to insert each gene in succession into the pUWL201PW
StreptomycesꢀE. coli shuttle vector43 to generate final vectors containing either creD and creE
(pUWL201PWꢀcreDE) or creD, creE, and creM (pUWL201PWꢀcreDEM) using the primers shown in
Table S1. All cloning was performed in E. coli TOP10 cells. Plasmids were sequenced for verification.
ACS Paragon Plus Environment

Page 13 of 23
1
2
3
The Journal of Organic Chemistry
The pUWL201PW vector confers ampicillin resistance (100 ꢀg/mL) in E. coli and thiostrepton
4
5
6
7
8
9
resistance (50 ꢀg/mL) in Streptomyces species. Briefly, creD was first cloned into pUWL201PW using
restriction sites NdeI (5’) and BamHI (3’). After purification and sequence verification of
pUWL201PWꢀcreD, the plasmid was digested with AvrII and BamHI while creE was cloned into the
plasmid using NheI (5’) and BamHI (3’) sites (AvrII and NheI have compatible ends) to generate
pUWL201PWꢀcreDE. After purification and sequence verification of pUWL201PWꢀcreDE, the plasmid
was digested using AvrII and BamHI while creM was cloned into the plasmid using NheI (5’) and
BamHI (3’) sites to generate pUWL201PWꢀcreDEM. The vector backbone contains a ribosome binding
site (RBS) for the first gene creD and a constitutive promoter, ermE, to drive transcription of the entire
creDEM operon. A RBS with the sequence GGAGGAGCC was inserted three nucleotides upstream of
both creE and creM for translation of these genes. Streptomyces lividans TK64 protoplasts were
generated and transformed with plasmid DNA according previous reports in Practical Streptomyces
Genetics.44 After protoplasts were transformed, the viable exꢀtransformants were streaked on R5 agar
plates with 50 ꢀg/mL thiostrepton to confirm resistance through the plasmid. Confirmed exꢀ
transformants were stored as spores or mycelial stocks in 25% glycerol at –80 °C.
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In vivo production of cremeomycin and 2,4-HMBA from S. lividans TK64 strains expressing CreD,
CreE, and CreM.
Stocks of S. lividans TK64[pUWL201PWꢀcreDE] and S. lividans TK64[pUWL201PWꢀ
creDEM] stored at –80 °C were streaked on R5 agar plates44 containing 50 µg/mL thiostrepton and
incubated at 30 °C for approximately 4–6 days until robust mycelial growth and sporulation occurred. S.
lividans TK64 wildꢀtype stored at –80 °C was streaked on R5 agar plates containing no antibiotics. The
mycelia/spores from approximately ¼ of an agar plate were removed with a cotton swab tip and used to
inoculate 50 mL of seed culture of R5, Q or FM liquid media containing 50 µg/mL thiostrepton (for
strains expressing CreDE or CreDEM) or no antibiotics (wildꢀtype strain). Cultures were incubated at 30
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 14 of 23
1
2
3
4
5
6
7
8
°C with shaking at 220 rpm for 3 days. These seed cultures were used to inoculate 50 mL of fresh R5, Q
or FM liquid media supplemented with 1 mM 3,2,4ꢀAHMBA and 50 ꢀg/mL thiostrepton for CreDEꢀ
and CreDEMꢀexpressing strains at an inoculum of 1:100 and incubated at 30 °C with shaking at 220 rpm
for 36 h. The pH of the buffered fermentation culture remained at pH 7 over the course of the
fermentation. After 36 h, 1 mL of culture was removed and centrifuged at 4 °C at 16,000 x g for 5 min.
A 200 ꢀL aliquot of clarified supernatant was removed and quenched with 400 ꢀL of acetone. This
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
mixture was centrifuged at 4 °C at 16,000 x g for 15 min. 200 ꢀL of clarified supernatant was removed
and analyzed by LCꢀMS as described in General Methods.
Non-enzymatic diazotization of 3,2,4-AHMBA in various bacterial growth media.
Lysogeny broth (LB), R544, Q36 and fermentation media33 (FM) media were prepared, adjusted to
pH ~7 and sterilized by autoclaving. To 500 ꢀL of each medium ampicillin was added at 100 µg/mL to
prevent bacterial growth during subsequent incubation plus 500 µM of 3,2,4ꢀAHMBA and 500 µM
sodium nitrite. Additional controls were prepared in which 3,2,4ꢀAHMBA or sodium nitrite was not
added. Samples were wrapped in tinfoil and incubated at 30 °C for 24 h. After 24 h, samples were
centrifuged at 4 °C at 16,000 x g for 15 min. A 200 ꢀL aliquot of clarified supernatant was removed and
quenched with 400 ꢀL of acetone. This mixture was centrifuged at 4 °C at 16,000 x g for 15 min. 200
ꢀL of clarified supernatant was removed and analyzed by LCꢀMS as described in General Methods.
Generation of E. coli BL21 strains expressing CreD, CreE, and CreM.
The genes creD, creE, and creM were cloned into various vectors (DUET vectors: pCOLA (kanamycin
resistance), pETDUET (ampicillin resistance), pACYC (chloramphenicol resistance) and maltose
binding protein fusion vector: pMALꢀc2x (ampicillin resistance)) via either PCR and ligation or
subcloning. Plasmids were subsequently transformed into E. coli BL21 and Tuner in a variety of
ACS Paragon Plus Environment

Page 15 of 23
1
The Journal of Organic Chemistry
combinations to generate Strains 1–8 as described in Table S2. Primers used for this cloning are given
in Table S3. All cloning was performed in E. coli TOP10 cells. After cloning and sequence verification
of each plasmid, the plasmid combinations were transformed into E. coli BL21 or Tuner via heat shock
or electroporation. The E. coli Strains 1–8 were stored in 25% glycerol stocks at –80 °C.
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In vivo production of 2,4-HMBA from E. coli strains expressing of CreD, CreE, and CreM.
Glycerol cell stocks of E. coli Strain 1–8, as well as wildꢀtype E. coli BL21 and Tuner strains, were used
to inoculate 5 mL of LB media containing the appropriate antibiotics (refer to Table S2 for resistance
profile of each strain) and incubated at 37 °C overnight on a rotating drum. These seed cultures were
used to inoculate 5 mL of fresh LB media containing the appropriate antibiotics at an inoculum of 1:100,
which were then incubated at 37 °C on a rotating drum. After approximately 2 h, the strains were
transferred to an incubator at 15 °C and incubated for approximately one additional hour with shaking.
When the OD600 of the cultures reached 0.5–0.6, 100 ꢀM of 3,2,4ꢀAHMBA and 500 ꢀM of IPTG was
added and the strains were incubated overnight at 15 °C with shaking. The pH of the E. coli culture
remained at pH 7 over the course of the fermentation. After approximately 15 h, the cultures were
centrifuged at 4 °C at 3,200 x g for 5 min. A 400 ꢀL aliquot of supernatant was removed and quenched
with 800 ꢀL of acetone. This mixture was centrifuged at 4 °C at 16,000 x g for 15 min. 200 ꢀL of
clarified supernatant was removed and analyzed by LCꢀMS as described in General Methods.
Cremeomycin degradation experiments in presence of E. coli in LB media.
A glycerol stock of E. coli BL21 was used to inoculate 5 mL of LB media and incubated at 37 °C
overnight on a rotating drum. This seed culture was used to inoculate 5 mL of fresh LB media at an
inoculum of 1:100 and incubated at 37 °C on a rotating drum. After approximately 2 h, the strain was
transferred to an incubator at 15 °C and incubated for approximately one additional hour with shaking.
When the OD600 of the culture reached 0.5–0.6, 500 µL of culture was transferred to an Eppendorf tube
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 16 of 23
1
2
3
4
5
6
7
8
(with holes punctured in the top to allow for aeration) and 500 µM of synthetically prepared
cremeomycin was added to the culture. The tube was covered in tinfoil and placed horizontally on the
platform in the incubator and allowed to shake overnight for 15 h. Additional controls were set up in
which cremeomycin was added to LB in the absence of E. coli cells or where cremeomycin was added
to water alone. After 15 h, the culture was centrifuged at 4 °C at 16,000 x g for 5 min. A 200 ꢀL aliquot
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
of supernatant was removed and quenched with 400 ꢀL of acetone. This mixture was centrifuged at 4 °C
at 16,000 x g for 15 min. 200 ꢀL of clarified supernatant was removed and analyzed by LCꢀMS as
described in General Methods.
Generation of E. coli BL21 strains for MBP-CreM expression experiments.
The creM gene was cloned into pET28ꢀMBP vector (constructed and received from Prof. Douglas
Mitchell, University of Illinois, UrbanaꢀChampaign) using the BamHI (5’) and XhoI (3’) restriction sites
to create an inꢀframe Nꢀterminal MBP fusion construct with creM. The gene creM was amplified from a
bacterial artificial chromosome (BAC) clone from an S. cremeus genomic library constructed previously
using the primers shown in Table S4.35 The gene creB was cloned into pET28a using the NdeI (5’) and
XhoI (3’) restriction sites to give an Nꢀterminal His6ꢀtagged protein. The gene creB was amplified from
S. cremeus genomic DNA using the primers shown in Table S4. The pET28ꢀMBPꢀcreM_E352A
plasmid was constructed by siteꢀdirected mutagenesis through whole plasmid amplification with the
primers shown in Table S4. All cloning was performed in E. coli TOP10 cells. After cloning and
sequence verification of each plasmid, the plasmids were transformed into chemicallyꢀcompetent E. coli
BL21 and stored in 25% glycerol stocks at –80 °C. For the ‘empty vector’ control, pET28a was
transformed into E. coli BL21 and for the ‘no vector’ control wildꢀtype E. coli BL21 was used.
In vivo production of cremeomycin and 2,4-HMBA from E. coli strains expressing MBP-CreM.
Glycerol cell stocks of the E. coli BL21 strains were used to inoculate 5 mL of LB media containing
ACS Paragon Plus Environment

Page 17 of 23
1
The Journal of Organic Chemistry
kanamycin (50 µg/mL final concentration) where appropriate and incubated at 37 °C overnight on a
rotating drum. These seed cultures were used to inoculate 5 mL of fresh LB media containing
kanamycin (50 µg/mL final concentration) where appropriate and incubated at 37 °C on a rotating
drum. After approximately 2 h, the strains were transferred to an incubator at 15 °C and incubated for
approximately one additional hour with shaking. When the OD600 of the cultures reached 0.5–0.6, 100
ꢀM of 3,2,4ꢀAHMBA, 200 µM sodium nitrite, and 250 ꢀM of IPTG were added, and the strains were
incubated overnight at 15 °C with shaking. The pH of the E. coli culture remained at pH 7 over the
course of the fermentation. After approximately 15 h, the cultures were centrifuged at 4 °C at 3,200 x g
for 5 min. A 400 ꢀL aliquot of supernatant was removed and quenched with 800 ꢀL of acetone. This
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
mixture was centrifuged at 4 °C at 16,000 x g for 15 min. 200 ꢀL of clarified supernatant was removed
and analyzed by LCꢀMS as described in General Methods.
Quantification of cremeomycin and 2,4-HBMA production levels from E. coli expressing MBP-
CreM.
Dilution curves for cremeomycin and 2,4ꢀHMBA were prepared using authentic standards in the
concentration ranges of 0.1 ꢀM – 25 ꢀM and 0.01 ꢀM – 25 ꢀM, respectively. Each concentration was
prepared in triplicate and analyzed by LCꢀMS to generate a standard curve for each compound as shown
in Figure S5. This standard curve was used to calculate the concentrations of cremeomycin and 2,4ꢀ
HMBA in triplicate samples in a typical in vivo experiment of E. coli BL21 expressing MBPꢀCreM.
Lysate diazotization experiments with E. coli BL21 expressing MBP-CreM.
Glycerol cell stocks of the E. coli BL21 strains were used to inoculate 5 mL of LB media
containing kanamycin (50 ꢀg/mL final concentration) where appropriate and incubated at 37 °C
overnight on a rotating drum. These seed cultures were used to inoculate 50 mL of fresh LB media
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 18 of 23
1
2
3
4
5
6
7
8
containing kanamycin (50 ꢀg/mL final concentration) where appropriate and incubated at 37 °C on a
rotating drum. After approximately 2 h, the strains were transferred to an incubator at 15 °C and
incubated for approximately one additional hour with shaking. When the OD600 of the cultures reached
0.5–0.6, 250 ꢀM of IPTG was added, and the strains were incubated overnight at 15 °C with shaking.
After approximately 15 h, the cultures were centrifuged at 4 °C at 3,200 x g for 5 min to collect the cells.
The pellets were each resuspended in 1 mL of iceꢀcold lysis buffer (25 mM TrisꢀHCl pH 7.5, 500 mM
NaCl, 10 mM MgCl2, Thermo Scientific Pierce Protease inhibitor cocktail Prod #A32963) and kept on
ice. The E. coli cell suspensions were lysed by sonication (Branson Digital Sonifier Model 450; Method:
10 s on, 30 s off, 40 s total, 25% duty cycle) on ice. Lysate assays were setup in 100 ꢀL reactions (final
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
volume) in buffer (50 mM TrisꢀHCl pH 7.0, 0.1 mM MgCl2, 10 mM NaCl) containing 100 ꢀM 3,2,4ꢀ
AHMBA, 200 ꢀM sodium nitrite, and 200 ꢀM ATP. Reactions were initiated with the addition of 20 ꢀL
nonꢀclarified E. coli cell lysate, mixed, and then incubated at room temperature overnight. After
approximately 12 h, the reactions were quenched with 200 ꢀL of acetone and vortexed to mix
vigorously. This mixture was centrifuged at 4 °C at 16,000 x g for 15 min. 150 ꢀL of clarified
supernatant was removed and analyzed by LCꢀMS as described in General Methods.
Cloning of N-terminal His6-CreM from S. lividans TK64.
The creM gene was cloned into the StreptomycesꢀE. coli shuttle vector pUWL201PW using the
restriction sites NdeI (5’) and EcoRI (3’). As the pUWL201PW encodes no purification tags, an Nꢀ
terminal His6 was engineered into the primers as shown in Table S5. All cloning was performed in E.
coli TOP10 cells. Plasmids were sequenced for verification. The pUWL201PW vector confers
ampicillin resistance (100 ꢀg/mL) in E. coli and thiostrepton resistance (50 ꢀg/mL) in Streptomyces
species. The vector backbone contains a ribosome binding site (RBS) for creM and a constitutive
promoter, ermE, to drive transcription. Streptomyces lividans TK64 protoplasts were generated and
ACS Paragon Plus Environment

Page 19 of 23
1
The Journal of Organic Chemistry
transformed with pUWL201PWꢀNꢀHis6ꢀcreM according to previous reports in Practical Streptomyces
Genetics.44 After protoplasts were transformed, the viable exꢀtransformants were streaked on R5 agar
plates with 50 ꢀg/mL thiostrepton to confirm resistance through the plasmid. Confirmed exꢀ
transformants were stored as spore or mycelial stocks in 25% glycerol at –80 °C.
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Expression and partial purification of N-terminal His6-CreM from S. lividans TK64.
Stocks of S. lividans TK64[pUWL201PWꢀNꢀHis6ꢀcreM] stored at –80 °C or from fresh
transformations were streaked on to R5 agar plates containing 50 ꢀg/mL thiostrepton and incubated at
30 °C for approximately 4–6 days until robust mycelial growth and sporulation occurred. The
mycelia/spores from approximately ¼ of an agar plate were removed with a cotton swab tip and used to
inoculate 4 x 50 mL seed cultures in R5 liquid media containing 50 ꢀg/mL thiostrepton. Cultures were
incubated at 30 °C with shaking at 220 rpm for 3 days. These seed cultures were then used to inoculate 4
x 500 mL of fresh R5 liquid media at an inoculum of 1:50 and incubated at 30 °C with shaking at 220
rpm for 3 days. After 3 days, cells from each culture were centrifuged separately at 4 °C at 6,500 x g for
30 min and taken through the following purification separately. The cell pellets were resuspended in 100
mL of iceꢀcold lysis buffer (25 mM TrisꢀHCl pH 7.5, 500 mM NaCl, 10 mM MgCl2, 1 mg/mL
lysozyme, 1 ꢀg/mL DNase, Thermo Scientific Pierce Protease inhibitor cocktail Prod #A32963) and
kept on ice. The cells were lysed by passage through a cell disruptor (Avestin EmulsiFlexꢀC3) twice at
20,000–25,000 psi and the lysate was clarified by centrifugation at 20,000 x g for 45 min. Imidazole (10
mM final concentration) was added to the supernatant and flowed over a glass column containing 5 mL
of NiꢀNTA resin and allowed to exit the column by gravity flow. The column was washed with 20 mL
each of wash buffers (25 mM TrisꢀHCl pH 7.5, 500 mM NaCl, 10 mM MgCl2) containing 25 mM
imidazole and 50 mM imidazole. Protein was eluted with elution buffer (25 mM TrisꢀHCl pH 7.5, 500
mM NaCl, 10 mM MgCl2) containing 200 mM imidazole. Collected fractions were analyzed by SDSꢀ
PAGE to ascertain the presence and purity of protein in each fraction. Fractions containing NꢀHis6ꢀ
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 20 of 23
1
2
3
4
5
6
7
8
CreM were pooled and the buffer was exchanged using an Illustra NAPꢀ10 desalting column (GE
Healthcare Prod #17ꢀ0854ꢀ02) into 25 mM TrisꢀHCl pH 7.5, 50 mM NaCl, 10% glycerol. This provided
a partiallyꢀpurified protein extract (Figure S9) for use in in vitro biochemical assays. Solutions
containing protein were frozen in liquid nitrogen and stored at –80 °C.
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In vitro biochemical reconstitution of diazotization of 3,2,4-AHMBA using partially-purified
CreM.
In vitro biochemical assays were setup in 100 ꢀL reactions (final volume) in buffer (50 mM Trisꢀ
HCl pH 7.0, 0.1 mM MgCl2, 10 mM NaCl) containing 100 ꢀM 3,2,4ꢀAHMBA, 200 ꢀM sodium nitrite,
and 200 ꢀM ATP. Reactions were initiated with the addition of 10 ꢀL of partially purified CreM, mixed,
and incubated at room temperature overnight. For the heated control, ~100 ꢀL of CreM was heated at 95
°C for 5 min and then centrifuged at 16,000 x g for 15 min. The resulting clarified supernatant was used
in the assay after cooling to room temperature. After approximately 12 h, the reactions were quenched
with 200 ꢀL of acetone and vortexed to mix vigorously. This mixture was centrifuged at 4 °C at 16,000
x g for 15 min. 150 ꢀL of clarified supernatant was removed and analyzed by LCꢀMS as described in
General Methods.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications Website at DOI:
Additional information and data related to S. lividans heterologous system, cremeomycin
degradation, nonꢀenzymatic diazotization, quantification of cremeomycin and 2,4ꢀHMBA
production levels in E. coli, 2,4ꢀHMBA production in E. coli, lysate and in vitro experiments,
multiple sequence alignment of CreM, structural homology model of CreM, and partial
1
13
purification of CreM. H and C NMR spectra for cremeomycin. Primers, plasmids and strain
descriptions.
AUTHOR INFORMATION
ACS Paragon Plus Environment

Page 21 of 23
1
The Journal of Organic Chemistry
Corresponding Author
2
3
4
5
*Eꢀmail: balskus@chemistry.harvard.edu
6
7
Notes
8
9
The authors declare no competing financial interest.
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACKNOWLEDGEMENTS
We acknowledge financial support from Harvard University, the Searle Scholars Program, and the NIH
(DP2 GM105434). A. J. W. acknowledges fellowship support from the NIH (GM095450) and the
BristolꢀMyers Squibb Graduate Fellowship in Synthetic Organic Chemistry. We thank Prof. Doug
Mitchell (University of Illinois UrbanaꢀChampaign) for providing the pET28ꢀMBP vector and Prof.
Wenjun Zhang (University of California at Berkeley) for providing Streptomyces lividans TK64.
1.
Acid Catalysis of C-C Bond Formation Using Diazoalkanes. Angew. Chem., Int. Ed. 2010, 49, 2290-2298.
2. Ye, T.; Mckervey, M. A., Organic Synthesis with a-Diazo Carbonyl Compounds. Chem. Rev. 1994,
94, 1091-1160.
Johnston, J. N.; Muchalski, H.; Troyer, T. L., To Protonate or Alkylate? Stereoselective Bronsted
3.
Davies, H. M. L.; Manning, J. R., Catalytic C-H Functionalization by Metal Carbenoid and
Nitrenoid Insertion. Nature 2008, 451, 417-424.
4.
abiological carbene and nitrene transfer reactions. Curr. Opin. Biotechnol. 2017, 47, 102-111.
5. Dydio, P.; Key, H. M.; Nazarenko, A.; Rha, J. Y.-E.; Seyedkazemi, V.; Clark, D. S.; Hartwig, J. F., An
artificial metalloenzyme with the kinetics of native enzymes. Science 2016, 354, 102-106.
6. Wang, Z. J.; Peck, N. E.; Renata, H.; Arnold, F. H., Cytochrome P450-Catalyzed Insertion of
Carbenoids into N-H Bonds. Chem. Sci. 2014, 5 (2), 598-601.
7. Kan, S. B. J.; Huang, X.; Gumulya, Y.; Chen, K.; Arnold, F. H., Genetically programmed chiral
organoborane synthesis. Nature 2017, 552 (7683), 132-136.
8. Kan, S. B. J.; Lewis, R. D.; Chen, K.; Arnold, F. H., Directed evolution of cytochrome c for carbon-
silicon bond formation: Bringing silicon to life. Science 2016, 354 (6315), 1048-1051.
9. Tyagi, V.; Bonn, R. B.; Fasan, R., Intermolecular carbene S-H insertion catalysed by engineered
myoglobin-based catalystsdagger. Chem Sci 2015, 6 (4), 2488-2494.
10. Coelho, P. S.; Wang, Z. L.; Ener, M. E.; Baril, S. A.; Kannan, A.; Arnold, F. H.; Brustad, E. M., A
Brandenberg, O. F.; Fasan, R.; Arnold, F. H., Exploiting and engineering hemoproteins for
serine-substituted P450 catalyzes highly efficient carbene transfer to olefins in vivo. Nat. Chem. Biol.
2013, 9, 485-487.
11.
Nawrat, C. C.; Moody, C. J., Natural Products Containing a Diazo Group. Nat. Prod. Rep. 2011, 28
(8), 1426-1444.
12.
Ellison, R. R.; Karnofsky, D. A.; Sternberg, S. S.; Murphy, M. L.; Burchenal, J. H., Clinical Trials of
O-diazoacetyl-L-serine (Azaserine) in Neoplastic Disease. Cancer 1954, 7 (801-814).
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 22 of 23
1
2
13.
Catane, R.; Von Hoff, D. D.; Glaubiger, D. L.; Muggia, F. M., Azaserine, DON, and Azotomycin:
3
4
5
6
7
8
Three Diazo Analogs of L-Glutamine with Clinical Antitumor Activity. Cancer Treat Rep. 1979, 63, 1033-
1038.
14.
Fusari, S. A.; Frohardt, R. P.; Ryder, A.; Haskell, T. H.; Johannes, D. W.; Elder, C. C.; Bartz, Q. R.,
Azaserine, a New Tumor-inhibitory Substance. Isolation and Characterization. J. Am. Chem. Soc. 1954,
76 (2878-2881).
9
15.
Dion, H. W.; Fusari, S. A.; Jakubowski, Z. L.; Zora, J. G.; Bartz, Q. R., 6-Diazo-5-oxo-L-norleucine,
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A New Tumor-Inhibitory Substance. II. Isolation and Characterization. J. Am. Chem. Soc. 1956, 78
(3075-3077).
16.
Inhibitory Substance: Studies with Crocker Mouse Sarcoma 180. Nature 1954, 173 (71-72).
17. Colis, L. C.; Woo, C. M.; Hegan, D. C.; Li, Z.; Glazer, P. M.; Herzon, S. B., The Cytotoxicity of (-)-
Lomaiviticin A Arises from Induction of Double-Strand Dreaks in DNA. Nat. Chem. 2014, 6, 504-10.
18. He, H.; Ding, W. D.; Bernan, V. S.; Richardson, A. D.; Ireland, C. M.; Greenstein, M.; Ellestad, G.
Stock, C. C.; Clarke, D. A.; Reilly, H. C.; Rhoads, C. P.; Buckley, S. M., Azaserine, A New Tumor-
A.; Carter, G. T., Lomaiviticins A and B, Potent Antitumor Antibiotics from Micromonospora
lomaivitiensis. J. Am. Chem. Soc. 2001, 123, 5362-5363.
19.
Lomaiviticin Aglycon. J. Am. Chem. Soc. 2011, 133, 7260-7263.
20. Herzon, S. B.; Woo, C. M., The Diazofluorene Antitumor Antibiotics: Structural Elucidation,
Biosynthetic, Synthetic, and Chemical Biological Studies. Nat. Prod. Rep. 2012, 29, 87-118.
21. Janso, J. E.; Haltli, B. A.; Eustaquio, A. S.; Kulowski, K.; Waldman, A. J.; Zha, L.; Nakamura, H.;
Herzon, S. B.; Lu, L.; Woo, C. M.; Gholap, S. L., 11-Step Enantioselective Synthesis of (-)-
Bernan, V. S.; He, H.; Carter, G. T.; Koehn, F. E.; Balskus, E. P., Discovery of the Lomaiviticin Biosynthetic
Gene Cluster in Salinispora pacifica. Tetrahedron 2014, 70, 4156-4164.
22.
Kersten, R. D.; Lane, A. L.; Nett, M.; Richter, T. K. S.; Duggan, B. M.; Dorrestein, P. C.; Moore, B.
S., Bioactivity-Guided Genome Mining Reveals the Lomaiviticin Biosynthetic Gene Cluster in Salinispora
tropica. Chembiochem 2013, 14, 955-962.
23.
Woo, C. M.; Li, Z.; Paulson, E. K.; Herzon, S. B., Structural Basis for DNA Cleavage by the Potent
Antiproliferative Agent (-)-Lomaiviticin A. Proc. Natl. Acad. Sci. USA 2016, 113, 2851-2856.
24.
25.
Maas, G., New Syntheses of Diazo Compounds. Angew. Chem., Int. Ed. 2009, 48, 8186-8195.
Myers, E. L.; Raines, R. T., A phosphine-mediated conversion of azides into diazo compounds.
Angew Chem Int Ed Engl 2009, 48 (13), 2359-63.
26.
27.
Gould, S. J., Biosynthesis of the Kinamycins. Chem. Rev. 1997, 97, 2499-2509.
Gould, S. J.; Melville, C. R., Kinamycin Biosynthesis. Synthesis, Detection, and Incorporation of
Kinobscurinone, a Benzo[b]fluorenone. Bioorg. Med. Chem. 1995, 5, 51-54.
28. Gould, S. J.; Melville, C. R.; Cone, M. C.; Chen, J.; Carney, J. R., Kinamycin Biosynthesis.
Synthesis, Isolation and Incorporation of Stealthin C, an Aminobenzo[b]fluorene. J. Org. Chem. 1997,
62, 320-324.
29.
Winter, J. M.; Jansma, A. L.; Handel, T. M.; Moore, B. S., Formation of the Pyridazine Natural
Product Azamerone by Biosynthetic Rearrangement of an Aryl Diazoketone. Angew. Chem., Int. Ed.
2009, 48 (4), 767-770.
30.
Type Smiles Rearrangement. J Am Chem Soc 2017, 139 (8), 2864-2867.
31. Huang, Z.; Wang, K. K.; Lee, J.; van der Donk, W. A., Biosynthesis of Fosfazinomycin is a
Convergent Process. Chem. Sci. 2015, 6 (2), 1282-1287.
32. Huang, Z.; Wang, K. K. A.; van der Donk, W. A., New Insights into the Biosynthesis of
Fosfazinomycin. Chem. Sci. 2016, 7, 5219-5223.
Wang, P.; Hong, G. J.; Wilson, M. R.; Balskus, E. P., Production of Stealthin C Involves an S-N-
ACS Paragon Plus Environment

Page 23 of 23
1
The Journal of Organic Chemistry
33.
Bergy, M. E.; Pyke, T. R. Cremeomycin and Process for Making. 3350269, October 31, 1967,
McGuire, J. N.; Wilson, S. R.; Rinehart, K. L., Cremeomycin, a Novel Cytotoxic Antibiotic from
2
3
4
5
1967.
34.
Streptomyces cremeus. Structure Elucidation and Biological Activity. J. Antibiot. (Tokyo) 1995, 48, 516-
519.
6
7
8
9
35.
Biosynthetic Gene Cluster Encodes a Pathway for Diazo Formation. Chembiochem 2015, 16, 2172-2175.
36. Sugai, Y.; Katsuyama, Y.; Ohnishi, Y., A Nitrous Acid Biosynthetic Pathway for Diazo Group
Formation in Bacteria. Nat. Chem. Biol. 2016, 12, 73-5.
37. Cho, J. Y.; Kwon, H. C.; Williams, P. G.; Jensen, P. R.; Fenical, W., Azamerone, a Terpenoid
Waldman, A. J.; Pechersky, Y.; Wang, P.; Wang, J. X.; Balskus, E. P., The Cremeomycin
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Phthalazinone from a Marine-Derived Bacterium Related to the Genus Streptomyces
(Actinomycetales). Org. Lett. 2006, 8, 2471-2474.
38.
Cremeomycin. Synthesis 2008, 22, 3601-3604.
39. Gulick, A. M., Conformational Dynamics in the Acyl-CoA Synthetases, Adenylation Domains of
Non-ribosomal Peptide Synthetases, and Firefly Luciferase. ACS Chem. Biol. 2009, 4 (10), 811-827.
40. Walsh, C. T.; Tu, B. P.; Tang, Y., Eight Kinetically Stable but Thermodynamically Activated
Molecules that Power Cell Metabolism. Chem Rev 2018, 118 (4), 1460-1494.
Varley, L. M.; Moody, C. J., First Synthesis of the Naturally Occurring Diazocarbonyl Compound
41.
71.
42.
Schmelz, S.; Naismith, J. H., Adenylate-forming enzymes. Curr Opin Struct Biol 2009, 19 (6), 666-
Chen, Y.; Sun, Y.; Song, H.; Guo, Z., Structural Basis for the ATP-dependent Configuration of
Adenylation Active Site in Bacillus subtilis o-Succinylbenzoyl-CoA Synthetase. J Biol Chem 2015, 290
(39), 23971-83.
43.
Doumith, D.; Weingarten, P.; Wehmeier, U. F.; Salah-Bey, K.; Benhamou, B.; Capdevila, C.;
Michel, J.-M.; Piepersberg, W.; Raynal, M.-C., Analysis of genes involved in 6-deoxyhexose biosynthesis
and transfer in Saccharopolyspora erythraea. Mol. Gen. Genet. 2000, 264, 477-485
44.
Kieser, T.; Bibb, M. J.; Buttner, M. J.; Chater, K. F.; Hopwood, D. A., Practical Streptomyces
Genetics. John Innes Foundation: Norwich, UK, 2000.
TOC Graphic:
ACS Paragon Plus Environment