Diverse Combinatorial Biosynthesis Strategies for C-H Functionalization of Anthracyclinones

Abstract

Streptomyces spp. are “nature’s antibiotic factories” that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in Streptomyces coelicolor M1152ΔmatAB that could produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues via two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and S. coelicolor M1152ΔmatAB expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant Streptomyces spp. hosts.

Full text

Diverse Combinatorial Biosynthesis Strategies for C−H
Functionalization of Anthracyclinones
Rongbin Wang,
⊥
Benjamin Nji Wandi,
⊥
Nora Schwartz,
⊥
Jacob Hecht,
⊥
Larissa Ponomareva,
Kendall Paige, Alexis West, Kathryn Desanti, Jennifer Nguyen, Jarmo Niemi, Jon S. Thorson,
Khaled A. Shaaban,*Mikko Metsä-Ketelä,*and S. Eric Nybo*
Cite This: https://doi.org/10.1021/acssynbio.4c00043
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sı Supporting Information
ABSTRACT: Streptomyces spp. are “nature’s antibiotic factories” that
produce valuable bioactive metabolites, such as the cytotoxic
anthracycline polyketides. While the anthracyclines have hundreds of
natural and chemically synthesized analogues, much of the chemical
diversity stems from enzymatic modifications to the saccharide chains
and, to a lesser extent, from alterations to the core scaold. Previous
work has resulted in the generation of a BioBricks synthetic biology
toolbox in Streptomyces coelicolor M1152ΔmatAB that could produce
aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone. In
this work, we extended the platform to generate oxidatively modified
analogues via two crucial strategies. (i) We swapped the ketoreductase
and first-ring cyclase enzymes for the aromatase cyclase from the
mithramycin biosynthetic pathway in our polyketide synthase (PKS)
cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-
hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed
improved plasmid vectors and S. coelicolor M1152ΔmatAB expression hosts to produce anthracyclinones. This work sets the stage for
the combinatorial biosynthesis of bespoke anthracyclines using recombinant Streptomyces spp. hosts.
KEYWORDS: BioBricks, synthetic biology, natural product biosynthesis, anthracyclinones, Streptomyces coelicolor, oxygenase, anticancer
■INTRODUCTION
Anthracyclines are glycosylated aromatic polyketides produced
by various soil bacteria in the actinomycete family.
Doxorubicin and aclarubicin are utilized as anticancer agents
for treating various human cancers, making them some of the
broadest spectrum antineoplastic agents used in the clinic.
1
Anthracyclines inhibit the proliferation of cancer cells through
two distinctive mechanisms: histone eviction and inhibition of
topoisomerase II, leading to the scission of DNA strands.
1,2
However, anthracyclines have limitations that diminish their
clinical utility. Cancer cells can develop drug resistance to the
anthracyclines, for example, by overexpressing the p-
glycoprotein ATP binding cassette.
3
Additionally, the long-
term use of anthracyclines is associated with cardiotoxicity.
4
These observations have motivated the systematic biosynthetic
modification of anthracyclines to achieve new analogues with
advantageous properties over currently used medications,
including increased potency, decreased drug resistance, and
reduced cardiotoxicity.
5
Anthracyclines are biosynthesized by polyketide synthase
(PKS) complexes, which are composed of a minimal PKS
(minPKS) consisting of a ketoacyl synthase (KSα), chain
length factor (CLF or KSβ), and acyl carrier protein (ACP)
that catalyzes the Claisen condensation of one molecule of
acetyl-CoA or propionyl-CoA to nine molecules of malonyl-
CoA (Figure 1A). The resulting poly β-keto thioester
decaketide undergoes controlled folding by 9-ketoreductase
(9-KR), aromatase (ARO), second-/third-ring cyclase (2/3-
CYC), and oxygenase (OXY) enzymes to generate the first
stable intermediates aklanonic acid and nogalonic acid. Further
reactions by methyltransferase (MET), fourth-ring cyclase (4-
CYC), and ketoreductase (7-KR) enzymes furnish the core
tetracyclic aromatic carbon skeletons (Figure 1A).
3
Previously,
we developed a BioBricks platform for the improved
biosynthesis of anthracyclinones.
6
We developed vectors to
produce four anthracyclinone scaolds: aklavinone (1), 9-epi-
aklavinone (2), auramycinone (3), and nogalamycinone (4)
(Figure 1A). In addition, endogenous activity from the host
Received: January 22, 2024
Revised: April 11, 2024
Accepted: April 16, 2024
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Figure 1. Metabolic engineering strategies for C−H functionalization of anthracyclinones. Biosynthesis of (A) the four anthracyclinone aglycones
1−4, (B) degradation products 5−8through the action of endogenous enzymatic activities of the host strain S. coelicolor and (C) 2-hydroxylated
target compounds 9−14 accessible via PKS cassette engineering utilizing stemycin B biosynthetic logic. Depiction of the diversity of post-PKS
tailoring steps on (D) doxorubicin, (E) rhodomycin, (F) komodoquinone, and (G) kosinostatin pathways.
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B

strain Streptomyces coelicolor M1152ΔmatAB led to the
production of 7-deoxygenated versions of these compounds,
including 7-deoxy-aklavinone (5), 7-deoxy-9-epi-aklavinone
(6), 7-deoxy-auramycinone (7), and 7-deoxy-nogalamycinone
(8) (Figure 1B).
Anthracycline biosynthetic gene clusters harbor extensive
gene sets to modify the core anthracyclinone structures further.
Particularly, gene products catalyzing redox chemistry for C−
H functionalization, which is an important component in the
chemodiversification of anthracyclines, are abundant. The
daunorubicin pathway harbors the FAD-dependent 11-
monooxygenase DnrF (Figure 1C).
7−9
RdmE also catalyzes
the same reaction on the rhodomycin pathway, containing the
15-methylesterase RdmC and the methyltransferase-like RdmB
for C-10 hydroxylation (Figure 1D).
7,9,10
The komodoquinone
pathway includes enzymes for 10-decarboxylation by the C-15
esterase EamC and the methyltransferase-like EamK (Figure
1E).
11,12
Finally, the kosinostatin biosynthetic gene cluster
encodes the short-chain aldol reductase KstA16 and the
cyclase-like KstA15 that jointly catalyze C-1 hydroxylation
(Figure 1F).
13
The reaction cascade is further extended to 4-
hydroxyl regioisomerization by the NmrA-like short-chain
dehydrogenase/reductase enzymes KstA11 and KstA10
(Figure 1F).
13
In this work, we were interested in combinatorial biosyn-
thesis and C−H functionalization of anthracyclinones using
two distinct approaches. The first strategy included reprogram-
ming the PKS cassettes to aord 2-hydroxylated analogues,
similar to stemycin biosynthesis.
14
This could plausibly be
achieved by excluding the KR gene and exchanging ARO/CYC
genes with those residing on aureolic acid biosynthetic
pathways (Figure 1C).
14−16
The second strategy included
diverse post-PKS tailoring genes from the kosinostatin,
13
rhodomycin,
9,10
doxorubicin,
8
and komodoquinone B path-
ways.
11
One significant goal of these studies was to
systematically evaluate the substrate promiscuity of post-PKS
tailoring enzymes toward alternative substrates 1−4.
The metabolic engineering presented here resulted in the
generation of 26 anthracyclinones, including nine novel
analogues with regiospecific C−H oxygenation. Chemical
characterization and bioactivity profiling revealed the im-
portance of 1-, 10-, and 11-hydroxylation in the cytotoxicity of
the anthracyclinones. Installing ketone, aldehyde, and alcohol
functional groups provides chemical handles for group-transfer
enzymes, such as methyltransferases, aminotransferases, and
glycosyltransferases.
12
This opens the door for rational
metabolic engineering to generate diverse glycosylated
anthracycline analogues in the future.
■RESULTS AND DISCUSSION
Development of Improved Strains and Vectors. The
yields of 1−4from the previous PKS cassettes ranged between
1 and 5 mg/L. We applied four distinct strategies to improve
production (Figure 2A,B).
17
First, we incorporated stronger
synthetic sp41, sp42, and sp44 promoters (Table S1)
18,19
together with ribozyme-based insulator parts (e.g., sp41-vtmoJ,
Figure 2. Four metabolic engineering strategies to increase the yields of anthracyclinones. (A) SBOL diagram of redesigned PKS cassettes.
Constructs encoded the simultaneous expression of eight to ten genes (depending on the anthracyclinone) under the expression of strong sp41,
sp42, and sp44 promoters. Constructs were insulated from external genomic promoter expression by incorporating tt-sbi-A and fd-term
transcriptional terminators. Promoters were fused to ribozyme-insulator parts to stabilize the expression of the three operons within the construct.
(B) Overexpression of ssgA,scbr2, and accA2BE for anthracyclinone enhancement. SsgA triggers sporulation and cell division, which works with the
ΔmatAB mutation to enhance biomass accumulation. Scbr2 is a pseudo-γ-butyrolactone response regulator that regulates glycolytic flux. AccA2BE
enhances the supply of malonyl-CoA for anthracyclinone biosynthesis. (C) Production titers of 9-epi-aklavinone in strains expressing pEN10002
and/or coexpressing pEAKV2 with scbr2, accA2BE, and/or ssgA. (D) Production titers of nogalamycinone in strains expressing pEN10004 and/or
coexpressing pNOG2 with scbr2, accA2BE, and/or ssgA.
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sp42-ltsvJ, and sp44-riboJ) to eliminate interference between
the promoters and ribosome initiation sites (Figure 2A).
6,20
Ribozyme-based insulators function by cleaving the 5′
untranslated region (5′-UTR) of the mRNA to form a hairpin
loop to stabilize the transcript. Furthermore, the cassettes were
bracketed with transcriptional terminators (e.g., fd phage and
ttsbi-A terminators)
21,22
The new PKS cassettes were cloned
into the pOSV802 vector, allowing single-copy chromosomal
expression in Streptomyces (Table S1).
23
Two vectors, pEAKV2
encoding the production of 9-epi-aklavinone and pNOG2
encoding the output of nogalamycinone, were expressed in S.
coelicolor M1152ΔmatAB (Table S2). The improved strains
were fermented in E1 liquid media and produced 4 mg/L of 2
and 12 mg/L of 4, which represented 1- and 6-fold
improvement over the previous vectors pEN10002 and
pEN10004, respectively (p< 0.001) (Figure 2C,D). The
enhanced transcriptional stability of the constructs appeared to
contribute to improved translation of the PKS machinery and
metabolic flux to the target molecules.
Second, we increased substrate availability via acetyl-CoA
carboxylase (e.g., accA2BE) overexpression (Figure 2B),
previously employed with tetracenomycin engineering to
achieve 3-fold yield enhancement.
24,25
The acetyl-CoA
carboxylase converts acetyl-CoA to malonyl-CoA, an essential
precursor for the biosynthesis of polyketides.
26
Integration of
the pOSV808-accA2BE expression cassette into the S. coelicolor
lines resulted in a 2-fold improvement in 2(4.4 to 9.2 mg/L, p
< 0.0001) and 4(12.1 to 23 mg/L, p< 0.0001) (Figure 2C,D).
Third, we sought to increase yields by overexpressing ssgA
from Streptomyces griseus (Figure 2B), which has been shown
to suppress sporulation and enhance the fragmented growth of
mycelia, thus resulting in faster growth kinetics and increased
production of biomass and cell products.
27,28
The over-
expression of ssgA using the pENSV3 expression vector
resulted in a nearly 3-fold increase in 2(4.4 to 11.2 mg/L, p
< 0.0001) and 4(12.1 to 31.0 mg/L, p< 0.0001) production
titers (Figure 2C,D). This result demonstrated that ssgA could
improve anthracyclinone production titers.
Fourth, we overexpressed a pseudo-γbutyrolactone (GBL)
receptor scbr2 in S. coelicolor M1152ΔmatAB::cos16F4iE
(Figure 2B). Scbr2 does not bind γ-butyrolactones but has
been shown to interact with numerous endogenous and
exogenous natural products.
29,30
The regulatory eects of
Scbr2 are mediated by promoting glycolysis via upregulation of
glyceraldehyde-3-phosphate dehydrogenase (gap1) and pyr-
uvate kinase (pyk2), which we reasoned could be important for
carbon flow and growth kinetics.
31
The scbr2 gene has been
deleted in S. coelicolor M1152 as part of the cpk cluster,
32
which
has led to increased oxidative metabolism and oxidative stress
(i.e., flux through the tricarboxylic acid cycle) based on a
genome-scale metabolic model.
33
To test this hypothesis, we
overexpressed scbr2 in S. coelicolor, which resulted in a 3-fold
Figure 3. Engineering of aromatase/cyclase enzymes to biosynthesize 2-hydroxylated anthracyclinones. (A−F) HPLC-UV/vis chromatograms of
dierent strains monitored at 430 nm engineered with two plasmids producing metabolites indicated with the numbered compound. (A) S.
coelicolor M1152::acc::A2C1 (2-hydroxy-aklanonic acid, 9); (B) S. coelicolor M1152::acc::S2C1 (2-hydroxy-nogalonic acid, 10); (C) S. coelicolor
M1152::acc::A2C1::A6 (2-hydroxy-aklavinone, 11); (D) S. coelicolor M1152::acc::A2C1::S6 (2-hydroxy-9-epi-aklavinone, 12); (E) S. coelicolor
M1152::acc::S2C1::A6 (2-hydroxy-auramycinone, 13); (F) S. coelicolor M1152::acc::S2C1::S6 (2-hydroxy-nogalamycinone, 14). (G) Production
titers of 2-hydroxylated anthracyclinones from strains engineered with two plasmids. (H) The production titers of strains engineered with and
without expression of the C-12 oxygenase (snoaB). (I) The 1H,13C-HMBC, 1H,1H−COSY, and 1H,1H-NOESY two-dimensional NMR correlations
for compounds 11, 12, 13, and 13b.
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improvement in 2(4.4 to 13.0 mg/L, p< 0.0001) and 4(12.1
to 30.0 mg/L, p< 0.0001) titers (Figure 2C,D). This result
demonstrated that overexpression of scbr2 may have balanced
glycolytic flux and relieved oxidative stress for improved
product formation, but the exact mechanism requires further
study.
Metabolic Engineering of PKS Cassettes for 2-
Hydroxylated Anthracyclinones. We sought to develop a
means for producing 2-hydroxylated analogues of 1−4by
reprogramming the PKS cassettes.
6
The polyketide-derived 2-
hydroxyl group is removed during anthracycline biosynthesis
by KR enzymes (Figure 1A) and, consistently, pathways
lacking these redox enzymes have resulted in the production of
2-hydroxy-aklavinone
34
and 2-hydroxy-nogalonic acid.
15,35
Here, we reasoned that coexpression of anthracycline minPKS
genes aknBCDE2For snoa123 together with ARO and 2/3-
CYC genes from nonreducing pathways,
36
such as mtmQY
involved in mithramycin biosynthesis, would result in the
production of 2-hydroxy-aklanonic acid (9) or 2-hydroxy-
nogalonic acid (10), respectively (Figure 1C). Cloning and
transformation of gene cassettes pA2C1 (aknBCDE2F+
mtmQY) and pS2C1 (snoa123 + mtmQY) resulted in the
production of 9and 10, respectively (Figures 3A,B and S1−
S2).
To extend the pathway, we cloned additional cassettes
encoding MET, 4-CYC, and 7-KR (e.g., aknGHU) and pTG1-
S6 (e.g., snoaCLF) to the host strain, which resulted in the 2-
hydroxy-aklavinone (11), 2-hydroxy-9-epi-aklavinone (12), 2-
hydroxy-auramycinone (13), and 2-hydroxy-nogalamycinone
(14) based on high-performance liquid chromatography-mass
spectrometry (HPLC-MS) (Figures 1C and 3C−F and Table
S3). Although product yields were reasonable (3−10 mg/L)
(Figure 3G), we hypothesized that additional coexpression of
an OXY gene could enhance the folding and shaping of the
unnatural 2-hydroxylated polyketides. Indeed, the coexpression
of snoaB on a separate multicopy expression vector
(pUWL201PW) under the control of the ermE*presulted in
a 50% increase in production of 11 and 13 (Figure 3H). Based
on these findings, we refactored our constructs to generate
improved versions of pHAKV2, pHEAKV2, pHAURA2, and
pHNOG2 (e.g., encoding the production of 11, 12, 13, and
14, respectively, Table S1). pHAKV2, pHEAKV2, and
pHAURA2 were used in subsequent scale-up fermentation
experiments (see the Methods Section).
To confirm the structures of 11,12, and 13, the
fermentations were scaled up in 5 L of E1 media, the
metabolites were extracted and purified using various
chromatographic techniques, and structurally characterized
based on high-resolution electrospray ionization-MS (HRESI-
MS) and NMR spectroscopy. The chemical characterization
revealed that the early pathway intermediate SEK15 (13b) was
accumulated as a side product in the scale-up fermentation of
pHAURA2. In addition, three new 2-hydroxy-anthracyclinones
(11−13) were isolated, based on one-dimensional (1D) (1H
and 13C NMR) and two-dimensional (2D) (COSY, HSQC,
HMBC, TOCSY, and NOESY) NMR spectroscopic measure-
Figure 4. Enzymatic assays and metabolic engineering of 11-hydroxylated anthracyclinones. (A) DnrF catalyzes 11-hydroxylation of 1−4to aord
15−18. (B) HPLC-UV/vis traces at 490 nm of enzymatic reactions of 1−4incubated with purified DnrF and no-enzyme controls. (C) HPLC-UV/
vis traces at 490 nm of S. coelicolor lines engineered with expression constructs encoding 1−4and dnrF or control lines producing only 1−4.
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ments (Tables S4−S5 and Figures 3I and S3−S46). The
molecular formulas of 11−13 were established as C22H20O9,
C22H20O9, and C21H18O9based on (+)-HRESIMS, with Δm/z
= 16 higher than those of aklavinone (1), 9-epi-aklavinone (2),
and auramycinone (3), respectively, indicative of the presence
of an extra oxygen atom in 11−13 (Figures S4, S17, and S27).
Comparison of the NMR data (1H and 13C NMR) of the new
compounds 11-13 with previously reported compounds 1-3
revealed that the main dierences were observed in the
aromatic ring A, where the trisubstituted aromatic rings in
compounds 1-3were converted to tetra-substituted aromatic
rings in compounds 11-13 (with two m-coupled protons, H-1/
H-3; Tables S4 and S5). The position of the hydroxy groups in
compounds 11-13 was established to be at 2-position based on
the observed HMBC correlations from H-1 to C-12/C-4a/
CH-3; 2-OH to CH-1/C-2/CH-3 and H-3 to CH-1/C-4a
(Figure 3I). All of the remaining 2D-NMR (1H, 1H−COSY,
HMBC, TOCSY, and NOESY) correlations fully agree with
structures 11−13 (Figure 3I and Supporting Information). As
new natural products and are closely related to 1−3,
compounds 11-13 were designated as 2-hydroxy-aklavinone
(11), 2-hydroxy-9-epi-aklavinone (12), and 2-hydroxy-auramy-
cinone (2-hydroxy-9-epi-nogalamycinone; 2-hydroxy-9-epi-no-
galavinone; 13), respectively. The structure of 14 is suggested
to be 2-hydroxy-nogalamycinone.
Anthracyclinone 11-Hydroxylation by DnrF. To probe
the substrate promiscuity of post-PKS tailoring enzymes for 1−
4, we carried out parallel investigations with purified enzymes
and gene expression studies. We first cloned the dnrF gene
37
from the doxorubicin pathway into the pBAD/His B vector for
expression in Escherichia coli TOP10. After the production and
purification of recombinant 11-hydroxylase DnrF, we assayed
the conversion of 1−4to the 11-hydroxylated species 15, 16,
17, and 18 (Figures 1D and 4A). The incubation of 1and 3
resulted in quantitative conversion to 15 and 17, whereas 2
and 4were converted to 16 and 18 with poor eciency <5%
(Figures 4B and S47−S75).
For the in vivo expression experiments, dnrF was fused to the
strong gapdhpEL promoter, cloned into expression vector
pENSV3, and transformed into cell lines producing 1−4.
Analysis of culture extracts demonstrated that 1was converted
with >90% eciency to 15 and maggiemycin, which is a shunt
product derived from 11-hydroxylation of aklaviketone.
38
Compound 3was also converted in >90% eciency to 17,
previously isolated from fermentations of Streptomyces
coeruleorubidis ATCC 31276.
39
However, similarly to the in
Figure 5. Enzymatic assays and metabolic engineering of 10-hydroxylated and 10-decarboxylated anthracyclinones. (A) EamC and EamK catalyze
10-decarboxylation of 1−4to aord 19−22. EamC and RdmB catalyze 10-hydroxylation of 1and 3to produce 23 and 24. (B) HPLC-UV/vis
traces at 430 nm of enzymatic reaction of 1−4incubated with purified EamC + RdmB, EamC + EamK, and no-enzyme controls. (C) HPLC-UV/
vis traces at 430 nm of S. coelicolor lines engineered with expression constructs encoding 1−4and either eamC +eamK or eamC +rdmB.
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vitro analyses, 2and 4were converted to 16 and 18 with poor
5−10% eciency (Figures 4C and S47−S75), respectively.
These results demonstrated that DnrF is more selective toward
9(R)-configured than 9(S)-configured anthracyclines.
Anthracyclinone 10-Hydroxylation by RdmB and 10-
Decarboxylation EamK. Both anthracycline 10-hydroxyla-
tion and 10-decarboxylation by the SAM-dependent hydrox-
ylase RdmB and decarboxylase EamK, respectively, require
initial 15-methylesterase activity (Figures 1E,F and 5A). Since
the rhodomycin enzymes have a preference for glycosylated
substrates,
11,40
which is in contrast to the komodoquinone
enzymes that convert aglycone substrates,
11
we utilized the 15-
methylesterase EamC in all experiments. Our in vitro (Figure
5B) and in vivo (Figure 5C) data were highly convergent and
demonstrated that 1−4were quantitatively converted by
EamC and EamK to 10-decarboxylated products 19−22
(Figures S76−S91). In contrast, only 1and 3were converted
to 10-hydroxylated species 23 and 24, respectively (Figures
5B,C and S92−S97), which demonstrates that RdmB exhibits
preference toward 9(R)-configured metabolites both in vitro
and in vivo.
Anthracyclinone 10- and 11-Hydroxylation by RdmE,
RdmC, and RdmB. We next sought to incorporate the
tailoring steps for concomitant 10 and 11-hydroxylation by the
entire RdmE, RdmC, and RdmB reaction cascade (Figures 1E
and 6A). We incubated 1−4with purified DnrF, EamC, and
RdmB, which resulted in the conversion of substrates 1and 3
to 25 and 26 (Figures 6B and S98−S104). Similarly, 1and 3
were converted to 25 and 26 in Streptomyces strains
coexpressing the appropriate PKS cassettes and a cassette
expressing rdmE, rdmC, and rdmB (Figures 6C and S98−
S104). Both in vitro and in vivo, the substrates 2and 4
underwent 10-decarboxylation toward 20 and 22 as the
primary metabolic route and were not processed by the
hydroxylating enzymes. The production titers of 25 and 26
from the engineered strains in SG-TES media were 30.3 ±10
and 9.3 ±4 mg/L, respectively (Figure S105). Altogether, this
indicates robust production of the β-rhodomycinone analogues
25 and 26 in the engineered microorganisms.
Anthracyclinone 1-Hydroxylation by KstA15 and
KstA16. We next utilized the two-component monooxygenase
system, KstA15 and KstA16, from the kosinostatin pathway for
Figure 6. Enzymatic assays and metabolic engineering of 10-hydroxylated and 11-hydroxylated anthracyclinones. (A) RdmE, EamC/RdmC, and
RdmB catalyze 11-hydroxylation and 10-hydroxylation of 1and 4to generate 25 and 26, respectively. (B) HPLC-UV/vis traces at 490 nm of
enzymatic reaction of 1and 3incubated with purified DnrF, EamC, RdmB, and no-enzyme controls. (C) HPLC-UV/vis traces at 490 nm of S.
coelicolor lines engineered with expression constructs encoding 1and 3and rdmE +rdmC +rdmB and control lines producing 1and 3.
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1-hydroxylation (Figures 1F and 7A).
13
KstA15 is a polyketide
cyclase-like enzyme,
41
while KstA16 belongs to short-chain
alcohol dehydrogenases.
42
The enzyme assays with substrates
1−4resulted in the quantitative conversion to 1-hydroxylated
Figure 7. Enzymatic assays and metabolic engineering of 1-hydroxylated and 4-regioisomerized anthracyclinones. (A) KstA15 and KstA16 catalyze
1-hydroxylation of 1−4yielding 27−30, KstA10 and KstA11 carry out asymmetric reduction and dearomatization, followed by a region-specific
reduction and dehydration yielding 31−34. (B) HPLC-UV/vis traces of enzymatic reaction of 1−4incubated with purified KstA15 and KstA16
and no-enzyme controls. (C) HPLC-UV/vis traces of S. coelicolor lines engineered with expression constructs encoding 1−4and kstA15 and kstA16
and control lines producing 1−4. (D) HPLC-UV/vis traces of enzymatic reaction of 1−4incubated with purified KstA15, KstA16, KstA11, and
KstA10 and no-enzyme controls. (E) HPLC-UV/vis traces of S. coelicolor lines engineered with expression constructs encoding 1−4and kstA15,
kstA16, kstA10, and kstA11 and control lines producing 1−4.
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H

anthracyclinones 27−30 (Figures 7B and S106−S120).
Analogously, the heterologous expression of a cassette
encoding kstA15 and kstA16 resulted in the production of 1-
hydroxylated species 27−30 (Figures 7C and S106−S120).
The compounds 27,29, and 30 have been generated in
previous studies,
41−43
but the anthracyclinone 28 has not been
reported in the literature. The 1-hydroxylation mechanism is a
necessary modification for 1-O-glycosylation of anthracyclines,
such as that occurs with the nogalamycin family of
compounds.
44
Anthracyclinone 4-Regioisomerization by KstA15,
KstA16, KstA11, and KstA10. KstA15 and KstA16 are part
of a four-enzyme cascade to generate a hydroxy regioiso-
merized anthracyclinone. Following the successful 1-hydrox-
ylation by KstA15 and KstA16, we reconstituted the entire 4-
regioisomerization pathway by incorporating KstA11 and
KstA10 (Figures 1F and 7A).
13
The in vitro assays with the
four enzymes converted 1−4to iso-anthracyclinones 31−34
(Figures 7D and S121−S132). Several putative intermediates
were detected in the enzymatic reactions, including the
dearomatized 1,4-diketone species (indicated with #) (Figure
7D). Similarly, coexpression of the four kst genes in
appropriate S. coelicolor strains producing 1−4led to
accumulation of 31−34 (Figures 7E and S121−S132) in
yields ranging from 8 to 30 mg/L (Figure S133). Within the
ESI-MS single ion monitoring traces, the parental substrates,
unknown intermediates (marked *), and products 31−34 were
detected with the expected mass ions (Table S3). These results
indicate that the kosinostatin enzymes are remarkably flexible
toward 9(R) and 9(S)-configured anthracyclinones. Previously,
31 has been isolated from strains engineered for isoanthracy-
cline production
45
and 33 is the native substrate of the
kosinostatin pathway,
13
but compounds 32 and 34 have not
been reported to date.
■HUMAN CANCER CELL VIABILITY ASSAYS
To investigate the potential anticancer activity of the
anthracyclinone extracts, the crude extracts were normalized
to 40 μg/mL concentration and were tested in a panel of
human cancer cells: A549 (nonsmall cell lung), PC3
(prostate), TC32 (Ewing sarcoma), and HCT116 (colorectal)
human cancer cell lines (Table 1 and Figure S134). As
previously reported, the anthracyclinone aglycones 3and 4are
inactive at IC50 values >30 μM in A549, PC3, MKL1, and
MCC26 cancer cell lines, though 1is slightly more active in
PC3 cells at 7 μM IC50 and A549 cells at 17 μM, respectively.
6
Most of the extracts were inactive against these cancer cell
lines; however, extracts from five engineered lines containing
compounds 15,17,18,22,25,26, and 30 exhibited
substantive cytotoxic activity in the cell lines tested (<20%
cell viability T/C) (Figures S134). Compounds 11,12, and 13
were inactive at concentrations of 3 μM (Figure S135), which
indicated that 2-hydroxylation does not enhance the
cytotoxicity of these aglycones.
Anthracyclines are considered to require the glycoside
moiety for binding to DNA and inhibition of DNA
topoisomerases.
5
Therefore, the significant cytotoxicity against
the human cancer cell lines in selected extracts can be
considered to be surprising (Table 1). The data provides
guidance regarding the rational engineering of anthracyclines
toward 11-hydroxylated, 10,11-dihydroxylated, or 1-hydroxy-
lated nogalamycinone derivatives. Indeed, anthracyclines with
C-1 or C-11 hydroxyl groups were shown to have increased
potency in L1210 leukemia cells,
46
while 11-hydroxylation of
aclacinomycins resulted in markedly improved cytotoxicity in
the NCI 60-cell line assay.
47
Rhodomycin A, which is a 10,11-
dihydroxylated anthracycline, was recently identified as an
ecient antagonist for knocking down Src-associated onco-
genes.
48
Table 1. Cytotoxic Activities of the Generated Strain Extracts in PC3, TC32, A549, and HCT116 Cell Lines
b
,
c
a
The samples were culture extracts from the strains producing the numbered compound and contain other impurities. Actinomycin D and H2O2
were used as positive controls at 20 μM and 1 mM concentration, respectively (0% viable cells, n= 3. SE = standard error).
b
The cell viability of
each line was determined as the percentage of cell viability relative to untreated controls.
c
% Viability values were obtained after 72 h incubation.
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I

■CONCLUSIONS
Anthracyclines have been a cornerstone of anticancer chemo-
therapy for several decades. Despite the success of these
molecules, severe side eects have made the continued
exploration of the chemical space around anthracyclines
necessary for discovering improved congeners. For example,
developing the semisynthetic idarubicin (4-demethoxy daunor-
ubicin) for acute myeloid leukemia has demonstrated the
importance of C−H functionalization of anthracyclines.
49
However, the regiospecific modification of the polyaromatic
anthracycline core has remained challenging using organic
synthesis. This is in contrast to the biosynthesis of
anthracyclines in Streptomyces bacteria, where several enzy-
matic systems have evolved for C−H functionalization in the
various natural metabolic pathways.
We have used our BioBricks metabolic engineering platform
to systematically probe 1-, 2-, 10-, and 11-hydroxylations, 10-
decarboxylation, and 4-hydroxyl regioisomerization. For the
first time, we analyzed the functionality of 10 tailoring genes
with the four possible anthracyclinone core structures 1−4
comparatively. The results demonstrate that the in vivo activity
remained consistently high in 10-decarboxylation, 1-hydrox-
ylation, and 4-hydroxyl regioisomerization (Figure 8). In
contrast, 10-hydroxylation and 11-hydroxylation were depend-
ent on the 9R-stereochemistry but were tolerant toward both
methyl and ethyl side chains at the same location. The dual 10-
and 11-hydroxylations proved the most challenging, which may
be because the 10-hydroxylase RdmB generally prefers
glycosylated substrates (Figure 8).
Our work demonstrates that tailoring steps in anthracycline
biosynthesis are well suited for combinatorial biosynthesis for
increasing the chemical diversity of natural products. We
utilized genetic material from five distinct pathways and their
use in combination with the four aglycone possibilities led to
the generation of nine novel anthracyclinones (11, 12, 13, 14,
16, 18, 24, 26, 28). It is noteworthy that even though all of the
tailoring enzymes have been extensively studied in the past,
including by structural analysis at atomic resolution, it was not
possible to predict a priori which gene combinations resulted
in new functional metabolic pathways. Therefore, the ability to
use multiplasmid expression systems in a robust heterologous
host in combination with an expanding modular BioBricks
library is essential to allow high-throughput combinatorial
work to compensate for the limitations of an unpredictable
design space. In addition, the highly concurrent in vitro and in
vivo data indicate that combinatorial enzymatic synthesis may
be an ecient preliminary tool to guide metabolic engineering
projects. Notably, the human cancer cell line viability assays
(Table 1) provide further direction for the rational engineering
of anthracyclines based on the 15,17, 25, and 26 scaolds.
These strains will serve as valuable chassis for combinatorial
biosynthesis of TDP-deoxysugar pathways to develop “new to
nature” anthracyclines, which could be developed into potent
antitumoral metabolites.
■METHODS
Bacterial Strains and Growth Conditions. E. coli
TOP10 and E. coli ET12567 were grown in LB broth or LB
agar at 37 °C as previously described.
50
E. coli TOP10 was
used for plasmid propagation, subcloning, and enzyme
expression. Enzymes were cloned into pBAD/His B vector
(Invitrogen) and enzymes were expressed using the arabinose-
inducible promoter.
51
E. coli ET12567/pUZ8002 was used as
the conjugation donor host for mobilizing expression vectors
into S. coelicolor as previously described.
52
When appropriate,
ampicillin (100 μg mL−1), kanamycin (25 μg mL−1),
apramycin (25 μg mL−1), viomycin (25 μg mL−1),
spectinomycin (100 μg mL−1), hygromycin (50 μg mL−1),
and nalidixic acid (30 μg mL−1) were supplemented to media
to select for recombinant microorganisms.
S. coelicolor derivative strains were routinely maintained on
Soya-Mannitol Flour (SFM) agar supplemented with 10 mM
MgCl2 and International Streptomyces Project medium #4
(ISP4) (BD Difco) at 30 °C as described previously.
52
For
liquid culturing, S. coelicolor derivative strains were grown in
TSB media (3 mL) to ferment the seed culture and then grown
in a modified 50 mL SG-TES liquid medium (soytone 10 g,
glucose 20 g, yeast extract 5 g, TES free acid 5.73 g, CoCl21
mg, per liter) or 50 mL E1 medium for production for four to
5 days.
53
All media and reagents were purchased from Thermo
Fisher Scientific.
Molecular Biology Procedures. Routine genetic cloning
and plasmid manipulation were carried out in E. coli DH10B
cells (New England Biolabs). E. coli ET12567/pUZ8002 was
used as the host for intergeneric conjugation with S. coelicolor
as previously described.
52
E. coli chemically competent cells
were prepared using the Mix and Go! E. coli Transformation
Kit (Zymo Research). E. coli was transformed with plasmid
DNA via chemically competent heat-shock transformation as
described previously. Plasmid DNA was isolated via the Wizard
Plus SV Minipreps DNA Purification System by following the
manufacturer’s protocols (Promega). All molecular biology
reagents and enzymes used for plasmid construction were
purchased from New England Biolabs. The conjugation donor
host E. coli ET12567/pUZ8002 was transformed with
constructs for mobilization into S. coelicolor strains, as
previously described. For each transformation, 9−12 inde-
pendent exconjugants were plated to DNA plates supple-
mented with antibiotics and grown for 4−5 days until the
formation of vegetative mycelium.
Preparation of In Vitro Samples and In Vivo Samples
for HPLC-MS Analysis. The in vitro samples’ conditions for
protein expression, purification, and enzyme assays are
described in the Supporting Information (Supporting Methods
1, 2, and 3). All reactions were checked by UHPLC (Shimadzu
Nexera LC-40 system with a diode array detector set to 430
Figure 8. Heat map of the in vivo activity of the tailoring gene
constructs utilized in this study when coexpressed with anthracycli-
none scaolds 1−4.
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and 490 nm wavelengths) using a Phenomenex Kinetex
Phenyl-Hexyl column (2.6 μm, 100 Å, 4.6 mm ×100 mm).
Method: Solvent A: 15% CH3CN/0.1% FA; solvent B:
CH3CN; flow rate: 0.5 mL/min; 0−2 min, 0% B; 2−20 min,
0−40% B; 20−24 min, 100% B; 24−29 min, 0% B.
For each in vivo experiment, four to six biological replicates
were grown in 50 mL of SG-TES liquid media in baed
Erlenmeyer flasks as previously described.
6,24,25
The shake flask
fermentations were grown in an orbital shaker for 5 days at 30
°C at 200 rpm. The entire cultures were extracted with 3
volumes of 0.1% formic acid in ethyl acetate and the extracts
were dried down in vacuo. The extracts were resuspended in 4
mL of methanol, filtered in a 0.5 μm nylon syringe filter, and
10 μL was analyzed via HPLC-MS.
The analysis of anthracyclinones was carried out on an
Agilent 1260 Infinity II LC/MSD iQ single quadrupole
instrument. In brief, 10 μL of the sample was injected via an
autosampler onto the sample loop, separated on a Poroshell
120 Phenyl-Hexyl Column (ID 2.7 μm, 4.6 mm ×100 mm),
and analyzed in gradients of solvent A (0.1% formic acid in
water) and solvent B (0.1% formic acid in acetonitrile). The
HPLC program used a constant flow rate of 0.5 mL per minute
and the following gradient steps: 0 min, 95% solvent A and 5%
solvent B; 0−10 min, 95% solvent A and 5% solvent B to 5%
solvent A and 95% solvent B; 10−13 min, held at 5% solvent A
and 95% solvent B; 13.1 min, reequilibrate to 95% solvent A
and 5% solvent B; 13.1−15.1 min, 95% solvent A and 5%
solvent B. The diode array detector (DAD) was set to monitor
UV/vis absorbance at 430 and 490 nm. The ESI-MS was set to
scan from 200 m/z−500 m/zfragments in positive and
negative ionization modes.
The yields were determined by comparing them to
authenticated external standard curves of 1and 4analyzed
via HPLC-MS. The conversion percentages were determined
by measuring the area under the curve of the anthracyclinone
metabolites at 430 and 490 nm wavelengths.
General Experimental Procedures. Ultraviolet−visible
(UV−vis) spectra were taken directly from analytical HPLC
runs and show relative intensities. The NMR spectra were
recorded on a Bruker Avance NEO 400 MHz (Bruker BioSpin
Corporation, Billerica, MA) (1H, 400.13 MHz; 13C, 100.25
MHz), Varian 500 MHz (Agilent, Santa Clara, CA) (1H, 500
MHz; 13C, 125.7 MHz), and/or Bruker Avance NEO 600
MHz NMR (1H, 600.37 MHz; 13C, 150.96 MHz)
spectrometer, equipped with triple-channel TCI 5 mm
cryoprobe (all spectra were processed using Bruker Topspin
4.1.4 version, and 2D spectra were apodized with QSINE or
SINE window functions and zero-filled to (2048 ×1024
points)). All of the spectra were analyzed and plotted using
Mnova [where δ-values were referenced to respective solvent
signals CD3OD, δH3.31 ppm, δC49.15 ppm; DMSO-d6,δH
2.50 ppm, δC39.51 ppm]. High-resolution electrospray
ionization (HRESI) mass spectra were recorded on the AB
SCIEX Triple TOF 5600 system (AB Sciex, Framingham,
MA). HPLC-UV/MS analyses were accomplished with an
Agilent InfinityLab LC/MSD mass spectrometer (MS Model
G6125B; Agilent Technologies, Santa Clara, CA) equipped
with an Agilent 1260 Infinity II Series Quaternary LC system
and a Phenomenex NX-C18 column (250 mm ×4.6 mm, 5
μm; Phenomenex, Torrance, CA) [Method A: solvent A:
H2O/0.1% formic acid, solvent B: CH3CN; flow rate: 0.5 mL
min−1; 0−30 min, 5−100% B (linear gradient); 30−35 min,
100% B; 35−36 min, 100%−5% B; 36−40 min, 5% B]. HPLC-
UV analyses were carried out in an Agilent 1260 system
equipped with a photodiode array detector (PDA) and a
Phenomenex C18 column (250 mm ×4.6 mm, 5 μm;
Phenomenex, Torrance, CA) [Method B: solvent A: H2O/
0.1% TFA, solvent B: CH3CN; flow rate: 1.0 mL min−1; 0−30
min, 5−100% B; 30−35 min, 100% B; 35−36 min, 100−5% B;
36−40 min, 5% B]. Semi-preparative HPLC were carried out in
a Agilent 1260 Infinity II (Prep HPLC) system equipped with
a Diode Array Detector (DAD) and a Gemini 5 μm C18 110 Å,
LC column 250 mm ×10 mm (Phenomenex, Torrance, CA)
[Method C: solvent A: H2O/0.025% TFA; solvent B:
CH3CN; flow rate: 5.0 mL min−1; 0−3 min, 25% B; 3−10
min, 25−75% B; 10−16 min, 75−100% B; 16−18 min, 100%
B; 18−19 min, 100−25% B; 19−20 min, 25% B]; [Method D:
solvent A: H2O/0.025% TFA; solvent B: CH3CN; flow rate:
5.0 mL min−1; 0−3 min, 25% B; 3−17 min, 25−100% B; 17−
18 min, 100% B; 18−19 min, 100−25% B; 19−20 min, 25%
B]; [Method E: solvent A: H2O/0.025% TFA; solvent B:
CH3CN; flow rate: 5.0 mL min−1; 0−3 min, 25% B; 3−17 min,
25−100% B; 17−22 min, 100% B; 22−23 min, 100−25% B;
23−27 min, 25% B]. All solvents used were of ACS grade and
purchased from Pharmco-AAPER (Brookfield, CT). Size
exclusion chromatography was performed on Sephadex LH-
20 (25−100 μm; GE Healthcare, Piscataway, NJ). A549, PC3,
and HCT116 cells were obtained from ATCC (Manassas,
VA). All other reagents used were of reagent grade and
purchased from Sigma-Aldrich (Saint Louis, MO), unless
otherwise noted.
Purification of Compounds 11−13 and 13b. The
reddish-brown oily crude extract (3.82 g) produced by Strain
1[S. coelicolor M1152ΔmatAB::pSET154BB-kasOp*-snoa123-
kasOp*-mtmQY-sp44-aknGHU] was dissolved in MeOH (10
mL) followed by Sephadex LH-20 (MeOH; 2.5 cm ×50 cm)
mentored by TLC to aord six fractions. LC-MS analysis
indicates that target metabolite 13 was mainly detected in
fractions F3 and F4. Semi-prep-HPLC purification (Method
C) of F3 and F4 aorded compound 13 (2-hydroxy-
auramycinone; 24.4 mg) in pure form as red solid, and
compound 13b (SEK15; 8.5 mg) in pure form as pale-yellow
solid.
The reddish-brown oily crude extract (3.20 g) produced by
Strain 2 [S. coelicolorM1152ΔmatAB::pHEAKV2] was frac-
tionated using silica gel column (DCM/0−50% MeOH; 2.5
cm ×30 cm) to aord six fractions F1 (DCM; 0.5 L), F2
(DCM/2% MeOH; 0.5 L), F3 (DCM/4% MeOH; 0.5 L), F4
(DCM/10% MeOH; 0.5 L), F5 (DCM/20% MeOH; 0.5 L),
and F6 (DCM/50% MeOH; 0.5 L), followed by LC-MS
analysis. The target compound was detected in F3 and F4.
Fractions F3−F4 were combined followed by semi-prep-HPLC
purification (Method D) to aord compound 12 (2-hydroxy-
9-epi-aklavinone; 4.2 mg) in pure form as red solid.
In the same manner, the crude extract (2.22 g, reddish-
brown oily) produced by Strain 3 [S. coelicolor M1152Δma-
tAB::pSET-A2M1A6] was fractionated using silica gel column
(DCM/0−50% MeOH; 2.0 cm ×25 cm) to yielded six
fractions F1 (DCM; 0.5 L), F2 (DCM/2% MeOH; 0.5 L), F3
(DCM/4% MeOH; 0.5 L), F4 (DCM/10% MeOH; 0.5 L), F5
(DCM/20% MeOH; 0.5 L), and F6 (DCM/50% MeOH; 0.5
L). LC-MS analysis of the obtained fractions indicates that the
target compound was detected in fractions F2−4, however,
with low yield. Fractions F2−4 have been combined, dissolved
in MeOH (2 mL) followed by Sephadex LH-20 (1 cm ×40
cm; MeOH) and semi-prep-HPLC purification (Method E) to
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K

give compound 11 (2-hydroxy-aklavinone; 1.56 mg) in pure
form as red solid.
Statistical Analyses. The statistical significance of the
impact of genetic manipulations and combinatorially assessed
variables on production was assessed via post hoc analysis.
One-way ANOVA, two-way ANOVA, and Student’s ttest
analyses were performed using GraphPad Prism version 10.2.1
for Mac OS X, GraphPad Software, San Diego, CA, www.
graphpad.com.
Cancer Cell Line Viability Assay. Mammalian cell line
cytotoxicity [A549 (nonsmall cell lung) and PC3 (prostate),
TC32 (Ewing sarcoma), and HCT116 (colorectal) human
cancer cell lines] assays were accomplished in triplicate
following our previously reported protocols.
24,54−57
Actino-
mycin D (A549 and PC3) was used as positive controls.
Physicochemical Properties of Compounds 11−13. 2-
Hydroxy-aklavinone (11).C22H20O9(428); red solid; HPLC-
Rt= 24.72 min (Supporting Information, Figures S9−S21);
UV/vis λmax 228, 250 (sh), 268, 290 (sh), 445 nm; 1H NMR
(DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz),
see Tables S1 and S2; (−)-ESI-MS: m/z427 [M −H]−;
(+)-ESI-MS: m/z411 [(M-H2O) + H]+, 393 [(M-H2O) +
H]+; (+)-HRESI-MS: m/z393.0942 [(M-2H2O) + H]+(calcd
for C22H17O7, 393.0969), 879.1973 [2M + Na]+(calcd for
C44H40O18Na, 879.2106).
2-Hydroxy-9-epi-aklavinone (12). C22H20O9(428); red
solid; HPLC-Rt= 22.47 min (Supporting Information, Figures
S22−S31); UV/vis λmax 228, 268, 290 (sh), 445 nm; 1H NMR
(DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz),
see Tables S1 and S2; (−)-ESI-MS: m/z427 [M −H]−;
(+)-ESI-MS: m/z393 [(M-2H2O) + H]+, 879 [2M + Na]+;
(+)-HRESI-MS: m/z393.0958 [(M-2H2O) + H]+(calcd for
C22H17O7, 393.0969), 451.0986 [M + Na]+(calcd for
C22H20O9Na, 451.0999), 879.2020 [2M + Na]+(calcd for
C44H40O18Na, 879.2106).
2-Hydroxy-auramycinone (2-Hydroxy-9-epi-nogalamyci-
none; 2-Hydroxy-9-epi-nogalavinone; 13). C21H18O9(414);
red solid; HPLC-Rt= 30.39 min (Supporting Information,
Figures S32−S40); UV/vis λmax 228, 258, 270, 290, 430 nm;
1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6,
150 MHz), see Tables S1 and S2; (−)-ESI-MS: m/z413 [M −
H]−; (+)-ESI-MS: m/z397 [(M-H2O) + H]+, 379 [(M-
2H2O) + H]+, 851 [2M + Na]+; (+)-HRESI-MS: m/z
379.0806 [(M-2H2O) + H]+(calcd for C21H15O7,
379.0812), 851.1746 [2M + Na]+(calcd for C42H36O18Na,
851.1793).
SEK15 (13b). C20H16O8(384); pale-yellow solid; HPLC-Rt
= 26.81 min (Supporting Information, Figures S42−S51);
UV/vis λmax 210, 290, 320 (sh) nm; 1H NMR (CD3OD, 600
MHz) and 13C NMR (CD3OD, 150 MHz), see Tables S1 and
S2; (−)-ESI-MS: m/z383 [M −H]−, 767 [2M −H]−;
(+)-ESI-MS: m/z385 [M + H]+; (+)-HRESI-MS: m/z
385.0893 [M + H]+(calcd for C20H17O8, 385.0918),
407.0679 [M + Na]+(calcd for C20H16O8Na, 407.0737),
769.1791 [2M + H]+(calcd for C40H32O16Na, 767.1763).
■ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acssynbio.4c00043.
Plasmid and strain information; chemical schemas;
NMR data for compounds 11−13 and SEK15; HPLC
chromatograph traces of the in vitro and in vivo
comparisons; mass spectra; high-resolution mass spectra,
and mammalian cell cytotoxicity data (PDF)
■AUTHOR INFORMATION
Corresponding Authors
Khaled A. Shaaban −Center for Pharmaceutical Research and
Innovation and Department of Pharmaceutical Sciences,
College of Pharmacy, University of Kentucky, Lexington,
Kentucky 40536, United States; orcid.org/0000-0001-
7638-4942; Email: Khaled_shaaban@uky.edu
Mikko Metsä-Ketelä −Department of Life Technologies,
University of Turku, FIN-20014 Turku, Finland;
orcid.org/0000-0003-3176-2908; Email: mianme@utu.fi
S. Eric Nybo −Department of Pharmaceutical Sciences,
College of Pharmacy, Ferris State University, Big Rapids,
Michigan 49307, United States; orcid.org/0000-0001-
7884-7787; Email: EricNybo@Ferris.edu
Authors
Rongbin Wang −Department of Life Technologies, University
of Turku, FIN-20014 Turku, Finland
Benjamin Nji Wandi −Department of Life Technologies,
University of Turku, FIN-20014 Turku, Finland;
orcid.org/0000-0003-1071-3111
Nora Schwartz −Department of Pharmaceutical Sciences,
College of Pharmacy, Ferris State University, Big Rapids,
Michigan 49307, United States
Jacob Hecht −Department of Pharmaceutical Sciences,
College of Pharmacy, Ferris State University, Big Rapids,
Michigan 49307, United States
Larissa Ponomareva −Center for Pharmaceutical Research
and Innovation and Department of Pharmaceutical Sciences,
College of Pharmacy, University of Kentucky, Lexington,
Kentucky 40536, United States
Kendall Paige −Department of Pharmaceutical Sciences,
College of Pharmacy, Ferris State University, Big Rapids,
Michigan 49307, United States
Alexis West −Department of Pharmaceutical Sciences, College
of Pharmacy, Ferris State University, Big Rapids, Michigan
49307, United States
Kathryn Desanti −Department of Pharmaceutical Sciences,
College of Pharmacy, Ferris State University, Big Rapids,
Michigan 49307, United States
Jennifer Nguyen −Department of Pharmaceutical Sciences,
College of Pharmacy, Ferris State University, Big Rapids,
Michigan 49307, United States; orcid.org/0000-0002-
4557-1638
Jarmo Niemi −Department of Life Technologies, University of
Turku, FIN-20014 Turku, Finland; orcid.org/0000-
0002-7447-8379
Jon S. Thorson −Center for Pharmaceutical Research and
Innovation and Department of Pharmaceutical Sciences,
College of Pharmacy, University of Kentucky, Lexington,
Kentucky 40536, United States; orcid.org/0000-0002-
7148-0721
Complete contact information is available at:
https://pubs.acs.org/10.1021/acssynbio.4c00043
Author Contributions
⊥
R.W., B.N.W., N.S., and J.H. contributed equally to this work.
M.M.-K, K.A.S., and S.E.N. conceived and designed the study.
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ACS Synth. Biol. XXXX, XXX, XXX−XXX
L

R.W. and B.N.W. performed cloning, protein expression, and
enzymatic assays. N.S., J.H., A.W., K.P., J.N., and K.S.
performed molecular biology, actinomycete transformation,
metabolic engineering, and chemical profiling of extracts from
engineered Streptomyces lines. S.E.N., J.H., and N.S. scaled up
fermentations and isolated compounds. K.A.S. purified and
performed NMR spectroscopic analyses and HRESI mass
spectrometric studies. L.P. carried out cancer cell line viability
assays and curated data. M.M.-K., K.A.S., S.E.N., L.P., and
J.S.T. wrote, edited, and revised the manuscript.
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
Research reported in this publication was supported by the
National Science Foundation under grant nos. ENG-2015951
and ENG-2321976 (S.E.N.), by the National Cancer Institute
of the National Institutes of Health under Award No.
R15CA252830 (S.E.N.), by National Institutes of Health
grant R37 AI052218 (J.S.T.), the Center of Biomedical
Research Excellence (COBRE) for Translational Chemical
Biology (CTCB, NIH P20 GM130456), the National Institute
of Food and Agriculture (USDA-NIFA-CBGP, Grant No.
2023-38821-39584), the University of Kentucky College of
Pharmacy, the University of Kentucky Markey Cancer Center,
and the National Center for Advancing Translational Sciences
(UL1TR000117 and UL1TR001998). This work was
supported by the Research Council of Finland (grants
340013 and 354998 to M.M.-K.) The authors also thank the
College of Pharmacy PharmNMR Center for analytical
support. NMR data was acquired on a Bruker AVANCE
NEO 400 MHz NMR spectrometer funded or a Bruker
AVANCE NEO 600 MHz high-performance digital NMR
spectrometer [supported, in part, by NIH grants P20
GM130456 (J.S.T.) and S10 OD28690].
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