Activation and Characterization of Bohemamine Biosynthetic Gene Cluster from Streptomyces sp. CB02009

Article abstract of DOI:10.1021/acs.orglett.0c01224

Bohemamines (BHMs) are bacterial alkaloids containing a pyrrolizidine core with two unusual methyl groups. Herein we report the activation of BHMs biosynthesis using a ribosome engineering approach. Characterization of the bhm gene cluster reveals that nonribosomal peptide synthetase BhmJ and Baeyer-Villiger monooxygenase BhmK are responsible for the formation of the pyrrolizidine core, which is further methylated on C-7 by methyltransferase BhmG. The 9-methyl group of BHMs is instead originated from a nonproteinogenic amino acid (2S,5S)-5-methylproline.

Full text of DOI:10.1021/acs.orglett.0c01224

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Letter
Activation and Characterization of Bohemamine Biosynthetic Gene
Cluster from Streptomyces sp. CB02009
Ling Liu, Sainan Li, Runze Sun, Xiangjing Qin, Jianhua Ju, Changsheng Zhang, Yanwen Duan,*
and Yong Huang*
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ABSTRACT: Bohemamines (BHMs) are bacterial alkaloids con-
taining a pyrrolizidine core with two unusual methyl groups. Herein
we report the activation of BHMs biosynthesis using a ribosome
engineering approach. Characterization of the bhm gene cluster
reveals that nonribosomal peptide synthetase BhmJ and Baeyer−
Villiger monooxygenase BhmK are responsible for the formation of
the pyrrolizidine core, which is further methylated on C-7 by
methyltransferase BhmG. The 9-methyl group of BHMs is instead
originated from a nonproteinogenic amino acid (2S,5S)-5-methyl-
proline.
acterial pyrrolizidine alkaloids, as well as structurally
A total of 11 rifampicin-resistant colonies, named CB02009-
R-1 to CB02009-R-11, were obtained from Streptomyces sp.
CB02009 using ribosome engineering (Table S3, Figures S3−
4). Compared to wild-type, two additional peaks were
observed in the fermentation extracts of the mutant
CB02009-R-5 with R440H mutation in RNA polymerase β-
subunit (RpoB) (Figure 1B, I−II). HRESIMS and NMR
analysis confirmed the production of BHMs in CB02009-R-5
and the presence of trace amounts of BHMs in several other
B
related lipocyclocarbamates and azetidomonamides, are
an emerging family of natural products with promising
1
−3
biological activities (Figures 1A, S1−2). They are produced
as quorum-sensing metabolites or used as self-defensive
4
−6
mechanisms against predation by amoebae.
Several
Streptomyces species produced bohemamines (BHMs, 1−4),
featuring a pyrrolizidine core with two unusual methyl
6
−14
groups.
from homospermidine,
alkaloids may involve nonribosomal peptide synthetases
NRPSs) for the formation of indolizidine intermediates,
followed by consecutive reactions to generate pyrrolizidine
In contrast to plant pyrrolizidines originated
2
,15−17
8
,10
the biosynthesis of bacterial
mutants (Figures S5−7).
The discovery of BHMs in CB02009-R-5 provided an
excellent opportunity to study their biosynthesis. In silico
analysis of the draft genome of Streptomyces sp. CB02009 using
antiSMASH 5.0 revealed a candidate bhm BGC based on its
(
1
,6,18
scaffolds by Baeyer−Villiger monooxygenases (BVMOs).
The biosynthesis of BHM derivative spithioneines was instead
similarity with those of legonmycins and pyrrolizixenamide
11
proposed to utilize a peptide/polyketide logic. However, the
bhm biosynthetic gene cluster (BGC) has not been identified
despite the isolation of 1−3 in 1980 and the recent genome
6,18,29
(
Figure 1D, Table S4).
The bhm BGC consists of 17
genes encoding NRPS, tailoring enzymes, precursor biosyn-
thetic protein complexes, regulatory, and resistance pro-
8
,13,19
sequencing of alternative BHMs producers.
30,31
teins.
Further genome mining revealed three additional
A typical actinomycete genome contains 20−40 BGCs, and
streptomycetes with similar BGCs, among which Streptomyces
most of them are silent under laboratory culture condi-
19
2
0,21
sp. TPA0873 is a known BHMs producer (Tables S5−7).
tions.
Ribosome engineering is a rapid and cost-effective
The BhmJ NRPS contains two distinct modules predicted for
strategy to improve the yields of many secondary metabolites
6
,18,32
22−28
serine and methyl-proline incorporation (Table S8).
To
or activate silent BGCs.
Herein we report the activation of
study if bhmJ is involved in the biosynthesis of BHMs we
the bhm BGC in Streptomyces sp. CB02009 from our strain
collection using this strategy. Using gene inactivation and
heterologous expression, we discovered that BHMs are
biosynthesized by BhmJ NRPS capable of incorporating a
nonproteinogenic amino acid (2S,5S)-5-methyl-proline (5-
Mepro). The tandem action of a BMVOs BhmK and a C-
methyltransferase BhmG is responsible for the pyrrolizidine
formation and C-7 methylation.
Received: April 6, 2020
©
XXXX American Chemical Society
https://dx.doi.org/10.1021/acs.orglett.0c01224
A
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Figure 1. Biosynthesis of BHMs. (A) Representative bacterial pyrrolizidine alkaloid derivatives. (B) HPLC analysis of BHMs production in
Streptomyces sp. CB02009 (I) and its mutants (II−V). (C) Single-crystal X-ray structures of 6, 8, and 9. (D) Organization of bhm BGC in
Streptomyces strains. (E) Proposed BHMs biosynthetic pathway. (F) HPLC profiles of YX3004 in shake flasks (I−III) and baffled shake flasks (IV−
VI). (G) HPLC profiles of YX3005 (I, IV), YX3006 (II, V), and YX3007 (II, VI) in shake flasks and baffled shake flasks for 3 days.
inactivated bhmJ, resulting in a mutant YX3001(ΔbhmJ)
without the production of 1−3 (Figure 1B, III; Figure S8).
The FAD-dependent monooxygenase gene bhmK is tran-
scriptionally coupled to bhmJ, due to the overlapping of the
start codon of bhmK with the stop codon of bhmJ. BhmK
shares high sequence identity with PxaB, LgnC, AzeC, and
LpiC, previously reported BMVOs involved in pyrrolizidine
(Tables S9−10). The production of 5−7 in YX3002(ΔbhmK)
suggests that BhmK is responsible for the formation of
pyrrolizidine core in BHMs through a Bayer-Villiger type
oxidation (Figure 1E). The storage of 6 or 7 in methanol
afforded methylated products 9 or 10, respectively (Tables
S10, 15). The structures of 6 and 9 were further confirmed
using single-crystal X-ray diffraction analysis (Figure 1C).
The methyltransferase BhmG resembles MdpB1 and
PokMT-1, responsible for C-methylation on aryl-CoA
3
,4,6,18
formation (Figure S9).
We next inactivated bhmK by
replacing it with a mutant copy in which bhmK was disrupted
35
by a thiostrepton-resistance gene with a kasOp* promoter
substrates (Figure S11). To ascertain if BhmG is responsible
for C-7 methylation in BHMs, a mutant strain YX3004-
(ΔbhmG) was similarly obtained (Figure S12). Compared to
CB02009-R-5, YX3004(ΔbhmG) abolished the production of
1−3, while also accumulating 5−7, as well as 4, 8, and 11
(Figure 1F). Note that 8 is a novel indolizidine alkaloid
3
3,34
(Figure S10).
The recombinants completely abolished the
production of 1−3, while they accumulated three new
metabolites 5−7 (Figure 1B, IV). A bhmK complementation
strain YX3003 restored BHMs production (Figure 1B, V).
Large scale fermentation of YX3002(ΔbhmK) led to the
isolation of 5−7, and their structures were deducedby
HRESIMS, CD spectra, and 1D and 2D NMR spectroscopy
36,37
containing a rare methylperoxyl moiety (Tables S11, 14).
Compound 11 has the characteristic BHM-type UV−vis
B
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Letter
Figure 2. Heterologous expression of bhmGJK and the biosynthetic origin of the C-9 methyl group in BHMs. (A) Heterologous expression of bhm
genes in S. albus J1074. I−II: Expression of pSET152 or bhmJK without (2S,5R)-5-MePro or (2S,5S)-5-MePro; III: Expression of bhmJK with
(
2S,5R)-5-MePro (2.5 mM); IV−VIII: Expression of various combinations of bhmJ, bhmK, and bhmG with (2S,5S)-5-MePro (2.5 mM). The
13
proposed structures of 13 and 14 were based on LCMS. (B) HRMS analysis of C-incorporation into BHMs in Streptomyces sp. CB02009-R-5 (I−
II) and YX3004 (III−VI) by feeding L-methionine or [methyl- CH ]-L-methionine.
1
3
3
+
6,18
adsorption and a distinctive [M + H] ion (m/z 235.1446),
which differed from 4 by one oxygen. Further NMR
characterization of 11 was hindered due to its intrinsic
instability. The production of 4−8, 11 in YX3004(ΔbhmG)
suggested that BhmG is only involved in C-7 methylation
substrate for BhmG-catalyzed C-7 methylation than 4.
In
fact, no hydroxyl congeners were reported for pyrrolixena-
mides, lipocyclocarbamates, azetidomonamides, or azabicy-
Taken together, the BMVOs involved in bacterial
pyrrolizidine biosynthesis might only execute a remarkable
3
,4,40
clene.
(
Figure 1E).
three-step cascade reaction, and the final hydroxylation
reaction catalyzed by LgnC may be an exception.
1
8
The different fermentation profiles of YX3004(ΔbhmG) in
either shake flasks (Erlenmeyer) or baffled flasks provide an
opportunity to understand the detailed catalytic activity and
mechanism of BhmK BMVO in vivo. First, compound 4 was
accumulated as the main product in baffled shake flasks for 7
days, while only trace amounts of 4 and 11 were present in
shake flasks (Figure 1F, VI vs III). These data suggested that
the catalytic activity of BhmK is dependent on the level of
dissolved oxygen during fermentation, consistent with previous
Compounds 6 and 7 would likely be produced by air-oxidation
of 5′. The enolization of 5′ may generate 5, since similar
indolizidines legonindolizidine A and B were also observed in
18
the ΔlgnC mutant.
Third, the production of 5−7 in YX3004(ΔbhmG) suggests
either the disruption of certain regulatory mechanisms for the
productions of BHMs and the intermediates or the
compromise of the catalytic activity of BhmK, due to the
gene inactivation of bhmG. Interestingly, the presence of a
functional BhmJ NRPS in YX3004(ΔbhmG) rules out any
polar effect resulting from bhmG gene replacement. Instead,
YX3004(ΔbhmG) produced at least >10-fold of 5−7 than
YX3002(ΔbhmK) (Figure 1B, IV vs Figure 1F, IV).
38,39
reports of other BMVOs.
Second, BhmK might only catalyze the ring expansion and
contraction from the putative intermediate 5′, because (1)
compound 4 was produced with longer cultivation in
YX3004(ΔbhmG) and (2) 11 is more likely to be the
C
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Therefore, in order to further study the function of BhmJ,
BhmK, and BmhG, we first prepared three constructs in
integrative vector pSET152 containing genes encoding BhmG,
and functionally impaired BhmG(G184R) and BhmG(Y140F),
based on the comparison with the amino acids in the active site
strains could be attributed to variable factors, such as lack of
pathway-specific transcriptional regulators, different protein
expression levels, substrate availability, and differences among
56
the metabolome and proteome of the hosts.
Finally, to study the effect of (2S,5S)-5-Mepro on BHMs
production in their native producer, it was fed into CB02009-
R-5 and resulted in 2-fold increase of BHMs production
(Figure S19). Feeding of other amino acids, including D-
proline and L-proline, resulted in no obvious change of BHMs
production. This result also suggests that (2S,5S)-5-Mepro is
incorporated into BHMs scaffold in the native producer. To
further reveal the biosynthetic origin of the methyl group in
4
1
of PokMT-1. The constructs were introduced into YX3004-
ΔbhmG), and the resultant strains YX3005−YX3007 were
(
fermented for 3 days. In baffled shake flasks, there was no
accumulation of 5−7 in all three strains, suggesting that BhmK
would be fully functional in the presence of BhmG and its
variants, as well as high levels of dissolved oxygen (Figure 1G,
IV−VI). Although the methylation activity of BhmG(G184R)
was completely shut off, abundant amounts of 4 and 11 were
still produced, further suggesting that the physical presence of
BhmG(G184R) is also critical for the activity of BhmK. Due to
the highly unstable nature of 11, the coordinated action of
BhmJ, BhmK, and BmhG might be one of Nature’s strategies
to avoid the accumulation of shunt metabolites 5−8 in
CB02009-R-5. Similar enzyme pairs have previously been
1
3
(2S,5S)-5-Mepro, [methyl- CH ]-L-methionine was next fed
3
into the culture of both CB02009-R-5 and YX3004(ΔbhmG)
(Figure 2B, II, IV; Figure S20). HRESIMS analysis showed
that only 8-methyl group, not the 9-methyl group in 1−3, was
1
3
originated from the C-labeled L-methionine. Both 4 and 11
were not labeled in YX3004(ΔbhmG), suggesting an
unidentified pathway for the biosynthesis of (2S,5S)-5-MePro
in CB02009-R-5. There are several conserved proteins, such as
BlmD, BlmH, and BhmI among the identified bhm BGCs, and
their role in (2S,5S)-5-MePro or BHM biosynthesis remains to
be determined.
4
2
43
identified in the biosynthesis of bacillaenes, microcin B17,
4
4
45
rebecamycin, and coenzyme Q. The identification of 12
known and 35 orphan NRPS/BVMOs pairs also suggests their
potential evolutionary advantage for bacterial pyrrolizidine
formation (Table S8, Figure S9).
Next, we established an expression platform in Streptomyces
46,47
In conclusion, the bhm BGC was discovered in CB02009-R-
5, assisted by a rapid ribosome engineering approach. The
biosynthetic mechanisms for two unusual methyl groups in
BHMs were also revealed, including (1) BhmG-catalyzed C-
methylation and (2) incorporation of a rare amino acid
(2S,5S)-5-MePro. Intriguingly, BhmJ NRPS or BhmK BMVO
are functionally competent enzymes in heterologous Strepto-
myces hosts, while it seemed that unique regulatory
mechanisms or certain protein−protein interactions among
BhmJ, BhmK, and BhmG are operational in the native strain
4
8,49
albus J1074 for the expression of BhmJ, BhmK, and BmhG.
Various methylated prolines are incorporated into peptides
through NRPS logic, including polyoxypeptin, nostopeptolide,
5
0−52
and griselimycins (Figures S13−14).
However, 5-Mepro
present in dozens of actinomycin congeners was suggested to
53−55
derive from a post NRPS-logic.
Unique to the previously
isolated BHMs analogs is the presence of a 9-methyl group. We
hypothesized that an unusual (2S,5S)-5-Mepro might be
incorporated in BHMs through M2-A adenylation domain in
BhmJ S. albus strain containing bhmJK in pSET152 with the
constitutive promoter ermEp* produced no BHMs (Figure 2A,
I−II). Since S. albus J1074 may be incapable to produce this
nonproteinogenic proline, (2S,5S)-5-Mepro was then fed into
S. albus J1074(bhmJK), which produced 4, 11, 13, and 14
(
Figure 1). The reasons could be that the native strain
CB02009-R-5 may need to develop intricate mechanisms to
enable the biosynthesis of proper amounts of 1−3, when ample
(
2S,5S)-5-Mepro could be provided (Figure S21). Further
study of BHM biosynthesis, such as in vitro characterization of
BhmGJK would provide more insights for this observation.
(
Figure 2A, IV). Compounds 13 and 14 were identified to be
the analogs of 4 and 11 with a 3-methyl-2-butylamide moiety
Figure S15). BhmO could catalyze the dehydrogenation of
the 3-methyl-2-butylamide moiety synthesized by BhmLMN
Figure 1E). These compounds could be similarly produced
ASSOCIATED CONTENT
sı Supporting Information
■
(
*
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c01224.
(
when Streptomyces coelicolor M145 and Streptomyces lividians
TK24 were used as hosts for bhmJK expression by
supplementing (2S,5S)-5-Mepro (Figure S16). In contrast,
no BHM analogs were produced when (2S,5R)-5-Mepro or
other proline analogs were used (Figure 2A, III; Figure S17).
Four S. albus strains containing bhmJ, bhmGJ, or bhmGJK
were also obtained and fermented. Intriguingly, the efficient
production of BHM analogs in these heterologous hosts
suggests that BhmJ, BhmK, and BhmG may function
independently (Figure 2A, V−VIII; Figure S18). Compounds
Experimental procedures and additional data and figures
including bioinformatic analysis of bhm BGCs, gene
inactivation, heterologous expression, NMR and
ESIHRMS data of BHM congeners (PDF)
Accession Codes
CCDC 1978350 and 1978354−1978355 contain the supple-
mentary crystallographic data for this paper. These data can be
obtained free of charge via www.ccdc.cam.ac.uk/data_request/
cif, or by emailing data_request@ccdc.cam.ac.uk, or by
contacting The Cambridge Crystallographic Data Centre, 12
Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
5
−7 were produced in the two S. albus strains when bhmJ or
bhmGJ were overexpressed. Only S. albus(bhmGJK) produced
a small amount of NP25302 (12), the methylation product of
1
1, suggesting the attenuated activity of BhmG. Higher gene
AUTHOR INFORMATION
Corresponding Authors
■
dosage of bhmG seemed detrimental to the activity of BhmG,
while several new metabolites unrelated to BHMs were
produced, suggesting the activation of certain cryptic BGCs
from S. albus (Figure 2A, V). The disruption of the
interdependency of BhmJ/BhmK/BhmG in these heterologous
Yanwen Duan − Xiangya International Academy of
Translational Medicine at Central South University, Changsha
410013, Hunan, China; Hunan Engineering Research Center of
D
https://dx.doi.org/10.1021/acs.orglett.0c01224
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Discovery, Changsha 410011, Hunan, China; National
Engineering Research Center of Combinatorial Biosynthesis for
Drug Discovery, Changsha 410011, Hunan, China;
Email: ywduan66@sina.com
Yong Huang − Xiangya International Academy of Translational
Medicine at Central South University, Changsha 410013,
Hunan, China; National Engineering Research Center of
Combinatorial Biosynthesis for Drug Discovery, Changsha
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Changsheng Zhang − Key Laboratory of Tropical Marine Bio-
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Complete contact information is available at:
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ACKNOWLEDGMENTS
■
This work was supported in parts by NSFC grants 81473124,
1530092, and the Chinese Ministry of Education 111 Project
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Central South University for NMR experiments. An early
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