Cloning, sequencing and characterization of the biosynthetic gene cluster of sanglifehrin A, a potent cyclophilin inhibitor

Article abstract of DOI:10.1039/c0mb00234h

Sanglifehrin A (SFA), a potent cyclophilin inhibitor produced by Streptomyces flaveolus DSM 9954, bears a unique [5.5] spirolactam moiety conjugated with a 22-membered, highly functionalized macrolide through a linear carbon chain. SFA displays a diverse range of biological activities and offers significant therapeutic potential. However, the structural complexity of SFA poses a tremendous challenge for new analogue development via chemical synthesis. Based on a rational prediction of its biosynthetic origin, herein we report the cloning, sequencing and characterization of the gene cluster responsible for SFA biosynthesis. Analysis of the 92776 bp contiguous DNA region reveals a mixed polyketide synthase (PKS)/non-ribosomal peptide synthetase (NRPS) pathway which includes a variety of unique features for unusual PKS and NRPS building block formation. Our findings suggest that SFA biosynthesis requires a crotonyl-CoA reductase/carboxylase (CCR) for generation of the putative unusual PKS starter unit (2R)-2-ethylmalonamyl-CoA, an iterative type I PKS for the putative atypical extender unit (2S)-2-(2-oxo-butyl)malonyl-CoA and a phenylalanine hydroxylase for the NRPS extender unit (2S)-m-tyrosine. A spontaneous ketalization of significant note, may trigger spirolactam formation in a stereo-selective manner. This study provides a framework for the application of combinatorial biosynthesis methods in order to expand the structural diversity of SFA. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c0mb00234h

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Cite this: Mol. BioSyst., 2011, 7, 852–861
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PAPER
Cloning, sequencing and characterization of the biosynthetic gene cluster
of sanglifehrin A, a potent cyclophilin inhibitorw
Xudong Qu,a Nan Jiang,a Fei Xu,a Lei Shao,a Gongli Tang,a Barrie Wilkinsonb and
Wen Liu*a
Received 15th October 2010, Accepted 16th November 2010
DOI: 10.1039/c0mb00234h
Sanglifehrin A (SFA), a potent cyclophilin inhibitor produced by Streptomyces flaveolus
DSM 9954, bears a unique [5.5] spirolactam moiety conjugated with a 22-membered, highly
functionalized macrolide through a linear carbon chain. SFA displays a diverse range of biological
activities and offers significant therapeutic potential. However, the structural complexity of SFA
poses a tremendous challenge for new analogue development via chemical synthesis. Based
on a rational prediction of its biosynthetic origin, herein we report the cloning, sequencing and
characterization of the gene cluster responsible for SFA biosynthesis. Analysis of the 92 776 bp
contiguous DNA region reveals a mixed polyketide synthase (PKS)/non-ribosomal peptide
synthetase (NRPS) pathway which includes a variety of unique features for unusual PKS and
NRPS building block formation. Our findings suggest that SFA biosynthesis requires a
crotonyl-CoA reductase/carboxylase (CCR) for generation of the putative unusual PKS starter
unit (2R)-2-ethylmalonamyl-CoA, an iterative type I PKS for the putative atypical extender unit
(2S)-2-(2-oxo-butyl)malonyl-CoA and a phenylalanine hydroxylase for the NRPS extender unit
(2S)-m-tyrosine. A spontaneous ketalization of significant note, may trigger spirolactam formation
in a stereo-selective manner. This study provides a framework for the application of combinatorial
biosynthesis methods in order to expand the structural diversity of SFA.
(2S)-m-tyrosine (m-Tyr, 1) and (3S)-3-carboxypiperazine (Pip, 3).
Introduction
Distinct from other known piperazic-acid-containing natural
Sanglifehrin A (SFA), along with 20+ biosynthetic congeners,
products isolated thus far, which exclusively have an amide
was isolated from Streptomyces flaveolus DSM 9954 (previously
bond linkage on the a-nitrogen, SFA utilizes the b-nitrogen
known as Streptomyces sp. A92-308110) as a result of screening
atom of Pip for amide formation.
microbial broth extracts for new cyclophilin A (CypA)
ligands.1,2 The highly complex structure of SFA is unprece-
SFA exhibits potent activity against lymphocyte (e.g. T and B
cells) proliferation3 and viral infection (e.g. HCV and HIV),2,4
dented (Fig. 1), featuring a [5.5] spirolactam moiety conjugated
and may be useful for promoting recovery after myocardial
infarction.5 The mechanism for immunosuppression remains
with a 22-membered macrolide by a linear carbon chain. The
spirolactam system, with a quaternary carbon at its center,
unclear, however it is distinct from other immunophilin-binding
drugs such as cyclosporin A (CsA), FK506 and rapamycin.6
possesses seven stereogenic centers (SFA has seventeen in
total), and the macrolide moiety displays rich structural
CsA binds to CypA to form an initial binary complex, whereas
diversity reflected in a 2-oxobutyl side-chain and a tripeptide
rapamycin and FK506 bind to a different receptor known as
moiety containing the unusual non-proteinogenic amino acids
FKBP12. The CsA–CypA and FK506–FKBP complexes
interact with the same target protein, calcineurin, to inhibit
its Ser/Thr phosphatase activity thereby arresting T-cell
a State Key Laboratory of Bio-organic and Natural Products
Chemistry, Shanghai Institute of Organic Chemistry, Chinese
proliferation in the G0–G1 stage, whilst the rapamycin–FKBP
complex acts on mTOR.7 Although SFA shows strong affinity
Academy of Sciences, Shanghai, 200032, China.
E-mail: wliu@mail.sioc.ac.cn; Fax: +86-21-64166128;
Tel: +86-21-54925111
for CypA binding (60-fold higher than that of CsA), the
ultimate target of the SFA–CypA complex remains elusive.6
b Biotica Technology Ltd, Chesterford Research Park, Cambridge
CB10 1XL, UK
SFA has no effect on the growth of human dendritic cells but
inhibits the production of IL-12,8 which plays a key role in
regulating Th1 and NK cell proliferation, and in linking innate
immunity with adaptive immunity, raising the possibility that its
bioactivity is independent of SFA–CypA complex formation.
w Electronic supplementary information (ESI) available: bacterial
strains and plasmids (Table S1); primers (Table S2); sequence aligment
(Fig. S1–S6 and S9); chromosome walking (Fig. S2); genotype
verification for the mutant strains (Fig. S7) and SDS-PAGE analysis
of the purified protein (Fig. S8). See DOI: 10.1039/c0mb00234h
c
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Fig. 1 Retro-biosynthetic analysis of SFA. MCoA: malonyl-CoA; mMCoA: (2S)-2-methylmalonyl-CoA; Gln/Glu: glutamine/glutamic acid; Phe:
phenylalanine and Val: valine. Key building blocks are highlighted within boxes.
Therapeutic treatment with the immunosuppressant agents
currently in clinical use can lead to severe side effects such as
renal and central nervous system toxicity,9 thus their use in
certain immune dysfunction diseases is hindered. For example,
calcineurin inhibition is the underlying cause of both the
immunosuppressive and toxic effects of CsA and FK506.
SFA, with a distinct mechanism of action, provides an
alternative lead structure for the generation of new potent
immunosuppressant drugs with lower toxicity. Since the
complex architecture of SFA poses a tremendous challenge
for chemical synthesis, combinatorial biosynthesis offers a
promising way to access structural diversity. However, no
in vivo or in vitro studies of SFA biosynthesis have been
reported so far, a prerequisite for this approach.
hybrid modular PKS/NRPS systems.10 Our initial attempts,
utilizing degenerate primers to amplify PKS and NRPS
encoding fragments as described previously,11,12 yielded a
group of distinct PCR products including 7 for PKS and 11
for NRPS. Unfortunately, gene disruption experiments using
each of these failed to eliminate SFA production in S. flaveolus
DSM 9954 (data not shown), suggesting that this SFA-
producing strain, like many actinobacteria, possesses the
genetic machinery for production of multiple PKS and NRPS
based metabolites.
Based on the a-alkylation, b-hydroxylation and double bond
geometry pattern of the polyketide chain, in conjunction with
the orientation of amide bond formation, we propose that SFA
assembly begins from the spirolactam moiety (Fig. 1). This
analysis further suggests that polyketide biosynthesis may end
with the incorporation of an unusual 6-carbon unit, (2S)-2-
(2-oxobutyl)malonyl-CoA (11). Dissection of the spirolactam
system to a linear chain allows prediction of a unique starter unit
(2R)-2-ethylmalonamyl-CoA (7) for PKS priming. Structurally,
both of these atypical building blocks are reminiscent of (2S)-2-
ethylmalonyl-CoA (5), a somewhat uncommon PKS building
block whose biogenesis typically arises in crotonyl-CoA (4), and
requires a-carboxylation of 4 by CCR in a NADPH dependent
manner.13,14 The ccr gene is generally clustered with the
genes required for producing polyketides with an a-ethyl
branch such as salinosporamide14 and monensin.15 These
combined observations indicate that screening for a ccr gene
may provide selective access to the SFA biosynthetic machinery.
Based on a rational prediction of its biosynthetic origin, we
now report the specific access to the SFA biosynthetic machinery
by cloning of the crotonyl-CoA reductase/carboxylase (CCR)
gene from S. flaveolus DSM 9954. Characterization of the
SFA gene cluster unveils a hybrid polyketide synthase (PKS)/
nonribosomal peptide synthetase (NRPS) system for assembly of
the SFA skeleton, and a number of new genes believed to be
responsible for the synthesis of unusual building blocks for
assembling the polyketide peptide hybrid backbone.
Results and discussion
Prediction of the biosynthetic origin
The chemical structure of SFA implies that its biosynthesis
consists of two phases (Fig. 1). The first phase yields a carbon
chain of variable saturation and alkyl substitution, and is typical
of polyketide biosynthesis. In the second phase the presumptive
polyketide intermediate can be further elongated by the addition
of three amino acid residues. Such a process is characteristic of
the polyketide/polypeptide backbones assembled by a range of
Cloning of the SFA biosynthetic gene cluster
We therefore designed a pair of degenerate PCR primers
according to the conserved motifs of known CCRs in a-ethyl
branched polyketide biosynthesis (Fig. S1w). Subsequent DNA
amplification from the genomic DNA of S. flaveolus gave a
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Fig. 2 Gene cluster and proposed biosynthetic pathway. A, Organization of the SFA biosynthetic genes, the deduced functions of which are
labeled in color and summarized in Table 1. B, PKS/NRPS-directed skeleton assembly. C–F, Pathways for building block biosynthesis: C, (2S)-
m-tyrosine (green); D, (2R)-2-ethylmalonamyl-CoA (cherry-red); E, (2S)-2-(2-oxobutyl)malonyl-CoA (blue); and F, (3S)-3-carboxypiperazine
(purple).
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single product band on an agarose gel with the expected size of
900 bp. The amplified DNA was cloned, and sequencing of
multiple clones resulted in three distinct ccr gene fragments
which were used as probes to screen a genomic library of
S. flaveolus by colony hybridization. This led to the finding of a
single genetic locus harboring both a ccr and type I modular
PKS genes. Chromosome walking from this locus led to the
identification of a 150 kb contiguous DNA region on the
chromosome (Fig. S2w). To validate that this locus is
responsible for SFA biosynthesis, an internal DNA fragment
(subsequently shown to encompass parts of sfaF and sfaH, and
all of sfaG) was replaced by the erythromycin antibiotic
resistance gene ermE via double-crossover homologous
recombination. The resulting mutant TL3001 completely lost
the ability to produce SFA (Fig. 3, trace VII), confirming the
essential nature of this locus for SFA production. Cosmids
pTL3102, pTL3104 and pTL3106 were then selected for
sequencing, yielding 118 372 bp of contiguous sequence with
an average GC content of 74.3%.
one putative regulatory gene (sfaC), and five additional genes
(sfaJ, L, O, sfaN and sfaS) whose function in SFA biosynthesis
could not be assigned based on sequence analysis alone. To
identify the boundaries of the SFA gene cluster, we inactivated
orf3 and orf4. The resulting mutant strains TL3002 and TL3003
produced SFA at a titer comparable to that of the wild strain
(Fig. 3, traces III and IV), indicating that the sfa gene cluster,
spanning 92 776 bp, encompasses the 19 ORFs from sfaA to sfaS.
Polyketide assembly
The five genes sfaE-I encode a type I PKS system comprising of a
mono-domain loading module and thirteen typical extension
modules, which are organized co-linearly with their activities in
the biosynthetic assembly process as shown in Fig. 2B. Each
module consists of ketoreductase (KS), acyltransferase (AT), and
acyl carrier protein (ACP) domains for carbon chain extension,
and optionally the dehydratase (DH), enoylreductase (ER) and
ketoreductase (KR) domains for reductive modifications to
process the b-oxo functionality. This is in agreement with our
biosynthetic prediction, which suggests the presence of unusual
starter and extender units.
Bioinformatic analysis revealed 19 open reading frames (ORFs)
(Fig. 2A and Table 1), which are proposed to constitute the sfa
gene cluster based upon functional assignment of the deduced
gene products. These ORFs have the same transcriptional
orientation and may be translationally coupled as judged by the
overlapping of many start and stop codons. Consistent with the
structure of SFA, 5 modular type I PKS genes (sfaEFGHI) and 1
NRPS gene (sfaD) for assembly of SFA are present. Two genes
(sfaAB) are suggested to be involved in biosynthesis of the non-
proteinogenic amino acids 1 and 3, and five genes (sfaMKPQR)
may be required for supplying the unusual starter and extender
unit building blocks 7 and 11 for polyketide biosynthesis. There is
Analysis of the amino acid motifs conferring substrate
selectivity16–19 shows that the AT domains of modules 4, 6,
7, 9, 10 and 11 are specific for malonyl-CoA, while the other
ATs (except for module 13) are specific for (2S)-2-
methylmalonyl-CoA (Fig. S3w). This is consistent with the
structure of SFA. In the motif C (GAGH) of the module 13
AT domain the usual Ser residue within YASH for (2S)-2-
methylmalonyl-CoA specificity is replaced by Gly. This
variation may broaden the substrate binding cavity,17
Table 1 Deduced functions of ORFs in the SFA biosynthetic gene cluster
% Identity/
similarity
Gene Sizea Protein homologue and originb
Proposed function
orf1
88
SACE_5995 (YP_001108100); from Saccharopolyspora erythraea
NRRL 2338
orf7 (AAA88561); from S. fradiae
SSEG_08631 (YP_002208581); from S. sviceus ATCC 29083
PAH (NP_000268); from Homo sapiens
KtzI (ABV56589); from Kutzneria sp. 744
orf4 (ABV56600); from Kutzneria sp. 744
80/62
Transglycosylase-associated protein
orf2
orf3
sfaA
sfaB
sfaC
sfaD
sfaE
sfaF
sfaG
sfaH
sfaI
146
155
276
446
231
72/69
55/41
53/37
63/47
50/41
55/38
64/52
56/45
63/53
65/54
60/49
38/27
47/34
40/32
57/40
49/27
60/40
76/64
68/55
91/86
72/60
81/65
Unknown protein
Conserved hypothetical protein
Phenylalanine hydroxylase
Lysine/ornithine-N-monooxygenase
Negative transcriptional regulator
NRPS
PKS
PKS
PKS
PKS
PKS
Enoyl reductase
Iterative PKS
Acyl transferase
Short chain dehydrogenase
3-Oxoacyl-(ACP) synthase III (KASIII)
ACP
Asparagine synthetase homologue
Thioesterase
3609 OciB (ABI26078); from Planktothrix agardhii NIVA-CYA 116
2217 MerB (ABJ97438); from S. violaceusniger
4206 ObsA (AAS00419); from Saccharopolyspora spinosa
3628 NlmA1 (AAS46341); from S. nanchangensis
8301 FscC (AAQ82564); from S. sp. FR-008
3405 NanA8 (AAP42874); from S. nanchangensis
388
1515 MxaC (AAK57187); from Stigmatella aurantiaca
sfaJ
sfaK
sfaL
MupE (AAM12917); from Pseudomonas fluorescens
425
PksE (AAO25884); from Micromonospora sp. 046/Eco11
AAur_3586 (YP_949278); from Arthrobacter aurescens TC1
EhpO (AAN40904); from Pantoea agglomerans
Snoa3 (CAA12019); from S. nogalater
OxyD (AAZ78328); from S. rimosus
RifR (AAG52991); from Amycolatopsis mediterranei
RimJ (AAR16523); from S. diastaticus
KtzJ (ABV56590); from Kutzneria sp. 744
orf7 (CAD18995); from S. cattleya
SSDG_02397 (YP_002199237); from S. pristinaespiralis ATCC 25486 63/53
Strop_2904 (YP_001159721); from Salinispora tropica CNB-440
Strop_3504 (YP_001160313); from Salinispora tropica CNB-440
sfaM 239
sfaN
sfaO
sfaP
sfaQ
sfaR
sfaS
orf4
orf5
orf6
orf7
340
84
616
259
453
71
151
152
144
177
Crotonyl-CoA reductase/carboxylase
MbtH like protein
Hypothetical protein
Conserved hypothetical protein
Hypothetical protein Strop_2904
Putative transposase IS4
57/45
63/52
a
b
Numbers are in amino acids. NCBI accession numbers are given in parentheses.
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Fig. 3 HPLC analysis of SFA production. Authentic SFA (I); wild type strain S. flaveolus (II); mutant strain TL3002 (Dorf3, III); mutant strain
TL3003 (Dorf4, IV); mutant strain TL3007 (DsfaK, V); mutant strain TL3005 (DsfaR, VI); mutant strain TL3001 (DsfaFBDsfaH, VII); mutant
strain TL3006 (DsfaA, VIII) and mutant strain TL3006 fed with 2 mM DL-m-tyrosine (IX). Solid triangle indicates SFA.
supporting an unusual function such as the ability to utilize the
putative extender unit 11 for the last polyketide chain extension.
Sequence analysis of the 12 KR domains, according to a
published model for predicting the stereochemistry of the
secondary hydroxyl-group products,20–22 allowed their classifi-
cation into three groups, and is consistent with the stereo-
chemistry of SFA (Fig. S4w). The DH domains in SfaF-M3
and SfaI-M13 are apparently inactive as both lack the essential
His and Gly residues in the conserved catalytic motif
(Fig. S5w).23,24 Finally, it is notable that the PKS loading module
housed in SfaE contains only an ACP domain, suggesting an ‘‘in
trans’’ priming process for selection and loading of the starter
unit. Taken together, SfaEFGHI appear sufficient for the
assembly of a nascent ACP-tethered polyketide intermediate
via 13 decarboxylative condensations.
sfaD encodes the only NRPS identified within the biosynthetic
gene cluster, supporting its role in tripeptide biosynthesis as
outlined in Fig. 2(A and B). SfaD consists of three typical NRPS
modules, each of which comprises an adenylation (A) domain, a
peptidyl carrier protein (PCP) domain and a condensation (C)
domain. The third module contains an additional C domain at
the C-terminus. To predict the substrates of SfaD the substrate
specificity-conferring motif, which encompass ten amino acids
for each A domain, were identified by sequence alignment with
the A domain of GrsA (Fig. S6a and bw).28–30 For SfaD-M14-A
the sequence DAYWVGGTFK is typical for selection of
L-valine as is found in SFA. For SfaD-M15-A the sequence
DIYCLGAAFK does not correlate with any known A domain,
as would be anticipated for incorporating the unusual 1 residue
of SFA (consistent with above DL-m-tyrosine feeding experiment
performed in the sfaA mutant strain TL3006). Finally, for
SfaD-M16-A the sequence DVQFSAHVVK fits most closely
with the specificity-conferring codes to A domains for L-proline
residue activation, in agreement with the structural similarity
between Pro and 3 residues.
Non-proteinogenic amino acid biosynthesis and peptide assembly
The gene products of sfaA and sfaB are likely to be involved in
biosynthesis of the non-proteinogenic amino acids m-Tyr (1)
and Pip (3) as shown in Fig. 2(C and F). SfaA belongs to
the family of phenylalanine hydroxylases and may act on
L-phenylalanine to introduce a hydroxyl group at the meta-
position to give 1.25,26 Inactivation of sfaA by gene
replacement with aac(3) IV (an apramycin resistant gene)
completely abolished SFA production, which could then be
restored by feeding of 2 mM DL-m-tyrosine into the resulting
mutant strain TL3006, strongly supporting the above
assumption (Fig. 3, VIII and IX). SfaB is highly homologous
to various lysine/ornithine N-mono-oxygenases and may
catalyze the flavin-dependent oxidation of an ornithine-like
precursor to initiate ring closure and form the g,d-
dehydropiperazoate precursor of 2 as has been proposed for
KtzI during kutznerides biosynthesis.27 Enamine to imine
tautomerization of 2 may be followed by a reduction,
resulting in 3 as the final building block required for
incorporation into the SFA backbone.
These combined data are consistent with an assembly line
process in which the PKS SfaEFGHI and NRPS SfaD are
together responsible for biosynthesis of SFA. In analogy to
(for example) rapamycin biosynthesis, release of the template
chain along with intra-molecular lactonization is likely to be
catalyzed by the C domain located at the C-terminus of SfaD,
rather than by the more commonly found thioesterase (TE)
domains.31
PKS starter unit biosynthesis
Our proposal for SFA biosynthesis requires (2R)-2-ethyl-
malonamyl-CoA (7) as the starter unit for PKS assembly
(Fig. 2D). The most logical biosynthetic scheme for (2R)-2-
ethylmalonamyl-CoA (7) starts with crotonyl-CoA (4) in
analogy to (2S)-2-ethylmalonyl-CoA (5) biosynthesis. As
discussed above, sfaR encodes a putative CCR capable of the
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reductive-carboxylation of 4 to give 5 in an NADPH
dependent manner.32 Epimerization of
into (2R)-2-
(Fig. 4A).32 By extension to other studied examples we assume
that the product of SfaR is (2S)-2-ethylmalonyl-CoA.35
5
ethylmalonyl-CoA (6) can be catalyzed by either a pathway
specific epimerase or a 2-methylmalonyl-CoA epimerase such
as that which has been characterized from the acetate
assimilation pathway of Rhodobacter sphaeroides.33 As no
putative epimerase gene could be identified in the SFA gene
cluster, we presume the latter option to be the most probable.
Amidation of 6 to form 7 could be catalyzed by SfaP, an
asparagine synthetase homologue. This is supported by the
observation that the highly homologous protein OxyD
performs the conversion of malonyl-CoA into malonamyl-
CoA during oxytetracycline biosynthesis.34 Finally, a ‘‘in
trans’’ process could be adopted to prime the starter unit
7 onto the C-terminal ACP module of SfaE (Fig. 2B).
Spirolactam formation
The spirolactam moiety of SFA is unique among natural
products, and the biosynthetic mechanism leading to its
formation is of significant interest. Experimental evidence
has revealed a hemiketal intermediate during formation of
the avermectin spiroketal moiety,36 and a similar stepwise
pathway is likely involved in SFA biosynthesis. During the
total synthesis of SFA, formation of the spirolactam moiety
was accomplished by spontaneous ketalization triggered by
acid deprotection of a linear polyketide chain which is similar
to the theoretical PKS bound intermediate of the module
4- ACP domain in SfaF (12, Fig. 2B and 5).37,38
In an attempt to validate our hypothesis for 7 biosynthesis
we inactivated sfaR by gene deletion. The resultant mutant
TL3005 produced SFA at significantly reduced levels (approx.
33%) versus the wild-type strain (Fig. 3, trace VI). This fact
suggests that the additional ccr homologues identified during
degenerate PCR amplification may participate in supplying
5 (>60%) for SFA biosynthesis. A similar result has been
reported for monensin biosynthesis.15 To confirm its function
as a CCR we then expressed sfaR in E. coli BL21 (DE3) and
assayed the purified SfaR in vitro (Fig. 4B). In the presence of
biocarbonate and NADPH, 4 was converted rapidly into the
carboxylated product 5 along with a small quantity of the
reduced product n-butyryl-CoA 9 (approx. 13%). When
biocarbonate was removed from the assay system, 4 was
totally converted into 9. These data clearly demonstrated that
SfaR is a CCR capable of generating 2-ethylmalonyl-CoA 5
from crotonyl-CoA 4 via a reductive carboxylation mechanism
Sequencing of the SFA gene cluster revealed no candidate genes
for functional involvement in formation of the spirolactam moiety,
and we speculate that a spontaneous process may occur with a
PKS-bound pentaketide intermediate as shown in Fig. 5. First, the
keto group at C37 is attacked by the C33 hydroxyl group to form
the hemiketal-containing intermediate 13, which can exist in
equilibrium with the oxonium intermediate 14. Chain elongation
of the pentaketide moiety 13 with no further modification would
lead to formation of the naturally occurring SFA congener
sanglifehrin E (Fig. 1). Intra-molecular cyclization of the C41
amide with the oxonium ion intermediate may then form 15,
and subsequent chain elongation leads to the formation of SFA.
Interestingly, this process appears to be stereoselective and
computational analysis has revealed an extra hydrogen bond
interaction between the amine and the hydroxyl group at C35
in the ‘‘pre-S’’ hemiketal.38 This presumably leads to pre-
organization so that nucleophilic attack by the amine is favoured
from the Si face to form the product with S-configuration at C37.
Similar results were also achieved by organic synthesis where the
ratio of epimers formed is 7 : 1 (S:R).38
Formation of the putative 6-carbon extender unit
Based on analysis of the SFA structure and PKS architecture it
appears that the final round of polyketide chain assembly
requires an unusual extender unit, which we suggest to be
(2S)-2-(2-oxobutyl)malonyl-CoA (11). Analysis of the SFA
gene cluster led to the identification of sfaK, encoding an
atypical PKS as a probable candidate for biosynthesis of an
11 precursor (Fig. 2, A and E). SfaK consists of four domains
of which the N-terminal KS and AT domains are characteristic
of known type I PKSs, and the remaining two domains have no
significant sequence homologies to any proteins of known
function. Based on secondary structural modeling by using
the software Phyre39 and web-based software (Motif Scan,
http://hits.isb-sib.ch/cgi-bin/PFSCAN)40 we predict that the
third domain encompassing B74 amino acids is in fact a
unique ACP that contains a catalytic motif GFDSL (the
central Ser residue is for phosphopantetheine attachment)
(Fig. S5cw), and that the fourth domain of B220 amino
acids at the C-terminus is a KR and DH like di-domain
(Fig. 2E, S4 and S9w). This domain architecture is similar to
that found for the iterative PKSs involved in enediyne
biosynthesis, e.g. SgcE which is required for antibiotic
Fig. 4 Characterization of SfaR as a CCR in vitro. A, mechanism of
SfaR- catalyzed reaction. B, product examination by HPLC. Standard
crotonyl-CoA (I); assay system contains crotonyl-CoA, NaHCO3,
NADPH, but no SfaR (II); assay system contains crotonyl-CoA,
NaHCO3, NADPH and SfaR (III); and assay system contains
crotonyl-CoA, NADPH and SfaR (IV).
c
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Fig. 5 Proposed cyclization mechanism for spirolactam formation.
C-1027 biosynthesis.41 In analogy to SgcE, SfaK can be
Materials and methods
envisaged to catalyze the assembly of a nascent linear
unsaturated triketide from acetyl-CoA and two malonyl-
CoA units. The conserved motif for malonyl-CoA selection
in the SfaK–AT domain supports this prediction (Fig. S3w).
How this generates the required a,b-olefin and g-keto groups is
unclear, but could involve some crosstalk with enzymes from
primary metabolism in fatty acid biosynthesis.
Bacterial strains, plasmids, and reagents
Bacterial strains and plasmids used in this study are
summarized in Table S1.w Biochemicals, chemicals, media,
restriction enzymes, and other molecular biological reagents
were obtained from standard commercial sources.
To convert the putative SfaK product to 11 requires the
reductive carboxylation of an a,b-unsaturated crotonyl-CoA
like substrate (it is also not clear how this unit might be
transferred to CoA or ACP) in a process similar to that
catalyzed by SalG during (2S)-2-(2-chloroethyl)malonyl-CoA
biosynthesis.14 Giving that the CCR gene sfaR within the sfa
gene cluster is not essential for SFA biosynthesis as shown
above, we assume that the reductive carboxylation reaction
required to generate 11 can be performed by one of the alter-
native ccr homologues identified in the genome. To determine
whether SfaK is essential for SFA biosynthesis, sfak was
removed by in frame deletion. SFA production was totally
abolished in the resultant mutant strain TL3007 (Fig. 3, trace V),
confirming its essential requirement for SFA biosynthesis and
consistent with our suggested role in SFA biosynthesis.
General biological methods and DNA sequencing
DNA isolation and manipulation in E. coli and Streptomyces
were carried out according to standard methods.42,43 For
Southern analysis, digoxigen labeling of DNA probes,
hybridization, and detection were performed according to the
protocols provided by the manufacturer (Roche Diagnostics
Corp., Indianapolis, IN, USA). T4 polymerase (Dalian
TaKaRa Biotechnology Co. Ltd., Dalian, China) was used
to generate blunt ends. PCR amplification was carried out on
an authorized cycler Eppendorf AG 22331 (Hamburg,
Germany) using either Taq DNA polymerase (Dingguo Co.
Ltd., China) for routine genotype verification or PrimeSTAR
HS DNA polymerase (Dalian TaKaRa Biotechnology Co.
Ltd., Dalian, China) for high fidelity amplification. Primer
synthesis and DNA sequencing were performed at Shanghai
Invitrogen Biotech Co., Ltd. (Shanghai, China) and the
Chinese National Human Genome Center (Shanghai, China).
Conclusion
We have uncovered the biosynthetic pathway of SFA by cloning,
sequencing and characterization of the sfa gene cluster in
S. flaveolus DSM 9954. SFA biosynthesis occurs by a mixed
PKS–NRPS system in a linear manner. The discovery of many
new enzymes, putatively involved in the synthesis of unusual
PKS and NRPS building blocks and formation of structurally
unique moieties, presents an excellent opportunity to investigate
their new chemistry and enzymology. SFA exhibits a diverse
range of biological effects with significant potential of
therapeutic values. Structurally, SFA is also a hybrid polyketide/
non-ribosomal peptide product of particular novelty and
complexity, and due to this the use of SFA as a drug lead for
optimization through semi-synthetic methods is technically
demanding. The availability of the SFA biosynthetic gene
cluster now provides the genetic basis for application of
biosynthetic medicinal chemistry methods for its optimization.
Sequence analysis
ORFs were identified using the FramePlot 4.0b program
(http://nocardia.nih.go.jp/fp4/). The corresponding deduced
proteins were compared with other known proteins in the
databases by using available BLAST methods (http://www.
ncbi.nlm.nih.gov/blast/). Amino acid sequence alignments
were performed by using the strap (http://www.
bioinformatics.org/strap/).
Fermentation, analysis and isolation of SFA
S. flaveolus DSM 9954 (German Collection of Microorganisms
and Cell Cultures, Braunschweig Germany) and recombinant
streptomyces strains generated in this study were routinely
grown at 28 1C on ISP-4 agar medium or TSB liquid medium.42
ISP-4 agar medium was used to prepare S. flaveolus spores. For
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fermentation of S. flaveolus DSM 9954 and recombinant
strains, frozen spore stocks (0.5 mL) were inoculated into
500 mL-baffled flasks containing 50 mL seed medium (4%
glycerol, 1% soy peptone, 2.1% malt extract; pH adjusted to
7.0 with 1.0 M NaOH) and shaken at 250 rpm and 27 1C. After
24 h an aliquot of seed culture (3 mL) was used to inoculate a
500 mL-production flask containing 50 mL of production
medium (2% toasted soy flour, 4% glycerol, 1.95% MES
hydrate, pH adjusted to 6.8 with 10 M NaOH) and shaken
at 250 rpm and 24 1C for 5 days. For feeding experiment,
DL-m-tyrosine (Sigma-aldrich Co.) was added into the 2 days
old production broth with a final concentration of 2 mM and
fermentation was continued for a further 3 days. Ethyl acetate
(50 mL) and fermentation broth (50 mL) were stirred
rigorously under sonication for 20 min and then clarified by
centrifugation. The upper organic phase was removed and the
solvent removed under reduced pressure and the residue
dissolved in methanol (1 mL). Analysis by HPLC-ESI-MS
was performed on a Thermo Fisher LTQ Fleet ESI mass
spectrometer (Thermo Fisher Scientific Inc., USA) coupled
to an Agilent 1200 HPLC (Agilent Technologies Inc., USA).
Chromatography was achieved over an Aglient Zorbax
column (SB-C18, 5 mm, 4.6 ꢀ 250 mm) eluting with a flow
rate of 1 mL minꢁ1 over a 30 min gradient as follows: T = 0,
40% B; T = 5, 40% B; T = 25, 90% B; T = 27, 90% B;
T = 29, 40% B, and T = 30, 40% B. A, H2O + 0.1% formic
acid; B, CH3CN + 0.1% formic acid. UV analysis was made at
242 nm. The retention time of SFA standard was
approximately 21.3 ꢂ 0.2 min. For HPLC-UV analysis the
same methods and conditions as above were used but the
formic acid was replaced with 0.05% TFA.
10 min. TSB (500 mL) was then added and the whole incubated
at 37 1C for 3–5 h. Spores were then recovered by
centrifugation and resuspended in LB medium (2 mL) to be
used as recipients. E. coli S17-1 containing the respective
plasmid was grown in LB medium with appropriate
antibiotics to an OD600 of 0.4–0.6. Cells from 50 mL of
culture were recovered by centrifugation, washed twice with
20 mL LB medium, and resuspended in LB medium (1 mL) for
use as the donor. Donor (100 mL) and recipients (100 mL) were
mixed and distributed onto MS plates supplemented with
10 mM MgCl2. The plates were incubated at 30 1C for
10–16 h then overlaid with 1 mL water of containing
nalidixic acid to select against E. coli and either apramycin
(Am) or erythromycin (Em) or kanamycin (Km) to select for
S. flaveolus exconjugants (the final concentration of antibiotics
is 50 mg mLꢁ1). Incubation was continued at 30 1C until
exconjugants appeared.
Generation of the gene replacement vector pKC5201
A 900 bp DNA fragment containing the neomycin resistance
cassette was amplified from SuperCos1 using primers neo-for
(50-AA GAG CTC GCA GAA ACG GTG CTG ACC-30, SacI
ꢁ ꢁ ꢁ ꢁ ꢁꢁ
site is underlined) and neo-rev (50-AA GAG CTC ATA GAA
ꢁ ꢁ ꢁ
ꢁ
ꢁ
GGC GGC GGT GGA-30, SacI site is underlined) and further
cloned into the pKC1139 SacI site for replacing the aac(3)IV
resistance gene to generate pKC5201. The neomycin resistant
gene in the resulted plasmid pKC5201 has the same transcription
direction to aac(3)IV of pKC1139.
Deletion of the pks (sfaKBsfaH)
A 1.8 kb EcoRI/StuI fragment containing the ermE resistance
cassette from pAGe-1 was cloned into the EcoRI/EcoRV site
of pSP72 to yield TL3201. Cosmid pTL3104 was digested with
BamHI to afford a 9.5 kb DNA fragment containing vector,
which further self-ligates to yield pTL3202. The 1.8 kb BglII
fragment containing the ermE resistance gene from pTL3201
was cloned into the BamHI site of pTL3202 to generate
plasmid pTL3203 which contains the same coding direction
for the ermE and pks genes. Finally, the 4.5 kb EcoRI fragment
was moved from pTL3203 into the EcoRI site of pKC1139 to
create pTL3111, which was subsequently used to replace part
of the pks locus with the ermE gene.
Cloning and isolation of the SFA biosynthetic gene cluster
A genomic library of S. flaveolus DSM 9954 was constructed in
SuperCos1 according to a method described previously.11 The
0.9 kb DNA probes were amplified from the S. flaveolus
genomic DNA by using the degenerate primers of ccr: CCR-
for (50-AG GAA TTC ATG GCC TCC KCS RTS AAC TAC
ꢁ ꢁ ꢁ ꢁꢁ ꢁ
AAY-30, EcoRI site is underlined) and CCR-rev (50-TC GGA
ꢁ ꢁ ꢁ
TCC GAT GAT GCG CTT SWS BKD CAT CCA -30, BamHI
ꢁ ꢁ ꢁ
site is underlined). The genomic library was screened by colony
hybridization with the mixture of three ccr fragments as the
probe (probe 1). The ccr positive cosmids were further screened
by PCR with degenerate PKS primers11 to identify those which
contain both ccr and pks genes. BamHI was then used to digest
the resulting cosmids to generate restriction maps. The 6.4 kb
BamHI fragment excised from the upstream end of pTL3102
(probe 4), 1.4 kb BamHI fragment derived from the upstream
end of pTL3104 (probe 5) and 4.8 kb BamHI fragment from
the upstream end of pTL3106 (probe 6), were used as probes
for chromosomal walking to isolate overlapping cosmids
covering the entire gene cluster (Fig. S2w).
Inactivation of orf3
Primers orf3L-for (50-AA GGA TCC GGA TGA CCT GTG
ꢁ ꢁ ꢁ ꢁ ꢁ ꢁ
ACA GCG AAC CTT G-30, BamHI site is underlined) and
orf3L-rev (50-AA CAT ATG GCT TGG ACA TGA AGT
ꢁ ꢁ ꢁ ꢁ ꢁ ꢁ
ATC CCC TCA T-30, NdeI site is underlined) were used to
amplify a 1.5 kb fragment from pTL3106 containing orf3. The
resultant blunt-ended fragment was cloned into the StuI site of
pANT841 to generate pTL3206 as the left arm for gene
replacement. The right arm for gene replacement was
achieved by cloning a 1.5 kb blunt-ended DNA fragment,
amplified from pTL3106 with primers orf3R-for (50-AA
Genetic manipulation of S. flaveolus
Genetic manipulation of S. flaveolus was carried out using
standard literature methods42 with minor modifications. In
brief, approximately 109 S. flaveolus spores were suspended in
TES (500 mL, 0.05 M, pH 8.0) and heat shocked at 50 1C for
CAT ATG GCG GGC GAG ATG CGG GAT GTC GAG
G-30, NdeI site is underlined) and orf3R-rev (50-AA GAA TTC
GCG GTA CAG GTT GGC GAA CCG GTC G-30, EcoRI
ꢁ ꢁ ꢁ ꢁ ꢁ ꢁ
ꢁ ꢁ ꢁ ꢁꢁ ꢁ
site is underlined), into the EcoRV site of pGEM 5zf to yield
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pTL3207. Finally, a three part ligation was achieved between
the 1.5 kb BamHI-NdeI fragment of pTL3206, the 1.5 kb NdeI-
EcoRI fragment from pTL3207, and BamHI-EcoRI digested
pKC1139 to yield pTL3114. This was the vector used for orf3
in frame deletion.
days to induce the single crossover. A single colony was then
inoculated into 3 mL TSB media without antibiotics and
incubated in a shaker at 37 1C for 4 days. This was used to
innoculate further TSB media (3 mL, 0.1% v/v) which was
incubated under same condition for 4 days. This step was
repeated 5 times to induce double crossover. 50 mL of the
resulting broths were then streaked on ISP-4 ager media
containing 50 mg mLꢁ1 Em for pTL3111, or 50 mg mLꢁ1
Am for pTL3113, or without any antibiotics for
pTL3114–pTL3117 and incubated at 37 1C until colonies were
seen. Verification of antibiotic resistance phenotype was per-
formed by replica streaking of colonies onto plates containing
different antibiotics and checked resistant phenotype. For
pTL3111, the double crossover mutant should be EmR and
AmS. For pTL3113 it should be AmR and KmS. For
pTL3114–pTL3117 the AmS phenotype indicated loss of the
vector backbone and could be either wild type strain or inframe
deletion mutants. Genotype verification by PCR was performed
to verify the mutants (see ESIw).
Inactivation of sfaA
Primers 201-2L (50-GTG GAA ATC GGC TCG GGC GCG
CCC GAA TTA ACC GCG TCG ATT CCG GGG ATC
CGT CGA CC-30) and 201-2R (50-AAT GGA TGT ATC
GTC GCA GGA CGC CCA GAA TTC ACC TGC TGT
AGG CTG GAG CTG CTT C-30) were used to amplify the
1.3 kb aac(3)IV-oriT cassette from pIJ773. pTL3205 was
constructed via lRED-mediated PCR targeting44 in order to
replace the sfaA gene of pTL3106 with the aac(3)-oriT cassette.
The 8 kb BglII fragment was then transferred from pTL3205
into the BamHI site of pKC5201 to afford plasmid pTL3113,
which was used for sfaA gene replacement.
Inactivation of sfaK
Synthesis of crotonyl-CoA
Similar to in frame deletion of orf3, the left arm is amplified
from pTL3102 by primers sfaKL-for (50-AT GAA TTC GTG
GCG GTC GTC GGC ATG GCC TG-30,with EcoRI
Crotonic acid (3 mg, 35 mmol, 3.5 eq., Aldrich Inc.), PyBOP
(8.4 mg, 16 mmol, 1.6 eq., GL Biochem Shanghai Ltd.) and
K2CO3 (5.6 mg, 40 mmol, 4 eq., China National Medicines Co.
Ltd.) were dissolved in anhydrous THF (3 mL, China National
Medicines Co. Ltd.) using vigorous stirring under argon at
room temperature for at least 10 min. A solution of CoASH
(7.67 mg, 10 mmol, 1 eq., Sigma Inc.) was then added slowly
and the reaction was stirred at room temperature for 1 h. The
reaction mixture was purified by Jasco LC-NetII HPLC
(Jasco, Inc. Japan) and an Aglient Zorbax column (SB-C18,
5 mm, 4.6 ꢀ 250 mm) monitoring at a wavelength of 260 nm
with condition of 5–40% B (CH3CN) and A (Water, 10 mM
NH4OAc) elution in 30 min and structurally determined by
ESI-MS (Thermo Fisher LTQ Fleet ESI and Agilent 1200
HPLC) for crotonyl CoA 835.90 (M + H+).
ꢁ ꢁ ꢁ ꢁꢁꢁ
underlined) and sfaKL-rev (50-AT GGA TCC CCT CGG
ꢁ ꢁ ꢁ ꢁ ꢁ ꢁ
TGC CGC GGA ACA GGA GC-3, with BamHI site
underlined) and cloned into the EcoRV site of pGEM 5zf to
achieve pTL3208. Right arm amplified from pTL3102 by
primers sfaKR-for (50-AT GGA TCC GGC GAT GAC
ꢁ ꢁ ꢁ ꢁ ꢁ ꢁ
CGA CTG GCT CGC CAC-30, with BamHI underlined)
and sfaKR-rev (50-AT AAG CTT GGT CCC CGA CGG
ꢁ ꢁ ꢁ ꢁꢁꢁ
TGA GCA GCA CC-30, with HindIII underlined) was cloned
into the EcoRV site of pGEM 5zf to generate pTL3209. The
EcoRI–BamHI fragment from pTL3208 and the
BamHI–HindIII fragment from pTL3209 were recovered and
then co-ligated into the EcoRI–HindIII site of pKC1139,
yielding pTL3113 for in frame deletion of sfaK.
Inactivation of sfaR
Overexpression, purification and biological assay of SfaR
The 4.7 kb BglII-KpnI fragment from pTL3102 was cloned into
the same sites of pSP72 to create pTL3210. The internal 674 bp
fragment of pTL3210 was then removed by EcoR72I digestion
and the rest part further self-ligated to yield pTL3211. The
BglII-HindIII fragment of pTL3211 was further cloned into
BamHI-HindIII sites of pKC1139 to afford plasmid pTL3116,
which is used for sfaR gene replacement.
The sfaR gene was amplified by PCR from cosmid TL3102
using primers (SfaR-for: 50-TC GAA TTC CAT ATG AAC
ꢁ ꢁ ꢁ ꢁꢁ ꢁ ꢁ ꢁ ꢁ ꢁ ꢁ ꢁ
GAG ATA CTC AGC G-30 and SfaR-rev: 50-TCA AAG CTT
ꢁ ꢁ ꢁ ꢁ ꢁꢁ
ATC AGA CCG GCT CTG TCT GC-30, restriction sites
EcoRI, NdeI and HindIII are underlined). This was then
cloned into the NdeI and HindIII sites of His6-tagged vector
pET28a and the resulting plasmid pTL3213 was transferred
into E. coli BL21 (DE3) for overexpression. SfaR with a
molecular weight of B50 kD was expressed at 25 1C
250 rpm for 10 h under 100 mM IPTG induction (IPTG was
added when OD600 reached 0.6) and purified to homogeneity
by Ni-NTA affinity chromatography (Fig. S9w). The elutent
containing SfaR was exchanged into stocking buffer (50 mM
Tris-HCl, 100 mM NaCl, and 10% (v/v) glycerol, pH = 8.0)
by PD-10 (GE healthcare), concentrated to 10 mg mLꢁ1 by
Vivaspin (GE healthcare) and stored at ꢁ80 1C. Protein
concentration was determined by standard Bradford
method.45 The standard assay system consists of 1 mM
NADPH, 60 mM NaHCO3, 20 mM SfaR and 500 mM
substrate in Tris-Cl buffer (75 mM, pH = 8.0) with
Inactivation of orf4
An internal 300 bp DNA fragment of orf4 was removed from
pTL3210 by ClaI-EcoRI double digestion. The remaining
major fragment of pTL3210 was treated with T4 DNA
polymerase to generate blunt ends and allowed to self-ligate
to generate pTL3212. The BglII-HindIII fragment from
pTL3212 was moved into BamHI-HindIII digested pKC1139
to yield pTL3117. This was used for orf4 gene deletion.
Generation of mutant strains
Each exconjugant colony was streaked onto ISP-4 agar media
containing 50 mg mLꢁ1 Am and incubated at 37 1C for several
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incubated at 30 1C. In order to further study the reduction step,
the same assay was used except NaHCO3 was omitted. The assay
system are analyzed by HPLC-ESIMS with a flow rate of
1 mL minꢁ1 over a 30 min gradient as follows T = 0, 5% B;
T = 30, 40% B (A: 10 mM NH4OAc in water; B: acetonitrile).
(2S)-2-ethylmalonyl-CoA (M + H+: 881.90) and butyryl-CoA
(M + H+: 837.86) were eluted at 6.6 min and 9.0 min, respectively.
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Acknowledgements
This work was supported in part by grants from NNSF
(30900021, 20902102, 20832009, 90713012 and 20921091),
MST (2009ZX09501-008), ‘‘973 program’’ (2010CB833200),
CAS (KJCX2-YW-H08 and KSCX2-YW-G-06), and STCSM
(09QH1402700) of China. We thank Nigel Coates, Matthew
Gregory, Teresa Foster, Steven Moss, Mahmood Piraee, Tony
Warneck and Ming Zhang at Biotica Technology Ltd. for help
with fermentation methods and analysis of strains.
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