Engineered biosynthesis of hybrid macrolide polyketides containing d-angolosamine and d-mycaminose moieties

Article abstract of DOI:10.1039/b807914e

The glycosylation of natural product scaffolds with highly modified deoxysugars is often essential for their biological activity, being responsible for specific contacts to molecular targets and significantly affecting their pharmacokinetic properties. In

Full text of DOI:10.1039/b807914e

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Engineered biosynthesis of hybrid macrolide polyketides containing
D-angolosamine and D-mycaminose moieties†
Ursula Schell,‡a Stephen F. Haydock,‡b Andrew L. Kaja,a Isabelle Carletti,a Rachel E. Lill,a Eliot Read,a
Lesley S. Sheehan,a Lindsey Low,a Maria-Jose Fernandez,b Friederike Grolle,b Hamish A. I. McArthur,c
Rose M. Sheridan,a Peter F. Leadlay,b Barrie Wilkinson*a and Sabine Gaisser*a,d
Received 9th May 2008, Accepted 24th June 2008
First published as an Advance Article on the web 28th July 2008
DOI: 10.1039/b807914e
The glycosylation of natural product scaffolds with highly modified deoxysugars is often essential for
their biological activity, being responsible for specific contacts to molecular targets and significantly
affecting their pharmacokinetic properties. In order to provide tools for the targeted alteration of
natural product glycosylation patterns, significant strides have been made to understand the
biosynthesis of activated deoxysugars and their transfer. We report here efforts towards the production
of plasmid-borne biosynthetic gene cassettes capable of producing TDP-activated forms of
D-mycaminose, D-angolosamine and D-desosamine. We additionally describe the transfer of these
deoxysugars to macrolide aglycones using the glycosyl transferases EryCIII, TylMII and AngMII,
which display usefully broad substrate tolerance.
We have previously described alteration of the glycosylation
pattern of the macrolide antibiotics erythromycin (1), tylosin
(2), and oleandomycin as well as the insecticidal spinosyns (3)
Introduction
The elucidation of high-resolution X-ray crystal structures for
(see Fig. 1).5–8 Here we report the successful use of biosynthetic
gene cassettes for the heterologous production and transfer of
several deoxyhexoses to polyketide aglycones in place of the
natural substituents. These are changes of potential relevance
for generating compounds with altered and desirable activities.
Such experiments have to some degree been previously hampered
by incomplete information about the biosynthetic pathways to
activated deoxyaminohexoses. For example, in the biosynthe-
sis of TDP-D-mycaminose (4) (see Fig. 2), the 3,6-dideoxy-3-
aminosugar component of 2, it was originally proposed that the
tylMIII gene from the biosynthetic gene cluster encoded a 3,4-
ketoisomerase responsible for converting TDP-4-keto-6-deoxy-D-
glucose (5) to its 3-keto isomer (6).9 This assignment was based
on its significant sequence similarity to genes governing TDP-D-
desosamine (7) biosynthesis in the erythromcyin biosynthetic gene
cluster.10 Studies reported by Liu and colleagues have since shown
that the biosynthetic route to TDP-D-desosamine does not require
3,4-keto tautomerisation.11 Moreover, it has now been shown
that TylMIII and homologues from other deoxyaminohexose
biosynthetic gene clusters are actually involved in the glycosylation
process itself, forming a complex with, and enhancing the activity
of, the cognate GT.12–14 The 3,4-ketoisomerase required for TDP-
D-mycaminose production is actually encoded by tyl1a, a previ-
ously unassigned open reading frame (ORF) within the tylosin
biosynthetic gene cluster of Streptomyces fradiae ATCC19609.15,16
This finding stemmed from the observation that a homologue
of tyl1a (fdtA) is required for TDP-3-acetamido-3,6-dideoxy-a-D-
galactose biosynthesis in Aneurinibacillus thermoaerophilus L420-
91T, and is responsible for the production of TDP-6-deoxy-D-
xylohex-3-ulose from TDP-6-deoxy-D-xylohex-4-ulose.17 Liu and
co-workers have used this insight to produce, in vivo, macrolide
analogues bearing a D-mycaminose moiety in place of the natural
clinically important antibiotics bound to their ribosomal targets
has revealed key molecular interactions and provided insights
into how such templates might be adapted through structure-
aided drug design to provide modified derivatives with activity
against drug-resistant organisms.1 Macrolide antibiotics consist
of a core aglycone, of polyketide origin, to which deoxysugar
(most commonly deoxyhexose) moieties are attached. It is the
deoxysugar moieties which confer potent and specific antibacterial
activity through key contacts to the ribosome. Indeed, the
importance of deoxysugar components for the activity of multiple
types of natural products has driven an increasing interest in
understanding the mechanisms and pathways by which they are
biosynthesised, and in characterising the substrate range and toler-
ances of the glycosyltransferases (GTs) which attach them to their
acceptor substrates. This has led to the development of powerful
methods for the manipulation of deoxysugar biosynthesis and
the production of new ‘non-natural natural products’ through
the application of glycosylation engineering.2,3 In particular, the
utility of biosynthetic gene cassettes for deoxysugar production
in heterologous Streptomyces hosts has been clearly demonstrated
by Salas, Me´ndez and co-workers.4
aBiotica Technology Ltd, Chesterford Research Park, Little Chesterford, Saf-
fron Walden, Essex, CB10 1XL, UK. E-mail: barrie.wilkinson@biotica.com
bDepartment of Biochemistry, University of Cambridge, 80 Tennis Court
Road, Cambridge, CB2 1GA, UK
cBioprocess Research, Central Research Division, Pfizer Inc., Groton, CT,
06340, USA
dHochschule Biberach, Karlstraße 11, 88400, Biberach/Riß, Germany.
E-mail: gaisser@fh-biberach.de
† Electronic supplementary information (ESI) available: Details of the
construction of gene cassettes, isolation of conversion vectors, and
construction of strains. See DOI: 10.1039/b807914e
‡ These authors made equal contributions.
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Fig. 1 Structures of selected polyketide macrolides.
Fig. 2 Biosynthetic pathways to the TDP-D-deoxyaminohexoses discussed. The proteins from each pathway responsible for the different chemical steps
are given (Des/Ery, desosamine pathways; Tyl, mycaminose pathway; Ang, angolosamine pathway; Spn, forosamine pathway).
D-desosamine.15 The requirement of tyl1a for TDP-D-mycaminose
biosynthesis was independently described in an international
patent application.18
Herein we report the cloning and sequencing of the biosyn-
thetic gene cluster from Streptomyces eurythermus ATCC23956
encoding production of the 16-membered macrolide antibiotic
angolamycin (8).19 8 exhibits relatively weak antibacterial activity
but is interesting due to the presence of an unusual 3-amino-2,3,6-
trideoxy-D-hexose moiety derived from TDP-D-angolosamine
(9). Further, we describe the construction of biosynthetic gene
cassettes that confer upon heterologous hosts the ability to
synthesise TDP-D-mycaminose, TDP-D-desosamine and TDP-D-
angolosamine, and examine the ability of several cloned GTs to
transfer these and other deoxyhexoses to polyketide aglycones to
produce a range of novel macrolides.
Results
Provision of genes for the assembly of biosynthetic gene cassettes
The genes responsible for TDP-D-mycaminose biosynthesis were
obtained from cloned DNA of the fully-sequenced tylosin
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Fig. 3 Representation of the angolamycin biosynthetic gene cluster: The diagrams A and B (above and below the PKS gene cluster) show enlargement
of the PKS flanking regions.
biosynthetic gene cluster of S. fradiae ATCC19609.9 The genes
required for the production and transfer of TDP-D-desosamine
were obtained from the fully-sequenced erythromycin gene cluster
of Saccharopolyspora erythraea NRRL2338.10,20 We additionally
used genes from the spinosyn biosynthetic gene cluster of
Saccharopolyspora spinosa NRRL18395 involved in biosynthesis
of the deoxyaminosugar TDP-D-forosamine (10) (Fig. 2).21 To
obtain the genes required for TDP-D-angolosamine biosynthesis
we identified, cloned and sequenced the biosynthetic gene cluster
for angolamycin (see below).
CoA) to be readily deduced.22 Examination of the b-keto group
processing domains present (b-ketoreductase (KR), dehydratase
(DH) and enoylreductase (ER)) allowed the level of reduction to
be deduced for each such extension. This analysis showed that
the constitution of Ang1–5 is wholly consistent with a role in
the assembly of tylactone (11). Ang1 encompasses the loading
domain and extension modules 1 and 2, Ang2 encompasses
module 3, Ang3 encompasses modules 4 and 5, Ang4 encompasses
module 6, and Ang5 encompasses module 7 plus the C-terminal
thioesterase (TE) domain required for cyclisation and release
of the polyketide chain from the PKS. This arrangement of
enzyme domains is exactly analogous to that found in the tylosin
PKS.9
Identification, cloning and sequence analysis of the angolamycin
biosynthetic gene cluster
Flanking the PKS-encoding genes are other ORFs involved in
the biosynthesis of 8. Numerous ORFs could be identified by
comparison to sequences in published databases and by direct
comparison with genes from the tylosin biosynthetic gene cluster.
The proposed functions of these putative ORFs together with
the best database matches are shown in Table 1 and include
those required for TDP-D-angolosamine biosynthesis. The genes
orf15 and orf13* most probably form the boundaries of the gene
cluster. Upstream of orf15 there is a strong RNA terminator
structure, adjacent to genes encoding another distinct modular
PKS. Downstream of orf13* there is a non-coding region of about
1 kbp, followed by genes whose products appear to be involved in
sporulation (data not shown).
A pathway for TDP-D-angolosamine biosynthesis is shown
in Fig. 2 and is based on our sequencing and heterologoues
expression experiments presented here. This is consistent with
published data for the hedamycin23 and medermycin24 biosynthetic
gene clusters. These polyketides also contain D-angolosamine
moieties. The genes angAI, angAII, angMIII, angB and angMI
seem to be exact counterparts of tylAI, tylAII, tylMIII, tylB
and tylMI involved in TDP-D-mycaminose biosynthesis. The
gene ang-ORF14 was identified to encode a TDP-4-keto-6-
deoxyhexose reductase, the counterpart of med-ORF14 and hedN
from the medermycin and hedamycin biosynthetic gene clusters
respectively.
S. eurythermus ATCC23956 was obtained from the American Type
Culture Collection (Manassas, Virginia, USA). Initial identifi-
cation of the angolamycin biosynthetic cluster was achieved by
end-sequencing 768 clones from a S. eurythermus cosmid library
generated in Supercos (Stratagene). Six clones were identified by
BLAST searching which exhibited very high sequence homology
to the genes of the tylosin biosynthetic cluster. The complete
DNA sequence of four of these cosmids, designated 2C3, 2D8,
4h12 and 5B2 respectively, was obtained by the assembly of
overlapping Sau3A fragments obtained from partial digestion of
the parent cosmid. Remaining sequence gaps were filled using
21mer synthetic oligonucleotides as primers. These four cosmid
clones were found to span an entire gene cluster containing
modular polyketide synthase (PKS) genes with striking similarity
to those of tylosin biosynthesis.9 The angolamycin biosynthetic
gene cluster sequence has been deposited as EU038272, EU220288
and EU232693.
The putative angolamycin (8) biosynthetic cluster lies within a
77 kbp locus (Fig. 3/Table 1). The biosynthesis of the polyketide
backbone of 8 is governed by 5 large ORFs, designated ang1 to
ang5, which are co-transcribed and which encode, respectively, the
multimodular PKS polypeptides Ang1 to Ang5. Examination of
their encoded acyltransferase (AT) domains allowed the nature
of the extender units (malonyl-, methylmalonyl-, or ethylmalonyl-
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Table 1 Proposed functions of genes encoded by the angolamycin biosynthetic gene cluster
No. of
amino
acids
Best match in
database
ORF
% Identity
Organism
Proposed function
Tylosin orthologue
TylB
15 (AngB)
373
333
415
211
272
385
79
Q7wt26
Q67g46
Q331r5
Q9s4d4
Q9s4d5
Q9s4d6
Q9shq2
Q5sfa6
Q9zhq4
Q9zhq5
Q9zhq6
77
49
66
72
79
63
51
63
71
69
57
Streptomyces sp
AM-7161
NDP-Hexose
aminotransferase
NDP-Hexose
4-ketoreductase
Cytochrome P450
monooxygenase
NDP-Hexose
3,(5)-epimerase
O-Methyltransferase
14
13
12
11
10
9
Streptomyces
griseoruber
Streptomyces sp.
KCTC0041BP
Streptomyces
fradiae
Streptomyces
fradiae
Streptomyces
fradiae
Streptomyces
fradiae
Streptomyces
fradiae
Streptomyces
fradiae
Streptomyces
fradiae
TylJ
TylF
Cytochrome P450
monooxygenase
Flavodoxin
TylH1
Tylodoxin
TylD
8
331
395
422
283
NDP-Hexose
4-ketoreductase
O-Methyltransferase
7
TylE
6
Glycosyltransferase
(deoxyallose)
Ribosomal
methylase
(resistance)
TylN
5
Streptomyces
fradiae
4 N-terminal
C-terminal
3 (AngAII)
2 (AngAI)
1
787
Q9jn57
Q76kz0
Q54144
Qfsfd2
Q59910
P95746
P95747
P95748
Q53865
Q00509
Q9jn56
Q9xc69
033936
63
60
70
74
80
45
67
65
93
36
53
54
67
Streptomyces
fradiae
Streptomyces
halstedii
Streptomyces
fradiae
Streptomyces
bikiniensis
Streptomyces
fradiae
Streptomyces
fradiae
Streptomyces
fradiae
Streptomyces
fradiae
Streptomyces
collinus
Type II thioesterase
NDP-Hexose
2,3-dehydratase
NDP-Hexose
4,6-dehydratase
NDP-Hexose
synthase
Cytochrome P450
monooxygenase
Activation of
tylCVI
tylAII
tylAI
332
295
414
447
424
240
455
641
447
333
334
1*
TylMIII
TylMII
TylMI
(AngMIII)
2* (AngMII)
AngMII
Glycosyltransferase
(angolosamine)
N-Methyltransferase
3* (AngMI)
4*
5*
6*
7*
8*
Crotonyl-CoA
reductase
Regulatory (SrmR)
Streptomyces
ambofaciens
Streptomyces
fradiae
Streptomyces
fradiae
Regulatory
TylR
NDP-Hexose
TylCIV
TylCII
4-ketoreductase
4-Keto-6-deoxy
NDP-kexose 2,3
reductase
Saccharopolyspora
erythraea
9*
410
404
202
388
577
Q9xc68
Q9xc67
Q9xc66
Q82m93
Q60248
78
64
60
29
65
Streptomyces
fradiae
Streptomyces
fradiae
Streptomyces
fradiae
Streptomyces
avermitilis
Streptomyces
halstedii
NDP-Hexose 3-C-
methyltransferase
Glycosyltransferase
TylCIII
TylCV
10*
11*
12*
13*
NDP-hexose
3,(5)-epimerase
Transaldolase
TylCVII
Exporter/resistance
Heterologous expression hosts
S. erythraea strain SGQ2 (BIOT-2175) is a quadruple mutant
lacking the eryA PKS genes, the two GTs eryCIII and eryBV,
and eryCVI, an N,N-dimethyltransferase gene required for the
biosynthesis of TDP-D-desosamine. All of these are essential for
erythromycin (1) biosynthesis and were successively mutated by
We previously described an in vivo glycosylation system based
upon defined S. erythraea mutant strains.5–8 In the present
study additional S. erythraea mutant strains were constructed.
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introducing deletions into the chromosome. S. erythraea Q42/1
(BIOT-2166) is a derivative of SGQ2 in which the biosynthesis
of TDP-L-mycarose (an additional deoxyhexose required for the
biosynthesis of 1) was disrupted by integration of a suicide vector
containing an internal fragment of eryBVI, a gene essential for its
biosynthesis. S. erythraea LB1 (BIOT-2634) is deleted in almost
the entire erythromcyin biosynthetic gene cluster, leaving only the
ermE and eryCI genes on one flank and the gene eryK encoding
a cyp450-dependent monooxygenase on the other flank. The host
strains described here all retain the ermE gene, which confers
resistance to macrolide antibiotics. A detailed description of host
preparation is given in the ESI† (also see ref. 18).
Three different GTs were cloned and separately introduced
into these host strains as required. When combined with appro-
priate aglycone substrates, these acted as reporter systems for
the production of activated deoxyhexoses and their subsequent
transfer to aglycone acceptor substrates. The first two GTs studied
were TylMII from S. fradiae and AngMII from S. eurythermus
(described above) both of which use tylactone (11) (see Fig. 4)
as their natural acceptor substrate. During tylosin biosynthesis
TylMII (in combination with TylMIII) catalyses transfer of D-
mycaminose to the C5-hydroxyl group of 11 to give 5-O-b-D-
mycaminosyltylactone (12) (Fig. 4). AngMII (plus AngMIII; this
study) is proposed to catalyze the analogous transfer of a D-
angolosamine moiety to 11 during biosynthesis of angolamycin.
The third GT utilized was EryCIII which (with EryCII) catalyses
attachment of D-desosamine to the C5-hydroxy group of 3-O-a-L-
mycarosylerythronolide B (13) during erythromycin biosynthesis.2
Construction of D-deoxyaminohexose biosynthetic gene cassettes
Biosynthetic gene cassettes were assembled in an integrative
expression vector using the cloning strategy outlined in detail
in the ESI†. This is an advancement of a procedure described
previously.7 In order to incorporate each gene into a cassette it
was amplified with an NdeI restriction site overlapping the start
codon and an XbaI restriction site adjacent to the stop codon.
These PCR products were then cloned into the conversion vector
pSGLit1 which was employed to introduce a Shine–Dalgarno
sequence/ribosome binding site and to incorporate an XbaI site
at the 5ꢀ-end of the gene that is sensitive to dam− methylation.
DNA from the resulting plasmids was isolated from a dam− strain
background, the gene isolated by XbaI digestion, and incorporated
into the gene cassette after XbaI digests. The presence of a single
methylation sensitive XbaI site allows for easy determination of the
direction of integration by restriction digest. A schematic overview
of the strategy is provided in the ESI as Fig. S1. This methodology
is extremely flexible and has been used extensively in other systems,
for example the combinatorial assembly of libraries of rapamycin
post-PKS gene cassettes.25
Production of novel macrolides containing D-angolosamine
A biosynthetic gene cassette for the production of TDP-D-
angolosamine (9) was assembled using genes from both the
angolamycin and the spinosyn biosynthetic gene clusters. Ang-
ORF4, a putative TDP-4-keto-6-deoxyhexose-2,3-dehydratase,
appears to be present as the C-terminal domain of a protein
whose N-terminal portion houses a thioesterase activity. To
avoid uncertainty being introduced by use of this apparently
bifunctional protein we substituted ang-ORF4 with spnO, which
encodes the counterpart gene required for TDP-D-forosamine (10)
biosynthesis in the spinosyn pathway of S. spinosa (see Fig. 2),21
and whose function has been confirmed.26 A gene cassette was
thus constructed comprising angAI, angAII, angMIII, spnO,
ang-ORF14, angB, angMI and angMII, and this cassette was
used to transform S. erythraea Q42/1 to give strain BIOT-2945.
The functions of these genes are described in Tables 1 and 2.
Tylactone (11) was added to growing cultures of BIOT-2945 and
after five days the fermentation broth was extracted with organic
solvent. LCMS analysis of the extracts revealed the presence of
a new 11-derived metabolite which was not present when 11 was
Fig.
4
Biotransformation of tylactone (11) to 5-O-b-D-angolo-
saminyltylactone (14) (BIOT-2945/2808) and 5-O-b-D-mycamino-
syltylactone (12) (BIOT-2879/3007); the structure of the previously
reported5 5-O-b-D-desosaminyltylactone (15) is given for comparison.
Table 2 Function of deoxysugar biosynthetic genes utilised/discussed
Gene
Strain
Accession number
Proposed function
tylAI
Streptomycs fradiae
U08223
U08223
X81885
X81885
X81885
U08223
U08223
Y11199
AM420293
Y14332
Y14332
Y11199
X51891
AY007564
TDP-D-glucose synthase
TDP-D-glucose dehydratase
N,N-Dimethyltransferase
Glycosyltransferase
Glycosyltransferase ancillary protein
Aminotransferase
3,4-Ketoisomerase
Glycosyltransferase
NDP-hexose 2,3-dehydratase
Glycosyltransferase ancillary protein
Glycosyltransferase
tylAII
tylMI
tylMII
tylMIII
tylB
Streptomycs fradiae
Streptomycs fradiae
Streptomycs fradiae
Streptomycs fradiae
Streptomycs fradiae
tylIa
Streptomycs fradiae
eryBV
eryBVI
eryCII
eryCIII
eryCVI
ermE
Saccharopolyspora erythraea
Saccharopolyspora erythraea
Saccharopolyspora erythraea
Saccharopolyspora erythraea
Saccharopolyspora erythraea
Saccharopolyspora erythraea
Saccharopolyspora spinosa
N,N-Methyltransferase
Methylase (erythromycin resistance)
NDP-hexose 2,3-dehydratase
spnO
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fed to the control strain S. erythraea Q42/1. The new compound
(14) contained a deoxyaminohexose whose fragment mass (158
amu) corresponded to the presence of a D-angolosamine moiety.
The new product represented ∼50% of the tylactone (11) supplied.
The experiment was then repeated using a gene cassette in which
the GT-encoding angMII was replaced with tylMII to give strain
BIOT-2808, and the same novel macrolide (14) was produced with
similar efficiency from the added 11. A more polar analogue was
also observed in each case whose LCMS data was consistent with
this being 5-O-b-D-glucosyltylactone produced by an unidentified
native D-glucosyltransferase of broad substrate range as described
previously.5
To confirm the identity of 14 the biotransformation using strain
BIOT-2808 was scaled up to provide 20 mg of purified compound.
Its structure was confirmed as 5-O-b-D-angolosaminyltylactone
(Fig. 4) using multi-dimensional NMR experiments. The data
are reported in the experimental section, but key features are
the lack of an oxygen substituent at C-2ꢀ and a hydroxyl group
at C-4ꢀ. Glycosidic linkage between C-5 and C-1ꢀ was firmly
established through observation of the appropriate correlation
in the HMBC spectrum. The analysis was aided by comparison
to the equivalent D-desosamine5 (15) and D-mycaminose27 (12)
containing analogues reported previously. This result confirmed
that the assembled gene cassette was indeed capable of producing
TDP-D-angolosamine (9) in the heterologous host strain, and also
that the function of AngMII during 8 biosynthesis is the transfer of
a D-angolosamine moiety to the hydroxy group at C-5 of tylactone.
To examine whether AngMII could transfer D-angolosamine
to other macrolide aglycones, erythronolide B (16) (the agly-
cone of the erythromycin biosynthetic pathway) and 3-O-a-L-
mycarosylerythronolide B (13) (the subsequent intermediate of
erythromycin biosynthesis) were added separately to growing cul-
tures of strain BIOT-2945. A new macrolide product was observed
(>90% bioconversion) only with 16 as substrate. Following scale-
up, analysis of the purified compound using a combination of
LCMS and NMR methods was straightforward and allowed its
structure to be confirmed as 3-O-b-D-angolosaminylerythronolide
B (17) (Fig. 5A). The D-angolosamine moiety was readily assigned,
and HMBC data clearly located the glycosidic linkage as C3–C1ꢀ.
This result demonstrates that AngMII is reasonably relaxed in
choosing its aglycone acceptor substrate. Glycosylation at the C3-
hydroxyl group to yield 17 was unanticipated as AngMII normally
transfers a D-angolosamine residue to the C5-hydroxyl group of
its native substrate tylactone.
Previous work has suggested that, in general, GTs are more
specific for their acceptor substrate than for the deoxysugar
donor substrate. Therefore, to examine the possibility of transfer-
ring D-angolosamine to 13, and produce modified erythromycin
analogues, angMII was substituted within the biosynthetic gene
cassette by the GT eryCIII. This cassette was then transferred
into S. erythraea Q42/1 to give strain BIOT-2943. This host
strain contains eryCII whose gene product is the activator partner
protein for EryCIII.18,28 Analysis by LCMS/MS indicated a series
of erythromycin analogues which were not present in extracts
when 13 was not fed, nor when 16 was fed. The LCMS profile
of these compounds was consistent with the transfer to 13 of
a deoxyaminohexose with the appropriate MS characteristics
consistent for D-angolosamine (loss of 158 amu by MS/MS).
The LCMS data for the erythromycin analogue 18 (Fig. 6) is
Fig. 5 A. Biotransformation of erythronolide B (16) to 3-O-b-D-angolo-
saminylerythronolide B (17) (BIOT-2945); B. Biotransformation of 3-O-
a-L-mycarosylerythronolide
B
(13) to 3,5-bis-O-a-L-mycarosylery-
thronolide B (20) (BIOT-2191).
Fig. 6 Biotransformation of 3-O-a-L-mycarosylerythronolide B (13) to
erythromycin analogues in which the 5-O-b-D-desosamine moiety has been
replaced with D-angolosamine (18) and D-mycaminose (19) by engineered
S. erythraea strains expressing eryCII/CIII and producing TDP-forms
of the D-deoxyhexoses in place of TDP-D-desosamine (7) (BIOT-2943 &
-3010/2794 respectively).
provided in the experimental section. Unfortunately the yield
of this biotransformation was poor (∼5% total conversion) and
the compound was not isolated in sufficient yield and purity for
complete NMR analysis. The poor yield is presumably because of
a restricted specificity of EryCIII for TDP-D-angolosamine, or of
the efficiency of EryCII/III to catalyse its transfer.
Production of novel macrolides containing D-mycaminose
A biosynthetic gene cassette for the production of TDP-D-
mycaminose (4) was assembled using the same approach as
described above. This cassette contained the genes tylAI, tylAII,
tylMIII, tylB, tyl1a, tylMI and tylMII and was used to transform
S. erythraea Q42/1 to give strain BIOT-2879. Tylactone (11) was
added to growing cultures and after five days the fermentation
broth was extracted and examined by LCMS/MS. A new com-
pound was produced only on addition of 11 (∼50% bioconversion)
whose retention time and MS/MS spectrum (loss of a 176 amu
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fragment) was identical with that of an authentic sample27 of
the anticipated product 5-O-b-D-mycaminosyltylactone (12) (see
Fig. 4). The same gene cassette was also used to transform S.
erythraea LB1 (lacking essentially all of the erythromycin gene
cluster) to give strain BIOT-3007. The efficient bioconversion of
11 to 12 by this strain was also confirmed by LCMS/MS.
EryCIII transfers L-mycarose to 3-O-a-L-mycarosylerythronolide
B (13)
Previous analysis of strains lacking in TDP-D-desosamine (7)
biosynthesis showed elevated levels of a compound with MS/MS
characteristics consistent with the addition of a second L-mycarose
moiety to 13.18 To confirm the identity of this compound, and to
determine whether EryCIII was responsible for the biosynthesis of
this compound, the plasmid pSGeryCIII was introduced in trans
into S. erythraea SGQ. pSGeryCIII contains eryCIII under the
control of a strong promoter. S. erythraea SGQ2, which lacks both
native erythromycin pathway GTs, was unable to biotransform
exogenously added 13 whereas S. erythraea SGQ2/pSGeryCIII
(BIOT-2191) converted ∼50% of exogenous 13 to the described
compound. This biotransformation was scaled up and the new
compound was isolated. NMR analysis clearly identified this as
3,5-bis-O-a-L-mycarosylerythronolide B (20) (Fig. 5B).
We then examined the possibility that EryCIII could transfer
D-mycaminose to 3-O-a-L-mycarosylerythronolide B (13) in order
to produce the equivalent erythromycin analogues, e.g. 19. Such
compounds would contain an additional 4ꢀꢀ-hydroxy moiety and
have been reported previously, including through semi-synthesis
starting from erythromycin.28,29 Thus, the tylMII gene at the 3ꢀ-end
of the TDP-D-mycaminose biosynthetic gene cassette described
above was replaced by eryCIII. This cassette was then used
to transform S. erythraea Q42/1 to give strain BIOT-3010.
After addition of 13 to growing cultures, LCMS/MS analysis
showed the production of new compounds formed by the addition
of a D-mycaminose moiety (e.g. 19; Fig. 6). This indicated
that D-mycaminose was transferred to yield a mixture of the
corresponding erythromycin analogues A–D. Unfortunately the
efficiency of this bioconversion was low, with the majority (∼90%)
of the substrate 13 remaining at the end of the biotransformation.
When the gene cassette was used to transform S. erythraea LB1,
which does not contain eryCII encoding the activator protein for
EryCIII, bioconversion of 13 was not observed.
Discussion
In order to modify the glycosylation pattern of macrolide
antibiotics through engineered biosynthesis, it is important to
understand both the biosynthetic pathways leading to the ac-
tivated deoxyhexose components and the substrate tolerance of
the cognate GTs. The majority of published studies have focused
on the 14-membered macrolides erythromycin (1), oleandomycin
and picromycin, and the 16-membered macrolide tylosin (2).2
As a result, the biosynthetic routes to TDP-D-desosamine (for
1, oleandomycin and pikromycin), TDP-L-mycarose (1 and 2),
TDP-D-mycaminose (for 2), and TDP-D-forosamine (for 3) are
relatively well understood (see Fig. 2). These studies have led to
the identification of individual enzyme functions.
The identification and cloning of the angolamycin (8) biosyn-
thetic gene cluster, in combination with the experiments reported
here, is consistent with a pathway for the biosynthesis of TDP-D-
angolosamine (9) proposed elsewhere23,24 (Fig. 2). The common
intermediate TDP-4-keto-6-deoxy-D-glucose (5) first undergoes
C2-deoxygenation through the action of a TDP-4-keto-6-deoxy-
D-glucose 2,3-dehydratase (believed to be Ang-ORF4 in S. eury-
thermus). The resulting compound 21 is then reduced by Ang-
ORF14, a deoxyhexose 4-ketoreductase, to give 22. The amino
function is introduced at C3 by the aminotransferase AngB,
followed by N,N-dimethylation catalysed by AngMI to yield
TDP-D-angolosamine (9). It is noteworthy that several of the
ang genes are clear counterparts to those required for TDP-D-
mycaminose production during tylosin biosynthesis. It is now
understood that among the group of TDP-D-deoxyaminohexoses
discussed here only the biosynthesis of TDP-D-mycaminose (4)
requires a 3,4-ketoisomerase activity, and that this activity is
encoded by tyl1a (see Fig. 2).15,16,18 For biosynthesis of TDP-
D-desosamine (7) and TDP-D-angolosamine (9), generation of
the 3-keto functionality required for introduction of an amino
group originates via the action of a TDP-4-keto-6-deoxyhexose-
2,3-dehydratase (biosynthesis of 9) or a novel 4-deoxygenation
mechanism involving C4-aminotransfer and a deaminase activity
(biosynthesis of 7).11 We used this combined information to
prepare biosynthetic gene cassettes which, when expressed in an
appropriate host strain, confer upon the resulting mutant the
ability to biotransform various polyketide aglycones to produce
Unexpected production of 5-des-O-b-D-desosaminyl-5-O-b-
D-mycaminosylerythromycin A (19)
As demonstrated by the production of 12, the biosynthetic gene
cassette containing the 3,4-ketoisomerase tyl1a and other tylM
genes are sufficient for efficient TDP-D-mycaminose biosynthesis
in both S. erythraea mutants Q41/1 and LB1. Further, when the
GT tylMII is replaced with eryCIII, transfer of D-mycaminose
to 3-O-a-L-mycarosylerythronolide B (13) was observed, although
the production level of erythromycins bearing a D-mycaminose
moiety in place of D-desosamine at C5 was low.
In the course of this work we also assembled a gene
cassette which contained the majority of the genes for
TDP-D-angolosamine biosynthesis, angAI, angAII, ang-ORF14,
angMIII, angB and angMI, but not the gene ang-ORF4 which
encodes the putative TDP-4-keto-6-deoxyhexose-2,3-dehydratase.
This gene cassette was transferred into the host strain S. erythraea
SGQ2, which still contains a functional copy of the native
TDP-4-keto-6-deoxyhexose-2,3-dehydratase-encoding gene ery-
BVI whose gene product is responsible for 2-deoxygenation during
TDP-L-mycarose biosynthesis. The gene cassette additionally
contained the GT-encoding gene eryCIII (eryCII was present in
the genome). When the resulting strain S. erythraea BIOT-2794
was fermented in the presence of 3-O-a-L-mycarosylerythronolide
B (13), analysis by LCMS/MS indicated the surprising presence
of compounds consistent with 19. The bioconversion efficiency
(∼20%) was significantly greater than that observed in the previous
example and, after scale-up, 19 (15 mg) was purified and its
structure confirmed by NMR analysis; the data for erythromycin,
5-O-b-D-mycaminosyltylactone (12) and related compounds aided
the assignment. Significantly, no metabolites could be observed
by LCMS/MS containing a D-angolosamine moiety in place of
D-mycaminose.
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novel macrolide structures. We believe that these gene cassettes
will have utility in systems other than those based on S. erythraea.
The construction of biosynthetic gene cassettes extended our
cloning strategy in which each gene is cloned using identical
restriction sites by virtue of iterative conversion of introduced
XbaI restriction sites to a methylation-sensitive sequence.7,18,25
This approach allows the same gene fragments to be used in
the construction of multiple cassettes without the limitations of
restriction site compatibility, and thus combinatorial assembly
processes are possible. Additionally, this method provides each
gene with an individual ribosome binding site.
When introduced into both S. erythraea strains Q42/1 and LB1
the biosynthetic gene cassette for TDP-D-mycaminose (4), con-
taining the cognate GT pairing of tylMII/tylMIII or the hybrid
pair tylMII/angMIII, conferred the ability to efficiently produce
and transfer D-mycaminose to exogenously added tylactone (11)
as anticipated (this is the native aglycone (acceptor) for both
of these GTs). Similarly, the cassette for TDP-D-angolosamine
(9) in combination with angMII/angMIII was able to efficiently
produce this D-deoxyaminohexose and transfer it to 11. These data
confirmed our biosynthetic gene cassette approach. Similarly, a
cassette was assembled for TDP-D-desosamine (7) biosynthesis
including the GT pairing of eryCII/eryCIII, and its effectiveness
was verified by transformation into S. erythraea LB1, which lacks
almost the entire erythromycin biosynthetic gene cluster (data not
shown). Addition of exogenous 3-O-a-L-mycarosylerythronolide
B (13) resulted in quantitative biotransformation to erythromycin
C as anticipated.
As described in this paper, all three GTs studied displayed
significant substrate flexibility and utilized both ‘non-native’ TDP-
deoxyhexose (donor) and polyketide aglycone (acceptor) sub-
strates. TylMII and AngMII were both able to accept and transfer
TDP-D-angolosamine to tylactone (11). TylMII has previously
been shown to efficiently transfer D-desosamine to 11.5 Thus
TDP-D-mycaminose (4), TDP-D-desosamine (7) and TDP-D-
angolosamine (9) are all effective deoxysugar donor substrates for
this enzyme. In addition, we found that TylMII is able to transfer
non-amino L-configured deoxyhexoses to 11 (Fig. 7). During our
investigations of biosynthetic gene cassette assembly we expressed
tylMII in a background lacking the aminotransferase angB. When
exogenous 11 was added to this strain, several novel compounds
were produced. Interestingly, two of these were only produced
when the GT helper gene tylMIII was co-expressed with tylMII,
even though the counterpart gene eryCII from the erythromycin
biosynthetic gene cluster was present in the genome. The total
bioconversion yield of these experiments was between 20–50% of
the added material at an analytical scale. After scale-up biotrans-
formation, 5 mg of the major new compound was isolated and
detailed NMR analysis confirmed it as 5-O-a-L-rhamosyltylactone
(23) (see experimental section). The MS spectrum of the closely
eluting minor new metabolite was identical to that of 23 and
most likely an analogue with a 6-deoxyhexose moiety attached
at C5 (possibly D-quinovose although sufficient material was not
obtained for complete characterization). The strain also produced
the previously reported 5-O-b-D-glucosyltylactone, which arises
through the action of an uncharacterized host GT.5 The TDP-L-
rhamnose required for 23 biosynthesis is believed to arise from
the background metabolism of the S. erythraea mutant host
as described previously.5,30 The synthesis of 23 was reported
previously during an in vitro study concerning the substrate
flexibility of the DesVII/DesVIII GT pairing.13
In addition to TDP-L-rhamnose, TylMII is also capable of
utilizing TDP-L-mycarose to make 5-O-a-L-mycarosyltylactone
(24) (Fig. 7). This material was observed at low levels in several
biotransformation experiments when tylactone (11) was fed to S.
erythraea mutants competent in TDP-L-mycarose (but not TDP-D-
desosamine) biosynthesis and which contained tylMII and either
tylMIII or angMIII. We were able to confirm the presence of
24 through the use of LCMS analysis and comparison to an
authentic sample.27 5-O-a-L-Mycarosyltylactone (24) is a known
and significant product, along with tylactone (11), of S. fradiae
mutants deficient in TDP-D-mycaminose biosynthesis.27 Thus,
TylMII shows a remarkably broad substrate tolerance towards
its deoxysugar donor substrate.
In addition to its natural 16-membered aglycone (acceptor)
substrate 11, AngMII was able to transfer D-angolosamine
to the C3-hydroxyl group of the 14-membered macrolide ery-
thronolide B (16). The corresponding activity has also been
observed for TylMII, in which an S. fradiae mutant blocked
in tylactone production31 biotransformed 16 to give 3-O-b-D-
mycaminosylerythronolide B (unpublished results32). The ability
of macrolide GTs to act on structurally related aglycones of
varying size may be a general phenomenon, and is a notable char-
acteristic of DesVII, which naturally transfers D-desosamine to
methonolide and narbonolide (12- and 14-membered aglycones re-
spectively) during methymycin/pikromycin biosynthesis.12 DesVII
can also glycosylate the 16-membered tylactone 11 both in vitro13
and in vivo.33
The biosynthetic gene cassettes for TDP-D-mycaminose (4) and
TDP-D-angolosamine (9) were also used in combination with
eryCIII. We were gratified to observe that EryCIII transferred
both of these D-deoxyaminohexoses to its native substrate 3-
O-a-L-mycarosylerythronolide B (13), although the transfer was
inefficient. These new macrolides were not produced in suffi-
cient yield for NMR characterization but LCMS/MS data was
consistent with the production of the anticipated erythromycin
analogues. As the biosynthetic gene cassettes utilized had been
shown to be efficient for the production of their respective
D-deoxyhexoses, we speculated that EryCIII may have limited
tolerance towards these substrates. Surprisingly, in a related
experiment we were able to produce the D-mycaminose-containing
erythromycin derivative 19 in an improved, albeit still modest,
yield. In this instance, a biosynthetic gene cassette was prepared
which contained the majority of the genes required for TDP-
D-angolosomine (9) biosynthesis, but lacked the TDP-4-keto-6-
deoxyhexose-2,3-dehydratase-encoding gene (neither ang-ORF4
Fig. 7 Structure of 5-O-a-L-rhamnosyltylactone (23) and 5-O-a-L-
mycarosyltylactone (24).
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nor the heterologous spnO gene were included). This was trans-
formed into S. erythraea SGQ2, which contains eryBVI, the native
TDP-4-keto-6-deoxyhexose-2,3-dehydratase required for TDP-L-
mycarose biosynthesis, and this cassette also contained eryCIII.
This mutant was examined for its ability (or not) to biosynthesise 9.
When 13 was added to fermentations we were surprised to observe
reasonable bioconversion to give novel erythromycins with an
LCMS/MS profile consistent with D-mycaminosylated products.
After scale-up and purification the major new product was shown
to have a structure consistent with 19. This result was surprising
as the mutant does not contain a 3,4-ketoisomerase activity which
is believed to be essential for TDP-D-mycaminose production.
No metabolites containing a D-angolosamine moiety could be
observed, and the host strain itself was not capable of producing
19 when fed 13. We have no current explanation for this result
but did observe that while a mutant transformed with an identical
gene cassette lacking only ang-ORF14, a TDP-deoxyhexose C4-
ketoreductase, was also able to produce 19, its biotransformation
capacity was much reduced.
The ability of EryCIII to utilize TDP-D-mycaminose (4) as a
substrate in vitro has been reported elsewhere, although the activity
with this substrate appeared significantly lower than with the
native TDP-D-desosamine (7).28 The tolerance of EryCIII towards
alternative deoxyhexose templates appears relatively broad as we
also demonstrated its ability to utilize TDP-D-angolosamine (9),
and, surprisingly, a TDP-L-deoxyhexose (TDP-L-mycarose) and
convert 3-O-a-L-mycarosylerythronolide B (13) to 3,5-bis-O-a-L-
mycarosylerythronolide B (20) when expressed in BIOT-2191. This
is consistent with observations that 20 is a direct fermentation
product of an S. erythraea strain disrupted in the biosynthesis of
TDP-D-desosamine.18
Experimental
General methods
All solvents used were HPLC grade. LCMS/MS analysis was
performed on an Agilent HP1100 HPLC system in combination
with a Bruker Daltonics Esquire 3000+ ion trap mass spectrometer
fitted with an electrospray source. The MS was operated in both
positive and negative ion modes. UV analysis was performed at
210, 258 and 280 nm on an Agilent DAD detector. High resolution
mass spectra were acquired on a Bruker BioApex II FTICR mass
spectrometer. NMR spectra were recorded on a Bruker Avance 500
spectrometer at 298 K operating at 500 MHz and 125 MHz for 1H
and 13C respectively. Standard Bruker pulse programs were used
1
to acquire the H–1H COSY, APT, HMQC and HMBC spectra.
Coupling constants are given in Hertz. NMR experiments were
referenced to the residual resonance of the solvent.
Escherichia coli XL1-Blue MR (Stratagene), E. coli DH10B
(GibcoBRL) and E. coli ET1256737 were grown in 2TY medium as
described elsewhere.38 Vectors pUC18, pUC19 and Litmus 28 were
obtained from New England Biolabs. E. coli transformants were
selected with ampicillin (100 lg cm−3). S. erythraea NRRL2338
strains were cultured as described previously20 unless expressly
described otherwise below. Expression vectors in S. erythraea were
derived from plasmid pSG142.7 Plasmid-containing S. erythraea
were selected with thiostrepton (40 lg cm−3) or apramycin
(50 lg cm−3). DNA manipulations, PCR and electroporation
procedures were carried out using standard procedures.38 Proto-
plast formation and transformation procedures of S. erythraea
were as described previously.10,20 Southern hybridizations were
carried out with probes labelled with digoxigenin using the DIG
DNA labelling kit (Boehringer, Mannheim). DNA sequencing was
performed in the Department of Biochemistry DNA Sequencing
Facility at the University of Cambridge. An Applied Biosystems
800 molecular biology CATALYST robot was used to apply Taq
dideoxy terminator sequencing reactions (Big Dye Terminator
kit, ABI) to an ABI 373A sequencer according to manufacturers’
protocols.
This apparent general ability of macrolide GTs to accept
both TDP-D-deoxyaminohexoses and TDP-L-deoxyhexoses was
highlighted by us previously during studies of SpnP, a GT whose
native deoxysugar substrate is TDP-D-forosamine (10) during
spinosyn (3) biosynthesis.6 SpnP was shown to accept TDP-
L-mycarose when expressed in a S. erythraea mutant and to
produce novel spinosyn analogues. We proposed that SpnP may
recognize the 4C1 conformation of TDP-L-mycarose in which
the hexose ring ‘flips’ to an alternative conformation where
the b-TDP moiety sits in an axial conformation and would
be more readily recognized by SpnP (D-deoxyhexoses favour
Cloning and sequencing of the angolamycin (8) biosynthetic gene
cluster
S. eurythermus ATCC23956 was propagated in tryptic soy broth
(TSB; Difco) at 30 ◦C. Supercos (Stratagene) was used for
construction of the cosmid library and transfections were per-
formed in E. coli XL-Blue MR (Stratagene USA). Sub-cloning
from recombinant cosmids was into plasmid pSG39739 and
recombinants were selected by growth in media supplemented with
chloramphenicol (50 lg cm−3).
Total DNA was isolated from S. eurythermus by ‘Procedure B’
from the John Innes Streptomyces manual.40 For the generation
of the cosmid library, total DNA was partially digested with
Sau3A, dephosphorylated with shrimp alkaline phosphatase,
ligated directly into pSuperCos and packaged with Gigapack
Gold packaging extract (Stratagene) without size fractiona-
tion. All procedures were performed in accordance with the
manufacturer’s recommendations. Identification of the putative
angolamycin biosynthetic cluster was by direct sequencing of
cosmid DNA. Briefly, 768 colonies of the S. eurythermus cosmid
library were individually grown in TSB cultures (1 cm3), shaken
4
the C1 conformation). Similar arguments have been forwarded
when discussing StaG required for staurosporine biosynthesis,
which also accepts L- and D-deoxyhexoses.34 Further publications
demonstrate that GTs in other engineered systems will accept both
NDP-D- and L-deoxyhexose substrates; notably ElmGT involved
in elloramycin biosynthesis35 and VinC involved in vicenistatin
biosynthesis.36
In summary, a procedure for the generation of biosynthetic
gene cassettes targeted towards the production of important D-
deoxyaminohexoses has been described. Such gene cassettes can be
expressed in appropriate heterologous hosts and, in combination
with macrolide GTs and their ancillary proteins. The resulting
organisms can be utilized as effective tools for the generation
of hybrid macrolide antibiotics. The macrolide GTs EryCIII,
AngMII and TylMII all exhibit usefully broad substrate tolerance,
accepting a range of TDP-D- and L-deoxyhexose donors and
several acceptor substrates.
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in eight 96-well plates for 24 h at 37 ◦C. An aliquot of each
culture (0.05 cm3) was transferred to corresponding labelled wells
on a 96-well microtitre plate using a multi-channel pipette, and
stored at −80 ◦C after addition of an equal volume of 40%
glycerol (v/v). These stock suspensions were later used to inoculate
larger volumes of culture once individual target clones had been
identified from sequence data. Small scale DNA preparations
were made from the remainder of each culture (0.9 cm3; done
as recommended by ABI) and the sequence of the ends of each
insert was obtained using T3 and T7 primers as described below.
Six clones were identified by BLAST searching with very high
sequence homology to the genes of the tylosin biosynthetic cluster.
Liquid cultures of these clones were grown in 2 × LB medium
with appropriate antibiotic selection, and cosmid DNA was
prepared from them using Qiagen midi-prep DNA purification
kits. The complete DNA sequence of four of these cosmids,
designated 2C3, 2D8, 4h12 and 5B2 respectively, was obtained
by assembly of overlapping Sau3A fragments obtained from
partial digestion of the parent cosmid. Briefly, DNA fragments
of 2–5 kbp from the Sau3A digest were eluted from an agarose
gel using the Gene Clean kit (Bio101, BioRad) and sub-cloned
into pSG397. Remaining sequence gaps were filled using 21mer
synthetic oligonucleotides as primers. Plasmid sequencing was
perfomed using pUC forward and reverse primers. SeqEd v 1.0.3
was used for sequence editing. Sequence assembly employed the
GAP (Genome Assembly Program) version 4.2.41 DNA sequence
translations were performed with the GCG software package.
Database searches used the BLAST algorithm.
Extraction and purification of new macrolides
The following general procedure was used unless described
otherwise below. The biotransformation broth was clarified by
centrifugation. The supernatant was adjusted to pH 9.0 (5 M
NaOH) and extracted three times with an equal volume of ethyl
acetate. The cell pellet was extracted twice with an equal volume
of acetone–methanol (1 : 1). The organic extracts were combined
and the solvents removed under reduced pressure. The resulting
aqueous fraction was extracted three times with an equal volume
of ethyl acetate and the combined organic solvent removed under
reduced pressure to leave a crude oily extract. This was dissolved
in methanol and chromatographed over base-deactivated Luna
C18 reversed-phase silica (5 micron particle size) using a Luna
HPLC column (250 × 21 mm; Phenomenex (Macclesfield, UK)).
A Gilson 315 binary HPLC system was used to deliver a linear
gradient of CH3CN–H2O (3 : 7) containing 10 mM ammonium
acetate to CH3CN–H2O (7 : 3) containing 10 mM ammonium
acetate at 21 cm3 min−1 over 20 min.
Production and isolation of 3,5-bis-O-a-L-mycarosylerythronolide
B (20)
S. erythraea SGQ2/pSGeryCIII (BIOT-2191) was grown initially
in TSB and this seed then used to inoculate SSDM medium (4 dm3;
5% inoculum) in a Applikon 7 dm3 bioreactor (30 ◦C; initial
pH 6.0–6.4; Rushton turbine agitator, tip speed 0.9–2.7 m s−1;
airflow 0.75 L L−1 min−1). 13 (100 mg, 0.183 mmol) dissolved in
methanol (1 cm3) was added after 24 h. After a further 46–68 h
the fermentation was harvested and clarified by centrifugation.
The supernatant was applied to a column (16 × 15 cm) of
Analytical chemistry
R
Diaionꢀ HP20 resin (Supelco), washed with 10% acetone in water
An aliquot of fermentation broth was shaken vigorously with
an equal volume of methanol (20 min) and then clarified by
centrifugation. Supernatants were analysed by LCMS/MS and
chromatography was achieved over base-deactivated Luna C18
reversed-phase silica (5 micron particle size) using a Luna HPLC
column (250 × 4.6 mm; Phenomenex (Macclesfield, UK)) heated
at 40 ◦C. Gradient elution was from 25% mobile phase B to 75%
mobile phase B over 19 min at a flow rate of 1 cm3 min−1. Mobile
phase A was CH3CN–H2O (1 : 9), containing 10 mM ammonium
acetate and 0.15% formic acid; mobile phase B was CH3CN–H2O
(9 : 1), containing 10 mM ammonium acetate and 0.15% formic
acid.
(2 × 2 dm3), and then eluted with acetone (3.5 dm3). The cells
were extracted twice with an equal volume of acetone–methanol
(1 : 1). The organic extracts were combined and the solvents
removed under reduced pressure. The resulting aqueous fraction
was extracted three times with an equal volume of ethyl acetate
and the combined organic solvent removed under reduced pressure
to leave a crude oily extract. This was dissolved in methanol and
chromatographed over base-deactivated Luna C18 reversed-phase
silica (5 micron particle size) using a Luna HPLC column (250 ×
21 mm; Phenomenex (Macclesfield, UK)). A Gilson 315 binary
HPLC system was used to deliver the mobile phase at 21 cm3 min−1.
Elution involved a linear gradient of 32.5% B to 63% B was used
to initially to partially purify the sample. Fractions containing 20
were combined and finally purified by isocratic elution with 30%
B. Mobile phase A was 20 mM ammonium acetate and mobile
phase B was CH3CN.
General procedure for analytical scale biotransformation
This was based on published procedures.20,42
General procedure for scale up of biotransformations
3,5-Bis-O-a-L-mycarosylerythronolide B (20)
S. erythraea strains were grown in TSB seed medium and then
sucrose–succinate (SSDM) production media using protocols
described previously.20,42 For a typical experiment the production
stage consisted of Erlenmeyer flasks (2 dm3) each containing
400 cm3 of SSDM medium (5% inoculum from seed). Substrate
aglycones were added as methanol solutions to achieve a final
concentration of 50 mg cm−3. These were added after the
production medium had been growing for 24 h.
Isolated yield, 15.8% (20 mg, 0.029 mmol). dH (500 MHz; CD3OD)
0.88 (dd, J 7.5 & 7.3, C(15)Me), 0.92 (d, J 7.2, C(12)Me), 0.97 (d,
J 7.0, C(10)Me), 1.01 (d, J 7.7, C(4)Me), 1.12 (d, J 7.0, C(8)Me),
1.20 (d, J 7.0, C(2)Me), 1.20 (s, C(3ꢀꢀ)Me), 1.22 (s, C(3ꢀ)Me), 1.23
(d, J 6.4, C(6ꢀꢀ)Me), 1.29 (d, J 6.2, C(6ꢀ)Me), 1.38 (dd, J 14.9
& 4.1, C(7a)H), 1.45 (s, C(6)Me), 1.49 (dqd, J 17.1, 7.5 & 4.5,
C(14a)H), 1.64 (dqd, J 10.2, 7.2, 1.0, C(12)H), 1.74 (ddq, J 17.1,
9.8 & 7.3, C(14b)H), 1.81 (dd, J 14.5 & 4.4, C(2ꢀꢀa)H), 1.86 (dd, J
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14.5 & 4.6, C(2ꢀa)H), 2.00 (dd, J 14.9 & 9.0, C(7b)H), 2.05 (dd, J
14.5 & 1.1, C(2ꢀꢀb)H), 2.12 (dd, J 14.5 & 1.5, C(2ꢀb)H), 2.20 (qdd,
J 7.7, 7.3 & 1.4, C(4)H), 2.78 (m, C(8)H), 2.93 (dq, J 10.2 & 7.0,
C(2)H), 2.97 (d, J 9.4, C(4ꢀ)H), 2.97 (d, J 9.8, C(4ꢀꢀ)H), 2.99 (qd,
J 7.0 & 1.5, C(10)H), 3.63 (d, J 7.3, C(5)H), 3.92 (dq, J 9.8 &
6.4, C(5ꢀꢀ)H), 3.94 (dd, J 10.2 & 1.5, C(11)H), 3.97 (dd, J 10.2 &
1.4, C(3)H), 4.09 (dq, J 9.4 & 6.2, C(5ꢀ)H), 4.98 (d, J 4.6, C(1ꢀ)H),
5.08 (d, J 4.4, C(1ꢀꢀ)H), 5.38 (ddd, J 9.8, 4.5 & 1.0, C(13)H); dC
(125 MHz; CDCl3) 9.5 (C10-Me), 9.8 (C12-Me), 10.4 (C4-Me),
11.0 (C15), 16.5 (C2-Me), 18.2 (C6ꢀꢀ), 18.8 (C8-Me), 19.1 (C6ꢀ),
26.5 (C7ꢀꢀ), 26.8 (C7ꢀ), 27.0 (C14), 27.8 (C6-Me), 39.3 (C7), 39.7
(C4), 41.2 (C10), 41.8 (C2ꢀꢀ), 41.8 (Cꢀ2), 42.1 (C12), 44.8 (C8), 46.1
(C2), 67.0 (C5ꢀꢀ), 67.3 (C5ꢀ), 70.5 (C3ꢀ), 70.8 (C11), 71.3 (C3ꢀꢀ), 74.7
(C6), 76.2 (C13), 77.7 (C4ꢀꢀ), 77.9 (C4ꢀ), 84.8 (C3), 87.4 (C5), 99.0
(C1), 100.5 (C1ꢀꢀ), 177.0 (C1), 220.4 (C9); MS (ES) m/z 1403.1
[2M + Na]+, 713.3 [M + Na]+, 672.7 [M − H2O + H]+, 529 [M −
H2O − mycarose + H]+.
C(6)Me), 1.34 (d, J 6.0, C(6ꢀ)H), 1.44 (dd, J 14.6 & 5.4, C(7a)H),
1.49 (m, C(2ꢀa)H), 1.51 (m, C(14a)H), 1.69 (m, C(12)H), 1.71 (qd,
J 7.2 & 2.2, C(14b)H), 1.92 (dd, J 14.6 & 5.4, C(7b)H), 1.99 (m
C(4)H), 2.00 (m, C(2ꢀb)H), 2.48 (td, J 10.2 & 3.5, C(3ꢀ)H), 2.69 (m,
C(8)H), 2.81 (dq, J 10.5 & 6.7, C(2)H), 2.91 (bq, J 6.6, C(10)H),
3.03 (dd, J 9.5 & 9.5, C(4ꢀ)H), 3.34 (dq, J 8.7 & 6.0, C(5ꢀ)H),
3.66 (dd, J 10.5 & 10.5, C(3)H), 3.69 (bs, C(5)H), 3.78 (d, J 10.0,
C(11)H), 4.61 (dd, J 9.2 & 1.6, C(1ꢀ)H), 5.40 (dd, J 9.5 & 9.3,
C(13)H); dC (125 MHz; CDCl3) 8.3 (C4-Me), 8.5 (C10-Me), 9.1
(C15), 10.4 (C12-Me), 15.2 (C2-Me), 16.9 (C8-Me), 17.5 (C6ꢀ),
26.6 (C6-Me), 25.8 (C14), 27.0 (C2ꢀ), 36.5 (C4), 38.3 (C7), 40.1
(C10), 40.2 (C12), 43.4 (C8), 44.5 (C2), 65.2 (C3ꢀ), 70.3 (C4ꢀ), 70.6
(C11), 73.9 (C5ꢀ), 75.2 (C6), 75.6 (C13), 81.5 (C5), 89.7 (C3), 103.0
(C1ꢀ), 176.3 (C1), 217.8 (C9); MS (ES) m/z 582.5 [M + Na]+,
560.5 [M + H]+, 425.3 [M − ang + Na]+, 158.0 [ang − OH]+: (ang,
angolosamine).
5-Des-O-b-D-desosaminyl-5-O-b-D-angolosaminylerythromycin A
(18)
5-O-b-D-Mycaminosyltylactone (12)
The identity of this compound was verified versus an authentic
standard.27 kmax(DAD)/nm 281; MS (ES) m/z 568.4 [M + H]+,
550.3 [M − H2O + H]+, 411.4 [M − myc + H]+, 174.0 [myc −
OH]+: (myc, mycaminose).
MS (ES) m/z 734.4 [M + H]+, 716.5 [M − H2O + H]+, 576.4
[M − cla + H]+, 558.3 [M − cla − H2O + H]+, 158.2 [ang −
OH]+: (cla, cladinose; ang, angolosamine). Additional peaks for
the erythromycin B analogue and other minor components were
also observed.
5-O-b-D-Angolosaminyltylactone (14)
Isolated yield, 17.9% (20 mg, 0.036 mmol). This was pu-
rified from 1.6 dm3 of biotransformation broth (80 mg
(0.203 mmol) of 11 was used as substrate). kmax(DAD)/nm 281;
dH (500 MHz; CDCl3) 0.83 (t, J 7.2, C(6)CH2CH3), 0.91 (d
J 7.2, C(4)Me), 0.91 (t, J 7.2, C(17)H), 1.05 (d, J 6.5, C(14)Me),
1.15 (d, J 6.8, C(8)Me), 1.30 (d, J 6.0, C(6ꢀ)H), 1.45 (m, C(7a)H),
1.48 (m, C(2ꢀa)H), 1.55 (m, C(7b)H), 1.55 (m, C(16a)H), 1.55
(m, C(6)CH2CH3), 1.56 (m, C(4)H), 1.76 (s, C(12)Me), 1.82 (m,
C(16b)H), 1.91 (d, J 16.8, C(2a)H), 2.05 (ddd, J 10.4, 3.9 & 1.6,
C(2ꢀb)H), 2.46 (dd, J 16.8 & 10.5, C(2b)H), 2.48 (s, C(3ꢀꢀ)NMe2),
2.68 (m, C(6)H), 2.70 (m, C(8)H), 2.70 (m, C(14)H), 2.89 (td,
J 10.4 & 3.9, H(3ꢀ)H), 3.16 (dd, J 9.6 & 9.0, C(4ꢀ)H), 3.26 (dq,
J 9.6 & 6.0, C(5ꢀ)H), 3.68 (dd, J 10.5 & 1.2, C(3)H), 3.76 (d,
J 10.3, C(5)H), 4.41 (d, J 8.6, C(1ꢀ)H), 4.68 (td, J 9.7 & 2.4,
C(15)H), 5.60 (d, J 10.4, C(13)H), 6.26 (d, J 15.5, C(10)H), 7.27
(d, J 15.5, C(11)H); dC (125 MHz; CDCl3) 9.4 (C17), 9.7 (C4-Me),
11.8 (C6-CH2CH3), 13.0 (C12-Me), 16.1 (C14-Me), 17.1 (C8-Me),
17.7 (C6ꢀ), 21.0 (C6-CH2CH3), 24.7 (C16), 28.0 (C2ꢀ), 33.6 (C7),
38.3 (C14), 38.7 (C6), 39.4 (C3ꢀꢀ-NMe2), 39.8 (C2), 40.4 (C4), 45.0
(C8), 65.8 (C3ꢀ), 66.9 (C3), 70.5 (C4ꢀ), 73.2 (C5ꢀ), 78.8 (C15), 80.7
(C5), 101.0 (C1ꢀ), 118.3 (C10), 133.5 (C12), 145.4 (C13), 157.7
(C11), 174.4 (C1), 203.9 (C9); MS (ES) m/z 552.3 [M + H]+, 534.3
[M − H2O + H]+, 395.4 [M − ang + H]+, 158.2 [ang − OH]+: (ang,
angolosamine).
5-Des-O-b-D-desosaminyl-5-O-b-D-mycaminosylerythromycin A
(19)
Isolated yield, 7.1% (15 mg, 0.013 mmol). This was purified from
4 dm3 of biotransformation broth (100 mg (0.183 mmol) of 13 was
used as substrate). dH (500 MHz; CDCl3) 0.83 (dd, J 7.4 & 7.4,
C(15)H), 1.03 (d, J 7.4, C(4)Me), 1.12 (s, C(12)Me), 1.14 (d, J 7.0,
C(10)Me), 1.16 (d, J 7.0, C(8)Me), 1.18 (d, J 7.1, C(2)Me), 1.23 (s,
C(3ꢀ)Me), 1.27 (d, J 6.2, (C(6ꢀ)H), 1.29 (d J 6.1, C(6ꢀꢀ)H), 1.44 (s,
C(6)Me), 1.47 (dqd, J 14.3, 11.0 & 7.2, C(14b)), 1.55 (dd, J 15.2
& 4.8, C(2ꢀb)H), 1.66 (dd, J 14.8 & 2.2, C(7a)H), 1.82 (dd, J 14.8
& 11.4, C(7b)H), 1.91 (ddq, J, 14.3, 7.5, & 2.2, C(14a)H), 2.00 (m,
C(4)H), 2.32 (dd, J 15.2 & 0.9, C(2ꢀb)H), 2.48 (dd, J 10.4 & 10.3,
C(3ꢀꢀ)H), 2.58 (s, C(3ꢀꢀ)NMe2), 2.69 (dqd, J 11.3, 7.0 & 2.2, C(8)H),
2.83 (dq, J 9.7 & 7.1, C(2)H), 3.01 (d, J 9.3, C(4ꢀ)H), 3.06 (qd, J 6.9
& 1.3, C(10)H), 3.09 (dd, J 9.9 & 9.0, C(4ꢀꢀ)H), 3.29 (s, C3ꢀ)OMe),
3.31 (dq, J 9.0 & 6.1, C(5ꢀꢀ)H), 3.53 (d, J 6.8, C(5)H), 3.56 (dd,
J 10.4 & 7.3, C(2ꢀꢀ)H), 3.81 (d, J 1.3, C(11)H), 3.91 (dd, J 9.7 & 1.6,
C(3)H), 3.99 (dq, J 9.3 & 6.2, C(5ꢀ)H), 4.43 (d, J 7.4, C(1ꢀꢀ)H), 4.87
(d, J 4.8, C(1ꢀ)H), 5.04 (dd, J 11.0 & 2.3, C(13)H); dC (125 MHz;
CDCl3) 9.7 (C4-Me), 10.6 (C15), 12.0 (C10-Me), 16.0 (C2-Me),
16.2 (C12-Me), 18.1 (C6ꢀꢀ), 18.3 (C8-Me), 18.5 (C6ꢀ), 21.1 (C14),
21.4 (C3ꢀ-Me), 26.6 (C6-Me), 34.9 (C2ꢀ), 38.0 (C10), 38.5 (C7),
39.1 (C4), 41.7 (C3ꢀꢀ-NMe2), 44.9 (C2), 44.9 (C8), 49.4 (C3ꢀ-OMe),
65.6 (C5ꢀ), 68.9 (C11), 70.2 (C4ꢀꢀ), 70.6 (C3ꢀꢀ), 71.3 (C2ꢀꢀ), 72.8 (C3ꢀ),
72.9 (C5ꢀꢀ), 74.6 (C12), 74.8 (C6), 76.8 (C13*), 77.8 (C4ꢀ), 80.0 (C3),
85.4 (C5), 96.4 (C1ꢀ), 103.3 (C1ꢀꢀ), 175.4 (C1), 221.6 (C9). (* This
carbon was assigned from the HMQC spectrum); MS (ES) m/z
750.5 [M + H]+, 732.5 [M − H2O + H]+, 592.4 [M − cla + H]+, 574
[M − cla − H2O + H]+, 174.1 [myc − OH]+: (cla, cladinose; myc,
mycaminose); HRMS (ES) m/z 750.4654 calcd for C37H68NO14
requires 750.4634.
3-O-b-D-Angolosaminylerythronolide B (17)
Isolated yield, 36% (30 mg, 0.054 mmol). This was purified from
1.2 dm3 of biotransformation broth (60 mg (0.149 mmol) of 11 was
used as substrate). dH (500 MHz; CDCl3) 0.89 (d, J 7.7, C(12)Me),
0.90 (d, J 7.7, C(15)H), 0.98 (d, J 7.7, C(10)Me), 1.06 (d, J 6.7,
C(4)Me), 1.16 (d, J 6.1, C(8)Me), 1.19 (d, J 6.9, C(2)Me), 1.30 (s,
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5-O-a-L-Rhamnosyltylactone (23)
8 S. Gaisser, R. Lill, G. Wirtz, F. Grolle, J. Staunton and P. F. Leadlay,
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11 L. Zhao, S. Borisova, S.-M. Yeung and H.-W. Liu, J. Am. Chem. Soc.,
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13 S. A. Borisova, C. Zhang, H. Takahashi, H. Zhang, A. W. Wong,
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2753.
14 C. Leimkuhler, M. Fridman, T. Lupoli, S. Walker, C. T. Walsh and D.
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18 PCT Appl. WO 054265, 2005.
Isolated yield, 5 mg. kmax(DAD)/nm 281; dH (500 MHz; CDCl3)
0.89 (t, J 7.5, C(6)CH2CH3), 0.91 (d, J 6.8, C(4)Me), 0.92 (t, J 7.4,
C(17)H), 1.05 (d, J 6.5, C(14)Me), 1.19 (d, J 6.8, C(8)Me), 1.25
(m, C(6)CH2CH3), 1.26 (d, J 6.0, C(6ꢀ)H), 1.53 (m, C(16a)H),
1.55 (m, C(4)H), 1.77 (s, C(12)Me), 1.83 (ddd, J 14.3, 7.4 & 2.5,
C(16b)H), 1.91 (d, J 16.8, C(2a)H), 2.45 (dd, J 16.8 & 10.3,
C(2b)H), 2.70 (m, C(8)H), 2.70 (m, C(14)H), 3.46 (dd, J 9.4 &
9.4, C(4ꢀ)H), 3.67 (m C(3)H), 3.68 (dd, J 9.4 & 3.1, C(3ꢀ)H),
3.74 (m, C(5)H), 3.76 (d, J 9.4, C(5ꢀ)H), 4.03 (dd, J 3.1 & 1.5,
C(2ꢀ)H), 4.68 (td, J 8.9, C(15)H), 4.73 (bs, C(1ꢀ)H), 5.61 (d, J 10.3,
C(13)H), 6.26 (d, 13.7, C(10)H), 7.28 (d, J 13.7, C(12)H); dC
(125 MHz; CDCl3) 9.5 (C17), 9.5 (C4-Me), 12.1 (C6-CH2CH3),
12.9 (C12-Me), 16.1 (C14-Me), 17.2 (C8-Me), 17.2 (C6ꢀ), 21.9 (C6-
CH2CH3), 24.7 (C16), 33.8 (C7), 38.4 (C14), 38.7 (C6), 39.8 (C2),
40.8 (C4), 44.9 (C8), 67.0 (C3), 68.6 (C5ꢀ), 71.1 (C2ꢀ), 71.9 (C3ꢀ),
73.1 (C4ꢀ), 79.2 (C15), 83.4 (C5), 102.6 (C1ꢀ), 118.4 (C10), 136.7
(C12), 145.5 (C13), 148.1 (C11), 174.3 (C1), 204.0 (C9); MS (ES)
m/z 563.5 [M + Na]+, 545.4 [M − H2O + Na]+, 579.5 [M + K]+,
561.5 [M − H2O + K]+.
19 R. Corbaz, L. Ettlinger, E. Ga¨umann, W. Keller-Schierlein, L. Neipp,
V. Prelog, P. Reusser and H. Za¨hner, Helv. Chim. Acta, 1955, 38, 1202–
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5-O-a-L-Mycarosyltylactone (24)
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21 C. Waldron, P. Matsushima, P. R. Rosteck Jr., M. C. Broughton, J.
Turner, K. Madduri, K. P. Crawford, D. J. Merlo and R. H. Baltz,
Chem. Biol., 2001, 8, 487–499.
The identity of this compound was verified versus an authentic
standard.27 kmax(DAD)/nm 281; MS (ES) m/z 561.5 [M + Na]+,
543.5 [M − H2O + Na]+.
22 S. F. Haydock, J. F. Aparicio, I. Molnar, T. Schwecke, L. E. Khaw,
A. Ko¨nig, A. F. A. Marsden, I. S. Galloway, J. Staunton and P. F.
Leadlay, FEBS Lett., 1995, 374, 246–248; C. D. Reeves, S. Murli,
G. W. Ashley, M. Piagentini, C. R. Hutchinson and R. McDaniel,
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Kendrew, L. Low, B. Wilkinson, R. Lill, J. Cortes, B. A. M. Rudd, J.
Staunton and P. F. Leadlay, J. Ind. Microbiol. Biotechnol., 2003, 30, 489–
494.
23 T. Bililign, C.-G. Hyun, J. S. Williams, A. M. Czisny and J. S. Thorson,
Chem. Biol., 2004, 11, 959–969.
24 K. Ichinose, M. Ozawa, K. Itou, K. Kunieda and Y. Ebizuka,
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25 PCT., WO007709, 2004;M. A. Gregory, H. Petkovic, R. E. Lill, S. J.
Moss, B. Wilkinson, S. Gaisser, P. F. Leadlay and R. M. Sheridan,
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R. E. Lill, S. Gaisser, H. Petkovic, L. Low, L. S. Sheehan, I. Carletti,
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Acknowledgements
Professor Eric Cundliffe (University of Leicester) is acknowl-
edged for numerous stimulating discussions regarding this
work. We thank the staff of the DNA sequencing facil-
ity at the Department of Biochemistry, University of Cam-
bridge. Tylactone, 5-O-b-D-mycaminosytylactone, 23-hydroxy-
5-O-b-D-mycaminosyltylactone and 5-O-a-L-mycarosyltylactone
were kind gifts of the ex-Bioprocess Research Group of
GlaxoWellcome (now GSK).23,27 Erythronolide B and 3-O-a-L-
mycarosylerythronolide B were kind gifts of Pfizer (Groton, USA).
The work was supported in part by a Marie Curie Industry Host
Fellowship to Biotica from the European Community (TORINO,
QLK2-CT-2001–60023), and a project grant to P.F.L. from the
European Community (GENOVA, QLRT-1999–00095).
26 L. Hong, Z. Zhao, C. E. Melanc¸on III, H. Zhang and H.–W. Liu, J. Am.
Chem. Soc., 2008, 130, 4954–4967.
27 Authentic tylactone, 5-O-b-D-mycaminosyltylactone, 23-hydroxy-5-O-
b-D-mycaminosyltylactone and 5-O-a-L-mycarosyltylactone were ob-
tained following patent procedures: US Pat. 4423148, 1983; US. Pat.
4440857, 1984.
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