Definition of the common and divergent steps in carbapenem β-lactam antibiotic biosynthesis

Article abstract of DOI:10.1002/cbic.201100366

Approximately 50 naturally occurring carbapenem β-lactam antibiotics are known. All but one of these have been isolated from Streptomyces species and are disubstituted structural variants of a simple core that is synthesized by Pectobacterium carotovorum

Full text of DOI:10.1002/cbic.201100366

DOI: 10.1002/cbic.201100366
Definition of the Common and Divergent Steps in
Carbapenem b-Lactam Antibiotic Biosynthesis
Micah J. Bodner, Rongfeng Li, Ryan M. Phelan, Michael F. Freeman, Kristos A. Moshos,
Evan P. Lloyd, and Craig A. Townsend*[a]
Approximately 50 naturally occurring carbapenem b-lactam an-
tibiotics are known. All but one of these have been isolated
from Streptomyces species and are disubstituted structural var-
iants of a simple core that is synthesized by Pectobacterium
carotovorum (Erwinia carotovora), a phylogenetically distant
plant pathogen. While the biosynthesis of the simple carbape-
nem, (5R)-carbapen-2-em-3-carboxylic acid, is impressively effi-
cient requiring only three enzymes, CarA, CarB and CarC, the
formation of thienamycin, one of the former group of metabo-
lites from Streptomyces, is markedly more complex. Despite
their phylogenetic separation, bioinformatic analysis of the en-
coding gene clusters suggests that the two pathways could be
related. Here we demonstrate with gene swapping, stereo-
chemical and kinetics experiments that CarB and CarA and
their S. cattleya orthologues, ThnE and ThnM, respectively, are
functionally and stereochemically equivalent, although their
catalytic efficiencies differ. The biosynthetic pathways, there-
fore, to thienamycin, and likely to the other disubstituted car-
bapenems, and to the simplest carbapenem, (5R)-carbapen-2-
em-3-carboxylic acid, are initiated in the same manner, but
share only two common steps before diverging.
Introduction
While semisynthetic penicillins and cephalosporins remain fre-
quently prescribed drugs in human medicine, the carbapenem
b-lactam antibiotics have assumed growing importance for
their broad-spectrum activity, potency and effectiveness
against resistant infections.[1] The family of naturally-occurring
carbapenems consists of the simplest core metabolite, carbap-
en-2-em-3-carboxylic acid (1) on the one hand, and approxi-
mately 50 more highly elaborated structures that differ by the
nature of the sulfur substituent at C2 and an alkyl group at C6,
often present at an elevated oxidation state, for example, 2
(Scheme 1A).[2] Prominent among the latter is thienamycin (3)
the amidine derivative of which, imipenem, is marketed as Pri-
maxin.[3] The two carbons of the hydroxyethyl side chain of 3
are known to be derived from methionine by presumably suc-
cessive C1-transfers.[4–6] Recent work has established that the
C2 side chain is derived by stepwise truncation of coenzyme A
(CoA) rather than by direct incorporation of cysteamine or cys-
teine, as previously thought.[7] The timing, however, of C2 and
C6 side-chain attachment among the overall biosynthetic
events leading to thienamycin is unknown.
methyl (2S,5S)-CMP disasteriomers 10 and 11 in a 1.2:1 ratio.[8]
Adenylation of 9 by carbapenam synthetase (CarA) is coupled
to b-lactam formation by the coordinated participation of an
active site Tyr–Glu dyad and a lysine residue to deftly catalyze
formation of this more highly strained bicyclic intermediate.[9]
In contrast to the strict stereospecificity of CarB, CarA will
accept and process epimers of (2S,5S)-CMP (9).[8a,9b] Finally, car-
bapenem synthase (CarC), a member of the nonheme iron a-
ketoglutarate (a-KG)-dependent oxygenase superfamily, carries
out the bridgehead epimerization of 12 to 13 and ring desatu-
ration to give 1.[10]
Identification of the thienamycin biosynthetic gene cluster
in Streptomyces cattleya opened the way to investigation of
the more structurally complex members of the carbapenem
antibiotic family.[6] It was immediately apparent that many
more proteins were involved in the biosynthesis of 3 than of 1
and, while orthologues of CarA and CarB were encoded by the
cluster, ThnE and ThnM bore only 37 and 25% amino acid se-
quence identity, respectively. Second, there was no orthologue
of CarC, suggesting that different biochemical solutions had
evolved to accomplish C2/3 desaturation and, by analogy to
the formation of 1, to carry out bridgehead inversion—both
essential to antibiotic activity. Support for at least one step in
a common pathway to both 3 and the simple carbapenem 1
The unsubstituted carbapenem 1 is created in three highly
efficient steps (Scheme 1B). Carboxymethylproline synthase
(CarB), a member of the crotonase superfamily, mediates the
decarboxylation of malonyl-CoA (MalCoA) and the stereospecif-
ic addition of the resulting enzyme-bound enolate to l-gluta-
mate g-semialdehyde (4) or l-pyrrolidine-5-carboxylic acid (l-
P5C, 5). The resulting (2S,5S)-carboxymethylproline coenzyme
A thioester (CMP-CoA, 6) partitions between hydrolysis and dif-
fusion from the active site to give the acid 9. CarB is also capa-
ble of catalyzing the same series of reactions with methyl-
malonyl-CoA (MeMalCoA), albeit less efficiently, to give the 6-
[a] Dr. M. J. Bodner, Dr. R. Li, R. M. Phelan, Dr. M. F. Freeman, Dr. K. A. Moshos,
E. P. Lloyd, Prof. Dr. C. A. Townsend
Department of Chemistry, The Johns Hopkins University
Baltimore, Maryland 21218 (USA)
E-mail: ctownsend@jhu.edu
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201100366.
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C. A. Townsend et al.
was recently reported when a truncated ThnE (D2–46) indeed
catalyzed the formation of (2S,5S)-CMP (9, Scheme 1B) from
MalCoA, like CarB. A 4:1 diaste-
reomeric mixture of 6-methyl-
(2S,5S)-CMPs 10 and 11 was also
observed from MeMalCoA.[11] We
have gathered compelling gene
swapping, stereochemical and
kinetic evidence that 3 and 1
share two biosynthetic steps,
despite substantial phylogenetic
separation of the producing or-
ganisms, before diverging, and
that introduction of the C6 side
chain in thienamycin biosynthe-
sis occurs after carbapenam 12
formation.
and a b-lactamase induction assay.[9b,13] To obtain higher ex-
pression and improved solubility of ThnE, the codons of the 21
Results and Discussion
In vivo analysis of ThnE and
ThnM
Despite the low overall identity
between ThnE and CarB, or
ThnM and CarA, many residues
in their respective active sites
appeared to be conserved, indi-
cating that ThnE and ThnM
could potentially carry out corre-
sponding reactions in the simple
carbapenem pathway. Their bio-
synthetic activities were first
Scheme 1. A) Representative carbapenems. B) The biosynthesis of carbapen-2-em-3-carboxylic acid (1). C) Carboxy-
methylprolines relevant to thienamycin (3) biosynthesis.
probed globally, therefore, by
analyzing their abilities to substi-
tute for their counterparts CarB
and CarA and support the bio-
synthesis of (5R)-carbapen-2-em-
3-carboxylic acid (1), in vivo
(Figure 1). Genetic replacements
by thnE and thnM were made
individually and pairwise in the
previously described plasmid,
pET24a(+)/carABC.[12] In this con-
struct, expression of carA, carB
and carC were controlled by a
T7 promoter and T7 terminator
as a single operon. It had been
shown that the coexpression of
carB, carA and carC in E. coli
BL21(DE3)(pLysS) reconstituted
the synthesis of carbapenem 1
at a titer comparable to that of
wild-type P. carotovorum, which
could be readily detected by
two sensitive bioassays by using
Figure 1. CarA and CarB orthologue replacement experiments. A) Inhibition of the growth of b-lactam super-sensi-
tive E. coli SC12155. B) Coexpression of biosynthetic enzymes; U: uninduced cellular fraction, I: induced cellular
a b-lactam super-sensitive E. coli fraction.
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Common Steps in Carbapenem Biosynthesis
N-terminal amino acids were optimized and the engineered
ThnE (ThnE*) showed elevated expression profiles (data not
shown). As illustrated in Figure 1, when substitutions were
made in this construct by either or both thnE or thnM, approxi-
mately equivalent levels of antibiotic production were ach-
ieved in the optimized medium, suggesting overlap of their
catalytic activities with both CarB and CarA, respectively.
The ability, however, of CarB and truncated ThnE (D2–46) to
carry out reactions with both MalCoA and MeMalCoA led us to
address more rigorously the reactions catalyzed by ThnE.[8a]
Similarly, the modest sequence identity between CarA and
ThnM raised questions about the substrate preferences of
both ThnE and ThnM, particularly with respect to C2 and C6
side-chain attachments and their timing in the biosynthetic
pathway. The thnE gene was cloned from S. cattleya genomic
DNA and the first 21 codons were optimized to favor expres-
sion in E. coli. The partially codon-optimized thnE* was inserted
into pET28b encoding an N-terminal His6-tag. The thnM gene
was similarly cloned and inserted into pET29b bearing a C-ter-
minal His6-tag. The recombinant proteins were over-produced
in E. coli Rosetta2(DE3) and purified by Ni-NTA affinity chroma-
tography.
Preparation of substrates
The substrates of CarB and CarA, l-P5C (5) and (2S,5S)-CMP (9),
as well as (2S,5R)-CMP (14, Scheme 1C) were synthesized by
using established methods.[8a,10b] In addition, (6R)-methyl and
(6R)-ethyl CMPs (10 and 15) were synthesized by extension of
a recently described route to carbapenems (Scheme 2).[14] 3-
Methyl and 3-ethyl (3R,4R)-azetidinones 16 and 17 were pre-
pared in catalytic, asymmetric reactions to set the absolute
configuration at C4 that is produced by ThnE and the configu-
ration at C3 that is observed in thienamycin (3) and all known
carbapenem metabolites of S. cattleya. The cis relation of the
C3/C4 substituents was preserved by Arndt–Eistert homologa-
tion of 24 and 25 in the synthesis of C2/3 unsubstituted carba-
penems.[15,16] The a,b-unsaturated esters 30 and 31 thus ob-
tained underwent reduction by selective conjugate hydride ad-
dition mediated by Stryker’s reagent to give the saturated car-
bapenams 32 and 33.[17] The newly created carbapenam C3
stereocenter was directed exclusively exo to the b-lactam ring,
matching the orientation present in 9. The C3 acids could be
deblocked by hydrogenolysis prior to hydrolysis of the b-
lactam ring. Alternatively, the b-lactam ring and ester could be
cleaved concurrently to give the C6-substituted carboxyme-
thylprolines 10 and 15. Epimerization of the thermodynamical-
ly less stable cis-C5/C6 configuration to the more stable trans-
Scheme 2. Synthesis of carboxymethylprolines. a) SmI2, iPrOH, Sm(0), THF,
80%; b) CH2Cl2, TBSOTf, Et3N, 92%; c) THF, H2, Pd/C, 99%; d) 1) CH2Cl2, cat.
DMF, oxalyl chloride, 2) Et2O, diazomethane, 85%; e) THF, 10% H2O, hv, 96%;
f) 1) CH3CN, carbonyl diimidazole, 2) Mg(mono-PNB malonate)2, 3) MeOH,
10% 1m HCl, 4) CH3CN, mesyl azide, Et3N, 50%; g) 1) benzene, Rh2(OAc)4,
808C, 2) THF/MeOH, NaBH4, À788C, 3) CH2Cl2, mesyl chloride, Et3N, 65%;
h) benzene, MeOH, [(PPh3)CuH]6, polymethylhydrosiloxane, 50%; i) 2:1 THF/
H2O, 2 equiv KOH, 99%; j) 2:1 THF/H2O, 1 equiv KHCO3, H2, Pd/C, 90%;
k) H2O, 1 equiv KOH, 99%.
1
C5/C6 configuration was not detected by H NMR spectrosco-
corresponding cleaved (2S,3R,5S)- and (2R,3R,5S)-2-cysteaminyl-
5-carboxy-ethylprolines 40 and 41 (Scheme 3 and the Support-
ing Information).
py.
The known (5S)-carbapenem 35 was treated with N-acetyl-
cysteamine to give a disastereotopic mixture of 36 and 37 in a
4:1 ratio, which was separated by chromatography on silica
gel and deprotected by hydrogenolysis.[16] The carboxylic acids
38 and 39 were purified by HPLC. The low concentration of
acid present during this step catalyzed b-lactam hydrolysis.
The individual stereoisomers upon lyophilization yielded the
Alternate substrate studies and kinetics
A more detailed analysis of ThnE function began with a com-
parison of its substrate specificity for MalCoA, the native sub-
strate of CarB, or MeMalCoA, a potential precursor of the thi-
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C. A. Townsend et al.
9, 6-methyl (2S,5S,6R)-CMP (10) and 6-ethyl
(2S,5S,6R)-CMP (15), the latter two bearing alkyl sub-
stituents matching the thienamycin (3) stereochemis-
try, were examined. As a further probe of pathway
timing and C5 stereoisomerization, ThnM-catalyzed
b-lactam formation with the epimeric (2S,5R)-CMP
(14) as substrate was also investigated. Of 9, 10, 15
and 14, ThnM showed reaction only with 9 and 10.
In an analogous manner the 2-cysteaminyl CMPs, 40
and 41 were assayed and also found to be inactive
with ThnM. It is apparent that, while a methyl sub-
stituent is tolerated, more sterically demanding sub-
stitutions at C2 or C6 are not.
Scheme 3. Synthesis of 2-N-acetylcysteaminyl-CMP diastereomers. a) N-acetylcysteamine,
DBU, CH3CN; b) H2, 10% Pd/C, KHCO3, 2:1 THF/H2O; c) HPLC separation/lyophilization.
To investigate the reactions of ThnM with 9 and 10
more precisely, their kinetic constants were deter-
mined under optimized conditions from a pH/rate
enamycin (3) hydroxyethyl group. The derivation of (R)- or (S)-
MeMalCoA or ethylmalonyl-CoA by C1-transfers to MalCoA by
S-adenosylmethionine is unprecedented, but is chemically fea-
sible.[18] However remote, this possibility was examined by in-
cubation of l-P5C (5) with MalCoA or MeMalCoA and monitor-
ing the course of reaction by HPLC. The ThnE-catalyzed reac-
tion between MalCoA and 5 was confirmed by Dowex purifica-
tion and characterization of (2S,5S)-CMP (9) from the enzymatic
reaction. ThnE also condensed MeMalCoA with 5 to give a 3:2
diastereomeric mixture of 6-methyl CMPs 10 and 11, respec-
tively, an unselective product ratio similar to that seen previ-
ously with CarB.[8a] No reaction was observed with ethylmalon-
yl-CoA as previously with a truncated ThnE.[11]
profile by using a continuous coupled-enzyme assay that
linked production of the terminal byproduct AMP to a
decrease in the concentration of NADH detectable at 340 nm
(Table 2 and the Supporting Information).[19] Three notable ob-
Table 2. Steady state kinetic parameters for ThnM-catalyzed reactions.
Substrate
KM [mm]
kcat [sÀ1
]
kcat/KM
[mmÀ1 sÀ1
]
(2S,5S)-CMP (9)
(2S,5S,6R)-6-methyl CMP (10) 5.78Æ1.00
0.115Æ0.014 0.30Æ0.01
0.260Æ0.033
0.046Æ0.004 0.008Æ0.002
With MeMalCoA as a substrate, ThnE also accumulated two
other products observable by HPLC. HPLC purification and ESI
mass spectrometric analysis (m/z 935.14 [MÀH+]) identified
them as CMP-CoA esters 7 and 8. The appearance of these
CMP-CoA ester intermediates was also observed in the CarB-
catalyzed reaction and could indicate a high degree of catalytic
similarity between the enzymes. To further probe the extent of
functional similarity, the kinetic constants for the ThnE-cata-
lyzed reaction between MalCoA or MeMalCoA and 5 were de-
termined (Table 1). ThnE is selective for MalCoA with a 66-fold
higher specificity constant (kcat/KM) for the reaction with 5 and
MalCoA than with MeMalCoA.
servations emerged from this analysis. First, based on the se-
lectivity constants (kcat/KM), the preferred substrate for ThnM,
like CarA, is 9. Second, while the KM of 9 is nearly identical for
ThnM and CarA, the kcat for the former is an order of magni-
tude lower, signaling a much less efficient enzyme.[9b] This
lower kcat might result from selective pressure on the host to
survive a more potent carbapenem product, or from down-
stream enzyme(s) that are overall rate-limiting to the flux of
the biosynthesis, hence reducing evolutionary pressure on
ThnM. Third, the kcat for 10 is approximately 50% greater than
the unsubstituted substrate. We propose that it is the eclipsed
relationship of the (6R)-methyl and the five-membered ring of
the adenylated CMP imposed by the active site that engenders
bond angle compression in the classic Thorpe–Ingold sense.
This steric compression raises the ground state energy leading
to b-lactam closure, hence accelerating the rate of this chemi-
cal step.[20] It is known from detailed kinetic analysis of CarA
that both formation of the four-membered ring and a protein
conformational change limit the overall rate of this enzyme.[9d]
By analogy to ThnM, the intrinsic magnitude of the Thorpe–
Ingold effect favoring b-lactam synthesis could be partially
masked by a corresponding protein conformational change.
Table 1. Steady state kinetic parameters for ThnE-catalyzed reactions.
Substrate
KM [mm]
kcat [sÀ1
]
kcat/KM
[mmÀ1 sÀ1
]
MalCoA
MeMalCoA
0.017Æ0.002
0.203Æ0.008
0.30Æ0.01
17.4Æ2.1
0.05Æ0.001
0.26Æ0.01
A more likely point of side-chain methylation can be envi-
sioned to occur at the stage of (2S,5S)-CMP (9) that would
then be a substrate for the b-lactam-forming enzyme, ThnM. In
a first experiment, potential substrates were screened in fixed
time assays (3 h) at 10 mm, a high concentration 50-times
greater than the KM of CarA with 9, its native substrate.[9b] Thus
Conclusions
The combined results for ThnE and ThnM, particularly the ki-
netic discrimination of each enzyme for variously methyl- and
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Common Steps in Carbapenem Biosynthesis
carA. The resulting plasmid with the RBS-carB at the same orienta-
tion as the carA was named pET24a(+)/carA-carB. The RBS-carC
was excised from pET24a(+)/carC as a XbaI–XhoI fragment. It was
blunt-ended and inserted into the blunt-ended XhoI site down-
stream of the carB in pET24a(+)/carA-carB to form the final vector
pET24a(+)/carABC. The co-overexpression of carA, carB and carC in
pET24a(+)/carABC was controlled by the T7 promoter and T7 ter-
minator as a single operon.
ethyl-substituted potential substrates, reveal unambiguous
preferences for the natural substrates of CarB and CarA in-
volved in the synthesis of the structurally simpler (5R)-carbap-
en-2-em-3-carboxylic acid (1). The low sequence identity evi-
dent for each pair of homologous enzymes raised the question
whether C1-alkylations or thioether side-chain introduction
could take place at this early stage in the biosynthesis of thi-
enamycin (3). One can envision that the active site(s) of CarB
and ThnE or CarA and ThnM could have easily been altered by
mutation to accommodate such charge–neutral steric changes
in their substrates. The experiments strongly suggest, however,
that this evolutionary alternative was not chosen despite the
large phylogenetic separation of the respective hosts, and that
thienamycin and simple carbapenem biosynthesis are function-
ally and stereochemically identical up to the formation of
(3S,5S)-carbapenam (12). At this point the pathways to the
simple 1 and 3 diverge (Scheme 4). In P. carotovorum the FeII/
a-ketoglutarate-dependent oxygenase CarC both inverts the
Cloning and partial codon optimization of thnE: Wild-type thnE
was amplified from genomic DNA (gDNA) of S. cattleya by using
the forward primer: GCATATGGGC GCGGCCGCCG GCGAG, and
reverse primer: CAAGCTTCAG CTCCGCCCGA TGACGCG. NdeI and
HindIII restriction sites were introduced to facilitate downstream
cloning experiments. The PCR reaction (100 mL) contained 10ꢁ
cloned Pfu DNA polymerase buffer (10 mL), dNTPs (2.5 mL, 10 mm),
each primer (2 mL, 20 nmolmLÀ1), DMSO (5 mL), gDNA
(85 ngmLÀ1, 1 mL), and Pfu DNA polymerase (2.5 UmLÀ1, 1 mL).
The reaction was preheated to 988C for 2 min before the addition
of Pfu DNA polymerase, then followed by 30 cycles of 45 s at
988C, 45 s at 698C, and 1 min at 728C with the final extension
time of 10 min at 728C. The PCR product was purified with the
GeneClean III kit (Q-BioGene, Solon, OH, USA) and ligated into
pBluescript II SK+ (Stratagene, La Jolla, CA, USA) to obtain pBS/
thnE. Once confirmed by DNA sequencing analysis, thnE was ex-
cised from pBS/thnE with NdeI/HindIII and ligated into pET24a(+)
to generate pET24a(+)/thnE.
bicyclic ring junction and desaturates C2/3 to give
1
(Scheme 1B). By contrast a more complex series of events
takes place to accomplish these steps and C2 and C6 side-
chain modification in 3 formation.[2] Recently described inser-
tional inactivation of thnL and thnP, two apparent radical SAM
methyltransferases in the thienamycin biosynthetic gene clus-
ter, gives rise to a detectable product the nominal mass of
which is at least consistent with the accumulation of 12.[21]
While this experiment does not strictly prove the identity or in-
termediacy of 12 in thienamycin biosynthesis, it is potentially
supportive of the conclusions drawn here. C6 alkylation and
C2 sulfur side-chain attachment each temper the reactivity of
the simple carbapenem-3-carboxylic acid in ways desirable for
antibiotic activity, a property that could account for the addi-
tional metabolic burden they entail. The timing and mecha-
nism of these events, and of bicyclic ring inversion and C2/3
desaturation, remain to be determined.
Experimental Section
Construction of co-overexpression vector pET24a(+)/carABC:
RBS-carB generated as a XbaI–HindIII fragment from pET24a(+)/
carB was blunted with T4 DNA polymerase and ligated into the
blunt-ended NotI site located downstream of carA in pET24a(+)/
To optimize the codons of the first 21 amino acids of thnE, two
oligonucleotides, Ecod-F and Ecod-R, harboring the optimized
codons and NdeI and BsrI restric-
tion ends were mixed in equal
concentrations. After being heated
at 1008C for 10 min, the mixture
was left to cool slowly at room
temperature
to
allow
the
oligonucleotides to anneal. Plas-
mid pET24a(+)/thnE was digested
with HindIII/BsrI to generate
a
821 bp fragment of thnE absent
the 64 bp 5’ region. A three-way li-
gation reaction containing NdeI/
HindIII digested pET24a(+), the
821 bp BsrI–HindIII thnE fragment
and the 64 bp NdeI–BsrI annealed
oligonucleotides was carried out
to generate plasmid pET24a(+)/
Scheme 4. Common biosynthetic steps to the carbapenem b-lactam antibiotics.
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C. A. Townsend et al.
thnE*. Gene thnE* was excised with NdeI/HindIII from pET24a(+)/
thnE* and inserted into pET28b(+) to give pET28b(+)/thnE* for ex-
pression of ThnE as N-His6 protein.
HPLC analysis of ThnE-catalyzed reactions: A previously reported
method was modified.[8a] HPLC conditions: Phenomenex Prodigy
5m ODS-3 analytical column, l=260 nm, 1 mLminÀ1; buffer A=
100 mm NH4H2PO4, 75 mm ammonium acetate, pH 4.65 with acetic
acid, buffer B=70% solvent A and 30% methanol. Method 1, t=
0 min 40% B, t=28 min 85% B, t=29 min 100% B, t=39 min
100% B, t=42 min 40% B, t=52 min 40% B. Method 2, t=0 min
40% B, t=20 min 80% B, t=21 min 40% B, t=30 min 40% B.
Construction of co-overexpression vector pET24a(+)/thnE*M-
carC: RBS-carC was excised from pET24a(+)/carC with XbaI/HindIII,
blunt-ended with Klenow DNA polymerase, and ligated into the
blunt ended NotI site of pET24a(+)/thnM to give plasmid
pET24a(+)/thnM-carC. The RBS-thnM-RBS-carC fragment was gener-
ated with XbaI/XhoI, blunt-ended and ligated downstream of thnE*
at the blunt-ended XhoI site to obtain the final expression vector
pET24a(+)/thnE*M-carC.
Relative rates of ThnE-catalyzed reactions with l-P5C (5) and
MalCoA or MeMalCoA: Reactions containing l-P5C (5, 0.5 mm),
0.3 mm MalCoA or MeMalCoA and ThnE (2.4 mgmLÀ1) as well as
the corresponding reactions without 5 and no-enzyme reactions
were assembled in potassium phosphate (100 mm, pH 7.8).[8a] At 0,
15, 30, 60, 120 min and overnight, samples (200 mL) were
quenched in HCl (200 mL, 0.2m) then vortexed with CCl4 (400 mL),
the top layer (300 mL) was removed from the CCl4 for HPLC analy-
sis.
Construction of co-overexpression vector pET24a(+)/thnE*-
carAC and pET28b(+)/thnE*-carAC: The RBS-carC fragment was
generated by digesting pET24a(+)/carC with XbaI/XhoI. It was
blunt-ended with T4 DNA polymerase and inserted into the blunt-
ended NotI site of pET24a(+)/carA to give the co-overexpression
vector pET24a(+)/carAC. RBS-carA-RBS-carC was excised as a XbaI–
XhoI fragment from pET24a(+)/carAC and blunt-ended with
Klenow DNA polymerase. Plasmids pET24a(+)/thnE* and
pET28b(+)/thnE* were digested with NotI, blunt-ended with
Klenow DNA polymerase and ligated with the RBS-carA-RBS-carC
fragment to generate co-overexpression vectors pET24a(+)/thnE*-
carAC and pET28b(+)/thnE*-carAC, respectively.
Purification and ESI-MS of CMP-CoA esters from ThnE reactions:
New products observable in the HPLC trace were collected from
multiple runs, lyophilized and desalted with a STRATA X column
(33 mm, 30 mgmLÀ1).[8a] The column was prewashed with methanol
(10 mL) and 0.1% TFA (10 mL). The lyophilized sample was taken
up in water (1 mL) and 0.1% TFA (2 mL) was added. The sample
was applied to the column and washed with 0.1% TFA (2 mL). It
was then eluted with methanol/0.1% TFA 4:1 (5 mL). Fractions
(1 mL) containing the desired CoA ester were identified by their
characteristic absorbance at 260 nm and lyophilized. The resulting
powder was taken up in 1:1 CH3CN/water with 0.1% NH4OH or
0.1% TFA and analyzed by ESI-MS.
Construction of co-overexpression vector pET24a(+)/thnM-
carBC: pET24a(+)/thnM-carC was digested with XbaI/XhoI to give a
fragment containing RBS-thnM-RBS-carC. It was then blunt-ended
with Klenow DNA polymerase and ligated into the blunt-ended
XhoI site of pET24a(+)/carB to give the final plasmid pET24a(+)/
thnM-carBC.
HPLC analysis of ThnM-catalyzed reactions: Reactions for the de-
termination of product formation (200 mL) were run in a buffer
containing HEPES (100 mm) and piperazine (80 mm) at pH 8.2 with
m=100 mm (KCl).[9b] Each reaction contained ATP (2 mm), DTT
(1 mm), MgCl2 (12.5 mm), substrate (10 mm) and ThnM (15.0 mm).
The reactions were run at 258C for 2 h and immediately frozen in
liquid N2 until analysis. Analysis was performed on an Agilent 1100
HPLC by using a Phenomenex Luna 5 m phenyl–hexyl analytical
column. Injections (50 mL) were run in an isocratic mobile phase
consisting of a phosphate buffer (50 mm) at pH 6.5 with a flow
rate of 2 mLminÀ1. Reactions were monitored at l=210 and
230 nm. Compounds produced in ThnM-catalyzed reactions were
identified by comparison to authentic product standards.
Small-scale co-overexpression of biosynthetic genes and heter-
ologous production of carbapenem in E. coli: Seed medium
(3 mL) containing kanamycin (50 mgmLÀ1) and chloramphenicol
(25 mgmLÀ1) was inoculated with a single colony of freshly trans-
formed Rosetta2(De3) or BL21(DE3)(pLysS) harboring recombinant
plasmid and grown, overnight, at 378C. Seed culture (100 mL) was
transferred into LB+ medium (50 mL; gLÀ1: Bacto-tryptone 10;
Bacto-yeast extract 5; NaCl 10; glutamate 10; NaOAc 1; FeSO4·7H2O
0.25; CoCl2·6H2O 0.01, pH 7.5) or modified carbapenem production
medium (gLÀ1: glutamate 5; NH4Cl 0.75; K2HPO4 2; NaCl 0.5; CaCO3
0.25; glucose 10, pH 7.6; 75 mg FeSO4·7H2O and 12.5 mL of 1m
MgSO4 were added after autoclaving). The secondary culture was
grown at 378C to OD600 =0.3–0.4. Expression of proteins was in-
duced with IPTG (1 mm) at 288C for 5 h.
Acknowledgements
Detection of carbapenems: Samples were withdrawn from cul-
tures every 60 min after co-overexpression was induced, and cen-
trifuged at 16000g for 10 min. Supernatant (250 mL) was added to
paper discs placed on a bioassay plate of Bacto-Nutrient agar
seeded with b-lactam supersensitive E. coli SC12155.[13b] The bioas-
say plates were incubated at 378C for 20 h. The production of car-
bapenems in the engineered E. coli was indicated by the inhibition
of super-sensitive E. coli growth in zones around the paper discs.
The production of carbapenem was also observed by Nitrocefin
colorimetric assay.[13a] The recombinant E. coli supernatant (300 mL)
was loaded on to paper discs sitting on BA2 agar (gLÀ1: BBL seed
agar 30.5; NaCl 5) seeded with Bacillus lichniformis ATCC14580. The
plates were incubated at 378C for 3 h and overlaid with Nitrocefin
solution (1.5 mL of 300mgmLÀ1). The production of carbapenems
was indicated by the formation of red zones around the paper
discs.
We are grateful to Dr. Anthony Stapon for preparation of CMPs,
and to Prof. Barbara Gerratana and Dr. Samantha O. Arnett for
guidance and early insightful discussions. We thank Dr. Mary L.
Raber for critical review of the kinetic data and the NIH
(AI014937) for financial support.
Keywords: beta-lactam
antibiotics
·
biosynthesis
·
carbapenems · enzyme catalysis · thienamycin
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Received: June 11, 2011
Published online on August 24, 2011
ChemBioChem 2011, 12, 2159 – 2165
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www.chembiochem.org
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