Please cite this article in press as: Huang et al., Biosynthesis of Neocarazostatin A Reveals the Sequential Carbazole Prenylation and Hydroxylation in
68ꢀC for 10 min. The obtained fragments were cloned into the HindIII/EcoRI
site of pKC1139 by using the In-fusion HD Cloning Kit (Clontech) to obtain
the in-frame deletion vector construct, which was then transferred into Strep-
tomyces sp. MA37 via E. coli-Streptomyces conjugation. Following a previ-
ously published procedure (Yu et al., 2009), the nzsA in-frame deletion mutant
strains were identified by screening out and designated as WDY633. The same
strategy was used to construct nzsB to nzsJ in-frame deletion mutants, except
that different primers were used to amplify the left and right arms of the target
genes (Table S3). The in-frame deletion mutant strains of nzsB to NzsJ were
designated as WDY640, WDY641, WDY642, WDY644, WDY646, WDY638,
WDY630, WDY648, and WDY635, respectively (Table S1).
Regenerated Cellulose 3,000 molecular weight cutoff). The samples were
then desalted by PD-10 Columns (GE Healthcare) according to the manufac-
turer’s instruction. The cleavage of the SUMO tag of the eluted recombinant
proteins was conducted using SUMO Protease (Invitrogen, catalog #12588-
018) in buffer composed of 50 mM Tris-HCl (pH 8.0) and 150 mM NaCl at
4ꢀC for 4 hr. The SUMO tag and SUMO Protease were finally removed from
the cleavage reaction by using 0.5 ml of Ni-Sepharose 6 Fast Flow (GE Health-
care). The purified protein was stored at À80ꢀC in storage buffer (50 mM Tris-
HCl [pH 8.0], 150 mM NaCl, 10% [w/v] glycerol, and 1 mM DTT).
NzsG Activity Assay
The enzyme assay of NzsG was carried out in 50 mM Tris-HCl buffer (pH 7.5)
with 5 mM MgCl2, containing 0.8 mg/ml NzsG, 1 mM substrate, and 0.2 mM
DMAPP, in a final volume of 50 ml. The optimal assay conditions were obtained
at 30ꢀC. After 30 min, the reaction was quenched by the addition of two equal
volumes of methanol and mixed by vortexing. The mixture was centrifuged at
15,000 rpm for 20 min to remove protein. The supernatant was then subjected
to LC-tandem MS (MS/MS) analysis under the same conditions as described
above.
Complementation of the Mutant Strains WDY633, WDY644,
WDY646, WDY638, WDY630, WDY648, and WDY635
To complement WDY633, a 1,257-bp fragment that contains the whole nzsA
gene sequence was amplified from genomic DNA of Streptomyces sp.
MA37 by high-fidelity PCR using the primers N-1-HB-F and N-1-HB-R (Table
S3). The amplification conditions were: initial denaturation at 95ꢀC for 5 min; 30
cycles of denaturation at 95ꢀC for 30 s, annealing at 58ꢀC for 30 s, and exten-
sion at 68ꢀC for 1 min; and gap infilling at 68ꢀC for 10 min. The obtained frag-
ment was cloned into the NdeI/EcoRI site of pIB139, which can integrate into
Streptomyces chromosome via the VC31 phage site. The construct obtained
was then transferred into Streptomyces sp. MA37 in-frame deletion mutant via
E. coli-Streptomyces conjugation. Following the procedure described previ-
ously (Kieser et al., 2000), the DnzsA complementation mutant strain was iden-
tified by screening out and designated as WDY634. The same strategy was
used to complement WDY644, WDY646, WDY638, WDY630, WDY648, and
WDY635, except that different pairs of primers were used for each comple-
mentation construct (Table S3). The complementation mutant strains of DnzsE,
DnzsF, and DnzsG–J were identified by screening out and designated as
WDY645, WDY647, WDY639, WDY631, WDY649, and WDY636, respectively.
Kinetic Studies of NzsG
Optimization of NzsG in vitro assays is described in the legend of Figure S4D.
The enzyme assays of NzsG were performed in a mixture (total volume 50 ml)
containing 50 mM Tris-HCl (pH 8.0), 5 mM Mg2+, 1 mM DMAPP, 1 mM DTT,
and 0.087–3 mM precarazostatin 5 at 30ꢀC for 10 min. Reactions were initiated
by the addition of enzyme (0.5 mM NzsG). An equal volume of methanol was
added to quench the reaction and remove proteins by centrifugation. The su-
pernatant was analyzed by HPLC. Kinetic analyses of NzsG reactions were
carried out as described in the legend of Figure S4F.
NzsA Activity Assay
Enzyme assay of NzsA activity was carried out on a 50-ml scale with substrates
(1 mM), NzsA (1 mg/ml), spinach ferredoxin (100 mg/ml), spinach ferredoxin-
NADP+ reductase (0.2 U/ml), NADPH (1.0 mM), glucose-6-phosphate
(10 mM), and glucose-6-phosphate dehydrogenase (10 U/ml) in Tris-HCl
buffer (50 mM, pH 7.5). After incubation at 30ꢀC for 30 min, the reaction was
quenched by the addition of two equal volumes of methanol and mixed by vor-
texing. The mixture was centrifuged at 15,000 rpm for 20 min to remove pro-
tein. The supernatant was then subjected to LC-MS/MS analysis under the
same conditions as described above.
Construction of NzsA and NzsG Overexpression Vector
To overexpress NzsA, we amplified a 1,257-bp fragment that contains the
whole nzsA from genomic DNA of Streptomyces sp. MA37 by PCR using the
primers NzsA_F/NzsA_R (Table S2). PCR was performed in 20 ml volume
with 5% DMSO and KOD DNA polymerase (Toyobo). The amplification condi-
tions were: initial denaturation at 95ꢀC for 5 min; 30 cycles of denaturation at
95ꢀC for 30 s, annealing at 58ꢀC for 30 s, and extension at 68ꢀC for 90 s;
and gap infilling at 68ꢀC for 10 min. The obtained fragments were cloned
into the KpnI/XhoI site of pHS_SUMO (Lv et al., 2015) using the In-fusion HD
Cloning Kit (Clontech) to yield the overexpression construct pWDY651. The
same strategy was used for nzsG cloning, except that the primers NzsG_F/
NzsG_R were used for amplification (Table S3). The NzsG overexpression
construct was designated pWDY650.
ACCESSION NUMBERS
The sequence of the nzs gene cluster from Streptomyces sp. MA37 has been
deposited in the GenBank database under the accession number NCBI:
KP657980.
Expression and Purification of NzsA and NzsG
The protein expression constructs pWDY638 and pWDY639 were individually
transformed into E. coli BL21 (lDE3) (Novagen) competent cells. Single
colonies from each transformation were inoculated to a starter culture (5 ml
of super optimal broth [SOB] medium containing 50 mg/ml kanamycin) and
cultivated at 37ꢀC and 200 rpm. When the A600 of the medium reached 0.5,
the culture was transferred to 500 ml of fresh SOB medium and incubated at
37ꢀC, 200 rpm. Isopropyl b-D-1-thiogalactopyranoside was added to a final
concentration of 1 mM when the A600 reached 0.6. After overnight culture at
16ꢀC, cells were harvested by centrifugation and frozen at À40ꢀC. All subse-
quent steps were performed at 4ꢀC. After thawing on ice, cells were sus-
pended in lysis buffer (200 mM Tris-HCl [pH 8.0], 500 mM NaCl, and 10 mM
imidazole). The cell suspension was lysed with a Nano Homogenize Machine
(AH100B; ATS Engineering). To separate the cellular debris from the soluble
protein, the lysate was centrifuged at 20,000 3 g at 4ꢀC for 20 min. The super-
natant was incubated with 1.5 ml Ni-Sepharose 6 Fast Flow (GE Healthcare)
which had been pre-equilibrated with equilibration buffer (200 mM Tris-HCl
[pH 8.0], 500 mM NaCl, and 10 mM imidazole) for 2 hr at 4ꢀC. The resin was
washed with 5 ml of the equilibration buffer, followed by twice washing with
buffer containing 25 mM imidazole. The recombinant protein was eluted with
5 ml of wash buffer containing 250 mM imidazole. The eluted recombinant pro-
teins were concentrated to 2.5 ml using Centrifugal Filter Units (Millipore
SUPPLEMENTAL INFORMATION
Supplemental Information includes six figures and five tables and can be found
ACKNOWLEDGMENTS
Y.Y. acknowledges the financial support from ‘‘973’’ Program
(2012CB721006) and National Natural Science Foundation of China
(31570033). R.E., K.K., H.D., and M.J. acknowledge the financial support of
the Leverhulme Trust-Royal Society Africa Award (AA090088).
Received: September 1, 2015
Revised: October 15, 2015
Accepted: October 21, 2015
Published: December 3, 2015
REFERENCES
Aziz, R.K., Bartels, D., Best, A.A., DeJongh, M., Disz, T., Edwards, R.A.,
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Chemistry & Biology 22, 1–10, December 17, 2015 ª2015 Elsevier Ltd All rights reserved