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chromosomal DNA by use of primer pairs P11/P12 and P13/P14, re-
spectively, and sequentially cloned into the pIJ2925 plasmid; the
B11–B12 fragment was then inserted into the suicide shuttle
vector pD2925. The pKC1139DgenB2 vector containing genB2 with
in-frame deletion was constructed by inserting two genB2-flanking
DNA fragments into the pKC1139 temperature-sensitive shuttle
vector. B21 and B22 fragments were amplified by PCR with use of
primer pairs P21/P22 and P23/P24 and sequentially cloned into the
pKC1139 plasmid. The pKC1139DgenP vector containing genP with
in-frame deletion was constructed as follows: I1 and I2 fragments
were amplified by use of primer pairs P1/P2 and P3/P4, respective-
ly, and sequentially cloned into the pIJ2925 vector; the I1–I2 frag-
ment was then inserted into the pKC1139 plasmid. All plasmids
were verified by sequencing.
by reduced pressure distillation, lyophilized, resuspended in
ddH2O, filtered through a 0.22 mm filter, and analyzed by HPLC per-
formed with an Ultimate LP-C18 column (4.6250 mm; Welch Ma-
terials, Inc., Maryland, USA) connected to an evaporative light scat-
tering detector. The mobile phase was trifluoroacetic acid (0.2m)/
À1
methanol (92:8 or 98:2, v/v) used at a flow rate of 0.6 mLmin
.
Production yields for all strains were calculated with the aid of an
external standard. The contents of the components were deter-
mined by area normalization with gentamicin C1a as the standard.
Separation of the components was performed by dissolving lyophi-
lized gentamicin samples in ddH2O and loading them onto a D152
+
NH4 ion-exchange column; elution with 0.01–2m gradient NH4Cl
was monitored by assaying the eluted fractions for anti-B. subtilis
activity. Fractions containing single components were combined,
concentrated by reduced pressure distillation, lyophilized, and ana-
lyzed by MS and NMR.
Construction of gene deletion mutants: E. coli ET12567
(pUZ8002) was transformed with the pD2925DgenB1,
pKC1139DgenB2, and pKC1139DgenP plasmids. The disruption
plasmids were introduced into M. echinospora through conjugal
transfer from E. coli on MS agar at 288C for 24 h, as described pre-
viously.[25] After overlaying of agar with apramycin (50 mgmLÀ1) and
pipemidic acid (PPA, 50 mgmLÀ1), incubation was continued for
eight days at 288C. Single-crossover recombinant colonies were
first selected for apramycin resistance and were then cultivated on
SSM plates without antibiotic at 378C. The apramycin-sensitive
strains were confirmed by PCR with appropriate primers (Fig-
ure S14) and by sequencing.
NMR analysis: All experiments were performed with a Bruker
Avance 600 MHz NMR spectrometer operating at 600.13 MHz (1 H)
and 150.90 MHz (13C) and equipped with the XWIN-NMR software,
version 3.0 (Bruker Analytik, Rheinstetten, Germany). For 1 H, 13C,
1H,1H COSY, HSQC, and NOESY NMR analyses, JI-20Ba (20 mg) or JI-
20B (20 mg) was dissolved in D2O (500 mL); for 1H NMR analysis,
ZL101 (5 mg) was dissolved in D2O (500 mL).
Biotransformation experiments: The DgenK mutant was cultured
in fermentation medium for 96 h, intermediates of the gentamicin
pathway were then added at the final concentration of 1 mgmLÀ1
,
and the culture was further incubated for 144 h at 348C. Each
experiment was repeated three times. Product extraction, purifica-
tion, and analysis were performed as described above.
The disruption plasmid pD2925DgenB1 was introduced into the
DgenB2 and DgenP strains through conjugal transfer to obtain
double mutants DgenB2DgenB1 and DgenPDgenB1. The disruption
plasmid pKC1139DgenB2 was introduced into the DgenP strain
through conjugal transfer to obtain the double mutant DgenPDg-
enB2 strain. The genomic DNA of the double mutants was ana-
lyzed by PCR with use of two primer pairs (Figure S14).
Cloning, overexpression, and purification of GenQ, GenB1, and
GenB2: The genB1, genB2, and genQ genes were amplified from
chromosomal DNA of the WT strain by PCR with use of primer
pairs exB1–1/exB1–2, exB2–1/exB2–2, and exQ-1/exQ-2, respective-
ly. PCR products inserted into the NcoI and HindIII sites of the
pET32a (+) vector were verified by nucleotide sequencing, and the
recombinant and empty plasmids were used to transform E. coli
BL21 (DE3). The recombinant strain BL21-pET32a was used for
blank control.
Complementation of single mutants: The pSPU241 and pEAP1
plasmids were used to construct gene complementation vectors.
The genB1, genB2, and genP genes were amplified from WT chro-
mosomal DNA by use of primer pairs CB1–1/exB1–2, CB2–1/exB2–
2, and CP-1/CP-2, respectively, and PCR products were inserted
into the BamHI and HindIII sites of pSPU241. The cloned genes
containing the PermE* promoter and To terminator were digested
with BglII and inserted into pEAP1 to generate pEAPB1, pEAPB2,
and pEAPP. The complementation plasmids were verified by se-
quencing and then introduced individually into DgenB1, DgenB2,
DgenP by conjugation. The plasmids were integrated into M. echi-
nospora chromosomal DNA by site-specific recombination. Com-
plemented exconjugants were identified by erythromycin resist-
ance and confirmed by PCR (Figure S15).
Transformed bacteria were cultured at 378C in LB liquid medium
supplemented with ampicillin (100 mgmLÀ1) until the optical densi-
ty at 600 nm was 0.4–0.6. Protein expression was induced with
IPTG (0.2 mm) at 288C for 6 h. The bacteria were harvested by cen-
trifugation, resuspended in binding buffer [20 mL, Tris·HCl (20 mm),
NaCl (0.5m), imidazole (5 mm), pH 8.0], and disrupted by sonication
for 10 min with use of a 3 s-on/5 s-off cycle. After centrifugation at
15480g at 48C for 10 min, the supernatant was loaded into Ni-Se-
pharose 6 Fast Flow (GE Healthcare), the column was washed with
washing buffer [Tris·HCl (20 mm), NaCl (0.5m), imidazole (10–
50 mm), pH 8.0], and His-tagged recombinant proteins were eluted
with elution buffer [Tris·HCl (20 mm), NaCl (0.5m), imidazole
(200 mm), pH 8.0].
Extraction, purification, separation, and analysis of metabolites
from M. echinospora strains: Fermentation cultures were adjusted
to pH 2.0 with H2SO4 (2m) and agitated for 30 min. After centrifu-
gation at 1600g for 10 min at 48C, the supernatant was neutralized
with NaOH (2m). Antibiotics were isolated from the fermentation
The purified enzymes were concentrated and dialyzed against
Tris·HCl (pH 8.0, 20 mm) containing KCl (50 mm) and glycerol (10%)
and stored at À208C. Purified proteins demonstrated single bands
in SDS-PAGE gels, and these were in agreement with the calculated
molecular weight (MWs) of 63.5, 63.2, and 73.4 KDa (Figure S11).
The blank control strain did not produce target proteins.
+
broth by ion-exchange chromatography with 732NH4 ion-ex-
change resin. The anion-exchange column was washed with
double-distilled (dd) H2O until the effluent was colorless, and
bound antibiotics were then sequentially eluted with eight column
volumes of HCl (0.4m), NH4Cl (0.4m), a mixture of HCl (0.2m) and
NH4Cl (0.62m), and again with ddH2O. The eluates were then
passed through 711H+ ion-exchange resin for discoloration, and
antibiotics were eluted with six column volumes of NH4Cl (5%).
The fractions with the activity against B. subtilis were concentrated
Enzymatic assays: The GenQ/GenB1 and GenQ/GenB2 coupled
assay was performed in Tris·HCl (pH 8.0, 20 mm) containing puri-
fied GenQ (30–50 mg), GenB1 (30–50 mg) or GenB2 (30–50 mg),
ChemBioChem 2015, 16, 1933 – 1942
1941
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