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Supplementary materials
Characterization of AvaR1, an autoregulator receptor that negatively
controls avermectins production in a high avermectin-producing strain
Jian-Bo Wang, Feng Zhang, Jin-Yue Pu, Juan Zhao, Qun-Fei Zhao and Gong-Li Tang*
State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of
Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Rd., Shanghai 200032, China
* To whom correspondence should be addressed: Shanghai Institute of Organic Chemistry, Chinese
Academy of Sciences, 345 Lingling Rd., Shanghai 200032, China. Gong-Li Tang,
Email: [email protected], Tel: 86-21-54925113, Fax: 86-21-64166128
Materials and methods.
Strain information, medium and culture condition
Streptomyces avermitilis M0, a high-Ave-prodcution strain, was used to produce avermectins (which
was separated in China, similar to S. avermitilis MA4680, and was developed to become an
avermectins high-yield strain through mutagenesis). LB broth or LB agar supplemented with the
appropriate antibiotics are used to culture Escherichia coli strains. MS agar medium and modified
liquid YEME medium with 25% sucrose were used for the sporulation and growth of S. avermitilis. If
necessary, apramycin was added to a concentration of 50 μg/mL in MS or 25 μg/mL in YEME. The
Stretopmyces strains were grown at 30 ℃ on MS medium for sporulation. MS supplemented with 10
mM MgCl2 was used for intergenic conjugation from E. coli into Streptomyces.
Gene knock out
Intergeneric conjugation between S. avermitilis and E. coli were performed as described previously by
Hopwood et al (Hopwood et al. 1985). Putative exconjugants were regenerated on MS medium
containing 50 μg mL-1 apramycin, 25 μg mL-1 nalidixic acid for 5 days to confirm resistance. To
eliminate autonomously replicating plasmid pTG2003, the exconjugants were incubated in YEME
culture containing 20 µg mL-1 apramycin, 25 μg mL-1 nalidixic acid for 3 days. The strains of
exconjugants were spread on MS agar plates containing apramycin. The plates were incubated at 37 °C
for 5 days. E. coli–Streptomyces shuttle plasmid pKC1139 has a temperature-sensitive replicon from
Streptomyces ghanaensis and can't replicate in Streptomyces when the temperature is higher than 34 °C.
Therefore, only the insertion mutant in which pTG2003 was integrated into the chromosome of S.
avermitilis by homologous recombination could grow on MS containing apramycin. For chromosomal
eviction of pTG2003, the inframe deletion mutants were incubated in YEME culture under
nonselective condition for four rounds and double crossover took place only in those colonies sensitive
to apramycin (Aprs). The mutants were confirmed by PCR.
Culture conditions for production of Ave
For the production of avermectins, S. avermitilis M0 and mutant strains were precultured in 250
mL baffled flasks containing 40 mL seed medium (30 g corn starch, 4 g yeast extract, 2 g soya
peptone, and 10 mg CoCl2·6H2O per liter of H2O). After growth for 40 h at 28 °C on a rotary
shaker with 180 rpm, 1.5 mL of the preculture was inoculated into 250 mL baffled flasks
containing 30 mL fermentation medium (100 g corn starch, 10 g yeast extract, 0.5 g
K2HPO4·3H2O, 0.5 g MgSO4·7H2O, 4 g KCl, 10 mg CoCl2·6H2O, and 0.8 g CaCO3 per liter of
H2O). In this medium, cells were cultured at 28 °C on a rotary shaker for 10 days.
Analysis of Ave
A 1.0 mL fermentation broth was extracted with 2.0 mL methanol for 30 min and centrifuged at
4,000×g for 10 min. The supernatant was directly applied to high performance liquid chromatography
(HPLC) analysis. For HPLC analysis, the column packed with C18 (10 μm; 4.6 i.d. mm×250 mm) was
equilibrated with methanol–water (85:15) and the flow rate was 0.85 mL/min under an isocratic
method. Detection at 246 nm by UV absorption was monitored, and control samples of avermectin B1a
were used as internal standards.
Supplementary Table 1. Strains and plasmids used in this study
Strains
or
Characteristics
Source
E. coli DH5α
Host strain for general clone
Invitrogen
E. coli ET12567
Host strain for conjugation
(MacNeil et al. 1992)
Streptomyces
Avermectins-producing strain
plasmids
Strains
avermitilis
M0
Parents strain
This laboratory
TG2003
avaR1 deletion mutant
This study
TG2004
complemented avaR1 in TG2002
This study
E. coli cloning vector
(Yanisch-Perron, Vieira, and
Plasmids
pGEM-3zf
Messing 1985)
pANT841
E. coli cloning vector
pET28a
Protein expression vector
pKC1139
E. coli - Streptomyces shuttle vector; repts; OriT
(Bierman et al. 1992)
pSET152
E.
(Bierman et al. 1992)
coli
-
Streptomyces
(Rajgarhia and Strohl 1997)
shuttle
vector;
site-specific integration; OriT
pTG2003
avaR1 deletion plasmid, derived from pKC1139
This study
pTG2004
avaR1 complemention plasmid, derived from
This study
pSET152, containing an ermE* promoter
pTG2009
AvaR1 expression plasmid
This study
Supplementary Table 2. Primers used in this study
Target gene operation
Designation
Sequence (5’-3’)
avaR1-deletion
Primer 1
TTTAAGCTTaccccttggcgaccgccgtc
HindIII
TTTTCTAGAtcgctcctgccgcgccacac
XbaI
TTTTCTAGAggcacggacgttggagtgac
XbaI
TTTGAATTCgtgcggatcgcgcgttcctg
EcoRI
Primer 2
Primer 3
Primer 4
avaR1-complemention
Primer 5
Primer 6
avaR1-disruption
confirmed
aveA1
aveA3
R1jdF
R1jdR
A1F
A1R
A3F
TTTAAGCTTcggagggtgcgtgtggcgc
HindIII
TTTTCTAGAgtcactccaacgtccgtgcc
XbaI
ACGAGAGTGCGGTGCGCTC
GATGACGCGCATGGGCAG
CGCTTCCGACGTCTTCCG
AACGACTGTGCTCCGGGG
GCCGATTGCGATTGTGGG
aveR
avaR1
aco
hrdB
aveC
AvaR1-expression
A3R
aveRF
aveRR
avaR1F
CCGCTACCGTTGCCGATGT
CCTCGGAAACCAGAAGAACTCA
CGGTGACGGTCAGGCAGTAG
CATTCGGACGCAGAC
avaR1R
TGGCGAAGGACGGCATCAG
acoF
acoR
hrdBF
hrDBR
aveCF
aveCR
Primer 7
ACCGCACTCTCGTACGAC
CGTCGGTGTGTGCAGATG
GCAGCCTCAACCAGATCCTC
TTGGCAGTCACCGTCTTCG
CAGCAAGGATACGGGGAC
ACCGAGCACGATGCCGATG
TTTCATATGgcgcggcaggagcgag
NdeI
TTTAAGCTTtaaCTCGAGctccaacgtccgtgccgc
HindIII
XhoI
ccatgcaccgcgagcagaaggaactccttcacc
tccatgcaccgcgcatcttatttctcctttttc
Primer 8
aveR-promoter
aco-promoter
Primer 9
Primer 10
Primer 11
Primer 12
tccatgcaccgcgacgca AGACAGGCCCGGTCCTGAC
tccatgcaccgcgacgca CGAGCTTCTCCCTGGCTTG
Supplementary Figure 1. Gene-deletion of avaR1 and PCR confirmed the geno-type of the
mutant. Ethidium bromide-stained agarose gels in the left, lane 1, 2, 5 the avaR1-deletion mutants;
lane3, the wild type; lane 4, DL 5000 DNA marker.
Supplementary Figure 2.
sequence alignment between AvaR1 and ScbR. According to the crystal
structure of CprB, their structures include ten α-helix, and the residues labeled with * are related to bind to the
target DNA.
supplementary Figure 3. HPLC and MS analysis of avermectins production by S. avermitilis M0
and the mutants, A: HPLC results (I) S. avermitilis M0; (II) S. avermitilis TG2003; (III) mutant S.
avermitilis TG2004; (), avermectin B2a; (), avermectin A2a;
A1a. B; Mass Spectra of avermectin components
A.
B.
(),avermectin B1a; (▼), avermectin
Supplementary data reference
Bierman M, Logan R, O'Brien K, Seno ET, Rao RN, Schoner BE (1992) Plasmid cloning vectors for
the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116 :43-9.
Hopwood DA, Bibb MJ, Kieser T, Bruton CJ, Kieser HM, Lydiate DJ, Smith CP, Ward JM, Schrempf
H (1985) Genetic manipulation of Streptomyces: a laboratory manual. John Innes Foundation, Norwich
MacNeil DJ, Gewain KM, Ruby CL, Dezeny G, Gibbons PH, and MacNeil T (1992). Analysis of
Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector.
Gene 111:61-8.
Rajgarhia VB, and Strohl WR (1997) Minimal Streptomyces sp. strain C5 daunorubicin polyketide
biosynthesis genes required for aklanonic acid biosynthesis. J Bacteriol 179 :2690-6.
Yanisch-Perron C, Vieira J, and Messing J (1985)Improved M13 phage cloning vectors and host strains:
nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33 :103-19.