* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download 10529_2013_1416_MOESM1_ESM
Survey
Document related concepts
Nucleic acid analogue wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Gene regulatory network wikipedia , lookup
Gene expression profiling wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Expanded genetic code wikipedia , lookup
Bottromycin wikipedia , lookup
E. coli long-term evolution experiment wikipedia , lookup
Two-hybrid screening wikipedia , lookup
Real-time polymerase chain reaction wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Molecular cloning wikipedia , lookup
Transformation (genetics) wikipedia , lookup
Transcript
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.