Figure 5 Analysis of anthramycin production by HPLC/MS. After separating anthramycin on an HPLC column, mass spectrometry was performed using 6520 Agilent Accurate-Mass Q-TOF LC/MS. Conclusions This study shows that by isolation of new strains and testing

several plasmids, a host-vector system in a fast-growing and moderately thermophilic Streptomyces species could be developed. Two antibiotic biosynthetic gene clusters from mesophilic and thermophilic Streptomyces were heterlogously expressed in one strain. We expect that by utilizing thermophilic Streptomyces-specific promoters, more genes and especially antibiotic genes clusters of mesophilic Streptomyces should be heterologously expressed. Methods Bacterial strains, plasmids, JAK inhibitor and general methods Strains used in this work are listed in Table 1. Plasmid isolation, transformation of E. coli DH5α and PCR amplification followed Sambrook et al. [42]. 4SC-202 clinical trial Streptomyces culture, plasmid isolation and preparation of protoplasts and transformation of Streptomyces lividans ZX7 followed Kieser et al. [6]. Plasmid trans-conjugation from E. coli ET12567/pUZ8002 into thermophilic Streptomyces strains followed Bierman et al.

[38]. KpnI-treated pTSC1 was cloned in pBluescript II SK to obtain pCWH100 and was sequenced by primer-walking at Shanghai Invitrogen Inc. Sequence comparisons were done with software from the National Center for Biotechnology Information http://​www.​ncbi.​nlm.​nih.​gov/​BLAST. The complete nucleotide sequence of pTSC1 was deposited in the GenBank database under no. GU271942. Isolation and identification of thermophilic Streptomyces strains Samples of P505-15 cost garden soil, weed compost and swine manure were collected from Shanghai city, Hunan, Hubei and Fujian provinces in the summers of 2005 and 2006. The samples 4-Aminobutyrate aminotransferase were dried at 100°C for 1 h and cultivated on SC medium (starch 10 g, casein 0.3 g, KNO3 2 g, MgSO4.7H2O 0.05 g, FeSO4.7H2O 0.01 g, CaCO3 0.02 g, agar 18 g, H2O to 1000 ml, pH7.2) [43] at 50°C for 3-5 d. Thermophilic Streptomyces strains were cultured in TSB (Oxoid tryptone soya broth powder, 30 g, H2O to 1000 ml)

liquid medium at 45°C for 1 d and genomic DNA was isolated followed the Kirby mix procedure [6]. 16S rRNA genes were amplified by PCR with primers (5′-AGAGTTTGATCCTGGCTCAG-3′ and 5′-TCAGGCTACCTTGTTACGACTT-3′). PCR conditions were: template DNA denatured at 95°C for 5 min, then 95°C 30 s, 55°C 30 s, 72°C 2 min, for 35 cycles. PCR products were cloned in pBluescript II SK and sequenced with its T7 and T3 primers. Strains were inoculated on MS (mannitol 20 g, soya flour 20 g, agar 20 g, H2O to 1000 ml, pH7) medium covered with cellophane disks. After 2 days incubation at 42°C, the cells were fixed with fresh 2% glutaraldehyde (pH7.2) and 1% osmium tetroxide. Spores were examined with a JSM-6360LV scanning electron microscopy (Jeol).