To quantitate the productivity of actinorhodin, equal amounts of spores of M145 and 4F containing pCWH74 were inoculated into R2YE liquid medium

lacking KH2PO4 and CaCl2, and 1 ml culture was harvested in a time-course. As shown in Figure 4, actinorhodin was produced in 4F at both 30 and 37°C, earlier than in M145 at 30°C. At 100 h, productivity of actinorhodin in 4F at 30°C was ~2.8 times higher than in M145 at 30°C. Strains M145 and 4F grew better in TSB than in R2YE liquid media (data no shown), but no actinorhodin was detected when cultured in TSB medium at 30 and 37°C. Growth curves of the two VX-680 molecular weight strains in R2 lacking KH2PO4 and CaCl2 at 30°C showed that their biomass values were similar from 20 to 120 hours (data not shown). Thus, better growth of M145 and 4F in TSB medium (Figure 3) did not correlate with delayed and less production of actinorhodin in R2YE medium (Figure 4). Like in 4F, M145 produced more actinorhodin in R2YE medium at 30°C than at 37°C, suggesting that expression of the actinorhodin biosynthetic genes might be temperature-dependent. Temperature-dependent antibiotic gene clusters have been reported in Streptomyces, for example, much higher productivity Smad phosphorylation of validamycin A produced by Streptomyces hygroscopicus was found at 37°C than at 30°C [40]. We infer that by replacement of thermophilic-specific promoters, many single genes and especially antibiotic

genes clusters of mesophilic Streptomyces should be heterologously expressed in the fast-growing and thermophilic Streptomyces. Heterologous expression of the anthramycin biosynthetic gene cluster of the

thermophilic S. refuineus subsp. thermotolerans in strain 4F Expression of the anthramycin biosynthetic genes of S. refuineus subsp. thermotolerans could be detected at high temperature (i.e. 47°C), but not at 30 or 37°C [22]. An integrating cosmid, 024COA-3, containing the whole anthramycin biosynthetic gene cluster was introduced by conjugation from E. coli into strain 4F. PCR amplification experiments confirmed the presence of the anthramycin genes in the clone of 4F. Aldehyde dehydrogenase After culturing in AP1 medium at 30, 37 and 47°C for 24 h, mycelium was extracted, dried and re-dissolved in MeOH. Thin-layer chromatography, followed by a bio-assay by overlaying with LB agar containing as indicator strain a Bacillus sp., revealed a zone of growth inhibition on 4F at 47°C, but no inhibition zone was found at 30 and 37°C (data not shown). A spot on a TLC plate was further purified for HPLC-MS analysis. As shown in Figure 5, an anthramycin-specific peak (ES+ = 316 Dalton, see ref [41]) was detected. Thus the anthramycin biosynthetic gene cluster of the thermophilic S. refuineus subsp. thermotolerans was heterologously expressed in strain 4F. We introduced the same cosmid 024COA-3 containing the anthramycin gene cluster into strain 2C, but no transformants were obtained.