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330 PROCEEDINGS OF INTERNATIONAL SCIENTIFIC CONFERENCE ON APPLIED BIOTECHNOLOGYof glucose (30 g/L), soybean meal (30 g/L), pharmamedia (5 g/L), yeast extract (1 g/L) and CaCO3 (3 g/L) [5]. A. hibisca P172-2 was incubated at 28%u00b0C, at 200 rpm for 7 days.2.2. Ribosome engineering of A. hibisca P172-2Ribosome engineering of A. hibisca P172-2 was done as described previously by Xu et al. [13]. To determine the minimum inhibitory concentration (MIC) of streptomycin against A. hibisca P172-2, spore suspensions (106 spores) were spread onto MS plates containing streptomycin at concentrations ranging from 0 to 50 %u03bcg/mL. After incubation at 28%u00b0C for 5 days, the MIC was determined as 20 %u03bcg/mL. Subsequently, spore suspensions (109 spores) were plated on MS plates with streptomycin concentrations of 200 %u03bcg/mL. Mutant colonies displaying the most dark-red pigment were selected.2.3. Analysis of streptomycin mutations in the rpsL geneA. hibisca P172-2 was harvested after incubating in ISP2 broth at 28%u00b0C, 200 rpm for 3 days. Total DNA was extracted using standard protocols as described by Nybo et al. [14]. PCR was performed for the analysis of streptomycin mutations in the rpsL gene using Promega master mix with two primers: rpsL-F: 5%u2019-GGCCGACAAACAGAACGT-3%u2019 and rpsL-R: 5%u2019-GTTCACCAACTGGGTGAC-3%u2019. PCR product was cloned in a pGEMT-easy vector and then was sequenced to detect the mutation of the rpsL gene.2.4. Pradimicin A production and extractionTo evaluate pradimicin A production, A. hibisca wild-type and mutant strains were cultured in 50 mL production medium at 28 %u00b0C for 7 days. The pH of the culture broth of these strains was adjusted to 2.0 using 5 N HCl, and then the culture broth was extracted with 100 mL of n-butanol and methanol (100:1).