Bioinformatics analysis of Med-ORF12 and HRMS spectra of two shunt products.

Figure A: Phylogenetic tree established using more homologues of Med-ORF12. These entries include proteins involved in biosynthesis of secondary metabolites: Med-ORF12 and Med-ORF6 (for MED 2 in Streptomyces sp AM-7161), ActVI-ORF1 and ActIII (for ACT 1 in S. coelicolor A3(2)), DauB (for aklaviketone in Streptomyces sp.), Gra-ORF5 and Gra-ORF6 (for GRA 3 in Streptomyces violaceoruber, AveF (for avermectin in Streptomyces avermitilis), DnrU(for daunorubicin in Streptomyces peucetius), Sa10 (for indigoidine/auricin in Streptomyces aureofaciens), 3HAD (3HAD_B) from human heart and a hypothetical protein WP_019763579 (encoded by a cluster highly in Streptomyces sp Wigar10, homologous to medermycin cluster). Additionally, several highly homologous proteins proposed to encode hydroxylacyl-CoA dehydrogenases were also analyzed: WP_033529872 from Streptomyces galbus (identity to Med-ORF12: 80%), WP_030735501from Streptomyces sp. (66%), WP_043385010 from Streptomyces mutabilis (66%), WP_030080416 from Streptomyces sp. (65%), WP_042193116 from Kibdelosporangium sp. (65%) and WP_030666394 from Streptomyces cellulosae (64%). The bar indicated the evolutionary distance. The numbers on branch nodes were percentages of 1000 sets of bootstrap supports. All homologies are divided into two families (3HAD: 3-hydroxyacyl-CoA dehydrogenase protein family; SDR: short-chain alcohol dehydrogenases family) and further into three groups. Figure B: Comparison of metabolite production between the wild type strain WT/pIJ8600 (A) and med-ORF12-deficient strain MS/pIJ8600 (B and C). Crude extracts isolated from the wild type and mutant strains respectively were subjected to HPLC analysis, indicated as UV absorption at 434 nm. The Y axils of B and C (both for mutant strain) were adjusted into different scales for better comparison with the wild type strain. In a contrast to the wild type strain (WT/pIJ8600, A), the mutant strain (MS/pIJ8600) could not produce MED 2 due to the deficiency of med-ORF12, but could produce many intermediates or shunt products (12–25 min), which were not present in the wild type strain. Among of them, two major components (deduced to be DMAC 5 and aloesaponarin II 6) were further analyzed in next experiments. Figure C: HRMS analysis of aloesaponarin II 6 from the control strain CH999/pRM5 (A and C) and med-ORF12-deficient strain MS/pIJ8600 (B and D). The parent ions were detected in A and B (HR-MS, A and B) using atmospheric pressure chemical ionization (APCI-), and then further fragmented (HR-MS2, C and D). Figure D: HRMS analysis of DMAC 5 from the control strain CH999/pRM5 (A) and med-ORF12-deficient strain MS/pIJ8600 (B). The parent ions were detected in A and B (HR-MS, APCI-).
(DOCX)