Rabelomycin

Strain improvement of Streptomyces dengpaensis XZHG99T was performed by combined UV mutagenesis and ribosome engineering, as well as fermentation optimization for enhanced angucycline production (rabelomycin and saquayamycin B1). First, four streptomycin-resistant mutants were obtained after screening of UV mutagenesis and ribosome engineering. Then a rpsL mutant (HTT7) with higher productivity of rabelomycin and saquayamycin B1 was selected according to genetic screening and HPLC/LC-MS analyses, whose maximum titers of rabelomycin and saquayamycin B1 were 3.6 ± 0.02 mg/L and 7.5 ± 0.04 mg/L, respectively, about fourfold higher than those produced by XZHG99T. Next, fermentation optimization of HTT7 was successively carried out by single-factor experiments in shake flasks. The titers of rabelomycin and saquayamycin B1 were increased to 11.2 ± 0.04 mg/L and 20.5 ± 0.02 mg/L after optimization of shake flask fermentation conditions, respectively, which was increased about sixfold compared with those produced by XZHG99T. Finally, the titers of rabelomycin and saquayamycin B1 reached 15.7 ± 0.05 mg/L and 39.9 ± 0.05 mg/L after the scaled-up fermentation, which was 7.8-fold and 11.4-fold higher than those produced by XZHG99T, respectively. These data demonstrate that the combined empirical strain-breeding approaches are still an effective and convenient pathway to improve strain production ability.

The bacteria of the genus Streptomyces are among the most important producers of biologically active secondary metabolites. Moreover, recent genomic sequence data have shown their enormous genetic potential for new natural products, although many new biosynthetic gene clusters (BGCs) are silent. Therefore, efficient and stable genome modification techniques are needed to activate their production or to manipulate their biosynthesis towards increased production or improved properties. We have recently developed an efficient markerless genome modification system for streptomycetes based on positive blue/white selection of double crossovers using the bpsA gene from indigoidine biosynthesis, which has been successfully applied for markerless deletions of genes and BGCs. In the present study, we optimized this system for markerless insertion of large BGCs. In a pilot test experiment, we successfully inserted a part of the landomycin BGC (lanFABCDL) under the control of the ermEp* promoter in place of the actinorhodin BGC (act) of Streptomyces lividans TK24 and RedStrep 1.3. The resulting strains correctly produced UWM6 and rabelomycin in twice the yield compared to S. lividans strains with the same construct inserted using the PhiBT1 phage-based integration vector system. Moreover, the system was more stable. Subsequently, using the same strategy, we effectively inserted the entire BGC for mithramycin (MTM) in place of the calcium-dependent antibiotic BGC (cda) of S. lividans RedStrep 1.3 without antibiotic-resistant markers. The resulting strain produced similar levels of MTM when compared to the previously described S. lividans RedStrep 1.3 strain with the VWB phage-based integration plasmid pMTMF. The system was also more stable. KEY POINTS: · Optimized genome editing system for markerless insertion of BGCs into Streptomyces genomes · Efficient heterologous production of MTM in the stable engineered S. lividans strain.

Actinomycete strain YIM PH20352, isolated from the rhizosphere soil sample of Panax notoginseng collected in WenShang, Yunnan Province, China, exhibited antifungal activity against some phytopathogenic fungi. The structures of bioactive molecules, isolated from the ethyl acetate extract of the fermentation broth of the strain, were identified as rabelomycin (1) and dehydrorabelomycin (2) based on extensive spectroscopic analyses. Compound 1 exhibited antifungal activity against four tested root-rot pathogens of the Panax notoginseng including Plectosphaerella cucumerina, Alternaria panax, Fusarium oxysporum, and Fusarium solani with the MIC values at 32, 64, 128, and 128 μg/mL, respectively. Compound 2 exhibited antifungal activity against F. oxysporum, P. cucumerina, F. solani, and A. panax with the MIC values at 64, 64, 128, and 128 μg/mL, respectively. Based on the phylogenetic analyses, the closest phylogenetic relative of strain YIM PH20352 is Streptomyces cellulosae NBRC 13027 T (AB184265) (99.88%), so strain YIM PH20352 was identified as Streptomyces cellulosae. To the best of our knowledge, this is the first report of rabelomycin and rabelomycin-type antibiotics from Streptomyces cellulosae and their antifungal activity against root-rot pathogens of the Panax notoginseng.