A γ-butyrolactone-sensing activator/repressor, JadR3, controls a regulatory mini-network for jadomycin biosynthesis.

Two regulatory genes, jadR2 and jadR3, in the jadomycin (jad) biosynthetic gene cluster of Streptomyces venezuelae encode homologues of γ-butyrolactone receptor. JadR2 was previously shown to be a pseudo γ-butyrolactone receptor. jadR3 is situated at the upstream of jadW123 encoding putative enzymes for γ-butyrolactone biosynthesis. Disruption of jadR3 resulted in markedly decreased production of jadomycin. Transcriptional analysis revealed that JadR3 represses jadW1, jadR2 and jadR3 but activates jadR1, the key activator gene for jadomycin biosynthesis. DNase I footprinting showed that JadR3 has four binding sites in the intergenic regions of jadR2-jadR1 and jadR3-jadW1. A JadR3 interactive molecule, SVB1, was purified from a large-scale fermentation and its structure found to be the same as SCB3, a γ-butyrolactone from Streptomyces coelicolor, and was absent from a jadW123 mutant lacking jadomycin production. Addition of SVB1 or extract from S. coelicolor to the mutant restored jadomycin production. Overall, our results revealed that the association of JadR3 and SVB1 plays an important role in controlling a regulatory mini-network governing jadomycin biosynthesis, providing new insights into the ways in which γ-butyrolactone/receptor systems modulate antibiotic biosynthesis in Streptomyces.

[Engineering and heterologous expression of a nikkomycin biosynthetic gene cluster].

OBJECTIVE: We expressed a nikkomycin biosynthetic gene cluster in the well-characterized surrogate Streptomyces coelicolor M1146. METHODS: By using PCR-targeting method, we replaced the promoters of sanG and sanF in pNIK, which contains nikkomycin biosynthetic gene cluster, with the hrdB promoter to generate pNIKm. We transferred pNIK and pNIKm into S. coelicolor M1146 by intergeneric conjugation and obtained M1146-NIK and M1146-NIKm, respectively. We then evaluated expression of the gene cluster in the heterologous host by RT-PCR. Furthermore, we also compared the antifugal activity and nikkomycin production of M1146-NIK and M1146-NIKm by bioassay against Alternaria longipes and HPLC analysis. RESULTS: M1146-NIK and M1146-NIKm exhibited antifungal activity, and they can produce a trace amount of nikkomycin X, nikkomycin Z and pseudo-Z. There was a substantial accumulation of uridine in M1146-NIK, whereas substantial accumulations of uridine, ribofuranosyl-4-formyl-4-imidazolone and pyridylhomothreonine were observed in M1146-NIKm. CONCLUSION: We successfully expressed the nikkomycin biosynthetic gene cluster in the heterologous host and identified nikkomycins and some of its key biosynthetic intermediates. This study will provide the basis for enzymatic reaction of the condensation between the two nikkomycin moieties and for the generation of hybrid antibiotics by combinatorial biosynthesis.

Novel nikkomycin analogues generated by mutasynthesis in Streptomyces ansochromogenes.

BACKGROUND: Nikkomycins are competitive inhibitors of chitin synthase and inhibit the growth of filamentous fungi, insects, acarids and yeasts. The gene cluster responsible for biosynthesis of nikkomycins has been cloned and the biosynthetic pathway was elucidated at the genetic, enzymatic and regulatory levels. RESULTS: Streptomyces ansochromogenes ΔsanL was constructed by homologous recombination and the mutant strain was fed with benzoic acid, 4-hydroxybenzoic acid, nicotinic acid and isonicotinic acid. Two novel nikkomycin analogues were produced when cultures were supplemented with nicotinic acid. These two compounds were identified as nikkomycin Px and Pz by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). Bioassays against Candida albicans and Alternaria longipes showed that nikkomycin Px and Pz exhibited comparatively strong inhibitory activity as nikkomycin X and Z produced by Streptomyces ansochromogenes 7100 (wild-type strain). Moreover, nikkomycin Px and Pz were found to be more stable than nikkomycin X and Z at different pH and temperature conditions. CONCLUSIONS: Two novel nikkomycin analogues (nikkomycin Px and Pz) were generated by mutasynthesis with the sanL inactivated mutant of Streptomyces ansochromogenes 7100. Although antifungal activities of these two compounds are similar to those of nikkomycin X and Z, their stabilities are much better than nikkomycin X and Z under different pHs and temperatures.

Improvement of gougerotin and nikkomycin production by engineering their biosynthetic gene clusters.

Nikkomycins and gougerotin are peptidyl nucleoside antibiotics with broad biological activities. The nikkomycin biosynthetic gene cluster comprises one pathway-specific regulatory gene (sanG) and 21 structural genes, whereas the gene cluster for gougerotin biosynthesis includes one putative regulatory gene, one major facilitator superfamily transporter gene, and 13 structural genes. In the present study, we introduced sanG driven by six different promoters into Streptomyces ansochromogenes TH322. Nikkomycin production was increased significantly with the highest increase in engineered strain harboring hrdB promoter-driven sanG. In the meantime, we replaced the native promoter of key structural genes in the gougerotin (gou) gene cluster with the hrdB promoters. The heterologous producer Streptomyces coelicolor M1146 harboring the modified gene cluster produced gougerotin up to 10-fold more than strains carrying the unmodified cluster. Therefore, genetic manipulations of genes involved in antibiotics biosynthesis with the constitutive hrdB promoter present a robust, easy-to-use system generally useful for the improvement of antibiotics production in Streptomyces.

Rapid isolation and purification of inotodiol and trametenolic acid from Inonotus obliquus by high-speed counter-current chromatography with evaporative light scatting detection.

INTRODUCTION: In Eastern Europe, especially Russia, the fruiting body of Inonotus obliquus has been used as a folk medicine for cancer since the sixteenth or seventeenth century. Inotodiol and trametenolic acid are considered to be the main bioactive compounds of the fruiting body of the mushroom. These compounds show various biological activities, including anti-tumour, anti-viral, hypoglycaemic, anti-oxidant and cyto-protective. However, effective methods for isolating and purifying inotodiol and trametenolic acid from the fruiting body of Inonotus obliquus are not currently available. OBJECTIVE: To develop a suitable preparative method in order to isolate inotodiol and trametenolic acid from a complex Inonotus obliquus extract by preparative high-speed counter-current chromatography (HSCCC). METHODOLOGY: Inotodiol and trametenolic acid were rapidly isolated and purified from the chloroform extract of Inonotus obliquus (Fr.) by HSCCC with evaporative light scatting detection (ELSD). The purity of the obtained target compounds was analysed by high-performance liquid chromatography (HPLC) with ELSD. The structures of the two compounds were identified by ¹H NMR and ¹³C NMR. RESULT: The target compounds were finally isolated and purified with a solvent system of hexane:ethyl acetate:methanol:water (1:0.4:1:0.4, v/v/v/v). In a single operation, 100 mg of the I. obliquus extracts yielded 13.0 mg of inotodiol and 7.0 mg of trametenolic acid. The entire separation and purification process took less than 5 h. The purities of obtained inotodiol and trametenolic acid were 97.51 and 94.04%, respectively. CONCLUSION: HSCCC-ELSD was an efficient and rapid method for the separation and purification of inotodiol and trametenolic acid from I. obliquus.

Genome engineering and direct cloning of antibiotic gene clusters via phage ϕBT1 integrase-mediated site-specific recombination in Streptomyces.

Several strategies have been used to clone large DNA fragments directly from bacterial genome. Most of these approaches are based on different site-specific recombination systems consisting of a specialized recombinase and its target sites. In this study, a novel strategy based on phage ϕBT1 integrase-mediated site-specific recombination was developed, and used for simultaneous Streptomyces genome engineering and cloning of antibiotic gene clusters. This method has been proved successful for the cloning of actinorhodin gene cluster from Streptomyces coelicolor M145, napsamycin gene cluster and daptomycin gene cluster from Streptomyces roseosporus NRRL 15998 at a frequency higher than 80%. Furthermore, the system could be used to increase the titer of antibiotics as we demonstrated with actinorhodin and daptomycin, and it will be broadly applicable in many Streptomyces.