Validamycin C

Paired homologs of γ-butyrolactone (GBL) biosynthesis gene afsA and GBL receptor gene arpA are located at different positions in genome of Streptomyces hygroscopicus 5008. Inactivation of afsA homologs dramatically decreased biosynthesis of validamycin, an important anti-fungal antibiotic and a critical substrate for antidiabetic drug synthesis, and the deletion of arpA homologs increased validamycin production by 26% (ΔshbR1) and 20% (ΔshbR3). By double deletion, the ΔshbR1/R3 mutant showed higher transcriptional levels of adpA-H (the S. hygroscopicus ortholog of the global regulatory gene adpA) and validamycin biosynthetic genes, and validamycin production increased by 55%. Furthermore, by engineering a high-producing industrial strain via tandem deletion of GBL receptor genes, validamycin production and productivity were enhanced from 19 to 24 g/L (by 26%) and from 6.7 to 9.7 g/L(-1) d(-1) (by 45%), respectively, which was the highest ever reported. The strategy demonstrated here may be useful to engineering other Streptomyces spp. with multiple pairs of afsA-arpA homologs.

Background:Streptomyces hygroscopicus 5008 has been used for the production of the antifungal validamycin/jinggangmycin for more than 40 years. A high yield of validamycin is achieved by culturing the strain at 37°C, rather than at 30°C for normal growth and sporulation. The mechanism(s) of its thermo-regulated biosynthesis was largely unknown.Results:The 10,383,684-bp genome of strain 5008 was completely sequenced and composed of a linear chromosome, a 164.57-kb linear plasmid, and a 73.28-kb circular plasmid. Compared with other Streptomyces genomes, the chromosome of strain 5008 has a smaller core region and shorter terminal inverted repeats, encodes more α/β hydrolases, major facilitator superfamily transporters, and Mg2+/Mn2+-dependent regulatory phosphatases. Transcriptomic analysis revealed that the expression of 7.5% of coding sequences was increased at 37°C, including biosynthetic genes for validamycin and other three secondary metabolites. At 37°C, a glutamate dehydrogenase was transcriptionally up-regulated, and further proved its involvement in validamycin production by gene replacement. Moreover, efficient synthesis and utilization of intracellular glutamate were noticed in strain 5008 at 37°C, revealing glutamate as the nitrogen source for validamycin biosynthesis. Furthermore, a SARP-family regulatory gene with enhanced transcription at 37°C was identified and confirmed to be positively involved in the thermo-regulation of validamycin production by gene inactivation and transcriptional analysis.Conclusions:Strain 5008 seemed to have evolved with specific genomic components to facilitate the thermo-regulated validamycin biosynthesis. The data obtained here will facilitate future studies for validamycin yield improvement and industrial bioprocess optimization.

Validamycin, known to interfere with fungal energy metabolism by inhibiting trehalase, has been extensively used to control plant diseases caused by Rhizoctonia spp. However, the effect of validamycin on controlling Fusarium graminearum has not been previously reported. In this study, when applied to F. graminearum in vitro, validamycin inhibited the synthesis of deoxynivalenol (DON), which is a mycotoxin and virulence factor, by decreasing trehalase activity and the production of glucose and pyruvate, which are precursors of DON biosynthesis. Because FgNTH encodes the main trehalase in F. graminearum, these effects were nullified in the FgNTH deletion mutant ΔFgNTH but restored in the complemented strain ΔFgNTHC. In addition, validamycin also increased the expression of pathogenesis-related genes (PRs) PR1, PR2, and PR5 in wheat, inducing resistance responses of wheat against F. graminearum. Therefore, validamycin exhibits dual efficacies on controlling Fusarium head blight (FHB) caused by F. graminearum: inhibition of DON biosynthesis and induction of host resistance. In addition, field trials further confirmed that validamycin increased FHB control and reduced DON contamination in grain. Control of FHB and DON contamination by validamycin increased when the antibiotic was applied with the triazole fungicide metconazole. Overall, this study is a successful case from foundational research to applied research, providing useful information for wheat protection programs against toxigenic fungi responsible for FHB and the consequent mycotoxin accumulation in grains.