Transcriptome analysis of the regulation of natamycin biosynthesis in Streptomyces natalensis HW-2 by fungal elicitor

Background Natamycin is a polyene macrolide polyketide antibiotics and used in 150 countries as a natural food preservative. Streptomyces natalensis is an important producer. Elicitation had been approved to be an effective method to improve the biosynthesis of secondary metabolites. Fungal elicitor from Penicillium chrysogenum AS 3.5163 showed inductive effect on the biosynthesis of natamycin in S. natalensis HW-2 fermentation. However, regarding the global gene expression of natamycin in response to fungal elicitor is not still reported. Results RNA-Seq analysis showed that there were 1265 differential expression genes (DEGs) at 40 h and 2196 DEGs at 80 h. The fungal elicitor had stronger effects on the transcription level of S. natalensis HW-2 at 80 h than that at 40 h. Gene Ontology (GO) enrichment analysis of DEGs showed significant enrichment in biological processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the fungal elicitor mainly affected the expression levels of some genes about cellular process, metabolism and genetic information, especially in pentose phosphate pathway (PPP), glycolytic pathway (EMP) and tricarboxylic acid cycle (TCA). KEGG pathway showed that fungal elicitor had a greater influence on the metabolism of branched-chain amino acids (BCAAs). Among them, 23 DEGs associated with BCAAs metabolism were up-regulated or down-regulated. The supplementation experiment with BCAAs confirmed that 0.2 g/L of L-Ile and 0.5 g/L of L-Val increased natamycin yield by 17.6% and 37.8%, respectively. Fungal elicitor also up-regulated the transcriptional levels of most of the enzymes associated with the biosynthesis of natamycin and two important transcription regulators ( pimR and pimM ). To confirm the accuracy of RNA-Seq, the results of qPCR showed that these gene expression levels were in agreement with the transcription changes by RNA-Seq. Conclusion In this study, the change of transcriptional levels in S. natalensis HW-2 under treated with the fungal elicitor was firstly reported. The major finding of our comparative transcriptome analysis is that the fungal elicitor improves the supply of precursor, and alters the expression of natamycin related genes and regulator of secondary metabolism. From our results, we conclude that regulatory alterations are important factors for the enhancement of natamycin.

Promoter Engineering Reveals the Importance of Heptameric Direct Repeats for DNA Binding byStreptomycesAntibiotic Regulatory Protein–Large ATP-Binding Regulator of the LuxR Family (SARP-LAL) Regulators inStreptomyces natalensis

ABSTRACTThe biosynthesis of small-size polyene macrolides is ultimately controlled by a couple of transcriptional regulators that act in a hierarchical way. AStreptomycesantibiotic regulatory protein–large ATP-binding regulator of the LuxR family (SARP-LAL) regulator binds the promoter of a PAS-LuxR regulator-encoding gene and activates its transcription, and in turn, the gene product of the latter activates transcription from various promoters of the polyene gene cluster directly. The primary operator of PimR, the archetype of SARP-LAL regulators, contains three heptameric direct repeats separated by four-nucleotide spacers, but the regulator can also bind a secondary operator with only two direct repeats separated by a 3-nucleotide spacer, both located in the promoter region of its unique target gene,pimM. A similar arrangement of operators has been identified for PimR counterparts encoded by gene clusters for different antifungal secondary metabolites, including not only polyene macrolides but peptidyl nucleosides, phoslactomycins, or cycloheximide. Here, we used promoter engineering and quantitative transcriptional analyses to determine the contributions of the different heptameric repeats to transcriptional activation and final polyene production. Optimized promoters have thus been developed. Deletion studies and electrophoretic mobility assays were used for the definition of DNA-binding boxes formed by 22-nucleotide sequences comprising two conserved heptameric direct repeats separated by four-nucleotide less conserved spacers. The cooperative binding of PimRSARPappears to be the mechanism involved in the binding of regulator monomers to operators, and at least two protein monomers are required for efficient binding.IMPORTANCEHere, we have shown that a modulation of the production of the antifungal pimaricin inStreptomyces natalensiscan be accomplished via promoter engineering of the PAS-LuxR transcriptional activatorpimM. The expression of this gene is controlled by theStreptomycesantibiotic regulatory protein–large ATP-binding regulator of the LuxR family (SARP-LAL) regulator PimR, which binds a series of heptameric direct repeats in its promoter region. The structure and importance of such repeats in protein binding, transcriptional activation, and polyene production have been investigated. These findings should provide important clues to understand the regulatory machinery that modulates antibiotic biosynthesis inStreptomycesand open new possibilities for the manipulation of metabolite production. The presence of PimR orthologues encoded by gene clusters for different secondary metabolites and the conservation of their operators suggest that the improvements observed in the activation of pimaricin biosynthesis byStreptomyces natalensiscould be extrapolated to the production of different compounds by other species.