An intricate regulation of WblA controlling production of silent tylosin analogues and abolishment of expressible nikkomycin.

Genome sequencing has revealed that actinomycetes possess the potential to produce many more secondary metabolites than previously thought. The existing challenge is to devise efficient methods to activate these silent biosynthetic gene clusters (BGCs). In Streptomyces ansochromogenes, disruption of wblA, a pleiotropic regulatory gene, activated the expression of cryptic tylosin analogues and abolished nikkomycin production simultaneously. Overexpressing pathway-specific regulatory genes tylR1 and tylR2 can also trigger the biosynthesis of silent tylosin analogues, in which TylR1 exerted its function via enhancing tylR2 expression. Bacterial one-hybrid system experiments unveiled that WblA directly inhibits the transcription of tylR1 and tylR2 to result in the silence of tylosin analogues BGC. Furthermore, WblA can activate the nikkomycin production through up-regulating the transcription of pleiotropic regulatory gene adpA. More interestingly, AdpA can activate sanG (an activator gene in nikkomycin BGC) but repress wblA. Our studies provide a valuable insight into the complex functions of pleiotropic regulators.

Activation of Cryptic Antibiotic Biosynthetic Gene Clusters Guided by RNA-seq Data from Both Streptomyces ansochromogenes and ΔwblA.

With the increase of drug resistance caused by the improper use and abuse of antibiotics, human beings are facing a global health crisis. Sequencing of Streptomyces genomes revealed the presence of an important reservoir of secondary metabolic gene clusters for previously unsuspected products with potentially valuable bioactivity. It has therefore become necessary to activate these cryptic pathways through various strategies. Here, we used RNA-seq data to perform a comparative transcriptome analysis of Streptomyces ansochromogenes (wild-type, WT) and its global regulatory gene disruption mutant ΔwblA, in which some differentially expressed genes are associated with the abolished nikkomycin biosynthesis and activated tylosin analogue compounds (TACs) production, and also with the oviedomycin production that is induced by the genetic manipulation of two differentially expressed genes (san7324 and san7324L) encoding RsbR. These results provide a significant clue for the discovery of new drug candidates and the activation of cryptic biosynthetic gene clusters.

Molecular mechanism of mureidomycin biosynthesis activated by introduction of an exogenous regulatory gene ssaA into Streptomyces roseosporus.

Mureidomycins (MRDs), a group of unique uridyl-peptide antibiotics, exhibit antibacterial activity against the highly refractory pathogen Pseudomonas aeruginosa. Our previous study showed that the cryptic MRD biosynthetic gene cluster (BGC) mrd in Streptomyces roseosporus NRRL 15998 could not be activated by its endogenous regulator 02995 but activated by an exogenous activator SsaA from sansanmycin's BGC ssa of Streptomyces sp. strain SS. Here we report the molecular mechanism for this inexplicable regulation. EMSAs and footprinting experiments revealed that SsaA could directly bind to a 14-nt palindrome sequence of 5'-CTGRCNNNNGTCAG-3' within six promoter regions of mrd. Disruption of three representative target genes (SSGG-02981, SSGG-02987 and SSGG-02994) showed that the target genes directly controlled by SsaA were essential for MRD production. The regulatory function was further investigated by replacing six regions of SSGG-02995 with those of ssaA. Surprisingly, only the replacement of 343-450 nt fragment encoding the 115-150 amino acids (AA) of SsaA could activate MRD biosynthesis. Further bioinformatics analysis showed that the 115-150 AA situated between two conserved domains of SsaA. Our findings significantly demonstrate that constitutive expression of a homologous exogenous regulatory gene is an effective strategy to awaken cryptic biosynthetic pathways in Streptomyces.

Co-expression of a SARP Family Activator ChlF2 and a Type II Thioesterase ChlK Led to High Production of Chlorothricin in Streptomyces antibioticus DSM 40725.

Chlorothricin (CHL), produced by Streptomyces antibioticus DSM 40725 (wild-type strain, WT), belongs to a growing family of spirotetronate antibiotics that have biological activities inhibiting pyruvate carboxylase and malate dehydrogenase. ChlF2, a cluster-situated SARP regulator, can activate the transcription of chlJ, chlC3, chlC6, chlE1, chlM, and chlL to control CHL biosynthesis. Co-expression of chlF2 and chlK encoding type II thioesterase in WT strain under the control of P kan led to high production of chlorothricin by 840% in comparison with that of WT. Since the inhibitory activity of CHL against several Gram-positive bacteria is higher than des-CHL, combinatorial strategies were applied to promote the conversion of des-CHL to CHL. Over-expression of chlB4, encoding a halogenase, combining with the supplementation of sodium chloride led to further 41% increase of CHL production compared to that of F2OE, a chlF2 over-expression strain. These findings provide new insights into the fine-tuned regulation of spirotetronate family of antibiotics and the construction of high-yield engineered strains.

Component Optimization of Neomycin Biosynthesis via the Reconstitution of a Combinatorial Mini-Gene-Cluster in Streptomyces fradiae.

Neomycin, a multicomponent aminoglycoside antibiotic, is mainly utilized in livestock husbandry and feed additives in animals. The antimicrobial potency of the main product neomycin B is higher than that of its stereoisomer neomycin C. However, the content of neomycin C as an impurity in the high-producing strain is relatively high, and its isolation or removal from neomycin B is quite difficult, which influences the widespread application of neomycin. In this work, the essential genes responsible for neomycin biosynthesis were evaluated and overexpressed to reduce the content of neomycin C. Among them, neoG and neoH are two novel regulatory genes for neomycin biosynthesis, aphA is a resistance gene, neoN encoding a radical SAM-dependent epimerase is responsible for the conversion of neomycin C to B using SAM as the cofactor, and metK is a SAM synthetase coding gene. We demonstrated that the reconstitution and overexpression of a mini-gene-cluster (PkasO*-neoN-metK-PkasO*-neoGH-aphA) could effectively reduce the accumulation of neomycin C from 19.1 to 12.7% and simultaneously increase neomycin B by ∼13.1% in the engineered strain Sf/pKCZ04 compared with the wild-type strain (Sf). Real-time quantitative polymerase chain reaction analysis revealed the remarkable up-regulation of the neoE, neoH, neoN, and metK genes situated in the mini-gene-cluster. The findings will pave a new path for component optimization and the large-scale industrial production of significant commercial antibiotics.

Important role of a LAL regulator StaR in the staurosporine biosynthesis and high-production of Streptomyces fradiae CGMCC 4.576.

Staurosporine, belonging to indolocarbazole compounds, is regarded as an excellent lead compound for synthesizing antitumor agents as a potent inhibitor against various protein kinases. In this study, two separate clusters (cluster A and cluster B), corresponding to biosyntheses of K-252c (staurosporine aglycone) and sugar moiety, were identified in Streptomyces fradiae CGMCC 4.576 and heterologously expressed in Streptomyces coelicolor M1146 separately or together. StaR, a cluster-situated LAL family regulator, activates staurosporine biosynthesis by binding to the promoter regions of staO-staC and staG-staN. The conserved sequences GGGGG and GCGCG were found through gradually truncating promoters of staO and staG, and further determined by mutational experiments. Overexpression of staR with the supplementation of 0.01 g L-1 FeSO4 increased staurosporine production to 5.2-fold compared with that of the parental strain Streptomyces fradiae CGMCC 4.576 in GYM medium. Our results provided an approach for improvement of staurosporine production mediated by a positive regulator and established the basis for dissecting the regulatory mechanisms of other indolocarbazole compounds with clinical application value.

Enhancement of neomycin production by engineering the entire biosynthetic gene cluster and feeding key precursors in Streptomyces fradiae CGMCC 4.576.

Neomycin, an aminoglycoside antibiotic, is widely used in the livestock husbandry due to its higher antimicrobial activity and availability of feed additives in animals. However, its production yield is relatively low and cannot meet the needs of developing market and clinical application. Here, the entire natural neo cluster was cloned from Streptomyces fradiae CGMCC 4.576 by φBT1 integrase-mediated site-specific recombination. Then, the rational reconstruction of the neo cluster was performed by using λ-Red-mediated PCR targeting for improving neomycin production. In order to coordinate with this attempt, the supplementation of suitable precursors was carried out. The constructed recombinant strain Sf/pKCZ03 has multi-copy of the neo cluster modified by disrupting the negative regulatory gene neoI and replacing the native promoter of the neoE-D with PkasO*. Compared to the yield (1282 mg/L) of Streptomyces fradiae CGMCC 4.576, the engineered strain Sf/pKCZ03 had a 36% enhancement of neomycin production. Quantitative real-time PCR analysis revealed the increased transcription of structural genes (neoE, neoB, neoL, aacC8) and regulatory genes (neoR, neoH) in Sf/pKCZ03. Additionally, under the supplementation of 1 g/L N-acetyl-D-glucosamine and 5 g/L L-glutamine, the yield of engineered strain Sf/pKCZ03 showed 62% and 107% improvements compared to that of the wild-type strain in the original medium, respectively. These findings demonstrated that engineering the antibiotic gene cluster in combination with precursors feeding was an effective approach for strain improvement, and would be potentially extended to other Streptomyces for large-scale production of commercialized antibiotics.