Important role of a LAL regulator Sta R 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-252 c(staurosporine aglycone) and sugar moiety, were identified in Streptomyces fradiae CGMCC 4.576 and heterologously expressed in Streptomyces coelicolor M1146 separately or together. Sta R, a cluster-situated LAL family regulator, activates staurosporine biosynthesis by binding to the promoter regions of sta O-sta C and sta G-sta N. The conserved sequences GGGGG and GCGCG were found through gradually truncating promoters of sta O and sta G, and further determined by mutational experiments. Overexpression of sta R with the supplementation of 0.01 g L^–1 Fe SO4 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.

Multi-omics Data Reveal the Effect of Sodium Butyrate on Gene Expression and Protein Modification in Streptomyces

Streptomycetes possess numerous gene clusters and the potential to produce a large amount of natural products.Histone deacetylase(HDAC)inhibitors play an important role in the regulation of histone modifications in fungi,but their roles in prokaryotes remain poorly understood.Here,we investigated the global effects of the HDAC inhibitor,sodium butyrate(SB),on marine-derived Streptomyces olivaceus FXJ 8.021,particularly focusing on the activation of secondary metabolite biosynthesis.The antiSMASH analysis revealed 33 secondary metabolite biosynthetic gene clusters(BGCs)in strain FXJ 8.021,among which the silent lobophorin BGC was activated by SB.Transcriptomic data showed that the expression of genes involved in lobophorin biosynthesis(ge00097–ge00139)and CoA-ester formation(e.g.,ge02824),as well as the glycolysis/gluconeogenesis pathway(e.g.,ge01661),was significantly up-regulated in the presence of SB.Intracellular CoA-ester analysis confirmed that SB triggered the biosynthesis of CoA-ester,thereby increasing the precursor supply for lobophorin biosynthesis.Further acetylomic analysis revealed that the acetylation levels on 218 sites of 190 proteins were up-regulated and those on 411 sites of 310 proteins were down-regulated.These acetylated proteins were particularly enriched in transcriptional and translational machinery components(e.g.,elongation factor GE04399),and their correlations with the proteins involved in lobophorin biosynthesis were established by protein–protein interaction network analysis,suggesting that SB might function via a complex hierarchical.

An intricate regulation of Wbl A 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 wbl A,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 Wbl A directly inhibits the transcription of tylR1 and tylR2 to result in the silence of tylosin analogues BGC.Furthermore,Wbl A can activate the nikkomycin production through up-regulating the transcription of pleiotropic regulatory gene adp A.More interestingly,Adp A can activate san G(an activator gene in nikkomycin BGC)but repress wbl A.Our studies provide a valuable insight into the complex functions of pleiotropic regulators.

Microbial quorum sensing signaling molecules and their roles in the biosynthesis of natural products

Microorganisms act as a double-edged sword that can bring benefits and diseases to humans.Their physiological and metabolic processes are under precise control of intricate regulatory networks,in which signaling systems are critical for cells coordinating or competing with each other to overcome unfavorable environmental conditions,including the prompt and acute responses to endogenous or exogenous stimuli.

Enhancement of salinomycin production by ribosome engineering in Streptomyces albus

Dear Editor,Streptomyces can produce a large variety of secondary metabolites as a major source of anti-infective, antitumor or immune-suppressive agents widely applied in clinical treatment. Antibiotics-resistant bacteria are spreading at alarming rates.

Biosynthesis and molecular regulation of secondary metabolites in microorganisms

Antibiotics are most important compounds in microbial secondary metabolites.As we know,streptomycetes are a particularly abundant source of antibiotics and related compounds,providing more than half of medically important antimicrobial and antitumor agents.Various environmental and physiological conditions influence the onset and level of antibiotic production.Because of improper use and abuse of antibiotics as well as the horizontal transfer of antibiotic resistance genes between bacteria by conjugation,transduction or transformation, these have led to the appearance of antibiotic resistance and the loss of antibiotic native efficiency.

Reconstruction of a hybrid nucleoside antibiotic gene cluster based on scarless modification of large DNA fragments

Genetic modification of large DNA fragments(gene clusters) is of great importance in synthetic biology and combinatorial biosynthesis as it facilitates rational design and modification of natural products to increase their value and productivity.In this study,we developed a method for scarless and precise modification of large gene clusters by using RecET/RED-mediated polymerase chain reaction(PCR) targeting combined with Gibson assembly.In this strategy,the biosynthetic genes for peptidyl moieties(HPHT) in the nikkomycin biosynthetic gene cluster were replaced with those for carbamoylpolyoxamic acid(CPOAA)from the polyoxin biosynthetic gene cluster to generate a^40 kb hybrid gene cluster in Escherichia coli with a reusable targeting cassette.The reconstructed cluster was introduced into Streptomyces lividans TK23 for heterologous expression and the expected hybrid antibiotic,polynik A,was obtained and verified.This study provides an efficient strategy for gene cluster reconstruction and modification that could be applied in synthetic biology and combinatory biosynthesis to synthesize novel bioactive metabolites or to improve antibiotic production.

Neomycin biosynthesis is regulated positively by AfsA-g and NeoR in Streptomyces fradiae CGMCC 4.7387

Neomycins are a group of aminoglycoside antibiotics with both clinical and agricultural applications.To elucidate the regulatory mechanism of neomycin biosynthesis,we completed draft genome sequencing of a neomycin producer Streptomyces fradiae CGMCC 4.7387 from marine sediments,and the neomycin biosynthesis gene cluster was identified.Inactivation of the afsA-g gene encoding a γ-butyrolactone(GBL) synthase in S.fradiae CGMCC 4.7387 resulted in a significant decrease of neomycin production.Quantitative RT-PCR analysis revealed that the transcriptional level of neoR and the aphA-neoGH operon were reduced in the afsA-g::aac(3)Ⅳ mutant.Interestingly,a conserved binding site of AdpA,a key activator in the GBL regulatory cascade,was discovered upstream of neoR,a putative regulatory gene encoding a protein with an ATPase domain and a tetratricopeptide repeat domain.When neoR was inactivated,the neomycin production was reduced about 40%in comparison with the WT strain.Quantitative RT-PCR analysis revealed that the transcriptional levels of genes in the aphA-neoGH operon were reduced clearly in the neoR::aac(3)Ⅳ mutant.Finally,the titers of neomycin were improved considerably by overexpression of qfsA-gand neoR in S.fradiae CGMCC 4.7387.

Establishment of a Genetic Transformation System and Its Application in Thermoanaerobacter tengcongensis

The whole-genome sequence of Thermoanaerobacter tengcongensis, an anaerobic thermophilic bacterium isolated from the Tengchong hot spring in China, was completed in 2002. However, in vivo studies on the genes of this strain have been hindered in the absence of genetic manipulation system. In order to establish such a system, the plasmid pBOL01 containing the replication origin of the T. tengcongensis chromosome and a kanamycin resistance cassette, in which kanamycin resistance gene expression was controlled by the tte1482 promoter from T. tengcongensis, was constructed and introduced into T. tengcongensis via electroporation. Subsequently, the high transformation efficiency occurred when using freshly cultured T. tengcongensis cells without electroporation treatment, suggesting that T. tengcongensis is naturally competent under appropriate growth stage. A genetic transformation system for this strain was then established based on these important components, and this system was proved to be available for studying physiological characters of T. tengcongensis in vivo by means of hisG gene disruption and complementation.

Biosynthesis and combinatorial biosynthesis of antifungal nucleoside antibiotics

There is an urgent need for new antifungal agents to treat or combat fungal infection in humans and plants.Antifungal nucleoside antibiotics are an important family of natural products with distinctive structural features.Understanding their biosynthetic machinery is of great importance for the improvement of antibiotics titers.More importantly,it is a requisite for combinatorial biosynthesis to create hybrid nucleoside antibiotics.We herein focus on findings on the natural and designed biosynthesis of this important family of nucleoside antibiotics.

A widespread response of Gram-negative bacterial acyl-homoserine lactone receptors to Gram-positive Streptomyces γ-butyrolactone signaling molecules

Cell-cell communication is critical for bacterial survival in natural habitats,in which miscellaneous regulatory networks are encompassed.However,elucidating the interaction networks of a microbial community has been hindered by the population complexity.This study reveals thatγ-butyrolactone(GBL)molecules from Streptomyces species,the major antibiotic producers,can directly bind to the acyl-homoserine lactone(AHL)receptor of Chromobacterium violaceum and influence violacein production controlled by the quorum sensing(QS)system.Subsequently,the widespread responses of more Gram-negative bacterial AHL receptors to Gram-positive Streptomyces signaling molecules are unveiled.Based on the cross-talk between GBL and AHL signaling systems,combinatorial regulatory circuits(CRC)are designed and proved to be workable in Escherichia coli(E.coli).It is significant that the QS systems of Gram-positive and Gram-negative bacteria can be bridged via native Streptomyces signaling molecules.These findings pave a new path for unlocking the comprehensive cell-cell communications in microbial communities and facilitate the exploitation of innovative regulatory elements for synthetic biology.

Molecular mechanism of mureidomycin biosynthesis activated by introduction of an exogenous regulatory gene ssa A 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 Ssa A 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 Ssa A 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 Ssa Awere essential for MRD production. The regulatory function was further investigated by replacing six regions of SSGG-02995 with those of ssa A.Surprisingly, only the replacement of 343–450 nt fragment encoding the 115–150 amino acids(AA) of Ssa A could activate MRD biosynthesis. Further bioinformatics analysis showed that the 115–150 AA situated between two conserved domains of Ssa A.Our findings significantly demonstrate that constitutive expression of a homologous exogenous regulatory gene is an effective strategy to awaken cryptic biosynthetic pathways in Streptomyces.

SCO3129,a TetR family regulator,is responsible for osmotic stress in Streptomyces coelicolor

Streptomyces are the soil-dwelling bacteria with a complex lifecycle and a considerable ability to produce a variety of secondary metabolites.Osmoregulation is important for their lifecycle in nature.In the genome of Streptomyces coelicolor M145,SCO3128(encodes a putative fatty acid desaturase),SCO3129(encodes a putative TetR family regulator)and SCO3130(encodes a putative L-carnitine dehydratase)constitute a transcriptional unit,and its transcript was found to be in response to osmotic stress.Disruption of SCO3130 led to a bald phenotype on MMG medium and the mycelia lysis on the edge of the colony when KCl/NaCl was added to the medium.These results indicated that SCO3130 is important for the osmotic stress resistance in S.coelicolor.Transcriptional analysis and electrophoretic mobility shift assays(EMSA)demonstrated that SCO3129 repressed the transcription of SCO3128-3130 operon through directly binding to the promoter region of SCO3128,indicating that SCO3129 regulates the transcription of SCO3128-3130 in response to osmotic stress.

Transcriptomic Analysis of Thermoanaerobacter tengcongensis Grown at Different Temperatures by RNA Sequencing

Genome sequencing of more than 80 thermophiles has pro- vided powerful databases for the preliminary determination of genes essential to thermal adaptation （Berka et al., 2003; Berezovsky and Shakhnovich, 2005; Andrews et al., 2010）. A laboratory strain of Thermoanaerobacter tengcongensis MB4 has been shown to grow at temperatures over the range 50℃ to 80℃, with an optimum of 75℃ （Xue et al., 2001） and its thermal adaptation mechanisms have been studied with the sequencing of its genome （Bao et al., 2002）. Using two- dimensional gel electrophoresis （2DE） and Matrix-Assisted Laser Desorption/Ionization Time of Flight （MALDI-TOF/ TOF） mass spectrometry, Wang et al.