Enhancing Ristomycin A Production by Overexpression of ParB-Like StrR Family Regulators Controlling the Biosynthesis Genes.

Amycolatopsis sp. strain TNS106 harbors a ristomycin-biosynthetic gene cluster (asr) in its genome and produces ristomycin A. Deletion of the sole cluster-situated StrR family regulatory gene, asrR, abolished ristomycin A production and the transcription of the asr genes orf5 to orf39. The ristomycin A fermentation titer in Amycolatopsis sp. strain TNS106 was dramatically improved by overexpression of asrR and a heterologous StrR family regulatory gene, bbr, from the balhimycin-biosynthetic gene cluster (BGC) utilizing strong promoters and multiple gene copies. Ristomycin A production was improved by approximately 60-fold, resulting in a fermentation titer of 4.01 g/liter in flask culture, in one of the engineered strains. Overexpression of AsrR and Bbr upregulated transcription of tested asr biosynthetic genes, indicating that these asr genes were positively regulated by AsrR and Bbr. However, only the promoter region of the asrR operon and the intergenic region upstream of orf12 were bound by AsrR and Bbr in gel retardation assays, suggesting that AsrR and Bbr directly regulated the asrR operon and probably orf12 to orf14 but no other asr biosynthetic genes. Further assays with synthetic short probes showed that AsrR and Bbr specifically bound not only probes containing the canonical inverted repeats but also a probe with only one 7-bp element of the inverted repeats in its native context. AsrR and Bbr have an N-terminal ParB-like domain and a central winged helix-turn-helix DNA-binding domain. Site-directed mutations indicated that the N-terminal ParB-like domain was involved in activation of ristomycin A biosynthesis and did not affect the DNA-binding activity of AsrR and Bbr. IMPORTANCE This study showed that overexpression of either a native StrR family regulator (AsrR) or a heterologous StrR family regulator (Bbr) dramatically improved ristomycin A production by increasing the transcription of biosynthetic genes directly or indirectly. The conserved ParB-like domain of AsrR and Bbr was demonstrated to be involved in the regulation of asr BGC expression. These findings provide new insights into the mechanism of StrR family regulators in the regulation of glycopeptide antibiotic biosynthesis. Furthermore, the regulator overexpression plasmids constructed in this study could serve as valuable tools for strain improvement and genome mining for new glycopeptide antibiotics. In addition, ristomycin A is a type III glycopeptide antibiotic clinically used as a diagnostic reagent due to its side effects. The overproduction strains engineered in this study are ideal materials for industrial production of ristomycin A.

Trichlorination of a Teicoplanin-Type Glycopeptide Antibiotic by the Halogenase StaI Evades Resistance.

Glycopeptide antibiotics (GPAs) include clinically important drugs used for the treatment of infections caused by Gram-positive pathogens. These antibiotics are specialized metabolites produced by several genera of actinomycete bacteria. While many GPAs are highly chemically modified, A47934 is a relatively unadorned GPA lacking sugar or acyl modifications, common to other members of the class, but which is chlorinated at three distinct sites. The biosynthesis of A47934 is encoded by a 68-kb gene cluster in Streptomyces toyocaensis NRRL 15009. The cluster includes all necessary genes for the synthesis of A47934, including two predicted halogenase genes, staI and staK In this study, we report that only one of the halogenase genes, staI, is necessary and essential for A47934 biosynthesis. Chlorination of the A47934 scaffold is important for antibiotic activity, as assessed by binding affinity for the target N-acyl-d-Ala-d-Ala. Surprisingly, chlorination is also vital to avoid activation of enterococcal and Streptomyces VanB-type GPA resistance through induction of resistance genes. Phenotypic assays showed stronger induction of GPA resistance by the dechlorinated compared to the chlorinated GPA. Correspondingly, the relative expression of the enterococcal vanA resistance gene was shown to be increased by the dechlorinated compared to the chlorinated compound. These results provide insight into the biosynthesis of GPAs and the biological function of GPA chlorination for this medically important class of antibiotic.

More than just recruitment: the X-domain influences catalysis of the first phenolic coupling reaction in A47934 biosynthesis by Cytochrome P450 StaH.

Glycopeptide antibiotic biosynthesis involves a complex cascade of reactions centred on a non-ribosomal peptide synthetase and modifiying proteins acting in trans, such as Cytochrome P450 enzymes. These P450s are responsible for cyclisation of the peptide via cross-linking aromatic amino acid side chains, which are a hallmark of the glycopeptide antibiotics. Here, we analysed the first cyclisation reaction in the biosynthesis of the glycopeptide antibiotic A47934. Our results demonstrate that the P450 StaH is recruited to the NRPS machinery through interaction with the X-domain present in the last A47934 NRPS module. We determined the crystal structure of StaH and showed that it is responsible for the first cyclisation in A47934 biosynthesis and additionally exhibits flexible substrate specificity. Our results further point out that the X-domain has an impact on the efficiency of the in vitro cyclisation reaction: hybrid PCP-X constructs obtained by domain exchange between A47934 and teicoplanin biosynthesis NRPS modules reveal that the X-domain from A47934 leads to decreased P450 activity and alternate stereochemical preference for the substrate peptide. We determined that a tight interaction between StaH and the A47934 X-domain correlates with decreased in vitro P450 activity: this highlights the need for glycopeptide antibiotic cyclisation to be a dynamic system, with an overly tight interaction interfering with substrate turnover in vitro.

Crystal structure of StaL, a glycopeptide antibiotic sulfotransferase from Streptomyces toyocaensis.

Over the past decade, antimicrobial resistance has emerged as a major public health crisis. Glycopeptide antibiotics such as vancomycin and teicoplanin are clinically important for the treatment of Gram-positive bacterial infections. StaL is a 3'-phosphoadenosine 5'-phosphosulfate-dependent sulfotransferase capable of sulfating the cross-linked heptapeptide substrate both in vivo and in vitro, yielding the product A47934, a unique teicoplanin-class glycopeptide antibiotic. The sulfonation reaction catalyzed by StaL constitutes the final step in A47934 biosynthesis. Here we report the crystal structure of StaL and its complex with the cofactor product 3'-phosphoadenosine 5'-phosphate. This is only the second prokaryotic sulfotransferase to be structurally characterized. StaL belongs to the large sulfotransferase family and shows higher similarity to cytosolic sulfotransferases (ST) than to the bacterial ST (Stf0). StaL has a novel dimerization motif, different from any other STs that have been structurally characterized. We have also applied molecular modeling to investigate the binding mode of the unique substrate, desulfo-A47934. Based on the structural analysis and modeling results, a series of residues was mutated and kinetically characterized. In addition to the conserved residues (Lys(12), His(67), and Ser(98)), molecular modeling, fluorescence quenching experiments, and mutagenesis studies identified several other residues essential for substrate binding and/or activity, including Trp(34), His(43), Phe(77), Trp(132), and Glu(205).

PCR screening reveals unexpected antibiotic biosynthetic potential in Amycolatopsis sp. strain UM16.

AIMS: To assess the antibiotic biosynthetic potential of Amycolatopsis sp. strain UM16 and eight other Amycolatopsis species. METHODS AND RESULTS: Amycolatopsis genomic DNA was screened by PCR for the glycopeptide, Type-II (aromatic) polyketide and ansamycin biosynthetic gene clusters. Amycolatopsis sp. strain UM16, which exhibits weak antitubercular activity, was shown to have the glycopeptide oxyB gene and the Type-II (aromatic) polyketide-synthase KSalpha-KSbeta tandem gene pair, but not the AHBA synthase gene. The ristocetin (glycopeptide) producer, Amycolatopsis lurida NRRL 2430(T), was shown to have the oxyB gene and the Type-II polyketide-synthase KSalpha-KSbeta tandem gene pair. Amycolatopsis alba NRRL 18532(T) was shown to have the glycopeptide oxyB gene and the AHBA synthase gene. Phylogenetic analyses using Amycolatopsis oxyB and KSalpha-KSbeta gene sequences were conducted. CONCLUSIONS: Amycolatopsis sp. strain UM16 appears to have the biosynthetic potential to produce glycopeptide and Type-II polyketide antibiotics, but not ansamycins. The potential to synthesize aromatic polyketides may be more widely distributed in Amycolatopsis than is currently recognized. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR screening is a very useful tool for rapidly identifying the biosynthetic potential of an antibiotic-producing actinomycete isolate. Advanced knowledge of the type of antibiotic(s) produced will allow appropriate methods to be selected for antibiotic purification.

Comparative analysis and insights into the evolution of gene clusters for glycopeptide antibiotic biosynthesis.

The bal, cep, dbv, sta and tcp gene clusters specify the biosynthesis of the glycopeptide antibiotics balhimycin, chloroeremomycin, A40926, A47934 and teicoplanin, respectively. These structurally related compounds share a similar mechanism of action in their inhibition of bacterial cell wall formation. Comparative sequence analysis was performed on the five gene clusters. Extensive conserved synteny was observed between the bal and cep clusters, which direct the synthesis of very similar compounds but originate from two different species of the genus Amycolatopsis. All other cluster pairs show a limited degree of conserved synteny, involving biosynthetically functional gene cassettes: these include those involved in the synthesis of the carbon backbone of two non-proteinogenic amino acids; in the linkage of amino acids 1--3 and 4--7 in the heptapeptide; and in the formation of the aromatic cross-links. Furthermore, these segments of conserved synteny are often preceded by conserved intergenic regions. Phylogenetic analysis of protein families shows several instances in which relatedness in the chemical structure of the glycopeptides is not reflected in the extent of the relationship of the corresponding polypeptides. Coherent branchings are observed for all polypeptides encoded by the syntenous gene cassettes. These results suggest that the acquisition of distinct, functional genetic elements has played a significant role in the evolution of glycopeptide gene clusters, giving them a mosaic structure. In addition, the synthesis of the structurally similar compounds A40926 and teicoplanin appears as the result of convergent evolution.

Assembling the glycopeptide antibiotic scaffold: The biosynthesis of A47934 from Streptomyces toyocaensis NRRL15009.

The glycopeptide antibiotics vancomycin and teicoplanin are vital components of modern anti-infective chemotherapy exhibiting outstanding activity against Gram-positive pathogens including members of the genera Streptococcus, Staphylococcus, and Enterococcus. These antibiotics also provide fascinating examples of the chemical and associated biosynthetic complexity exploitable in the synthesis of natural products by actinomycetes group of bacteria. We report the sequencing and annotation of the biosynthetic gene cluster for the glycopeptide antibiotic from Streptomyces toyocaensis NRRL15009, the first complete sequence for a teicoplanin class glycopeptide. The cluster includes 34 ORFs encompassing 68 kb and includes all of the genes predicted to be required to synthesize and regulate its biosynthesis. The gene cluster also contains ORFs encoding enzymes responsible for glycopeptide resistance. This role was confirmed by insertional inactivation of the d-Ala-d-lactate ligase, vanAst, which resulted in the predicted -sensitive phenotype and impaired antibiotic biosynthesis. These results provide increased understanding of the biosynthesis of these complex natural products.

Inhibition of sporulation, glycopeptide antibiotic production and resistance in Streptomyces toyocaensis NRRL 15009 by protein kinase inhibitors.

Production of the glycopeptide antibiotic A47934 by Streptomyces toyocaensis NRRL 15009 begins in the late exponential phase in liquid culture and peaks in the early stationary phase. The pattern of cellular phosphoprotein production changes upon onset of A48934 production with the appearance of several novel phosphoproteins only when an antibiotic is being produced. Phosphoamino acid analysis revealed that S. toyocaensis NRRL 15009 produces proteins phosphorylated on His, Ser, Thr and Tyr, with most being membrane-associated. Addition of the isoflavones genistein or quercetin abolishes A47934 production in liquid culture and sporulation on solid medium. Furthermore, genistein slows the onset of inducible glycopeptide antibiotic resistance in S. toyocaensis NRRL 15009. These results support the participation of protein kinase pathways in A47934 biosynthesis and resistance and cell differentiation in S. toyocaensis NRRL 15009.

Biosynthetic studies on antibiotic A47934.

A47934, a peptide antibiotic produced by Streptomyces toyocaensis, belongs to the glycopeptide class of compounds which includes ristocetin and vancomycin. Incorporation studies with radioisotope-labeled substrates indicated that tyrosine, p-hydroxyphenylglycine, p-hydroxyphenylglyoxylate, acetate, and sulfate were efficiently incorporated into A47934. This is consistent with the reported biosynthesis of other glycopeptide antibiotics. Prototrophic mutants blocked in antibiotic biosynthesis were isolated at a low frequency (0.4%) after mutagenesis. Secretor-convertor pairings of the 36 mutants obtained demonstrated that they belonged to three classes: two groups of secretor-convertor pairs and a larger group of mutants that did not make antibiotic under any condition tested. Neither the secretor-convertor studies not supplementation of the cultures with putative biosynthetic intermediates was useful in identifying the location of the biosynthetic blocks. All studies to determine the timing of the sulfate addition step in the biosynthesis indicated that the sulfate is added prior to the formation of intermediates that possess antimicrobial activity.

A47934, a novel glycopeptide-aglycone antibiotic produced by a strain of Streptomyces toyocaensis taxonomy and fermentation studies.

A47934, a novel glycopeptide-aglycone antibiotic, is produced by a strain of Streptomyces toyocaensis, NRRL 15009. A47934 is unique among reported glycopeptides in that it contains a sulfate ester. Like several other glycopeptides, the majority of the A47934 produced remained associated with the producing biomass, from which it could be released into aqueous media by alkalization. Antibiotic biosynthesis was depressed when initial levels of phosphate phosphorus in the medium exceeded the normal level of 35 micrograms/ml. Enrichment of the fermentation medium with tyrosine depressed A47934 yields while enrichment with p-hydroxyphenylglycine or p-hydroxyphenylglyoxylic acid stimulated antibiotic biosynthesis.

[Selection of the medium for the simultaneous synthesis of an antibiotic, protease and pigment by a Nocardia fructiferi culture]. / Podbor sredy dlia odnovremennogo sinteza kul'turoi Nocardia fructiferi antibiotika, proteaz i pigmenta.

Using method of mathematical planning of experiment the culture medium ensuring the simultaneous intensive production of ristomycin, protease and violet pigment by Nocardia fructiferi has been worked out.

[Properties of the extracellular proteases of Nocardia fructiferi var. ristomycini]. / Izuchenie nekotorykh svoistv vnekletochnykh proteaz Nocardia fructiferi var. ristomycini.

A complex of proteases was isolated from the culture fluid of Nocardia fructiferi var. ristomycini, strain 76. The complex showed caseinolytic activity and was capable of coagulating milk for 30 minutes. It was stable at a temperature of 50 degrees C and pH 8.0. The maximum level of casein hydrolysis was observed at 37 degrees C and pH 8.0. The protease complex preserved its properties for 3 months at 4 degrees C and for 1 month at room temperature.

[Effect of various carbon and nitrogen sources on the biosynthesis of ristomycin, protease and pigments by a culture of Nocardia fructiferi var. ristomycini]. / Vliianie razlichnykh istochnikov ugleroda i azota na biosintez ristomitsina, proteazy i pigmentov kul'turoi Nocardia fructiferi var. ristomycini.

The effect of various sources of carbon and nitrogen on the biosynthesis of ristomycin, protease and pigments by Nocardia fructiferi was studied. It was shown that the carbon sources had the most significant effect on the biosynthesis of the antibiotic. The maximum biosynthetic activity of the Nocardia was observed in the medium containing 1-2 per cent of soybean meal and 2 per cent of glycerol. Under such conditions all the three biologically active substances formed. The contents of ristomycin, protease and pigments amounted to 562-649 microgram/ml, 26-30 PU/ml and 0.45-0.63 conditional units, respectively.

[Action of novobiocin on the capacity of Nocardia fructiferi var. ristomycini for ristomycin biosynthesis]. / Deistvie novobiotsina na sposobnost' Nocardia fructiferi var. ristomycini k biosintezu ristomitsina.

The effect of novobiocin in concentrations of 0.05 and 0.2--0.3 microgram/ml on morphological variation of N. fructifer and its capacity for ristomycin production was studied. It was found that the number of colonies with the maximum activity increased under the effect of novobiocin used in a concentration of 0.05 microgram/ml. An increase in the level of novobiocin in the medium up to 0.2--0.3. microgram/ml markedly increased the number of the colonies with low antibiotic productivity.