Identification of the streptothricin and tunicamycin biosynthetic gene clusters by genome mining in Streptomyces sp. strain fd1-xmd.

The genus Streptomyces have been highly regarded for their important source of natural products. Combined with the technology of genome sequencing and mining, we could identify the active ingredients from fermentation broth quickly. Here, we report on Streptomyces sp. strain fd1-xmd, which was isolated from a soil sample collected in Shanghai. Interestingly, the fermentation broth derived from this strain demonstrated broad-spectrum antimicrobial activity against gram-positive bacteria, gram-negative bacteria, and eukaryotes. To identify the antimicrobial substances and their biosynthetic gene clusters, we sequenced the fd1-xmd strain and obtained a genome 7,929,999 bp in length. The average GC content of the chromosome was 72.5 mol%. Knockout experiments demonstrated that out of eight biosynthetic gene clusters we could identify, two are responsible for the biosynthesis of the antibiotics streptothricin (ST) and tunicamycin (TM). The ST biosynthetic gene cluster from fd1-xmd was verified via successful heterologous expression in Streptomyces coelicolor M1146. ST production had a yield of up to 0.5 g/L after the optimization of culture conditions. This study describes a novel producer of ST and TM and outlines the complete process undertaken for Streptomyces sp. strain fd1-xmd genome mining.

tRNA-Dependent Aminoacylation of an Amino Sugar Intermediate in the Biosynthesis of a Streptothricin-Related Antibiotic.

UNLABELLED: The antibiotic streptothricin (ST) possesses an amino sugar bound to an l-ß-lysine (ß-Lys) residue via a peptide bond. The peptide bond formation has been shown to be catalyzed by a nonribosomal peptide synthetase (NRPS) during ST biosynthesis. The focus of this study is the closely related ST analogue BD-12, which carries a glycine-derived side chain rather than a ß-Lys residue. Here, in Streptomyces luteocolor NBRC13826, we describe our biosynthetic studies of BD-12, which revealed that the peptide bond between the amino sugar and the glycine residue is catalyzed by a Fem-like enzyme (Orf11) in a tRNA-dependent manner rather than by an NRPS. Although there have been several reports of peptide bond-forming tRNA-dependent enzymes, to our knowledge, Orf11 is the first enzyme that can accept an amino sugar as a substrate. Our findings clearly demonstrate that the structural diversity of the side chains of ST-type compounds in nature is generated in an unusual manner via two distinct peptide bond-forming mechanisms. Moreover, the identification and functional analysis of Orf11 resulted in not only the production of new ST-related compounds, but also the provision of new insights into the structure-activity relationship of the ST-related antibiotics. IMPORTANCE: The antibiotic streptothricin (ST) possesses an amino sugar bound to an l-ß-lysine (ß-Lys) side chain via a peptide bond formed by a nonribosomal peptide synthetase (NRPS). BD-12, an analogue of ST, carries a glycine-derived side chain rather than ß-Lys, and here, we describe the BD-12-biosynthetic gene cluster from Streptomyces luteocolor NBRC13826, which contains the orf11 gene encoding a novel tRNA-dependent peptide bond-forming enzyme. The unique Fem-like enzyme (Orf11) accepts the amino sugar as a substrate and mediates the peptide formation between the amino sugar intermediate and glycine. Our studies demonstrate that the structural diversity of the side chains of ST-related compounds in nature is generated via two distinct peptide bond-forming mechanisms.

[Progress in streptothricin antibiotics - A review].

Streptothricins are a group of the earliest discovered antibiotics with broad antimicrobial spectrum, and have been used for crop protection. We reviewed the studies on streptothricin resistance, biosynthesis of the three components (streptolidine, carbamoylated D-glucosamine and poly ß-lysine chain) and chemical synthesis of streptothricins. The important aspects for future streptothricin researches were also discussed.

Biosynthesis of the carbamoylated D-gulosamine moiety of streptothricins: involvement of a guanidino-N-glycosyltransferase and an N-acetyl-D-gulosamine deacetylase.

Streptothricins (STNs) are atypical aminoglycosides containing a rare carbamoylated D-gulosamine (D-GulN) moiety, and the antimicrobial activity of STNs has been exploited for crop protection. Herein, the biosynthetic pathway of the carbamoylated D-GulN moiety was delineated. An N-acetyl-D-galactosamine is first attached to the streptolidine lactam by the glycosyltransferse StnG and then epimerized to N-acetyl-D-gulosamine by the putative epimerase StnJ. After carbamoylation by the carbamoyltransferase StnQ, N-acetyl-D-GulN is deacetylated by StnI to furnish the carbamoylated D-GulN moiety. In vitro studies characterized two novel enzymes: StnG is an unprecedented GT-A fold N-glycosyltransferase that glycosylates the imine nitrogen atom of guanidine, and StnI is the first reported N-acetyl-D-GulN deacetylase.

A stand-alone adenylation domain forms amide bonds in streptothricin biosynthesis.

The streptothricin (ST) antibiotics, produced by Streptomyces bacteria, contain L-ß-lysine ((3S)-3,6-diaminohexanoic acid) oligopeptides as pendant chains. Here we describe three unusual nonribosomal peptide synthetases (NRPSs) involved in ST biosynthesis: ORF 5 (a stand-alone adenylation (A) domain), ORF 18 (containing thiolation (T) and condensation (C) domains) and ORF 19 (a stand-alone A domain). We demonstrate that ST biosynthesis begins with adenylation of L-ß-lysine by ORF 5, followed by transfer to the T domain of ORF 18. In contrast, L-ß-lysine molecules adenylated by ORF 19 are used to elongate an L-ß-lysine peptide chain on ORF 18, a reaction unexpectedly catalyzed by ORF 19 itself. Finally, the C domain of ORF 18 catalyzes the condensation of L-ß-lysine oligopeptides covalently bound to ORF 18 with a freely diffusible intermediate to release the ST products. These results highlight an unusual activity for an A domain and unique mechanisms of crosstalk within NRPS machinery.

Functional analysis of methyltransferases participating in streptothricin-related antibiotic biosynthesis.

Streptothricin (ST) and its related compounds produced by Streptomyces strains are broad-spectrum antibiotics that consist of carbamoylated d-gulosamine, amino-acid side chain, and streptolidine lactam moieties. BD-12, a streptothricin-related antibiotic, has a glycine-derived side chain and two N-methyl groups, whereas ST-F carrying the l-ß-lysine side chain has no methyl group. In our previous studies, we identified and characterized the BD-12 and ST biosynthetic gene clusters. Here we report the functional analysis of two methyltransferase genes (orf 6 and orf 13) in the BD-12 biosynthetic gene cluster. Combinatorial biosynthesis using these two methyltransferase genes and the ST biosynthetic gene cluster resulted in the production of three methylated forms of ST-F. Among them, N,N'-dimethyl-ST-F, a novel compound generated in the present study, showed bacteria-specific antibiotic activities, although ST-F exhibits antibiotic activities against both prokaryotes and eukaryotes. Our findings also demonstrated that the orf 6 and orf 13 genes are responsible for the N-methylations of the amide bonds in the streptolidine lactam and in the amino-acid side chain linkage, respectively, and that N-methyl modification of the streptolidine lactam confers resistance in part against an ST hydrolase, SttH.

Separation of Streptothricin Antibiotics from Culture Broth with Colorimetric Determination Using Dipicrylamine.

Our earlier method for the detection and separation of ε-poly-L-lysine using a yellow anionic dye, the dipicrylamine (DPA-) anion, was herein optimized for streptothricin antibiotics (ST), which contains the ß-lysine oligopeptides moiety, H-[NH-(CH2)3-CH(NH2)-CH2-CO]n-. We then applied this method to the detection and separation of ST in a commercially available nourseothricin, a mixture of ST species with n = 1, 2, 3, and 4. The ST species were precipitated with the DPA- anion. The precipitate was found to consist of the salts of the fully protonated ST species, STz+ (z = n + 1), with the DPA- anion. The ST(DPA)z precipitate was re-dissolved in acetonitrile. The solution was yellowish, and gave an absorption maximum at around 420 nm. Thus, the equivalent concentration of the ST species referred to the charge numbers of STz+ can be determined colorimetrically. By the addition of bis(triphenylphosphoranylidene)ammonium chloride, the ST species could be re-precipitated from the acetonitrile solution as hydrochloride salts. All of the ST species were found in the precipitate with high yields. The method was thus successfully applied to the detection and separation of ST species from the culture broth.

Nourseothricin (streptothricin) inactivated by a plasmid pIE636 encoded acetyl transferase of Escherichia coli: location of the acetyl group.

Escherichia coli strains harbouring the plasmid pIE636 are able to synthesize acetylcoenzyme A: streptothricin acetyltransferase (ACSAT). The (enzymatic) N-acetylation of streptothricin F is known to contribute significantly towards the loss of antibacterial activity. 13C-NMR analysis of [14C]N-acetyl-labelled streptothricin F, produced by ACSAT-catalysed acetylation of streptothricin F and subsequent purification by various chromatographical steps, unequivocally revealed streptothricin F to be acetylated at the beta-amino group (C16) (and not at the epsilon-amino group (C19)).

Biosynthesis of streptolidine moiety of streptothricins by Streptomyces noursei JA 3890b.

The incorporation of uniformly 14C-labeled compounds into the streptothricin-type antibiotic nourseothricin was studied with a strain of Streptomyces noursei JA 3890b. 6.5% of radioactivity from U-14C-L-arginine was incorporated into the antibiotic, while glutamic acid, aspartic acid, alanine, proline, glycine and leucine displayed much lower incorporations. Furhtermore, 95% of the activity incorporated from arginine was located in the streptolidine moiety supporting the suggestion that this subunit of streptothricin antibiotics is formed via the dehydroarginine pathway.

Glycinothricin, a new streptothricin-class antibiotic from Streptomyces griseus.

Glycinothricin is a streptothricin-class antibiotic obtained for the first time from the culture broth of a strain of Streptomyces griseus. Glycinothricin, the deformimino derivative of antibiotic LL-AB664, gives N-methylstreptolidine, N-methyl-D-glucosamine and glycine on acid hydrolysis. In comparison with LL-AB664, glycinothricin is less active against gram-positive and gram-negative bacteria and less toxic to mice.

Antiviral antibiotic S15-1. Taxonomy of the producing strain and study of conditions for production of the antibiotic.

Strain S15-1 which produces antiviral antibiotic S15-1 belonging to the streptothricin group of antibiotics was isolated from a soil sample. Cell analysis, colony morphology, the absence of sporangia-like vesicles, the formation of spores in chains of the Rectus Flexibilis type, and the ability to produce melanoid pigment, indicate that this strain belongs to the genus Streptomyces Waksman and Henrici. A comparison of the characteristics of strain S15-1 with related Streptomyces show that it should be identified as Streptomyces purpeofuscus Yamaguchi and Saburi. The investigation of cultural conditions show that soluble starch and meat extract are the most suitable carbon and organic nitrogen sources for the production of antibiotic S15-1. Strain S15-1 grew poorly on media with no organic nitrogen sources, and did not produce the antibiotic. Antiviral antibiotic S15-1 is accumulated at the highest level after 3 or 4 days growth of the producing organism.

Neo-enactin, a new antifungal antibiotic potentiating polyene antifungal antibiotics. II. Taxonomic studies of the producing microorganism and simultaneous production of bleomycin group and streptothricin-like antibiotics.

A new antifungal antibiotic, named neo-enactin, was produced mainly in the mycelia of strain H 829-MY 10. Strain H 829-MY 10 was identified as a Streptoverticillium, determined to be nonchromogenic, and fits in the white color-series. Although Streptoverticillium olivoreticuli is known to be chromogenic, strain H 829-MY 10 is most related to that species. Thus, strain H 829-MY 10 is named as Streptoverticillium olivoreticuli subsp. neoenacticus. Besides neo-enactin, two bleomycin-group antibiotics and two streptothricin-like antibiotics were simultaneously produced by strain H 829-MY 10.

Model aided dynamic process analysis and optimization for the nourseothricin fermentation.

The relation between product formation and growth kinetics could be characterized by two facts: the specific product formation rate depends on the ageing of the population and on the specific growth rate. These relation was formulated and quantified by a mathematical model, which was fitted to experimental data of a representative fermentation run und used to predict an optimal fermentation mode. In the result of this discussion cyclic fed batch fermentation was found to be optimal.

Metabolism of phosphate-limited cultures of Streptomyces. IV. Protein-phosphorylation of antibiotics-producing cultures.

It has been demonstrated that in the cellular proteins of Streptomyces hygroscopicus JA 6599 and Streptomyces noursei JA 3890 b, the producers of the antibiotics turimycin and nourseothricin, respectively, phosphorylated proteins are present. Numbers and concentrations of phosphorylated proteins decreased during the idiophase as characterized by phosphate limitation, antibiotic biosynthesis and phosphatase formation. Phosphoamino acids of serine, threonine and tyrosine were found in the hydrolysates of proteins. Protein tyrosyl kinase was demonstrated in the cellular extracts. The results supports the hypothesis that protein phosphorylation possesses a function in the regulation of growth and secondary product formation.

In situ monitoring of streptothricin production by Streptomyces rochei F20 in soil and rhizosphere.

The onset of streptothricin (ST) biosynthesis in Streptomyces rochei F20 was studied by using reverse transcription-PCR (RT-PCR) to detect transcripts of ST genes during growth in liquid medium, soil, and the rhizosphere. In situ results correlated with those obtained in vitro, illustrating the growth phase-dependent manner of ST production by F20. Maximal transcription of ST resistance (sttR) and biosynthesis (sttA) genes occurred during the transition between the exponential and stationary phases of growth, when the specific growth rate (micro) started to decline. A higher level of gene expression of sttR versus sttA was observed in all experiments. In liquid culture, maximal transcript accumulation of the sttA gene was only ca. 40% that of the sttR gene. sttA and sttR mRNAs were detected in soil containing approximately 10(6) CFU of growing cells g of soil(-1). sttR mRNA was detected in sterile and nonsterile rhizosphere colonized with growing mycelium of F20 at 1.2 x 10(6) and 4.0 x 10(5) CFU g of soil(-1), respectively. However, neither sttR nor sttA transcripts were detected by RT-PCR in the rhizoplane, which supported a lower population density of F20 than the rhizosphere.

[Metabolism of phosphate-limited Streptomyces cultures. III. The ambivalent effect of phosphates in nourseothricin-producing cultures of Streptomyces noursei JA 3890b]. / Untersuchungen zum Stoffwechsel phosphatlimitierter Streptomycetenkulturen. III. Mitteilung: Uber die ambivalente Wirkung des Phosphates in Nourseothricin-bildenden Kulturen von Streptomyces noursei JA 3890b.

A common condition in the evolution of organisms and their metabolism seems to be a latent lack of available phosphate in the natural environment. Accordingly, the phosphate dependent metabolisms of the soil-living streptomycetes should be stamped by lack of phosphate, too. The biosynthesis of the streptothricin antibiotic nourseothricin by Streptomyces noursei 3890b is initiated by limitation of soluble phosphate in the fermentation medium. At the other side is shown that a certain rate of feeding of phosphate during the fermentation increases the nourseothricin biosynthesis. An ambivalente role of phosphate on the secondary metabolite biosynthesis is stated. The limitation of phosphate leads to a special physiological state of the producer, characterized by secondary product formation and dephosphorylating activities in cells. This state is temporally stabilized by the presence of a sufficient phosphate supply, realized by enzymatic hydrolysis of complex phosphate-containing substrates or by a direct feeding of inorganic phosphate to the fermentations. The occurrence of different physiological states in respect to the phosphate-dependent metabolism is described by S-shaped functions of the relationship between specific growth rate and the phosphate concentration in the medium. The special behaviour of Streptomyces noursei cells at phosphate limitation is discussed to be the result of the dephosphorylating activities in cells, hydrolyzing phosphoester-bonds of regulatory metabolites as well as energy-rich compounds.

Directed selection of differentiation mutants of Streptomyces noursei using chemostat cultivation.

A nourseothricin-producing Streptomyces noursei strain was continuously cultivated in a chemostat equipped with a stirrer for mechanical fractionation of the mycelium. Different cultivation conditions allowed the selection of six types of differentiation mutants after the culture had reached a population genetically stationary state. The mutants showed an altered control pattern of sporulation as well as altered antibiotic biosynthesis and antibiotic resistance. In addition, the stability of the recombinant plasmid pIJ385 in several differentiation type mutants as host strains was tested. The results suggest that there exists a strong correlation between the cultivation conditions employed and the type of differentiation mutants selected.

Influence of inorganic phosphate on the lipid synthesis of a phosphate-deregulated mutant of Streptomyces noursei.

Phosphate-dependent changes of the mycelial lipid composition were studied in the streptothricin-producing parental strain Streptomyces noursei JA 3890 b/2 and its mutant RG 2. In contrast to its ancestor, the mutant was capable of producing the antibiotic nourseothricin even when large quantities of inorganic phosphate were present in the medium. The apparent insensitivity of the secondary metabolism to phosphate inhibition corresponds to a decreased level of phospholipids in the presence of excessive inorganic phosphate and, during phosphate limitation, to a much higher production of the alkaline phosphatases. A model is discussed which proposed the control by a common genetic element of both the phospholipid and antibiotic production.