Comparative metabolomics reveals the mechanism of avermectin production enhancement by <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine

It was found that S-adenosylmethionine (SAM) could effectively improve avermectin titer with 30–60 μg/mL addition to FH medium. To clearly elucidate the mechanism of SAM on intracellular metabolites of Streptomyces avermitilis, a GC–MS-based comparative metabolomics approach was carried out. First, 230 intracellular metabolites were identified and 14 of them remarkably influenced avermectin biosynthesis were discriminative biomarkers between non-SAM groups and SAM-treated groups by principal components analysis (PCA) and partial least squares (PLS). Based on further key metabolic pathway analyses, these biomarkers, such as glucose, oxaloacetic acid, fatty acids (in soybean oil), threonine, valine, and leucine, were identified as potentially beneficial precursors and added in medium. Compared with single-precursor feeding, the combined feeding of the precursors and SAM markedly increased the avermectin titer. The co-feeding approach not only directly verified our hypothesis on the mechanism of SAM by comparative metabolomics, but also provided a novel strategy to increase avermectin production.     

The ROK-family regulator Rok7B7 directly controls carbon catabolite repression,antibiotic biosynthesis,and morphological development in Streptomyces avermitilis

Carbon catabolite repression (CCR) is a common phenomenon in bacteria that modulates expression of genes involved in uptake of alternative carbon sources. In the filamentous streptomycetes, which produce half of all known antibiotics, the precise mechanism of CCR is yet unknown. We report here that the ROK-family regulator Rok7B7 pleiotropically controls xylose and glucose uptake, CCR, development, as well as production of the macrolide antibiotics avermectin and oligomycin A in Streptomyces avermitilis. Rok7B7 directly repressed structural genes for avermectin biosynthesis, whereas it activated olmRI, the cluster-situated activator gene for oligomycin A biosynthesis. Rok7B7 also directly repressed the xylose uptake operon xylFGH, whose expression was induced by xylose and repressed by glucose. Both xylose and glucose served as Rok7B7 ligands. rok7B7 deletion led to enhancement and reduction of avermectin and oligomycin A production, respectively, relieved CCR of xylFGH, and increased co-uptake efficiency of xylose and glucose. A consensus Rok7B7-binding site, 5′-TTKAMKHSTTSAV-3′, was identified within aveA1p, olmRIp, and xylFp, which allowed prediction of the Rok7B7 regulon and confirmation of 11 additional targets involved in development, secondary metabolism, glucose uptake, and primary metabolic processes. Our findings will facilitate methods for strain improvement, antibiotic overproduction, and co-uptake of xylose and glucose in Streptomyces species.     

Stimulation of avermectin B1a biosynthesis in Streptomyces avermilitis by feeding glucose and propionate

The biosynthetic ability of avermectin B1a in Streptomyces avermilitis was improved with propionate addition and glucose feeding. The results showed that B1a production was increased by 12.8–13.8% through supplement of 0.8% propionate at 24 h of cultivation. A stronger stimulation on B1a biosynthesis in S. avermilitis was observed by 3.0% glucose feeding at 5 d of cultivation. The B1a biosynthesis could be further enhanced by repeated glucose fed-batch process in a 10-l bench-top fermentor. A maximal B1a concentration of 780.0 mg/l was obtained by repeated 1.0% glucose addition on 4 d, 5 d and 6 d, which was 2.1-fold higher than that in a control fermentation. Corresponding with this, an additional 6.8% increase of B1a proportion was observed in comparison with the control process. This stimulation on avermectin B1a production was obvious even in a 2000-l fermentor under suitable control of aeration.     

Structural characterization of BVU_3255, a methyltransferase from human intestine antibiotic resistant pathogen Bacteroides vulgatus

Methylation is important for various cellular activities. To date, there is no report of any methyltransferase structure from the human intestine antibiotic resistant pathogen Bacteroides vulgatus. The protein BVU_3255 from B. vulgatus ATCC 8482 belongs to a SAM-dependent methyltransferase. Here, we report the crystal structure of apo BVU_3255, and its complexes with SAM and SAH, which revealed a typical class I Rossmann Fold Methyltransferase. Isothermal titration calorimetric studies showed that both SAM and SAH bind with equal affinity. The structural and sequence analysis suggested that BVU_3255 is a small molecule methyltransferase and involved in methylating the intermediates in ubiquinone biosynthesis pathway.     

Overexpression of the ABC transporter AvtAB increases avermectin production in Streptomyces avermitilis

Avermectins are 16-membered macrocyclic polyketides with potent antiparasitic activities, produced by Streptomyces avermitilis. Upstream of the avermectin biosynthetic gene cluster, there is the avtAB operon encoding the ABC transporter AvtAB, which is highly homologous to the mammalian multidrug efflux pump P-glycoprotein (Pgp). Inactivation of avtAB had no effect, but increasing the concentration of avtAB mRNA 30-500-fold, using a multi-copy plasmid in S. avermitilis, enhanced avermectin production about two-fold both in the wild-type and in a high-yield producer strain on agar plates. In liquid industrial fermentation medium, the overall productivity of avermectin B1a in the engineered high-yield producer was improved for about 50%, from 3.3 to 4.8?g/l. In liquid YMG medium, moreover, the ratio of intracellular to extracellular accumulation of avermectin B1a was dropped from 6:1 to 4.5:1 in response to multiple copies of avtAB. Additionally, the overexpression of avtAB did not cause any increased expression of the avermectin biosynthetic genes through RT-PCR analysis. We propose that the AvtAB transporter exports avermectin, and thus reduces the feedback inhibition on avermectin production inside the cell. This strategy may be useful for enhancing the production of other antibiotics.     

Comparison of the Efficacies of Chloromethane, Methionine, and S-Adenosylmethionine as Methyl Precursors in the Biosynthesis of Veratryl Alcohol and Related Compounds in Phanerochaete chrysosporium

The effect on veratryl alcohol production of supplementing cultures of the lignin-degrading fungus Phanerochaete chrysosporium with different methyl-(sup2)H(inf3)-labelled methyl precursors has been investigated. Both chloromethane (CH(inf3)Cl) and l-methionine caused earlier initiation of veratryl alcohol biosynthesis, but S-adenosyl-l-methionine (SAM) retarded the formation of the compound. A high level of C(sup2)H(inf3) incorporation into both the 3- and 4-O-methyl groups of veratryl alcohol occurred when either l-[methyl-(sup2)H(inf3)]methionine or C(sup2)H(inf3)Cl was present, but no significant labelling was detected when S-adenosyl-l-[methyl-(sup2)H(inf3)]methionine was added. Incorporation of C(sup2)H(inf3) from C(sup2)H(inf3)Cl was strongly antagonized by the presence of unlabelled l-methionine; conversely, incorporation of C(sup2)H(inf3) from l-[methyl-(sup2)H(inf3)]methionine was reduced by CH(inf3)Cl. These results suggest that l-methionine is converted either directly or via an intermediate to CH(inf3)Cl, which is utilized as a methyl donor in veratryl alcohol biosynthesis. SAM is not an intermediate in the conversion of l-methionine to CH(inf3)Cl. In an attempt to identify the substrates for O methylation in the metabolic transformation of benzoic acid to veratryl alcohol, the relative activities of the SAM- and CH(inf3)Cl-dependent methylating systems on several possible intermediates were compared in whole mycelia by using isotopic techniques. 4-Hydroxybenzoic acid was a much better substrate for the CH(inf3)Cl-dependent methylation system than for the SAM-dependent system. The CH(inf3)Cl-dependent system also had significantly increased activities toward both isovanillic acid and vanillyl alcohol compared with the SAM-dependent system. On the basis of these results, it is proposed that the conversion of benzoic acid to veratryl alcohol involves para hydroxylation, methylation of 4-hydroxybenzoic acid, meta hydroxylation of 4-methoxybenzoic acid to form isovanillic acid, and methylation of isovanillic acid to yield veratric acid.     

Mutational analysis of Encephalitozoon cuniculi mRNA cap (guanine-N7) methyltransferase, structure of the enzyme bound to sinefungin, and evidence that cap methyltransferase is the target of sinefungin's antifungal activity

Cap (guanine-N7) methylation is an essential step in eukaryal mRNA synthesis and a potential target for antiviral, antifungal, and antiprotozoal drug discovery. Previous mutational and structural analyses of Encephalitozoon cuniculi Ecm1, a prototypal cellular cap methyltransferase, identified amino acids required for cap methylation in vivo, but also underscored the nonessentiality of many side chains that contact the cap and AdoMet substrates. Here we tested new mutations in residues that comprise the guanine-binding pocket, alone and in combination. The outcomes indicate that the shape of the guanine binding pocket is more crucial than particular base edge interactions, and they highlight the contributions of the aliphatic carbons of Phe-141 and Tyr-145 that engage in multiple van der Waals contacts with guanosine and S-adenosylmethionine (AdoMet), respectively. We purified 45 Ecm1 mutant proteins and assayed them for methylation of GpppA in vitro. Of the 21 mutations that resulted in unconditional lethality in vivo,14 reduced activity in vitro to < or = 2% of the wild-type level and 5 reduced methyltransferase activity to between 4 and 9% of wild-type Ecm1. The natural product antibiotic sinefungin is an AdoMet analog that inhibits Ecm1 with modest potency. The crystal structure of an Ecm1-sinefungin binary complex reveals sinefungin-specific polar contacts with main-chain and side-chain atoms that can explain the 3-fold higher affinity of Ecm1 for sinefungin versus AdoMet or S-adenosylhomocysteine (AdoHcy). In contrast, sinefungin is an extremely potent inhibitor of the yeast cap methyltransferase Abd1, to which sinefungin binds 900-fold more avidly than AdoHcy or AdoMet. We find that the sensitivity of Saccharomyces cerevisiae to growth inhibition by sinefungin is diminished when Abd1 is overexpressed. These results highlight cap methylation as a principal target of the antifungal activity of sinefungin.     

S-Adenosylmethionine (SAM) and antibiotic biosynthesis: effect of external addition of SAM and of overexpression of SAM biosynthesis genes on novobiocin production in Streptomyces

The production of antibiotics in different Streptomyces strains has been reported to be stimulated by the external addition of S-adenosylmethionine (SAM) and by overexpression of the SAM synthetase gene metK. We investigated the influence of SAM addition, and of the expression of SAM biosynthetic genes, on the production of the aminocoumarin antibiotic novobiocin in the heterologous producer strain Streptomyces coelicolor M512 (nov-BG1). External addition of SAM did not influence novobiocin accumulation. However, overexpression of a SAM synthase gene stimulated novobiocin formation, concomitant with an increase of the intracellular SAM concentration. Streptomyces genomes contain orthologs of all genes required for the SAM cycle known from mammals. In contrast, most other bacteria use a different cycle for SAM regeneration. Three secondary metabolic gene clusters, coding for the biosynthesis of structurally very different antibiotics in different Streptomyces strains, were found to contain an operon comprising all five putative genes of the SAM cycle. We cloned one of these operons into an expression plasmid, under control of a strong constitutive promoter. However, transformation of the heterologous novobiocin producer strain with this plasmid did not stimulate novobiocin production, but rather showed a detrimental effect on cell viability in the stationary phase and strongly reduced novobiocin accumulation.     

Effect of the phosphorus concentration on novobiocin formation by the producer Act. spheroides]

According to the literature data biosynthesis of novobiocin by Act. spheroides unlike other antibiotics does not practically depend on the phosphorus levels in the medium. In the present paper it is shown that production of novobiocin in natural media is sensitive to the concentration of mineral phosphorus in the medium. The optimal concentration of phosphorus for biosynthesis of novobiocin is almost within the same ranges as that for biosynthesis of streptomycin, tetracyclines and oleandomycin.     

Oleandomycin and its natural and synthetic analogs. II. Structure and properties of oleandomycin B, a new compound of the oleandomycin group

The revealed regularities of mass spectroscopic disintegration of oleandomycin and its derivatives made it possible to determine analytic criteria for identification of compounds related by their structure to oleandomycin. Analysis of the extracts from oleandomycin fermentation broth filtrates on the basis of the selected group of diagnostic ions showed that along with the main antibiotic there formed during the biosynthesis oleandomycin B, a structurally close minor component. The structure of the substance was assigned and its physico-chemical and biological properties were studied.     

Regulation of sinefungin biosynthesis by the wild-type strain and mutants of Streptomyces incarnatus.

Sinefungin, an antifungal and antiparasitic antibiotic, is produced efficiently from ammonium citrate by prototrophic strains of Streptomyces incarnatus. The regulation of the biosynthesis of this nucleoside, composed of adenosine and ornithine, was studied by using auxotrophic mutants and a resting-cell system. Mutants blocked in arginine synthesis were not able to produce sinefungin. A uridine-negative mutant produced sinefungin in the presence of ATP, but this production was strongly inhibited when amino acids of the urea cycle were added. The same mutant produced sinefungin from aspartic acid, and this production was enhanced by ornithine. Our results show that the ornithine part of the molecule originates from arginine, liberated by either anabolic or catabolic processes.     

Regulation of sinefungin biosynthesis by the wild-type strain and mutants of Streptomyces incarnatus

Sinefungin, an antifungal and antiparasitic antibiotic, is produced efficiently from ammonium citrate by prototrophic strains of Streptomyces incarnatus. The regulation of the biosynthesis of this nucleoside, composed of adenosine and ornithine, was studied by using auxotrophic mutants and a resting-cell system. Mutants blocked in arginine synthesis were not able to produce sinefungin. A uridine-negative mutant produced sinefungin in the presence of ATP, but this production was strongly inhibited when amino acids of the urea cycle were added. The same mutant produced sinefungin from aspartic acid, and this production was enhanced by ornithine. Our results show that the ornithine part of the molecule originates from arginine, liberated by either anabolic or catabolic processes.     

Functional analysis of desVIII homologues involved in glycosylation of macrolide antibiotics by interspecies complementation

The DesVIII is an auxiliary protein which enhances the transfer of TDP-d-desosamine catalyzed by DesVII glycosyltransferase in the biosynthesis of macrolide antibiotics, neomethymycin, methymycin and pikromycin, in Streptomyces venezuelae ATCC 15439. Homologues of the desVIII gene are present in a number of aminosugar-containing antibiotic biosynthetic gene clusters including eryCII from the erythromycin producer Saccharopolyspora erythraea, oleP1 from the oleandomycin producer Streptomyces antibioticus, dnrQ from the doxorubicin producer Streptomyces peucetius, and tylMIII from the tylosin producer Streptomyces fradiae. In order to gain further insight into the function of these DesVIII homologues, interspecies complementation experiments were carried out by expressing each gene in a desVIII deletion mutant strain of S. venezuelae. Complementation by expressing EryCII, OleP1, and DnrQ in this mutant strain restored the production of glycosylated macrolides to an approximate level of 66%, 26% and 26%, respectively, compared to self-complementation by DesVIII. However, expression of TylMIII did not restore the antibiotic production. These results suggest that the DesVIII homologues (except for TylMIII) can functionally replace the native DesVIII for glycosylation to proceed in vivo and their functions are similar in acting as glycosyltransferase auxiliary proteins. The requirement of glycosyltransferase auxiliary protein seems to be more widespread in polyketide biosynthetic pathways than previously known.     

Susceptibility to macrolide antibiotics of Bordetella pertussis and Bordetella parapertussis strains isolated from whooping cough patients in 1968 and in 1997-99]

The activity of erythromycin, azithromycin, clarithromycin, dirithromycin, oleandomycin, roxithromycin, spiramycin and josamycin against 21 and 34 B. pertussis strains and against 6 and 8 B. parapertussis strains isolated respectively in the years 1968 and 1997-99 was examined. The antibiotic agar dilution method was used. The minimum concentration of macrolides which inhibited growth of B. pertussis and B. parapertussis was calculated for 50% (MIC50) and 90% (MIC90) of isolates. The susceptibility to macrolides of B. pertussis and B. parapertussis strains isolated in the years 1968 and 1997-99 did not differ significantly. The MIC90 values of erythromycin were the same for B. pertussis (MIC90 = 0.125 mg/l) and B. parapertussis strains (MIC90 = 0.25 mg/l) recovered in 1968 as for those recovered in the years 1997-99. The most active antibiotic against all strains was azithromycin (MIC90 = 0.06 mg/l). The least active antibiotics were oleandomycin (MIC90 = 2-4 mg/l) and spiramycin (MIC90 = 8 mg/l). The study showed that erythromycin remains the antibiotic of choice for treatment of whooping cough and in case of emergence of B. pertussis and/or B. parapertussis strains erythromycin resistant, can be replaced by azithromycin.     

Identification of a gene cluster encoding meilingmycin biosynthesis among multiple polyketide synthase contigs isolated from<Emphasis Type="Italic"> Streptomyces nanchangensis</Emphasis> NS3226

A cluster encoding genes for the biosynthesis of meilingmycin, a macrolide antibiotic structurally similar to avermectin and milbemycin 11, was identified among seven uncharacterized polyketide synthase gene clusters isolated from Streptomyces nanchangensis NS3226 by hybridization with PCR products using primers derived from the sequences of aveE, aveF and a thioesterase domain of the avermectin biosynthetic gene cluster. Introduction of a 24.1-kb deletion by targeted gene replacement resulted in a loss of meilingmycin production, confirming that the gene cluster encodes biosynthesis of this important anthelminthic antibiotic compound. A sequenced 8.6-kb fragment had aveC and aveE homologues (meiC and meiE) linked together, as in the avermectin gene cluster, but the arrangement of aveF (meiF) and the thioesterase homologues differed. The results should pave the way to producing novel insecticidal compounds by generating hybrids between the two pathways.     

S‐adenosyl‐l‐methionine usage during climacteric ripening of tomato in relation to ethylene and polyamine biosynthesis and transmethylation capacity

S‐adenosyl‐l ‐methionine (SAM) is the major methyl donor in cells and it is also used for the biosynthesis of polyamines and the plant hormone ethylene. During climacteric ripening of tomato (Solanum lycopersicum ‘Bonaparte’), ethylene production rises considerably which makes it an ideal object to study SAM involvement. We examined in ripening fruit how a 1‐MCP treatment affects SAM usage by the three major SAM‐associated pathways. The 1‐MCP treatment inhibited autocatalytic ethylene production but did not affect SAM levels. We also observed that 1‐(malonylamino)cyclopropane‐1‐carboxylic acid formation during ripening is ethylene dependent. SAM decarboxylase expression was also found to be upregulated by ethylene. Nonetheless polyamine content was higher in 1‐MCP‐treated fruit. This leads to the conclusion that the ethylene and polyamine pathway can operate simultaneously. We also observed a higher methylation capacity in 1‐MCP‐treated fruit. During fruit ripening substantial methylation reactions occur which are gradually inhibited by the methylation product S‐adenosyl‐l ‐homocysteine (SAH). SAH accumulation is caused by a drop in adenosine kinase expression, which is not observed in 1‐MCP‐treated fruit. We can conclude that tomato fruit possesses the capability to simultaneously consume SAM during ripening to ensure a high rate of ethylene and polyamine production and transmethylation reactions. SAM usage during ripening requires a complex cellular regulation mechanism in order to control SAM levels.     

Substantia nigra degeneration and tyrosine hydroxylase depletion caused by excess S-adenosylmethionine in the rat brain

The major symptoms of Parkinson's disease (PD) are tremors, hypokinesia, rigidity, and abnormal posture, caused by degeneration of dopamine (DA) neurons in the substantia nigra (SN) and deficiency of DA in the neostriatal dopaminergic terminals. Norepinephrine, serotonin, and melanin pigments are also decreased and cholinergic activity is increased. The cause of PD is unknown. Increased methylation reactions may play a role in the etiology of PD, because it has been observed recently that the CNS administration of S-adenosyl-l-methionine (SAM), the methyl donor, caused tremors, hypokinesia, and rigidity; symptoms that resemble those that occur in PD. Furthermore, many of the biochemical changes seen in PD resemble changes that could occur if SAM-dependent methylation reactions are increased in the brain, and interestingly,l-DOPA, the most effective drug used to treat PD, reacts avidly with SAM. So methylation may be important in PD; an idea that is of particular interest because methylation reactions increase in aging, the symptoms of PD are strikingly similar to the neurological and functional changes seen in advanced aging, and PD is age-related. For methylation to be regarded as important in PD it means that, along with its biochemical reactions and behavioral effects, increased methylation should also cause specific neuronal degeneration. To know this, the effects of an increase in methylation in the brain were studied by injecting SAM into the lateral ventricle of rats. The injection of SAM caused neuronal degeneration, noted by a loss of neurons, gliosis, and increased silver reactive fibers in the SN. The degeneration was accompanied with a decrease in SN tyrosine hydroxylase (TH) immunoreactivity, and degeneration of TH-containing fibers. At the injection site in the lateral ventricle it appears that SAM caused a weakening or dissolution of the intercellular substances; observed as a disruption of the ependymal cell layer and the adjacent caudate tissues. SAM may also cause brain atrophy; evidenced by the dilation of the cerebral ventricle. Most of the SAM-induced anatomical changes that were observed in the rat model are similar to the changes that occur in PD, which further support a role of SAM-dependent increased methylation in PD.     

Ketoolivosyl-tetracenomycin C: a new ketosugar bearing tetracenomycin reveals new insight into the substrate flexibility of glycosyltransferase ElmGT

A new tetracenomycin analog, 8-demethyl-8-(4'-keto)-α-L-olivosyl-tetracenomycin C, was generated through combinatorial biosynthesis. Streptomyces lividans TK 24 (cos16F4) was used as a host for expression of a 'sugar plasmid' (pKOL) directing the biosynthesis of NDP-4-keto-L-olivose. This strain harbors all of the genes necessary for production of 8-demethyl-tetracenomycin C and the sugar flexible glycosyltransferase ElmGT. To the best of our knowledge, this report represents the first characterization of a tetracenomycin derivative decorated with a ketosugar moiety. Also, as far as we know, 4-keto-L-olivose has only been described as an intermediate of oleandomycin biosynthesis, but has not been described before as an appendage for a polyketide compound. Furthermore, this report gives further insight into the substrate flexibility of ElmGT to include an NDP-ketosugar, which is unusual and is rarely observed among glycosyltransferases from antibiotic biosynthetic pathways.