Identification of a plausible biosynthetic enzyme for the IM-2-type autoregulator in Streptomyces antibioticus

Virginiae butanolides (VBs) and IM-2 are members of Streptomyces hormones called ‘butyrolactone autoregulators’ which regulate the antibiotic production in Streptomyces species at nanomolar concentrations. Cell-free extract of a VB-A overproducer, Streptomyces antibioticus NF-18, is capable of catalyzing the final step of the autoregulator biosynthesis, namely, the NADPH-dependent reduction of 6-dehydroVB-A. However, physico-chemical analyses of the purified enzymatic products revealed that, in addition to the VB-type isomer [(2R,3R,6S)-enantiomer], IM-2-type isomers [(2R,3R,6R)- and (2S,3S,6S)-enantiomers] were also produced from (±)-6-dehydroVB-A, suggesting the existence of several 6-dehydroVB-A reductases with respective stereoselectivities. The reductase activity of the crude extracts was separated into two activity peaks, peak I (major) and peak II (minor), by DEAE-5PW HPLC. Chiral HPLC analyses demonstrated that peak I enzyme and peak II enzyme catalyzed the production of (2R,3R,6S), (2R,3R,6R) and (2S,3S,6S) isomers at ratios of 46:1:3.2 and 4.9:1:1.5, respectively, indicating clearly that S. antibioticus NF-18 possesses at least two 6-dehydroVB-A reductases: one much favored toward VB-A biosynthesis, the other with relaxed stereoselectivity capable of synthesizing both VB-type and IM-2-type autoregulators.     

Identification and characterization of 6-dehydroVB-A reductase from Streptomyces antibioticus

Streptomyces antibioticus NF-18 is a hyperproducing strain of a Streptomyces hormone, virginiae butanolide A (VB-A), that induces virginiamycin production of S. virginiae at nanomolar concentrations. To characterize the biosynthetic pathway of VB-A, we identified and characterized for the first time the 6-dehydro VB-A reductase that is responsible for the final reduction step in the biosynthesis. Assay protocols and stabilization conditions were established. The 6-dehydro VB-A reductase was found to require NADPH, not NADH, as a coenzyme. The K(m) values of the enzyme for NADPH and (+/-)-6-dehydro VB-A were determined to be 50 +/- 2 microM and 100 +/- 5 microM, respectively. Ultracentrifugation experiments revealed that 6-dehydro VB-A reductase was present almost exclusively in the 100,000 x g supernatant fraction, indicating that the enzyme is a cytoplasmic-soluble protein. The M(r) of the native 6-dehydro VB-A reductase was estimated to be 82,000 +/- 3000 by molecular sieve HPLC. The optimal pH was found to be 6.7 +/- 0.2.     

Gene replacement analysis of the butyrolactone autoregulator receptor (FarA) reveals that FarA acts as a Novel regulator in secondary metabolism of Streptomyces lavendulae FRI-5.

IM-2 [(2R,3R,1'R)-2-1'-hydroxybutyl-3-hydroxymethyl gamma-butanolide] is a gamma-butyrolactone autoregulator which, in Streptomyces lavendulae FRI-5, switches off the production of D-cycloserine but switches on the production of a blue pigment and several nucleoside antibiotics. To clarify the in vivo function of an IM-2-specific receptor (FarA) in the IM-2 signaling cascade of S. lavendulae FRI-5, a farA deletion mutant was constructed by means of homologous recombination. On several solid media, no significant difference in morphology was observed between the wild-type strain and the farA mutant (strain K104), which demonstrated that the IM-2-FarA system does not participate in the morphological control of S. lavendulae FRI-5. In liquid media, the farA mutant overproduced nucleoside antibiotics and produced blue pigment earlier than did the wild-type strain, suggesting that the FarA protein acts primarily as a negative regulator on the biosynthesis of these compounds in the absence of IM-2. However, contrary to the IM-2-dependent suppression of D-cycloserine production in the wild-type strain, overproduction of D-cycloserine was observed in the farA mutant, indicating for the first time that the presence of both IM-2 and intact FarA are necessary for the suppression of D-cycloserine biosynthesis.     

Purification and characterization of the IM-2-binding protein from Streptomyces sp. strain FRI-5.

IM-2 [(2R,3R,1'R)-2-(1'-hydroxybutyl)-3-(hydroxymethyl)butanolide] of Streptomyces sp. strain FRI-5 is one of the butyrolactone autoregulators of Streptomyces species and triggers production of blue pigment as well as the nucleoside antibiotics showdomycin and minimycin. A tritium-labeled IM-2 analogue, 2,3-trans-2(1'-beta-hydroxy-[4',5'-3H]pentyl)-3-(hydroxymethyl)butano lide ([3H]IM-2-C5; 40 Ci/mmol), was synthesized for a competitive binding assay, and an IM-2-specific binding protein was found to be present in the crude cell extract of Streptomyces sp. strain FRI-5. During cultivation for 24 h, the specific IM-2-binding activity increased rapidly, reached a plateau at 10 to 14 h, and declined sharply thereafter, showing only 6% activity after 24 h of cultivation. A Scatchard plot of the binding data demonstrated that the dissociation constant (Kd) for [3H]IM-2-C5 was 1.3 nM, while the Kd for a 3H-labeled virginiae butanolide (VB) analogue, 2-(1'-alpha-hydroxy-[6',7'-3H]heptyl)-3-(hydroxymethyl)butanolide ([3H]VB-C7), another butyrolactone autoregulator possessing the opposite configuration at C-1' was 35 nM. Furthermore, at a 15-fold molar excess, the effectiveness of several autoregulators as nonlabeled competitive ligands against [3H]IM-2-C5 was IM-2 type > VB-C type >> A-factor type, indicating that the binding protein in Streptomyces sp. strain FRI-5 is highly specific toward IM-2. Ultracentrifugation showed that the IM-2-binding protein is present almost exclusively in the 100,000 x g supernatant fraction, indicating that the binding protein is a cytoplasmic soluble protein. The binding protein was purified by ammonium sulfate precipitation, DEAE-Sephacel chromatography, Sephacryl S-100 HR gel filtration, DEAE-5PW high-performance liquid chromatography (HPLC), and phenyl-5PW HPLC. The apparent Mr of the native IM-2-binding protein as determined by molecular sieve HPLC was about 60,000 in the presence of 0.5, 0.3, or 0.1 M KCl, while by sodium dodecyl sulfate-polyacrylamide gel electrophoresis it was about 27,000, suggesting that the native binding protein is present in the form of a dimer.     

barS1, a gene for biosynthesis of a gamma-butyrolactone autoregulator,a microbial signaling molecule eliciting antibiotic production in Streptomyces species

From Streptomyces virginiae, in which production of streptogramin antibiotic virginiamycin M(1) and S is tightly regulated by a low-molecular-weight Streptomyces hormone called virginiae butanolide (VB), which is a member of the gamma-butyrolactone autoregulators, the hormone biosynthetic gene (barS1) was cloned and characterized by heterologous expression in Escherichia coli and by gene disruption in S. virginiae. The barS1 gene (a 774-bp open reading frame encoding a 257-amino-acid protein [M(r), 27,095]) is situated in the 10-kb regulator island surrounding the VB-specific receptor gene, barA. The deduced BarS1 protein is weakly homologous to beta-ketoacyl-acyl carrier protein/coenzyme A reductase and belongs to the superfamily of short-chain alcohol dehydrogenase. The function of the BarS1 protein in VB biosynthesis was confirmed by BarS1-dependent in vitro conversion of 6-dehydro-VB-A to VB-A, the last catalytic step in VB biosynthesis. Of the four possible enantiomeric products from racemic 6-dehydro-VB-A as a substrate, only the natural enantiomer of (2R,3R,6S)-VB-A was produced by the purified recombinant BarS1 (rBarS1), indicating that rBarS1 is the stereospecific reductase recognizing (3R)-isomer as a substrate and reducing it stereospecifically to the (6S) product. In the DeltabarS1 mutant created by homologous recombination, the production of VB as well as the production of virginiamycin was lost. The production of virginiamycin by the DeltabarS1 mutant was fully recovered by the external addition of VB to the culture, which indicates that the barS1 gene is essential in the biosynthesis of the autoregulator VBs in S. virginiae and that the failure of virginiamycin production was a result of the loss of VB production.     

Functional analysis of OleY L-oleandrosyl 3-O-methyltransferase of the oleandomycin biosynthetic pathway in Streptomyces antibioticus

Oleandomycin, a macrolide antibiotic produced by Streptomyces antibioticus, contains two sugars attached to the aglycon: L-oleandrose and D-desosamine. oleY codes for a methyltransferase involved in the biosynthesis of L-oleandrose. This gene was overexpressed in Escherichia coli to form inclusion bodies and in Streptomyces lividans, producing a soluble protein. S. lividans overexpressing oleY was used as a biotransformation host, and it converted the precursor L-olivosyl-erythronolide B into its 3-O-methylated derivative, L-oleandrosyl-erythronolide B. Two other monoglycosylated derivatives were also substrates for the OleY methyltransferase: L-rhamnosyl- and L-mycarosyl-erythronolide B. OleY methyltransferase was purified yielding a 43-kDa single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme showed a molecular mass of 87 kDa by gel filtration chromatography, indicating that the enzyme acts as a dimer. It showed a narrow pH range for optimal activity, and its activity was clearly stimulated by the presence of several divalent cations, being maximal with Co(2+). The S. antibioticus OleG2 glycosyltransferase is proposed to transfer L-olivose to the oleandolide aglycon, which is then converted into L-oleandrose by the OleY methyltransferase. This represents an alternative route for L-oleandrose biosynthesis from that in the avermectin producer Streptomyces avermitilis, in which L-oleandrose is transferred to the aglycon by a glycosyltransferase.     

Generation of hybrid elloramycin analogs by combinatorial biosynthesis using genes from anthracycline-type and macrolide biosynthetic pathways

Elloramycin and oleandomycin are two polyketide compounds produced by Streptomyces olivaceus Tü2353 and Streptomyces antibioticus ATCC11891, respectively. Elloramycin is an anthracycline-like antitumor drug and oleandomycin a macrolide antibiotic. Expression in S. albus of a cosmid (cos16F4) containing part of the elloramycin biosynthetic gene cluster produced the elloramycin non-glycosylated intermediate 8-demethyl-tetracenomycin C. Several plasmid constructs harboring different gene combinations of L-oleandrose (neutral 2,6-dideoxyhexose attached to the macrolide antibiotic oleandomycin) biosynthetic genes of S. antibioticus that direct the biosynthesis of L-olivose, L-oleandrose and L-rhamnose were coexpressed with cos16F4 in S. albus. Three new hybrid elloramycin analogs were produced by these recombinant strains through combinatorial biosynthesis, containing elloramycinone or 12a-demethyl-elloramycinone (= 8-demethyl-tetracenomycin C) as aglycone moiety encoded by S. olivaceus genes and different sugar moieties, coded by the S. antibioticus genes. Among them is L-olivose, which is here described for the first time as a sugar moiety of a natural product.     

Characterization of SgcE6, the flavin reductase component supporting FAD-dependent halogenation and hydroxylation in the biosynthesis of the enediyne antitumor antibiotic C-1027

The C-1027 enediyne antitumor antibiotic from Streptomyces globisporus possesses an ( S )-3-chloro-5-hydroxy-β-tyrosine moiety, the chloro- and hydroxy-substituents of which are installed by a flavin-dependent halogenase SgcC3 and monooxygenase SgcC, respectively. Interestingly, a single flavin reductase, SgcE6, can provide reduced flavin to both enzymes. Bioinformatics analysis reveals that, similar to other flavin reductases involved in natural product biosynthesis, SgcE6 belongs to the HpaC-like subfamily of the Class I flavin reductases. The present study describes the steady-state kinetic characterization of SgcE6 as a strictly NADH- and FAD-specific enzyme.     

Resistance to oleandomycin in Streptomyces antibioticus, the producer organism

Resistance to oleandomycin in Streptomyces antibioticus, the producer organism, was studied. The organism was highly resistant in vivo to the antibiotic but sensitive to other macrolides and lincosamides. Protein synthesis in vivo by mycelium of S. antibioticus was more resistant to oleandomycin than that by mycelium of Streptomyces albus G, an oleandomycin-sensitive strain, and this resistance was dependent on the age of the culture, older mycelium of S. antibioticus being more resistant to oleandomycin than young mycelium. [3H]Oleandomycin was capable of binding to the same extent to the 50S subunits of the ribosomes of both organisms. Oleandomycin also inhibited in vitro protein synthesis by ribosomes obtained from an oleandomycin-production medium at the time when maximum levels of oleandomycin were being produced. A clear difference between the ability of the two organisms to incorporate exogenous oleandomycin was observed. Thus, while S. albus G took up oleandomycin, S. antibioticus showed a decreased permeability to the antibiotic, suggesting a role for cell permeability in self-resistance.     

链霉菌Streptomyces antibioticus NRRL 8167中Naphthgeranine A生物合成基因簇的分析鉴定

【背景】微生物来源的天然产物是小分子药物或药物先导物的重要来源。对链霉菌Streptomyces antibioticus NRRL 8167的基因组分析显示,其包含多个次级代谢产物的生物合成基因簇,具有产生多种新化合物的潜力。【目的】对链霉菌S. antibioticus NRRL 8167中次级代谢产物进行研究,以期发现结构新颖或生物活性独特的化合物,并对相应产物的生物合成基因簇和生物合成途径进行解析。【方法】利用HPLC图谱结合特征性紫外吸收和LC-MS方法,排除S. antibioticus NRRL 8167产生的已知化合物,确定具有特殊紫外吸收的化合物作为挖掘对象,然后利用正、反相硅胶柱色谱、高效液相色谱等技术对次级代谢产物进行分离纯化,分离化合物。利用质谱及核磁共振光谱技术对化合物结构进行解析和鉴定;提取链霉菌S. antibioticus NRRL 8167基因组DNA,利用PacBio测序平台进行基因组测序;利用生物信息学对基因组进行注释,并对合成该化合物的基因簇进行定位分析,推导其生物合成途径。【结果】确定这个化合物是NaphthgeranineA,属于聚酮类化合物。全基因组序列分析发现S.antibioticusNRRL8167基因组含有28个次级代谢产物生物合成基因簇,其中基因簇20可能负责Naphthgeranine A的生物合成,并对其生物合成途径进行了推导。【结论】基于紫外吸收光谱和质谱特征,从S. antibioticus NRRL 8167菌株的发酵提取物中分离鉴定了一个聚酮类化合物Naphthgeranine A。该菌株的全基因组测序为其生物合成基因簇的鉴定提供了前提,对Naphthgeranine A生物合成基因簇和生物合成途径的推测为进一步研究这个化合物的生物合成机制奠定了基础。     

Actinomycin synthesis in Streptomyces antibioticus: enzymatic conversion of 3-hydroxyanthranilic acid to 4-methyl-3-hydroxyanthranilic acid.

A methyltransferase which utilizes 3-hydroxyanthranilic acid (HAA) as a substrate was identified in detergent-treated extracts of the bacterium Streptomyces antibioticus. The enzyme catalyzes the transfer of methyl groups from [14C]S-adenosylmethionine to HAA, but does not catalyze the methylation of 3-hydroxy-DL-kynurenine. Enzyme, substrate, time, and pH dependencies for the methyl transfer reaction were examined. Reaction products obtained from scaled-up reaction mixtures were fractionated by chromatography on Dowex 1, and the Dowex 1 fractions were examined by paper and thin-layer chromatography. One Dowex fraction was shown to contain a radioactive product with the chromatographic properties of 4-methyl-3-hydroxyanthranilic acid (MHA), a known intermediate in the biosynthesis of actinomycin. Available evidence indicates that the conversion of HAA to MHA is an early step in the biosynthesis of actinomycin by S. antibioticus and other actinomycin-producing streptomycetes.     

Two glycosyltransferases and a glycosidase are involved in oleandomycin modification during its biosynthesis by Streptomyces antibioticus

A 5.2 kb region from the oleandomycin gene cluster in Streptomyces antibioticus located between the oleandomycin polyketide synthase gene and sugar biosynthetic genes was cloned. Sequence analysis revealed the presence of three open reading frames (designated oleI , oleN2 and oleR ). The oleI gene product resembled glycosyltransferases involved in macrolide inactivation including the oleD product, a previously described glycosyltransferase from S. antibioticus . The oleN2 gene product showed similarities with different aminotransferases involved in the biosynthesis of 6-deoxyhexoses. The oleR gene product was similar to several glucosidases from different origins. The oleI , oleR and oleD genes were expressed in Streptomyces lividans . OleI and OleD intracellular proteins were partially purified by affinity chromatography in an UDP-glucuronic acid agarose column and OleR was detected as a major band from the culture supernatant. OleI and OleD showed oleandomycin glycosylating activity but they differ in the pattern of substrate specificity: OleI being much more specific for oleandomycin. OleR showed glycosidase activity converting glycosylated oleandomycin into active oleandomycin. A model is proposed integrating these and previously reported results for intracellular inactivation, secretion and extracellular reactivation of oleandomycin.     

Geometrical E/Z isomers of (6R)- and (6S)-neoxanthin and biological implications

9'Z-(3S,5R,6R,3'S,5'R,6'S)-Neoxanthin reisolated from spinach (Spinacea oleracea) and characterized by HPLC, VIS, MS, and 2D (1)H NMR, has been submitted to photoinduced stereomutation in the presence of iodine or diphenyl diselenide at conditions not involving isomerization of the allenic bond. The six individual geometrical isomers, all-E,9Z,9'Z,13Z,13'Z,15Z and three minor di-Z-isomers, presumably 9,9'-di-Z,9',13-di-Z and 9',13'-di-Z, present in the equilibrium mixture have been characterized by HPLC, VIS data, 1H NMR and reversibility tests. Judged by the quantitative composition of the equilibrium mixture the naturally occurring 9'Z-isomer is thermodynamically less stable than the all-E-isomer. The availability of these isomers facilitates future search in natural sources. 9'Z-(6R90% of total neoxanthin in spinach and broccoli (Brassica oleracea var. italica), consistent with previous findings of its abundance in chloroplasts. all-E90% of total violaxanthin in the same sources. It is postulated that a neoxanthin Delta9'-isomerase is present and involved in the biosynthesis of abscisic acid in higher plants. Allenic S-isomers are of interest as postulated biosynthetic precursors of R-allenes. All-E-(6S)- and 9'Z-(6S)-neoxanthin were available as semi-synthetic model compounds. The allenic (6S)-diastereomers could not be detected in spinach or broccoli.     

Cyclic adenosine-3',5-monophosphate in Streptomyces antibioticus and its possible role in regulating oleandomycin biosynthesis and the growth of the culture

When glucose is substituted for sucrose in the fermentation medium for Streptomyces antibioticus, the pH of the cultural broth becomes more acidic, the rate of protein synthesis in the mycelium rises, and the rate of oleandomycin synthesis decreases abruptly. The dynamics of cAMP (cyclic monophosphate) accumulation was studied in the process of biosynthesis by the culture in different media. Most of the synthesized cAMP (80-90%) was shown to be excreted into the medium. Glucose stimulates cAMP synthesis and excretion from the mycelium by a factor of 1.5-3. No distinct correlation was found between cAMP content in S. antibioticus cells and the level of oleandomycin biosynthesis. A correlation between changes in the concentration of exocellular cAMP and protein synthesis in the mycelium suggests that the excreted cAMP may be involved in regulating the growth of the culture producing the antibiotic.     

Sigma-E is required for the production of the antibiotic actinomycin in Streptomyces antibioticus

The phsA gene encodes phenoxazinone synthase (PHS), which catalyses the penultimate step in the pathway for actinomycin biosynthesis in Streptomyces antibioticus . The phsA promoter strikingly resembles a putative Streptomyces σ^E cognate promoter, and purified Eσ^E holoenzyme transcribed the phsA promoter in vitro . However, the phsA promoter was still active in an S. antibioticus sigE null mutant and the level of PHS activity was unaffected. Despite this, disruption of sigE blocked actinomycin production completely. The loss of actinomycin production correlated with a 10-fold decrease in the activity of actinomycin synthetase I, the enzyme which catalyses the activation of the precursor of the actinomycin chromophore.     

An 86Y-labeled mirror-image oligonucleotide: influence of Y-DOTA isomers on the biodistribution in rats

A mirror-image oligonucleotide (L-RNA) was radiolabeled with the positron emitting radionuclide (86)Y (t(1/2) = 14.7 h) via the bifunctional chelator approach. DOTA-modification of the L-RNA (sequence: 5'-aminohexyl UGA CUG ACU GAC-3'; MW 3975) was performed using (S)-p-SCN-Bn-DOTA. (86)Y radiolabeling of the DOTA-L-RNA produced more than one species as evidenced by HPLC radiometric detection. For the identification of the (86)Y-labeled L-RNA, the structural analogue nonradioactive precursor [Y((S)-p-NH2-Bn-DOTA)](-) was synthesized. Two coordination isomers were separated via HPLC adopting the square antiprismatic (SAP) and the twisted square antiprismatic (TSAP) geometry, respectively. Their stereochemical configuration in the solution state was assessed by NMR and circular dichroism spectroscopy. Both [Y((S)-p-NH2-Bn-DOTA)](-) isomers were converted into isothiocyanate derivatives [Y((S)-p-SCN-Bn-DOTA)](-) and conjugated to the L-RNA. The identity of the [(86)Y-DOTA]-L-RNA species was finally established by comparison of the radiometric ((86)Y) and UV-visible chromatographic profiles. Biodistribution studies in Wistar rats showed minor changes in the biodistribution profile of the [(86)Y((S)-p-NH2-Bn-DOTA)](-) complex isomers, while no significant differences were observed for the [(86)Y-DOTA]-L-RNA isomers. High renal excretions were found for the [(86)Y((S)-p-NH 2-Bn-DOTA)](-) complex isomers as well as for the L-RNA isomers.     

High-frequency fusion of Streptomyces parvulus or Streptomyces antibioticus protoplasts induced by polyethylene glycol.

Conditions were established for the regeneration of protoplasts of Streptomyces parvulus and Streptomyces antibioticus to the mycelial form. Regeneration was accomplished with a hypertonic medium that contained sucrose, CaCl2, MgCl2, and low levels of phosphate. High-frequency fusion of protoplasts derived from auxotrophic strains of S. parvulus or S. antibioticus was induced by polyethylene glycol 4,000 (42%, wt/vol). The frequency of genetic transfer by the fusogenic procedure varied with the auxotrophic strains examined. Fusion with auxotrophic strains of S. parvulus resulted in the formation of true prototrophic recombinants. Similar studies with S. antibioticus revealed that both stable prototrophic recombinants and heterokaryons were formed.