A defined system for hybrid macrolide biosynthesis in Saccharopolyspora erythraea

The biological activity of polyketide antibiotics is often strongly dependent on the presence and type of deoxysugar residues attached to the aglycone core. A system is described here, based on the erythromycin-producing strain of Saccharopolyspora erythraea, for detection of hybrid glycoside formation, and this system has been used to demonstrate that an amino sugar characteristic of 14-membered macrolides (D-desosamine) can be efficiently attached to a 16-membered aglycone substrate. First, the S. erythraea mutant strain DM was created by deletion of both eryBV and eryCIII genes encoding the respective ery glycosyltransferase genes. The glycosyltransferase OleG2 from Streptomyces antibioticus, which transfers L-oleandrose, has recently been shown to transfer rhamnose to the oxygen at C-3 of erythronolide B and 6-deoxyerythronolide B. In full accordance with this finding, when oleG2 was expressed in S. erythraea DM, 3-O-rhamnosyl-erythronolide B and 3-O-rhamnosyl-6-deoxyerythronolide B were produced. Having thus validated the expression system, endogenous aglycone production was prevented by deletion of the polyketide synthase (eryA) genes from S. erythraea DM, creating the triple mutant SGT2. To examine the ability of the mycaminosyltransferase TylM2 from Streptomyces fradiae to utilise a different amino sugar, tylM2 was integrated into S. erythraea SGT2, and the resulting strain was fed with the 16-membered aglycone tylactone, the normal TylM2 substrate. A new hybrid glycoside was isolated in good yield and characterized as 5-O-desosaminyl-tylactone, indicating that TylM2 may be a useful glycosyltransferase for combinatorial biosynthesis. 5-O-glucosyl-tylactone was also obtained, showing that endogenous activated sugars and glycosyltransferases compete for aglycone in these cells.     

Studies in phytosterol biosynthesis. Mechanism of biosynthesis of cycloartenol

1. The mechanism of cycloartenol biosynthesis in leaves of Solanum tuberosum was investigated with the use of [2-¹⁴C,(4R)-4-³H₁]mevalonic acid. 2. The ³H/¹⁴C atomic ratio in cycloartenol was 6:6, the same as that in squalene; this eliminates lanosterol as a possible biosynthetic precursor of cycloartenol, and indicates that a hydrogen migration from C-9 to C-8 occurs. 3. Chemical isomerization of the cycloartenol to lanosterol (³H/¹⁴C ratio 5:6) and parkeol (³H/¹⁴C ratio 6:6) confirms the hydrogen migration from C-9 to C-8. 4. Possible mechanisms for the biosynthesis of cycloartenol and parkeol are discussed. 5. The ³H/¹⁴C ratio for 24-methylenecycloartanol was 6:6, demonstrating that the hydrogen atom at C-24 is retained during alkylation of the cycloartenol side chain.     

The biosynthesis of streptomycin. The origin of the C-formyl group of streptose

1. The biosynthesis of streptomycin in Streptomyces griseus has been studied by adding d-[3,4-(14)C(2)]glucose or d-[1,3-(14)C(2)]glucose to the growth medium and degrading the streptomycin produced. 2. The results suggest that the C-3' branch carbon atom of l-streptose arises from C-3 of d-glucose. 3. The mechanism of biosynthesis of streptose from glucose is discussed. It probably involves an intramolecular rearrangement of a 6-deoxy-4-oxyhexose derivative, and it is suggested that the nucleoside diphosphate sugar derivative hitherto recognized as an intermediate in the biosynthesis of l-rhamnose might participate in such a rearrangement.     

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.     

Cytochrome P450 homolog is responsible for C-N bond formation between aglycone and deoxysugar in the staurosporine biosynthesis of Streptomyces sp. TP-A0274

The staurosporine biosynthetic gene cluster in Streptomyces sp. TP-A0274 consists of 15 sta genes. In the cluster, it was predicted that staN, which shows high similarity to cytochrome P450 is involved in C-N bond formation between the nitrogen at N-12 of aglycone and the carbon at C-5' of deoxysugar. The staN disruptant produced holyrine A instead of staurosporine. The structure of holyrine A is aglycone linking to 2,3,6-trideoxy-3-aminoaldohexose between N-13 and C-1' of deoxysugar. Holyrine A was converted to staurosporine by the staD disruptant. These results indicate that StaN, cytochrome P450 is responsible for C-N bond formation. This is the first example of C-N bond formation catalyzed by cytochrome P450. In addition, holyrine A was confirmed to be an intermediate of staurosporine biosynthesis, which suggests that the N- and O-methylation at C-3' and C-4' takes place after the formation of the C-N bond between C-5' and N-12 in the biosynthetic pathway.     

An unusual UMP C-5 methylase in nucleoside antibiotic polyoxin biosynthesis

Polyoxin is a group of structurally-related peptidyl nucleoside antibiotics bearing C-5 modifications on the nucleoside skeleton. Although the structural diversity and bioactivity preference of polyoxin are, to some extent, affected by such modifications, the biosynthetic logic for their occurence remains obscure. Here we report the identification of PolB in polyoxin pathway as an unusual UMP C-5 methylase with thymidylate synthase activity which is responsible for the C-5 methylation of the nucleoside skeleton. To probe its molecular mechanism, we determined the crystal structures of PolB alone and in complexes with 5-Br UMP and 5-Br dUMP at 2.15 Å, 1.76 Å and 2.28 Å resolutions, respectively. Loop 1 (residues 117–131), Loop 2 (residues 192–201) and the substrate recognition peptide (residues 94–102) of PolB exhibit considerable conformational flexibility and adopt distinct structures upon binding to different substrate analogs. Consistent with the structural findings, a PolB homolog that harbors an identical function from Streptomyces viridochromogenes DSM 40736 was identified. The discovery of UMP C5-methylase opens the way to rational pathway engineering for polyoxin component optimization, and will also enrich the toolbox for natural nucleotide chemistry.     

The biosynthesis of natural and unnatural polyoxins by Streptomyces cacaoi

The biosynthesis of the pyrimidine moiety and the uronic acid moiety of the polyoxins and the formation of unnatural polyoxins has been studied in Streptomyces cacaoi. Experimental evidence is provided for the biosynthesis of thymine via a pathway that is independent of thymidylate synthetase. This new thymine pathway is based on two experimental approaches. First, two known inhibitors of DNA synthesis (1-formylisoquinoline thiosemicarbazide and 5-fluoro-2′-deoxyuridine), when added to polyoxin-producing cultures of S. cacaoi, inhibit the synthesis of TMP from exogenously supplied uracil but do not inhibit the synthesis of the thymine or hydroxymethyluracil in the polyoxin complex. Second, exogenously supplied thymine and hydroxymethyluracil are taken up by S. cacaoi but are not incorporated into the thymine or hydroxymethyluracil of the polyoxin complex. The thymine is incorporated into the DNA. The uracil in polyoxin L could be the parent pyrimidine chromophore with C-1 additions occurring at carbon-5 to form thymine and hydroxymethyluracil. Carbon-3 of serine but not the methyl group of methionine is a one-carbon source for the formation of the thymine and hydroxymethyluracil in the polyoxin complex.S. cacaoi can synthesize unnatural polyoxins, as evidenced by the incorporation of 5-fluoro, 5-bromo, and 6-azauracil into the polyoxins; 5-iodo-, 2-thio-, or 4-thiouracil is not a substrate. Two new polyoxin analogs synthesized and characterized when 5-fluorouracil is added to the cultures are 5-fluoropolyoxin L and 5-fluoropolyoxin M. There is a marked change in the molar ratio of the uracil:thymine:hydroxymethyluracil chromophores in the polyoxin complex following the incorporation of 5-fluoro-, 5-bromo-, or 6-azauracil. Apparently, the unnatural polyoxins inhibit the addition of the C-1 unit to carbon-5 of uracil in the polyoxin complex. Polyoxin L and polyoxin C do not inhibit Escherichia coli and Streptococcus faecalis, but 5-fluoropolyoxin L and 5-fluoropolyoxin C inhibit both these organisms. There is little or no difference in the inhibition of the fluorinated and natural polyoxins against leukemia L-1210 cells. The fluoro group on carbon-5 of the uracil ring does not affect the enzyme-inhibition complex with chitin synthetase since the inhibition constant of fluoropolyoxins L is the same as has been reported for polyoxins A, D, and L.The ¹⁴C-labeling pattern in the 5′-amino-5′-deoxy-d-allofuranosyluronic acid moiety of the polyoxins from ¹⁴C-labeled glucose, allose, and glycerol suggests that the formation of this unique C-6 uronic acid in the polyoxins does not proceed via the direct oxidation of either d-glucose or d-allose to the -onic or -uronic acids. Glucose is converted to two three-carbon trioses, followed by either (i) the oxidation of one of the trioses to a threecarbon acid and subsequent condensation with another three-carbon sugar to form the C-6 uronic or (ii) an 80:20 equilibrium of the two trioses followed by condensation to a hexose which is then oxidized to the C-6 uronic acid.     

Proof that the ACTVI genetic region of Streptomyces coelicolor A3(2) is involved in stereospecific pyran ring formation in the biosynthesis of actinorhodin

Pyran ring formation in the biosynthesis of actinorhodin in Streptomyces coelicolor A3(2) was studied using the act cluster deficient strain, CH999, carrying pRM5-based plasmids harbouring combinations of the actVI genes. The strain, CH999/pIJ5660 (pRM5 + actVI-ORF1), produced a chiral intermediate, (S)-DNPA, suggesting that the actVI-ORF1 product is a reductase determining the C-3 stereochemical centre.     

Anionic carbohydrate-containing polymers of cell walls in two streptoverticille genospecies.

The cell walls of two streptoverticille genospecies which belong to a historically isolated group of the genus Streptomyces contain anionic polymers of different structure. Streptomyces hachijoensis VKM Ac-191T and Streptomyces cinnamoneus subsp. azacoluta VKM Ac-606T assigned to one genospecies on the basis of DNA--DNA hybridization [5] contain 37% of an identical sugar-1-phosphate polymer. The repeating disaccharide units of the polymer, 2-amino-2-deoxy-alpha-D-glucopyranosyl-(1-->6)-2-acetamido-2-deoxy-al pha-D-glucopyranose, are linked at C-1 and C-6' by phosphodiester bonds. The cell walls of Streptomyces biverticillatus VKM Ac-891T and Streptomyces baldaccii VKM Ac-821T, members of another genospecies, contain about 30% 1,3-poly(glycerol phosphate) completely substituted by 2-amino-2-deoxy-alpha-D-glucopyranosyl residues at C-2. Due to the presence of an amino sugar with a free amino group in the repeating unit, the polymers exhibit neutral properties. Polymer structures were determined by chemical methods and NMR spectroscopy. The data indicate taxonomic specificity of anionic polymers in streptoverticille cell walls.     

Structure of anionic carbohydrate-containing cell wall polymers in several representatives of the order actinomycetales

A new teichoic acid was identified in the cell walls of Streptomyces griseoviridis VKM Ac-622T, Streptomyces sp. VKM Ac-2091, and Actinoplanes campanulata VKM Ac-1319T. The polymer is poly(glycosylglycerol phosphate). The repeating units of the polymer, alpha-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-beta-galactopyran+ ++ osyl-(1-->1)-glycerols, are in phosphodiester linkage at C-3 of glycerol and C-6 of galactose. The structures of cell wall teichoic acids in the strains Streptomyces chryseus VKM Ac-200T and "Streptomyces subflavus" VKM Ac-484 similar in morphology and growth characteristics are also identical: 1,5-poly(ribitol phosphate) substituted at C-4(2) by 2-acetamido-2-deoxy-beta-glucopyranosyl residues and 1,3-poly(glycerol phosphate). The taxonomic aspects of these results are discussed.     

A tryptophan C-methyltransferase involved in streptonigrin biosynthesis in Streptomyces flocculus.

A C-methyltransferase that catalyses the transfer of a methyl group from S-adenosylmethionine to C-3 of tryptophan, resulting in beta-methyltryptophan, has been identified in cell-free extracts of streptonigrin-producing Streptomyces flocculus. The absolute configuration of the product was shown to be (2S,3R)-beta-methyltryptophan by high-pressure liquid chromatography and reactivity with D- and L-amino acid oxidases. In shake culture, maximum specific activity occurs after S. flocculus enters stationary phase, but before significant streptonigrin accumulates.     

The biosynthesis of l-rhamnose of plum-leaf polysaccharides.

1. The utilization of d-[1-(14)C]- and d-[6-(14)C]-glucose in the biosynthesis of l-rhamnose units of plum-leaf polysaccharides has been studied. 2. After the precursors had been metabolized in the leaves, polysaccharide fractions were prepared therefrom and the constituent l-rhamnose was isolated and purified. 3. Both the specific activity and the distribution of (14)C along the carbon chain of l-rhamnose from two polysaccharide fractions from each experiment were determined. 4. The results indicated a close affinity between l-rhamnose and pectin, and show that biosynthesis of the 6-deoxyhexose from d-glucose occurs in the main without scission or inversion of the carbon chain. 5. A degradation scheme for l-rhamnose via l-rhamnitol was described which gives the labelling at C-1, C-2+C-3+C-4,C-5 and C-6 on a 0.3millimole scale.     

The O-methyltransferase SrsB catalyzes the decarboxylative methylation of alkylresorcylic acid during phenolic lipid biosynthesis by Streptomyces griseus

Streptomyces griseus contains the srs operon, which is required for phenolic lipid biosynthesis. The operon consists of srsA, srsB, and srsC, which encode a type III polyketide synthase, an O-methyltransferase, and a flavoprotein hydroxylase, respectively. We previously reported that the recombinant SrsA protein synthesized 3-(13'-methyltetradecyl)-4-methylresorcinol, using iso-C(16) fatty acyl-coenzyme A (CoA) as a starter substrate and malonyl-CoA and methylmalonyl-CoA as extender substrates. An in vitro SrsA reaction using [(13)C(3)]malonyl-CoA confirmed that the order of extender substrate condensation was methylmalonyl-CoA, followed by two extensions with malonyl-CoA. Furthermore, SrsA was revealed to produce an alkylresorcylic acid as its direct product rather than an alkylresorcinol. The functional SrsB protein was produced in the membrane fraction in Streptomyces lividans and used for the in vitro SrsB reaction. When the SrsA reaction was coupled, SrsB produced alkylresorcinol methyl ether in the presence of S-adenosyl-l-methionine (SAM). SrsB was incapable of catalyzing the O-methylation of alkylresorcinol, indicating that alkylresorcylic acid was the substrate of SrsB and that SrsB catalyzed the conversion of alkylresorcylic acid to alkylresorcinol methyl ether, namely, by both the O-methylation of the hydroxyl group (C-6) and the decarboxylation of the neighboring carboxyl group (C-1). O-methylated alkylresorcylic acid was not detected in the in vitro SrsAB reaction, although it was presumably stable, indicating that O-methylation did not precede decarboxylation. We therefore postulated that O-methylation was coupled with decarboxylation and proposed that SrsB catalyzed the feasible SAM-dependent decarboxylative methylation of alkylresorcylic acid. To the best of our knowledge, this is the first report of a methyltransferase that catalyzes decarboxylative methylation.     

Studies on the biosynthesis of 16-membered macrolide antibiotics using carbon-13 nuclear magnetic resonance spectroscopy.

The origin of the skeletal carbons in the lactone ring of 16-membered macrolide antiobiotics has been studied. 13C-labeled antibiotics leucomycin and tylosin, have been obtained from the culture broth of Streptomyces kitasatoensis 66-14-3 and Streptomyces fradiae C-373, respectively in the presence of appropriate 13C-labeled precursors, and 13C NMR spectra of the antibiotics thus obtained have been measured. It was shown that the aglycone of leucomycin A3 is derived from five acetates, one propionate, one butyrate, and an unknown precursor corresponding to two carbons. The formyl carbon which is characteristic of the basic 16-membered macrolides orginates from C-4 butyrate. On the other hand, the aglycone of tylosin is formed from two acetates, five propionates and one butyrate. Butyric acid and ethylmalonic acid are metabolized to propionyl-CoA or methylmolonyl-CoA through a pathway involving methylmalonyl-CoA mutase, and subsequently incorporated into the lactone ring of tylosin.     

Cloning, Expression, and Biological Function of a dTDP-Deoxyglucose Epimerase (gerF) Gene from Streptomyces sp. GERI-155

GERI-155 is a macrolide antibiotic containing two deoxyhexose molecules which has antimicrobial activities against gram-positive bacteria. The deoxyhexose biosynthetic gene cluster of GERI-155 from Streptomyces sp. GERI-155 genome has now been isolated. Four orf were identified and a putative orf, supposed to code for the dTDP-deoxyglucose epimerase gene, was designated as gerF. gerF was expressed in E. coli using recombinant expression vector pHJ3. The recombinant protein expressed in a soluble form. The enzyme was purified by Ni-affinity column using imidazole buffer as eluents. The molecular mass of the expressed protein correlated with the predicted mass (36,000 Da) deduced from the cloned gene sequence data. The purified enzyme produced maltol from dTDP-4-keto-6-deoxyglucose and it was confirmed that the expressed protein was dTDP-deoxyglucose epimerase catalyzing epimerization of C-3 and C-5 or C-3 of dTDP-4-keto-6-deoxyglucose.     

The pur3 gene from the pur cluster encodes a monophosphatase essential for puromycin biosynthesis in Streptomyces

The pur3 gene of the puromycin (pur) cluster from Streptomyces alboniger is essential for the biosynthesis of this antibiotic. Cell extracts from Streptomyces lividans containing pur3 had monophosphatase activity versus a variety of mononucleotides including 3'-amino-3'-dAMP (3'-N-3'-dAMP), (N6,N6)-dimethyl-3'-amino-3'-dAMP (PAN-5'-P) and AMP. This is in accordance with the high similarity of this protein to inositol monophosphatases from different sources. Pur3 was expressed in Escherichia coli as a recombinant protein and purified to apparent homogeneity. Similar to the intact protein in S. lividans, this recombinant enzyme dephosphorylated a wide variety of substrates for which the lowest Km values were obtained for the putative intermediates of the puromycin biosynthetic pathway 3'-N-3'-dAMP (Km = 1.37 mM) and PAN-5'-P (Km = 1.40 mM). The identification of this activity has allowed the revision of a previous proposal for the puromycin biosynthetic pathway.     

The transfer of hydrogen from C-24 to C-25 in ergosterol biosynthesis

1. A convenient synthesis of 3-hydroxytrisnorlanost-8-en-24-al and its conversion into [24-(3)H]lanosterol and [26,27-(14)C(2)]lanosterol is described. 2. A method for the efficient incorporation of lanosterol into ergosterol by the whole cells of Saccharomyces cerevisiae is also described. 3. It is shown that in the biosynthesis of ergosterol from doubly labelled lanosterol the C-24 hydrogen atom of lanosterol is retained in ergosterol. 4. On the basis of unambiguous degradations it is shown that the C-alkylation step in ergosterol biosynthesis is accompanied by the migration of a hydrogen atom from C-24 to C-25. 5. The mechanism for the biosynthesis of the ergosterol side chain is presented. 6. Mechanisms of other C-alkylation reactions are also discussed.     

Stereochemistry of a methyl-group rearrangement during the biosynthesis of lanosterol.

1. (3RS,6R)-[6-2H1,6-3H1,6-14C], (3RS,6S)-[6-2H1,6-3H1,6-14C] and (3RS)-[6-3H1,6-14C]mevalonolactones were synthesised from R-[2H1,3H1,2-14C], S-[2H1,3H1,2-14C] and [3h1,2-14C]acetic acids respectively. 2. Each mevalonate was converted into cholesterol by a rat liver preparation. 3. Each cholesterol specimen was converted into androsta-1,4-diene-3,17-dione by incubation with Mycobacterium phlei in the presence of 2,2'.dipyridyl. Each specimen of androsta-1,4-diene-3,17-dione was converted into androsta-1,4-dien-3-one-17-ethylene ketail. 4. The samples of androsta-1,4-dien-3-one-17-ethylene ketal were each converted chemically into oestrones in which the methyl group at C-18 is the only carbon atom that originated from C-6 in mevalonolactone. 5. The oestrone from (3RS)-[6-3H1,6-14C]mevalonolactone was oxidised chemically to acetic acid which was converted into p-bromophenacyl acetate and the 3H/14C ratio was measured. 6. There was no overall loss of tritium from the methyl group of acetic acid, as measured by determining the 3H/14C ratios of the p-bromophenacyl esters, when the synthetic and degradative procedures 1 -- 5 were tested with [3H1,2-14C]acetic acid. 7. The oestrones derived from the 6R and 6S-mevalonolactones were oxidised. The chiralities of the resulting acetates were determined by an established procedure whereby the acetates were converted into 2S-malates which were examined for loss of tritium on equilibration with fumarate hydratase. 8. The oestrone from (3RS,6R)-[6-2H1,6-3H1,6-14C]mevalonate gave acetic acid which was converted into 2S-malate that retained 68.6% of its tritium after treatment with fumarate hydratase; the configuration of this acetic acid was R. 9. The oestrone from (3RS,6S)-E16-2H1,6-3H1,6-14C]mevalonate was oxidised to acetic acid which was converted into 2S-malate that retained 31.9% of its tritium after treatment with fumarate hydratase; the configuration of this acetic acid was S. 10. There was no overall change in the configuration of a chiral methyl group between C-6 of mevalonate and C-18 of oestrone. It is cncluded that the intramolecular migration of a chiral methyl group from C-15 in 2,3-oxidosqualene to C-13 in lanosterol is stereospecific and occurs with overall retention of configuration.     

Directed biosynthesis of avermectins

Avermectin homologs are produced by Streptomyces avermitilis when externally supplied with sodium 2-methylpentanoate and sodium 2-methylhexanoate. The homologs carry 2-pentyl and 2-hexyl groups, respectively, at C-25 of the aglycone moiety as opposed to the 2-butyl group of "a" components and the isopropyl group of "b" components of natural avermectins. The new homologs designated as avermectin "c" and "d" components, respectively, possess potent anthelmintic and insecticidal activity.