A new mycinosyl rosamicin derivative produced by an engineered Micromonospora rosaria mutant with a cytochrome P450 gene disruption introducing the d-mycinose biosynthetic gene

Genetic engineering of post-polyketide synthase-tailoring genes can be used to generate new macrolide analogs through manipulation of the genes involved in their biosynthesis. Rosamicin, a 16-member macrolide antibiotic produced by Micromonospora rosaria IFO13697, contains a formyl group and an epoxide at C-20 and C-12/13 positions which are formed by the cytochrome P450 enzymes RosC and RosD, respectively. The d-mycinose biosynthesis genes in mycinamicin II biosynthesis gene cluster of Micomonospora guriseorubida A11725 were introduced into the rosC and rosD disruption mutants of M. rosaria IFO13697. The resulting engineered strains, M. rosaria TPMA0054 and TPMA0069, produced mycinosyl rosamicin derivatives, IZIV and IZV, respectively. IZIV was identified as a novel mycinosyl rosamicin derivative, 23-O-mycinosyl-20-deoxo-20-dihydrorosamicin.     

Production of rosamicin derivatives in <Emphasis Type="Italic">Micromonospora rosaria</Emphasis> by introduction of <Emphasis Type="SmallCaps">d</Emphasis>-mycinose biosynthetic gene with <Emphasis Type="Italic">Φ</Emphasis>C31-derived integration vector pSET152

Some of the polyketide-derived bioactive compounds contain sugars attached to the aglycone core, and these sugars often impart specific biological activity to the molecule or enhance this activity. Mycinamicin II, a 16-member macrolide antibiotic produced by Micromonospora griseorubida A11725, contains a branched lactone and two different deoxyhexose sugars, d-desosamine and d-mycinose, at the C-5 and C-21 positions, respectively. The d-mycinose biosynthesis genes, mycCI, mycCII, mycD, mycE, mycF, mydH, and mydI, present in the M. griseorubida A11725 chromosome were introduced into pSET152 under the regulation of the promoter of the apramycin-resistance gene aac(3)IV. The resulting plasmid pSETmycinose was introduced into Micromonospora rosaria IFO13697 cells, which produce the 16-membered macrolide antibiotic rosamicin containing a branched lactone and d-desosamine at the C-5 position. Although the M. rosaria TPMA0001 transconjugant exhibited low rosamicin productivity, two new compounds, IZI and IZII, were detected in the ethylacetate extract from the culture broth. IZI was identified as a mycinosyl rosamicin derivative, 23-O-mycinosyl-20-deoxo-20-dihydro-12,13-deepoxyrosamicin (MW 741), which has previously been synthesized by a bioconversion technique. This is the first report on production of mycinosyl rosamicin-derivatives by a engineered biosynthesis approach. The integration site ΦC31attB was identified on M. rosaria IFO13697 chromosome, and the site lay within an ORF coding a pirin homolog protein. The pSETmycinose could be useful for stimulating the production of “unnatural” natural mycinosyl compounds by various actinomycete strains using the bacteriophage ΦC31 att/int system.     

Synthesis of 3-O-β-d-xylopyranosyl-l-serine (xylosylserine) andO-β-d-galactopyranosyl-(1-4)-O-β-d-xylopyranosyl-l-serine (galactosylxylosylserine) and use of the synthetic products for detection of galactosyltransferase I activity in rat liver

3-O-β-d-Xylopyranosyl-l-serine (xylosylserine) was synthesized by the following three-step procedure: 1) 2,3,4-tri-O-benzoyl-α-d-xylopyranosyl bromide (benzobromoxylose) was condensed withN-carbobenzoxy-l-serine benzyl ester using the silver triflate-collidine complex as promoter; 2) theN-carbobenzoxy and benzyl ester groups in the resultant glycoside were cleaved by transfer hydrogenation with palladium black as catalyst and ammonium formate as hydrogen donor; and 3) the benzoyl groups were removed with methanolic ammonia. Xylosylserine was obtained in an overall yield of 70%. O-β-d-Galactopyranosyl-(1-4)-O-β-d-xylopyranosyl-(1-3)-l-serine (galactosylxylosylserine) was also synthesized by this methodology and was characterized by 2-dimensional (2D) NMR spectroscopy techniques. The two serine glycosides (xylosylserine and galactosylxylosylserine) were used in detection and partial purification of galactosyltransferase I (UDP-d-galactose:d-xylose galactosyltransferase) from adult rat liver.     

Five new galactolipids from the freshwater microalga Porphyridium aerugineum and their nitric oxide inhibitory activity

Chemical investigation of the freshwater rhodophyte microalga Porphyridium aerugineum led to the isolation of five new galactolipids, namely, (2S)-1-O-eicosapentaenoyl-2-O-arachidonoyl-3-O-β-d-galactopyranosylglycerol (1), (2S)-1-O-eicosapentaenoyl-2-O-linoleoyl-3-O-β-d-galactopyranosylglycerol (2), (2S)-1-O-arachidoyl-2-O-palmitoyl-3-O-(β-d-galactopyranosyl-6-1α-d-galactopyranosyl)-glycerol (6), (2S)-1-O-eicosapentaenoyl-2-O-arachidoyl-3-O-(β-d-galactopyranosyl-6-1α-d-galactopyranosyl)-glycerol (7), and (2S)-1-O-eicosapentaenoyl-2-O-linoleoyl-3-O-(β-d-galactopyranosyl-6-1α-d-galactopyranosyl)-glycerol (8) together with five known galactolipids. The stereo-structures of all new galactolipids were elucidated by spectroscopic analyses and both enzymatic and chemical degradation methods. This is the first report of galactolipids from P. aerugineum. The newly isolated galactolipids showed strong and dose-dependent nitric oxide (NO) inhibitory activity against lipopolysaccharide-induced NO production in RAW264.7 macrophage cells. Both galactolipids 1 and 2 possessed stronger NO inhibitory activity than N ^G-methyl-l-arginine acetate salt, a well-known NO inhibitor used as a positive control. Further study suggested that these galactolipids inhibit NO production through downregulation of inducible nitric oxide synthase expression.     

Protodioscin-glycosidase-1 hydrolyzing 26-O-β-d-glucoside and 3-O-(1 → 4)-α-l-rhamnoside of steroidal saponins from Aspergillus oryzae

A novel protodioscin-(steroidal saponin)-glycoside hydrolase, named protodioscin-glycosidase-1 (PGase-1), was purified and characterized from the Aspergillus oryzae strain. The molecular mass of this enzyme was determined to be about 55 kDa based on SDS-polyacrylamide gel electrophoresis. PGase-1 was able to hydrolyze the terminal 26-O-β-d-glucopyranoside of protodioscin (furostanoside) to produce dioscin (spirostanoside), and then further hydrolyze the terminal 3-O-(1?→?4)-α-l-rhamnopyranoside of dioscin to form progenin III. However, PGase-1 could hardly hydrolyze the 3-O-(1?→?2)-α-l-rhamnopyranoside of progenin III, 3-O-β-d-glucoside of trillin, and the 1-O-glycosides of ophiopogonin D (steroidal saponin). In addition, PGase-1 also could hydrolyze the α-d-galactopyranoside, β-d-glucopyranoside, and β-d-galactopyranoside of p-nitrophenyl-glycosides, but the enzyme could not hydrolyze the α-d-mannopyranoside, α-l-arabinopyranoside, α-d-glucopyranoside, β-d-xylopyranoside, and α-l-rhamnopyranoside of p-nitrophenyl-glycosides. These new properties of PGase-1 were significantly different from those of previously described steroidal saponin-glycosidases and the glycosidases currently described in Enzyme Nomenclature by the NC-IUBMB. The gene (termed as pgase-1) encoding PGase-1 was cloned, sequenced, and expressed in Pichia pastoris GS115. The complete nucleotide sequence of pgase-1 consists of 1,725 bp. The recombinant PGase-1 from recombinant P. pastoris GS115 strain also showed the activity hydrolyzing glycosides of steroidal saponins which was similar to that of the wild-type PGase-1 from A. oryzae. The PGase-1 gene is highly similar to Aspergilli α-amylase (EC 3.2.1.1), and PGase-1 should be classified as glycoside hydrolase family 13 by the method of gene sequence-based classification. But the enzyme properties of PGase-1 are different from those of α-amylase in this family.     

Synthesis of 2-(p-trifluoroacetamidophenyl)ethylO-α-l-fucopyranosyl-(1–3)-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl) (1–3)-O-β-d-galactopyranosyl-(1–4)-β-d-glucopyranoside,a tetrasaccharide fragment of a tumour-associated glycosphingolipid

The tetrasaccharide 2-(p-trifluoroacetamidophenyl)ethylO-α-l-fucopyranosyl-(1–3)-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1–3)-O-β-d-galactopyranosyl-(1–4)-β-d-glucopyranoside was synthesized from thioglycoside intermediates. The key step was a methyl triflate promoted glycosidation of a lactose-derived 3′,4′-diol with a disaccharide thioglycoside to give a β(1–3)-linked tetrasaccharide derivative in 67% yield.     

Isomerization ofd-glycero-d-galacto-heptose tod-manno-heptulose by selected yeasts

Selected eight yeast strains isomerized-glycero-d-galacto-heptose tod-manno-heptulose. The conversion is 7–10%. Under identical conditions, the reverse isomerization ofd-manno-heptulose tod-glycero-d-galacto-heptose ord-glycero-d-talo-heptose does not take place.     

Effect of sugars on the morphogenesis ofAureobasidium pullulans

The dominance of individual elements of the vegetative fructification of five selected strains of the polymorphic organismAureobasidium pullulans (de Baby)Arnaud was studied in media with basic assimilable sugars,d-glucose,d-galactose,d-xylose, maltose, sucrose, lactose and a mixture ofl -arabinose andd-mannitol. Pronounced differences between cultures grown in the presence of monosaccharides and those cultivated in the presence of disaccharides were detected.     

A combinatorial approach to the structure elucidation of a pyoverdine siderophore produced by a Pseudomonas putida isolate and the use of pyoverdine as a taxonomic marker for typing P. putida subspecies

The structure of a pyoverdine produced by Pseudomonas putida, W15Oct28, was elucidated by combining mass spectrometric methods and bioinformatics by the analysis of non-ribosomal peptide synthetase genes present in the newly sequenced genome. The only form of pyoverdine produced by P. putida W15Oct28 is characterized to contain α-ketoglutaric acid as acyl side chain, a dihydropyoverdine chromophore, and a 12 amino acid peptide chain. The peptide chain is unique among all pyoverdines produced by Pseudomonas subspecies strains. It was characterized as –l-Asp-l-Ala-d-AOHOrn-l-Thr-Gly-c[l-Thr(O-)-l-Hse-d-Hya-l-Ser-l-Orn-l-Hse-l-Ser-O-]. The chemical formula and the detected and calculated molecular weight of this pyoverdine are: C₆₅H₉₃N₁₇O₃₂, detected mass 1624.6404 Da, calculated mass 1624.6245. Additionally, pyoverdine structures from both literature reports and bioinformatics prediction of the genome sequenced P. putida strains are summarized allowing us to propose a scheme based on pyoverdines structures as tool for the phylogeny of P. putida. This study shows the strength of the combination of in silico analysis together with analytical data and literature mining in determining the structure of secondary metabolites such as peptidic siderophores.     

Exposition of antitumour activity of a chemically characterized exopolysaccharide from a probiotic Lactobacillus plantarum MTCC 9510

As majority of chemical compounds identified as anti-cancerous are toxic to normal cells, the discovery and identification of new safe drugs is a necessity in the biomedical field. The antioxidant, antitumour and immunomodulating properties of an exopolysaccharide of sequence -α-d-glucose, α-d-mannose and β-d-glucose-, purified from a probiotic Lactobacillus plantarum was studied. The immunostimulation of the compound in human lymphocytes was seen at a concentration of 0.1 mg/mL with 20% proliferation rate. The antitumour studies by morphological apoptosis determination and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay on breast adenocarcinoma cell line (MCF-7) exhibited an IC₅₀ of 10 mg/mL for the compound.     

On the structure of the peptidoglycan of cell walls from Myxobacter AL-1 (Myxobacterales)

Basically the peptidoglycan of Myxobater AL-1 consists of alternating β-1,4-linked N-acetylglucosamic-N-acetylmuramic acid chains. After splitting the aminosugar backbone with a specific algal enzyme three subunits arise: a monomer, a dimer and a trimer. Investigation of the monomer with specific enzymes and comparison of the degradation products to standards derived from other bacterial peptidoglycans suggest the following structure of the monomer peptide: l-alanyl-d-glutamic-l-meso-diaminopimelic-d-alanine. A d-alanyl-d-meso-diaminopimelic acid bond is the bridgebond between the peptides of the subunits.     

The mannobiose-forming exo-mannanase involved in a new mannan catabolic pathway in Bacteroides fragilis

We have proposed a new mannan catabolic pathway in Bacteroides fragilis NCTC 9343 that involves a putative mannanase ManA in glycoside hydrolase family 26 (BF0771), a mannobiose and/or sugar transporter (BF0773), mannobiose 2-epimerase (BF0774), and mannosylglucose phosphorylase (BF0772). If this hypothesis is correct, ManA has to generate mannobiose from mannans as the major end product. In this study, the BF0771 gene from the B. fragilis genome was cloned and expressed in Escherichia coli cells. The expressed protein was found to produce mannobiose exclusively from mannans and initially from manno-oligosaccharides. Production of 4-O-β-d-glucopyranosyl-d-mannose or 4-O-β-d-mannopyranosyl-d-glucose from mannans was not detectable. The results indicate that this enzyme is a novel mannobiose-forming exo-mannanase, consistent with the new microbial mannan catabolic pathway we proposed.     

Accumulation of galloyl derivatives in a green alga, Spirogyra varians, in response to cold stress

The green alga Spirogyra varians accumulated antioxidative compounds in response to cold stress. When the algae were transferred from 20°C to 4°C, the amount of phenolic contents and flavonoids in the cell increased 17 times and 30 times, respectively, in 2 months. At this time, the radical scavenging activity of the methanolic extract of S. varians was 238 times higher than that of initial culture. To identify the responsible antioxidants, the methanolic extract was obtained from the algae grown at 4°C. HPLC analysis of the extract showed six compounds newly produced or increased over time. Four of the compounds were successfully purified, and the structures were identified using ¹H NMR spectroscopy. The compounds were galloyl derivatives—methyl gallate, 1-O-Galloyl-β-d-glucose, 1,2,3,6-tetra-O-Galloyl-β-d-glucose and 1,2,3,4,6-penta-O-Galloyl-β-d-glucose which are intermediates of the shikimate pathway.     

Properties of the sugar carrier in Baker's yeast

Incubation ofSaccharomyces cerevisiae cells withd-galactose induced the formation of galactose-utilizing enzymes, among them a monosaccharide carrier, apparently synthesized as a proteinde novo. The synthesis of the carrier preceded that of galactokinase by as much as 2 h. The inducible carrier shows a preference for monosaccharides with an axial hydroxyl group at carbon 4 of theC1 chair conformation or at carbon 2 of the1C chair conformation. Through its mediation, some sugars normally poorly transported (d-galactose,d-fucose,l-xylose,l-arabinose) can enter into the entire cell water, occupying then one more kinetic (and morphological ?) compartment than before induction. Some other monosaccharides, readily transported even by a constitutive carrier system (e.g.l-sorbose,d-xylose,d-arabinose) share the newly synthesized carrier.     

Biosynthesis of ethylene glycol in Escherichia coli

Ethylene glycol (EG) is an important platform chemical with steadily expanding global demand. Its commercial production is currently limited to fossil resources; no biosynthesis route has been delineated. Herein, a biosynthesis route for EG production from d-xylose is reported. This route consists of four steps: d-xylose?→?d-xylonate?→?2-dehydro-3-deoxy-d-pentonate?→?glycoaldehyde?→?EG. Respective enzymes, d-xylose dehydrogenase, d-xylonate dehydratase, 2-dehydro-3-deoxy-d-pentonate aldolase, and glycoaldehyde reductase, were assembled. The route was implemented in a metabolically engineered Escherichia coli, in which the d-xylose?→?d-xylulose reaction was prevented by disrupting the d-xylose isomerase gene. The most efficient construct produced 11.7 g?L^?1 of EG from 40.0 g?L^?1 of d-xylose. Glycolate is a carbon-competing by-product during EG production in E. coli; blockage of glycoaldehyde?→?glycolate reaction was also performed by disrupting the gene encoding aldehyde dehydrogenase, but from this approach, EG productivity was not improved but rather led to d-xylonate accumulation. To channel more carbon flux towards EG than the glycolate pathway, further systematic metabolic engineering and fermentation optimization studies are still required to improve EG productivity.     

Lipids isolated from the cultivated red alga Chondrus crispus inhibit nitric oxide production

A MeOH extract of cultivated Chondrus crispus showed dose-dependent nitric oxide (NO) inhibition of lipopolysaccharide-induced NO production in macrophage RAW264.7 cells. NO inhibition-guided fractionation of the extract led to identification of eicosapentaenoic acid (EPA, 1), arachidonic acid (AA, 2), lutein (3), and eight galactolipids as active components. Based on spectral analysis, the isolated galactolipids were identified as (2S)-1,2-bis-O-eicosapentaenoyl-3-O-β-d-galactopyranosylglycerol (4), (2S)-1-O-eicosapentaenoyl-2-O-arachidonoyl-3-O-β-d-galactopyranosylglycerol (5), (2S)-1-O-(6Z,9Z,12Z,15Z-octadecatetranoyl)-2-O-palmitoyl-3-O-β-d-galactopyranosylglycerol (6), (2S)-1-O-eicosapentaenoyl-2-O-palmitoyl-3-O-β-d-galactopyranosylglycerol (7), (2S)-1,2-bis-O-arachidonoyl-3-O-β-d-galactopyranosylglycerol (8), (2S)-1-O-arachidonoyl-2-O-palmitoyl-3-O-β-d-galactopyranosylglycerol (9), (2S)-1-O-eicosapentaenoyl-2-O-palmitoyl-3-O-(β-d-galactopyranosyl-6-1α-d-galactopyranosyl)-glycerol (10), and (2S)-1-O-arachidonoyl-2-O-palmitoyl-3-O-(β-d-galactopyranosyl-6-1α-d-galactopyranosyl)-glycerol (11). All the isolated compounds showed significant NO inhibitory activity. This is the first report of the isolation and identification of individual galactolipids from C. crispus. Moreover, (2S)-1,2-bis-O-arachidonoyl ?3-O-β-d-galactopyranosylglycerol (8) is a novel compound.     

Mono- and digalactosyldiacylglycerols: potent nitric oxide inhibitors from the marine microalga Nannochloropsis granulata

Chemical investigation of a marine microalga, Nannochloropsis granulata, led to the isolation of four digalactosyldiacylglycerols namely, (2S)-1-O-eicosapentaenoyl-2-O-palmitoyl-3-O-(β-d-galactopyranosyl-6-1α-d-galactopyranosyl)-glycerol (1), (2S)-1-O-eicosapentaenoyl-2-O-palmitoleoyl-3-O-(β-d-galactopyranosyl-6-1α-d-galactopyranosyl)-glycerol (2), (2S)-1-O-eicosapentaenoyl-2-O-myristoyl-3-O-(β-d-galactopyranosyl-6-1α-d-galactopyranosyl)-glycerol (3), and (2S)-1,2-bis-O-eicosapentaenoyl-3-O-(β-d-galactopyranosyl-6-1α-d-galactopyranosyl)-glycerol (4), together with their monogalactosyl analogs (5–8). Among the isolated galactolipids 2 and 3 were new natural products. Complete stereochemistry of 1, 4, 5, 7, and 8 was determined for the first time by both spectroscopic techniques and classical degradation methods. Both mono- and digalactosyldiacylglycerols isolated from N. granulata possessed strong nitric oxide (NO) inhibitory activity against lipopolysaccharide-induced NO production in RAW264.7 macrophage cells through downregulation of inducible nitric oxide synthase expression indicating the possible use as anti-inflammatory agents.     

Effects of mutations at threonine-654 on the insoluble glucan synthesized by Leuconostoc mesenteroides NRRL B-1118 glucansucrase

Twelve different amino acids were each substituted for threonine-654 in a cloned glucansucrase from Leuconostoc mesenteroides NRRL B-1118. Both the native and the cloned enzyme with threonine at position 654 produced a water-insoluble glucan containing approximately 44 mol% 1,3-disubstituted α-d-glucopyranosyl units and 29 mol% 1,6-disubstituted α-d-glucopyranosyl units. Several substitutions yielded an enzyme that produced an increased percentage of 1,3-disubstituted α-d-glucopyranosyl units, with corresponding decreases in 1,6-disubstituted α-d-glucopyranosyl units. Only one substitution, tyrosine, resulted in a significant increase in the percentage of 1,6-disubstituted α-d-glucopyranosyl units, with a concomitant increase in glucan yield. The mutated enzymes that produced the highest levels of 1,3-disubstituted α-d-glucopyranosyl units were also significantly activated by the addition of dextran, but glucan yields were also lower in these mutants.     

The effect of arabinogalactan proteins on regeneration potential of juvenile citrus explants used for genetic transformation by Agrobacterium tumefaciens

A possible role of arabinogalactan proteins in control of shoot regeneration from stem explants of two citrus cultivars, Carrizo citrange and ‘Duncan’ grapefruit, was investigated. Treatment of explants with (β-d-Glc)₃ Yariv phenylglycoside, able to bind specifically to AGPs, led to a decrease of cumulative regeneration potential of both Carrizo citrange and ‘Duncan’ grapefruit. For Carrizo, lower cumulative regeneration potential on (β-d-Glc)₃ Yariv phenylglycoside-treated explants was the result of both lower number of shoots on the explants that had shoots (explant regeneration potential) and decreased percentage of explants with shoots. In the case of ‘Duncan’, treatment with (β-d-Glc)₃ Yariv phenylglycoside reduced cumulative regeneration potential only by lowering the percentage of explants with shoots, but it did not affect the number of shoots on the explants with shoots. Citrus explants treated with (α-d-Man)₃ Yariv phenylglycoside, which does not bind AGPs, responded similarly to untreated explants. Transformability of cells on the cut ends of explants was also lower for both cultivars following the treatment of explants with (β-d-Glc)₃ Yariv phenylglycoside. Our data suggest that arabinogalactan proteins play important role in processes controlling differentiation and genetic transformation of citrus cells by Agrobacterium.