Production of antibiotic carbomycin from Streptomyces graminofaciens with high lipid content mutation.

The most important tools in killing and overcoming on the microbes and pathogens that cause diseases in medicine and/or in agriculture are the antibiotics. The discovery and synthesis of the microbial natural products or antibiotics has greatly developed genetically and biotechnologically quickly in the last decades. It is necessary to access this great genetic diversity by finding ways to increase the level of expression of these biosynthetic pathways. In this study, we carried out an improvement in the antibiotic production of weak Streptomyces graminofaciens strain NBR9 that has high lipid content; using Ultra-Violet irradiation mutagenesis. This strain was isolated from the Northern Region in the kingdom of Saudi Arabia and identified biochemically and confirmed genetically by sequencing of the 16S rRNA gene as Streptomyces graminofaciens NBR9; Accession No. (MN640578). The resultant mutant strain showed increasing in their antimicrobial activities. The methods and techniques used for the antibiotic extraction, purification, characterization and identification proved that the obtained antibiotic is same with antibiotic Carbomycin.

Artificial control of the multistep oxidation reactions catalyzed by the cytochrome P450 enzyme RosC.

The cytochrome P450 monooxygenase RosC catalyzes the three-step oxidation reactions, which leads to the formation of a hydroxy, formyl, and carboxy group at C-20 during rosamicin biosynthesis in Micromonospora rosaria IFO13697. To determine if amino acid substitutions in RosC could allow for the control of the multistep oxidation reactions, we screened RosC random mutants. The RosC mutant RM30, with five amino acid substitutions (P107S, L176Q, S254N, V277A, and I319N), catalyzed only the first step of the oxidation reaction. Whole-cell assays using Escherichia coli cells expressing RosC mutants with single and double amino acid substitutions derived from RM30 indicated that P107S/L176Q, P107S/V277A, P107S/I319N, L176Q/V277A, L176Q/I319N, and S254N/V277A significantly reduced the catalytic activity of the second reaction, which is alcohol oxidation. Of the previously mentioned mutants, double mutants containing L176Q, which was presumed to occur in the FG loop region, lost the total catalytic activity of the third reaction (aldehyde oxidation). Additionally, an engineered M. rosaria strain with rosC disruption, which introduced the gene encoding the RosC mutants P107S/L176Q and P107S/V277A preferentially produced 20-dihydrorosamicin, which is formed after the first oxidation reaction of RosC.

Identification of two regulatory genes involved in carbomycin biosynthesis in Streptomyces thermotolerans.

Carbomycins are 16-membered macrolide antibiotics produced by Streptomyces thermotolerans ATCC 11416T. To characterize gene cluster responsible for carbomycin biosynthesis, the draft genome sequences for strain ATCC 11416T were obtained, from which the partial carbomycin biosynthetic gene cluster was identified. This gene cluster was approximately 40 kb in length, and encoding 30 ORFs. Two putative transcriptional regulatory genes, acyB2 and cbmR, were inactivated by insertion of the apramycin resistance gene, and the resulting mutants were unable to produce carbomycin, thus confirming the involvement of two regulatory genes in carbomycin biosynthesis. Overexpression of acyB2 greatly improved the yield of carbomycin; however, overexpression of cbmR blocked carbomycin production. The qPCR analysis of the carbomycin biosynthetic genes in various mutants indicated that most genes were highly expressed in acyB2-overexpressing strains, but few expressed in cbmR-overexpressing strains. Furthermore, acyB2 co-expression with 4″-isovaleryltransferase gene (ist), resulted in efficient biotransformation of spiramycin into bitespiramycin in S. lividans TK24, whereas ist gene regulated by acyB2 and cbmR would cause the lower efficiency of spiramycin biotransformation. These results indicated that AcyB2 was a pathway-specific positive regulator of carbomycin biosynthesis. However, CbmR played a dual role in the carbomycin biosynthesis by acting as a positive regulator, and as a repressor at cbmR high expression levels.

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.

Function of cytochrome P450 enzymes RosC and RosD in the biosynthesis of rosamicin macrolide antibiotic produced by Micromonospora rosaria.

The cytochrome P450 enzyme-encoding genes rosC and rosD were cloned from the rosamicin biosynthetic gene cluster of Micromonospora rosaria IFO13697. The functions of RosC and RosD were demonstrated by gene disruption and complementation with M. rosaria and bioconversion of rosamicin biosynthetic intermediates with Escherichia coli expressing RosC and RosD. It is proposed that M. rosaria IFO13697 has two pathway branches that lead from the first desosaminyl rosamicin intermediate, 20-deoxo-20-dihydro-12,13-deepoxyrosamicin, to rosamicin.

[Screening of nonpathogenic strains of Enterococcus faecium with antagonistic activity].

Forty two strains of enterococci were isolated from feces of healthy adolescents. Strains were selected according to their antagonistic effects associated with bacteriocinogenic and microcinogenic activity. Resistance of enterococci to antibiotics, various concentrations of hydrochloric acid and bile, their level of production of organic acids and adhesiveness were determined. Characteristics related to pathogenicity were investigared in 5 microcinogenic strains of E. faecium with broad spectrum of antagonistic activity. Non-pathogenic microcin-producing strains of E. faecium resistant to physiological concentrations of hydrochloric acid and bile with broad spectrum of antagonistic activity against obligatory pathogenic and opportunistic microorganisms can be considered as possessing probiotic activity.

Transposition mutagenesis in Streptomyces fradiae: identification of a neutral site for the stable insertion of DNA by transposon exchange.

We explored transposition in Streptomyces fradiae (Sf) as a means to insert a second copy of the tylF gene to improve tylosin (Ty) production. Transposons Tn5096 and Tn5099 transposed relatively randomly in Sf, and many of the insertions caused no deleterious effects on Ty production yields. Tn5098, a derivative of Tn5096 containing tylF and tylJ genes, recombined into the chromosome into the tyl gene cluster and transposition was not observed. However, following the tagging of a neutral site (NS) by Tn5099 transposition, tylF was effectively inserted into the NS by homologous recombination (transposon exchange). Recombinants obtained by transposon exchange produced higher yields of Ty.

Production of a hybrid macrolide antibiotic in Streptomyces ambofaciens and Streptomyces lividans by introduction of a cloned carbomycin biosynthetic gene from Streptomyces thermotolerans.

The structurally related macrolide antibiotics carbomycin (Cb) and spiramycin (Sp) are produced by Streptomyces thermotolerans and Streptomyces ambofaciens, respectively. Both antibiotics contain 16-membered lactone rings to which deoxysugars are attached. There are three sugars in Sp (forosamine, mycaminose and mycarose) and two sugars in Cb (mycaminose and a derivative of mycarose containing an isovaleryl group at position 4). We have identified the gene from S. thermotolerans (designated carE), which appears to encode an enzyme that acylates this mycarose sugar, and have shown that recombinant strains containing carE can use Sp as a substrate and convert it to the hybrid antibiotic, isovaleryl Sp (ivSp). Expression of carE was demonstrated in two heterologous hosts: in S. ambofaciens, where endogenously synthesized Sp was converted to ivSp, and in Streptomyces lividans where exogenously added Sp was converted to ivSp. The carE gene was isolated on a cosmid that also encodes genes required for Cb-lactone formation. These genes reside on a DNA segment of about 70 kb and are part of a Cb biosynthetic gene cluster that is flanked by two Cb-resistance genes, carA and carB. Mapping studies and nucleotide sequence analysis revealed that carE is located at one end of this gene cluster, immediately adjacent to the carB gene. Genes carB and carE are transcribed convergently and may share a common transcriptional terminator sequence.

Inhibition of the biosynthesis of leucomycin, a macrolide antibiotic, by cerulenin.

Cerulenin, an inhibitor of fatty acid synthesis, specifically inhibits the biosynthesis of leucomycin, a 16-membered macrolide antibiotic, in both growing cells and resting cells of Streptomyces kitasatoensis. In growing cells, the production of leucomycin was inhibited as long as cerulenin remained in the culture. In resting cells, 50 percent inhibition was achieved with a cerulenin concentration of 1.5 microgram/ml. Cells in which leucomycin synthesis was inhibited for 9 h remained capable of leucomycin synthesis upon removal of the inhibitor. Cerulenin specifically inhibits the incorporation of [14C]acetate into leucomycin but does not affect total protein or RNA synthesis. The uptake of [14C]acetate was not inhibited under conditions which completely inhibited the incorporation of acetate into leucomycin. Since cerulenin is known to block the condensation of malonyl-CoA subunits in the formation of fatty acids, it can be concluded that the aglycone of leucomycin is synthesized via the polyketide pathway by condensation steps similar to those involved in fatty acid biosynthesis.

Biosynthesis of the macrolide antibiotic tylosin. A preferred pathway from tylactone to tylosin.

The efficiencies of bioconversion of twenty-three potential intermediates in the biosynthesis of tylosin were determined with a mutant strain blocked only in tylactone biosynthesis. The results indicated that tylactone, the first intermediate excreted by Streptomyces fradiae, is converted to tylosin by a preferred sequence of reactions which include: (1) addition of mycaminose to the C-5 hydroxyl position of the lactone; (2) hydroxylation of the C-20 methyl group to a hydroxymethyl group; (3) dehydrogenation of the C-20 hydroxymethyl group to a formyl group; (4) hydroxylation of the C-23 methyl group to a hydroxymethyl; (5) addition of 6-deoxy-D-allose to the C-23 hydroxymethyl group; (6) addition of mycarose to the 4'-hydroxyl group of mycaminose; (7) addition of a methyl group to the 2"'-hydroxyl position of demethylmacrocin, and (8) addition of a methyl group to the 3"'-hydroxyl position of macrocin to produce tylosin. The intermediates which lacked both neutral sugars (mycarose and 6-deoxy-D-allose) were biologically unstable, and substantial quantities of these compounds were degraded during standard bioconversion experiments. However, the amount of one such intermediate (O-mycaminosyltylonolide) degraded was substantially reduced when low concentrations of the compound were used for bioconversion, and under these conditions, much higher efficiencies of bioconversion to tylosin were obtained. We have shown that a mutant blocked in hydroxylation of the C-20 methyl group is also blocked in the further dehydrogenation of the C-20 hydroxymethyl group to a formyl group, and have confirmed in in vitro studies that the 2"'-O-methylation of demethylmacrocin must proceed the 3"'-O-methylation of macrocin to produce tylosin.

Genetic and biochemical features of spiramycin biosynthesis in Streptomyces ambofaciens--curing, protoplast regeneration and plasmid transfer.

Spiramycin-producing Streptomyces ambofaciens KA-1028 harboring the pSA1 plasmid gave rise to spiramycin non-producing variants at high frequencies by various curing treatments. However, a number of the spiramycin non-producing progeny obtained by treatment with acridine dyes, still harbored plasmid DNAs which could not be differentiated from plasmid pSA1 by contour length, cleavage patterns and heteroduplex analysis. By treatment with mitomycin C, plasmid pSA1 was cured at high efficiency and spiramycin non-producing strains were obtained. Strain U-1717R obtained by regeneration of protoplasts of plasmid-cured strain U-1717 regained spiramycin production on growth on solid medium only. Furthermore, transconjugants obtained by mating between strain KA-1028 and U-1717R-24 (streptomycin-resistant) regained spiramycin production in both liquid and solid media. We conclude that the genes for the biosynthesis of spiramycin are encoded in a replicon other than plasmid pSA1 but that this plasmid plays a role in the regulation of spiramycin production.

Isolation and structures of nitrogen-free platenolide glycosides. I. The 5-O-(deoxy-3'-C-acetyl-beta-D-hexopyranosyl)-platenolides I and II.

Four novel nitrogen-free glycosides of platenolides I and II were isolated as secondary shunt metabolites of the turimycin biosynthesis from the culture broth of an industrial strain of Streptomyces hygroscopicus IMET JA 6599. By spectral (MS, 1H and 13C NMR) studies the structures of the glycosides have been settled as 5-O-(4',6'-dideoxy-3'-C-acetyl-beta-D-hexopyranosyl)-platenolide I (DDAH-Pl-I), 5-O-(4',6'-dideoxy-3'-C-acetyl-beta-D-hexopyranosyl)-platenolide II (DDAH-Pl-II), 5-O-(4',6'-dideoxy-3'-C-acetyl-beta-D-hexopyranosyl)-14-hydroxyl-platenolide II (DDAH-OH-Pl-II) and 5-O-(6'-deoxy-3'-C-acetyl-beta-D-hexopyranosyl)-platenolide II (DAH-Pl-II). A fifth glycoside, 5-O-(6'-deoxy-3'-C-acetyl-beta-D-hexopyranosyl)-platenolide I (DAH-Pl-I) was identified through its MS data.

Isolation and structures of nitrogen-free platenolide glycosides. II. The 5-O-(alpha-mycarosyl)- and 5-O-(3'-demethyl-beta-mycaroxyl)-platenolides I and II.

Three novel glycosides of platenolides I and II containing either mycarose (2,6-dideoxy-3-C-methyl-L-ribohexopyranose) or 3-demethyl-mycarose (2,6-dideoxy-L-ribohexopyranose) were isolated as the shunt products of turimycin biosynthesis by an industrial strain of Streptomyces hygroscopicus IMET JA 6599. By means of MS, 13C and 1H NMR spectroscopic studies, their structures were assigned as 5-O-(alpha-mycarosyl)-platenolide I (MYC-Pl-I), 5-O-(alpha-mycarosyl)-platenolide II (MYC-Pl-II) and 5-O-(3'-demethyl-beta-mycarosyl)-platenolide II (DM-MYC-Pl-II). The occurrence of 3-demethyl-mycaroside amongst the shunt metabolites is discussed in terms of its biosynthesis.

Mechanism of nitrogen regulation of protylonolide biosynthesis in Streptomyces fradiae.

A resting cell system was used to study the sites of inhibition by NH4+ of protylonolide biosynthesis by a blocked mutant, strain 261, of Streptomyces fradiae. With 14C-labeled succinate or valine as an exogenous substrate, labeled protylonolide formation by high-NH4+ grown mycelia of strain 261 was lower than by low-NH4+ grown mycelia. When 14C-labeled palmitate or acetate + labeled propionate + butyrate were used as the substrates, protylonolide production by mycelia grown under the two NH4+ conditions was nearly at the same rates. It is suggested that the metabolism of succinate and valine to lower fatty acid precursors is subject to NH4+ regulation, whereas condensation of acid precursors and related steps leading to protylonolide are insensitive to NH4+ concentration.

New tylosin analogs produced by mutants of Streptomyces fradiae.

2'''-Demethoxytylosin (component IIIc), 2'''-demethoxy-4'''-epi-tylosin (component IIId) and 2'''-O-demethyltylosin (component Vb) were produced by blocked mutant strains of Streptomyces fradiae. Fermentation, isolation, structure determination and biosynthetic considerations of these tylosin analogs are described.

Ammonium ions suppress the amino acid metabolism involved in the biosynthesis of protylonolide in a mutant of Streptomyces fradiae.

Protylonolide is a lactonic precursor of tylosin aglycone, produced by a mutant of Streptomyces fradiae. It originates from n-butyrate, propionate and acetate units. Studies were carried out using a protylonolide-producing mutant on the correlation between protylonolide biosynthesis, regulation by NH4+ and amino acid metabolism. Protylonolide production decreased in a defined medium containing high levels of NH4+, but was restored by adding lower fatty acids expected to serve as precursors of protylonolide biosynthesis. Resting cell studies demonstrated that 14C-labeled valine, threonine, leucine, isoleucine and alanine, but not lysine, were efficiently incorporated into protylonolide, indicating that these amino acids are metabolized to lower fatty acids. The incorporation of amino acids into protylonolide was reduced when the mutant strain was previously grown under high NH4+ conditions. We suggest that NH4+ suppresses the relevant amino acid metabolism, thereby reducing protylonolide formation.

Biosynthesis of the antitumor antibiotic CC-1065 by Streptomyces zelensis.

The biosynthesis of the antitumor antibiotic, CC-1065, has been investigated by radioactive isotope techniques, in combination with chemical degradation of CC-1065. Tyrosine, dopa, serine and methionine (S-CH3 group) have been shown to be precursors of CC-1065. Tyrosine is proposed to be a precursor of all three benzodipyrrole subunits, while dopa is only apparently incorporated into subunits B and C. Serine is postulated to contribute three 2C units, with loss of C-1, to all three subunits of CC-1065. The S-CH3 group of methionine probably contributes four C-1 units to CC-1065 of which one is incorporated with considerable loss of tritium, most probably into the cyclopropane ring of subunit A.

Phenotypic changes associated with loss of expression of tylosin biosynthesis and resistance genes in Streptomyces fradiae.

Two mutants of the tylosin-producing Streptomyces fradiae defective in the biosynthesis of the macrolide antibiotic tylosin were isolated from colonies derived from regenerated protoplasts. Both strains were unable to carry out any of at least seven tylosin biosynthetic steps and were sensitive to tylosin. One strain, JS82, was also more sensitive to chloramphenicol (Cm), mitomycin C (Mc), hygromycin B (Hm) and kanamycin (Km) than its parent strain. The other strain, JS87, was also more sensitive to Cm than wild type but expressed normal levels of resistance to Mc and Hm. Both strains expressed genetic instabilities associated with auxotrophy or expression of antibiotic resistance. Since the genetic instabilities were not due to defective error-free or error-prone DNA repair, they appear to be due to genetic rearrangements associated with the deletion or amplification of sequences linked to and perhaps encompassing tylosin biosynthesis genes.

Effect of ammonium ion, inorganic phosphate and amino acids on the biosynthesis of protylonolide, a precursor of tylosin aglycone.

The production of tylosin by Streptomyces fradiae KA-427 in a defined medium was inhibited by ammonium ions and by inorganic phosphate. The production of protylonolide, an early lactonic intermediate of tylosin biosynthesis with the same carbon skeleton as tylosin aglycone, by a mutant of strain KA-427 was also reduced by these two kinds of ions. In contrast, the bioconversion of protylonolide to tylosin by another mutant was less susceptible to ammonium ions but was sensitive to inorganic phosphate. The addition of protylonolide to a culture of S. fradiae KA-427 increased the tylosin yield, suggesting that aglycone synthesis is limiting under the conditions used. When L-valine, L-leucine, L-isoleucine, L-threonine, or the corresponding 2-keto acid was added to the culture medium, the protylonolide titer increased. The addition of [14C]valine gave rise to [14C]protylonolide. 13C NMR spectroscopic analysis revealed that iso-butyrate, which is a valine metabolite, was incorporated into protylonolide at the carbons known to originate from propionate and n-butyrate. Taking account of these findings, the regulation of tylosin biosynthesis in S. fradiae by ammonium ion is discussed in relation to amino acid metabolism.

Hybrid biosynthesis of derivatives of protylonolide and M-4365 by macrolide-producing microorganisms.

Biotransformation of a macrolide antibiotic and a related compound was studied using various macrolide-producing microorganisms grown in the presence of cerulenin, an inhibitor of de novo synthesis of the aglycone moiety. Protylonolide (1) was transformed into 5-O-(4'-O-propionylmycarosyl)protylonolide (2) by a leucomycin-producing strain, Streptoverticillium kitasatoensis KA-429. M-4365 G2 (3) was bioconverted into M-4365 G3 (4), 9-dihydro M-4365 G3 (5), 3-O-acetyl M-4365 G3 (6) and 3-O-acetyl-9-dihydro M-4365 G3 (7) by a spiramycin-producing strain, Streptomyces ambofaciens KA-1028. Forosaminylated derivatives of M-4365 G2 were not obtained using this microorganism. M-4365 G2 was converted into 3-O-acetyl M-4365 G2 (8) by Stv. kitasatoensis strain KA-429 and a carbomycin-producing strain, S. thermotolerans KA-442. These results suggest that the substrate specificity of mycaminose- and forosamine-binding enzymes is high in Stv. kitasatoensis and S. ambofaciens, respectively, while that of the 3-hydroxyl acylating enzyme and mycarose-binding enzyme is low in these microorganisms. The bioconversion products showed lower antibacterial and antimycoplasmal activities than those of M-4365 G2.