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Neomycin, a multicomponent aminoglycoside antibiotic, is mainly utilized in livestock husbandry and feed additives in animals. The antimicrobial potency of the main product neomycin B is higher than that of its stereoisomer neomycin C. However, the content of neomycin C as an impurity in the high-producing strain is relatively high, and its isolation or removal from neomycin B is quite difficult, which influences the widespread application of neomycin. In this work, the essential genes responsible for neomycin biosynthesis were evaluated and overexpressed to reduce the content of neomycin C. Among them, neoG and neoH are two novel regulatory genes for neomycin biosynthesis, aphA is a resistance gene, neoN encoding a radical SAM-dependent epimerase is responsible for the conversion of neomycin C to B using SAM as the cofactor, and metK is a SAM synthetase coding gene. We demonstrated that the reconstitution and overexpression of a mini-gene-cluster (PkasO*-neoN-metK-PkasO*-neoGH-aphA) could effectively reduce the accumulation of neomycin C from 19.1 to 12.7% and simultaneously increase neomycin B by ∼13.1% in the engineered strain Sf/pKCZ04 compared with the wild-type strain (Sf). Real-time quantitative polymerase chain reaction analysis revealed the remarkable up-regulation of the neoE, neoH, neoN, and metK genes situated in the mini-gene-cluster. The findings will pave a new path for component optimization and the large-scale industrial production of significant commercial antibiotics.

The methyl groups of [ methyl-( S)]- and [ methyl-( R)]-[ methyl-D,T]-l-methionine fed to Streptomyces fradiae were incorporated into fosfomycin, which was chemically degraded to chiral AcONa. The enzymatic test gave the ( S)-configuration for the chiral AcONa derived from methionine with the ( S)-[D,T]methyl group ( F = 31.7) and ( R) for the one derived from methionine with the ( R)-[D,T]methyl group ( F = 83.0). The radical SAM methyltransferase transfers the methyl group of MeCbl to HEP-CMP with inversion of configuration.

The S-adenosyl-l-methionine-dependent O-methyltransferases TylE and TylF catalyze the last two methylation reactions in the tylosin biosynthetic pathway of Streptomyces fradiae. It has long been known that the TylE-catalyzed C2‴-O-methylation of the 6-deoxy-d-allose bound to demethylmacrocin or demethyllactenocin precedes the TylF-catalyzed C3‴-O-methylation of the d-javose (C2‴-O-methylated 6-deoxy-d-allose) attached to macrocin or lactenocin. This study reveals the unexpected substrate promiscuity of TylE and TylF responsible for the biosynthesis of d-mycinose (C3‴-O-methylated d-javose) in tylosin through the identification of a new minor intermediate 2‴-O-demethyldesmycosin (2; 3‴-methyl-demethyllactenocin), which lacks a 2‴-O-methyl group on the mycinose moiety of desmycosin, along with 2‴-O-demethyltylosin (1; 3‴-methyl-demethylmacrocin) that was previously detected from the S. fradiae mutant containing a mutation in the tylE gene. These results unveil the unique substrate flexibility of TylE and TylF and demonstrate their potential for the engineered biosynthesis of novel glycosylated macrolide derivatives.

The importance of unusual deoxysugars in biology has become increasingly apparent over the past decade. Some, for example, play key roles in the physiological activities of the natural products to which they are attached. Here we describe a study of TylM1, a dimethyltransferase from Streptomyces fradiae involved in the production of dTDP-mycaminose. From this investigation, the manner in which the enzyme binds its dimethylated product has been revealed. More significantly, by providing the enzyme with an alternative substrate, it was possible to produce a monomethylated product not observed in nature. This has important ramifications for the production of unique carbohydrates that may prove useful in drug design.

In this work, for the first time, we report pKa values of the amino functions in a target-bound aminoglycoside antibiotic, which permitted dissection of the thermodynamic properties of an enzyme-aminoglycoside complex. Uniformly enriched 15N-neomycin was isolated from cultures of Streptomyces fradiae and used to study its binding to the aminoglycoside phosphotransferase(3')-IIIa (APH) by 15N NMR spectroscopy. 15N NMR studies showed that binding of neomycin to APH causes upshifts of approximately 1 pKa unit for the amines N2' and N2' '' while N6' experienced a 0.3 pKa unit shift. The pKa of N6' '' remained unaltered, and resonances of N1 and N3 showed significant broadening upon binding to the enzyme. The binding-linked protonation and pH dependence of the association constant (Kb) for the enzyme-aminoglycoside complex was determined by isothermal titration calorimetry. The enthalpy of binding became more favorable (negative) with increasing pH. At high pH, binding-linked protonation was attributable mostly to the amino functions of neomycin; however, at neutral pH, functional groups of the enzyme, possibly remote from the active site, also underwent protonation/deprotonation upon formation of the binary enzyme-neomycin complex. The Kb for the enzyme-neomycin complex showed a complicated dependence on pH, indicating that multiple interactions may affect the affinity of the ligand to the enzyme and altered conditions, such as pH, may favor one or another. This work highlights the importance of determining thermodynamic parameters of aminoglycoside-target interactions under different conditions before making attributions to specific sites and their effects on these global parameters.

A targeted search for glycosyltransferase (GT) encoding genes in the gene cluster of the urdamycin A producer Streptomyces fradiae Tu2717 resulted in the discovery of urdGT2, a GT encoding gene located approximately 7 kb downstream of the minimal polyketide synthase (PKS) encoding genes. Subsequent inactivation of this gene created a mutant strain, which produces completely different metabolites than the wild-type strain, consisting of the three new urdamycins I, J, and K. Their structures provide new insight into the important C-glycosyl-transfer step of the urdamycin biosynthetic pathway. The structures indicate that the corresponding gene product UrdGT2 catalyzes the C-glycosyl transfer of activated d-olivose to an angucyclinone precursor, which already bears the angular 12b-OH group. The structures of the new urdamycins could not have arisen without the involvement of substrate flexible post-PKS modifying genes, i.e., glycosyltransferases and oxidoreductases. This work proves that targeted gene disrup...

In order to investigate mid and early biosynthetic steps of angucycline group antibiotics, approximately 400 mutants ofthe urdamycin producer Streptomyces fradiae (strain Tu 2717) were prepared, of which ca. 10% were selected for further investigations. The selection criterion, i.e., the consideration of only pale-colored metabolite-producing blocked mutants, yielded several mutants whose block was in close proximity to the known late-stage biosynthetic steps

Experiments with 18 O 2 and [1- 13 C, 18 O 2 ]acetate determined the biosynthetic origin of the oxygen atoms of aquayamycin (1), which is the aglycon moiety of the urdamycins A (2, kerriamycin B) and G (3, OM 4842) and the most frequently occuring aglycon among the angucyclines

Analysis of the tylosin gene cluster in Streptomyces fradiae uncovered an ORF, tyl1a, homologous to a hexose 3,4-isomerase found in Aneurinibacillus thermoaerophilus. Inclusion of the tyl1a gene along with other mycaminose biosynthetic genes (tylB, tylM1, tylM2, tylM3) identified in previous studies in an in vivo expression system successfully reconstituted the mycaminose pathway. Expression of tyl1a alone in the S venezuelae KdesI mutant converted a desosamine pathway to a mycaminose pathway. These results strongly support the role of Tyl1a as a TDP-4-keto-6-deoxy-d-glucose 3,4-isomerase.