paromamine and 2-deoxystreptamine

Paromamine is a vital and common intermediate in the biosynthesis of 4,5 and 4,6-disubstituted 2-deoxystreptamine (DOS)-containing aminoglycosides. Our aim is to develop an engineered Escherichia coli system for heterologous production of paromamine.. We have constructed a mutant of E. coli BL21 (DE3) by disrupting glucose-6-phosphate isomerase (pgi) of primary metabolic pathway to increase glucose-6-phosphate pool inside the host. Disruption was carried out by lambda Red/ET recombination following the protocol mentioned in the kit. Recombinants bearing 2-deoxy-scyllo-inosose (DOI), DOS and paromamine producing genes were constructed from butirosin gene cluster and heterologously expressed in engineered host designed as E. coli BL21 (DE3) Delta pgi. Secondary metabolites produced by the recombinants fermentated in 2YTG medium were extracted, and analysis of the extracts showed there is formation of DOI, DOS and paromamine.. Escherichia coli system is engineered for heterologous expression of paromamine derivatives of aminoglycoside biosynthesis.. This is the first report of heterologous expression of paromamine gene set in E. coli. Hence a new platform is established in E. coli system for the production of paromamine which is useful for the exploration of novel aminoglycosides by combinatorial biosynthesis of 4,5- and 4,6-disubtituted route of DOS-containing aminoglycosides.

Topics: Aminoglycosides; Bacillus; Escherichia coli; Escherichia coli Proteins; Gene Expression; Glucose-6-Phosphate Isomerase; Hexosamines; Inositol; Multigene Family; Protein Engineering

The 2-deoxystreptamine and paromamine are two key intermediates in kanamycin biosynthesis. In the present study, pSK-2 and pSK-7 recombinant plasmids were constructed with two combinations of genes: kanABK and kanABKF and kacA respectively from kanamycin producer Streptomyces kanamyceticus ATCC12853. These plasmids were heterologously expressed into Streptomyces lividans TK24 independently and generated two recombinant strains named S. lividans Sk-2/SL and S. lividans SK-7/SL, respectively. ESI/ MS and ESI-LC/MS analysis of the metabolite from S. lividans SK-2/SL showed that the compound had a molecular mass of 163 [M + H]+, which corresponds to that of 2-deoxystreptamine. ESI/MS and MS/MS analysis of metabolites from S. lividans SK-7/SL demonstrated the production of paromamine with a molecular mass of 324 [M + H]+. In this study, we report the production of paromamine in a heterologous host for the first time. This study will evoke to explore complete biosynthetic pathways of kanamycin and related aminoglycoside antibiotics.

Topics: Aminoglycosides; Anti-Bacterial Agents; Biotechnology; Genetic Engineering; Hexosamines; In Vitro Techniques; Kanamycin; Mass Spectrometry; Molecular Weight; Species Specificity; Streptomyces lividans

Since the first use of streptomycin as an effective antibiotic drug in the treatment of tuberculosis, aminoglycoside antibiotics have been widely used against a variety of bacterial infections for over six decades. However, the pathways for aminoglycoside biosynthesis still remain unclear, mainly because of difficulty in genetic manipulation of actinomycetes producing this class of antibiotics. Gentamicin belongs to the group of 4,6-disubstituted aminoglycosides containing a characteristic core aminocyclitol moiety, 2-deoxystreptamine (2-DOS), and the recent discovery of its biosynthetic gene cluster in Micromonospora echinospora has enabled us to decipher its biosynthetic pathway. To determine the minimal set of genes and their functions for the generation of gentamicin A(2), the first pseudotrisaccharide intermediate in the biosynthetic pathway for the gentamicin complex, various sets of candidate genes from M. echinospora and other related aminoglycoside-producing strains were introduced into a nonaminoglycoside producing strain of Streptomyces venezuelae. Heterologous expression of different combinations of putative 2-DOS biosynthetic genes revealed that a subset, gtmB-gtmA-gacH, is responsible for the biosynthesis of this core aminocyclitol moiety of gentamicin. Expression of gtmG together with gtmB-gtmA-gacH led to production of 2'-N-acetylparomamine, demonstrating that GtmG acts as a glycosyltransferase that adds N-acetyl-d-glucosamine (GLcNA) to 2-DOS. Expression of gtmM in a 2'-N-acetylparomamine-producing recombinant S. venezuelae strain generated paromamine. Expression of gtmE in an engineered paromamine-producing strain of S. venezuelae successfully generated gentamicin A(2), indicating that GtmE is another glycosyltransferase that attaches d-xylose to paromamine. These results represent in vivo evidence elucidating the complete biosynthetic pathway of the pseudotrisaccharide aminoglycoside.

Topics: Aminoglycosides; Base Sequence; Chromatography, High Pressure Liquid; Disaccharides; Drug Resistance, Bacterial; Gene Expression; Genes, Bacterial; Gentamicins; Hexosamines; Micromonospora; Molecular Sequence Data; Multigene Family; N-Acylsphingosine Galactosyltransferase; Spectrometry, Mass, Electrospray Ionization; Streptomyces