Aims: 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. Methods and Results: 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. Conclusions: Escherichia coli system is engineered for heterologous expression of paromamine derivatives of aminoglycoside biosynthesis. Significance and Impact of the Study: 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.