Nagendra Prasad Kurumbang

Biosynthesis of paromamine derivatives in engineered Escherichia coli by heterologous expression

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.

Biosynthesis of Ribostamycin Derivatives by Reconstitution and Heterologous Expression of Required Gene Sets

Ribostamycin is a 4,5-disubstituted 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotics and naturally produced by Streptomyces ribosidificus ATCC 21294. It is also an intermediate in the biosynthesis of butirosin and neomycin. In the biosynthesis of ribostamycin, DOS is glycosylated to generate paromamine which is converted to neamine by successive dehydrogenation followed by amination, and finally ribosylation of neamine gives ribostamycin. Here, we report the biosynthesis of 6′-deamino-6′-hydroxyribostamycin (a ribostamycin derivative or pseudoribostamycin) in Streptomyces venezuelae YJ003 by reconstructing gene cassettes for direct ribosylation of paromamine. A trace amount of pseudoribostamycin was detected with ribostamycin in the isolates of ribostamycin cosmid heterologously expressed in Streptomyces lividans TK24. It has also indicated that the ribosyltransferase can accept both neamine and paromamine. Thus, the present in vivo modification of ribostamycin could be useful for the production of hybrid compounds to defend against bacterial resistance to aminoglycosides.

Heterologous production of ribostamycin derivatives in engineered Escherichia coli.

Aminoglycosides are a class of important antibiotic compounds used for various therapeutic indications. In recent times, their efficacy has been curtailed due to the rapid development of bacterial resistance. There is a need to develop novel derivatives with an improved spectrum of activity and higher sensitivity against pathogenic bacteria. Although efforts have been focused on the development of newer therapeutic agents by chemical synthesis, to our knowledge, there has been no attempt to harness the potential of microorganisms for this purpose. Escherichia coli affords a widely studied cellular system that could be utilized not only for understanding but also for attempting to engineer the biosynthetic pathway of secondary metabolites. The primary metabolic pathway of E. coli can be engineered to divert the precursor pool required for the biosynthesis of secondary metabolites. Utilizing this approach previously, we engineered E. coli host and generated E. coli M1. Here, we produced a ribostamycin derivative in the engineered host by heterologous expression of the recombinants constructed from the genes encoding the biosynthetic pathway in aminoglycoside-producing strains. The products obtained from the transformants were isolated, analyzed and verified to be ribostamycin derivatives. The study further demonstrated the importance of E. coli as surrogate antibiotic producer and also offered future possibility for the production of other aminoglycoside derivatives through genetic engineering and expression in a heterologous background.