2-Deoxy-scyllo-inosose (DOI) has been a valuable starting natural product for the manufacture of pharmaceuticals or chemical engineering resources such as pyranose catechol. DOI synthase, which uses glucose-6-phosphate (Glc6P) as a substrate for DOI biosynthesis, is indispensably involved in the initial stage of the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics including butirosin, gentamicin, kanamycin, and tobramycin. A number of metabolically engineered recombinant strains of Bacillus subtilis were constructed here; either one or both genes pgi and pgcA that encode Glc6p isomerase and phosphoglucomutase, respectively, was (or were) disrupted in the sugar metabolic pathway of the host. After that, three different DOI synthase–encoding genes, which were artificially synthesized according to the codon preference of the B. subtilis host, were separately introduced into the engineered recombinants. The expression of a natural btrC gene, encoding DOI synthase in butirosin-producing B. circulans, in the heterologous host B. subtilis (BSDOI-2) generated approximately 2.3 g/L DOI, whereas expression of an artificially codon-optimized tobC gene, derived from tobramycin-producing Streptomyces tenebrarius, into the recombinant of B. subtilis (BSDOI-15) in which both genes pgi and pgcA are disrupted significantly enhanced the DOI titer: up to 37.2 g/L. Fed-batch fermentation by the BSDOI-15 recombinant using glycerol and glucose as a dual carbon source yielded the highest DOI titer (38.0 g/L). The development of engineered microbial cell factories empowered through convergence of metabolic engineering and synthetic biology should enable mass production of DOI. Thus, strain BSDOI-15 will surely be a useful contributor to the industrial manufacturing of various kinds of DOI-based pharmaceuticals and fine chemicals.

Aminoglycosides (AGs) are the first and foremost classical antibiotics that are biosynthesized by attachment of amino-sugar units via glycosidic bonds to aminocyclitol derivatives [1]. They are recognized to inhibit prokaryotic protein synthesis by binding to the 16S rRNA subunit of the 30S bacterial ribosome, thus causing irreversible bactericidal effects against the pathogens [2]. According to the chemical features of the aminocyclitol moiety and the core aglycone unit, AGs have been categorized into well-known types containing 2-deoxystreptamine (DOS), (including 4,5-disubstituted butirosins and neomycins or 4,6-disubstituted gentamicins and kanamycins) and 1,4diaminocyclitol-containing AGs [3]. In the late 1970s, the above 1,4-diaminocyclitol-containing AGs, namely fortimicins (FORs) and istamycins (ISTs), were isolated from Micromonospora olivasterospora and Streptomyces tenjimariensis, respectively [4,5]. The two share an unusual pseudodisaccharide moiety that is different from a common