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Using a simple method to introduce genetic modifications into the chromosome of naturally nontransformable Bacillus, a set of marker-free inosine-producing and 5-aminoimidazole-4-carboxamide (AICA) ribonucleoside-producing Bacillus amyloliquefaciens strains has been constructed. These strains differ in expression levels of the genes responsible for nucleoside export. Overexpression of B. amyloliquefaciens pbuE and heterologous expression of Escherichia coli nepI, which encode nucleoside efflux transporters, each notably enhanced inosine production by a B. amyloliquefaciens nucleoside-producing strain. pbuE overexpression was found to increase AICA ribonucleoside accumulation, indicating that the substrate specificity of the PbuE pump extends to this nucleoside. These results demonstrate that identifying genes whose products facilitate transport of a desired nucleoside out of cells and enhancing their expression can improve the performance of strains used for industrial production.

The pbuE (ydhL) gene from Bacillus subtilis is known to encode the purine base efflux pump, and its expression is controlled by an adenine-dependent riboswitch. We cloned the pbuE gene from Bacillus amyloliquefaciens and examined gene expression by its own cis-acting regulatory elements in Escherichia coli. Regulation of pbuE expression, previously found in B. subtilis, was retained in this heterologous expression: it was induced by adenine and activated by a mutation in the 5' untranslated region, which disrupted transcription termination. This observation supports the model that the adenine-dependent riboswitch directly regulates pbuE expression, without requiring additional factors. Overexpression of the PbuE pump conferred upon the E. coli strain resistance to higher concentrations of inosine, adenosine and guanosine, and increased exogenous inosine accumulation by E. coli cells deficient in purine nucleoside phosphorylase. Overexpression of the PbuE pump also enhanced hypoxanthine excretion by the E. coli hypoxanthine-producing strain and inosine excretion both by the E. coli and B. amyloliquefaciens nucleoside-producing strains. Thus, for the first time, we obtained direct evidence for the involvement of PbuE in efflux of not only purine bases, but also purine ribonucleosides. A possible new role for the pump in cell physiology is discussed.

A two-step enzymatic synthesis process of 4-hydroxyisoleucine is suggested. In the first step, the aldol condensation of acetaldehyde and alpha-ketobutyrate catalyzed by specific aldolase results in the formation of 4-hydroxy-3-methyl-2-keto-pentanoate (HMKP). In the second step, amination of HMKP by the branched-chain amino acid aminotransferase leads to synthesis of 4-hydroxyisoleucine. An enzyme possessing HMKP aldolase activity (asHPAL) was purified 2500-fold from a crude extract of Arthrobacter simplex strain AKU 626. Sequencing of the asHPAL structural gene showed that the purified enzyme belongs to the HpcH/HpaI aldolase family. The 4-hydroxyisoleucine was synthesized in vitro from acetaldehyde, alpha-ketobutyrate and l-glutamate using a coupled aldolase/branched-chain amino acid aminotransferase bienzymatic reaction.