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Bacillus strains are used for the industrial production of the purine nucleosides inosine and guanosine, which are raw materials for the synthesis of the flavor enhancers disodium inosinate and disodium guanylate. An important precursor of purine nucleosides is 5-phospho-α-D: -ribosyl-1-pyrophosphate, which is synthesized by phosphoribosyl pyrophosphate synthetase (PRS, EC 2.7.6.1). Class I PRSs are widespread in bacteria and mammals, are highly conserved among different organisms, and are negatively regulated by two end products of purine biosynthesis, adenosine 5'-diphosphate (ADP) and guanosine 5'-diphosphate (GDP). The D52H, N114S, and L129I mutations in the human PRS isozyme I (PRS1) have been reported to cause uric acid overproduction and gout due to allosteric deregulation and enzyme superactivity. In this study, to find feedback-resistant Bacillus amyloliquefaciens PRS, the influence of the D58H, N120S, and L135I mutations (corresponding to the D52H, N114S, and L129I mutations in PRS1, respectively) on PRS enzymatic properties has been studied. Recombinant histidine-tagged wild-type PRS and three mutant PRSs were expressed in Escherichia coli, purified, and characterized. The N120S and L135I mutations were found to release the enzyme from ADP and GDP inhibition and significantly increase its sensitivity to inorganic phosphate (P(i)) activation. In contrast, PRS with the D58H mutation exhibited nearly identical sensitivity to ADP and GDP as the wild-type protein and had a notably greater P(i) requirement for activation. The N120S and L135I mutations improved B. amyloliquefaciens and Bacillus subtilis purine nucleoside-producing strains.

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.

Overproduction of noncanonical amino acids norvaline and norleucine by Escherichia coli with inactivated acetohydroxy acid synthases was demonstrated. The cultivation conditions for the overproduction of noncanonical amino acids were studied. The effect of the restoration of acetohydroxy acid synthase activity, increased expression of the leuABCD operon, and inactivation of the biosynthetic threonine deaminase on norvaline and norleucine synthesis was studied. When grown under valine limitation, E. coli cells with inactivated acetohydroxy acid synthases and an elevated level of expression of the valine operon were shown to accumulate norvaline and norleucine (up to 0.8 and 4 g/l, respectively). These results confirm the existing hypothesis of norvaline and norleucine formation from 2-ketobutyrate by leucine biosynthesis enzymes.

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.

The yicM gene of Escherichia coli was found by selection for resistance to 6-mercaptopurine. Translation and transcription initiation sites of yicM were determined. Overexpression of yicM increased resistance of sensitive cells to inosine and guanosine, decreased E. coli growth rate in medium containing these ribonucleosides as the sole carbon source, led to inosine accumulation by the E. coli strain deficient in purine nucleoside phosphorylase and enhanced the rate of inosine excretion by an inosine-producing strain. These results suggest that yicM encodes a purine ribonucleoside exporter and we have accordingly renamed it nepI (for 'nucleoside efflux permease-inosine').

Gamma-aminobutyraldehyde dehydrogenase (ABALDH) from wild-type E. coli K12 was purified to apparent homogeneity and identified as YdcW by MS-analysis. YdcW exists as a tetramer of 202+/-29 kDa in the native state, a molecular mass of one subunit was determined as 51+/-3 kDa. Km parameters of YdcW for gamma-aminobutyraldehyde, NAD+ and NADP+ were 41+/-7, 54+/-10 and 484+/-72 microM, respectively. YdcW is the unique ABALDH in E. coli K12. A coupling action of E. coli YgjG putrescine transaminase and YdcW dehydrogenase in vitro resulted in conversion of putrescine into gamma-aminobutyric acid.