Enhancement of extracellular purine nucleoside accumulation by Bacillus strains through genetic modifications of genes involved in nucleoside export

Abstract

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.

Reconstruction of Purine Metabolism in Bacillus subtilis to Obtain the Strain Producer of AICAR: A New Drug with a Wide Range of Therapeutic Applications

“…The mechanism of AICAR excretion remains unknown, although data was obtained indicating that the membrane protein encoded by the pbuE gene is involved in its export from bacilli cells [41]. …”

Expression and purification of the 5′-nucleotidase YitU from Bacillus species: its enzymatic properties and possible applications in biotechnology

“…The main desirable features for the application of many Bacillus species as microbial cell factories are their generally recognized as safe (GRAS) status, probiotic properties, absence of exotoxins and endotoxin production, fully sequenced genomes, well-studied secretion pathways, and fairly simple cultivation conditions; their available transcriptome, metabolome, and proteome analysis data, and advanced genetic engineering tools are suitable for use with these species. B. subtilis and B. amyloliquefaciens strains have been successfully designed to produce riboflavin (RF), adenosine, inosine, guanosine, and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), which are widely used in food technology and the pharmaceutical industry (Stepanov et al 1984;Perkins et al 1999;Asahara et al 2010;Lobanov et al 2011;Sheremet et al 2011;Zhang et al 2015). Since the compounds listed can be synthesized from their immediate phosphorylated precursors, flavin mononucleotide (FMN), AMP, IMP, GMP, and 5-aminoimidazole-4-carboxamide-1-β-Dribofuranosyl 5′-monophosphate (AICAR-P), respectively, Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00253-020-10428-y) contains supplementary material, which is available to authorized users.…”

The filamentous fungus Ashbya gossypii as a competitive industrial inosine producer

“…The majority of these industrial bacterial strains were obtained by conventional random mutagenesis, a process that inevitably accumulate unidentifiable and undesirable secondary mutations. Therefore, during the last years, considerable effort has been made to obtain super-producer strains by rational gene manipulation of B. subtilis (Asahara et al, 2010;Li et al, 2011;Sheremet et al, 2011) and E. coli (Shimaoka et al, 2007). To date, metabolic engineering approaches in these bacteria have targeted the de novo purine metabolism, the central metabolism, the salvage purine pathway, some regulator genes and the nucleotide and nucleoside transporters (reviewed in Ledesma-Amaro et al, 2013).…”