| Summary: | <p>Abstract</p> <p>Background</p> <p>Proteomics was recently used to reveal enzymes whose expression is associated with the production of the glycopeptide antibiotic balhimycin in <it>Amycolatopsis balhimycina </it>batch cultivations. Combining chemostat fermentation technology, where cells proliferate with constant parameters in a highly reproducible steady-state, and differential proteomics, the relationships between physiological <it>status </it>and metabolic pathways during antibiotic producing and non-producing conditions could be highlighted.</p> <p>Results</p> <p>Two minimal defined media, one with low Pi (0.6 mM; LP) and proficient glucose (12 g/l) concentrations and the other one with high Pi (1.8 mM) and limiting (6 g/l; LG) glucose concentrations, were developed to promote and repress antibiotic production, respectively, in <it>A. balhimycina </it>chemostat cultivations. Applying the same dilution rate (0.03 h<sup>-1</sup>), both LG and LP chemostat cultivations showed a stable steady-state where biomass production yield coefficients, calculated on glucose consumption, were 0.38 ± 0.02 and 0.33 ± 0.02 g/g (biomass dry weight/glucose), respectively. Notably, balhimycin was detected only in LP, where quantitative RT-PCR revealed upregulation of selected <it>bal </it>genes, devoted to balhimycin biosynthesis, and of <it>phoP</it>, <it>phoR</it>, <it>pstS </it>and <it>phoD</it>, known to be associated to Pi limitation stress response. 2D-Differential Gel Electrophoresis (DIGE) and protein identification, performed by mass spectrometry and computer-assisted 2 D reference-map <url>http://www.unipa.it/ampuglia/Abal-proteome-maps</url> matching, demonstrated a differential expression for proteins involved in many metabolic pathways or cellular processes, including central carbon and phosphate metabolism. Interestingly, proteins playing a key role in generation of primary metabolism intermediates and cofactors required for balhimycin biosynthesis were upregulated in LP. Finally, a bioinformatic approach showed PHO box-like regulatory elements in the upstream regions of nine differentially expressed genes, among which two were tested by electrophoresis mobility shift assays (EMSA).</p> <p>Conclusion</p> <p>In the two chemostat conditions, used to generate biomass for proteomic analysis, mycelia grew with the same rate and with similar glucose-biomass conversion efficiencies. Global gene expression analysis revealed a differential metabolic adaptation, highlighting strategies for energetic supply and biosynthesis of metabolic intermediates required for biomass production and, in LP, for balhimycin biosynthesis. These data, confirming a relationship between primary metabolism and antibiotic production, could be used to increase antibiotic yield both by rational genetic engineering and fermentation processes improvement.</p>
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