Differential proteomic analysis highlights metabolic strategies associated with balhimycin production in Amycolatopsis balhimycina chemostat cultivations.
BACKGROUND
Proteomics was recently used to reveal enzymes whose expression is associated with the production of the glycopeptide antibiotic balhimycin in Amycolatopsis balhimycina 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 status and metabolic pathways during antibiotic producing and non-producing conditions could be highlighted.


RESULTS
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 A. balhimycina chemostat cultivations. Applying the same dilution rate (0.03 h-1), 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 bal genes, devoted to balhimycin biosynthesis, and of phoP, phoR, pstS and phoD, 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 http://www.unipa.it/ampuglia/Abal-proteome-maps 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).


CONCLUSIONS
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.
Università di Palermo, Dipartimento di Biologia Cellulare e dello Sviluppo, Viale delle Scienze, Parco d'Orleans II, 90128 Palermo, Italy. giuseppe.gallo@unipa.it
Giuseppe
G
Gallo
Rosa
R
Alduina
Giovanni
G
Renzone
Jette
J
Thykaer
Linda
L
Bianco
Anna
A
Eliasson-Lantz
Andrea
A
Scaloni
Anna Maria
AM
Puglia
D000192:Actinomycetales / Q000254:growth & development / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002244:Carbon / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005227:Fatty Acids / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D014176:Protein Biosynthesis; D020543:Proteome / Q000032:analysis*; D014640:Vancomycin / Q000031:analogs & derivatives* / Q000096:biosynthesis
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D005227:Fatty Acids; D020543:Proteome; C087127:balhimycin; D014640:Vancomycin; D002244:Carbon; D005947:Glucose