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 2D 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). Conclusion 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

Identification of a Transcription Factor Controlling pH-Dependent Organic Acid Response in Aspergillus niger.

Acid formation in Aspergillus niger is known to be subjected to tight regulation, and the acid production profiles are fine-tuned to respond to the ambient pH. Based on transcriptome data, putative trans-acting pH responding transcription factors were listed and through knock out studies, mutants exhibiting an oxalate overproducing phenotype were identified. The yield of oxalate was increased up to 158% compared to the wild type and the corresponding transcription factor was therefore entitled Oxalic Acid repression Factor, OafA. Detailed physiological characterization of one of the ΔoafA mutants, compared to the wild type, showed that both strains produced substantial amounts of gluconic acid, but the mutant strain was more efficient in re-uptake of gluconic acid and converting it to oxalic acid, particularly at high pH (pH 5.0). Transcriptional profiles showed that 241 genes were differentially expressed due to the deletion of oafA and this supported the argument of OafA being a trans-acting transcription factor. Furthermore, expression of two phosphoketolases was down-regulated in the ΔoafA mutant, one of which has not previously been described in fungi. It was argued that the observed oxalate overproducing phenotype was a consequence of the efficient re-uptake of gluconic acid and thereby a higher flux through glycolysis. This results in a lower flux through the pentose phosphate pathway, demonstrated by the down-regulation of the phosphoketolases. Finally, the physiological data, in terms of the specific oxygen consumption, indicated a connection between the oxidative phosphorylation and oxalate production and this was further substantiated through transcription analysis

Disruption of the NADPH-dependent glutamate dehydrogenase affects the morphology of two industrial strains of Penicillium chrysogenum

New morphological aspects of Penicillium chrysogenum were found during physiological characterisation of two NADPH-dependent glutamate dehydrogenase mutant strains. A morphological characterisation of the previously constructed strains, together with the two beta-lactam producing industrial recipient strains, was conducted. The reference strains showed a compact structure with highly branched hyphal elements whereas the morphology of the Delta gdhA strains consisting of long elongated hyphal elements with few branches. On solid medium, the hyphal growth unit (length) increased from an average of 47 mu m tip(-1) in the reference strains to 117 mu m tip(-1) in the Delta gdhA strains and in submerged cultures a decrease of 18% in branching frequency was measured due to the gdhA deletion. P. chrysogenum Wis 54-1255, the ancestor of most production strains was also characterised and this strain showed morphology similar to the industrial strains. Interestingly, the constructed strains showed morphology similar to wild type Aspergillus nidulans another species carrying the penicillin biosynthetic cluster. Thus, the results showed that elimination of glutamate dehydrogenase activity in high producing strains of P chrysogenum has a radical impact on morphology. (c) 2009 Elsevier B.V. All rights reserved

Physiological coefficients from chemostat cultivations.

<p>Physiological coefficients from chemostat cultivations.</p

Metabolic map illustrating the response of central carbon metabolism caused by the <i>oafA</i> deletion.

<p>A red box indicates up-regulation of a gene encoding an enzyme catalyzing this reaction. Green box indicates down-regulation. The dotted line indicates fructose-6-phosphate phosphoketolase, a reaction not previously described within the fungal kingdom.</p

Primers used for deletion of <i>oafA</i> in <i>A. niger</i>.

<p>Lower-case letters indicate overlapping genetic elements used for fusion PCR.</p

Representative profiles of chemostat cultivations for wild type (top) and <i>ΔoafA</i> (bottom).

<p>Two steady states were obtained for each chemostat cultivation performed.</p

Representative profiles of the biomass concentration, sugar concentration, carbon dioxide formation and acid formation during batch cultivations at pH 6.0 with the WT-strain (left) and and the <i>ΔoafA</i> strain.

<p>The maximum specific growth rate was estimated trough a logarithmic plot of the biomass concentration as a function of time. Yield coefficients were calculated as overall yields based on the accumulated biomass or metabolite concentration in stationary phase related to the amount of consumed glucose. The volumetric oxalate formation rate was estimated as the slope of a linear regression of the oxalate titer as a function of time.</p