Differential proteomic analysis reveals novel links between primary metabolism and antibiotic production in Amycolatopsis balhimycina

Abstract

A differential proteomic analysis, based on 2-DE and MS procedures, was performed on Amycolatopsis balhimycina DSM5908, the actinomycete producing the vancomycin-like antibiotic balhimycin. A comparison of proteomic profiles before and during balhimycin production characterized differentially and constitutively expressed protein isoforms, which were associated with 203 ORFs in the A. balhimycina genome. These data, providing insights on the major metabolic pathways/molecular processes operating in this organism, were used to compile 2-DE reference maps covering 3-10, 4-7 and 4.5-5.5 pH gradients available over the World Wide Web as interactive web pages (http://www.unipa.it/ampuglia/Abal-proteome-maps). Functional clustering analysis revealed that differentially expressed proteins belong to functional groups involved in central carbon metabolism, amino acid metabolism and protein biosynthesis, energetic and redox balance, sugar/amino sugar metabolism, balhimycin biosynthesis and transcriptional regulation or with hypothetical and/or unknown function. Interestingly, proteins involved in the biosynthesis of balhimycin precursors, such as amino acids, amino sugars and central carbon metabolism intermediates, were upregulated during antibiotic production. qRT-PCR analysis revealed that 8 out of 14 upregulated genes showed a positive correlation between changes at translational and transcriptional expression level. Furthermore, proteomic analysis of two nonproducing mutants, restricted to a sub-set of differentially expressed proteins, showed that most proteins required for the biosynthesis of balhimycin precursors are downregulated in both mutants. These findings suggest that primary metabolic pathways support anabolic routes leading to balhimycin biosynthesis and the differentially expressed genes are interesting targets for the construction of high-yielding producer strains by rational genetic engineering.

Differential proteomic analysis highlights metabolic strategies associated with balhimycin production in Amycolatopsis balhimycina chemostat cultivations

“…The upregulation of glycolytic enzymes is also in agreement with the upregulation of TCA cycle enzymes aconitate hydratase (ACO), succinate dehydrogenase flavoprotein subunit (SudA), LpdA1 and 2, SucB, succinyl-CoA ligase alpha and beta subunit (A-and B-SCS) and malate dehydrogenase (MDH) (Figure 5; Additional file 1 Table 3S). These findings also correlated with the increased production yield of CO 2 in LP condition (Figure 1D) and also with the upregulation of ThiS and ThiC, involved in thiamine synthesis and previously associated with balhimycin production [23]. Thiamine diphosphate (TDP) is reported to be a co-factor of the upregulated TrK, pyruvate and alpha-ketoglutarate dehydrogenase complexes [34].…”

“…This investigation revealed 72 upregulated and 12 downregulated protein spots in LP condition, showing at least 1.5 fold increased or decreased abundance (calculated as spot Vol) and at least a probability for null hypothesis (p) < 0.05 (calculated using the ana lysis of var iance or ANOVA test). These proteins were identified by MS analysis or by automatic gel-matching using the A. balhimycina protein 2 D reference-maps, available over the World Wide Web as interactive pages at http://www.unipa.it/ampuglia/Abal-proteome-maps[23,24] (Additional file 1 Table 3S, 4S, 5S and 6S). The reliability of this analysis was firstly demonstrated by the identification of groups of proteins showing similar expression profiles and whose corresponding genes are arranged in putative operons (Additional file 1 Table 6S), which are characterized by a maximum of 60 bp between two adjacent ORFs.…”

“…In this context, we have recently used a differential proteomic approach to demonstrate that balhimycin production in batch culture is associated with the upregulation of enzymes involved in the biosynthesis of antibiotic precursors, thus suggesting that the metabolic apparatus is orientated to sustain balhimycin production [23,24]. Accordingly, an increased precursor availability, in particular tyrosine, was recently showed to be beneficial for balhimycin production [25].…”

Differential proteomic profiling reveals regulatory proteins and novel links between primary metabolism and spinosad production in Saccharopolyspora spinosa

“…Black dot indicated a enzyme owned a peak in T2 when compared to other three phases; black triangle signified a protein up-regulated in T2, T3 and T4 while compared to T1 and had a peak in T4. Reactions are reported according to KEGG metabolic pathway databases and Gallo et al [10]. Enzyme name abbreviations refer to Additional file 3: Table S5.…”

“…More traditional methods for separating protein mixtures include two-dimensional gel electrophoresis (2D-GE) or two-dimensional fluorescence-difference gel electrophoresis (2D-DIGE), followed by enzymatic digestion and mass spectrometry (MS) [9,10]. Another popular method uses gel-enhanced liquid chromatography-tandem mass spectrometry (LC-MS/MS), wherein proteins in a mixture are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with LC-MS/MS and label-free semi-quantification [11] or isobaric tags for relative and absolute quantitation (iTRAQ) [12].…”