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Review
, 2012, 105109

Proteomics Shows New Faces for the Old Penicillin Producer Penicillium Chrysogenum

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Review

Proteomics Shows New Faces for the Old Penicillin Producer Penicillium Chrysogenum

Carlos Barreiro et al. J Biomed Biotechnol.

Abstract

Fungi comprise a vast group of microorganisms including the Ascomycota (majority of all described fungi), the Basidiomycota (mushrooms or higher fungi), and the Zygomycota and Chytridiomycota (basal or lower fungi) that produce industrially interesting secondary metabolites, such as β-lactam antibiotics. These compounds are one of the most commonly prescribed drugs world-wide. Since Fleming's initial discovery of Penicillium notatum 80 years ago, the role of Penicillium as an antimicrobial source became patent. After the isolation of Penicillium chrysogenum NRRL 1951 six decades ago, classical mutagenesis and screening programs led to the development of industrial strains with increased productivity (at least three orders of magnitude). The new "omics" era has provided the key to understand the underlying mechanisms of the industrial strain improvement process. The review of different proteomics methods applied to P. chrysogenum has revealed that industrial modification of this microorganism was a consequence of a careful rebalancing of several metabolic pathways. In addition, the secretome analysis of P. chrysogenum has opened the door to new industrial applications for this versatile filamentous fungus.

Figures

Figure 1
Figure 1
Scheme showing the industrial strain improvement program of P. chrysogenum, Barreiro et al., 2011.
Figure 2
Figure 2
Examples of two well-known modifications that occurred during the strain improvement program. (a) Amplification of the DNA region containing the penicillin biosynthetic gene cluster in high-producing strains. LEB: left end border; REB: right end border; TRU: tandem repetition union. (b) Modification in the metabolic fluxes through the homogentisate pathway for phenylacetic acid catabolism and the penicillin biosynthetic pathway. Thickness of arrows indicates the flux rate through a specific enzyme. α-AAA: α-aminoadipic acid; ACV: δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine; CoA: coenzyme A; Cys: cysteine; Val: Valine, Barreiro et al., 2011.
Figure 3
Figure 3
Schematic representation of the optimized method for mycelia and secreted proteins collection of P. chrysogenum in order to analyze the intracellular and extracellular proteomes. Green/purple-coloured arrows show the common steps for both methods. Green arrows represent the specific method for secreted proteins isolation. Purple arrows show those steps specific for intracellular proteome isolation, Barreiro et al., 2011.
Figure 4
Figure 4
Optimization of the protein extraction protocol from mycelia of P. chrysogenum. (a) Extraction and purification of protein samples with ‘‘Clean-Up Kit” (GE Healthcare), based on Kniemeyer and coworkers [66] and silver stained. As sample buffer was used, 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 0.8% (v/v) IPG buffer pH 3–10 nonlinear (NL) (GE Healthcare), 40 mM Tris, 1 mM EDTA, and 20 mM DTT [75]. (b) The extraction is based on the method described by Fernández-Acero and coworkers [63] by using mortar gridding and phosphate buffer, plus blue silver Coomassie colloidal staining [76]. As sample buffer was used, 8 M urea, 2% (w/v) CHAPS, 0.5% (v/v) IPG buffer pH 3–10 NL (GE Healthcare), 20 mM DTT, and 0.002% bromophenol blue [69]. Precision plus protein standards (Bio-Rad) were used as markers. The molecular mass is indicated in kilo-Daltons (kDa). Note the problematic regions observed with the extraction method A, which are highlighted by arrowed square brackets (left and bottom), Barreiro et al., 2011.
Figure 5
Figure 5
Pathways and networks modified during the strain improvement program. Font size is in concordance with concentration and differences; the thickness of arrows indicates the flux through a specific pathway. α-AAA: α-aminoadipic acid; Cys: cysteine; N: nucleus; NADPH: reduced form of nicotinamide adenine dinucleotide phosphate; P: peroxisome; PAA: phenylacetic acid; PenG: benzylpenicillin; PPP: pentose phosphate pathway; Val: valine, Barreiro et al., 2011.

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