Identification of mycotoxins by UHPLC-QTOF MS in airborne fungi and fungi isolated from industrial paper and antique documents from the Archive of Bogotá.

Mold deterioration of historical documents in archives and libraries is a frequent and complex phenomenon that may have important economic and cultural consequences. In addition, exposure to toxic fungal metabolites might produce health problems. In this work, samples of broths of fungal species isolated from the documentary material and from indoor environmental samples of the Archive of Bogotá have been analyzed to investigate the presence of mycotoxins. High resolution mass spectrometry made possible to search for a large number of mycotoxins, even without reference standards available at the laboratory. For this purpose, a screening strategy based on ultra-high pressure liquid chromatography coupled to quadrupole-time of flight mass spectrometry (UHPLC-QTOF MS) under MS(E) mode was applied. A customized home-made database containing elemental composition for around 600 mycotoxins was compiled. The presence of the (de)protonated molecule measured at its accurate mass was evaluated in the samples. When a peak was detected, collision induced dissociation fragments and characteristic isotopic ions were also evaluated and used for tentative identification, based on structure compatibility and comparison with literature data (if existing). Up to 44 mycotoxins were tentatively identified by UHPLC-QTOF MS. 34 of these tentative compounds were confirmed by subsequent analysis using a targeted LC-MS/MS method, supporting the strong potential of QTOF MS for identification/elucidation purposes. The presence of mycotoxins in these samples might help to reinforce safety measures for researchers and staff who work on reception, restoration and conservation of archival material, not only at the Archive of Bogotá but worldwide.

Penicillium chrysogenum, a Vintage Model with a Cutting-Edge Profile in Biotechnology.

The discovery of penicillin entailed a decisive breakthrough in medicine. No other medical advance has ever had the same impact in the clinical practise. The fungus Penicillium chrysogenum (reclassified as P. rubens) has been used for industrial production of penicillin ever since the forties of the past century; industrial biotechnology developed hand in hand with it, and currently P. chrysogenum is a thoroughly studied model for secondary metabolite production and regulation. In addition to its role as penicillin producer, recent synthetic biology advances have put P. chrysogenum on the path to become a cell factory for the production of metabolites with biotechnological interest. In this review, we tell the history of P. chrysogenum, from the discovery of penicillin and the first isolation of strains with high production capacity to the most recent research advances with the fungus. We will describe how classical strain improvement programs achieved the goal of increasing production and how the development of different molecular tools allowed further improvements. The discovery of the penicillin gene cluster, the origin of the penicillin genes, the regulation of penicillin production, and a compilation of other P. chrysogenum secondary metabolites will also be covered and updated in this work.

Transcriptional and bioinformatic analysis of the 56.8 kb DNA region amplified in tandem repeats containing the penicillin gene cluster in Penicillium chrysogenum.

High penicillin-producing strains of Penicillium chrysogenum contain 6-14 copies of the three clustered structural biosynthetic genes, pcbAB, pcbC, and penDE [Barredo, J.L., Díez, B., Alvarez, E., Martín, J.F., 1989. Large amplification of a 35-kb DNA fragment carrying two penicillin biosynthetic genes in high penicillin producing strains of Penicillium chrysogenum. Curr. Genet. 16, 453-459; Smith, D.J., Bull, J.H., Edwards, J., Turner, G., 1989. Amplification of the isopenicillin N synthetase gene in a strain of Penicillium chrysogenum producing high levels of penicillin. Mol. Gen. Genet. 216, 492-497.] . The cluster is located in a 56.8 kb DNA region bounded by a conserved TGTAAA/T hexanucleotide that undergoes amplification in tandem repeats [Fierro, F., Barredo, J.L., Díez, B., Gutiérrez, S., Fernández, F.J., Martín, J.F., 1995. The penicillin gene cluster is amplified in tandem repeats linked by conserved hexanucleotide sequences. Proc. Natl. Acad. Sci. USA 92, 6200-6204; Newbert, R.W., Barton, B., Greaves, P., Harper, J., Turner, G., 1997. Analysis of a commercially improved Penicillium chrysogenum strain series: involvement of recombinogenic regions in amplification and deletion of the penicillin biosynthesis gene cluster. J. Ind. Microbiol. Biotechnol. 19, 18-27]. Transcriptional analysis of this amplified region (AR) revealed the presence of at least eight transcripts expressed in penicillin producing conditions. Three of them correspond to the known penicillin biosynthetic genes, pcbAB, pcbC, and penDE. To locate genes related to penicillin precursor formation, or penicillin transport and regulation we have sequenced and analyzed the 56.8 kb amplified region of P. chrysogenum AS-P-78, finding a total of 16 open reading frames. Two of these ORFs have orthologues of known function in the databases. Other ORFs showed similarities to specific domains occurring in different proteins and superfamilies which allowed to infer their probable function. ORF11 encodes a D-amino acid oxidase that might be responsible for the conversion of D-amino acids in the tripeptide L-alpha-aminoadipyl-L-cysteinyl-D-valine or other beta-lactam intermediates to deaminated by-products. ORF12 encodes a predicted protein with similarity to saccharopine dehydrogenases that seems to be related to biosynthesis of the penicillin precursor alpha-aminoadipic acid. A deletion mutant, P. chrysogenum npe10 lacking the entire AR including ORF12, shows a partial requirement of L-lysine for growth. ORF13 encodes a putative protein containing a Zn(II)2-Cys6 fungal-type DNA-binding domain, probably a transcriptional regulator. Although some of the ORFs in the AR may play roles in increasing penicillin production, none of the 13 ORFs other than pcbAB, pcbC, and penDE seem to be strictly indispensable for penicillin biosynthesis. The genes located in the P. chrysogenum AR have been compared with those found in the Aspergillus nidulans 50 kb DNA region adjacent to the penicillin gene cluster, showing no conservation between these two fungi.