Application of an <em>In vitro</em> DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

Oxidative stress is an unavoidable byproduct of  aerobic life.  Molecular oxygen is  essential for terrestrial metabolism, but it also takes part in many ...

Delft University of Technology

Industrial strain improvement via classical mutagenesis is a black box approach. In an attempt to learn from and understand the mutations introduced, we cloned and characterized the amplified region of industrial penicillin production strains. Upon amplification of this region Penicillium chrysogenum is capable of producing an increased amount of antibiotics, as was previously reported [Barredo, J.L., Diez, B., Alvarez, E., MartÃ­n, J.F., 1989a. Large amplification of a 35-kb DNA fragment carrying two penicillin biosynthetic genes in high yielding strains of Penicillium chrysogenum. Curr. Genet. 16, 453-459; 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 gene cluster. J. Ind. Microbiol. 19, 18-27]. Bioinformatic analysis of the central 56.9kb, present as six direct repeats in the strains analyzed in this study, predicted 15 Open Reading Frames (ORFs). Besides the three penicillin biosynthetic genes (pcbAB, pcbC and penDE) only one ORF has an orthologue of known function in the database: the Saccharomyces cerevisiae gene ERG25. Surprisingly, many genes known to encode direct or indirect steps beta-lactam biosynthesis like phenyl acetic acid CoA ligase and transporters are not present. Detailed analyses reveal a detectable transcript for most of the predicted ORFs under the conditions tested. We have studied the role of these in relation to penicillin production and amplification of the biosynthetic gene cluster. In contrast to what was expected, the genes encoding the three penicillin biosynthetic enzymes alone are sufficient to restore full beta-lactam synthesis in a mutant lacking the complete region. Therefore, the role of the other 12 ORFs in this region seems irrelevant for penicillin biosynthesis.

Functional characterization of the penicillin biosynthetic gene cluster of Penicillium chrysogenum Wisconsin54-1255.

Am&eacute;lioration de la souche industrielle par mutagen&egrave;se classique est une approche de la bo&icirc;te noire. Dans le but d&#39;apprendre et de comprendre les mutations introduites, nous avons clon&eacute; et caract&eacute;ris&eacute; la r&eacute;gion amplifi&eacute;e de souches de production industrielle de la p&eacute;nicilline. Lors de l&#39;amplification de cette r&eacute;gion de Penicillium chrysogenum est capable de produire une plus grande quantit&eacute; d&#39;antibiotiques, comme indiqu&eacute; pr&eacute;c&eacute;demment [Barredo, J.L., Diez, B., Alvarez, E., Mart&iacute;n, J.F., 1989 a. Des fragments de grande amplification d&#39;un ADN de Ko 35 porteurs de deux g&egrave;nes de biosynth&egrave;se de la p&eacute;nicilline dans les souches &agrave; rendement &eacute;lev&eacute;s de Penicillium chrysogenum. Curr. Genet. 16, 453-459 ; Newbert, R.W., Barton, B., Greaves, P., Harper, J., Turner, g., 1997. Analyse de commercialement am&eacute;lior&eacute;e Penicillium chrysogenum souche s&eacute;ries, implication des r&eacute;gions recombinog&eacute;nique en amplification et supprimer le groupe de g&egrave;nes de la p&eacute;nicilline. J. ind. Microbiol. 19, 18-27]. Analyse bioinformatique du central 56,9 Ko, pr&eacute;sent lorsque six direct est r&eacute;p&eacute;t&eacute;e dans les souches analys&eacute;es dans cette &eacute;tude, pr&eacute;dit 15 cadres ouverts de lecture (ORF). Outre les g&egrave;nes de biosynth&egrave;se de la trois p&eacute;nicilline (pcbAB, pcbC et penDE) seule ORF a un orthologue de fonction connue dans la base de donn&eacute;es : g&egrave;ne Saccharomyces cerevisiae ERG25. &Eacute;tonnamment, les nombreux g&egrave;nes connus pour coder la biosynth&egrave;se de b&ecirc;ta-lactame mesures directes ou indirectes comme la ligase d&#39;ACO ph&eacute;nyl ac&eacute;tique acide et les transporteurs ne sont pas pr&eacute;sents. Des analyses d&eacute;taill&eacute;es r&eacute;v&egrave;lent une transcription d&eacute;tectable pendant la majeure partie de l&#39;ORF pr&eacute;vue dans les conditions test&eacute;es. Nous avons &eacute;tudi&eacute; le r&ocirc;le de ceux-ci dans la production de la p&eacute;nicilline et l&#39;amplification de l&#39;amas de g&egrave;nes de biosynth&egrave;se. Contrairement &agrave; ce qui &eacute;tait attendu, les g&egrave;nes codant les enzymes biosynth&eacute;tiques trois p&eacute;nicilline seules sont suffisants pour restaurer la synth&egrave;se compl&egrave;te des b&ecirc;ta-lactamines chez un mutant d&eacute;pourvu de la r&eacute;gion compl&egrave;te. Le r&ocirc;le de la 12 autres ORF dans cette r&eacute;gion semble donc pas pertinent pour la biosynth&egrave;se de la p&eacute;nicilline.

Caract&eacute;risation fonctionnelle du groupe de g&egrave;nes de biosynth&egrave;se de la p&eacute;nicilline de Penicillium chrysogenum Wisconsin54-1255.

Miglioramento del ceppo industriale tramite mutagenesi classica &egrave; un approccio di scatola nera. Nel tentativo di imparare e capire le mutazioni introdotte, abbiamo clonato e caratterizzato la regione amplificata di ceppi di produzione industriale di penicillina. All&#39;amplificazione di questa regione Penicillium chrysogenum &egrave; in grado di produrre una maggiore quantit&agrave; di antibiotici, come gi&agrave; precedentemente segnalato [Barredo, J.L., Diez, B., Alvarez, E., Mart&iacute;n, J.F., 1989Un. Grande amplificazione di un 35-kb di DNA frammento portando due geni biosintetici penicillina in alti rendimento ceppi di Penicillium chrysogenum. Curr. Genet. 16, 453-459; Newbert, R.W., Barton, B., ciccioli, P., Harper, J., Turner, g., 1997. Analisi di una migliorata commercialmente Penicillium chrysogenum ceppo serie, coinvolgimento delle regioni recombinogenic per l&#39;amplificazione e l&#39;eliminazione del cluster genico di penicillina. J. ind. Microbiol. 19, 18-27]. Analisi bioinformatica della centrale 56,9 kb, presente come sei diretta ripete in ceppi analizzati in questo studio, predetto 15 Open Reading frame (ORF). Oltre i geni biosintetici tre penicillina (pcbAB, pcbC e penDE) sola ORF ha un orthologue della funzione nota nel database: gene Saccharomyces cerevisiae ERG25. Sorprendentemente, molti geni codificano biosintesi beta-lattamici passaggi diretti o indiretti come ligasi CoA di fenil acido acetico ed i trasportatori non sono presenti. Analisi dettagliate rivelano una trascrizione rilevabile per la maggior parte del ORFs previsto nelle condizioni testate. Abbiamo studiato il ruolo di questi in relazione alla produzione di penicillina e amplificazione del cluster gene biosintetico. Contrariamente a ci&ograve; che ci si aspettava, dei geni che codificano enzimi biosintetici tre penicillina soli sono sufficienti a ripristinare la sintesi completa beta-lattamici in un mutante manca la regione completa. Pertanto, il ruolo di altri 12 ORFs in questa regione sembra irrilevante per la biosintesi di penicillina.

Caratterizzazione funzionale di ammasso di Penicillium chrysogenum Wisconsin54-1255 gene biosintetico della penicillina.

Penicillium chrysogenum was successfully engineered to produce a novel carbamoylated cephalosporin that can be used as a synthon for semi-synthetic cephalosporins. To this end, genes for Acremonium chrysogenum expandase/hydroxylase and Streptomyces clavuligerus carbamoyltransferase were expressed in a penicillinG high-producing strain of P.chrysogenum. Growth of the engineered strain in the presence of adipic acid resulted in production of adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid (ad7-ACCCA) and of several adipoylated pathway intermediates. A combinatorial chemostat-based transcriptome study, in which the ad7-ACCCA-producing strain and a strain lacking key genes in beta-lactam synthesis were grown in the presence and absence of adipic acid, enabled the dissection of transcriptional responses to adipic acid per se and to ad7-ACCCA production. Transcriptome analysis revealed that adipate catabolism in P.chrysogenum occurs via beta-oxidation and enabled the identification of putative genes for enzymes involved in mitochondrial and peroxisomal beta-oxidation pathways. Several of the genes that showed a specifically altered transcript level in ad7-ACCCA-producing cultures were previously implicated in oxidative stress responses.

Engineering of Penicillium chrysogenum for fermentative production of a novel carbamoylated cephem antibiotic precursor.

Penicillium chrysogenum a &eacute;t&eacute; correctement con&ccedil;u pour produire une c&eacute;phalosporine de roman carbamoyl&eacute;e qui peut &ecirc;tre utilis&eacute;e comme un synthon des c&eacute;phalosporines semi-synth&eacute;tiques. &Agrave; cette fin, les g&egrave;nes pour Acremonium chrysogenum expandase/hydroxylase et Streptomyces clavuligerus carbamyl transf&eacute;rase sont exprim&eacute;s dans une souche de grandes productrices de penicillinG de P.chrysogenum. Croissance de la souche modifi&eacute;e en pr&eacute;sence d&#39;acide adipique a entra&icirc;n&eacute; la production de l&#39;acide adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic (ad7-ACCCA) et de plusieurs interm&eacute;diaires de voie d&#39;adipoylated. Une &eacute;tude de la combinatoire transcriptome ch&eacute;mostat-bas&eacute;, o&ugrave; la souche productrice ad7-ACCCA et une souche d&eacute;pourvues des g&egrave;nes cl&eacute;s dans la synth&egrave;se des b&ecirc;ta-lactamines ont &eacute;t&eacute; cultiv&eacute;es en pr&eacute;sence et en absence d&#39;acide adipique, activ&eacute; la dissection des r&eacute;ponses transcriptionnelles acide adipique en soi et la production ad7-ACCCA. Analyse du transcriptome a r&eacute;v&eacute;l&eacute; que l&#39;adipate de catabolisme dans P.chrysogenum s&#39;effectue via la b&ecirc;ta et permis l&#39;identification de g&egrave;nes putatifs des enzymes impliqu&eacute;s dans les voies de mitochondries et peroxysomes b&ecirc;ta. Plusieurs des g&egrave;nes qui ont montr&eacute; un niveau de transcription sp&eacute;cialement modifi&eacute;s en ad7 ACCCA-productrices de cultures &eacute;taient d&eacute;j&agrave; impliqu&eacute;s dans les r&eacute;actions de stress oxydatif.

Ing&eacute;nierie de Penicillium chrysogenum pour production fermentative d&#39;un pr&eacute;curseur aux antibiotiques du c&eacute;phem roman carbamoyl&eacute;e.

Penicillium chrysogenum con successo &egrave; stato progettato per produrre una cefalosporina carbamoylated romanzo che pu&ograve; essere utilizzata come un synthon per cefalosporine semisintetico. A tal fine, geni Acremonium chrysogenum expandase/idrossilasi e Streptomyces clavuligerus carbamiltrasferasi sono stati espressi in un ceppo di alto-produzione penicillinG di P.chrysogenum. Crescita del ceppo ingegnerizzato in presenza di acido adipico portato nella produzione di acido adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic (ad7-ACCCA) e di diversi prodotti intermedi del percorso adipoylated. Uno studio del transcriptome combinatoria basati su chemostat, in cui il ceppo ad7-ACCCA-producendo e un ceppo privo di geni chiave nella sintesi di beta-lattamici sono state coltivate in presenza e in assenza di acido adipico, abilitato la dissezione delle risposte di transcriptional di acido adipico per s&eacute; e per produzione di ad7-ACCCA. Transcriptome analisi hanno rivelato che adipato catabolismo in P.chrysogenum avviene attraverso metaboliti e permesso l&#39;identificazione di geni putativi per enzimi coinvolti nelle vie mitocondriale e perossisomiale metaboliti. Molti dei geni che hanno mostrato un livello di trascrizione specificamente alterati nelle culture ad7-ACCCA-producendo in precedenza sono stati implicati nelle risposte di stress ossidativo.

Ilja Westerlaken

Department of Bionanoscience,
Kavli Institute of Nanoscience,
Department of Bionanoscience, Kavli Institute of Nanoscience

Delft University of Technology

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

Ilja Westerlaken

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Bionanoscience,Kavli Institute of Nanoscience,Department of Bionanoscience, Kavli Institute of Nanoscience

Delft University of Technology

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

Department of Bionanoscience,
Kavli Institute of Nanoscience,
Department of Bionanoscience, Kavli Institute of Nanoscience