scholarly journals Genome Editing in Filamentous Fungus Penicillium Verruculosum Using the CRISPR/Cas9 System

2021 ◽  
Author(s):  
Valeriy Kislitsin ◽  
Andrey Chulkin ◽  
Ivan Zorov ◽  
Yuri Denisenko ◽  
Arkadiy Sinitsyn Sinitsyn ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Genome Editing ◽  
Filamentous Fungus ◽  
Penicillium Verruculosum ◽  
Selective Marker ◽  
Double Knockout ◽  
Nitrate Reductase Gene ◽  
Reductase Gene ◽  
Industrial Strains ◽  
First Time

Abstract PurposeTo adapt CRISPR/Cas9 genome editing method for use in filamentous fungus Penicillium verruculosum, which is industrial producer of carbohydrases. ResultsFor the first time the CRISPR/Cas9 method was adapted for genome editing in the filamentous fungi Penicillium verrucullosum. Using the nitrate reductase gene (niaD) as a selective marker with the CRISPR/Cas9 system we performed double knockout of niaD and cellobiohydrolase 1 (cbh1) genes. The efficiency of double editing was 50%. At the same time, it was unexpected that the specific cellobiohydrolase activity rised after knockout of cbh1 gene due to the increase CBH2 expression. ConclusionWe developed effective method for genome editing in P. verruculosum, which can be used to improve qualities of industrial strains.

scholarly journals Inactivation of gltB Abolishes Expression of the Assimilatory Nitrate Reductase Gene (nasB) in Pseudomonas putida KT2442

2000 ◽  
Vol 182 (12) ◽  
pp. 3368-3376 ◽  
Author(s):  
Leo Eberl ◽  
Aldo Ammendola ◽  
Michael H. Rothballer ◽  
Michael Givskov ◽  
Claus Sternberg ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Pseudomonas Putida ◽  
Nitrogen Starvation ◽  
Nitrate Assimilation ◽  
Glutamate Synthase ◽  
Nitrogen Sources ◽  
Nucleotide Sequence Analysis ◽  
Nitrate Reductase Gene ◽  
Physiological Characterization ◽  
Reductase Gene

ABSTRACT By using mini-Tn5 transposon mutagenesis, random transcriptional fusions of promoterless bacterial luciferase,luxAB, to genes of Pseudomonas putida KT2442 were generated. Insertion mutants that responded to ammonium deficiency by induction of bioluminescence were selected. The mutant that responded most strongly was genetically analyzed and is demonstrated to bear the transposon within the assimilatory nitrate reductase gene (nasB) of P. putida KT2442. Genetic evidence as well as sequence analyses of the DNA regions flanking nasBsuggest that the genes required for nitrate assimilation are not clustered. We isolated three second-site mutants in which induction ofnasB expression was completely abolished under nitrogen-limiting conditions. Nucleotide sequence analysis of the chromosomal junctions revealed that in all three mutants the secondary transposon had inserted at different sites in the gltB gene of P. putida KT2442 encoding the major subunit of the glutamate synthase. A detailed physiological characterization of thegltB mutants revealed that they are unable to utilize a number of potential nitrogen sources, are defective in the ability to express nitrogen starvation proteins, display an aberrant cell morphology under nitrogen-limiting conditions, and are impaired in the capacity to survive prolonged nitrogen starvation periods.

scholarly journals Cloning of a third nitrate reductase gene from the cyanobacterium Anacystis nidulans R2 using a shuttle cosmid library

Gene ◽  
1984 ◽  
Vol 31 (1-3) ◽  
pp. 109-116 ◽  
Author(s):  
C.J. Kuhlemeier ◽  
V.J.P. Teeuwsen ◽  
M.J.T. Janssen ◽  
G.A. van Arkel
Keyword(s):  
Nitrate Reductase ◽  
Anacystis Nidulans ◽  
Cosmid Library ◽  
Nitrate Reductase Gene ◽  
Reductase Gene

Characterization of the Tuber borchii nitrate reductase gene and its role in ectomycorrhizae

2003 ◽  
Vol 269 (6) ◽  
pp. 807-816 ◽  
Author(s):  
M. Guescini ◽  
R. Pierleoni ◽  
F. Palma ◽  
S. Zeppa ◽  
L. Vallorani ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reductase Gene ◽  
Tuber Borchii ◽  
Reductase Gene
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