Gene targeting in a nonhomologous end joining deficient Hypocrea jecorina.
The industrially applied ascomycete Hypocrea jecorina (synonym: Trichoderma reesei) exhibits a low rate of exogenous DNA integration by homologous recombination (HR). This hinders the high-throughput generation of strains by gene replacement and is therefore impeding systematic functional gene analyses towards, e.g. strain improvement for protein or enzyme production. To increase the rate of HR events during fungal transformation we identified and deleted the orthologue of the human KU70 in H. jecorina, which is required for the nonhomologous end joining (NHEJ) pathway and responsible for ectopic DNA integration. The effect of the absence of the H. jecorina tku70 on gene targeting was tested by deletion of two so far uncharacterized genes encoding a short chain dehydrogenase and a fungal specific transcription factor. Efficiency of gene targeting for both genes was >95% in a Deltatku70 strain when 1kb homologous flanking regions were used in the deletion construct. This is a significant increase in targeting efficiency compared to the parental - non-tku70 deleted - strain TU-6 where a gene knock-out frequency of only 5-10% was observed. Together with the recently annotated genomic sequence of H. jecorina, this system provides a useful tool for a genome-wide functional gene analysis on a high-throughput scale to improve the biotechnological potential of this fungus.
Guangtao
Zhang
Z
Molecular Biotechnology, Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, TU Wien, Getreidemarkt 9, A-1060 Wien, Austria.
Hartl
Lukas
L
Schuster
Andre
A
Polak
Stefan
S
Schmoll
Monika
M
Wang
Tianhong
T
Seidl
Verena
V
Seiboth
Bernhard
B
D045643:DNA Repair Enzymes / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D055786:Gene Knockout Techniques; D018390:Gene Targeting / Q000379:methods*; D005800:Genes, Fungal; D035901:Hypocrea / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D010641:Phenotype; D010692:Phleomycins / Q000494:pharmacology; D011995:Recombination, Genetic; D014170:Transformation, Genetic; D014466:Ultraviolet Rays
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D010692:Phleomycins; D008741:Methyl Methanesulfonate; D045643:DNA Repair Enzymes