Development of a highly efficient gene targeting system allowing rapid genetic manipulations in Penicillium decumbens

• Published in: Applied microbiology and biotechnology Vol. 87; no. 3; pp. 1065 - 1076
• Main Authors: Li, Zhong-Hai, Du, Chun-Mei, Zhong, Yao-Hua, Wang, Tian-Hong
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01-07-2010; Springer; Springer Nature B.V
• Subjects: Applied Genetics and Molecular Biotechnology; Biological and medical sciences; Biomedical and Life Sciences; Biorefineries; Biotechnology; Cellulase; Chemical agents; Cloning; DNA polymerase; Efficiency; Enzymes; Fundamental and applied biological sciences. Psychology; Fungal Proteins - genetics; Fungi; Gene Targeting - methods; Genes; Genetic engineering; Genetic recombination; Genomes; Genomics; Hydrogen peroxide; Kinases; Life Sciences; Lignocellulose; Microbial Genetics and Genomics; Microbiology; Mutagenesis; Penicillin; Penicillium - genetics; Penicillium decumbens; Recombination, Genetic; Sequence Deletion; Studies; Pku70; Gene targeting; Nonhomologous end-joining; Homologous recombination; Fungi; Targeting; Gene; Penicillium decumbens; Genetics; Fungi Imperfecti
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-010-2566-7

Penicillium decumbens
is an important industrial filamentous fungus and has been widely used in biorefinery due to its high production of cellulase and hemicellulase. However, molecular engineering has still rarely been applied for strain improvement in
P
.
decumbens
. It has been proven that gene targeting manipulation in many filamentous fungi is hampered by nonhomologous end-joining (NHEJ) pathway. To improve gene targeting efficiency in
P. decumbens
, the putative
pku70
encoding the Ku70 homologue involved in the NHEJ pathway was identified and deleted. The
Δpku70
strain showed no apparent defect in vegetative growth, conidiation, and cellulase production, and displayed similar sensitivity to chemical agents of hygromycin B, ethyl methane sulfonate, and H
2
O
2
at different concentrations compared with the wild-type strain. The effect of the absence of
pku70
on gene targeting was tested by disruption of
creA
encoding a putative carbon catabolite repressor and
xlnR
encoding a putative transcriptional activator. Efficiency of gene targeting for both genes was 100% in the
Δpku70
strain, compared with the low efficiency in the wild-type recipient. Furthermore, the integration types for three single targeting cassettes and the cotransformation of two independent targeting cassettes were primarily investigated in
P. decumbens
. The highly efficient gene targeting system established in this study will open the way to large-scale functional genomic analysis in
P. decumbens
and contribute to the study of the mechanism of lignocellulose degradation by
P
.
decumbens
.