Improved gene disruption method and Cre-loxP mutant system for multiple gene disruptions in Hansenula polymorpha.
In H. polymorpha, there is still a lack of a highly efficient gene disruption method. To help address this issue, we presented a simple and efficient method for both single and multiple gene disruptions in H. polymorpha. The knockout system combined a variation of sticky-end polymerase chain reaction method (SEP), split marker deletion method, co-transformation of single-stranded DNA and mutant Cre-loxP system. Using a slightly modified LiAc/SS-DNA/PEG procedure, the co-transformation double-stranded split marker constructs together with single-stranded split marker constructs resulted in at least 70% homologous recombination events when the homologous genomic DNA fragment had a size of approximately 500bp. Our evidence suggested that single-stranded DNA may be responsible for the increased gene disruption efficiency. We demonstrated the effectiveness of the method for gene disruption by constructing both single and double gene disruptions at the ALG3 and URA5 loci in the same genetic background. The method described here presents an improved strategy for gene disruption and a potential application for investigation of biological processes in other yeast strains.
Qian
Weidong
W
Center for Agricultural Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Beijing 100101, People's Republic of China.
Song
Haolei
H
Liu
Yueyong
Y
Zhang
Chaozheng
C
Niu
Zhendong
Z
Wang
Hui
H
Qiu
Bingsheng
B
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004277:DNA, Single-Stranded; D055786:Gene Knockout Techniques / Q000379:methods*; D005800:Genes, Fungal; D005819:Genetic Markers; D019426:Integrases / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010843:Pichia / Q000235:genetics*; D011995:Recombination, Genetic; D014170:Transformation, Genetic
D004277:DNA, Single-Stranded; D005819:Genetic Markers; C045073:Cre recombinase; D019426:Integrases