Development of a genome-wide random mutagenesis system using proofreading-deficient DNA polymerase delta in the methylotrophic yeast Hansenula polymorpha

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Title
Development of a genome-wide random mutagenesis system using proofreading-deficient DNA polymerase delta in the methylotrophic yeast Hansenula polymorpha
Author(s)
Oh Cheol Kim; Sang Yoon Kim; Dong Hyeon Hwang; Doo-Byoung Oh; H A Kang; Oh Suk Kwon
Bibliographic Citation
Journal of Microbiology and Biotechnology, vol. 23, no. 3, pp. 304-312
Publication Year
2013
Abstract
The thermotolerant methylotrophic yeast Hansenula polymorpha is attracting interest as a potential strain for the production of recombinant proteins and biofuels. However, only limited numbers of genome engineering tools are currently available for H. polymorpha. In the present study, we identified the HpPOL3 gene encoding the catalytic subunit of DNA polymerase δ of H. polymorpha and mutated the sequence encoding conserved amino acid residues that are important for its proofreading 3'-->5' exonuclease activity. The resulting HpPOL3* gene encoding the error-prone proofreading-deficient DNA polymerase δ was cloned under a methanol oxidase promoter to construct the mutator plasmid pHIF8, which also contains additional elements for site-specific chromosomal integration, selection, and excision. In a H. polymorpha mutator strain chromosomally integrated with pHIF8, a URA3(-) mutant resistant to 5-fluoroorotic acid was generated at a 50-fold higher frequency than in the wild-type strain, due to the dominant negative expression of HpPOL3*. Moreover, after obtaining the desired mutant, the mutator allele was readily removed from the chromosome by homologous recombination to avoid the uncontrolled accumulation of additional mutations. Our mutator system, which depends on the accumulation of random mutations that are incorporated during DNA replication, will be useful to generate strains with mutant phenotypes, especially those related to unknown or multiple genes on the chromosome.
ISSN
1017-7825
Publisher
South Korea
DOI
http://dx.doi.org/10.4014/jmb.1211.11048
Type
Article
Appears in Collections:
Division of Bio Technology Innovation > SME Support Center > 1. Journal Articles
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