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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

Authors 

Oh Cheol KimSang Yoon KimDong Hyeon HwangDoo-Byoung OhH A KangOh Suk Kwon

Publisher 

The Korean Society for Applied Microbiology

Issue Date 

2013

Citation 

Journal of Microbiology and Biotechnology, vol. 23, no. 3, pp. 304-312

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

Link 

http://dx.doi.org/10.4014/jmb.1211.11048

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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