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

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dc.contributor.authorOh Cheol Kim-
dc.contributor.authorSang Yoon Kim-
dc.contributor.authorDong Hyeon Hwang-
dc.contributor.authorDoo-Byoung Oh-
dc.contributor.authorH A Kang-
dc.contributor.authorOh Suk Kwon-
dc.date.accessioned2017-04-19T09:37:01Z-
dc.date.available2017-04-19T09:37:01Z-
dc.date.issued2013-
dc.identifier.issn1017-7825-
dc.identifier.uri10.4014/jmb.1211.11048ko
dc.identifier.urihttps://oak.kribb.re.kr/handle/201005/11231-
dc.description.abstractThe 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.-
dc.publisherKorea Soc-Assoc-Inst-
dc.titleDevelopment of a genome-wide random mutagenesis system using proofreading-deficient DNA polymerase delta in the methylotrophic yeast Hansenula polymorpha-
dc.title.alternativeDevelopment of a genome-wide random mutagenesis system using proofreading-deficient DNA polymerase delta in the methylotrophic yeast Hansenula polymorpha-
dc.typeArticle-
dc.citation.titleJournal of Microbiology and Biotechnology-
dc.citation.number3-
dc.citation.endPage312-
dc.citation.startPage304-
dc.citation.volume23-
dc.contributor.affiliatedAuthorOh Cheol Kim-
dc.contributor.affiliatedAuthorSang Yoon Kim-
dc.contributor.affiliatedAuthorDong Hyeon Hwang-
dc.contributor.affiliatedAuthorDoo-Byoung Oh-
dc.contributor.affiliatedAuthorOh Suk Kwon-
dc.contributor.alternativeName김오철-
dc.contributor.alternativeName김상윤-
dc.contributor.alternativeName황동현-
dc.contributor.alternativeName오두병-
dc.contributor.alternativeName강현아-
dc.contributor.alternativeName권오석-
dc.identifier.bibliographicCitationJournal of Microbiology and Biotechnology, vol. 23, no. 3, pp. 304-312-
dc.identifier.doi10.4014/jmb.1211.11048-
dc.subject.keywordHansenula polymorpha-
dc.subject.keywordRandom mutagenesis-
dc.subject.keywordDNA polymerase δ-
dc.subject.keywordMutator-
dc.subject.localHansenulapolymorpha-
dc.subject.localhansenula polymorpha-
dc.subject.localHansenula polymorpha-
dc.subject.localHansenula polymorpha (Pichia angusta)-
dc.subject.localRandom mutagenesis-
dc.subject.localrandom mutagenesis-
dc.subject.localDNA polymerase δ-
dc.subject.localMutator-
dc.description.journalClassY-
Appears in Collections:
Aging Convergence Research Center > 1. Journal Articles
Division of Bio Technology Innovation > SME Support Center > 1. Journal Articles
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