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논문 기본 정보
- 자료유형
- 학술저널
- 저자정보
- 저널정보
- 한국미생물생명공학회 Journal of Microbiology and Biotechnology Journal of Microbiology and Biotechnology 제23권 제3호
- 발행연도
- 2013.1
- 수록면
- 304 - 312 (9page)
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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 δwas 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.
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참고문헌 (22)
참고문헌 신청
Abe, H / 2009 / Development of valuable yeast strains using a novel mutagenesis technique for the effective production of therapeutic glycoproteins / Glycobiology 19 : 428 ~
Abe, H / 2009 / Ethanol-tolerant Saccharomyces cerevisiae strains isolated under selective conditions by over-expression of a proofreading-deficient DNA polymerase delta / J.
Abe, H / 2009 / Upregulation of genes involved in gluconeogenesis and the glyoxylate cycle suppressed the drug sensitivity of an Nglycan- deficient Saccharomyces cerevisiae m
Blazhenko, O. V / 2006 / Accumulation of cadmium ions in the methylotrophic yeast Hansenula polymorpha / Biometals 19 : 593 ~ 599
Boeke, J. D / 1984 / A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-Fluoro-orotic acid resistance / Mol. Gen. Genet 197 :
Abe, H / 2009 / Ethanol-tolerant Saccharomyces cerevisiae strains isolated under selective conditions by over-expression of a proofreading-deficient DNA polymerase delta / J.
Abe, H / 2009 / Upregulation of genes involved in gluconeogenesis and the glyoxylate cycle suppressed the drug sensitivity of an Nglycan- deficient Saccharomyces cerevisiae m
Blazhenko, O. V / 2006 / Accumulation of cadmium ions in the methylotrophic yeast Hansenula polymorpha / Biometals 19 : 593 ~ 599
Boeke, J. D / 1984 / A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-Fluoro-orotic acid resistance / Mol. Gen. Genet 197 :
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