원문정보
초록
영어
In the present study, we identified the HpPOL3 gene encoding the catalytic subunit of DNA polymerase δ of the thermotolerant methylotrophic yeast Hansenula polymorpha and mutated the sequence encoding conserved amino acid residues that are important for its proofreading 3’ à 5’ exonuclease activity. The resulting HpPOL3* gene encoding error-prone proofreading-deficient DNA polymerase 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, 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*. 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. Supported by the Intelligent Synthetic Biology Global Frontier Program and the Next-Generation BioGreen 21 Program.