title: Development of a genome-wide random mutagenesis system using proofreading-deficient DNA polymerase δ in the methylotrophic yeast Hansenula polymorpha.
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.
authors: Systems and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Korea.
authors: Oh Cheol
authors: OC
authors: Kim
authors: Sang-Yoon
authors: SY
authors: Kim
authors: Dong Hyeon
authors: DH
authors: Hwang
authors: Doo-Byoung
authors: DB
authors: Oh
authors: Hyun Ah
authors: HA
authors: Kang
authors: Ohsuk
authors: O
authors: Kwon
mesh_terms: D000595:Amino Acid Sequence; D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism; D005092:Exonucleases / Q000235:genetics / Q000378:metabolism; D005827:Genetics, Microbial / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D059645:Mutation Rate; D010843:Pichia / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment
chemical_list: D050505:Mutant Proteins; D004258:DNA Polymerase III; D005092:Exonucleases