Development of a random mutagenesis system for the methylotrophic yeast Hansenula polymorpha

The non-conventional yeast Hansenula polymorpha is a thermotolerant methylotrophic yeast that has been regarded as an attractive model organism for basic research and biotechnological applications. However, the currently available genome engineering tools for H. polymorpha are limited to those depending mainly on techniques based on homologous recombination, such as gene disruption based on Cre-loxP, gene targeting based on a non-homologous endjoining-deficient mutant, and random insertion techniques based on restriction enzyme-mediated integration. In this study, we identified the DNA polymerase δ of H. polymorpha, created a Pol3* allele encoding an error-prone proofreading-deficient DNA polymerase δ, and developed a mutator vector containing the POL3* gene. We also developed and experimentally verified a strategy for genome-wide random mutagenesis, which consists of a mutator strain created by the chromosomal integration of a mutator vector through targeted homologous recombination, Pol3*-based random mutagenesis, and stabilization of the obtained strain by the removal of the mutator allele by a second round of homologous recombination. 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 a grant from the Intelligent Synthetic Biology Global Frontier Program of the National Research Foundation and by the Next-Generation BioGreen 21 Program grant from the Rural Development Administration, Republic of Korea.