Abstract
In contrast to model yeasts, gene targeting efficiencies of non-conventional yeasts are usually low, which greatly limits the research and applications of these organisms. In this study, we aimed to enhance the gene targeting efficiency of non-conventional yeasts by improving the fitness of mutant strains, particularly by increasing the genetic redundancy of host cells. To demonstrate this process, OCH1 gene deletion in Pichia pastoris was performed. Extra copies of the OCH1 gene on a helper plasmid were provided for the P. pastoris GS115 strain before the native OCH1 gene in the genomic DNA was knocked out. The redundancy in OCH1 gene significantly eliminated the growth defects of the och1 mutant and increased the deletion efficiency of the OCH1 gene by two orders of magnitude with the same length of homologous flanks. The same strategy was used to delete the KU70 and SGS1 genes. The targeting efficiencies of KU70 and SGS1 were increased by 1- and 23-fold, respectively. Therefore, this study provided an efficient strategy for the deletion of “stubborn” genes in non-conventional yeasts. This study further showed that cellular fitness is potentially an important factor that can limit the efficiency of gene targeting.
Highlights
Gene targeting is one of the main molecular tools used in yeast science, which helps in understanding gene functions and interactions as well as cellular and molecular processes in yeasts [1]
Disruption cassettes with short flanking regions that range from 30 bp to 50 bp can integrate with high efficiencies via homologous integration at the correct genomic locus in S. cerevisiae [2]
Homologous arms varying from 200 bp to approximately 2000 bp are usually required to ensure efficient gene replacement in non-conventional yeasts [1]
Summary
Gene targeting (e.g., targeted gene replacement) is one of the main molecular tools used in yeast science, which helps in understanding gene functions and interactions as well as cellular and molecular processes in yeasts [1]. Model yeasts, such as Saccharymyces cerevisiae and Schizosaccharomyces pombe, have very efficient gene targeting systems. Homologous arms varying from 200 bp to approximately 2000 bp are usually required to ensure efficient gene replacement in non-conventional yeasts [1]. The low targeting efficiencies of non-conventional yeasts greatly limits the researches and applications of these industrially important microorganisms
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