Variation among Metschnikowia pulcherrima Isolates for Genetic Modification and Homologous Recombination

Area of Science:

• Biotechnology
• Microbiology
• Molecular Biology

Background:

• Metschnikowia pulcherrima is a non-conventional yeast with significant biotechnological potential, particularly for utilizing low-cost feedstocks.
• Current limitations in molecular research tools and genetic modification hinder the full exploitation of this yeast's capabilities.

Purpose of the Study:

• To establish and optimize a reliable genetic modification protocol for Metschnikowia pulcherrima.
• To assess the amenability of different M. pulcherrima strains to genetic transformation and recombination.
• To investigate methods for improving homologous recombination efficiency and marker gene removal.

Main Methods:

• Screened ten Metschnikowia pulcherrima strains for genetic modification susceptibility.
• Developed and applied a lithium acetate/polyethylene glycol (LiAc/PEG)-based transformation protocol for introducing heterologous DNA.
• Investigated non-homologous and homologous integration pathways, including the effect of non-homologous end joining (NHEJ) inhibition.
• Assessed the efficiency of selective marker removal using flippase recombinase.

Main Results:

• A LiAc/PEG transformation protocol was successfully established, enabling the introduction of foreign DNA into M. pulcherrima.
• Non-homologous DNA integration was broadly successful across tested strains.
• Homologous recombination was achieved in two specific strains, with a modest improvement observed upon chemical inhibition of NHEJ.
• Flippase-mediated marker excision was effective except when targeting the native URA3 locus, indicating potential genome-specific interference.

Conclusions:

• The developed transformation protocol provides essential tools for molecular research and genetic engineering of Metschnikowia pulcherrima.
• The success of homologous recombination in select strains opens avenues for targeted genetic manipulation.
• Further research is needed to understand and overcome locus-specific challenges, such as those at the URA3 locus, for enhanced genetic engineering efficacy.