Abstract
Metschnikowia pulcherrima is a non-conventional yeast with the potential to be used in biotechnological processes, especially involving low-cost feedstock exploitation. However, there are a lack of tools for researching it at a molecular level and for producing genetically modified strains. We tested the amenability to genetic modification of ten different strains, establishing a transformation protocol based on LiAc/PEG that allows us to introduce heterologous DNA. Non-homologous integration was broadly successful and homologous recombination was successful in two strains. Chemical inhibition of non-homologous end joining recombination had a modest effect on the improvement of homologous recombination rates. Removal of selective markers via flippase recombinase was successful across integrated loci except for those targeted to the native URA3 locus, suggesting that the genome sequence or structure alters the efficacy of this system.
Highlights
Modification and HomologousNovel yeasts and their natural variants offer exceptional promise for improved industrial biotechnology, from utilization of sustainable feedstocks through to production of high-value compounds [1,2]
Whilst exploiting natural strains is an important strategy, developing genetic tools in these novel yeasts is critical for establishing flexibility and opening the applications for specific yeast products and, importantly, to understand the basic biology of these yeasts [3]
While it is well recognized that developing genetic tools for a novel species can be challenging, variation within a species is often ignored in discussions around applications within yeasts [3]
Summary
Modification and HomologousNovel yeasts and their natural variants offer exceptional promise for improved industrial biotechnology, from utilization of sustainable feedstocks through to production of high-value compounds [1,2]. Whilst exploiting natural strains is an important strategy, developing genetic tools in these novel yeasts is critical for establishing flexibility and opening the applications for specific yeast products and, importantly, to understand the basic biology of these yeasts [3]. The first main challenges are: (1) getting detectably functional DNA into the yeast; (2) targeting the DNA within the genome; (3) having a system that allows more than one round of modification. These issues are not likely to be monolithic within a species and if a major goal is to exploit natural variation, it is important to determine how tools differ across strains within a species as well in the novel species.
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