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bioRxiv · 10.1101/2023.07.11.548570

Efficient PCR-based gene targeting in isolates of the non-conventional yeast Debaryomyces hansenii

Alhajouj, S.·Turkolmez, S.·Abalkhail, T.·Alwan, Z. H. O.·Gilmour, D. J.·Mitchell, P. J.·Hettema, E. H.
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Abstract

Debaryomyces hansenii is a yeast with considerable biotechnological potential as an osmotolerant, stress tolerant oleaginous microbe. However, targeted genome modification tools are limited and require a strain with auxotrophic markers. Gene targeting by homologous recombination has been reported to be inefficient but here we describe a set of reagents and a method that allows gene targeting at high efficiency in wild type isolates. It uses a simple PCR-based amplification that extends a completely heterologous selectable marker with 50 bp flanks identical to the target site in the genome. Transformants integrate the PCR product through homologous recombination at high frequency (>75%). We illustrate the potential of this method by disrupting genes at high efficiency and by expressing a heterologous protein from a safe chromosomal harbour site. These methods should stimulate and facilitate further analysis of D. hansenii strains and open the way to engineer strains for biotechnology.

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Alhajouj, S., Turkolmez, S., Abalkhail, T., Alwan, Z. H. O., Gilmour, D. J., Mitchell, P. J., Hettema, E. H.. 2023-07-11. Efficient PCR-based gene targeting in isolates of the non-conventional yeast Debaryomyces hansenii. https://doi.org/10.1101/2023.07.11.548570

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