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

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.

Peroxisomal NAD(H) Homeostasis in the Yeast Debaryomyces hansenii Depends on Two Redox Shuttles and the NAD+ Carrier, Pmp47

“…D. hansenii total DNA isolation was based on the method described by [ 13 ] with some minor modifications, as described in [ 5 ].…”

“…The PCR product was cloned into pBluescript KS+ ( Figure S1 ). Additionally, pHygR and pKanR plasmids have been described in [ 5 ].…”

“…For the deletion of the DhPMP47 or DhNPY1 ORFs, 50 bp flanking regions were introduced by PCR using primers that anneal on the selectable cassette and contain a 50 nt 5′ extension identical to the flanking regions of the ORFs (see Figure S1B ). PCR products were transformed using electroporation as previously described [ 19 ] with some small adaptations as described in [ 5 ]. These gene deletion mutants were generated in the strain NCYC3363.…”

“…All potential gene deletion mutants were screened for the presence of the gene deletion and for the absence of the WT copy before they were functionally analysed. The expression of mCherry-PTS1 was targeted to the ARG1 locus in NCYC102 wild-type (as described in [ 5 ]) and NCYC102 pex3::sat1 .…”

“…Genome-editing tools for D. hansenii have been limited. To overcome this, we recently developed a PCR-based method for gene disruption based on homologous recombination and described a genomic safe landing site for the expression of heterologous proteins [ 5 ], which allows the systematic exploration of the organization of lipid metabolism pathways in this yeast.…”