Acetamidase as a dominant recyclable marker for Komagataella phaffii strain engineering

Abstract

We have investigated the use of the gene coding for acetamidase (amdS) as a recyclable dominant marker for the methylotrophic yeast Komagataella phaffii in order to broaden its genetic toolbox. First, the endogenous constitutive AMD2 gene (a putative acetamidase) was deleted generating strain LA1. A cassette (amdSloxP) was constructed bearing a codon-optimized version of the Aspergillus nidulans amdS gene flanked by loxP sites for marker excision with Cre recombinase. This cassette was successfully tested as a dominant selection marker for transformation of the LA1 strain after selection on plates containing acetamide as a sole nitrogen source. Finally, amdSloxP was used to sequentially disrupt the K. phaffii ADE2 and URA5 genes. After each disruption event, a Cre-mediated marker recycling step was performed by plating cells on medium containing fluoroacetamide. In conclusion, amdS proved to be a suitable tool for K. phaffii transformation and marker recycling thus providing a new antibiotic-free system for genetic manipulation of this yeast.

Construction and characterization of centromeric plasmids for Komagataella phaffii using a color-based plasmid stability assay

“…Plasmid pPICH [ 27 ], derived from pPICHOLI (MoBiTec), contains the K . phaffii ARS1 replicating sequence [ 1 ].…”

“…phaffii strains were derived from X-33 (Invitrogen). The LA2 strain (amd2 ade2) was described in previous work [27]. Yeast was routinely grown in YPD medium (10 g L -1 yeast extract, 20 g L -1 peptone and 20 g L -1 glucose).…”

“…Strain LA2 [27] was transformed with an ADE3 deletion cassette and had the marker recycled before transformation with the centromeric plasmids. The construction of this deletion cassette used PCR reactions assembled by an In-Fusion cloning reaction.…”

“…Plasmid pPICH [27], derived from pPICHOLI (MoBiTec), contains the K. phaffii ARS1 replicating sequence [1]. This sequence is originally located on K. phaffii GS115 chromosome 2, coordinates 413701-413856 [2].…”

“…have been deleted in S. cerevisiae in order to create auxotrophic strains, while in K. phaffii studies have only focused on ADE1 and ADE2 [33,34]. K. phaffii LA2, a mutant ade2 strain [27], was used as a starting point for the construction of a strain that would allow plasmid stability verification. The deletion of the ADE2 gene results in cells that are auxotrophic for adenine and accumulate a red intermediate [32]; the additional deletion of genes located upstream in the adenine synthesis pathway, such as ADE1 or ADE3, should prevent the formation of such pigment [33].…”