Acetamidase as a dominant recyclable marker for Komagataella phaffii strain engineering

作者：Piva Luiza Cesca; De Marco Janice Li**oa; Pepe de Moraes Lidia Maria; Branco Reis Viviane Castelo; Goncalves Torres Fernando Araripe

来源：Applied Microbiology and Biotechnology, 2018, 102(6): 2753-2761.

DOI：10.1007/s00253-018-8824-9

• Acetamidase
• Marker recycling
• amdS
• Dominant marker
• Komagataella phaffii

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.

• 出版日期2018-3