Acetamidase as a dominant recyclable marker for Komagataella phaffii strain engineering
Luiza Cesca Piva1, Janice Lisboa De Marco1, Lidia Maria Pepe de Moraes1
1Instituto de Ciências Biológicas, Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, 70910-900, Brazil.
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.
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