Acetamidase as a dominant recyclable marker for Komagataella phaffii strain engineering.
Appl Microbiol Biotechnol
; 102(6): 2753-2761, 2018 Mar.
Article
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| MEDLINE
| ID: mdl-29435619
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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1
Banco de datos:
MEDLINE
Asunto principal:
Selección Genética
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Transformación Genética
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Ingeniería Genética
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Saccharomycetales
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Amidohidrolasas
Idioma:
En
Revista:
Appl Microbiol Biotechnol
Año:
2018
Tipo del documento:
Article
País de afiliación:
Brasil