Title:	Multicopy plasmid integration in Komagataella phaffii mediated by a defective auxotrophic marker
Authors:	Ocampo Betancur, Maritza Reis, Viviane Castelo Branco Nicola, André Moraes De Marco, Janice Lisboa Moraes, Lídia Maria Pepe de Torres, Fernando Araripe Gonçalves
Assunto::	Proteínas Células Leucina Leveduras
Issue Date:	8-Jun-2017
Publisher:	Microb Cell Fact
Citation:	OCAMPO BETANCUR, Maritza et al. Multicopy plasmid integration in Komagataella phaffii mediated by a defective auxotrophic marker. Microbial Cell Factories, v. 16, n. 1, Article 99, 8 jun. 2017. Disponível em: <https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-017-0715-8>. Acesso em: 19 jun. 2017. doi: 10.1186/s12934-017-0715-8.
Abstract:	Background: A commonly used approach to improve recombinant protein production is to increase the levels of expression by providing extra-copies of a heterologous gene. In Komagataella phaffii (Pichia pastoris) this is usually accomplished by transforming cells with an expression vector carrying a drug resistance marker following a screening for multicopy clones on plates with increasingly higher concentrations of an antibiotic. Alternatively, defective auxotrophic markers can be used for the same purpose. These markers are generally transcriptionally impaired genes lacking most of the promoter region. Among the defective markers commonly used in Saccharomyces cerevisiae is leu2-d, an allele of LEU2 which is involved in leucine metabolism. Cells transformed with this marker can recover prototrophy when they carry multiple copies of leu2-d in order to compensate the poor transcription from this defective allele. Results: A K. phaffii strain auxotrophic for leucine (M12) was constructed by disrupting endogenous LEU2. The resulting strain was successfully transformed with a vector carrying leu2-d and an EGFP (enhanced green fluorescent protein) reporter gene. Vector copy numbers were determined from selected clones which grew to different colony sizes on transformation plates. A direct correlation was observed between colony size, number of integrated vectors and EGFP production. By using this approach we were able to isolate genetically stable clones bearing as many as 20 integrated copies of the vector and with no significant effects on cell growth. Conclusions: In this work we have successfully developed a genetic system based on a defective auxotrophic which can be applied to improve heterologous protein production in K. phaffii. The system comprises a K. phaffii leu2 strain and an expression vector carrying the defective leu2-d marker which allowed the isolation of multicopy clones after a single transformation step. Because a linear correlation was observed between copy number and heterologous protein production, this system may provide a simple approach to improve recombinant protein productivity in K. phaffii.
Licença::	© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
DOI:	https://dx.doi.org/10.1186/s12934-017-0715-8
Appears in Collections:	Artigos publicados em periódicos e afins

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