Dual gene expression cassette vectors with antibiotic selection markers for engineering in Saccharomyces cerevisiae

C E Vickers; S F Bydder; Yuchan Zhou; L K Nielsen

doi:10.1186/1475-2859-12-96

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Dual gene expression cassette vectors with antibiotic selection markers for engineering in Saccharomyces cerevisiae

Journal article

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Dual gene expression cassette vectors with antibiotic selection markers for engineering in Saccharomyces cerevisiae

C E Vickers, S F Bydder, Yuchan Zhou and L K Nielsen

Microbial Cell Factories, Vol.12(1), 96

2013

DOI: https://doi.org/10.1186/1475-2859-12-96

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Abstract

saccharomyces cerevisiae

expression vector

antibiotic selection

G418

phleomycin

yeast

Background: Manipulations in Saccharomyces cerevisiae classically depend on use of auxotrophy selection markers. There are several disadvantages to this in a microbial cell factory setting: (1) auxotrophies must first be engineered in prototrophic strains, and many industrial strains are polyploid/aneuploid prototrophs (2) available strain auxotrophies must be paired with available repair plasmids (3) remaining auxotrophies must be repaired prior to development of industrial bioprocesses. Use of dominant antibiotic resistance markers can circumvent these problems. However, there are relatively few yeast antibiotic resistance marker vectors available; furthermore, available vectors contain only one expression cassette, and it is often desirable to introduce more than one gene at a time.Results: To overcome these problems, eight new shuttle vectors have been developed. The plasmids are maintained in yeast under a 2 μm ori and in E. coli by a pUC ori. They contain two yeast expression cassettes driven by either (1) the constitutive TEF1 and PGK1 promoters, or (2) the constitutive TEF1 promoter and the inducible GAL10 or HXT7 promoters. Expression strength of these promoters over a typical production time frame in glucose/galactose medium was examined, and identified the TEF1 and HXT7 promoters as preferred promoters over long term fermentations. Selection is provided by either aphA1 (conferring resistance to G418 in yeast and kanamycin/neomycin in E. coli) or ble (conferring resistance to phleomycin in both yeast and E. coli). Selection conditions for these plasmids/antibiotics in defined media were examined, and selection considerations are reviewed. In particular, medium pH has a strong effect on both G418 and phleomycin selection.Conclusions: These vectors allow manipulations in prototrophic yeast strains with expression of two gene cassettes per plasmid, and will be particularly useful for metabolic engineering applications. The vector set expands the (currently limited) selection of antibiotic marker plasmids available for use in yeast, and in addition makes available dual gene expression cassettes on individual plasmids using antibiotic selection. The resistance gene cassettes are flanked by loxP recognition sites to allow CreA-mediated marker removal and recycling, providing the potential for genomic integration of multiple genes. Guidelines for selection using G418 and phleomycin are provided. © 2013 Vickers et al.; licensee BioMed Central Ltd.

Details

Title: Dual gene expression cassette vectors with antibiotic selection markers for engineering in Saccharomyces cerevisiae
Authors: C E Vickers (Author) - University of Queensland
S F Bydder (Author) - University of Queensland
Yuchan Zhou (Author) - University of Queensland
L K Nielsen (Author) - University of Queensland
Publication details: Microbial Cell Factories, Vol.12(1), 96
Publisher: BioMed Central Ltd.
Date published: 2013
DOI: 10.1186/1475-2859-12-96
ISSN: 1475-2859
Copyright note: Copyright © 2013 Vickers et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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.
Organisation Unit: Australian Centre for Pacific Islands Research; University of the Sunshine Coast, Queensland
Language: English
Record Identifier: 99449239202621
Output Type: Journal article

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Web Of Science research areas: Biotechnology & Applied Microbiology