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

Vickers C.E., Bydder S.F., Zhou Y. and Nielsen L.K. (2013) Dual gene expression cassette vectors with antibiotic selection markers for engineering in Saccharomyces cerevisiae. Microbial Cell Factories, 12 96: 96.1-96.11. doi:10.1186/1475-2859-12-96


Author Vickers C.E.
Bydder S.F.
Zhou Y.
Nielsen L.K.
Title Dual gene expression cassette vectors with antibiotic selection markers for engineering in Saccharomyces cerevisiae
Formatted title
Dual gene expression cassette vectors with antibiotic selection markers for engineering in Saccharomyces cerevisiae
Journal name Microbial Cell Factories   Check publisher's open access policy
ISSN 1475-2859
Publication date 2013-10-25
Year available 2013
Sub-type Article (original research)
DOI 10.1186/1475-2859-12-96
Open Access Status DOI
Volume 12
Issue 96
Start page 96.1
End page 96.11
Total pages 11
Place of publication London, United Kingdom
Publisher BioMed Central
Language eng
Abstract 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.
Formatted abstract
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.
Keyword Antibiotic selection
Expression vector
G418
Phleomycin
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2014 Collection
Australian Institute for Bioengineering and Nanotechnology Publications
 
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