Efficient selection of hygromycin-B-resistant Yarrowia lipolytica transformants

Otero, Ricardo Cordero; Gaillardin, Claude

doi:10.1007/s002530050796

Cited by 32 publications

(15 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These results suggested that the lack of HYG gene expression in C. tropicalis was due to translation of the CTG codons as serines rather than leucines. Expression of the HYG gene failed in C. albicans (Leuker et al 1992), while it succeeded in S. cerevisiae (Gritz and Davies 1983) and Yarrowia lipolytica (Otero and Gaillardin 1996). It has been reported that the CTG codon, a universal leucine codon, is read as serine among several Candida species.…”

Section: Sensitivity Toward Hygromycin B Of C Tropicalismentioning

confidence: 96%

See 1 more Smart Citation

A mutated hygromycin resistance gene is functional in the n-alkane-assimilating yeast Candida tropicalis

et al. 2000

View full text Add to dashboard Cite

Development of a transformation system in the n-alkane-assimilating diploid yeast Candida tropicalis requires an antibiotic resistance gene in order to establish a selectable marker. The resistance gene for hygromycin B has often been used as a selectable marker in yeast transformation. However, C. tropicalis harboring the hygromycin resistance gene (HYG) was as sensitive to hygromycin B as the wild-type strain. Nine CTG codons were found in the ORF of the HYG gene. This codon has been reported to be translated as serine rather than leucine in Candida species. Analysis of the tRNA gene in C. tropicalis with the anticodon CAG [tRNA(CAG) gene], which is complementary to the codon CTG, showed that the sequence was highly similar to that of the C. maltosa tRNA(CAG) gene. In C. maltosa, the codon CTG is read as serine and not leucine. These results suggested that the HYG gene was not functional due to the nonuniversal usage of the CTG codon. Each of the nine CTG codons in the ORF of the HYG gene was changed to a CTC codon, which is read as leucine, by site-directed mutagenesis. When a plasmid containing the mutated HYG gene (HYG#) was constructed and introduced into C. tropicalis, hygromycin-resistant transformants were successfully obtained. This mutated hygromycin resistance gene may be useful for direct selection of C. tropicalis transformants.

show abstract

Section: Sensitivity Toward Hygromycin B Of C Tropicalismentioning

confidence: 96%

“…Resistance to G418 or hygromycin B is commonly used as a selectable marker in the selection of yeast transformants (Jimenez and Davies 1980;Gritz and Davies 1983;Otero and Gaillardin 1996). Unfortunately, C. tropicalis is highly resistant to G418.…”

mentioning

confidence: 99%

A mutated hygromycin resistance gene is functional in the n-alkane-assimilating yeast Candida tropicalis

et al. 2000

View full text Add to dashboard Cite

show abstract

“…36,37) One can obtain positive transformants at high frequency by targeted integration, since there is low homology between the drug-resistance marker and the yeast genome. Moreover, those markers make it possible to transform wild-type strains that have not been subjected to heavy mutagenesis, and they provide a great advantage for use in the case of a high-ploidy yeast like C. utilis.…”

Section: Discussionmentioning

confidence: 99%

Efficient Gene Disruption in the High-Ploidy YeastCandida utilisUsing the Cre-loxPSystem

Ikushima¹,

Fujii²,

Kobayashi³

2009

Bioscience, Biotechnology, and Biochemistry

View full text Add to dashboard Cite

“…We have sequenced, assembled, and annotated the initial non-homologous end-joining mutant strain used for construction of all the strains described herein allowing for accurate design of genetic constructs. Although some of the resources we have developed have been previously described in other genetic backgrounds in Y. lipolytica [31], including auxotrophs [7], utilization of hph [114] and gfp [115], and replicating and integrating plasmids, they have guided the design of these strains and plasmids. Having all these tools, along with the fluorescent organelle atlas in an isogenic non-homologous end-joining mutant will remove data differences caused by genetic background, and expedite functional genetics in Y. lipolytica .…”

Section: Discussionmentioning

confidence: 99%

A molecular genetic toolbox for Yarrowia lipolytica

Bredeweg

Pomraning

Dai

et al. 2017

Biotechnol Biofuels

View full text Add to dashboard Cite

Background Yarrowia lipolytica is an ascomycete yeast used in biotechnological research for its abilities to secrete high concentrations of proteins and accumulate lipids. Genetic tools have been made in a variety of backgrounds with varying similarity to a comprehensively sequenced strain.ResultsWe have developed a set of genetic and molecular tools in order to expand capabilities of Y. lipolytica for both biological research and industrial bioengineering applications. In this work, we generated a set of isogenic auxotrophic strains with decreased non-homologous end joining for targeted DNA incorporation. Genome sequencing, assembly, and annotation of this genetic background uncovers previously unidentified genes in Y. lipolytica. To complement these strains, we constructed plasmids with Y. lipolytica-optimized superfolder GFP for targeted overexpression and fluorescent tagging. We used these tools to build the “Yarrowia lipolytica Cell Atlas,” a collection of strains with endogenous fluorescently tagged organelles in the same genetic background, in order to define organelle morphology in live cells.ConclusionsThese molecular and isogenetic tools are useful for live assessment of organelle-specific protein expression, and for localization of lipid biosynthetic enzymes or other proteins in Y. lipolytica. This work provides the Yarrowia community with tools for cell biology and metabolism research in Y. lipolytica for further development of biofuels and natural products.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0687-7) contains supplementary material, which is available to authorized users.

show abstract

Efficient selection of hygromycin-B-resistant Yarrowia lipolytica transformants

Cited by 32 publications

References 20 publications

A mutated hygromycin resistance gene is functional in the n-alkane-assimilating yeast Candida tropicalis

A mutated hygromycin resistance gene is functional in the n-alkane-assimilating yeast Candida tropicalis

Efficient Gene Disruption in the High-Ploidy YeastCandida utilisUsing the Cre-loxPSystem

A molecular genetic toolbox for Yarrowia lipolytica

Contact Info

Product

Resources

About