skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Deletion of the KU70 homologue facilitates gene targeting in Lipomyces starkeyi strain NRRL Y-11558

Abstract

Recently, several transformation methods have been established for the oleaginous yeast Lipomyces starkeyi, a model strain being explored for use in the production of renewable fuels and chemicals in Lipomyces species. However, targeted gene disruption and/or integration in L. starkeyi have been impeded by its preference for the non-homologous end-joining (NHEJ) pathway. To augment homologous recombination (HR) by repressing the NHEJ function, the Lsku70 gene was identified and disrupted in L. starkeyi strain NRRL Y-11558. Targeted disruption efficiency of four selected genes (gsy1, mfe1, pex10, and tgl4) was increased to 50 to 100% in the Lsku70 mutant strain with 0.8 to 1.3 kb homologous flanking fragments. In contrast, HR frequency was 0 to 11% in the parent strain even with longer (1.34 kb) homologous flanking fragments. Furthermore, the minimum length of flanking homologous DNA fragments was about 0.6 kb for high-efficiency tgl4 gene disruption in the Lsku70 mutant strain, Site-specific gene insertion at intergenic regions near gsy1, ku70, mfe1, and pex10 genes were examined in the Lsku70 mutant and found to be 100% in all cases. Finally, the deletion of Lsku70 did not perturb the growth, sugar consumption, and total lipid production in the Lsku70 mutant. Therefore, the Lsku70 mutantmore » can serve as a useful platform strain for targeted gene manipulation and improvement of fuel and value-added chemical production.« less

Authors:
ORCiD logo; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1501755
Report Number(s):
PNNL-SA-130042
Journal ID: ISSN 0172-8083
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Journal Name:
Current Genetics
Additional Journal Information:
Journal Volume: 65; Journal Issue: 1; Journal ID: ISSN 0172-8083
Country of Publication:
United States
Language:
English

Citation Formats

Dai, Ziyu, Pomraning, Kyle R., Deng, Shuang, Hofstad, Beth A., Panisko, Ellen A., Rodriguez, Diana, Butcher, Mark G., Culley, David E., and Magnuson, Jon K. Deletion of the KU70 homologue facilitates gene targeting in Lipomyces starkeyi strain NRRL Y-11558. United States: N. p., 2018. Web. doi:10.1007/s00294-018-0875-z.
Dai, Ziyu, Pomraning, Kyle R., Deng, Shuang, Hofstad, Beth A., Panisko, Ellen A., Rodriguez, Diana, Butcher, Mark G., Culley, David E., & Magnuson, Jon K. Deletion of the KU70 homologue facilitates gene targeting in Lipomyces starkeyi strain NRRL Y-11558. United States. doi:10.1007/s00294-018-0875-z.
Dai, Ziyu, Pomraning, Kyle R., Deng, Shuang, Hofstad, Beth A., Panisko, Ellen A., Rodriguez, Diana, Butcher, Mark G., Culley, David E., and Magnuson, Jon K. Sat . "Deletion of the KU70 homologue facilitates gene targeting in Lipomyces starkeyi strain NRRL Y-11558". United States. doi:10.1007/s00294-018-0875-z.
@article{osti_1501755,
title = {Deletion of the KU70 homologue facilitates gene targeting in Lipomyces starkeyi strain NRRL Y-11558},
author = {Dai, Ziyu and Pomraning, Kyle R. and Deng, Shuang and Hofstad, Beth A. and Panisko, Ellen A. and Rodriguez, Diana and Butcher, Mark G. and Culley, David E. and Magnuson, Jon K.},
abstractNote = {Recently, several transformation methods have been established for the oleaginous yeast Lipomyces starkeyi, a model strain being explored for use in the production of renewable fuels and chemicals in Lipomyces species. However, targeted gene disruption and/or integration in L. starkeyi have been impeded by its preference for the non-homologous end-joining (NHEJ) pathway. To augment homologous recombination (HR) by repressing the NHEJ function, the Lsku70 gene was identified and disrupted in L. starkeyi strain NRRL Y-11558. Targeted disruption efficiency of four selected genes (gsy1, mfe1, pex10, and tgl4) was increased to 50 to 100% in the Lsku70 mutant strain with 0.8 to 1.3 kb homologous flanking fragments. In contrast, HR frequency was 0 to 11% in the parent strain even with longer (1.34 kb) homologous flanking fragments. Furthermore, the minimum length of flanking homologous DNA fragments was about 0.6 kb for high-efficiency tgl4 gene disruption in the Lsku70 mutant strain, Site-specific gene insertion at intergenic regions near gsy1, ku70, mfe1, and pex10 genes were examined in the Lsku70 mutant and found to be 100% in all cases. Finally, the deletion of Lsku70 did not perturb the growth, sugar consumption, and total lipid production in the Lsku70 mutant. Therefore, the Lsku70 mutant can serve as a useful platform strain for targeted gene manipulation and improvement of fuel and value-added chemical production.},
doi = {10.1007/s00294-018-0875-z},
journal = {Current Genetics},
issn = {0172-8083},
number = 1,
volume = 65,
place = {United States},
year = {2018},
month = {8}
}