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Journal of Bacteriology, September 2001, p. 5102-5109, Vol. 183, No. 17
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.17.5102-5109.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Insertional Mutagenesis in the n-Alkane-Assimilating Yeast Yarrowia lipolytica: Generation of Tagged Mutations in Genes Involved in Hydrophobic Substrate Utilization

Stephan Mauersberger,1 Hui-Jie Wang,2 Claude Gaillardin,2 Gerold Barth,1 and Jean-Marc Nicaud2,*

Laboratoire de Microbiologie et de Génétique Moléculaire, UR INRA 216, URA CNRS 1925, Institut National Agronomique Paris-Grignon, F-78850 Thiverval-Grignon, France,2 and Institut für Mikrobiologie, Technische Universität Dresden, D-01062 Dresden, Germany1

Received 22 March 2001/Accepted 13 June 2001

Tagged mutants affected in the degradation of hydrophobic compounds (HC) were generated by insertion of a zeta-URA3 mutagenesis cassette (MTC) into the genome of a zeta-free and ura3 deletion-containing strain of Yarrowia lipolytica. MTC integration occurred predominantly at random by nonhomologous recombination. A total of 8,600 Ura+ transformants were tested by replica plating for (i) growth on minimal media with alkanes of different chain lengths (decane, dodecane, and hexadecane), oleic acid, tributyrin, or ethanol as the C source and (ii) colonial defects on different glucose-containing media (YPD, YNBD, and YNBcas). A total of 257 mutants were obtained, of which about 70 were affected in HC degradation, representing different types of non-alkane-utilizing (Alk-) mutants (phenotypic classes alkA to alkE) and tributyrin degradation mutants. Among Alk- mutants, growth defects depending on the alkane chain length were observed (alkAa to alkAc). Furthermore, mutants defective in yeast-hypha transition and ethanol utilization and selected auxotrophic mutants were isolated. Flanking borders of the integrated MTC were sequenced to identify the disrupted genes. Sequence analysis indicated that the MTC was integrated in the LEU1 locus in N083, a leucine-auxotrophic mutant, in the isocitrate dehydrogenase gene of N156 (alkE leaky), in the thioredoxin reductase gene in N040 (alkAc), and in a peroxine gene (PEX14) in N078 (alkD). This indicates that MTC integration is a powerful tool for generating and analyzing tagged mutants in Y. lipolytica.

* Corresponding author. Mailing address: Laboratoire de Microbiologie et de Génétique Moléculaire, Institut National Agronomique Paris-Grignon, BP 01, F-78850 Thiverval-Grignon, France. Phone: 33 01 30 81 54 50. Fax: 33 01 30 81 54 57. E-mail: jean-marc.nicaud{at}grignon.inra.fr.

Journal of Bacteriology, September 2001, p. 5102-5109, Vol. 183, No. 17
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.17.5102-5109.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Mlickova, K., Roux, E., Athenstaedt, K., d'Andrea, S., Daum, G., Chardot, T., Nicaud, J.-M. (2004). Lipid Accumulation, Lipid Body Formation, and Acyl Coenzyme A Oxidases of the Yeast Yarrowia lipolytica. Appl. Environ. Microbiol. 70: 3918-3924 [Abstract] [Full Text]  


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