Biotechnology Letters 26: 849–854, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands. 849 Preparation of dodecanol-tolerant strains of Yarrowia lipolytica Martha S. Smit 1,∗ , Masego M. Mokgoro 1 , Evodia Setati 1 & Jean-Marc Nicaud 2 1 Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, P.O Box 339, Bloemfontein 9300, South Africa 2 Laboratoire de Microbiologie et G´ en´ etique Mol´ eculaire, INRA INA-PG CNRS, UMR 2585, Centre de Biotechno- logie Agro-Industrielle, B.P. 01, 78850 Thiverval-Grignon, France ∗ Author for correspondence (Fax: +27-51-4443219; E-mail: smitms.sci@mail.uovs.ac.za) Received 22 January 2004; Revisions requested 13 February 2004; Revisions received 15 March 2004; Accepted 15 March 2004 Key words: alkane degradation, hydrophobic substrate, solvent-tolerant yeast, toxicity, Yarrowia lipolytica Abstract Dodecanol (1% v/v) and dodecanoic acid (1% w/v) inhibited growth of Yarrowia lipolytica in complex media supplemented with glucose but dodecanedioic acid (1% w/v) was not toxic. Dodecanol-tolerant strains were prepared from the wild type strain H222 as well as the acyl-CoA oxidase deleted (POX2, POX3, POX5) strain MTLY35. These strains grew in rich media containing up to 10% (v/v) dodecanol. Dodecanol-tolerant strains remained dodecanol tolerant after they had been cultured in rich media without dodecanol. No significant amount of dodecanedioic acid was accumulated by the dodecanol-tolerant strains when grown on glucose in the presence of dodecanol. Introduction Alkane utilizing yeasts, such as Candida maltosa and Yarrowia lipolytica, grow well on the terminal and diterminal oxidation products of alkanes, i.e. 1- alkanols, fatty aldehydes, fatty acids, ω-hydroxy fatty acids and dicarboxylic acids, although the shorter chain lengths (< C 10 ) of these substrates are toxic for these yeasts or inhibit growth on alkanes (Mauersber- ger et al. 1996, Barth & Gaillardin 1996). However, Green et al. (2000) showed that decane and dodecane as well as fatty acids of chain lengths C10, C12, C14 and C16 were toxic for a dicarboxylic acid- accumulating strain of C. cloacae. This toxicity could be alleviated by the addition of 5% (v/v) pristane. [C. = cloacae is a synonym of C. maltosa (Mauers- berger et al. 1996).] We are investigating the different steps in the al- kane degradation pathway of the industrial yeast Y. lipolytica. We have investigated the toxicity of do- decanol, dodecanoic acid and dodecanedioic acid to- wards Y. lipolytica. Growth experiments were conduc- ted with Y. lipolyitca H222, the strain used by Mauers- berger et al. (2001), to construct tagged mutants deficient in alkane degradation. After establishing that dodecanol and dodecanoic acid were toxic, but not dodecanedioic acid, we also investigated the prepara- tion of dodecanol-tolerant strains of Y. lipolytica. Both Y. lipolytica H222 as well as an acyl-CoA oxidase triple-deleted strain Y. lipolytica MTLY35 (POX2, POX3, POX5) (Wang et al. 1999) were used for the preparation of dodecanol-tolerant strains. The pos- sibility that dodecanol-tolerant strains accumulate do- decanedioic acid as a means of detoxification, was also investigated. Materials and methods Microorganisms Yarrowia lipolytica wild type strain H222 (Wang et al. 1999) was obtained from Dr S. Mauersberger, Insti- tute of Microbiology, Dresden University of Techno- logy, Dresden, Germany and the acyl-CoA oxidase triple-deleted strain MTLY35 (POX2, POX3, POX5) (Wang et al. 1999) was obtained from the Laboratoire