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Applied and Environmental Microbiology, August 2000, p. 3283-3289, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Autocloning and Amplification of LIP2 in Yarrowia lipolytica

Georges Pignède,1 Hui-Jie Wang,2 Franck Fudalej,1 Michel Seman,1 Claude Gaillardin,2 and Jean-Marc Nicaud2,*

Laboratoire Mayoly-Spindler, Service Recherche, 78401 Chatou Cedex,1 and Laboratoire de Microbiologie et Génétique Moléculaire, INRA Centre de Grignon, 78850 Thiverval-Grignon,2 France

Received 13 December 1999/Accepted 20 May 2000

We synthesized a Yarrowia lipolytica strain overproducing lipase for industrial applications by using long terminal repeat (zeta ) of the Y. lipolytica retrotransposon Ylt1 and an allele of URA3 with a promoter deletion to construct JMP3. JMP3 is a derivative of plasmid pHSS6 carrying a NotI-NotI cassette which contains a defective URA3 allele, a polylinker sequence, and the zeta  region for targeting to multiple sites in the genome of the recipient. We inserted the LIP2 gene (encoding extracellular lipase) under the control of the strong POX2 promoter into JMP3 to generate JMP6. The pHSS6 region was removed by NotI digestion prior to transformation. Two Y. lipolytica strains transformed with the JMP6 LIP2 cassette had a mean of 10 integrated copies devoid of the Escherichia coli region, corresponding to an autocloning event. The copy number in the transformants was stable even after 120 generations in nonselective and lipase-inducing conditions. The resulting strains could produce 0.5 g of active lipase per liter in the supernatant, 40 times more than the single-copy strain with the LIP2 promoter. This work provides a new expression system in Y. lipolytica that results in strains devoid of bacterial DNA and in strains producing a high level of lipase for industrial uses, waste treatment, and pancreatic insufficiency therapy.

* Corresponding author. Mailing address: Laboratoire de Microbiologie et Génétique Moléculaire, INRA Centre de Grignon, BP 01, 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.

Applied and Environmental Microbiology, August 2000, p. 3283-3289, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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