Isolation and characterization of the TRP1 gene from the yeast Yarrowia lipolytica and multiple gene disruption using a TRP blaster

The TRP1 gene encoding N‐(5′‐phosphoribosyl)‐anthranilate isomerase was isolated from the yeast Yarrowia lipolytica, in which only a few genetic marker genes are available. The Y. lipolytica TRP1 gene (YlTRP1) cloned by complementation of Y. lipolytica trp1 mutation was found to be a functional homologue of Saccharomyces cerevisiae TRP1. Since YlTRP1 could be used for counterselection in medium containing 5‐fluoroanthranilic acid (5‐FAA), we constructed TRP blasters that contained YlTRP1 flanked by a direct repeat of a sequence and allowed the recycling of the YlTRP1 marker. Using the TRP blasters the sequential disruption of target genes could be carried out within the same strain of Y. lipolytica. The nucleotide sequence of the YlTRP1 gene has been deposited at GenBank under Accession No. AF420590. Copyright © 2003 John Wiley & Sons, Ltd.

[1]  G. Braus,et al.  A consensus transcription termination sequence in the promoter region is necessary for efficient gene expression of the TRP1 gene of Saccharomyces cerevisiae , 1988, Molecular and General Genetics MGG.

[2]  D. Ryu,et al.  High Cell Density Culture of Yarrowialipolytica Using a One‐Step Feeding Process , 2000, Biotechnology progress.

[3]  W. Kunau,et al.  Structure and metabolic control of the Yarrowia lipolytica peroxisomal 3-oxoacyl-CoA-thiolase gene. , 1993, European journal of biochemistry.

[4]  G. Barth,et al.  Physiology and genetics of the dimorphic fungus Yarrowia lipolytica. , 1997, FEMS microbiology reviews.

[5]  C. Szent-Gyorgyi,et al.  A simplified method for the repeated replacement of yeast chromosomal sequences with in vitro mutations , 1996, Yeast.

[6]  P. Ross-Macdonald,et al.  A shuttle mutagenesis system for tagging genes in the yeast Yarrowia lipolytica. , 1998, Gene.

[7]  J. Nicaud,et al.  Multiple-copy integration in the yeast Yarrowia lipolytica , 1994, Current Genetics.

[8]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[9]  J. Nicaud,et al.  Utilization of an auxotrophic strain of the yeast Yarrowia lipolytica to improve γ‐decalactone production yields , 1997, Letters in Applied Microbiology.

[10]  C. S. Park,et al.  Improvement of heterologous protein productivity using recombinant Yarrowia lipolytica and cyclic fed-batch process strategy. , 1998, Biotechnology and bioengineering.

[11]  Jeong-Yoon Kim,et al.  Expression, Secretion, and Processing of Rice α-Amylase in the Yeast Yarrowia lipolytica* , 1997, The Journal of Biological Chemistry.

[12]  Jeong-Yoon Kim,et al.  Recombinant bioprocess optimization for heterologous protein production using two-stage, cyclic fed-batch culture , 1998, Applied Microbiology and Biotechnology.

[13]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[14]  Jean-Marc Nicaud,et al.  Evaluation of Acyl Coenzyme A Oxidase (Aox) Isozyme Function in the n-Alkane-Assimilating YeastYarrowia lipolytica , 1999, Journal of bacteriology.

[15]  M. Seman,et al.  Characterization of an Extracellular Lipase Encoded by LIP2 in Yarrowia lipolytica , 2000, Journal of bacteriology.

[16]  G. Fink,et al.  A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance , 1984, Molecular and General Genetics MGG.

[17]  D. Irwin,et al.  Isogenic strain construction and gene mapping in Candida albicans. , 1993, Genetics.

[18]  D. J. Glover,et al.  The extracellular acid protease gene of Yarrowia lipolytica: sequence and pH-regulated transcription. , 1996, Microbiology.

[19]  C. S. Park,et al.  Molecular cloning of YlPMR1, a S. cerevisiae PMR1 homologue encoding a novel P-type secretory pathway Ca2+ -ATPase, in the yeast Yarrowia lipolytica. , 1998, Gene.

[20]  R. Mortimer,et al.  Genetics of Extracellular Protease Production in SACCHAROMYCOPSIS LIPOLYTICA. , 1977, Genetics.

[21]  Y Nagata,et al.  Isolation and characterization of acetoacetyl-CoA thiolase gene essential for n-decane assimilation in yeast Yarrowia lipolytica. , 2001, Biochemical and biophysical research communications.

[22]  J. Aitchison,et al.  PAY4, a gene required for peroxisome assembly in the yeast Yarrowia lipolytica, encodes a novel member of a family of putative ATPases. , 1994, The Journal of biological chemistry.

[23]  J. Toyn,et al.  A counterselection for the tryptophan pathway in yeast: 5‐fluoroanthranilic acid resistance , 2000, Yeast.

[24]  B. Gicquel,et al.  Use of fluorescence induction and sucrose counterselection to identify Mycobacterium tuberculosis genes expressed within host cells. , 1999, Microbiology.

[25]  H. Dalbøge,et al.  Comparison of expression systems in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha, Klyveromyces lactis, Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica , 1998, Yeast.

[26]  J. Nicaud,et al.  Peroxisomal β-oxidation activities and γ-decalactone production by the yeast Yarrowia lipolytica , 1998, Applied Microbiology and Biotechnology.

[27]  J. Nicaud,et al.  Vectors for gene expression and amplification in the yeast Yarrowia lipolytica , 2001, Yeast.

[28]  L. Davidow,et al.  The Yarrowia lipolytica LEU2 gene , 2004, Current Genetics.

[29]  J. Nicaud,et al.  Comparison of promoters suitable for regulated overexpression of β-galactosidase in the alkane-utilizing yeastYarrowia lipolytica , 2000 .