Integrative transformation of the yeast Yarrowia lipolytica

Davidow, Lance S.; Apostolakos, Diane; O'Donnell, M.M.; Proctor, Alan R.; Ogrydziak, David M.; Wing, Rod A.; Stasko, Irene; DeZeeuw, John R.

doi:10.1007/bf00418492

Cited by 113 publications

(36 citation statements)

References 33 publications

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“…Among alkane-or fatty acid-utilizing yeasts, transformation of C. tropicalis appears to be most similar to that reported for Yarrowia lipolytica (9,10). Both yeasts are transformed at a high frequency by DNA vectors containing auxotrophic marker genes, and vectors integrate mostly by homologous recombination between sequences shared by vector and host genomes.…”

Section: Methodsmentioning

confidence: 54%

Development of an integrative DNA transformation system for the yeast Candida tropicalis

1990

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We developed the alkane and fatty-acid utilizing yeast Candida tropicalis as a host for DNA transformations. The system is based on an auxotrophic mutant host of C. tropicalis which is defective in orotidine monophosphate decarboxylase (ura3). The ura3 host was isolated by mutagenesis and a double-selection procedure that combined nystatin enrichment selection and 5-fluoro-orotic acid resistance selection. As a selectable marker, we isolated and characterized the C. tropicalis URA3 gene. Plasmid vectors that contained the C. tropicalis URA3 gene transformed the C. tropicalis mutant host at a frequency of 10(3) to 10(4) transformants per micrograms of plasmid DNA. Vectors that contained the Saccharomyces cerevisiae URA3 gene could not transform C. tropicalis. DNA transfer was accomplished by modified versions of either spheroplast generation (CaCl2-polyethylene glycol)-fusion or cation (LiCl) procedures developed for S. cerevisiae. Plasmid vectors that had been cut within the C. tropicalis URA3 fragment integrated by homologous recombination at the URA3 locus.

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Section: Methodsmentioning

confidence: 54%

Development of an integrative DNA transformation system for the yeast Candida tropicalis

1990

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“…To test whether homologous integration occurs at the Leu ϩ locus, as has been described previously (12,67), and to obtain a physical map of the inserted DNA, we digested the DNAs of JC80-1 and JC110-1. After electrophoresis and Southern transfer, these DNAs were hybridized with a LEU2 probe.…”

Section: Isolation and Characterization Of Morphological Mutantsmentioning

confidence: 99%

“…lipolytica is a dimorphic heterothallic yeast (3) that is unusual in both the structure of its genes coding for rRNA (17) and in its relationship to other yeasts (2). Y. lipolytica is amenable to genetic analysis (48), and DNA-mediated integration (12,20) and autonomous transformation systems (18) have been developed. Mutations in the SEC14 gene (42) and deletion of XPR6 (16) have strong effects on the yeast-to-hypha transition.…”

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confidence: 99%

HOY1, a Homeo Gene Required for Hyphal Formation inYarrowia lipolytica

Torres-Guzmán

Domínguez

1997

Molecular and Cellular Biology

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The dimorphic fungus Yarrowia lipolytica grows to form hyphae either in rich media or in media with GlcNAc as a carbon source. A visual screening, called FIL (filamentation minus), for Y. lipolytica yeast growth mutants has been developed. The FIL screen was used to identify three Y. lipolytica genes that abolish hypha formation in all media assayed. Y. lipolytica HOY1, a gene whose deletion prevents the yeast-hypha transition both in liquid and solid media, was characterized. HOY1 is predicted to encode a 509-amino-acid protein with a homeodomain homologous to that found in the chicken Hox4.8 gene. Analysis of the protein predicts a nuclear location. These observations suggest that Hoy1p may function as a transcriptional regulatory protein. In disrupted strains, reintroduction of HOY1 restored the capacity for hypha formation. Northern blot hybridization revealed the HOY1 transcript to be approximately 1.6 kb. Expression of this gene was detected when Y. lipolytica grew as a budding yeast, but an increase in its expression was observed by 1 h after cells had been induced to form hyphae. The possible functions of HOY1 in hyphal growth and the uses of the FIL screen to identify morphogenetic regulatory genes from heterologous organisms are discussed.The yeast-to-hypha morphological transition (dimorphism) is typical of many pathogenic fungi (62). The dimorphic transition is a freely reversible process that can be induced by changes in many parameters (56). Much attention has been focused on Candida albicans as a model for analyzing dimorphism because it is the most frequently isolated fungal pathogen in humans. However, although several groups have reported the isolation of mutants which persist in either the yeast (10) or hyphal (21, 30) form, analysis of these mutants is hampered by the difficulties inherent to genetic manipulations of this asexual diploid organism.Two approaches have been successfully used to carry out the analysis of dimorphism. With one of them, differential hybridization screening (7), several genes that are differentially expressed during morphogenesis have been isolated in C. albicans (i.e., ECE1, expressed in association with cell elongation, and PHR1, which is differentially regulated in response to the pH of the growth medium). Deletion of PHR1 results in a pH-conditional defect in morphogenesis (59).The second approach consists of cloning Candida homologs of Saccharomyces cerevisiae genes known to regulate pseudofilamentous growth. Under conditions of nutrient limitation (i.e., nitrogen starvation), S. cerevisiae undergoes a dimorphic transition to growth of pseudohyphae, linear chains of elongated cells in which the daughters remain attached to the mothers (23, 24). In this context, several C. albicans genes that are members of a Candida map kinase (MAPK) cascade, i.e., CPH1 (40) or ACPR (43), HST7 (11), and CST20 (36, 39), have been isolated by complementation of the corresponding Saccharomyces mutants, i.e., STE12, STE7, and STE20, respectively (11,36,39,40,43). When C. albicans st...

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“…lipolytica-tetrad analysis (50), transformation (17,26), cloning of genes by complementation (19), gene replacement (J. DeZeeuw, personal communication), and expression of heterologous genes (25). Recently, a small ribonucleoprotein homologous to signal recognition particle has been isolated…”

mentioning

confidence: 99%

Intracellular precursors and secretion of alkaline extracellular protease of Yarrowia lipolytica.

Matoba¹,

Fukayama²,

Wing³

et al. 1988

Mol. Cell. Biol.

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Processing and secretion of the alkaline extracellular protease (AEP) from the yeast Yarrowia lipolytica was studied by pulse-chase and immunoprecipitation experiments. Over half of newly synthesized AEP was secreted by 6 min. Over 99% of AEP activity which was external to the cytoplasmic membrane was located in the supernatant medium. Polypeptides of 55, 52, 44, 36, and kifodaltons derived from the proregion of AEP indicated that one major processing pathway was 55K-*52K-*32K. The gene coding for AEP (XPR2) was cloned and sequenced. The sequence and the immunoprecipitation results suggest that AEP is originally synthesized with an additional preprol-proII-proIlI amino-terminal region. Processing definitely involves cleavage(s) after pairs of basic amino acids and the addition of one N-linked oligosaccharide. Signal peptidase cleavage, dipeptidyl aminopeptidase cleavages, and at least one additional proteolytic cleavage may also be involved.

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Integrative transformation of the yeast Yarrowia lipolytica

Cited by 113 publications

References 33 publications

Development of an integrative DNA transformation system for the yeast Candida tropicalis

Development of an integrative DNA transformation system for the yeast Candida tropicalis

HOY1, a Homeo Gene Required for Hyphal Formation inYarrowia lipolytica

Intracellular precursors and secretion of alkaline extracellular protease of Yarrowia lipolytica.

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