Genetic Analysis of Lipase Low-producing Mutants of Yarrowia lipolytica

Nga, B. H.; Gaillardin, Claude; Fournier, P.; Heslot, H.

doi:10.1099/00221287-135-9-2439

Cited by 3 publications

(3 citation statements)

References 14 publications

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“…This is supported by the lack of success of attempts to obtain mutants unable to grow on TG. The mutants isolated were affected only in lipase levels (32). Such multicomponent gene families have been observed in other yeasts.…”

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confidence: 74%

Characterization of an Extracellular Lipase Encoded by LIP2 in Yarrowia lipolytica

et al. 2000

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We isolated the LIP2 gene from the lipolytic yeast Yarrowia lipolytica. It was found to encode a 334-amino-acid precursor protein. The secreted lipase is a 301-amino-acid glycosylated polypeptide which is a member of the triacylglycerol hydrolase family (EC 3.1.1.3). The Lip2p precursor protein is processed by the KEX2-like endoprotease encoded by XPR6. Deletion of the XPR6 gene resulted in the secretion of an active but less stable proenzyme. Thus, the pro region does not inhibit lipase secretion and activity. However, it does play an essential role in the production of a stable enzyme. Processing was found to be correct in LIP2A (multiple LIP2 copy integrant)-overexpressing strains, which secreted 100 times more activity than the wild type, demonstrating that XPR6 maturation was not limiting. No extracellular lipase activity was detected with the lip2 knockout (KO) strain, strongly suggesting that extracellular lipase activity results from expression of the LIP2 gene. Nevertheless, the lip2 KO strain is still able to grow on triglycerides, suggesting an alternative pathway for triglyceride utilization in Y. lipolytica. This yeast naturally secretes several proteins, depending on the growth conditions (21). For example, if the pH is higher than 6, it secretes an alkaline extracellular protease (AEP) (33,38). Under optimal conditions, up to 1 g of AEP is secreted per liter (37). AEP is encoded by XPR2 (11,27,33). This gene codes for a 454-amino-acid (aa) prepro enzyme precursor containing a 15-aa signal sequence and a stretch of nine X-Ala or X-Pro dipeptides, followed by a 124-aa pro region that includes a glycosylation site (Asn 123 ) and a Lys 156 -Arg 157 processing site, and finally the mature form itself. The AEP precursor undergoes complex processing (27). A diaminopeptidase processes the stretch of nine dipeptides (X-Ala or X-Pro) (28,29), and the endoprotease encoded by the XPR6 gene is required to cleave the pro region, releasing the mature form (14). The pro region is involved in both the inhibition of protease activity and the folding of the propeptide into a conformation compatible with secretion, and secretion at 28°C depends on the glycosylation of the pro region (15, 28).Several enzymes are secreted by Y. lipolytica, and lipase and esterase activities have been detected and analyzed in various studies. Lipase secretion was first reported in 1948 by Peters and Nelson (41, 42), who described a single type of glucoserepressible activity. An extracellular and two cell-bound types of activity corresponding to lipase I (39 kDa) and lipase II (44 kDa) were described by Ota and coworkers (39, 47). The extracellular lipase required oleic acid as a stabilizer-activator, whereas the cell-bound lipases did not and differed in several properties from the extracellular enzyme (40). The rates of production of the extracellular and cell-bound enzymes were reported to depend on the carbon and nitrogen composition of the medium. Extracellular lipase was only detected in cultures grown with an organic nitrogen so...

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confidence: 74%

Characterization of an Extracellular Lipase Encoded by LIP2 in Yarrowia lipolytica

et al. 2000

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“…Mutants unable to use tributyrin as a carbon source were isolated after nystatin enrichment [105], and were shown to define three complementation groups [106]. A gene encoding a secretory protein highly homologous to fungal lipases has been recently cloned in an independent approach (A. Dominguez, personal communication): this should rapidly permit assessing if there is more than one lipase activity in Y. lipolytica .…”

Section: Physiologymentioning

confidence: 99%

Physiology and genetics of the dimorphic fungusYarrowia lipolytica

Barth¹,

Gaillardin²

1997

FEMS Microbiol Rev

442

100

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The ascomycetous yeast Yarrowia lipolytica (formerly Candida, Endomycopsis, or Saccharomyces lipolytica) is one of the more intensively studied 'non-conventional' yeast species. This yeast is quite different from the well-studied yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe with respect to its phylogenetic evolution, physiology, genetics, and molecular biology. However, Y. lipolytica is not only of interest for fundamental research, but also for biotechnological applications. It secretes several metabolites in large amounts (i.e. organic acids, extracellular proteins) and the tools are available for overproduction and secretion of foreign proteins. This review presents a comprehensive overview on the available data on physiology, cell biology, molecular biology and genetics of Y. lipolytica.

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“…Other groups isolated mutants unable to utilize short-chain triglycerides after nystatin enrichment using tributyrin as a carbon source (19,20) or unable to utilize the long-chain oleic acid (C 18 ) in a screen of genes involved in peroxisome biogenesis using a rapid immunofluorescence assay (22). Some of the Pex mutants exhibited pleiotropic phenotypes affecting peroxisome biogenesis, secretion, and morphology (32).…”

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

et al. 2001

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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.

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Genetic Analysis of Lipase Low-producing Mutants of Yarrowia lipolytica

Cited by 3 publications

References 14 publications

Characterization of an Extracellular Lipase Encoded by LIP2 in Yarrowia lipolytica

Characterization of an Extracellular Lipase Encoded by LIP2 in Yarrowia lipolytica

Physiology and genetics of the dimorphic fungusYarrowia lipolytica

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

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