ofYarrowia Lipolytica Research Articles

Insertional mutagenesis in the n-alkane-assimilating yeast Yarrowia lipolytica: generation of tagged mutations in genes involved in hydrophobic substrate utilization.

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.

Dependence of yeast permeabilization with Triton X-100 on the cell age

Acid phosphatase activity and protein release were determined in cell suspensions ofYarrowia lipolytica andTorulospora delbrueckii at different stages of their growth by permeabilization with Triton X-100. The effect of the surfactant on the cell permeability did not depend on the cell age.

Effect of ethanol concentration on the maximal specific growth rate and biomass composition ofYarrowia lipolytica mutant strain No. 1

the effect of ethanol concentration on the maximal specific growth rate and biomass composition ofYarrowia lipolytica No. 1 was studied during culturing in the pH-auxostat mode. Growth inhibition set in starting from a 2.64 g/liter residual concentration of ethanol. The constant of ethanol inhibition was 11.0 g/liter. Growth inhibition with ethanol was associated with changes of the fatty-acid composition of lipids and a resultant reduction of lipid unsaturation.

Drug resistance inYarrowia lipolytica

Six strains ofYarrowia lipolytica tested here were resistant to 10–20 g erythromycin or chloramphenicol per L glycerol-agar medium. Cells tolerating 4 g chloramphenicol per L were very rare and reverted rapidly to the highest resistance. In analogy with EryR mutants ofKluyveromyces lactis, our strains did not grow at 36°C but did not lose their viability at that temperature. Two levels of resistance were found with oligomycin and antimycin A,i.e. 10 and 3 mg/L in the former and 10 and 2 mg/L in the latter. The higher resistance levels segregated mitotically and were, therefore, controlled extrachromosomally. The lower resistance levels showed very frequent changes from sensitivity to resistance that prevented the genetic analysis of this resistance. An almost continuous range of tolerance to <5–400 μg mucidin per L was found in populations of the strains analyzed. Newly formed MucR cells were established only in the presence of the antibiotic. Pure cultures of MucR cells showed an extremely high instability caused by their lower viability and very low growth rate in the absence of mucidin. No loss of resistance to antimycin A was found, although AntR cells revealed similar negative selection. Mutability MucS»MucR and MucR»MucS was higher in AntR cells than in AntS ones.

Effect of lipids and detergents on the production and solubilization of extracellular lipase inYarrowia lipolytica

Triglycerides, oleic acid but not fatty acid-containing, nonionic detergents (Spans, Tweens) were able to stimulate the synthesis of cell-bound and soluble lipase ofYarrowia lipolytica grown in a complex medium containing citrate and urea. The optimal concentration of olive oil for induction was 0.5% (W/V). The combined effect of a high ionic strength (0.75 mol/L KCl) and of digitonin (2 mmol/L) at pH 7.6 resulted in solubilization of 80% of the cell-bound lipase and a significant activation of the enzyme. Comparison of twoY. lipolytica strains showed the effects of Mg2+ and Fe3+ concentrations in the medium on the synthesis of the enzyme to be strongly strain-dependent.

Cloning of theHIS3 gene ofYarrowia lipolytica

The HIS3 gene of the yeast Yarrowia lipolytica has been cloned from a genomic library by complementation of the his3 mutation of Saccharomyces cerevisiae. The gene was subsequently subcloned in Escherichia coli and characterized by restriction enzyme mapping.

On the false positive urease activity ofYarrowia lipolytica

The ability of Yarrowia lipolytica to produce ammonia from urea was found variable on some media. The colour change of the indicator in Christensen's urea agar was not due to the urease activity of this species but was a non-specific alkalization reaction. Rapid urea broth was reliable giving no false positive results. It was found that Y. lipolytica is a urease negative yeast species.

Selection of spontaneous mutants ofYarrowia lipolytica by inositol-less death

A procedure was developed for the selection of spontaneous mutants of the yeastYarrowia lipolytica. An inositol-requiring mutant of a wild-typeY. lipolytica, YB 3-122, was derived by mutagenesis and screening. The mutant had a reversion frequency of less than 6×10−9. A mutant selection procedure based on inositolless death was then developed using this mutant strain. The selection procedure killed growingY. lipolytica cells and enriched for mutants yielding cultures that consisted of 60–98% spontaneous mutants after two rounds of inositol-less death. The procedure enriched for four classes of mutants, strains that were auxotrophic, metabolite analog sensitive, temperature sensitive, or unable to grow on citric acid as the sole carbon source. Since strain YB 3-122 is now available to yeast researchers, inositol-less death will be useful for the routine isolation of spontaneous mutants ofY. lipolytica.

Genetic regulation of isocitrate lyase in the yeastYarrowia lipolytica

Isocitrate lyase-less (Icl−) mutants were selected from genetically defined strains ofYarrowia lipolytica to investigate the regulation of synthesis of isocitrate lyase (ICL). Eighteen mutations were localized in the geneICLI, which is most probably the structural gene of ICL in this yeast. Atrans-acting positive element (ICL2) could be identified. This gene is not linked toICL1. One mutation was detected in anotherICL gene (ICL3) which is linked to geneICL1. This mutation lowered the ICL activity incis but not intrans position to geneICLI.