Production of squalene by lactose-fermenting yeast Kluyveromyces lactis with reduced squalene epoxidase activity.

UNLABELLED: Utilization of yeast as squalene source for commercial use is limited by relatively high production costs. The ability of Kluyveromyces lactis to grow on cheap lactose-containing diary industry wastes could improve the economy of the production process. We therefore tested the potential of this yeast for squalene production. Accumulation of squalene was induced by partial inhibition of squalene epoxidase by a specific inhibitor terbinafine. Kluyveromyces lactis cultivated on glucose and lactose media showed similar growth sensitivity to terbinafine as Saccharomyces cerevisiae. The effect of terbinafine on neutral lipid pattern was tested at concentrations with low, moderate and strong growth inhibition (2·5, 5 and 7·5 µg ml(-1) , respectively). Compared to S. cerevisiae, treatment with subinhibitory terbinafine doses had a weaker effect on steryl ester levels and total ergosterol levels in K. lactis. Quantification of squalene levels in terbinafine-treated K. lactis cells revealed high accumulation of squalene particularly in cells treated with 7·5 µg ml(-1) terbinafine in lactose medium. Terbinafine treatment stimulated the development of lipid droplets as lipid storage organelles and this effect was different in K. lactis grown on glucose or lactose media. Present report is the first attempt to utilize lactose-fermenting yeast K. lactis for production of a high-value lipid and it proves squalene epoxidase as a promising target for squalene overproduction in this yeast. SIGNIFICANCE AND IMPACT OF THE STUDY: Squalene is a natural substance with wide applications in food, cosmetic and pharmaceutic industries. The suitability of lactose-fermenting yeast Kluyveromyces lactis for the production of squalene was tested in the study. Partial inhibition of squalene epoxidase by specific inhibitor terbinafine resulted in high accumulation of squalene in K. lactis grown on glucose or lactose comparable to values found in terbinafine-treated Saccharomyces cerevisiae. Our results prove that K. lactis is a promising micro-organism for genetic manipulations aimed at the production of squalene on industrial waste like whey as the growth substrate.

Regulation of phosphatidylglycerolphosphate synthase in aerobic yeast Kluyveromyces lactis.

The KlPGS1 gene encoding phosphatidylglycerolphosphate synthase (PGPS) is essential for the viability and multiplication of Kluyveromyces lactis. Regulation of PGPS expression by factors affecting mitochondrial development (C source, growth phase) and general phospholipid biosynthesis was followed. PGS1 mRNA levels were not altered as cells progressed from the exponential to the stationary phase of growth in glucose. PGS1 mRNA abundance was nearly identical in cells growing in a medium with glucose or glycerol as the sole C source during the different growth phases. Regulation of PGS1 expression by exogenous myo-inositol and choline was not mediated at the transcriptional level, the PGPS activity dropped to 70 % after myo-inositol addition.

Impact of mitochondrial function on yeast susceptibility to antifungal compounds.

Saccharomyces cerevisiae pell and crd1 mutants deficient in the biosynthesis of mitochondrial phosphatidylglycerol (PG) and cardiolipin (CL) as well as Kluyveromyces lactis mutants impaired in the respiratory chain function (RCF) containing dysfunctional mitochondria show altered sensitivity to metabolic inhibitors. The S. cerevisiae pell mutant displayed increased sensitivity to cycloheximide, chloramphenicol, oligomycin and the cell-wall perturbing agents caffeine, caspofungin and hygromycin. On the other hand, the pel1 mutant was less sensitive to fluconazole, similarly as the K. lactis mutants impaired in the function of mitochondrial cytochromes. Mitochondrial dysfunction resulting either from the absence of PG and CL or impairment of the RCF presumably renders the cells more resistant to fluconazole. The increased tolerance of K. lactis respiratory chain mutants to amphotericin B, caffeine and hygromycin is probably related to a modification of the cell wall.

Cross-resistance to strobilurin fungicides in mitochondrial and nuclear mutants of Saccharomyces cerevisiae.

In yeast the resistance to kresoxim-methyl and azoxystrobin, like the resistance to strobilurin A (mucidin) is under the control of both mitochondrial cob gene and the PDR network of nuclear genes involved in multidrug resistance. The mucidin-resistant mucl (G137R) and muc2 (L275S) mutants of Saccharomyces cerevisiae containing point mutations in mtDNA were found to be cross-resistant to kresoxim-methyl and azoxystrobin. Cross-resistance to all three strobilurin fungicides was also observed in yeast transformants containing gain-of-function mutations in the nuclear PDR3 gene. On the other hand, nuclear mutants containing disrupted chromosomal copies of the PDR1 and PDR3 genes or the PDR5 gene alone were hypersensitive to kresoxim-methyl, azoxystrobin and strobilurin A. The frequencies of spontaneous mutants selected for resistance either to kresoxim-methyl, azoxystrobin or strobilurin A were similar and resulted from mutations both in mitochondrial and nuclear genes. The results indicate that resistance to strobilurin fungicides, differing in chemical structure and specific activity, can be caused by the same molecular mechanism involving changes in the structure of apocytochrome b and/or increased efflux of strobilurins from fungal cells.

Role of the PDR gene network in yeast susceptibility to the antifungal antibiotic mucidin.

Yeast strains disrupted in the PDR1, PDR3, or PDR5 gene, but not in SNQ2, exhibited higher sensitivity to mucidin (strobilurin A) than did the isogenic wild-type strains. Different gain-of-function mutations in the PDR1 and PDR3 genes rendered yeast mutants resistant to this antibiotic. Mucidin induced PDR5 expression, but the changes in the expression of SNQ2 were only barely detectable. The results indicate that PDR5 provides the link between transcriptional regulation by PDR1 and PDR3 and mucidin resistance of yeast.

Growth of eukaryotic cells in relation to the structure of mitochondrial membranes and mitochondrial genome.

Viability of petite-negative yeast, such as Kluyveromyces lactis, is dependent on functional mitochondrial genome encoding essential components of both mitochondrial protein synthesizing system and oxidative phosphorylation. We have isolated several nuclear mutants impaired in mitochondrial functions that were unable to grow on non-fermentable carbon and energy sources. They were used for the isolation and molecular characterization of the three genes encoding apocytochrome c, apocytochrome c1 and the protein involved in the biogenesis of cytochrome oxidase. All cytochrome-deficient mutants were viable and did not survive the ethidium bromide mutagenesis. Petite-positive Saccharomyces cerevisiae requires intact mitochondrial genome when its phosphatidylglycerolphosphate synthase was inactivated due to mutation in the PEL1 gene. Using PEL-lacZ fusion genes it was demonstrated that Pel1p is a mitochondrial protein (expressed in response to myo-inositol and choline). The pel1 mutant was deficient in phosphatidylglycerol (PG) and cardiolipin (CL) and its rho-/rho0 mutants grew extremely slowly on complex medium with glucose. Under the same conditions the growth rate of the crd1 rho- double mutants was similar to that of its parent crd1 mutant deficient in cardiolipin synthase and accumulating PG. The results demonstrate that the petite negativity in yeast is not dependent on an intact respiratory chain or functional oxidative phosphorylation. The presence of the negatively charged PG or CL seems to be essential for the maintenance of specific mitochondrial functions required for the normal mitotic growth of yeast cells.

Phosphatidylglycerolphosphate synthase encoded by the PEL1/PGS1 gene in Saccharomyces cerevisiae is localized in mitochondria and its expression is regulated by phospholipid precursors.

The PEL1/PGS1 gene of the yeast Saccharomyces cerevisiae is essential for the viability of rho-/rho degrees mutants and the normal cardiolipin content of cells. The PEL1-GFP fusion gene has been found to complement the pel1/pgs1 mutation and its fluorescent protein was localized to mitochondria similarly to the beta-galactosidase activity of a protein encoded by the PEL1-lacZ fusion gene. The expression of the PEL1-lacZ reporter gene was repressed in cells grown in the presence of inositol and choline, reduced in the ino2 and ino4 strains, but constitutive in the opi1 null-mutant strain. The results demonstrate that Pel1p, playing a vital role in cells impaired in the mitochondrial DNA, is localized in the mitochondria and expressed in response to inositol and choline.

The pel1 mutant of Saccharomyces cerevisiae is deficient in cardiolipin and does not survive the disruption of the CHO1 gene encoding phosphatidylserine synthase.

Cells of the pel1 mutant of Saccharomyces cerevisiae were found to contain an extremely low content of cardiolipin, a decreased level of phosphatidylcholine and an increased level of phosphatidylinositol. Disruption of the PEL1 gene in cells containing a null mutation in the CHO1 gene was lethal. Despite its putative functional homology with CHO1, the overexpression of the PEL1 gene in the cho1 null mutant did not restore the wild-type properties of the transformed cells and failed to stimulate the incorporation of L-[3-3H]serine into total lipids of the intact yeast cells.

Energy-dependent mitochondrial mutagenicity of antibacterial ofloxacin and its recombinogenic activity in yeast.

Ofloxacin, a specific inhibitor of bacterial topoisomerase II, is known to inhibit the growth of yeast cells and to induce rho- mutants in the yeast S. cerevisiae. The frequency of ofloxacin-induced petite mutants under non-growth conditions was found to be strongly diminished when the cells were depleted in intramitochondrial ATP. Under optimal conditions of mitochondrial mutagenesis the drug induced mitotic recombination and reverse mutation in diploid strains but failed to cure either killer plasmids or the 2 microns DNA of dividing cells. The sensitivity to ofloxacin of the strains deficient in the DNA strand-break repair pathway (rad52) was significantly higher then that of the wild-type strains and of the mutants deficient in excision or mutagenic DNA repair. The results are compatible with the idea that the cytotoxic and genetic activity of ofloxacin in yeast probably results from the inhibited DNA ligation function of topoisomerase II creating DNA breaks that are reparable through the recombination repair pathway.

Induction of respiration-deficient mutants in Saccharomyces cerevisiae by chelerythrine.

Chelerythrine and sanguinarine, two structurally related benzo/c/phenanthridine alkaloids, prevented growth of yeast cells in medium containing either glucose or non-fermentable carbon sources. At concentrations permitting growth of the yeast Saccharomyces cerevisiae, chelerythrine, but not sanquinarine, induced cytoplasmic respiration-deficient mutants. The petite clones that were analysed exhibited suppressiveness and contained different fragments of the wild-type mitochondrial genome.

High-level resistance to cycloheximide resulting from an interaction of the mutated pdr3 and cyh genes in yeast.

In addition to pdr3-1, the S. cerevisiae nuclear pleiotropic drug resistance mutant 2D was found to contain another recessive nuclear mutation, cyh, conferring specific resistance to cycloheximide only. The cycloheximide resistance level due to either the pdr3-1 or the cyh mutation alone was low and was not altered by the ogd1 mutation which increased the physiological acidification of the culture. When pdr3-1 and cyh mutations occurred simultaneously in the haploid yeast strain their interaction was synergistic and resulted in high-level resistance to cycloheximide.

Ofloxacin induces cytoplasmic respiration-deficient mutants in yeast Saccharomyces cerevisiae.

Ofloxacin, a new quinolone with potent antibacterial activity, was also found to be effective against yeast. At relatively high concentrations, and at mild alkaline pH, ofloxacin inhibited the growth of yeast cells in medium containing glucose, and prevented growth on glycerol, as carbon and energy source. The cells growing in the presence of ofloxacin exhibited abberrantly budded forms, lost their viability and many of them converted to cytoplasmic respiration-deficient mutants. Induction of mutants was also observed under non-growing conditions. The petite clones analysed exhibited suppressiveness and contained different fragments of the wild-type mitochondrial genome.