Subcellular distribution of enzymes in the yeast Saccharomycopsis lipolytica, grown on n-hexadecane,with special reference to the ω-hydroxylase

The subcellular localization of the ω-hydroxylase of Saccharomycopsis lipolytica was assessed by the analytical fractionation technique, originally described by de Duve C., Pressman, B.C., Gianetto, R., Wattiaux, R. and Appelmans, F., and hitherto little, if at all, applied to yeast. Protoplasts were separated in six fractions by differential centrifugation. Some of these fractions were further fractioned by density gradient centrifugation. The distribution of ω-hydroxylase and 15 other constituents chosen as possible markers of its subcellular membranes has been established. ω-Hydroxylase resulted in being bound to a membrane that containes also cytochrome P-450 and NADPH-cytochrome c reductase. This membrane clearly differs from five other subcellular entities. (1) Mitochondria were characterized by particulate malate dehydrogenase, particulate Antimycin A-insensitive NADH-cytochrome c reductase, oligomycin-sensitive and K⁺-stimulated ATPase pH 9. (2) Most if not all of the catalase and urate oxidase is peroxisomal. (3) Free ribosomes account for most RNA. (4) Nucleoside diphosphatase is for the first time reported in a yeast and appears to belong to an homogeneous population of small membranes. (5) The soluble compartment contains magnesium pyrophosphatase, alkaline phosphatase, 5′-nucleotidase and part of the NADH-cytochrome c reductase. Latent arylesterase and ATPase pH7 have an unspecific distribution. Alkaline phosphodiesterase I has not been detected.     

Importance of the methyl-citrate cycle on glycerol metabolism in the yeast Yarrowia lipolytica

A novel approach to trigger lipid accumulation and/or citrate production in vivo through the inactivation of the 2-methyl-citrate dehydratase in Yarrowia lipolytica was developed. In nitrogen-limited cultures with biodiesel-derived glycerol utilized as substrate, the Δphd1 mutant (JMY1203) produced 57.7 g/L of total citrate, 1.6-fold more than the wild-type strain, with a concomitant glycerol to citrate yield of 0.91 g/g. Storage lipid in cells increased at the early growth stages, suggesting that inactivation of the 2-methyl-citrate dehydratase would mimic nitrogen limitation. Thus, a trial of JMY1203 strain was performed with glycerol under nitrogen-excess conditions. Compared with the equivalent nitrogen-limited culture, significant quantities of lipid (up to ∼31% w/w in dry weight, 1.6-fold higher than the nitrogen-limited experiment) were produced. Also, non-negligible quantities of citric acid (up to ∼26 g/L, though 0.57-fold lower than the nitrogen-limited experiment) were produced, despite remarkable nitrogen presence into the medium, indicating the construction of phenotype that constitutively accumulated lipid and secreted citrate in Y. lipolytica during growth on waste glycerol utilized as substrate.     

Saccharomycopsis fibuligera and its applications in biotechnology

Saccharomycopsis fibuligera is found to actively accumulate trehalose from starch and the gene responsible for biosynthesis of trehalose has been cloned and its expression has been characterized. This yeast is also found to secrete a large amount of amylases, acid protease and β-glucosidase which have highly potential applications in fermentation industry. The genes encoding amylases, acid protease and β-glucosidase in S. fibuligera have been cloned and characterized. It is also used to produce ethanol from starch, especially cassava starch by co-cultures of Saccharomyces cereviase or Zymomonas mobilis.     

Development of the genetic map of the yeast Saccharomycopsis lipolytica

Summary Tetrad and random spore analyses have been used to further develop the genetic map of Saccharomycopsis lipolytica. Mutations in 23 new nuclear genes have been isolated. Eight genes have been located on linkage fragment 1, 4 on fragment 2, 2 on fragment 5 and 3 on fragment 6. Linkage fragments 3 and 4 have been shown to be linked, and this fragment now contains 12 markers. A tentative map of the linkage fragments 1 and 3 is presented (Fig. 1). Markers exhibiting possible centromere linkage have been identified. Interference estimates suggest that there is little interference in S. lipolytica.     

Regulation of extracellular protease production in Candida lipolytica

Production of extracellular protease by Candida lipolytica NRRL Y-1094 was depressed upon transfer to carbon-, nitrogen- or sulphur-free medium but not upon transfer to phosphorus-free medium. The protease activities produced under the three nutrient limitations had alkaline pH optima and similar substrate and inhibitor specificities. Any one of the following three conditions wass found to be sufficient for depression of extracellular protease: (1) “poor” carbon source, (b) cysteine intracellular pool below 0.5 μmol/g dry weight cells and (c) ammonia intracellular pool below 10 μmol/g dry weight cells. Thus, extracellular protease production in C. lipolyutica was subject to at least three different regulatory controls, carbon, sulphur and nitrogen repression. Intracellular cysteine and ammonia appeared to be the metabolic signals for sulphur and nitrogen repression, respectively. Anabolic glutamate dehydrogenase did not act as a regulatory protein mediating nitrogen repression. Exogenous protein had an inductive effect on extracellular protease production.     

Cyanide-resistant respiration in yeast. II. Characterization of a cyanide-insensitive NAD(P)H oxidoreductase

A single protein species isolated from yeast (preceding paper) which catalyzes the cyanide-resistant reduction of molecular oxygen by reduced pyridine nucleotides has been characterized using spectral and chemical criteria. This NAD(P)H:O₂ oxidoreductase is a metalloflavoprotein containing FMN as a prosthetic group and Cu²⁺ as the metal ion. The enzyme which is devoid of nonheme iron centers is inhibited by the chelators m-chlorohydroxamic acid; salicylhydroxamic acid, and bathophenathroline sulfonate. Of the many substrates tested only NADH and NADPH were found to be active with the latter having a higher apparent K_m (2.1 of 10^?5 vs 1.4 × 10^?6m). Stopped-flow kinetics established that the $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for these substrates was 0.41 and 0.32, respectively. It is suggested that the function of this enzyme is associated with the nonmitochondrial P-450 system. A tentative pathway of electron flow has been proposed to NADPH → FMN → Cu²⁺ → H₂O₂.     

A study of cyanide-insensitive respiration in the genus Dekkera and Brettanomyces

Dekkera intermedia and Brettanomyces custersii were shown to have a respiratory pathway resistant to cyanide, antimycin A, and azide. This respiration remained sensitive to salicylhydroxamic acid (SHAM). The "cyanide-resistant" respiration was induced mainly at the end of the growth phase and could reach 50% of total respiratory capacity. The mitochondrial "petite colony" mutation had no effect on this oxidation pathway. The presence of this respiration pathway in these strains constitutes a compensation mechanism for the reducing activity of acetaldehyde dehydrogenase. This alternate pathway would thus be a fundamental element of the Custer effect, a characteristic feature of these strains.     

Modeling hexanal production in oxido-reducing conditions by the yeast Yarrowia lipolytica

Hexanal produced by cells of a recombinant Yarrowia lipolytica yeast expressing the hydroperoxide lyase (HPL) from green bell pepper fruit was studied under oxido-reducing conditions using the reducing dithiotreitol and oxidizing potassium ferricyanide compounds. The combined effect of pH, linoleic acid 13-hydroperoxides concentration, temperature and oxido-reducing molecules on the hexanal production was studied. Significant positive effects for the hexanal production were found using high concentrations of hydroperoxides (100 mM, 30 g/L). Adding reducing molecules enhanced significantly hexanal production while the oxidizing molecules had an inhibitory effect. Combined effects of 13-hydroperoxides and dithiotreitol were optimised by a central composite design and a model was proposed. Finally, 6 mM (600 mg/L) of hexanal was obtained when 119 mM of 13-hydroperoxides (37 g/L) and 50 mM of dithiotreitol were introduced directly in the biocatalytic medium of the yeast Y. lipolytica.