Genetic studies on the yeast Saccharomycopsis lipolytica Inactivation and mutagenesis

Spontaneous mutants of Saccharomycopsis lipolytica were selected and partially characterized. Several antibiotics and antimetabolites were used for selection of spontaneous resistant mutants from Saccharomycopsis lipolytica. The frequencies of such mutants were mainly arranged between 1 × 10^?7 and 5 × 10^?6 mutants per cell. But one class of glucosamine resistant mutants (GAM^RA) occurred more frequently. Among the resistant mutants different types of dominant and recessive resistant mutants could be observed. UV light was used for inactivation of cells and induction of mutants from S. lipolytica. Comparing four haploid strains only small differences were detected in sensitivity to UV light. UV light at a dosage of 135 J/m² was applied to increase the mutant frequencies in three haploid strains. Besides auxotrophic, temperature sensitive and colony morphology mutants, some new mutant types like small colony forming mutants, red-brown coloured mutants, allylalcohol, glucosamine, 2-deoxyglucose or nystatin resistant mutants, hitherto not described for S. lipolytica, were isolated and partially characterized.     

Genetic and biochemical studies of N-alkane non-ultilzing mutants of Saccharomycopsis lipolytica

Summary Alkane non-utilizing mutants of the yeast Saccharomycopsis lipolytica were induced by ultraviolet light. Thirtyfour of the mutants were found to be alkaline-negative and fatty acid-positive (Phenotypes A and C) indicating a defect in n-alkane uptake or in hydroxylase complex activity. The hydroxylase complex is a microsomal aggregate composed of the first three enzymes of n-alkane catabolism. Leaky and non-mating mutants were eliminated leaving 21 mutants which were analyzed genetically. All 21 of the mutations showed a 1:1 pattern of segregation indicating they are chromosomal and all but one were recessive. Analyses of inter-mutant complementation and recombination showed that the 21 mutations represent 18 genes.Several of the mutants had pleiotropic phenotypes in addition to alkane non-utilization. These phenotypes included a loss of mating function, an inability to sporulate, a changed colony and cellular morphology, osmotic sensitivity and a lack of extracellular protease.The hydroxylase complex activities of mutants and wild type were assayed in cell-free extracts prepared by protoplast lysis. A small amount of detergent was necessary for the extraction of hydroxylase complex activity. The hydroxylase complex was inducible by n-decane and incubation was complete by 6 h. Hydroxylase complex activities in the mutants varied from 2.8% to 46.5% of wild type. The hydroxylase complex activities of two temperature sensitive mutants were as stable as wild type at the non-permissive temperature. These mutants showed temperature sensitive induction suggesting that the uptake of n-alkanes is temperature dependent in these strains.     

Isolation and characterization of mitochondrial DNA from the alkane yeast Saccharomycopsis lipolytica

Summary Mitochondrial (mt) DNA of the alkane yeast, Saccharomycopsis lipolytica, was isolated. Its buoyant density in CsCl was found to be of 1.687 g/cm³, indicating a GC content of 27.5% and its melting point T_m = 79.5 °C, indicating a GC content of 24.9%. The corresponding values for nuclear (n) DNA, are 1.709 g/cm³ (GC: 49.5%) and T_m = 90.5 (GC: 51.7%) respectively. Electron microscopy revealed that mtDNA has a circular structure with a contour length of about 14.5 µm corresponding to 45.5 kb per molecule. The size estimated from restriction analyses performed with 7 endonucleases was 48.35 kb/molecule. A restriction map was constructed, using the cleavage data of 4 endonucleases.     

Analysis of genetic markers in new breeding stocks of the yeast Saccharomycopsis lipolytica

For development of breeding stocks of S. lipolytica for genetic investigations we isolated and identified several auxotrophic mutants. After selection of strains with normal meiotic segregation pattern for the markers metA, lysA, argA, leuA, uraA, hisA it was shown that the loci metA and lysA and the loci argA and leuA are linked, crossing over frequencies are 25 ± 0.9% and 30 ± 1.2%, respectively. We identified several complementation groups. The marker uraA is allel to ura3 from strains of the Donner Laboratory (USA). These results are the basis for selection of special mutants for forthcoming investigations. The existence of linkage fragments makes it possible to use these strains as tester strains for genetic analysis.     

Development of breeding stocks of the yeast Saccharomycopsis lipolytica by methods of moderate inbreeding

Sporulation parameters of genetically labelled strains, derived from a wild strain of the alkane-utilizing yeast Saccharomycopsis lipolytica were improved by a breeding program using brother-ssister crosses. Sporulation frequency, the number of four-spored asci and viability of ascospores could be significantly enhanced. To date a number of genetically well-defined strains is available that have good sporulation parameters and show a 1:1 segregation pattern of markers suitable for genetic analysis.     

Genetic analysis of sexual hybrids and protoplast fusion products of the yeast Saccharomycopsis lipolytica

Protoplast fusion of genetically marked Saccharomycopsis lipolytica strains was carried out succesfully. Mitotic segregation was induced to prove the presence of parental markers in fusion products. Methylbenzimidazol-2-yl-carbamate has been shown to induce efficiently mitotic segregation in this yeast. The segregation pattern of genetic markers in a sexual hybrid was compared to that of a parasexual hybrid.     

Gene organization of the mitochondrial DNA of yeasts: Kluyveromyces lactis and Saccharomycopsis lipolytica

Summary Restriction fragment maps have been constructed for the mitochondrial DNA from two petitenegative yeasts, Kluyveromyces lactis and Saccharomycopsis lipolytica (Candida lipolytica). On these circular genomes, we localized the sequences homologous to the S. cerevisiae mtDNA fragments carrying known genes. The arrangement of genes for ATPase subunit proteins, ribosomal RNA and 4S RNA shows a common feature with respect to S. cerevisiae mitochondrial genome.Abbreviations bp base pairs - mtDNA mitochondrial DNA - tRNA transfer RNA - rRNA ribosomal RNA     

Gemeinsame Verwertung von Glucose und n-Alkanen bei der Citronensäuresynthese durch Saccharomycopsis lipolytica

Fermentations for the overproduction of citrate and isocitrate with S. lipolytica in media containing both glucose and n-alkanes as mixed C-source have been performed. Biomass and product yields strongly depend on the C-source of the inoculation culture. If the inoculation culture had been taken from media containing glucose as sole C-source both glucose and n-alkanes were utilized for cell growth in the main culture whereas only glucose was utilized if the inoculation medium contained only n-alkanes. For idiophasic citrate and isocitrate production both glucose and n-alkanes were consumed independently of the C-source of the inoculum but that C-source was preferentially utilized which has been the C-source of the inoculation culture. These findings are reflected by the activities of the isocitrate lyase and the pyruvate carboxylase, respectively. In S. lipolytica both anaplerotic pathways are coexisting but the C-source of the inoculation culture determines the level of the specific activities even if the ratio of the cell-mass of the inoculum to the cell-mass of the main culture at the end of the growth phase is about 1:35.     

Einfluß des Sauerstoffpartialdrucks auf die Citronensäuresynthese durch Saccharomycopsis lipolytica aus n-Paraffinen

The dependence of growth formation of citric acids (citrate: isocitrate = 1:1) on oxygen partial pressure of an alkane utilising yeast Saccharomycopsis lipolytica was investigated. During growth oxygen corresponds to a MICHAELIS-MENTEN-kinetics (K_s = 2.0 · 10^?6 m). The respiration quotient RQ for a dissolved oxygen concentration in the range of 10–100% (air saturation) is 0.46 ± ± 0.04 The phase of product formation is characterized by 3 sections. Immediately after N-exhaustion the cell activities are the highest. They decline during the first 30 hours of production. Besides the production of reserve material in this first section the highest production rate for citrate and isocitrate is observed. The rate of citric acid production depends on the oxygen partial pressure and is governed by MICHAELIS-MENTEN-kinetics. The specific production rate and the rate of oxygen consumption correspond to K_s-values of 4.0 · 10^?5 and 3.3 · 10^?5 M, respectively The RQ-value declines to a constant value of 0.23 ± 0.02 and is not influenced by oxygen partial pressures in the range of 10–100% (related to air saturation) During the second section cell activities remain nearly constant for about 100 h. Due to this constancy the following equation could be derived: 14 O₂ + C₁₅H₃₂ 2 C₆H₈O₇ + 3 CO₂ - ₈ H₂O. In the third section the cell activities decline again.     

Identification of mutations preventing n-hexadecane uptake among 26 n-alkane non-utilizing mutants of Yarrowia (Saccharomycopsis) lipolytica

Summary Genetic analyses of n-alkane non-utilizing mutants of the yeast Yarrowia (Saccharomycopsis) lipolytica were continued. By analyses of inter-mutant complementation and recombination a total of 26 genetic loci have been identified. Mutations representing these loci have phenotypes characteristic of defects in substrate uptake or in one or more of the enzymatic activities making up the hydroxylase complex. Tests of ¹⁴C n-hexadecane uptake by a set of alkane-negative mutants representing the 26 loci show that 16 of the mutations cause a significant reduction in n-alkane uptake. N-alkane uptake by Y. lipolytica is shown to be inducible and to be inhibited by the metabolic poisons 2–4 dinitrophenol and KCN. The latter observation indicates that n-alkane uptake of Y. lipolytica is due to active transport.     

Alcohol dehydrogenase (ADH) in yeasts II. NAD+-and NADP+-dependent alcohol dehydrogenases in Saccharomycopsis lipolytica

In Sm. lipolytica one NAD⁺-dependent and three NADP⁺-dependent alcohol dehydrogenases are detectable by polyacrylamide gelelectrophoresis. The NAD⁺-dependent ADH (ADH I), with a molecular weight of 240,000 daltons, reacts more intensively with long-chain alcohols (octanol) than with short-chain alcohols (methanol, ethanol). The ADH I is not or only minimally subject to glucose repression. Besides the ADH I band no additional inducible NAD⁺-dependent ADH band is gel-electrophoretically detectable during growth of yeast cells in medium containing ethanol or paraffin. The ADH I band is very probably formed by two ADH enzymes with the same electrophoretic mobility. The NADP⁺-dependent alcohol dehydrogenases (ADH II–IV) react with methanol, ethanol and octanol with different intensity. In polyacrylamide gradients two bands of NADP⁺-dependent ADH are detectable: one with a molecular weight of 70,000 daltons and the other with 120,000 daltons. The occurrence of the three NADP⁺-dependent alcohol dehydrogenases is regulated by the carbon source of the medium. Sm. lipolytica shows a high tolerance against allylalcohol. Resistant mutants can be isolated only at concentrations of 1 M allyalcohol in the medium. All isolates of allylalcohol-resistant mutants show identical growth in medium containing ethanol as the wild type strain.     

Biochemical studies on sporulation in blue-green algae I. Glycogen accumulation

Glycogen accumulation in vegetative cells of Anabaena sp. is demonstrated to be a light-dependent process. No glycogen accumulation is found in dark or in cultures supplemented with 10^?5 M DCMU in light. Large quantities of glycogen acccumulate in cells undergoing sporulation and the amount increased with the onset of maturation of spores.     

Biochemical studies on sporulation in blue-green algae II. Factors affecting glycogen accumulation

Inorganic nitrogen sources like nitrate, nitrite enhanced sporulation and glycogen accumulation in Anabaena sp. but ammonium chloride neither influenced sporulation nor glycogen accumulation. Acetate and citrate also stimulated early sporulation and glycogen level was higher over nitrogen free control. Nitrogen and carbon sources in combination proved to be useful in inducing early sporulation and increased content of glycogen. Phosphate and calcium also affected glycogen accumulation significantly, although, the sporulation was found to be of the same order as in nitrogen free medium. Sulphate initiated early sporulation, the mechanism of which is not known.     

Clinical, microbiological, and experimental animal studies of Candida lipolytica.

Candida lipolytica was recovered from six patients in three different clinical centers. The index isolate caused a persistent fungemia with catheter-associated Candida thrombophlebitis, the second isolate was from a polymicrobial sinusitis, and the remaining four isolates were involved in tissue colonization. These and 20 other isolates were consistent in their morphological and physiological characteristics. All formed true hyphae and blastoconidia on cornmeal-Tween 80 agar and all assimilated glucose, glycerol, and erythritol. In a murine model of disseminated candidiasis, the index isolate that caused clinical fungemia caused no mortality and produced only two lesions on a kidney, as determined at necropsy. The nine isolates selected for in vitro antifungal susceptibility studies had intermediate susceptibilities to amphotericin B but were susceptible to ketoconazole. We conclude that C. lipolytica is a weakly virulent pathogen which may require an intravascular foreign body to cause fungemia.     

Substructural study of sporogenesis in Streptomyces griseus

A Streptomyces griseus strain deficient in the formation of aerial mycelium and arthrospore development (Amy^?) was studied by electron microscopy and compared with the sporulating parental strain (Amy⁺). The investigations were performed with colonies grown on solid medium. The substructural characteristics of the essential events of sporogenesis could clearly been demonstrated in the aerial mycelium of the Amy⁺ colonies. The mycelium of the surface region of the Amy^? colonies showed altered features. The most pronounced alteration was the absence of the surface sheath, normally present on the aerial hyphae of the parental strain. The cross wall type II, characteristic of sporogeneous hyphae, was not discernible in the Amy^? hyphae. Some substructural features resembling the essential events of normal sporogenesis were evident in the Amy^? strain, albeit diminished and interfered with by abnormalities. The resulting propagative cells were of different size and feature.     

Biochemical studies on sporulation in blue-green algae. III. Effect of amino acids on glycogen accumulation

The effect of 21 amino acids was studied on glycogen accumulation during sporulation in the blue-green alga Anabaena sp. All the amino acids enhanced the initial level of glycogen on the 4th day. The maximum amount of glycogen, on the 20th day, was noticed from l-methionine, l-tyrosine, glycine, and l-histidine supplemented cultures. Others like l-serine, l-valine, l-asparagine, dl-alanine, l-glutamic acid, l-phenylalanine, l-aspartic acid, l-arginine, and dl-lysine come next in the order. On the other hand, l-cysteine and l-cystine, although upto the 16th day they exhibited higher values of glycogen, did not show much variation in glycogen content over nitrogen-free medium. Except in these two amino acids, in all others initiation of sporulation occurred on the 4th day, resulting in free spores on the 8th day. But in case of l-serine, l-asparagine, l-isoleucine, and l-phenylalanine free spores were noticed only on the 12th day while in dl-lysine they were seen on the 16th day. l-cysteine and l-cystine supplemented media showed free spores on the 20th day as in nitrogen-free control.     

Untersuchungen zur Regulation der Enzyme des Glyoxylatzyklus bei Saccharomycopsis lipolytica I. Einfluß der C-Quelle auf die Aktivität von Isocitratlyase und Malatsynthase

Comperative studies on the activities of isocitrate lyase (ICL) and malate synthase (MS) were carried out with Saccharomycopsis lipolytica incubating the yeast on media with different carbon sources. When cells were incubated in minimal medium with glucose, the activities of both enzymes were very low. In contrast, in minimal medium with acetate enhanced enzyme activities could be demonstrated. It is probably that the synthesis of ICL is repressed in presence of glucose. Furthermore the activity of ICL was inhibited by tricarboxylic acid cycle intermediates like succinic acid and oxalacetic acid. It was concluded that the syntheses of enzymes are derepressed. When cells of Sm. lipolytica were incubated in minimal medium with acetate, a high enzyme activity is evident. Synthesis of ICL on acetate was inhibited by cycloheximid and actinomycin D. The results were discussed comparing them with data obtained from other organisms.