It is acknowledged that some microbes have interrupted distributions, yet these distributions have rarely been correlated with environmental variables. The wild biogeography of the fermenting yeasts Saccharomyces cerevisiae and Saccharomyces paradoxus are explored in this study, considering multiple environmental variables as potential effectors of each species' geographical distributions. I demonstrate that summer temperatures predict maximum species distribution limits for both S. paradoxus and S. cerevisiae on oak bark, and that S. paradoxus is more likely to be isolated from larger, older trees. Modelling these data predicts a generally denser southern European population of S. paradoxus, with S. cerevisiae being scarce on oak bark throughout Europe. It was not possible to recover ancient Saccharomyces DNA sequences from samples of sub-fossilized oaks, from Greco-Roman and North African amphora residues, or from North African 6th-14th Century pottery residues, which may be a consequence of the low concentration of these species in comparable modern environmental samples. Even from air dried breads and recent wines, Saccharomyces aDNA was not recovered as part of this study, although ancient DNA sequences from plants and other yeasts were identified in other samples via the same methods. Any future recovery of ancient Saccharomyces sequences may therefore be challenging. Novel plant sequences possibly belonging to the Musaceae family and Pinus genus were identified from 6th-14th century AD North African pottery; as well as a Vicia-like DNA sequence from a 13th-12th century BC North African amphora.

Experiments have been conducted to identify the fouling mechanism during the crossflow filtration of a model yeast fermentation broth of Vinyl Acetate particles suspended in a Bovine Serum Albumin (BSA) solution. These have been conducted with filter modules, to obtain quantitative data for the rate and the extent of flux decline due to membrane fouling, and with filter coupons, to obtain quantitative data for the build up of the fouling layer with each individual system and the mixed system. The data from the individual systems have been analysed and then used to determine their fouling mechanisms; this information has been used to predict the fouling mechanism for the mixed system. Finally, this prediction has been compared to the actual fouling mechanism determined by analysis of the mixed system data. For the model particulate suspension, the fouling was due to the build up of a cake layer, as with dead end filtration; however, fouling was limited by membrane scouring. For the model macromolecular solution, a four part fouling mechanism was identified: initially aggregates formed within the pores; the concentration at the membrane surface increased until protein came out of solution as strands; the strands disappeared causing increased aggregation in the pores; finally, a mesh formed on the membrane surface. For the mixed system, fouling was due to the formation of a particle cake on the membrane surface with protein aggregates forming in the pores. The fouling kinetics could be predicted by considering the results from the individual systems; however, the fouling mechanism could not be predicted without using visualisation experiments due to the interactions between the particles and the macromolecules.

The brewing industry constantly experiences problems in trying to maintain the quality of beer produced. Unfavourable conditions during fermentation may alter the performance of the yeast strain Saccharomyces cerevisiae, resulting in a "poor" end-product. It has been established that high concentrations of extracellular ethanol, when added to the fermentation medium inhibit yeast activity. It has been recently suggested that increased carbon dioxide pressure could inactivate the yeast activity adding to further brewing problems. The aim of this study was to investigate the effect of extracellular carbon dioxide pressure and ethanol addition, on yeast performance when added to a fermentation medium, and to establish whether an inhibitory relationship existed between ethanol and carbon dioxide pressure, when combined and added to the fermentation medium. Dissolved C0₂ in the medium, medium pH and substrate utilisation were analysed daily during a fermentation, as were membrane fatty acid composition. These parameters were used to assess the effect of ethanol and carbon dioxide on the yeast performance and consequently the final end-product. Supplementing the medium with extracellular ethanol, even as low as 5%, was shown to inhibit yeast performance during fermentation. This effect was even more marked as the ethanol concentration was increased, with almost total inhibition of yeast activity occuring after the addition of 15% ethanol (v/v). A similar effect was observed when elevated C0₂ pressures were applied to the medium, and although low C0₂ pressures initially induced the synthesis of saturated yeast membrane fatty acids, elevated C0₂ pressures (greater than 1,0 atm.) was shown to follow a similar inhibitory trend, if not as dramatic, as ethanol. A combination of both ethanol and C0₂ pressure showed a further increase in the level of yeast inhibition, although the low C0₂ pressure appeared to initially inhibit the toxicity of ethanol on the yeast. Increasing the levels of the C0₂/ethanol treatment (1,0 atm.), showed a synergistic effect on yeast performance. The results of this study indicate that both extracellular ethanol and carbon dioxide do appear to inhibit yeast performance and affect membrane fatty acid composition of the cells by inhibiting the synthesis of the respective fatty acid. This affect has a significant bearing on the general metabolism of the yeast cell.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ABC transporter family protein that acts as an ion channel. Mutations in CFTR cause the most common genetic disease in Caucasian populations, cystic fibrosis (CF). The high-resolution X-ray crystal structure of CFTR is now needed to aid the design of CFTR-targeted drugs for CF treatment and also to elucidate the molecular mechanisms behind its unique function as an ATP-ligand gated ion channel. However, until now, such structural studies have been severely limited by the lack of sufficient quantities of purified full-length CFTR protein. This thesis reports the novel over-expression and purification of milligram quantities of the chicken orthologue of CFTR protein from a Saccharomyces cerevisiae (yeast) expression system. A green fluorescent protein (GFP) tag fused to the CFTR C-terminus allowed rapid detection of the protein throughout the purification procedure. CFTR was expressed under an inducible promoter and appeared localised at, or near to, the plasma membrane, where it represented around 1 % of total protein after isolation in yeast microsomes. CFTR was solubilised from microsomes and purified using the detergents dodecylmaltoside (DDM) and lyso-phosphatidyl glycerol (LPG), by nickel affinity and size exclusion chromatography (SEC) to yield 1-2 mg of CFTR protein per 18 L fermentation culture. CFTR thermal stability was probed using fluorescent measurements to reveal a two-state cooperative unfolding transition around 40 °C for the DDM-purified protein, but no such transition was observed for the LPG-purified material. Light scattering and electron microscopy techniques revealed that, in LPG, CFTR was a homogenous population of monomeric particles around 60-Å in length that were soluble up to 8 mg/ml protein concentration. In DDM, CFTR was only soluble below 0.4 mg/ml protein concentration where is existed as a very heterogenous population of different sized amorphous particles, including dimeric particles around 180-Å in length. The DDM-purified CFTR protein could be crystallised as monomers in two-dimensional (2D) crystals with similar lattice parameters to 2D crystals of CFTR purified from mammalian cells. The ATPase activity of DDM-purified and reconstituted CFTR was similar to already published rates, at around 13 nmol Pi/min/mg integrated over a reaction time of 60 min, with an apparent affinity Km for ATP of 0.14 mM. Such a low ATPase rate compared to other ABC transporters may be due to the observed rapid run-down of activity with time and correlation with published CFTR channel gating kinetics. CFTR showed reduced ATPase activity after purification in LPG, suggesting a structural destabilisation in this detergent. The protocols presented here can now be used to provide sufficient quantities of purified CFTR protein for novel biochemical and biophysical studies. The tendency of CFTR to aggregate in a mild detergent remains a major obstacle towards 3D crystallisation trials and a high-resolution structure.

In the near future, biomass-derived energy is predicted to substantially complement that generated from petroleum. However, certain types of biomass employed as substrates in the microorganism-mediated production of renewable fuelethanol contain significant amounts of the recalcitrant hexose sugar galactose. The consumption of galactose in hexose sugar-fermenting yeasts is often delayed with respect to other sugars, such as glucose and mannose, because of an intrinsic preference for carbon sources requiring less energy in the preparatory reactions preceding glycolysis. This work comprised the search for, and characterization of anethanologenic yeast capable of efficiently assimilating galactose. Screening experiments conducted with wild-type Saccharomyces cerevisiae strains identified one isolate (Y-1528) exhibiting exceptionally fast galactose fermentation. The absence of conventional glucose repression, including a preference for galactose as carbon source and notable delays in the utilization of glucose and mannose, was demonstrated in mixed sugar fermentations. Endogenous extracellular glucose was observed during double sugar fermentations of galactose and mannose. This glucose was traced to supplied galactose by radioisotope labeling, suggesting involvement of UDP-galactose 4-epimerase in the responsible reaction mechanism(s).Sub-cellular fractionation was employed in an attempt to ascertain enzyme localization in Y-1528. Fermentations of lignocellulosic substrate mixtures by Y-1528 illustrated better performance than that accomplished by a reference yeast strain, and again showed a preference for galactose. Mixed cultures of Y-1528 and the same reference strain demonstrated accelerated hexose sugar consumption, and no detrimental effects from competition, during synthetic and lignocellulosic substrate fermentations. Glucose repression was absent in mixed culture fermentations. Fermentations of synthetic sugar mixtures augmented with lignocellulosic inhibitory compounds showed Y-1528 to have better performance than a reference yeast strain, despite a global detrimental effect relative to inhibitor-free fermentations. Cell recycle batch fermentations of spent sulfite liquor illustrated the toxic effect of the hardwood variant, as well as a net loss of performance from all strains tested. Y-1528 was taxonomically confirmed as S. cerevisiae. UDP-galactose 4-epimerase chromatographic purification was unsuccessful, but a partial sequence of the enzyme, showing complete identity with type sequence, was obtained by electrophoretic separation, liquid chromatography, and mass spectrometry. A significantly mutated UDP-galactose 4-epimerase gene was successfully sequenced. / Forestry, Faculty of / Graduate

The crystal structure of yeast (Saccharomyces cerevisiae) iso-1- cytochrome c has been determined through molecular replacement techniques, and refined against X-ray diffraction data in the resolution range 6.0-1.23 Å to a crystallographic R-factor of 0.192. The yeast iso-1-cytochrome c molecule has the typical cytochrome c fold, with the polypeptide chain organized into five α-helices and a series of loops which serve to enclose almost completely the heme prosthetic group within a hydrophobic pocket Comparison of the structures of yeast iso-1-, tuna and rice cytochromes c shows that the polypeptide backbone fold, intramolecular hydrogen bonding, conformation of side chains and particularly packing within the heme crevice of protein groups against the heme moiety are very similar in the three proteins. Significant structural differences among the three cytochromes c can be explained by differences in amino acid sequence. X-ray crystallographic techniques have also been used to study the effect of single-site amino acid substitutions at Phe82 and at Arg38 in iso-1-cytochrome c. The structures of the various variant iso-1-cytochromes c have been determined at nominal resolutions in the range 2.8 to 1.76 Å. Conspicuous structural perturbations in the neighborhood of the substituted side chain are evident in all of the variant proteins. In wild-type iso-1-cytochrome c, the phenyl ring of Phe82 is positioned adjacent and approximately parallel to the heme group, and occupies a non-polar cavity within the heme crevice. In the Ser82 variant, a channel extending from the surface of the molecule down into the heme crevice is created. In the Gly82 variant, the polypeptide backbone has refolded into the space formerly occupied by the phenyl ring of Phe82. Steric conflicts prevent both the phenolic ring of Tyr82 and the side chain of Ile82 from being completely accommodated within the pocket normally occupied by a phenyl ring. Substitution of alanine at position 38 causes a slight reorganization of the hydrogen bonding network in which Arg38 normally participates, and also exposes to external solvent a normally buried propionic acid group of the heme. The altered functional properties of the position 82 variant proteins have been interpreted with respect to the observed structural perturbations. The drop in reduction potential, most notably for the Ser82 and Gly82 variants, can be explained by the elevated heme environment polarity arising from the increased access of solvent or polar protein groups to the heme pocket The reduced stability of the heme crevice, as indicated by lowered pKa's for alkaline isomerization, is likely due to the disruption of stabilizing packing forces formed by the Phe82 phenyl ring within its hydrophobic cavity. The lowered activity, in comparison to the wild-type protein and the Tyr82 variant, for electron transfer with Zn+-cytochrome c peroxidase is attributed to the loss of an aromatic group positioned adjacent to the heme group. The altered surface topography of the variant proteins (particularly the Gly82, Tyr82 and Ile82 variants) may further hinder productive complex formation between cytochrome c and its redox partners. These results suggest that the invariant Phe82 contributes in at least three ways to the proper functioning of cytochrome c. It has an important structural role in maintaining the integrity of the heme crevice and in establishing the appropriate heme environment The phenyl ring of Phe82 may also be required for efficient movement of an electron to and from the heme of cytochrome c. Finally, Phe82 may have a role in forming intermolecular interactions with enzymic redox partners of cytochrome c. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Diploid organisms including yeast, most animals, and humans, typically carry two copies of each gene. Variation above or below two copies can however sometimes occur. When gene copy number reduction from two to one causes a disadvantage, that gene is considered haploinsufficient (HI). In the first part of my work, I identified associations between Saccharomyces cerevisiae gene properties and genome-scale HI phenotypes from earlier work. I compared HI profiles against 23 gene properties and found that genes with (i) greater numbers of protein interactions, (ii) greater numbers of genetic interactions, (iii) greater gene sequence conservation, and (iv) higher protein expression were significantly more likely to be HI. Additionally, HI showed negative relationships with (v) cell cycle regulation and (vi) promoter sequence conservation. I exploited the aforementioned associations using Linear Discriminant Analysis (LDA) to predict HI in existing data and guide experimental identification of 6 novel HI phenotypes, previously undetected in genome-scale screenings. I also found significant relationships between HI and two gene properties in Schizosaccharomyces pombe, relationships that hold despite the lack of conserved HI between S. cerevisiae and Sz. pombe orthologue gene pairs. These data suggest associations between HI and gene properties may be conserved in other organisms. The relationships and model presented here are a step towards understanding HI and its underlying mechanisms. Increases in copy number can occur through gene duplication. When duplication produces two functional gene copies, both experience relaxed selection and rapid mutation. This sometimes leads to interesting evolutionary events such as gain of novel function (neofunctionalisation). Previous work shows an ancient ancestor of S. cerevisiae underwent whole genome duplication (WGD) followed by massive redundant gene loss. Interestingly some duplicate pairs show retention of both copies, including the pair TUB1 and TUB3. Existing sequence data shows that TUB3 has experienced a very high rate of evolution post-WGD, suggesting neofunctionalisation. To characterise TUB3, I have carried out experiments measuring fitness effects of varying TUB1, TUB2 and TUB3 copy number across many environments. In ethanol media, some TUB1 and TUB3 null mutants interestingly show severe defects. Other data suggest stress response, ethanol tolerance, protein degradation and/or regulatory roles, which may involve the regulatory Snf1p protein kinase complex.

Protoplasts were prepared from two yeast strains P. rhodozyma (ATCC 24202) and K. fragilis (ATCC 8455). Protoplasts prepared from P. rhodozyma were facilitated by prior growth of the cells in a media containing S-(2-aminoethyl)-L-cysteine. Protoplasts from these two yeast genera were fused either by the use of electrofusion or polyethylene glycol treatment. Stable carotenoid producing cell lines were selected by growth at 30°C on yeast nitrogen base plus galactose. Selected single fusants display taxonomic characteristics common to both genera with a cellular morphology and a carotenoid composition similar to that of P. rhodozyma. / Land and Food Systems, Faculty of / Graduate

Orientador: Adilma Regina Pippa Scamparini / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-03T16:03:30Z (GMT). No. of bitstreams: 1 Maldonade_IrianiRodrigues_D.pdf: 4259808 bytes, checksum: a80eab15ad28ff36edff81d33fd716be (MD5) Previous issue date: 2003 / Resumo: Este trabalho teve como objetivo isolar e selecionar leveduras produtoras de carotenóides de ecossistemas brasileiros. As leveduras pigmentadas foram isoladas de amostras de solos, flores, folhas, frutos da região de Campinas-SP e de alimentos processados. As amostras foram colocadas em frascos de erlenmeyer de 50 mL, contendo 20 mL de meio de Extrato de Malte e Levedura (YM), e incubadas a 30° C por 48 horas. Após 48 horas, as amostras foram inoculadas em placas de petri contendo meio ágar-YM e incubadas a 30°C, por 120 horas. As colônias de leveduras que apresentaram coloração entre amarelo e vermelho, foram transferidas para tubos de ensaio contendo meio ágar YM inclinado e incubadas a 30°C até crescimento satisfatório. Estas leveduras foram reisoladas, pelo método de estrias de esgotamento, em placas de petri contendo meio ágar YM (30°C por 72 horas) e, posteriormente, transferidas para tubos de ensaios contendo ágar GYMP inclinado. As culturas pigmentadas foram codificadas do seguinte modo: L12, isolada como contaminante em massa de tomate; L108, isolada de solo da região da Universidade Estadual de Campinas; L125, isolada a partir de folhas da cana-de-açúcar; L135 e L137 isoladas de solo em Holambra-SP. Através das características morfológicas, de reprodução, testes fisiológicos e bioquímicos as leveduras foram identificadas como: L12, L108, L135 e L137 como Rhodotorula mucilaginosa, e L125 como Rhodotorula graminis. A composição de carotenóides, das leveduras isoladas no Brasil, foi estudada. As culturas de leveduras foram cultivadas em 200 mL de meio YM a 200 rpm em shaker, a 25°C por 5 dias. Cromatografia de coluna aberta, cromatografia de camada delgada e cromatografia líquida de alta eficiência foram utilizadas para separar os carotenóides obtidos das leveduras, a fim de identificá-los e quantificá-los. A linhagem de Rhodotorula glutinis foi a que apresentou maior concentração total de carotenóide (881 mg/L), seguido por Rhodotorula graminis (594 mg/L), Rhodotorula mucilaginosa-137 (590 mg/L) e Rhodotorula mucilaginosa-135 (545 mg/L). Rhodotorula minuta e Sporobolomyces tiveram a menor concentração de carotenóides (168 mg/L and 237 mg/L, respectivamente). Os principais pigmentos encontrados nestas linhagens foram toruleno e b-caroteno. b-Caroteno foi o carotenóide predominante em Rhodotorula grarninis-125, Rhodotorula glutinis e Sporobolornyces, enquanto que o toruleno foi o carotenóide principal nas leveduras de Rhodotorula rnucilaginosa. Em termos de produção específica de cartenóides (_g/g de células secas), Rhodotorula glutinis foi a que obteve maior concentração de carotenóides 132 mg/g. Duas linhagens foram selecionadas para otimização da produção de carotenóides, R. mucilaginosa-137 e R. glutinis. Estas duas culturas foram cultivadas em shaker a 200 rpm, a 25°C por 5 dias, sem iluminação. Utilizou-se planejamento experimental e análise de superfície de resposta para estudar o efeito do pH inicial, concentração de glicose, extrato de levedura, sais de fosfato e sulfato de magnésio na produção de carotenóides, de biomassa e proteína celular. Para cada linhagem, foram realizados 2 planejamentos fatoriais, sendo 1 fracionário e 1 completo.Para a linhagem de R. mucilaginosa-137, o extrato de levedura foi a variável de maior influência na produção de carotenóides, enquanto que os sais de sulfato e fosfato tiveram efeito negativo. O pH inicial não teve efeito significativo tanto na produção de carotenóides como na biomassa. Através dos resultados obtidos pelo planejamento completo, observou-se que a máxima concentração de carotenóides foi de 745 mg/L com 15 g/L de extrato de levedura e 20 g/L de glicose. Em relação a produção específica de carotenóides, a máxima concentração foi de 152 mg/g com 5 g/L de extrato de levedura e 15 g/L de glicose. A concentração de extrato de leveduras e glicose também foram importantes na produção da biomassa, que atingiu o valor máximo de 8 g/L, na faixa de concentração de 15 a 17,1 g /L de extrato de levedura e de 15 a 20 g/L de glicose. Para a linhagem de Rhodotorula glutinis as variáveis de maior influência na produção de carotenóides foram pH inicial, extrato de levedura e glicose. Os sais de sulfato e fosfato não tiveram efeito significativo. Através do planejamento fatorial completo 23 com três pontos centrais, observou-se que na produção de carotenóides, apenas a glicose teve efeito positivo significativo. Na produção específica de carotenóides, o pH inicial, glicose e extrato de levedura tiveram efeito positivo. A máxima concentração de carotenóides obtida foi de 1.269 mg/L com pH inicial 4, 4 g/L de extrato de levedura e 17 g/L de glicose. Na produção específica de carotenóides, a máxima concentração foi de 337 mg/g com pH inicial 4, a 4 g/L de extrato de levedura e 7 g/L de glicose. O crescimento celular foi afetado pelo pH inicial, concentração de extrato de levedura e glicose. Entretanto, o modelo matemático referente a biomassa não apresentou uma regressão satisfatória, devendo ser utilizado apenas para estabelecer tendência da resposta / Abstract: Pigmented yeasts were collected from soils, flowers, leaves, fruits from Campinas-SP region and industrialized foods. The samples were put in 50 mL erlenmeyers flasks, containing YM broth, and they were incubated at 30°C for 48 hours. After 48 hours, these samples were inoculated in Petri plates with YM agar, and incubated at 30°C for 120 hours. The yeasts colonies that had color between red and yellow were transferred to tubes, containing YM agar, and incubated at 30°C. These yeasts were reisolated by screening in Petri plates with YM agar (30°C for 72 hours) and then, transferred into tubes containing GYMP agar. After the selection, the pigmented yeasts were identified by a code: L12, was isolated from tomato sauce; L108, from soils of State University of Campinas; L125, from leaves of sugar cane; L135 e L137, from soils of Holambra-SP. The yeasts were identified by their morphology characteristics, reproduction characteristics, physiology and biochemical tests. The yeasts L12, L108, L135 and L137 were identified as Rhodotorula mucUaginosa and L125 as Rhodotorula graminis. The carotenoid composition of yeasts isolated in Brazil was studied. The yeasts were cultured in 200 mL broth yeast malt at 200 rpm in rotary shaker, 25°C for 5 days without illumination. Open column, thin layer chromatography and high performance liquid chromatography were used to separate, identify and determine carotenoid concentrations. The yeast Rhodotorula glutinis had the highest total carotenoid concentration (881 mg/L), followed by Rhodotorula graminis (594 mg/L), Rhodotorula mucUaginosa-137 (590 mg/L) and Rhodotorula mucilaginosa-135 (545 mg/L). Rhodotorula minuta and Sporobolomyces had the lowest carotenoid contents (168 mg/L and 237 mg/L, respectively). The principal pigments found in these yeasts were torulene and b-carotene. b-Carotene predominated in Rhodotorula graminis-125, Rhodotorula glutinis and Sporobolomyces, while torulene was the major carotenoid in Rhodotorula mucilaginosa. In specific carotenoid production (mg/g of dried cells), Rhodotorula glutinis had a total carotenoid concentration of 132 mg/g. Two of these strains were selected to optimize the carotenoid production, Rhodotorula mucilaginosa-137 and Rhodotorula glutinis. The cultures were cultivated into 200 mL broth yeast malt at 200 rpm in rotary shaker, 25°C for 5 days without illumination. Response surface design was used to study the effects of initial pH and concentrations of glucose, yeast extract, magnesium sulfate and potassium phosphate. Two statistical designs were used for each strain. For the strain of Rhodotorula mucilaginosa-137, the yeast extract the most important variable in terms of enhancing carotenoid formation; magnesium sulfate and potassium phosphate had a negative influence. The initial pH had no significant effect on carotenoid formation or on cell production. Analysis of the quadratic surfaces showed that after 5 days of cultivation at 25°C, the maximum carotenoid concentration of 745 mg/L appeared at 15 g/L of yeast extract and 20 g/L of glucose. The maximum concentration of specific carotenoid production was 152 mg/g at 5 g/L of yeast extract and 10 g/L of glucose. The concentrations of yeast extract and glucose were also important on biomass production, which reached maximum value of 8.0 g/L at a range of 15 to 17.1 glL of yeast extract and 15 to 20 g/L of glucose. The variables that had most influence on carotenoid production by Rhodotorula glutinis were initial pH, yeast extract and glucose. Magnesium sulfate and potassium phosphate had no influence. The carotenoid production was described by second order p01ynomial equation. Analysis of the 23 factorial design surfaces showed that after 5 days of cultivation at 25°C, the maximum carotenoid concentration of 1,269 mg/L with initial pH 4, 4 g/L of yeast extract and 17 g/L de glucose. The maximum specific carotenoid production was 337 j..tglg with initial pH 4, 4 g/L of yeast extract and 7 g/L of glucose. Moreover, carotenoid production in mg/g per liter was more sensitive to changes in yeast extract than to changes in glucose concentrations, in the vicinity of the optimum point of carotenoid production. The growth of the microorganism was affected by initial pH and concentration of yeast extract and glucose. However, the model obtained for biomass from the experimental designs had not a good correlation and because of that it should be used only to study the tendency of response / Doutorado / Doutor em Ciência de Alimentos

The oleaginous yeasts have an ability to accumulate an increased number of lipids, under certain circumstances. These microbial lipids differentiate in the number of fat acids present, which enables their wide application in biotechnological industry. This master’s thesis is aimed on lipid production, number of the fat acid groups present, and squalene production by Metschnikowia yeasts, based on the cultivating conditions. Biomass and lipid production was observed in separate cultivation media, with the addition of the different waste substrates. Production properties were observed by method of the gas chromatography. For the squalene production observation, a HLPC method was chosen. All production groups were able to accumulate lipids on the waste substrate, although in different values. These values were very individual, especially in the areas of the specific groups and growth on the given substrate. The lipid composition was different, which was caused by differences in the waste substrates. With regards to the squalene production – the yeasts from the Metschnikowia family were not able to produce squalene in the presence of the terbinafine and its increasing concentration. Therefore, the same procedure was chosen, as it was for the Yarrowia lipolytica yeast, with the difference in the sterol synthesis, however squalene was still not produced this way.