Efficiency of synthesis of extracellular polysaccharides strains of Lipomyces yeast

The formation of extracellular polysaccharides is a fairly well-studied property of bacteria that is used for the industrial production of such extracellular bacterial as xanthan, dextran, gellan, hyaluronan, etc.. Polysaccharides synthesized by fungi are also widely used, such as schizophillan and scleroglucan. However, polysaccharides synthesized by yeast and yeast-like fungi have not yet found wide industrial application, with the exception of pullulan produced by Aureobasidium pullulans yeast, although there are a number of promising developments in the use of yeast polysaccharides in medicine. Yeast synthesizes polymers that contain mannans, glucans, phosphomannans, galactomannans, and glucuronoxylmannans. Polysaccharides produced by different species, and sometimes even by different strains of the same species, may differ in chemical composition and structure. Such a variety of composition and properties opens up great prospects for their use in various fields: medicine, chemical, food and cosmetic industries, as well as feed additives. In this regard, the search for new producers of polysaccharides is very relevant. Yeast of the genus Lipomyces is found in the soils of the southern and northern hemispheres of the Earth, except in the high-mountainous regions and tundra soils, where soil formation processes are in early stages of development, but the soils are rich in steppe and forest zones. As a result of the research, it turns out that from the point of view of biomass growth on the presented nutrient medium at the temperatures studied, the strains of the Lipomyces lipofer yeast КБП Y-6267 and КБП Y-6265 attract the most attention, especially at low temperatures. With an increase in temperature, the increase in biomass in these yeasts decreases markedly. As producers of extracellular polysaccharide, it is worth noting the КБП Y-6267 and КБП Y-6264 strains at 20 °C and the КБП Y-6268 strains and the КБП Y-6234 at 30 °C, which indicates the possibility of using for these purposes different species of the genus Lipomyces. At 30 °C, Lipomyces lipofer strains of the КБП Y-6268 and Lipomyces kononenkoae КБП Y-6234 had the highest enzyme activities, however, there was no relationship between enzyme activities, biomass gains and polysaccharide yields at low temperatures.

Biocatalytic Studies of the Furanocoumarins Angelicin and Chalepensin

Bioconversion studies conducted on the furanocoumarins angelicin and chalepensin have revealed that angelicin was metabolized by Lipomyces lipofer (ATCC 10742) to give the corresponding reduced acid 3 and chalepensin was transformed by Cunninghamella blakesleeana (SC-2138) to give the diol metabolite 4. The structures of the new metabolites have been established on the basis of spectral data. The absolute stereochemistry around C-2″ in 4 was confirmed by Horeau's method. Angelicin and chalepensin were shown to possess antifungal activity against Candida albicans and Cryptococcus neoformans.

Lipid synthesis in Lipomyces lipofer

The synthesis of lipids by cell-free fractions of Lipomyces lipofer was investigated by measuring the incorporation of acetate-14C and other radioactive precursors into lipid-soluble material. On the basis of cofactor requirements, the reaction to inhibitors, and the ability to incorporate appreciable quantities of malonate-14C, it was concluded that the malonyl-CoA pathway was the principal route of fatty acid synthesis in this yeast. By employing a means of cell dispersion less rigorous than previously used with yeasts, it was shown that the soluble portion (high-speed supernatant) of the cell was primarily involved in lipid synthesis. The mitochondrial fraction and the high-speed pellet (96 000 × g, 1 h) were inactive although the latter fraction was required for the maximum activity of the high-speed supernatant. The stimulatory activity of this pellet was very labile and showed a lag period before it became effective. Several lines of evidence suggested that the carboxylation of acetyl-CoA is the rate-limiting step in lipid synthesis which is accelerated by the high-speed pellet.

THE LIPIDS OF LIPOMYCES LIPOFER

As a preliminary to the study of lipid synthesis in Lipomyces lipofer, the nature of the lipid accumulated by this organism was investigated. Neutral lipids were found to predominate, especially in older cultures of high lipid content. The triglyceride fraction was the major component, with smaller quantities of hydrocarbon, free and esterified ergosterol, monoglyceride, diglyceride, and free fatty acid also present. Phosphatid-y-linositol constituted almost one-quarter of the phospholipid fraction. The phosphoglycerides of choline, serine and ethanolamine were also detected. Gas chromatographic evidence indicated that 16:0, 16:1, 18:0, 18:1, 18:2, 18:3 were the principal fatty acid components. The 18:1 fraction was shown by oxidative-cleavage studies to be primarily oleic acid. This fraction predominated, reaching a level of 69% of the total fatty acids in older cultures.

PREPARATION AND PROPERTIES OF PROTOPLASTS FROM LIPOMYCES LIPOFER

Lipomyces lipofer was grown aerobically in chemically defined liquid media containing glucose or citrate as a carbon source. Protoplasts were prepared by digestion of young cells with the intestinal secretions from Helix pomatia in the presence of 10% (w/v) mannitol. Differences in substrate utilization between citrate- and glucose-grown cells were noted and compared with the substrate utilization of the corresponding protoplasts. Examination of protoplasts derived from glucose- and citrate-grown cells showed that the citrate-grown organisms yielded morphologically and metabolically more stable protoplasts. The effects of hypotonic conditions on the metabolic activity of protoplasts as well as the consequences of supplementation of these protoplasts with cofactors have been described.

MITOCHONDRIA FROM LIPOMYCES LIPOFER

Protoplasts of Lipomyces lipofer were ruptured by decompression in a French pressure cell. A particulate fraction sedimenting at 17,000 × g in saline or sucrose media contained the bulk of the substrate-dependent oxidative activity and was capable of phosphorylation. Particle fractions isolated in sucrose required supplementation with ATP and Mg2+while fractions from saline isolations required, in addition, NAD and cytochrome c. NADP and thiamine pyrophosphate had small or negligible effects in the presence of the other cofactors. Oxidative phosphorylation occurred most efficiently in particles isolated in sucrose, but in no case did the P/O ratio exceed 1.6. Electron microscopic examination of the sucrose-isolated fraction showed it to be a relatively homogeneous preparation of mitochondria which appear more "native" after incubation with substrate than immediately after isolation.

MITOSIS IN THE YEAST LIPOMYCES LIPOFER

A study of mitosis in Lipomyces has been carried out because preliminary observations by Ganesan and Roberts, 1959 (9), had indicated that the nucleus of this yeast might be unusually favourable for morphological observations. This impression has proved correct. The chromosomes of Lipomyces are visible as separate, countable bodies for the greater part of mitosis. The pattern of mitosis differs from the common one in that in Lipomyces the proper distribution of sister chromosomes is accomplished without the help of a spindle apparatus. At the end of prophase sister chromosomes are found in pairs which align themselves parallel to one another to form a palisade or stack whose long axis coincides with the axis of the impending division. At anaphase-telophase the stack of paired chromosomes fuses into a seemingly homogeneous cord which divides by constriction.