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Albany 2019: 20th Conversation - Abstracts

category image Albany 2019
Conversation 20
June 11-15 2019
Adenine Press (2019)

Modifications in the oxidative and constructive metabolism of yeast Yarrowia lipolytica caused by limitation the growth by nitrogen or thiamine

The study of oxidative and constructive metabolism under conditions of limitation of growth of microbial cultures is important from the point of view of the directional production of practically valuable metabolites, enzymes, biomass with the special composition.

The object of the study was the yeast Yarrowia lipolytica , the uniqueness of which lies in the ability to synthesize significant quantities of organic acids, lipids and enzymes from a wide range of carbon sources (Zinjarde, 2014; Morgunov, Kamzolova, 2015; Lazar et al. 2018). Ethanol was used as carbon source. Ethanol as a substrate for growth of producers of practically valuable compounds has several advantages over other substrates. It does not contain harmful impurities, it is well assimilated by yeast and dissolves in water in any proportions. Since ethanol is used in the human diet, the products derived from it do not require additional purification from the residual substrate. As reported by Weusthuis et al. (2011) the several companies in the USA and Switzerland created the food products based on microbial biomass produced from ethanol.

In the course of the research, it was established that under conditions of unlimited (exponential) growth of Yarrowia lipolytica VKM Y-2373 the efficiency of ethanol oxidation attained a maximum. Under those conditions the cells contained the maximum amount of protein, and the excretion of metabolites (citric, isocitric, α-ketoglutaric and other acids) did not occur (Table). In other words, the metabolism of Yarrowia lipolytica was relatively balanced.

On the contrary, under conditions of Yarrowia lipolytica VKM Y-2373 growth limitation by nitrogen or thiamine, the complex of functional changes takes place (Table), of which the most significant modifications are following:

1) the orientation of biosynthetic processes shifts into the direction of a sharp decline in protein synthesis;
2) the intensive excretion of cellular metabolites became active; the composition and quantity of the excreted organic acids depend on the nature of the growth-limiting component: the limitation with a nitrogen stimulated the excretion of citric and isocitric acids (6.8 and 11.2 g/L, respectively), while with a thiamine deficiency cells synthesize α-ketoglutaric acid (11.0 g/L).
3) the ratio of the enzymes responsible for the metabolism of the excreted products changes. Under unlimited growth the activity of all enzymes involved in primary oxidation of ethanol, Krebs and glyoxylate cycles was sufficiently high. Under conditions of nitrogen limitation, a high activities of citrate synthase and aconitate hydratase, necessary for the intensive synthesis of citric and isocitric acids, in distinction from the subsequent enzyme of Krebs cycle, NAD-isoсitrate dehydrogenase, were observed. Hence, these acids formed in Krebs cycle can be presumably excreted from the yeast cell rather than metabolized via the cycle. Similar data were previously obtained for Yarrowia lipolytica - producers of citric- and isocitric acids from biodiesel-derived waste (Morgunov, Kamzolova, 2015). In contrast, in the case of thiamine deficiency, the metabolic flux is blocked at the level of the thiamine-dependent enzyme, 2-oxoglutarate dehydrogenase, and α-ketoglutaric acid is excreted into the cultural medium rather than metabolized via the cycle. The continuous formation of excreted products was ensured by the functioning of the glyoxylate cycle, with is confirmed by the high activities of citrate synthase, aconitate hydratase and isocitrate lyase which operate both in the glyoxylate cycle and in Krebs cycle.

Table. The effect of limiting factors on the growth and product synthesis in Y. lipolytica VKM Y-2373

morgu-table2.gif

References

    Lazar, Z., Liu, N., Stephanopoulos, G. (2018) Holistic approaches in lipid production by Yarrowia lipolytica. Trends Biotechnol. 36(11), 1157-1170. doi: 10.1016/j.tibtech.2018.06.007

    Morgunov, I.G., Kamzolova, S.V. (2015) 166 Metabolism of biodiesel-derived waste in the yeast Yarrowia lipolytica – producers of citric- and isocitric acids. J. Biomol. Struct. Dyn. 33(sup1), 108-109, doi: 10.1080/07391102.2015.1032803

    Weusthuis, R., Aarts, J., Sanders, J. (2011) From biofuel to bioproduct: is bioethanol a suitable fermentation feedstock for synthesis of bulk chemicals? Biofuels, Bioprod. Biorefin. 5, 486-494.

    Zinjarde, S.S. (2014) Food-related applications of Yarrowia lipolytica. Food Chem. 152, 1-10. doi: 10.1016/j.foodchem.2013.11.117

Svetlana V. Kamzolova
Julia N. Lunina
Nadezhda N. Stepanova
Ramil K. Allayarov
Igor G. Morgunov

Federal Research Center Pushchino Center for Biological Research
Russian Academy of Sciences
G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms
RAS Pushchino
Moscow region 142290, Russia

Ph: (007) 9264145620
Fx: (007) 495-9563370
Email: morgunovs@rambler.ru