Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
Yarrowia Lipolytica Yeast Possesses An Atypical Catabolite Repression
Catabolite repression was thoroughly studied in the bacterium Escherihia coli and, though not so well, in the yeast Saccharomyces cerevisiae (1, 2). Glucose and related sugars repress the transcription of genes encoding enzymes required for the utilization of alternative carbon sources. The different sugars produce signals which modify the conformation of certain proteins that, in turn, directly or through a regulatory cascade affect the expression of the genes subject to catabolite repression. These genes are not all controlled by a single set of regulatory proteins (3, 4), but there are different circuits of repression for different groups of genes (2). Catabolite repression allows the respective microorganisms effectively use carbohydrate substrates, which first assimilate one of the two available substrates (commonly, a carbohydrate), whereas the assimilation of the other substrate starts only after the first substrate is fully consumed from the medium. The degree of catabolite repression varies very significantly in microorganisms. For example, glucose suppresses the expression of invertase in Saccharomyces cerevisiae by 800 times, whereas the expression of aconitate hydratase, cytochrome c oxidase, and isocitrate dehydrogenase, are suppressed not more than by 10 times (1).
The non-conventional yeast Yarrowia lipolytica is an organism of great biotechnological interest due to its ability to excrete organic acids and proteins to the medium. The phenomenon of catabolite repression in Yarrowia lipolytica yeast is poorly studied.
The aim of this work was to study the metabolism of Yarrowia lipolytica yeast in media containing two different carbon sources: glycerol/oleic acid; glucose/oleic acid; glucose/hexadecane and its regulation.
Igor G. Morgunov
G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms
Figure. The growth of Yarrowia lipolytica in the two-substrate medium (left) and key enzymes (write) in these conditions. Variation of ● biomass, ▲ glycerol,○ oleic acid, □ glucose and ■ hexadecane (expressed in g/l); enzyme activity (expressed in U/mg of protein) of GK (glycerolkinase), PC (pyruvate carboxylase), PDH (pyruvate dehydrogenase), IL (isocitrate lyase) and MS (malate synthase).
It is evident from the Figure that when Yarrowia lipolytica was cultivated on the mixture of glycerol and oleic acid, the concentration of these substrates started to decrease just from the first hours of cultivation. Moreover, the utilization of these two substrates went concurrently, although glycerol was utilized at a higher rate than oleic acid. Glycerol kinase (the key enzyme of glycerol metabolism) and two key enzymes of the glyoxylate cycle responsible for the metabolism of fatty acids (isocitrate lyase and malate synthase) were induced from the fist hours of cultivation and remained active to the end of the cultivation period. These results suggest that glycerol is more easily utilizable substrate than oleic acid and probably other fatty acids; however, glycerol does not suppress the metabolism of fatty acids. In contrast, upon the cultivation of Yarrowia lipolytica on the mixture of glucose and oleic acid, the latter substrate began to be utilized only when the concentration of glucose decreased from 10 to less than 2.5 g/L (on the 12th hour of cultivation). The glycolytic enzymes pyruvate dehydrogenase and pyruvate carboxylase were induced from the first hours of cultivation and remained at high levels until the exhaustion of glucose in the medium. At the same time, the activities of isocitrate lyase and malate synthase were very low during the metabolism of glucose, but were rapidly induced after the exhaustion of glucose in the medium. These data can be interpreted in such a manner that glucose at rather high concentrations suppresses enzymes involved in the metabolism of fatty acids.
When Yarrowia lipolytica was grown on the mixture of glucose and hexadecane, the dynamics of growth and substrate consumption was typical of the diauxie phenomenon. Indeed, the utilization of hexadecane began only in several hours after the time when glucose was completely exhausted in the cultivation medium. In this case, the exhaustion of glucose arrested growth and the culture resumed growth only after a lag period. The assay of enzymes showed that the glycolytic enzymes pyruvate dehydrogenase and pyruvate carboxylase were active during the phase of growth on glucose, whereas the enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, were active during the phase of growth on hexadecane.
Thus, experiments on the cultivation of Yarrowia lipolytica on two substrate pairs: glycerol/oleic acid and glucose/oleic acid and glucose/hexadecane indicated that glycerol does not suppress the assimilation of oleic acid, whereas glucose suppresses it in such a manner that oleic acid begins to be consumed only after the concentration of glucose in the medium falls to about zero.