Effect of the cancer specific shorter form of human 6-phosphofructo-1-kinase on the metabolism of the yeast Saccharomyces cerevisiae

Abstract

BackgroundAt first glance, there appears to be a high degree of similarity between the metabolism of yeast (the Crabtree effect) and human cancer cells (the Warburg effect). At the root of both effects is accelerated metabolic flow through glycolysis which leads to overflows of ethanol and lactic acid, respectively. It has been proposed that enhanced glycolytic flow in cancer cells is triggered by the altered kinetic characteristics of the key glycolytic regulatory enzyme 6-phosphofructo-1-kinase (Pfk). Through a posttranslational modification, highly active shorter Pfk-M fragments, which are resistant to feedback inhibition, are formed after the proteolytic cleavage of the C-terminus of the native human Pfk-M. Alternatively, enhanced glycolysis is triggered by optimal growth conditions in the yeast Saccharomyces cerevisiae. ResultsTo assess the deregulation of glycolysis in yeast cells, the sfPFKM gene encoding highly active human shorter Pfk-M fragments was introduced into pfk-null S. cerevisiae. No growth of the transformants with the sfPFKM gene was observed on glucose and fructose. Glucose even induced rapid deactivation of Pfk1 activities in such transformants. However, Pfk1 activities of the sfPFKM transformants were detected in maltose medium, but the growth in maltose was possible only after the addition of 10 mM of ethanol to the medium. Ethanol seemed to alleviate the severely unbalanced NADH/NADPH ratio in the sfPFKM cells. However, the transformants carrying modified Pfk-M enzymes grew faster than the transformants with the human native human Pfk-M enzyme in a narrow ecological niche with a low maltose concentration medium that was further improved by additional modifications. Interestingly, periodic extracellular accumulation of phenylacetaldehyde was detected during the growth of the strain with modified Pfk-M but not with the strain encoding the human native enzyme.ConclusionsHighly active cancer-specific shorter Pfk-M fragments appear to trigger several controlling mechanisms in the primary metabolism of yeast S. cerevisiae cells. These results suggest more complex metabolic regulation is present in S. cerevisiae as free living unicellular eukaryotic organisms in comparison to metazoan human cells. However, increased productivity under broader growth conditions may be achieved if more gene engineering is performed to reduce or omit several controlling mechanisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-017-0362-5) contains supplementary material, which is available to authorized users.

Another Form of Modified, Highly-Active 6-Phosphofructo-1-Kinase in Cancer Cells

“…After 15 minutes of incubation, the cells were collected by centrifugation and cell-free homogenates were prepared. Similar to the results observed previously with the sfPFKM strain [14], no Pfk1 activity was detected in the extract of the sfPfk-L strain after a short incubation in glucose. Also, no Pfk1 activity was observed in the extract of the wild-type transformant with the inserted sfPfk-L gene.…”

“…Growth characteristics of the sfPfk-L strain in SMM with the addition of fermentative sugars, were similar to results to those of the sfPFKM strain encoding shorter 47-kDa sfPfk-M fragments [14]. Although both strains were unable to grow on glucose, they showed higher growth coefficients in respect to the wild-type strain in the SMM with a low maltose concentration and 10 mM ethanol.…”

“…Transformants were grown on Supplemented Minimal Medium (SMM), as reported previously [14]. The substrate contained synthetic dropout medium without uracil (Sigma-Aldrich; St. Louis, MO, USA) containing 2% glycerol and 2% ethanol as non-fermentable carbon sources.…”

“…For the different carbon source tests, identical SMM were used, but the nonfermentable sugars were replaced with glucose or maltose. Growth kinetics were monitored by measuring optical density (OD 600 ) with a spectrophotometer (Lambda 25, Perkin-Elmer; Boston, MA, USA) and growth coefficients were determined as previously described [14].…”

“…The nPFKL strain grew well in the SMM with glucose or fructose, with a growth rate coefficient of about 0.25 (h -1 ), while no growth of the sfPfk-L strain was observed in such media. Similar to the sfPFKM yeast transformant encoding the muscle type human shorter 47-kDa fragments, the sfPfk-L strain grew on the maltose medium; however, 10 mM ethanol must have been added [14]. At the best growth conditions (0.1% maltose), the transformant-encoding liver type shorter 70-kDa isoenzyme reached a growth rate coefficient of 0.19 h -1 that surmounted that of the transformant encoding native nPfk-L (0.14 h -1 ) (Figure 5A) and the muscle type 47-kDa sfPfk-M isoenzyme (0.125 h -1 ) (Figure 5B).…”