Thialysine utilization by thialysine resistant CHO cells

Thialysine resistant CHO cells utilize thialysine added to the culture medium to a lesser extent than the parental cells. Thialysine is utilized in protein synthesis and it is incorporated into proteins in place of lysine. The parental strain substitutes up to 11% of protein lysine by thialysine, while variant cells substitute a maximum of 5% of protein lysine.     

Biochemical characterization of a thialysine-resistant clone of CHO cells

The intracellular transport and the activation of lysine, thialysine and selenalysine have been investigated in a thialysine-resistant CHO cell mutant strain in comparison with the parental strain. The cationic amino acid transport system responsible for the transport of these 3 amino acids shows no differences between the 2 strains as regards its affinity for each of these amino acids. On the other hand the Vmax of the transport system in the mutant is about double that in the parental strain. The lysyl-tRNA synthetase, assayed both as ATP = PPi exchange reaction and lysyl-tRNA synthesis, shows a lower affinity for thialysine and selenalysine than for lysine in both strains; in the mutant, however, the difference is even greater. Thus the thialysine resistance of the mutant is mainly due to the properties of its lysyl-tRNA synthetase, which shows a greater difference of the affinities for lysine and thialysine with respect to the parental strain.     

Thialysine- and selenalysine-resistance in a E. coli mutant

A thialysine-resistant mutant of E. coli strain KL16 also shows a lower sensitivity to selenalysine, the lysine analog containing selenium. No difference between the mutant and the parental strain has been shown regarding the affinities of the transport systems and the lysyl-tRNA synthetase for selenalysine, thialysine and lysine as well as the inhibitory effects of these three aminoacids on the activity of the lysine biosynthetic pathway. A marked difference between the two strains has been evidenced in the AK III repression: in the mutant the repression by selenalysine, thialysine and lysine is much lower than in the parental strain.     

Aspartokinase III repression and lysine analogs utilization for protein synthesis

The extents of thialysine and selenalysine incorporation into cell proteins were compared in E. coli KL16 and in a mutant able to grow equally well in the presence or in the absence of both lysine analogs. The mutant differs from the parental strain in the repression of aspartokinase III (AKIII), the first enzyme of the lysine biosynthetic pathway. No analog incorporation into proteins was observed in mutant cells grown in the presence of either analog, whereas a marked analog incorporation was observed in the parental strain, where up to 17% and 12% of protein lysine can be substituted by thialysine and selenalysine respectively. In the parental strain grown in media containing either analog at different concentration the extent of analog incorporation into proteins is related to the extent of AKIII repression.     

Aspartokinase III repression in a thialysine-resistant mutant of E. coli

A thialysine-resistant mutant of the E. coli KL16 strain was isolated. It can grow equally well in the presence and in the absence of thialysine. The properties of the two lysine transport systems, of the lysyl-tRNA synthetase and of the aspartokinase III (AK III) were studied in the mutant and in the parent strain. AK III is the first enzyme of the lysine biosynthetic pathway and its activity is involved in the regulation of lysine biosynthesis by feed-back and repression mechanism. No difference between the two strains was evidenced as regards 1) the affinity of the transport systems for lysine and thialysine 2) the activity of the lysyl-tRNA synthetase 3) the allosteric inhibition of the AK III by lysine and thialysine. A marked difference between the two strains has been evidenced in the AK III repression: in the mutant the enzyme is much less repressed both by lysine and thialysine. The possible correlation between the activity of AK III and the thialysine-resistance is discussed in this paper.     

Thialysine utilization by a lysine-requiring Escherichia coli mutant

Summary Thialysine cannot completely substitute lysine as growth factor for a lysine-requiring E. coli mutant. However it can be utilized for growth in the presence of limiting amounts of lysine, in substitution of, and in competition with this latter. The effects of thialysine on growth rate, protein synthesis rate and cell viability, and its incorporation into proteins were studied in function of lysine and thialysine concentration in the culture media. Up to 60% of protein lysine substitution by thialysine is observed, without appreciable effects on cell viability.     

Lysine overproduction mutations in the yeast Saccharomyces cerevisiae and its transfection into industrial Yeast strains ]

Yeast mutants resistant to a toxic lysine analog, thialysine were obtained by a method described in the literature. A strain excreting the maximum amount of lysine (0.45 g/l) was selected from these mutants. The intracellular content of lysine was also increased by 30%. The genetic nature of lysine overproduction was studied in this strain. An increase in the amount of excreted lysine was shown to be determined by at least two genes, one of which carries a mutation of thialysine resistance manifesting the pleiotropic effect of lysine overproduction (Th1R) and the other is involved in the regulation of lysine production (PRL). Linkage groups of these genes were determined: the first gene was mapped to the IV chromosome and the second, to the XV chromosome. Both genetic characters were introduced into industrial baker's yeast strains via a series of backcrosses. The stabilization of the genome in the newly derived strains was confirmed by electrokaryotyping.     

Selenalysine and protein synthesis.

Selenalysine is a lysine analog having the gamma-methylene group substituted by a selenium atom. It has been demonstrated that selenalysine is activated and transferred to tRNAlys by either Escherichia coli or rat liver aminoacyl-tRNA synthetases, and inhibits lysine incorporation into polypeptides in protein-synthesizing systems from E. coli, rat liver or rabbit reticulocytes. All tests were performed in comparison with thialysine, a lysine analog having the gamma-methylene group substituted by a sulfur atom. In all the reactions studied, both thialysine and selenalysine act as competitive inhibitors of lysine. With respect to thialysine, selenalysine act as competitive inhibitors of lysine. With respect to thialysine, selenalysine shows a slightly lower activity as lysine inhibitor.     

Inhibition of growth and aspartokinase activity of Salmonella typhimurium by thialysine

Thialysine (S-2-aminoethyl cysteine) is an analog of lysine and has been reported to inhibit the lysyl-tRNA synthetase activity of Escherichia coli. This analog inhibits the growth of Salmonella typhimurium when added to glucose minimal medium at concentrations of 1.25 mM or greater. The addition of lysine with thialysine restores the normal growth rate, whereas, methionine, valine, or leucine each enhances the growth inhibition caused by thialysine. Enzyme assays demonstrate that thialysine inhibits not only the lysyl-tRNA synthetase from S. typhimurium, but also the aspartokinase activity. Lysine and thialysine appear to inhibit the same 40% of the total aspartokinase because simultaneous addition of the two compounds to the reaction mixture does not increase the inhibition caused by either alone. Furthermore, the slow growth of cells in the presence of 2.5 mM thialysine decreases the level of aspartokinase activity, suggesting that thialysine causes repression of enzymes synthesis as well as inhibition of activity.     

Degradation of thialysine- or selenalysine-containing abnormal proteins in CHO cells

CHO cells can incorporate thialysine and selenalysine in their proteins in substitution of lysine. Data are reported in the present paper showing that proteins containing either thialysine or selenalysine are unstable and quite rapidly degraded. The degradation rate is strictly related to the extent of protein lysine substitution. At similar extent of substitution, selenalysine-containing proteins are more unstable that thialysine-containing ones.     

Inhibition of growth and aspartokinase activity of Salmonella typhimurium by thialysine.

Thialysine (S-2-aminoethyl cysteine) is an analog of lysine and has been reported to inhibit the lysyl-tRNA synthetase activity of Escherichia coli. This analog inhibits the growth of Salmonella typhimurium when added to glucose minimal medium at concentrations of 1.25 mM or greater. The addition of lysine with thialysine restores the normal growth rate, whereas, methionine, valine, or leucine each enhances the growth inhibition casued by thialysine. Enzyme assays demonstrate that thialysine inhibits not only the lysyl-tRNA synthetase from S. typhimurium, but also the aspartokinase activity. Lysine and thialysine appear to inhibit the same 40% of the total aspartokinase because simultaneous addition of the two compounds to the reaction mixture does not increase the inhibition caused by either alone. Furthermore, the slow growth of cells in the presence of 2.5 mM thialysine decreases the level of aspartokinase activity, suggesting that thialysine causes repression of enzyme synthesis as well as inhibition of activity.     

Lysine Overproduction Mutations in the YeastSaccharomyces cerevisiae Are Introduced into Industrial Yeast Strains

Yeast mutants resistant to a toxic lysine analog, thialysine were obtained by a method described in the literature [1]. A strain excreting the maximum amount of lysine (0.45 g/l) was selected from these mutants. The intracellular content of lysine was also increased by 30%. The genetic nature of lysine overproduction was studied in this strain. An increase in the amount of excreted lysine was shown to be determined by at least two genes, one of which carries a mutation of thialysine resistance manifesting the pleiotropic effect of lysine overproduction (Th1 ^R) and the other is involved in the regulation of lysine production (PRL). Linkage groups of these genes were determined: the first gene was mapped to the IV chromosome and the second, to the XV chromosome. Both genetic characters were introduced into industrial baker's yeast strains via a series of backcrosses. The stabilization of the genome in the newly derived strains was confirmed by electrokaryotyping.     

Thialysine and selenalysine utilization for protein synthesis by E. coli in comparison with lysine

Utilization of thialysine and selenalysine for protein synthesis by a lysine requiring E. coli mutant was studied. Incorporation into proteins of thialysine or selenalysine, added to culture medium together with lysine, becomes evident when the amount of available lysine in the medium is highly reduced, that is the mutant utilizes the isologs only after all the available natural aminoacid has been utilized. Compared to selenalysine, thialysine is better utilized; when both isologs are present in the medium at equal concentrations, up to 46% of protein lysine is substituted by thialysine and only 12% by selenalysine.     

Thialysine utilization for protein synthesis by CHO cells

Chinese Hamster Ovary (CHO) cells utilize thialysine when added to the culture medium. Thialysine utilization is prevented by increasing lysine concentration in the medium, thus indicating that thialysine is utilized in substitution for and in competition with lysine. Almost all thialysine disappeared from the medium is recovered in cell protein hydrolysates. Thialysine is used for protein synthesis in substitution for lysine, and up to 10% of lysine can be substituted.     

Utilization of lysine analogs by a lysine-requiring E. coli mutant

Thialysine and selenalysine cannot substitute lysine as a growth factor for a lysine-requiring E. coli mutant, but can nevertheless be utilized for protein synthesis in the presence of lysine. In order to have information about the effects of lysine on the utilization of the two analogs, the extent of the incorporation of the three aminoacids into newly synthesized proteins has been determined. The analog starts to be utilized by cells growing in a medium containing either analog and lysine when lysine concentration becomes very low. Of the two analogs, thialysine is more easily utilized. In fact thialysine can be utilized when the lysine/thialysine ratio in the medium is 1/25. Selenalysine starts to be utilized when the lysine/selenalysine ratio is 1/200.     

Thialysine-Resistant Mutant of SALMONELLA TYPHIMURIUM with a Lesion in the thrA Gene

A mutant of Salmonella typhimurium was selected for its spontaneous resistance to the lysine analog, thialysine (S-2-aminoethyl cysteine). This strain, JB585, exhibits a number of pleiotropic properties including a partial growth requirement for threonine, resistance to thiaisoleucine and azaleucine, excretion of lysine and valine, and inhibition of growth by methionine. Genetic studies show that these properties are caused by a single mutation in the thrA gene which encodes the threonine-controlled aspartokinase-homoserine dehydrogenase activities. Enzyme assays demonstrated that the aspartokinase activity is unstable and the threonine-controlled homoserine dehydrogenase activity absent in extracts prepared from the mutant. These results explain the growth inhibition by methionine because the remaining homoserine dehydrogenase isoenzyme would be repressed by methionine, causing a limitation for threonine. The partial growth requirement for threonine during growth in glucose minimal medium may also, by producing an isoleucine limitation, cause derepression of the isoleucine-valine enzymes and provide an explanation for both the valine excretion, and azaleucine and thiaisoleucine resistance. The overproduction of lysine may confer the thialysine resistance.     

Thialysine and selenalysine incorporation into CHO cell proteins and its effects on cell growth

CHO cells can incorporate into proteins both thialysine and selenalysine when both are present together in the culture medium. Thialysine and selenalysine inhibit cell growth and cell viability. The inhibitory effect of either analog is additive. The inhibition of cell viability is related to the extent of protein lysine substitution by thialysine or selenalysine; it is however irrelevant whether lysine is substituted by one or the other analog or by both.     

Thialysine utilization for protein synthesis by an exponentially growing E. coli culture

The extent of protein lysine substitution by thialysine in E. coli cells grown in media containing the analog depends on the time interval the cells are grown in the presence of analog and on the analog concentration in the medium. By calculating the percent of lysine substitution in newly synthesized proteins it was shown that this reaches, after one cell doubling in the presence of analog, a maximum which is 17% in the cells grown with 0.1 or 0.2 mM thialysine and 8% in cells grown with 0.05 mM thialysine. Proteins synthesized in the presence of analog in the concentration range 0.05-0.2 mM show similar stability to those synthesized in the absence of analog. The extent of analog incorporation into newly synthesized proteins, as regards both the time course and the dependence on analog concentration in the medium, is strictly related to the extent of the repression of AK III, the first enzyme of lysine biosynthetic pathway.     

Genetic and biochemical properties of thialysine-resistant mutants of Saccharomyces cerevisiae

Three groups of lysine-excreting, thialysine-resistant mutants of Saccharomyces cerevisiae were derived from the wild-type strain (X2180) by mutagenic treatment and selected on the basis of a cross-feeding assay. Mutants MNNG2-9, MNNG2-27, MNNG2-39 and MNNG2-62 (group 1) exhibited a 2:2 segregation for thialysine resistance following mating with a wild-type strain and a lower than wild-type lysyl-tRNA synthetase activity; the thialysine-resistant phenotype was dominant in specific hybrids. Mutant MNNG2-2 (group II) was similar to group I mutants except that the thialysine-resistant phenotype was recessive in the hybrid. Mutant MNNG3-142 (group III) exhibited an irregular ratio of segregation of thialysine resistance and a significantly lower lysyl-tRNA synthetase activity; the thialysine-resistant phenotype was recessive in the hybrid. The growth of both group I and group III mutants was temperature-sensitive. The thialysine-resistant mutants exhibited pleiotropic properties including the increased production and excretion of lysine, thermosensitive growth and an impairment of lysyl-tRNA synthetase activity.     

Intracellular transport of thialysine and selenalysine in CHO cells

The intracellular transport of thialysine and selenalysine in CHO cells has been studied. Data have been obtained indicating that the two lysine analogs can be transported by both the cationic aminoacid transport system and by the L transport system. The affinity of the cationic aminoacid transport system is similar for the two lysine analogs but lower than that for lysine and the affinity of the L transport system for the two lysine analogs is lower than that for leucine.