The Lys20 homocitrate synthase isoform exerts most of the flux control over the lysine synthesis pathway in Saccharomyces cerevisiae

Abstract

SummaryIn Saccharomyces cerevisiae, the first committed step in the lysine (Lys) biosynthetic pathway is catalysed by the Lys20 and Lys21 homocitrate synthase (HCS) isoforms. Overexpression of Lys20 resulted in eightfold increased Lys, as well as 2-oxoglutarate pools, which were not attained by overexpressing Lys21 or other pathway enzymes (Lys1, Lys9 or Lys12). A metabolic control analysis-based strategy, by gradually and individually manipulating the Lys20 and Lys21 activities demonstrated that the cooperative and strongly feedback-inhibited Lys21 isoform exerted low control of the pathway flux whereas most of the control resided on the non-cooperative and weakly feedback-inhibited Lys20 isoform. Therefore, the higher control of Lys20 over the Lys flux represents an exception to the dogma of higher pathway control by the strongest feedback-inhibited enzyme and points out to multi-site engineering (HCS isoforms and supply of precursors) to increase Lys synthesis.

The 2‐oxoglutarate supply exerts significant control on the lysine synthesis flux in Saccharomyces cerevisiae

“…Such an effect was not observed by over-expressing other LYS genes, including LYS21 [3]. The high control by Lys20 is in agreement with results found in the present work, in which disruption of the LYS20 gene (but not LYS21) elicited a substantial decrease in Lys free content to 34% of the WT level.…”

“…3A) [12,28]. Remarkably, no significant differences in Lys prot were observed for all these mutants ( Table 3), suggesting that demand for this amino acid for protein synthesis does not significantly control the pathway flux, as previously demonstrated for HCS mutants [3].…”

“…In our previous work [3], it was demonstrated that, within the a-AAP, Lys20 activity was the main controlling step of the flux to Lys free because its over-expression in the cells led to an increase in the intracellular Lys free pool and flux. Such an effect was not observed by over-expressing other LYS genes, including LYS21 [3].…”

“…Recently, we reported that the Lys synthesis flux is mostly controlled by the activity of the Lys20 HCS isoform, whereas the control exerted by protein synthesis (Lys demand) is low as neither the cellular protein content nor the amount of Lys present in proteins varied when the Lys pool increased sevenfold [3]. However, the contribution of the 2-OG supply to the flux of Lys synthesis was not determined, and the possibility remained that a proportion of the control by Lys20 may be accounted for by the 2-OG supply.…”

“…1). In Saccharomyces cerevisiae, 2-OG is at a sub-saturating concentration (0.46 mM), whereas cytosolic acetyl CoA is saturating (230 lM) for the two HCS isoforms present in this yeast, Lys20 and Lys21 (K m(2-OG) = 4.6 and 9 mM; K m(acetyl CoA) = 7.7 and 19 lM, respectively) [1][2][3]. Therefore, higher cellular 2-OG contents may increase the rate of the HCS isoforms and hence may increase the Lys synthesis flux.…”