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.