Mechanism of metabolites distribution between 2,3-butanediol and branched-chain amino acid synthesis pathways in Klebsiella pneumoniae

Klebsiella pneumoniae is a commonly known 2,3-butanediol producer. 2,3-Butanediol synthesis and branched-chain amino acid (BCAA) synthesis pathways share the same step of α-acetolactate synthesis from pyruvate. Those two pathways do not interfere with each other in the wild-type strain. Knocking out budA (encoding α-acetolactate decarboxylase) blocks the 2,3-butanediol synthesis pathway. Meanwhile, metabolites of the BCAA synthesis pathway (valine, 2-ketoisovalerate, 2,3-dihydroxyisovalerate and 2-hydroxyisovalerate) are accumulated. However, the mechanism underlying the metabolite changes resulting from the inactivation of budA remains unclear. In this study, both ex vivo and in vitro experiments were conducted to elucidate this mechanism. Kinetic parameters of BudA and acetohydroxy acid isomeroreductase (IlvC) were determined. BudA has a higher affinity toward α-acetolactate and has a higher catalytic constant (K_m = 3.66 mM, k_cat = 7.8 s^-1) compared to IlvC (K_m = 17.98 mM, k_cat = 0.68 s^-1). ex vivo experiments showed that IlvC activities were not influenced by knocking out budA and vice versa. IlvC activities were improved in the cells in which ilvC was overexpressed, but this did not lead to the accumulation of metabolites of the BCAA synthesis pathway. The activities of IlvC in the cell were not affected by the accumulation of 2,3-dihydroxyisovalerate, 2-ketoisovalerate, or valine in the broth. These results indicated that the competitiveness of BudA and IlvC in the cell determines the metabolites distribution between those two pathways. The inactivation of BudA and intact IlvC led to the exceeded α-acetolactate flow into the BCAA synthesis pathway, which caused the accumulation of metabolites of the BCAA synthesis pathway.