Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of l -Lysine, l -Valine, and 2-Ketoisovalerate

ABSTRACT Exchange of the native Corynebacterium glutamicum promoter of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutated dapA promoter variants led to a series of C. glutamicum strains with gradually reduced growth rates and PDHC activities. Upon overexpression of the l -valine biosynthetic genes ilvBNCE , all strains produced l -valine. Among these strains, C. glutamicum aceE A16 (pJC4 ilvBNCE ) showed the highest biomass and product yields, and thus it was further improved by additional deletion of the pqo and ppc genes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities, C. glutamicum aceE A16 Δ pqo Δppc (pJC4 ilvBNCE ) produced up to 738 mM (i.e., 86.5 g/liter) l -valine with an overall yield ( Y P/S ) of 0.36 mol per mol of glucose and a volumetric productivity ( Q P ) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene ( ilvE ) and overexpression of ilvBNCD instead of ilvBNCE transformed the l -valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with a Y P/S of 0.24 mol per mol of glucose and a Q P of 6.9 mM per h [0.8 g/(liter × h)]. The replacement of the aceE promoter by the dapA -A16 promoter in the two C. glutamicum l -lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate that C. glutamicum strains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.