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 Δpqo Δppc (pJC4 ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter) l-valine with an overall yield (YP/S) of 0.36 mol per mol of glucose and a volumetric productivity (QP) 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 YP/S of 0.24 mol per mol of glucose and a QP 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.

Fig 1

Enzymes of the central metabolism with the biosynthetic pathway of 2-ketoisovalerate/l-valine and l-lysine in C. glutamicum. Abbreviations: AHAIR, acetohydroxyacid isomeroreductase; AHAS, acetohydroxyacid synthase; AK, acetate kinase; DHAD, dihydroxyacid dehydratase; PCx, pyruvate carboxylase; PDHC, pyruvate dehydrogenase complex; PEP, phosphoenolpyruvate; PEPCk, PEP carboxykinase; PEPCx, PEP carboxylase; PK, pyruvate kinase; PTA, phosphotransacetylase; PQO, pyruvate:quinone oxidoreductase; TA, transaminase B; TCA, tricarboxylic acid.

Fig 2

(A) Growth of C. glutamicum WT and its derivatives with reduced (A16, A23, A25, and L1) or abolished (C. glutamicum ΔaceE) PDHC activity in shake flasks containing CGXII medium with 222 mM glucose. □, C. glutamicum WT; ▲, C. glutamicum ΔaceE; △, C. glutamicum aceE A16; ●, C. glutamicum aceE A25; ♢, C. glutamicum aceE A23; ■, C. glutamicum aceE L1. (B) Specific PDHC activities of C. glutamicum WT and its derivatives with reduced PDHC activity grown in shake flasks containing CGXII medium with 222 mM glucose. Three independent fermentations were performed. Error bars show standard deviations.

Fig 3

Growth, glucose consumption, and l-alanine and l-valine formation by C. glutamicum aceE A16 (pJC4 ilvBNCE) cultivated in shake flasks with CGXII medium containing 222 mM glucose. ▼, OD₆₀₀; □, glucose; ♢, l-alanine; ●, l-valine. Three independent fermentations were performed. Error bars show standard deviations.

Fig 4

Representative fed-batch fermentation of C. glutamicum aceE A16 Δpqo Δppc (pJC4 ilvBNCE) in CGXII medium initially containing about 333 mM glucose. ▼, OD₆₀₀; □, glucose; ♢, l-alanine; ●, l-valine; ○, phosphate.

Fig 5

Representative fed-batch fermentation of C. glutamicum aceE A16 Δpqo Δppc ΔilvE (pJC4 ilvBNCD) in CGXII medium initially containing about 333 mM glucose, 1% (wt/vol) yeast extract, and 10 mM l-valine, l-isoleucine, and l-leucine. ▼, OD₆₀₀; □, glucose; ♢, pyruvate; ●, 2-ketoisovalerate.