title: 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.
authors.affiliation: Institute of Biochemical Engineering, University of Stuttgart, Stuttgart, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Buchholz
authors.forename: Andreas
authors.initials: A
authors.lastname: Schwentner
authors.forename: Britta
authors.initials: B
authors.lastname: Brunnenkan
authors.forename: Christina
authors.initials: C
authors.lastname: Gabris
authors.forename: Simon
authors.initials: S
authors.lastname: Grimm
authors.forename: Robert
authors.initials: R
authors.lastname: Gerstmeir
authors.forename: Ralf
authors.initials: R
authors.lastname: Takors
authors.forename: Bernhard J
authors.initials: BJ
authors.lastname: Eikmanns
authors.forename: Bastian
authors.initials: B
authors.lastname: Blombach
mesh_terms: D018533:Biomass; D048230:Corynebacterium glutamicum / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D015536:Down-Regulation; D017353:Gene Deletion; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D045782:Hemiterpenes; D007651:Keto Acids / Q000378:metabolism*; D008239:Lysine / Q000096:biosynthesis*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics; D011401:Promoter Regions, Genetic; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics*; D011995:Recombination, Genetic; D014633:Valine / Q000096:biosynthesis*
chemical_list: D045782:Hemiterpenes; D007651:Keto Acids; D011768:Pyruvate Dehydrogenase Complex; C001505:alpha-ketoisovalerate; D014633:Valine; D005947:Glucose; D008239:Lysine