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 ⌬pqo ⌬ppc (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.

A novel aceE mutation leading to a better growth profile and a higher l-serine production in a high-yield l-serine-producing Corynebacterium glutamicum strain

“…The aceE-deleted strains were deprived of PDHC activity completely and could not grow on glucose without acetate [1][2][3], and the resulting aceE-deleted strains displayed growth-decoupled production. Besides performing gene inactivation, the aceE expression was also weakened by being regulated by a weak promoter PdapA16, and the resulting strain showed an 80 % reduction in PDHC activity and a 31 % reduction in growth rate [5]. All the strains with the aceE deletion or promoter replacement showed a significant enhancement of production but a slower growth rate.…”

“…Protein concentrations were measured with a Bio-Rad protein assay kit (Bio-Rad Laboratories) [4]. PDGH activity was measured according to a previous report with some modifications [5]. It was measured in 50 mM phosphate buffer (pH 7.0) containing 12.5 mM MgCl 2 , 0.18 mM thiamine pyrophosphate, 0.175 mM CoA, 2 mM NAD + , and 5 mM pyruvate.…”

A giant market and a powerful metabolism: l-lysine provided by Corynebacterium glutamicum

“…Pyruvate dehydrogenase is part of a multienzyme complex identified in Corynebacteriales consisting of at least five proteins which has also oxo-glutarate dehydrogenase activity (Hoffelder et al 2010;Niebisch et al 2006). Reduction of aceE expression, encoding the E1p subunit of the pyruvate dehydrogenase, resulted in increased L-lysine synthesis and other pyruvate-derived products (Buchholz et al 2013). It is not surprising that reduction of acetohydroxy acid synthase consuming two pyruvate molecules for L-valine synthesis also results in increased L-lysine formation (Blombach et al 2009).…”

Corynebacterium glutamicum als Produzent von Pyruvat-abgeleiteten Produkten

“…Um die Acetatabhängigkeit beim Wachstum zu umgehen und möglicherweise die Produktionsphase auf die Wachstumsphase auszuweiten, generierten J. Buchholz et al [13] durch Austausch des nativen aceEPromotors im Genom von C. glutamicum gegen schwächere Promotoren Stämme mit abgestufter, abnehmender PDHC-Aktivität und versahen einen dieser Stämme (C. glutamicum aceEA16) mit den oben beschriebenen Modifikationen für die L-Valin-bzw. die KIVProduktion.…”

“…Angegeben sind die Genotypen der konstruierten Stämme. A, C modifiziert aus [7,12], © American Society of Microbiology; B, D modifiziert aus [9,13]. …”