Metabolic engineering of Corynebacterium glutamicum for 2-ketoisocaproate production

Abstract

2-Ketoisocaproate (KIC) is used as a therapeutic agent, and a KIC-producing organism may serve as a platform for products deriving from this 2-keto acid. We engineered Corynebacterium glutamicum for the production of KIC from glucose by deletion of ltbR and ilvE, encoding the transcriptional repressor LtbR and transaminase B, respectively, and additional overexpression of ilvBNCD, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. The KIC-producing strain was improved by deletion of the methylcitrate synthase genes and by decreasing citrate synthase activity by exchange of the native promoter of the citrate synthase gene. In shake-flask fermentations under L-leucine limitation, the newly constructed strain C. glutamicum VB (pJC4ilvBNCD) produced 31 ± 2 mM (4.0 ± 0.3 g l(-1)) KIC and showed a product yield of about 0.26 ± 0.02 mol per mole (0.19 ± 0.01 g per gram) of glucose. As by-product, the strain formed about 33 mM 2-ketoisovalerate, which is a precursor of KIC. KIC production was further improved by additional expression of an isopropylmalate synthase allele (leuA (EC-G462D)), encoding an enzyme resistant towards L-leucine inhibition, and by addition of acetate as additional substrate. With glucose and acetate, the newly constructed strain produced 71 ± 3.2 mM (9.2 ± 0.4 g l(-1)) KIC with a yield of 0.24 ± 0.01 mol C (KIC) per mole C (in both substrates) and with nearly no 2-ketoisovalerate by-product formation (<2 mM). Investigating the activities and regulation of the native isopropylmalate synthase and dehydratase of C. glutamicum, we observed competitive and noncompetitive inhibition, respectively, by KIC.

Metabolic engineering of Corynebacterium glutamicum for the production of itaconate

“…In the past decades, C. glutamicum strains were developed for a broad spectrum of other metabolites (Becker and Wittmann, 2012). Besides various amino acids, such as L-serine (Stolz et al, 2007), L-valine (Blombach et al, 2007;Hasegawa et al, 2013;Radmacher et al, 2002), L-isoleucine (Vogt et al, 2014c), or L-leucine (Vogt et al, 2014a), this spectrum also included organic acids (Wieschalka et al, 2013), such as D-lactate (Okino et al, 2008b), succinate (Litsanov et al, 2012a(Litsanov et al, , 2012b(Litsanov et al, , 2013(Litsanov et al, , 2014Okino et al, 2008a), pyruvate (Wieschalka et al, 2012), 2-ketoisovalerate (Krause et al, 2010), or 2-ketoisocaproate (Bückle-Vallant et al, 2014;Vogt et al, 2014b). The production parameters of several of these strains were highly competitive with alternative production hosts or represented best-in-class examples, which motivated us to study itaconate production with C. glutamicum.…”

One‐step Synthesis of α‐Keto Acids from Racemic Amino Acids by A Versatile Immobilized Multienzyme Cell‐free System

“…[9,12,35] The production of a-keto acids by whole-cells of Corynebacterium glutamicum, Rhodococcus opacus DSM 43 250 and E. coli expressing an engineered l-amino acid oxidase (LAAO)f rom Proteous vulgaris has been previously reported. Although, strains are only modified by overexpression or knockout of operons or particularg enes, andi nm any cases without achieving an efficient regulation of the metabolic pathways.…”

“…[7] Consequently, there is considerable interest in the developmento fe co-friendly alternatives fort he production of a-keto acids. [9] The use of soluble enzymest op roduce aketo acids has as one of the main disadvantages the previous purification of the enzyme that increases the cost of the bioconversion regarding the whole-cell method. [8] In general,d irect fermentation uses low-priced carbon sources, but it has low productivity.W hole-cellb iocatalyst has several advantages such as high yield and productivity.H owever,t his methodr equires the genetic manipulationo fm etabolic pathways of am icroorganism through metabolic engineering.…”

Corynebacterium glutamicum als Produzent von Pyruvat-abgeleiteten Produkten

“…1). Aus diesem Grund wurde ein KIC-Produzent auf der Basis eines C. glutamicum-Stamms mit abgeschwächter Citrat-Synthase entwickelt [11]. Dem letztlich entwickelten Stamm fehlen die Gene für die Transaminase B, für die beiden Methylcitrat-Synthasen und für einen Regulator, der die Expression der Gene für die Leucin-Biosyntheseenzyme reprimiert.…”