Disruption of genes for the enhanced biosynthesis of α-ketoglutarate in Corynebacterium glutamicum

Abstract

The development of microbial strains for the enhanced production of α-ketoglutarate (α-KG) was investigated using a strain of Corynebacterium glutamicum that overproduces of l-glutamate, by disrupting three genes involved in the α-KG biosynthetic pathway. The pathways competing with the biosynthesis of α-KG were blocked by knocking out aceA (encoding isocitrate lyase, ICL), gdh (encoding glutamate dehydrogenase, l-gluDH), and gltB (encoding glutamate synthase or glutamate-2-oxoglutarate aminotransferase, GOGAT). The strain with aceA, gltB, and gdh disrupted showed reduced ICL activity and no GOGAT and l-gluDH activities, resulting in up to 16-fold more α-KG production than the control strain in flask culture. These results suggest that l-gluDH is the key enzyme in the conversion of α-KG to l-glutamate; therefore, prevention of this step could promote α-KG accumulation. The inactivation of ICL leads the carbon flow to α-KG by blocking the glyoxylate pathway. However, the disruption of gltB did not affect the biosynthesis of α-KG. Our results can be applied in the industrial production of α-KG by using C. glutamicum as producer.

Crystal structure of the 2‐iminoglutarate‐bound complex of glutamate dehydrogenase from Corynebacterium glutamicum

“…GDH from C. glutamicum (CgGDH) is an NADPH-dependent enzyme [13], suggesting that CgGDH contributes to the overproduction of glutamate. This notion is supported by two reports: one showing that high level expression of CgGDH in C. glutamicum cells increased the intracellular glutamate pool [14] and the other showing that disruption of the CgGDH gene resulted in 2-OG accumulation accompanied by a simultaneous decrease in glutamate production [15]. Therefore, CgGDH is considered to be one of the key enzymes in the industrial fermentation of glutamate.…”

Bio‐based production of organic acids with Corynebacterium glutamicum

“…An enzymatic process to synthesize 2‐ketoglutarate from glutamate via the coupled reactions of glutamate dehydrogenase and NADH oxidase has been established (Ödmann et al ., ), however, this bioconversion seems not very efficient. Therefore, Jo and colleagues () very recently used a glutamate‐overproducing mutant of C. glutamicum for the construction of a 2‐ketoglutarate‐producer. Inactivation of the genes encoding glutamate dehydrogenase, glutamate synthase and isocitrate lyase ( gdh , gltB , and aceA respectively) led to a drastic reduction of glutamate formation (< 10 mM) and concomitantly to 2‐ketoglutarate accumulation to concentrations of up to 325 mM (47.5 g l −1 ) after 120 h of cultivation in medium containing glucose, molasses, glutamate, and soybean hydrolysate (Jo et al ., ).…”

“…Therefore, Jo and colleagues () very recently used a glutamate‐overproducing mutant of C. glutamicum for the construction of a 2‐ketoglutarate‐producer. Inactivation of the genes encoding glutamate dehydrogenase, glutamate synthase and isocitrate lyase ( gdh , gltB , and aceA respectively) led to a drastic reduction of glutamate formation (< 10 mM) and concomitantly to 2‐ketoglutarate accumulation to concentrations of up to 325 mM (47.5 g l −1 ) after 120 h of cultivation in medium containing glucose, molasses, glutamate, and soybean hydrolysate (Jo et al ., ). To our knowledge, there were no other approaches to produce 2‐ketoglutarate by fermentation with any other bacterium.…”

White Biotechnology for Organic Acids

“…80 When derivative recombinant strains Y. lipolytica ACS1 and ACL, which overexpress genes encoding acetyl-CoA synthetase and ATP-citrate lyase, respectively, were used, the α-KG concentrated produced increased up to 56.5 g/L; however, a high amount of pyruvic acid (20.2 g/L) was also produced as a byproduct. 82 A comparison of the yield and titer of α-KG produced by different strains is shown in Table 11.4. Although producing α-KG through microbial fermentation generates less environmental pollution than that produced by chemical synthesis, this approach is currently limited to laboratory research because of the high production cost.…”