Cofactor engineering through heterologous expression of an NADH oxidase and its impact on metabolic flux redistribution in Klebsiella pneumoniae

Abstract

BackgroundAcetoin is an important bio-based platform chemical. However, it is usually existed as a minor byproduct of 2,3-butanediol fermentation in bacteria.ResultsThe present study reports introducing an exogenous NAD+ regeneration sysytem into a 2,3-butanediol producing strain Klebsiella pneumoniae to increse the accumulation of acetoin. Batch fermentation suggested that heterologous expression of the NADH oxidase in K. pneumoniae resulted in large decreases in the intracellular NADH concentration (1.4 fold) and NADH/NAD+ ratio (2.0 fold). Metabolic flux analysis revealed that fluxes to acetoin and acetic acid were enhanced, whereas, production of lactic acid and ethanol were decreased, with the accumualation of 2,3-butanediol nearly unaltered. By fed-batch culture of the recombinant, the highest reported acetoin production level (25.9 g/L) by Klebsiella species was obtained.ConclusionsThe present study indicates that microbial production of acetoin could be improved by decreasing the intracellular NADH/NAD+ ratio in K. pneumoniae. It demonstrated that the cofactor engineering method, which is by manipulating the level of intracellular cofactors to redirect cellular metabolism, could be employed to achieve a high efficiency of producing the NAD+-dependent microbial metabolite.

Involvement of the NADH oxidase-encoding noxA gene in oxidative stress responses in Corynebacterium glutamicum

“…Considering the role of C. glutamicum cells in metabolite overproduction and the importance of cofactor regeneration such as NAD + /NADH cycling in metabolic engineering (Hou et al 2014;Ji et al 2013;Rocha-Martin et al 2011;Zhang et al 2014), the noxA gene may be of importance in the field of biotechnology. Toward fully elucidating the function of noxA in C. glutamicum physiology, additional investigations are underway.…”

Enhanced production of 2,3‐butanediol by overexpressing acetolactate synthase and acetoin reductase in Klebsiella pneumoniae

“…Except for KG-rs, all the recombinant mutants exhibited slow growth rate and high by-product yield; such factors may be the reasons why the 2,3-BD yields did not significantly increase. Previous studies have shown that protein overproduction does not always increase the corresponding product yields; on the contrary, this overproduction may be detrimental to cell growth [18,31]. The same phenomenon was observed in other strains, such as Saccharomyces cerevisiae [25], in which only the constitutive expression of protein can dramatically increase the cell growth and improve the fermentation efficiency.…”

“…Metabolic engineering is widely used to improve the properties of microbial strains, such as manipulating the key enzyme levels through the amplification, disruption, or addition of metabolic pathways [18,[24][25][26]. This study demonstrates that the overexpression of the key enzymes for 2,3-BD biosynthesis caused by a strong promoter increases 2,3-BD production in K. pneumoniae.…”

“…The intracellular NADH and nicotinamide adenine dinucleotide (NAD + ) concentrations were measured by procedures presented by Ji et al [18]. The concentrations of glucose, 2,3-BD, acetic acid, lactic acid, alcohol, and acetoin were determined using high-performance liquid chromatography (Agilent 1260 Infinity LC; Agilent, Palo Alto, CA, USA; Shodex RI-101 Refractive Index Detector, G1362A; Agilent, Palo Alto, CA, USA;…”

Improved bioethanol production from metabolic engineering of Enterobacter aerogenes ATCC 29007

“…An interesting benefit of this ldhA deletion was an increase in microbial cell growth [22]. Generally, a metabolically engineered microorganism has reduced cell growth compared with the wild type strain [32,33]. The increased microbial cell growth of the mutant could be largely attributed to the reduced production of lactic acid, which lowers the acidification rate of the media [34].…”