Metabolic flux analysis of wild-type Escherichia coli and mutants deficient in pyruvate-dissimilating enzymes during the fermentative metabolism of glucuronate

Abstract

The fermentative metabolism of D-glucuronic acid (glucuronate) in Escherichia coli was investigated with emphasis on the dissimilation of pyruvate via pyruvate formate-lyase (PFL) and pyruvate dehydrogenase (PDH). In silico and in vivo metabolic flux analysis (MFA) revealed that PFL and PDH share the dissimilation of pyruvate in wild-type MG1655. Surprisingly, it was found that PDH supports fermentative growth on glucuronate in the absence of PFL. The PDHdeficient strain (Pdh") exhibited a slower transition into the exponential phase and a decrease in specific rates of growth and glucuronate utilization. Moreover, a significant redistribution of metabolic fluxes was found in PDH-and PFL-deficient strains. Since no role had been proposed for PDH in the fermentative metabolism of E. coli, the metabolic differences between MG1655 and Pdh" were further investigated. An increase in the oxidative pentose phosphate pathway (ox-PPP) flux was observed in response to PDH deficiency. A comparison of the ox-PPP and PDH pathways led to the hypothesis that the role of PDH is the supply of reducing equivalents. The finding that a PDH deficiency lowers the NADH : NAD + ratio supported the proposed role of PDH. Moreover, the NADH : NAD + ratio in a strain deficient in both PDH and the ox-PPP (Pdh"Zwf") was even lower than that observed for Pdh". Strain Pdh"Zwf" also exhibited a slower transition into the exponential phase and a lower growth rate than Pdh". Finally, a transhydrogenase-deficient strain grew more slowly than wild-type but did not show the slower transition into the exponential phase characteristic of Pdh" mutants. It is proposed that PDH fulfils two metabolic functions. First, by creating the appropriate internal redox state (i.e. appropriate NADH : NAD + ratio), PDH ensures the functioning of the glucuronate utilization pathway. Secondly, the NADH generated by PDH can be converted to NADPH by the action of transhydrogenases, thus serving as biosynthetic reducing power in the synthesis of building blocks and macromolecules.

Identification of a High-Affinity Pyruvate Receptor in Escherichia coli

“…coli strains have focused on metabolic engineering, aiming to understand how processes such as elevated intracellular pyruvate levels affect metabolite distribution 30, 31 or how central mutations trigger/alter metabolic fluxes 32–34 . In recent years, technological advances have permitted detailed analyses of in vivo metabolism 35 , revealing its complexity and its influence on virulence and pathogenesis 36 .…”

Improvement of 2,3-Butanediol Yield in Klebsiella pneumoniae by Deletion of the Pyruvate Formate-Lyase Gene

“…1). In E. coli, the pflB deletion and its metabolic effects have been intensively investigated to enhance the yield of target products (35)(36)(37). Therefore, flask cultivation of pflB-deficient E. coli was compared with that of K. pneumoniae under microaerobic conditions for 24 h (Fig.…”

Metabolic engineering of Escherichia coli for the production of 1,2‐propanediol from glycerol

“…PDH, which converts pyruvate to acetyl-CoA with the generation of CO 2 and NADH, is typically active under respiratory conditions and the absence of activity under anaerobic conditions has been explained by its sensitivity to NADH inhibition (de Graef et al, 1999;Sawers and Clark, 2004). However, it has recently been shown that PDH plays a significant role in fermentative metabolism of glucose and glucuronate indicating that it can function under anaerobic conditions (Murarka et al, 2010a(Murarka et al, , 2010b. In addition, the highly reduced nature of the product distribution in the engineered E. coli strain (Table IV) may result in a lower NADH/NAD ratio, which could relieve some of the NADH inhibition on PDH.…”

“…The activity of methylglyoxal synthase was determined using a colorimetric assay (Berrios-Rivera et al, 2003). The activity of pyruvate dehydrogenase was measured as previously reported (Murarka et al, 2010a).…”