Salt stress effects on the central and carnitine metabolisms of Escherichia coli

Abstract

The aim was to understand how interaction of the central carbon and the secondary carnitine metabolisms is affected under salt stress and its effect on the production of L-carnitine by Escherichia coli. The biotransformation of crotonobetaine into L-carnitine by resting cells of E. coli O44 K74 was improved by salt stress, a yield of nearly twofold that for the control being obtained with 0.5 M NaCl. Crotonobetaine and the L-carnitine formed acted as an osmoprotectant during cell growth and biotransformation in the presence of NaCl. The enzyme activities involved in the biotransformation process (crotonobetaine hydration reaction and crotonobetaine reduction reaction), in the synthesis of acetyl-CoA/acetate (pyruvate dehydrogenase, acetyl-CoA synthetase [ACS] and ATP/acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid cycle (isocitrate dehydrogenase [ICDH]) and glyoxylate shunt (isocitrate lyase [ICL]) were followed in batch with resting cells both in the presence and absence of NaCl and in perturbation experiments performed on growing cells in a high density cell recycle membrane reactor. Further, the levels of carnitine, crotonobetaine, gamma-butyrobetaine and ATP and the NADH/NAD(+) ratio were measured in order to know how the metabolic state was modified and coenzyme pools redistributed as a result of NaCl's effect on the energy content of the cell. The results provided the first experimental evidence of the important role played by salt stress during resting and growing cell biotransformation (0.5 M NaCl increased the L-carnitine production in nearly 85%), and the need for high levels of ATP to maintain metabolite transport and biotransformation. Moreover, the main metabolic pathways and carbon flow operating during cell biotransformation was that controlled by the ICDH/ICL ratio, which decreased from 8.0 to 2.5, and the phosphotransferase/ACS ratio, which increased from 2.1 to 5.2, after a NaCl pulse fivefold the steady-state level. Resting E. coli cells were seen to be made up of heterogeneous populations consisting of several types of subpopulation (intact, depolarized, and permeabilized cells) differing in viability and metabolic activity as biotransformation run-time and the NaCl concentration increased. The results are discussed in relation with the general stress response of E. coli, which alters the NADH/NAD(+) ratio, ATP content, and central carbon enzyme activities.

Model identification in presence of incomplete information by generalized principal component analysis: Application to the common and differential responses of Escherichia coli to multiple pulse perturbations in continuous, high‐biomass density culture

“…Although the latter justifies the production of formate, it cannot explain the observed correlation with ATP. Point (ii) is explained because ATP is required for crotonobetaine transport and for carnitine biosynthesis (Cánovas et al, 2006(Cánovas et al, , 2007. Point (iii) implies that, under the tested conditions, carnitine biosynthesis is far from being limited by the CHR enzyme.…”

“…wiley.com. ] In our case, the possible existence of some systematic bias in the pulse experiments (Cánovas et al, 2006(Cánovas et al, , 2007 led us to disregard any dissimilarity due to differences in the response intensities. Instead, we focused in comparing the structure of their covariance across the perturbation conditions.…”

“…Using alternative methods, we herein assess the experimental data series gathered by Cánovas et al (2006Cánovas et al ( , 2007 and already analyzed by us (Guebel et al, 2009). Detailed descriptions of the microorganism (E. coli O74K74), the culture technique (anaerobic chemostat with recycling biomass), the pulse perturbations applied (glycerol, fumarate, acetate, crotonobetaine, hypersaline plus high-glycerol basal medium, and crotonobetaine plus hypersaline basal medium) are given in these reports.…”

“…The experimental conditions are: Group 1 (glycerol pulse); Group 2 (fumarate pulse); Group 3 (crotonobetaine pulse); Group 4 (acetate pulse); Group 5 (hypersaline (NaCl) pulse plus high glycerol basal medium); Group 6 (Crotonobetaine pulse plus hypersaline (NaCl) basal medium). Raw experimental data are taken from Cánovas et al (2006Cánovas et al ( , 2007. For each monitored variable (V j , 1 j 11) we computed the percent of variance (var(V j ) E(k) .100/var(V j ) W(k) ) that can be extracted after a cycle of deflation by a common GPCA factor (E ðkÞ ¼ W ðkÞ À t superscoreðkÞ p T ðkÞ ; where 1 k 6).…”

Cofactor Engineering Enhances the Physiological Function of an Industrial Strain

“…In Streptococcus, mutants deficient in the minimally conserved bacterial signal recognition particle (SRP) elements remain viable but are more sensitive to environmental stress because the SRP deficiency decreases ATPase activity and limits the ATP supply [40,41] . It was found that industrial strain grown on high environmental stress have a high ATP demand [42] and increase ATP supply [43] could well facilitate the resistance to stress. However, most researches are focused on ATP-related mechanisms or phenomenon description during resistance to different stresses.…”