fumarates and crotonic-acid-betaine

The aim of this work was to understand the steps controlling the biotransformation of trimethylammonium compounds into L(-)-carnitine by Escherichia coli. The high-cell density reactor steady-state levels of carbon source (glycerol), biotransformation substrate (crotonobetaine), acetate (anaerobiosis product) and fumarate (as an electron acceptor) were pulsed by increasing them fivefold. Following the pulse, the evolution of the enzyme activities involved in the biotransformation process of crotonobetaine into L(-)-carnitine (crotonobetaine hydration), in the synthesis of acetyl-CoA (ACS: acetyl-CoA synthetase and PTA: ATP: acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid (ICDH: isocitrate dehydrogenase) and glyoxylate (ICL: isocitrate lyase) cycles was monitored. In addition, the levels of carnitine, the cell ATP content and the NADH/NAD(+) ratio were measured in order to assess the importance and participation of these energetic coenzymes in the catabolic system. The results provided an experimental demonstration of the important role of the glyoxylate shunt during biotransformation and the need for high levels of ATP to maintain metabolite transport and biotransformation. Moreover, the results obtained for the NADH/NAD(+) pool indicated that it is correlated with the biotransformation process at the NAD(+) regeneration and ATP production level in anaerobiosis. More importantly, a linear correlation between the NADH/NAD(+) ratio and the levels of the ICDH and ICL (carbon and electron flows) and the PTA and ACS (acetate and ATP production and acetyl-CoA synthesis) activity levels was assessed. The main metabolic pathway operating during cell metabolic perturbation with a pulse of glycerol and acetate in the high-cell density membrane reactor was that related to ICDH and ICL, both regulating the carbon metabolism, together with PTA and ACS enzymes (regulating ATP production).

Topics: Acetates; Adenosine Triphosphate; Betaine; Biomedical Engineering; Bioreactors; Biosynthetic Pathways; Biotechnology; Biotransformation; Carnitine; Coenzymes; Escherichia coli; Fumarates; Glycerol; NAD; Trimethyl Ammonium Compounds

A simple unstructured model, which includes carbon source as the limiting and essential substrate and oxygen as an enhancing substrate for cell growth, has been implemented to depict cell population evolution of two Escherichia coli strains and the expression of their trimethylammonium metabolism in batch and continuous reactors. Although the model is applied to represent the trans-crotonobetaine to L-(-)-carnitine biotransformation, it is also useful for understanding the complete metabolic flow of trimethylammonium compounds in E. coli. Cell growth and biotransformation were studied in both anaerobic and aerobic conditions. For this reason we derived equations to modify the specific growth rate, mu, and the cell yield on the carbon source (glycerol), Y(xg), as oxygen increased the rate of growth. Inhibition functions representing an excess of the glycerol and oxygen were included to depict cell evolution during extreme conditions. As a result, the model fitted experimental data for various growth conditions, including different carbon source concentrations, initial oxygen levels, and the existence of a certain degree of cell death. Moreover, the production of enzymes involved within the E. coli trimethylammonium metabolism and related to trans-crotonobetaine biotransformation was also modeled as a function of both the cell and oxygen concentrations within the system. The model describes all the activities of the different enzymes within the transformed and wild strains, able to produce L-(-)-carnitine from trans-crotonobetaine under both anaerobic and aerobic conditions. Crotonobetaine reductase inhibition by either oxygen or the addition of fumarate as well as its non-reversible catalytic action was taken into consideration. The proposed model was useful for describing the whole set of variables under both growing and resting conditions. Both E. coli strains within membrane high-density reactors were well represented by the model as results matched the experimental data.

Topics: Algorithms; Betaine; Bioreactors; Carnitine; Computer Simulation; Escherichia coli; Fumarates; Membranes; Models, Biological; Multienzyme Complexes; Oxidoreductases; Time Factors

L(-)-carnitine was produced from D(+)-carnitine by resting cells of Escherichia coli O44 K74. Oxygen did not inhibit either the carnitine transport system or the enzymes involved in the biotransformation process. Aerobic conditions led to higher product yield than anaerobic conditions. The biotransformation yield depended both on biomass and initial substrate concentrations used; the selected values for these variables were 4.30 g l-1 cells and 100 mmol l-1 D(+)-carnitine. Under these conditions the L(-)-carnitine production rate was 0.55 g l-1 h-1, the process yield was 44%, and the productivity was 0.22 g l-1 h-1 after a 30 h incubation period. Crotonobetaine production, besides L(-)-carnitine, showed that the action of more than one enzyme occurred during the biotransformation process. On the other hand, the addition of fumarate at high substrate concentrations (250 and 500 mmol l-1) led to a higher metabolic activity, which meant an increment of L(-)-carnitine production.

Topics: Aerobiosis; Anaerobiosis; Betaine; Biomass; Biotransformation; Carnitine; Chromatography, High Pressure Liquid; Escherichia coli; Fumarates; Hydrogen-Ion Concentration; Temperature; Time Factors