The aim of this work was to understand the steps controlling the process of biotransformation of trimethylamonium compounds into L(-)-carnitine by Escherichia coli and the link between the central carbon or primary and the secondary metabolism expressed. Thus, the enzyme activities involved in the biotransformation process of crotonobetaine into L(-)-carnitine (crotonobetaine hydration reaction and crotonobetaine reduction reaction), in the synthesis of acetyl-CoA (pyruvate dehydrogenase, acetyl-CoA synthetase, and ATP:acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid (isocitrate dehydrogenase) and glyoxylate (isocitrate lyase) cycles, were followed in batch with both growing and resting cells and during continuous cell growth in stirred-tank and high-cell-density membrane reactors. In addition, the levels of carnitine, crotonobetaine, gamma-butyrobetaine, ATP, NADH/NAD(+), and acetyl-CoA/CoA ratios were measured to determine how metabolic fluxes were distributed in the catabolic system. The results provide the first experimental evidence demonstrating the important role of the glyoxylate shunt during biotransformation of resting cells and the need for high levels of ATP to maintain metabolite transport and biotransformation (2.1 to 16.0 mmol L cellular/mmol ATP L reactor h). Moreover, the results obtained for the pool of acetyl-CoA/CoA indicate that it also correlated with the biotransformation process. The main metabolic pathway operating during cell growth in the high cell-density membrane reactor was that related to isocitrate dehydrogenase (during start-up) and isocitrate lyase (during steady-state operation), together with phosphotransacetylase and acetyl-CoA synthetase. More importantly, the link between central carbon and L(-)-carnitine metabolism at the level of the ATP pool was also confirmed.
The biotransformation of crotonobetaine and D(+)-carnitine into L(-)-carnitine is affected by salt stress in the resting cells of E. coli O44 K74 and the transformed E. coli K38 pT7-5KE32. A yield of 65 and 80% of L(-)-carnitine, respectively, were obtained with 0.5 M NaCl with the wild and transformed strain compared with the 40% obtained with the control. Higher salt levels reduced the conversion. In L(-)-carnitine transport studies using both strains, the transformed strain presented slightly lower apparent K(m) and V values. Arsenate reduced both the transport and biotransformation of crotono-betaine in the presence or absence of 0.5 M NaCl, whereas vanadate only inhibited these processes under salt stress conditions. Hg(II) inhibited both the transport and biotransformation and Pb(II) reduced the biotransformation only under salt stress conditions. Cu(II) produced a significantly higher decrease than Pb(II) in the biotransformation with both substrates in the absence of salt stress conditions, but only affected transport in the presence of such conditions. Furthermore, salt stress affected the CaiT transporter for L(-)-carnitine and crotonobetaine and induced ProU and ProP in the absence of the inducer of the L(-)-carnitine metabolism. It is highly likely that the increase in L(-)-carnitine production was not only due to improved transport but also to the permeabilization effect caused by NaCl, as transport and 1-N-phenylnaphthylamine uptake studies revealed.
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