Link between primary and secondary metabolism in the biotransformation of trimethylammonium compounds by escherichia coli

Abstract

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.

Plasmid maintenance and physiology of a genetically engineered Escherichia coli strain during continuous l-carnitine production

“…Furthermore, cell state and biotransformation capacity were correlated, maximum productivities being observed in actively growing cells, coinciding with the ''healthier'' cell state. This effect, observed for both freely suspended and immobilized cells, can easily be interpreted by bearing in mind the energydependence of transport and activation of trimethylammonium compounds (Cánovas et al 2003), which makes the biotransformation more likely to operate in actively growing cells.…”

“…The complex and minimal media based on bacteriological peptone and glycerol (carbon source) and only glycerol (carbon source, respectively, employed have been described previously (Cánovas et al 2003). The media were supplemented with 100 mM crotonobetaine as inducer and substrate for the biotransformation; ampicillin and kanamycin were used at 100 and 50 mg/ml.…”

“…L-Carnitine and crotonobetaine concentrations were determined enzymatically with the carnitine acetyl transferase method as already described (Cánovas et al 2003).…”

“…Crotonobetaine (dehydrated carnitine) was employed as substrate and fumarate as alternative electron acceptor and inhibitor of crotonobetaine reductase activity (Cánovas et al 2003). Optimized conditions included the use of minimal medium, 378C and 30% oxygen saturation (Cánovas et al 2005).…”

“…The cai operon products are involved in L-carnitine metabolism (Eichler et al 1994;Elssner et al 2001), the biotransformation being possible provided that the formation of the by-product c-butyrobetaine is inhibited by fumarate or O 2 ( Fig. 1; Cánovas et al 2003Cánovas et al , 2005.…”