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Aims:  Characterization of the role of CaiC in the biotransformation of trimethylammonium compounds into l(−)‐carnitine in Escherichia coli. Methods and Results:  The caiC gene was cloned and overexpressed in E. coli and its effect on the production of l(−)‐carnitine was analysed. Betaine:CoA ligase and CoA transferase activities were analysed in cell free extracts and products were studied by electrospray mass spectrometry (ESI‐MS). Substrate specificity of the caiC gene product was high, reflecting the high specialization of the carnitine pathway. Although CoA‐transferase activity was also detected in vitro, the main in vivo role of CaiC was found to be the synthesis of betainyl‐CoAs. Overexpression of CaiC allowed the biotransformation of crotonobetaine to l(−)‐carnitine to be enhanced nearly 20‐fold, the yield reaching up to 30% (with growing cells). Higher yields were obtained using resting cells (up to 60%), even when d(+)‐carnitine was used as substrate. Conclusions:  The expression of CaiC is a control step in the biotransformation of trimethylammonium compounds in E. coli. Significance and Impact of the Study:  A bacterial betaine:CoA ligase has been characterized for the first time, underlining its important role for the production of l‐carnitine with Escherichia coli.

BackgroundL(-)-carnitine production has been widely studied because of its beneficial properties on various diseases and dysfunctions. Enterobacteria possess a specific biotransformation pathway which can be used for the enantioselective production of L(-)-carnitine. Although bioprocesses catalyzed by enzymes or whole cells can overcome the lack of enantioselectivity of chemical methods, current processes for L(−)-carnitine production still have severe disadvantages, such as the low yields, side reactions and the need of high catalyst concentrations and anaerobic conditions for proper expression of the biotransformation pathway. Additionally, genetically engineered strains so far constructed for L(-)-carnitine production are based on plasmids and, therefore, suffer from segregational unstability.ResultsIn this work, a stable, high yielding strain for L(-)-carnitine production from low cost substrates was constructed. A metabolic engineering strategy was implemented in a multiple mutant for use in both growing and resting cells systems. The effect of mutations on gene expression and metabolism was analyzed to characterize the productivity constraints of the wild type and the overproducer strains. Precise deletion of genes which encode proteins of central and carnitine metabolisms were performed. Specifically, flux through the TCA cycle was increased by deletion of aceK (which encodes a bifunctional kinase/phosphatase which inhibits isocitrate dehydrogenase activity) and the synthesis of the by-product γ-butyrobetaine was prevented by deletion of caiA (which encodes a crotonobetainyl-CoA reductase). Both mutations led to improve the L(-)-carnitine production by 20 and 42%, respectively. Moreover, the highly regulated promoter of the cai operon was substituted by a constitutive artificial promoter increasing the biotransformation rate, even under aerobic conditions. Resting cells of the BW ΔaceK ΔcaiA p37cai strain produced 59.6 mmol l-1 · h-1 of L(−)-carnitine, doubling the productivity of the wild type strain. In addition, almost total conversion was attained in less than two hours without concomitant production of the side product γ–butyrobetaine.ConclusionsL(-)-carnitine production has been enhanced by strain engineering. Metabolic engineering strategies herein implemented allowed obtaining a robust and high yielding E. coli strain. The new overproducer strain attained almost complete conversion of crotonobetaine into L(-)-carnitine with growing and resting cells, and even under aerobic conditions, overcoming the main environmental restriction to carnitine metabolism expression. So far, this is the best performing L(-)-carnitine production E. coli strain described.

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