Development of a Biosensor for Crotonobetaine-CoA Ligase Screening Based on the Elucidation of

l-Carnitine is essential in the intermediary metabolism of eukaryotes and is involved in the β-oxidation of medium- and long-chain fatty acids; thus, it has applications for medicinal purposes and as a dietary supplement. In addition, l-carnitine plays roles in bacterial physiology and metabolism, which have been exploited by the industry to develop biotechnological carnitine production processes. Here, on the basis of studies of l-carnitine metabolism in Escherichia coli and its activation by the transcriptional activator CaiF, a biosensor was developed. It expresses a fluorescent reporter gene that responds in a dose-dependent manner to crotonobetainyl-CoA, which is an intermediate of l-carnitine metabolism in E. coli and is proposed to be a coactivator of CaiF. Moreover, a dual-input biosensor for l-carnitine and crotonobetaine was developed. As an application of the biosensor, potential homologues of the betaine:CoA ligase CaiC from Citrobacter freundii, Proteus mirabilis, and Arcobacter marinus were screened and shown to be functionally active CaiC variants. These variants and the developed biosensor may be valuable for improving l-carnitine production processes.