APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY Reconstruction of the carnitine biosynthesis pathway from Neurospora crassa in the yeast Saccharomyces cerevisiae Jaco Franken 1 & Anita Burger 1,2 & Jan H. Swiegers 1 & Florian F. Bauer 1 Received: 15 February 2015 /Revised: 11 March 2015 /Accepted: 19 March 2015 /Published online: 9 April 2015 # Springer-Verlag Berlin Heidelberg 2015 Abstract Industrial synthesis of L-carnitine is currently per- formed by whole-cell biotransformation of industrial waste products, mostly D-carnitine and cronobetaine, through spe- cific bacterial species. No comparable system has been established using eukaryotic microorganisms, even though there is a significant and growing international demand for either the pure compound or carnitine-enriched consumables. In eukaryotes, including the fungus Neurospora crassa, L-car- nitine is biosynthesized through a four-step metabolic conver- sion of trimethyllysine to L-carnitine. In contrast, the industrial yeast, Saccharomyces cerevisiae lacks the enzymes of the eu- karyotic biosynthesis pathway and is unable to synthesize car- nitine. This study describes the cloning of all four of the N. crassa carnitine biosynthesis genes and the reconstruction of the entire pathway in S. cerevisiae. The engineered yeast strains were able to catalyze the synthesis of L-carnitine, which was quantified using hydrophilic interaction liquid chroma- tography electrospray ionization mass spectrometry (HILIC- ESI-MS) analyses, from trimethyllysine. Furthermore, the yeast threonine aldolase Gly1p was shown to effectively cat- alyze the second step of the pathway, fulfilling the role of a serine hydroxymethyltransferase. The analyses also identified yeast enzymes that interact with the introduced pathway, including Can1p, which was identified as the yeast transporter for trimethyllysine, and the two yeast serine hydroxymethyltransferases, Shm1p and Shm2p. Together, this study opens the possibility of using an engineered, carnitine-producing yeast in various industrial applications while providing insight into possible future strategies aimed at tailoring the production capacity of such strains. Keywords Carnitine . Biosynthesis . Neurospora crassa . Saccharomyces cerevisiae Introduction L-carnitine is a vitamin-like molecule with generally recog- nized as safe (GRAS) status that plays an essential and con- served shuttling function in eukaryotes by transferring activat- ed acyl residues across intracellular membranes (Bieber 1988). This shuttling function is supported by the activities of various carnitine acyltransferases and transporters, whose activity is collectively referred to as the carnitine shuttle (reviewed in Ramsay et al. 2001). The importance of this compound and of the carnitine shuttle in eukaryotic energy metabolism has been highlighted by many studies linking di- verse metabolic diseases to a dysfunction of carnitine-related metabolism (reviewed in Longo et al. 2006). These disorders are, in many cases, effectively treated through dietary carni- tine supplementation (Winter 2003). Studies investigating the impact of carnitine supplementa- tion have furthermore indicated that advantageous effects can be observed in patients with certain types of cardiological, neurological and/or renal disorders (Calabrese et al. 2006; DiNicolantonio et al. 2013; Bonafé et al. 2014; Chen et al. 2014). Aside from carnitine’s therapeutic potential, the Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6561-x) contains supplementary material, which is available to authorized users. * Florian F. Bauer fb2@sun.ac.za 1 Department of Viticulture and Oenology, Institute for Wine Biotechnology, Stellenbosch University, Matieland 7602, South Africa 2 Present address: Industrial Biotechnology Sector Unit, Technology Innovation Agency, PO Box 172, Pretoria 0063, South Africa Appl Microbiol Biotechnol (2015) 99:6377–6389 DOI 10.1007/s00253-015-6561-x