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Reconstruction of the carnitine biosynthesis pathway from Neurospora crassa in the yeast Saccharomyces cerevisiae

  • Applied genetics and molecular biotechnology
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Abstract

Industrial synthesis of l-carnitine is currently performed by whole-cell biotransformation of industrial waste products, mostly d-carnitine and cronobetaine, through specific 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-carnitine is biosynthesized through a four-step metabolic conversion of trimethyllysine to l-carnitine. In contrast, the industrial yeast, Saccharomyces cerevisiae lacks the enzymes of the eukaryotic biosynthesis pathway and is unable to synthesize carnitine. 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 chromatography electrospray ionization mass spectrometry (HILIC-ESI-MS) analyses, from trimethyllysine. Furthermore, the yeast threonine aldolase Gly1p was shown to effectively catalyze 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.

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Acknowledgments

Jaco Franken was supported by an NRF NPPD postdoctoral fellowship (National Research Foundation of SA, grant number 78691 (http://www.nrf.ac.za/)).

Conflict of interest

The authors declare that they have no conflict of interest.

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Author notes

  1. Anita Burger

    Present address: Industrial Biotechnology Sector Unit, Technology Innovation Agency, PO Box 172, Pretoria, 0063, South Africa

Authors and Affiliations

  1. Department of Viticulture and Oenology, Institute for Wine Biotechnology, Stellenbosch University, Matieland, 7602, South Africa

    Jaco Franken, Anita Burger, Jan H. Swiegers & Florian F. Bauer

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  1. Jaco Franken
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  2. Anita Burger
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Correspondence to Florian F. Bauer.

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Franken, J., Burger, A., Swiegers, J.H. et al. Reconstruction of the carnitine biosynthesis pathway from Neurospora crassa in the yeast Saccharomyces cerevisiae . Appl Microbiol Biotechnol 99, 6377–6389 (2015). https://doi.org/10.1007/s00253-015-6561-x

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