caa a22 4500 605506817 CHVBK 20210128100629.0 cr unu---uuuuu 210128e20150801xx s 000 0 eng 10.1007/s00253-015-6561-x doi (NATIONALLICENCE)springer-10.1007/s00253-015-6561-x Reconstruction of the carnitine biosynthesis pathway from Neurospora crassa in the yeast Saccharomyces cerevisiae [Elektronische Daten] [Jaco Franken, Anita Burger, Jan Swiegers, Florian Bauer] 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. Springer-Verlag Berlin Heidelberg, 2015 Carnitine nationallicence Biosynthesis nationallicence Neurospora crassa nationallicence Saccharomyces cerevisiae nationallicence Franken Jaco Department of Viticulture and Oenology, Institute for Wine Biotechnology, Stellenbosch University, 7602, Matieland, South Africa aut Burger Anita Department of Viticulture and Oenology, Institute for Wine Biotechnology, Stellenbosch University, 7602, Matieland, South Africa aut Swiegers Jan Department of Viticulture and Oenology, Institute for Wine Biotechnology, Stellenbosch University, 7602, Matieland, South Africa aut Bauer Florian Department of Viticulture and Oenology, Institute for Wine Biotechnology, Stellenbosch University, 7602, Matieland, South Africa aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/15(2015-08-01), 6377-6389 0175-7598 99:15<6377 2015 99 253 https://doi.org/10.1007/s00253-015-6561-x text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6561-x text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Franken Jaco Department of Viticulture and Oenology, Institute for Wine Biotechnology, Stellenbosch University, 7602, Matieland, South Africa aut NATIONALLICENCE 700 1- Burger Anita Department of Viticulture and Oenology, Institute for Wine Biotechnology, Stellenbosch University, 7602, Matieland, South Africa aut NATIONALLICENCE 700 1- Swiegers Jan Department of Viticulture and Oenology, Institute for Wine Biotechnology, Stellenbosch University, 7602, Matieland, South Africa aut NATIONALLICENCE 700 1- Bauer Florian Department of Viticulture and Oenology, Institute for Wine Biotechnology, Stellenbosch University, 7602, Matieland, South Africa aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/15(2015-08-01), 6377-6389 0175-7598 99:15<6377 2015 99 253