MilliporeSigma
  • Home
  • Search Results
  • Reconstruction of the carnitine biosynthesis pathway from Neurospora crassa in the yeast Saccharomyces cerevisiae.

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

Applied microbiology and biotechnology (2015-04-09)
Jaco Franken, Anita Burger, Jan H Swiegers, Florian F Bauer
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.

MATERIALS
Product Number
Brand
Product Description

SAFC
Sodium chloride solution, 5 M
Sigma-Aldrich
Sodium chloride, BioPerformance Certified, ≥99% (titration), suitable for insect cell culture, suitable for plant cell culture
Sigma-Aldrich
Methanol, HPLC Plus, ≥99.9%, poly-coated bottles
Sigma-Aldrich
3-Mercaptopropionic acid, ≥99%
Sigma-Aldrich
Sodium chloride, 99.999% trace metals basis
Sigma-Aldrich
Acetonitrile solution, contains 10.0% acetone, 40.0% 2-propanol, 0.05% formic acid
Sigma-Aldrich
Methanol, NMR reference standard
Sigma-Aldrich
Acetonitrile solution, contains 0.05 % (w/v) ammonium formate, 0.1 % (v/v) formic acid, 5 % (v/v) water, suitable for HPLC
Sigma-Aldrich
Boric acid-11B, ≥99 atom % 11B
Sigma-Aldrich
Sodium phosphate dibasic solution, BioUltra, 0.5 M in H2O
Sigma-Aldrich
Acetonitrile solution, contains 0.05 % (v/v) trifluoroacetic acid
Sigma-Aldrich
Acetonitrile solution, contains 0.1 % (v/v) trifluoroacetic acid, suitable for HPLC
Sigma-Aldrich
Sodium chloride solution, 0.85%
Sigma-Aldrich
D-Glucose-12C6, 16O6, 99.9 atom % 16O, 99.9 atom % 12C
Sigma-Aldrich
Methanol, anhydrous, 99.8%
Sigma-Aldrich
Sodium chloride, AnhydroBeads, −10 mesh, 99.999% trace metals basis
Sigma-Aldrich
Sodium azide, BioUltra, ≥99.5% (T)
Sigma-Aldrich
Sodium chloride, AnhydroBeads, −10 mesh, 99.99% trace metals basis
Sigma-Aldrich
Sodium chloride-35Cl, 99 atom % 35Cl
Sigma-Aldrich
Sodium chloride, random crystals, optical grade, 99.9% trace metals basis
Sigma-Aldrich
Methanol, suitable for HPLC, gradient grade, 99.93%
Sigma-Aldrich
Acetonitrile solution, contains 0.1 % (v/v) formic acid, suitable for HPLC
Sigma-Aldrich
Sodium chloride, BioUltra, for molecular biology, ≥99.5% (AT)
Sigma-Aldrich
3-Mercaptopropionic acid, ≥99.0% (HPLC)
Sigma-Aldrich
Sodium azide, purum p.a., ≥99.0% (T)
Sigma-Aldrich
D-(+)-Glucose, ≥99.5% (GC)
Sigma-Aldrich
Sodium chloride, BioXtra, ≥99.5% (AT)
Sigma-Aldrich
D-(+)-Glucose, BioXtra, ≥99.5% (GC)
Sigma-Aldrich
Sodium chloride, for molecular biology, DNase, RNase, and protease, none detected, ≥99% (titration)
Sigma-Aldrich
Sodium chloride, meets analytical specification of Ph. Eur., BP, USP, 99.0-100.5%