title: Biosynthetic production of 13C-labeled amino acids with site-specific enrichment.
abstract: We have developed two Escherichia coli strains for the production of specifically labeled amino acids suitable for high resolution nuclear magnetic resonance experiments. The 13C atoms from the enriched carbon sources, [1-13C]lactate, [1,4-13C2]succinate, and [1-13C]-acetate, are incorporated into the amino acids producing multilabeled molecules with relatively few instances of adjacent enriched carbons. This greatly simplifies the resultant spectra as compared to the extensively spin-coupled spectra of uniformly enriched samples. No isotopic enrichment was found at carbon positions expected to be unenriched by consideration of the major biosynthetic pathways. Utilizing 90% enriched precursors, most positions were enriched to approximately 85% except for those positions derived from acetate and those affected by the carbon interchange of the pentose phosphate shunt. In both of these cases, the enrichment level fell to 70%. The only result enrichment at the delta-methyl carbon of isoleucine. For the bacterial strain in question, the main pathway of isoleucine biosynthesis appears not to be from threonine but from an alternate precursor, possibly glutamate.
mesh_terms: D000596:Amino Acids / Q000096:biosynthesis*; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry; D009154:Mutation; D013045:Species Specificity
chemical_list: D000596:Amino Acids; D002247:Carbon Isotopes