Biosynthetic production of 13C-labeled amino acids with site-specific enrichment.
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.
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
D000596:Amino Acids; D002247:Carbon Isotopes