Metabolic mutants of glutamic acid bacteria have been employed in the microbial production of certain amino acids, such as L-lysine, L-glutamic acid, and L-homoserine. Amino acid overproduction is prevented in wild-type strains by feedback regulation of the natural amino acid metabolic network. Hence, overproducing mutants are generally selected to possess an enzymatic block at a metabolic branch point, leading to auxotrophic requirements for metabolites downstream of the blockage. In an optimal fermentation, the feed of such metabolites is carefully controlled to ensure adequate supply for the cellular metabolic needs and, at the same time, minimal feedback regulation. Monitoring the metabolites for which auxotrophic requirements exist becomes then of critical importance. For the production of L-lysine, mutants deficient in homoserine dehydrogenase and thus auxotrophic for both L-threonine and L-methionine (or L-homoserine) are used.'-3 It is known that, in glutamic acid bacteria, L-threonine, in concert with L-lysine, inhibits aspartate kinase, the enzyme at the entrance to the pathway leading to L-lysine product i ~n . ~. ~. ~ Therefore, the level of L-threonine in the medium must be regulated in any L-lysine fermentation process.
Methods of amino acid detection, such as highperformance liquid chromatography (HPLC)6-9 and enzymatic assays,"-'* have been discussed in the literature. However, none are suitable for the task of quantifying low levels (mg/L) of L-threonine in the presence of high levels (g/L) of L-lysine and other fermentation media components such as glucose and ammonia.
Amino acid detection via HPLC is generally used on mixtures where the relative concentrations of the different amino acids are of the same order of magnitude. This is particularly important because quantification by precolumn derivatization requires reaction with a derivatizing agent (such as o-phthaldialdehyde (OPA), phenylisothiocyanate (PITC), or ninhydrin) which itself must be present in ex-* To whom all correspondence should be addressed.