l-Isoleucine Production with Corynebacterium glutamicum: Further Flux Increase and Limitation of Export.

The synthesis of l-isoleucine with Corynebacterium glutamicum involves 11 reaction steps, in at least five of which activity or expression is regulated. We used four genes and alleles encoding feedback-resistant enzymes (Fbr) in various combinations to assay flux increase through the sequence. During strain construction, the order of genes overexpressed was important. Only when ilvA(Fbr) was first overexpressed could hom(Fbr) be introduced. This succession apparently prevents the toxic accumulation of biosynthesis intermediates. The best strain constructed (SM13) was characterized by high-level expression of hom(Fbr), thrB, and ilvA(Fbr). With this strain a yield of 0.22 g of l-isoleucine per g of glucose was obtained, with a maximal specific productivity of 0.10 g of l-isoleucine per g (dry weight) per h. In strain SM13, with the high metabolite flux through the reaction sequence, effects on (i) other enzyme levels, (ii) time-dependent variations with process time, and (iii) concentrations of cytosolic intermediates were quantified. Most importantly, the intracellular l-isoleucine concentration is always higher at all process times than the extracellular concentration. The intracellular concentration rises to 110 mM, whereas extracellularly only 60 mM is accumulated. Also the immediate l-isoleucine precursor 2-ketomethyl valerate accumulates in the cell. Therefore, in the high-level l-isoleucine producer SM13, the export of this amino acid is the major limiting reaction step and therefore is a new target of strain design for biotechnological purposes.