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 i s regulated, We used four genes and alleles encoding feedback-resistan t enzymes (Fbr) in various combinations to assay flux increase through the sequence, During strain construction, the order of genes overexpr essed 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 construct ed (SM13) was characterized by high-level expression of horn(Fbr), thr B, and ilvA(Fbr). With this strain a yield of 0.22 g of L-isoleucine p er 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 alw ays higher at all process times than the extracellular concentration, The intracellular concentration rises to 110 mM, whereas extracellular ly only 60 mM is accumulated, Also the immediate L-isoleucine precurso r 2-ketomethyl valerate accumulates in the cell, Therefore, in the hig h-level L-isoleucine producer SM13, the export of this amino acid is t he major limiting reaction step and therefore is a new target of strai n design for biotechnological purposes.