ENGINEERING THE HOMOSERINE DEHYDROGENASE AND THREONINE DEHYDRATASE CONTROL POINTS TO ANALYZE FLUX TOWARDS L-ISOLEUCINE IN CORYNEBACTERIUM-GLUTAMICUM

The synthesis of L-isoleucine by Corynebacterium glutamicum involves 1 1 reaction steps with at least 5 of them regulated in activity or expr ession. Using gene replacement we constructed a vector-free C. glutami cum strain having feedback-resistant aspartate kinase and feedback-res istant homoserine dehydrogenase activity. Isogenic strains carrying in addition one or several copies of feedback-resistant threonine dehydr atase were made and their product accumulations compared. With strain SM1, with high threonine dehydratase activity, accumulation of 50 mM L -isoleucine was achieved, whereas with the parent strain only 4 mM L-i soleucine was obtained. Applying a closed-loop control fed-batch strat egy to strain SM1 a final titre of 138 mM L-isoleucine was achieved wi th an integral molar yield of 0.11 mol/mol, and a maximal specific pro ductivity of 0.28 mmol (g h)(-1). This shows that high L-isoleucine yi elds can be obtained in the presence of one copy of feedback-resistant homoserine dehydrogenase by applying the appropriate fermentation str ategy. In addition, the specific profiles of 2-oxoglutarate and pyruva te accumulation during fermentation revealed a major transition of the metabolism of C. glutamicum during the fermentation process.