USE OF FEEDBACK-RESISTANT THREONINE DEHYDRATASES OF CORYNEBACTERIUM-GLUTAMICUM TO INCREASE CARBON FLUX TOWARDS L-ISOLEUCINE

The biosynthesis of L-isoleucine proceeds via a highly regulated react ion sequence connected with L-lysine and L-threonine synthesis. Using defined genetic Corynebacterium glutamicum strains characterized by di fferent fluxes through the homoserine dehydrogenase reaction, we analy zed the influence of four different ilvA alleles (encoding threonine d ehydratase) in vectors with two different copy numbers on the total fl ux towards L-isoleucine. For this purpose, 18 different strains were c onstructed and analyzed. The result was that unlike ilvA in vectors wi th low copy numbers, ilvA in high-copy-number vectors increased the fi nal L-isoleucine yield by about 20%. An additional 40% increase in L-i soleucine yield was obtained by the use of ilvA alleles encoding feedb ack-resistant threonine dehydratases. The strain with the highest yiel d was characterized by three hom(Fbr) copies encoding feedback-resista nt homoserine dehydrogenase and ilvA(Fbr) encoding feedback-resistant threonine dehydratase on a multicopy plasmid. It accumulated 96 mM L-i soleucine, without any L-threonine as a by-product. The highest specif ic productivity was 0.052 g of L-isoleucine per g of biomass per h. Th is comparative flux analysis of isogenic strains showed that high leve ls of L-isoleucine formation from glucose can be achieved by the appro priate balance of homoserine dehydrogenase and threonine dehydratase a ctivities in a strain background with feedback-resistant aspartate kin ase. However, still-unknown limitations are present within the entire reaction sequence.