The fermentative production of L-threonine and L-isoleucine with Corynebacterium glutamicum is usually accompanied by the by-production of L-lysine, which shares partial biosynthesis pathway with L-threonine and L-isoleucine. Since the direct precursor for L-lysine synthesis, diaminopimelate, is a component of peptidoglycan and thus essential for cell wall synthesis, reducing L-lysine by-production could be troublesome. Here, a basal strain with eliminated L-lysine production was constructed from the wild type C. glutamicum ATCC13869 by deleting the chromosomal ddh and lysE. Furthermore, the basal strain as well as the ddh single mutant strain was engineered for L-threonine production by over expressing lysC1, hom1 and thrB, and for L-isoleucine production by over-expressing lysC1, hom1, thrB and ilvA1. Fermentation experiments with the engineered strains showed that (i) deletion of ddh improved L-threonine production by 17%, and additional deletion of lysE further improved L-threonine production by 28%; (ii) deletion of ddh improved L-isoleucine production by 8% and improved cell growth by 21%, whereas additional deletion of lysE had no further influence on both L-isoleucine production and cell growth; (iii) L-lysine by-production was reduced by 95% and 86% in L-threonine and L-isoleucine production, respectively, by deletion of ddh and lysE. This is the first report on improving L-threonine and L-isoleucine production by deleting ddh and lysE in C. glutamicum. The results demonstrate deletion of ddh and lysE as an effective strategy to reduce L-lysine by-production without surrendering the cell growth of C glutamicum. (C) 2016 Elsevier Inc. All rights reserved.