Abstract: This study aimed to rationally identify new targets for improving isoleucine production. The transcription levels of the key genes and intermediate metabolite levels involved in the isoleucine synthesis pathway of Corynebacterium glutamicum ATCC 13032 and C. glutamicum YILW, a isoleucine-producing strain derived from the parental strain ATCC 13032, were compared. The gene pyc was down-regulated, which might consequently lead to reduced supply of oxaloacetate. Then pyc was overexpressed in C. glutamicum YILW (denoted as YILW-1), resulting in increased oxaloacetate concentration and isoleucine production (from 1.32 to 3.32 μmol/g (md) and from 5.18 to 5.81g/L) but higher accumulation of lysine and intracellular 2-ketobutyrate as byproduct. The ilvBNC operon was further overexpressed in YILW-1 (denoted as YILW-2), resulting in production of up to 6.63 g/L isoleucine. To enhance exportation and consequently further increase the production of isoleucine, the isoleucine exporter genes brnE and brnF was overexpressed in YILW-2 (denoted as YILW-3), leading to increased production of isoleucine (7.31 g/L) by 10.3% as compared to that of YILW-2. The strategy resulted in 41.1% higher isoleucine production (from 5.18 to 7.31 g/L) and 40.0% higher yield (from 0.10 to 0.14 g/g glucose) together with lower by-product lelvels by YILW-3 as compared to C. glutamicum YILW. It could be concluded that overexpression of the pyc, ilvBNC operon as well as brnE and brnF based on transcription and metabolite pool analysis could significantly elevate isoleucine production and decrease by-product concentration levels.

Key words: Corynebacterium glutamicum, isoleucine, metabolite pool, oxaloacetate