Glucose-controlled L-isoleucine fed-batch production with recombinant strains of Corynebacterium glutamicum

L-Isoleucine was produced in a fed-batch bioprocess with L-leucine auxotrophic Corynebacterium glutamicum strains developed by genetic engineering. An efficient supply with nutrients was achieved by applying closed-loop control of glucose as the main carbon source, with a model-based, parameter-adaptive control strategy. This control strategy is based on an extended, semi-continuous Kalman filter for process identification and a minimum variance controller. The lab scale fed-batch process with C. glutamicum SM1 and C. glutamicum DR17 pECM3::ilvA38 was characterized with respect to biomass, product and by-product accumulation. A differential analysis of growth, specific productivities, and selectivities was performed to characterize the carbon flow over process time. Characterization of L-isoleucine transport steps across the cell membrane resulted in a balance of L-isoleucine transport over process time. Up to an extracellular L-isoleucine concentration of 140 mM the cytosolic L-isoleucine, provided by the biosynthesis, was quantitatively excreted into the medium via the export carrier system. Optimized feeding profiles for L-leucine and phosphate in correlation with the on-line estimated glucose consumption were achieved up to the pilot scale (300-1 stirred tank reactor). The maximum L-isoleucine concentration was 150 mM (21 g l^-1) with a space-time yield of 4.3 mmol l^-1 h^-1. With a 98% closed carbon balance the selectivity for isoleucine was 14%, for biomass 13%, and for CO₂ 68%.