Systems Metabolic Engineering of Corynebacterium glutamicum towards improved Lysine Production

L-lysine is an essential amino acid which is mainly applied as supplement in animal feed. With a world market of around 1,000,000 t/a it is one of the most important fermentation products. The gram positive soil bacterium Corynebacterium glutamicum thereby serves as major production organism. The aim of the present work was rational strain engineering of lysine production by C. glutamicum using a systems-oriented approach. Detailed studies on this organism exposed lysine biosynthesis, NADPH metabolism, TCA cycle and precursor supply as key pathways. The central role for strain characterization can here be assigned to 13C metabolic flux analysis, which was also applied in this work to obtain a detailed insight into the cellular physiology. Based on an existing model, the network for flux determination was extended in the present study to increase the number of estimable flux parameters. With regard to strain optimization, targeted modulation of enzyme activities within the cell is crucial to increase or reduce carbon conversion of the selected pathways. The common toolbox such as over expression by promoter exchange or gene deletion was complemented with the method of start codon exchange allowing permanent modulation of enzyme activity. As host-strains for target evaluation, genetically defined, wild type based lysine producers were used which all express a feedback-deregulated variant of aspartate kinase. The most beneficial modifications identified in this work comprised over expression of diaminopimelate dehydrogenase, down-regulation of TCA cycle by attenuation of isocitrate dehydrogenase and engineering of the NADPH metabolism. By subsequent implementation of beneficial targets in a single strain, a rationally designed lysine hyper-producer was created. This exhibited remarkable production properties such as a lysine-HCl titre of 120 g L-1 and a carbon conversion yield of up to 55 %.