Effect Of The Glyoxylate Cycle On L-Glutamate Synthesis By Corynebacterium Glutamicum

Since the discovery of Corynebacterium glutamicum as an efficient glutamate-overproducing microorganism in 1957, the production of L-amino acids by the fermentative method has become one of the most important research-target of industrial microbiology over the last four decades. Nevertheless, the mechanism of L-glutamate production and excretion by the microorganism is still unclear.It has been suggested that metabolic flux through the anaplerotic pathways could be limiting for amino acids synthesis. The carbon flux supplying oxaloacetate is very complicated in coryneform bacteria because several potential anaplerotic enzymes / pathways exist: phophoenolpyruvate carboxylase (PEPC), pyruvate carboxylase (PC), and the glyoxylic cycle which all contribute to carbon flux through oxaloacetate. Less research reports about the relationship between the glyoxylate cycle and L-glutamate synthesis are available up to the present and their results are contradictory. It will help elucidate the mechanism of L-glutamate overproduction and guide the breeding of L-glutamate high yield strains to investigate the role played by the glyoxylate cycle during L-glutamate synthesis.A series of research work was carried out about the importance of the glyoxylate cycle during L-glutamate synthesis and main results are as follows:1. Since L-glutamate production decreased when isocitrate lyase was partially inhibited and much less L-glutamate was produced and excreted when the glyoxylate cycle was blocked, the glyoxylate cycle of Corynebacterium glutamicum was active and played an important role during L-glutamate production when grown on glucose as a sole carbon source. It was extremely possible that the glyoxylate cycle played an important anaplerotic function during L-glutamate overproduction especially under biotin limitation, because pyruvate carboxylase is less active when biotin concentration in the culture medium is limited.2. Although isocitrate lyase activity increased by six fold when ace A gene was overexpressed, L-glutamate production increased by 12.82%. It seemed that the glyoxylate cycle played a bifunctional role for L-glutamate production: anaplerotic function and divergent flux.3. Although succinate partially inhibited isocitrate lyase, L-glutamate production and residual glucose increased, cell growth decreased with the increase of succinate concentration in the fermentative broth. The reason for these may be thatsuccinyl coenzyme A inhibited the 2-ketoglutarate dehydrogenase.4. Addition of acetate (?ï¿¡ lOg/L when concentration of glucose was 50g/L) in the culture medium promoted cell growth but was disadvantageous to L-glutamate production. This effect might be due to the enhancement of TCA cycle and 2-ketoglutarate dehydrogenase by acetate and the decrease of flux into L-glutamate synthesis. However, both cell growth and L-glutamate were detrimentally affected with further addition of acetate. It might be because of the acetate function as an uncoupler of the transmembrane pH gradient.5. Simultaneous supplement of acetate and succinate promoted L-glutamate production. It seemed that TCA cycle was strengthened and succinyl coenzyme A inhibited the 2-ketoglutarate dehydrogenase, hence the metabolic flux was directed to L-glutamate synthesis.6. A temperature and lysozyme sensitive strain C.glutamicum WT^IL was constructed. It was induced to overproduce L-glutamate after a fermentative temperature shift from 30D to 38D under the presence of an excess of biotin. Simultaneous supplement of acetate and succinate were also effective during L-glutamate production by the temperature and lysozyme sensitive strain C.glutamicum WT A L.7. Isocitrate lyase, isocitrate dehydrogenase, 2-ketoglutarate dehydrogenase and glutamate dehydrogenase were analysed. The results indicated that the increase of the flux from citrate to 2-ketoglutarate was the main reason for the cells to produce more L-glutamate if the anaplerotic function was assured.