6. Corynebacterium glutamicum Gram-positive, non-pathogenic, fast growing soil bacterium Pathways leading to amino acids are much simpler in their regulation when compared to E. Coli: -- very few enzymes are feedback controlled -- no isoenzymes detected -- genome sequenced Problems: 1) vector system are far from ideal (E.coli vectors will not work) 2) Transformation rates are low
18. C. glutamicum uses succinylase and dehydrogenase variant One enzyme controlled: aspartate kinase! Aspartate family Split pathway to diaminopimelate (DAP) Lysine production
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20. [NH 4 + ] determines flux partitioning high NH 4 + : 50-70% via dehydrogenase low NH 4 + : only succinylase C. glutamicum uses succinylase and dehydrogenase variant Lysine production
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23. Enzyme reactions important in the assimilation of ammonia. GS = glutamine. synthetase; GOGAT: glutamate synthase; AS: asparagine synthetase; AAT: aspartateaminotransferase.
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26. Process Overview Emphasis on complete metabolic networks rather than on individual reactions Two defining steps: Synthesis: Applied molecular biology Analysis: Engineering component
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28. Engineering an organism to produce a specific product. Once an organism is chosen, how do we modify it? 1. Channel nutrients down existing metabolic pathways that produce the desired product. 2. If option 1 is not possible, use recombinant DNA to actually change genome and create new metabolic pathways
29. Channeling nutrients down specific pathways Goal Restrict or add excess of specified nutrients for the purpose of inhibiting unwanted reactions and driving forward desired reactions Problem Nature has designed organisms with strict metabolic regulating mechanisms. Solution Knowledge of major and minor metabolic pathways aid in determining what variables/nutrients can be changed and how to go about changing them
30. The specific objectives are as follows: 1. To optimize the fermentation process for lysine production. 2. To optimize the downstream processing for lysine separation. 3. To prepare the techno-economic feasibility study of commercial lysine plant.
