Enhancing Glutamate Fermentation Stability And Yield By Techniques Of Metabolic Enzymology And Model

Glutamate is the amino acid with the largest production in the world. In China, biotinauxotroph Corynebacterium glutamicum strain is widely used in glutamate production,glutamate concentration and conversion yield (from sugar) vary at the levels of10%~12%and55%~60%. Glutamate production technology in China has a history of more than50years,but the major technological index, conversion yield in particular, is much lower thantheoretical value (81%) and the levels (68%) in advanced foreign countries. In industrialglutamate fermentation, the relatively rough method for medium preparation easily causes thefluctuations in medium components (biotin), and thus severe deteriorates the fermentationstability. In addition, the fermentation features or characteristics of the strain sometimes variesbatch by batch, leading to the lower resistant ability against the environmental change and thedecreased glutamate synthesis ability, which results in glutamate synthesis stoppage aftercarbon source feeding and thus fermentation instability. In this thesis, the efficient strategiesfor increasing the conversion yield and for stabilizing the fermentations to deal with the initialmedium components variations and change of strain characteristics, were proposed andexperimentally testified, using C. glutamicum S9114with the aids of metabolic enzymologyand model techniques. The mechanisms of the fermentation improvements when adopting theproposed strategies were also analyzed and explored theoretically. The main results of thisdissertation were summarized as follows:(1) Activities changes of the key enzymes at metabolic nodes of pyruvate, isocitrate andα-ketoglutarate, when initial biotin content varied, and when the content was at improperlevel but faults-rescue measures were adopted, were investigated. When initial biotin wasin shortage, isocitrate dehydrogenase (ICDH) activity was weakened and amount ofglutamate precursor reduced. α-oxoglutarate dehydrogenase complex (ODHC) activitywas inactivated and energy metabolism completely relied on glyoxylate shuttle. Wheninitial biotin was in shortage but biotin was adaptively supplemented, ICDH acitivityrebounded to higher level, and TCA cycle turned to be the main energy metabolism routeonce again. When initial biotin was in excess, activities of the key enzymes associatedwith glutamate synthesis (ICDH and glutamate dehydrogenase) decreased but that relatedwith energy metabolism (ODHC) was stimulated. When biotin was in excess and Tween40was adaptively added, pyruvate dehydrogenase (PDH) and ICDH remained at highlevels, but activities of ODHC and isocitrate lyase (ICL) declined to the normal levels.With the aids of the faults-rescue measures, the failure-likelihood fermentations due toimproper initial biotin variations could be recovered back to normal, and final glutamateconcentrations could reach the normal levels of75~80g·L-1.(2) The transcriptional levels of key enzymes for glutamate synthesis and glutamate transportprotein (TP) under varied initial biotin contents and adaptively adding Tween40, wereinvestigated. When initial biotin was in shortage, the transcriptional levels of genesencoding the key enzymes and TP were all down-regulated during the main production phase, especially that of ICDH, resulting in a very low glutamate concentration (53g·L-1),although glutamate efflux was not affected. When biotin was in excess, the transcriptionallevels of key enzymes were all at comparable levels as those of control but with lowICDH. In this case, the impermeable cellular membrane stopped the vitro glutamatesecretion even though TP expression was about10-fold of control, glutamate could not beaccumulated intracellularly and extracellularly. When initial biotin was in excess butadaptively adding Tween40stimulated the expression of all key enzymes and TP, inducedcell morphological transformation, increased intracellular glutamate content, resultingfinal glutamate concentration back to normal level (75~80g·L-1).(3) The strategy of mixed-carbon sources was proposed to stabilize fermentation performance.The results demonstrated that, if co-feeding glucose with sorbitol/glycerol at a weightratio of5:1or adding10~15g·L-1of sorbitol/glycerol in the initial medium, glutamatesynthesis could continue after substrate(s) feeding and final glutamate concentration couldbe recovered back to normal level. Under these environments, the NAD+/NADH ratio,ORP, the activities of PDH, ICDH and cytochrome c oxidase could be maintained athigher levels. Sorbitol and glycerol could not be used as carbon sources for thefermentation, they were considered to be the effective protective agents to increase cellsresistant ability against environmental changes and maintain key enzymes activities.(4) A new fermentation technology of adaptively regulating pH and NaHCO3addition wasproposed, aiming at increasing glutamate conversion yield from sugar. Fermentationswhen singly and coordinately regulating pH or/and NaHCO3addition were conducted andtheir performance was compared. The results indicated that, the amounts of glucoseconsumption and CO2released decreased significantly and the conversion yield increasedby34%~36%as compared with control, by raising pH before or at the same time as thecommencement of NaHCO3addition. At the same time, comparably high glutamateproductivity could be maintained. Enzymatic activities analysis revealed that increasingPC activity alone could not increase the yield and the yield could be enhanced only whenall key enzymes for glutamate synthesis worked coordinately.(5) A novel metabolic model integrating directed signal flow diagram and enzymatic activitiesdata was proposed to interpret the yield enhancement. The simulation and experimentalresults revealed that singly regulating each individual enzyme could not increase theconversion yield, and the yield could be enhanced only when six key enzymes of PC,PDH, ICDH, ICL, GDH and ODHC works in a coordinated way. Namely, relativeactivities ratios of enzymatic pairs of PC/PDH should be controlled at moderate level of6:4, while those of ICDH/ICL and GDH/ODHC at higher level of8:2simultaneously. Themodel could cluster data pairs of conversion yields and enzymatic activities obtainedunder different operation conditions into different categories, indicating its abilities inguiding optimal enzyme regulation ways for fermentations characterized with multipleenzymatic reactions and closed reaction loops.