Metabolic Engineering Of Corynebacterium Glutamicum For Cellulosic Glutamic Acid And γ-aminobutyric Acid Fermentation

Glutamic acid is one of the most important food additives and chemical raw materials,and its decarboxylation product γ-aminobutyric acid is an important bioactive and polymer monomer compound.Glutamic acid and γ-aminobutyric acid fermentation using the most available,cheap and renewable lignocellulose biomass instead of food starch-based feedstocks can lay a solid foundation for their large-scale application as chemical raw materials or polymer monomers.Howerver,few studies focused on their production from lignocellulose biomass,and no practical results had ever been reported.This may be due to the key factors in the complicated lignocellulose system that affect glutamic acid and γ-aminobutyric acid fermentation are still unclear.In this paper,in order to achieve a real breakthrough in the fermentation of cellulosic glutamic acid and γ-aminobutyric acid,their fermentation by Corynebacterium glutamicum using corn stover hydrolysate that prepared based on dry biorefinery process was studied.First,the key factors that affect glutamate production were investigated,and excessive biotin in corn stover hydrolysate was demonstrated to be the key factor for no glutamic acid accumulation.Then metabolic engineering was applied to trigger efficient glutamic acid production in corn stover hydrolysate by C.glutamicum,and the glutamic acid production performance was significantly improved.Finally,metabolic engineering was applied to solve the key problems and obstacles in γ-aminobutyric acid fermentation,and efficient γ-aminobutyric acid production from corn stover hydrolysate was successfully achieved.The first part of this study focused on glutamic acid production in corn stover hydrolysate,and better cell growth but no glutamic acid accumulation was observed when C.glutamicum cells were cultured in corn stover hydrolysate.Further experiment discovered excessive biotin in 15%(w/w)corn stover hydrolysate.The biotin concentration was as high as 22.5±4.3 μg/L,which was about ten-fold higher than that of "sub-optimal" level for glutamic acid accumulation.A series of experiments further demonstrated that excessive biotin in corn stover hydrolysate was the key factor for no glutamic acid accumulation.The rich existence of biotin was found to be a common phenomenon in a wide range of lignocellulose biomass,and most of biotin remain stable during the dry biorefining chain and thus creates an excessive condition for glutamic acid accumulation in corn stover hydrolysate.We also found that the maj or vitamin B compounds were under high concentration levels even after harsh pretreatment,and they may act as potential nutrients to biorefining fermentations.The second part of this study tried to achieve efficient glutamic acid accumulation by metabolic engineering of C.glutamicum S9114.Among various metabolic engineering strategies we tried or evaluated,activating glutamic acid secretion and decreasing α-oxoglutarate dehydrogenase complex(ODHC)activity were the two most effective methods.First,we modified the glutamate secretion channel MscCG to activate the glutamic acid secretion,and successfully achieved constitue glutamic acid accumulation in the biotin excessive corn stover hydrolysate with a final glutamic acid titer of 9.2 g/L.Then the ODHC activity was attenuated by regulating odhA RBS sequence,and glutamic acid accumulation was significantly improved to more than five folds.55.7 g/L glutamic acid was accumulated.16.8%and 55.6%improvement in glutamic acid titer productivity were achieved compared to that of penicillin trigged glutamic acid fermentation by the starting strain.Efficient cellulosic glutamic acid production was successfully reached.The final strain reached a highest glutamic acid titer of 65.2 g/L with a yield of 0.63 g/g glucose in fed-batch fermentation,and the practical cellulosic glutamate fermentation was successfully achieved for the first time.The third part of this thesis focused on metabolic engineering of C.glutamicum to overcome key problems of low titer,yield and productivity in y-aminobutyric acid fermentation.First,a modified glutamate decarboxylase from E.coli was heterologously expressed in C.glutamicum to achieve γ-aminobutyric acid production under glutamate overproducing condition.Then we facilitated the extracellular decarbonxylation reaction by secreting expression of glutamate decarboxylase through the Sec pathway,and more than 4-folds improvement in y-aminobutyric acid titer was achieved.Consequent expression promoter optimization and gabP gene knockout further improved the y-aminobutyric acid titer.Fed-batch fermentation of the final strain produced 77.6 g/L of GABA with the yield of 0.44 g/g glucose and productivity of 1.21 g·L-1·h-1 in complex medium.This is the highest result ever reported for γ-aminobutyric acid production by C.glutamicum.Significant improvement in γ-aminobutyric acid titer,yield and productivity was achieved.Finally,39 g/L γ-aminobutyric acid was produced in corn stover hydrolysate by the final strain with the yield of 0.44 g/g glucose,successfully achieved cellulosic γ-aminobutyric acid production.Based on the above studies,the key problems in glutamic acid and γ-aminobutyric acid production from lignocellulose biomass were identified and solved,and pratical cellulosic glutamic acid and γ-aminobutyric acid fermentation was successfully reached.These results laied a solid foundation for promoting glutamate and γ-aminobutyrate as monomers use in industrial production of polyesters and polyamides.