Metabolic Engineering Of Corynebacterium Glutamicum For γ-aminobutyric Acid Production

Gamma-aminobutyric acid（GABA）is an important nonprotein amino acid,and was widely applied in the area of food,pharmaceuticals,feed additives and clinic research.Traditional production of GABA is mainly using chemical synthesis and enzyme catalysis.Currently,microbial metabolic engineering and synthetic biology strategy were employed to produce GABA.In this study,the wild-type Corynebacterium glutamicum ATCC 13032 was used as the host to construct GABA-producing strain by introducing Lactobacillus brevis-derived glutamate decarboxylase.Next,the GABA synthetic pathway was optimized using metabolic engineering;ultimately,a high production strain of GABA was obtained.This strain was constructed by whole-genome expression method and plasmid-free system,it is not necessary to add any antibiotics and inducer during fermentation.Therefore,this production procedure demonstrates simple,economic and eco-friendly.The mainly results were showed as following:（1）An effective CRISPR/Cas9-assisted genome editing system was constructed in Corynebacterium glutamicum with following characters:The all elements for genome editing including Cas9,sg RNA and HRarms were integrated into a single plasmid;The recombinant plasmid for target gene editing could be constructed by once in E.coli;The plasmid contains temperature-sensitive replicon p BL1^P47S,which is stable in cells during 25-28℃,and can be cured by cultured at 37°C;The genome editing can be done by once electrotransformation in Corynebacterium glutamicum.The efficiency could reach 20%-95%.There are no extra sequences remain on the genome after gene editing.The whole editing work was done in 8-9days,so this method is fast and high effective.（2）Lactobacillus brevis-derived gene gad B2 encoding glutamate decarboxylase was introduced into Corynebacterium glutamicum genome to generate a GABA-producing strain.The gene cluster gab TDP was related to GABA intracellular-extracellular transportation and catabolism,its deletion improves the GABA accumulation.The effects of gdh A and gad B2copies in genome on GABA production were studied.E.coli W3110-derived gene gdh A encoding glutamate dehydrogenase was introduced into Corynebacterium glutamicum genome to enhance the precursor L-Glutamic acid production,the L-Glutamic acid synthetic ability was improved after gdh A insertion.The copy of gdh A and gad B2 were increased to promote intracellular glutamate dehydrogenase and glutamate decarboxylase activity,and optimized to 2:3 in genome.The generated strain CGY700 could produce GABA 20.10±0.50g/L when fermentation for 72 h in shake flask.In this study,gdh A and gad B2 was inserted the genes referring to the metabolic pathway of acetic acid,lactic acid and GABA transport（eut D,pox B,gab P,lld D and ald B2）,partial carbon bypass pathways were deleted following with the promotion of GABA synthesis.（3）The pathway of CO₂ anaplerotic reaction was constructed in generated strain CGY700 to improve carbon conversion rate and GABA production.Citric acid synthesis from pyruvic acid is the bottleneck of glycolysis and TCA cycle,in this study,E.coli W3110-derived ppc1（encoding PEP carboxylase）,pck（encoding PEP carboxykinase）,aotologous pyc（encoding pyruvate carboxylase）and E.coli W3110-derived glt A（encoding citrate synthase）were co-expressed in Corynebacterium glutamicum.There are three expression mode were designed:ppc1-glt A,pck-glt A and pyc-glt A,the results showed that ppc1-glt A was optimization for L-Glu and GABA production.Through using different promoter P_tac/P_{tac M} to optimize the co-expression of ppc1 and glt A,the optimal expression mode of P_{tac M-}ppc1-P_tac-glt A was constructed in plasmid p JYW-5.Deleting gene ldh,pkn G,ala T,and introducing plasmid p JYW-5-P_{tac M-}ppc1-P_tac-glt A in strain CGY700 to generate a new strain CGY710E,which could produce GABA 32.83±0.69 g/L when fermentation for72 h in shake flask.（4）The co-expression of PEPC and citrate synthase was optimized through chromosomal editing.On genomic level of Corynebacterium glutamicum,the native promoter of autologous PEP carboxylase gene ppc2 was replaced by strong constitutive promoter P_tacor P_{tac M},and integrated E.coli W3110-derived gene glt A.The optimal mode still was P_{tac M-}ppc2-P_tac-glt A after the co-expression was optimized.Co-deleting ldh,pkn G,and overexpressing gln A2encoding glutamine synthetase to obtain strain CGY-PG-304,which could produce GABA41.17±0.17 g/L at 72 h when fermentation in shaking flask,and 58.33 g/L GABA was obtained by Fed-Batch fermentation with a yield of 0.30 g/g glucose.