| γ-aminobutyric acid（GABA）is a four-carbon non-protein amino acid with a variety of physiological and biochemical functions,which is widely used in pharmaceutical,chemical and food fortification fields.As a food-safe strain,Corynebacterium glutamicum can be used industrially for mass production of L-glutamic acid and its derivatives.To achieve efficient synthesis of GABA in C.glutamicum,in this study,the activity was improved by rational design of glutamate dehydrogenase（GDH）,and the production of glutamic acid was enhanced.Based on this,we rationally engineered the catalytic p H range of glutamate decarboxylase（GAD）that is the rate-limiting enzyme for the production of GABA,and further combined with metabolic modification methods,recombinant C.glutamicum was obtained,which has increased yield and production efficiency of GABA.The improved production efficiency of GABA may facilitate its large-scale production.The main findings were as follows:（1）Screening of GDH from different sources and rational design.In this study,the GDHs from seven different strains were expressed in E.coli BL21（DE3）by plasmid p ET-28a after gene synthesis.SDS-PAGE verification showed that the proteins were all successfully expressed.After purification of the obtained proteins,They were used for enzymatic properties analysis.It was found that the optimum p H of each enzyme was generally in the alkaline range.The optimal p H of GDH from A.xylanus was 8.5,and the maximum specific enzyme activity was 273.06±6.48 U·mg-1,which was lower than that of the enzyme from C.glutamicum.Considering the coenzyme dependence and enzyme specific-activity,the GDH from A.xylanus was taken as the object,and the Supercharge algorithm of Rosetta software was used for rational design.Ten mutation points were designed:V55D,S150D,T188D,R255D,L257D,Q282D,L302E,K314E,R344E,and T389E.Construct corresponding recombinant E.coli,purify the expressed enzyme of each mutant strain,and determine each mutant enzyme’s relative activity under specific p H,the final optimal mutant enzyme Ax GDHS150D/T188D/L257D/L302E was obtained by combining beneficial mutation points.Then the optimum catalytic conditions,stability,and substrate bonding of Ax GDHS150D/T188D/L257D/L302E were compared with the wild enzyme by enzymatic properties determination and molecular dynamics simulation.The results showed that the optimal p H of Ax GDHS150D/T188D/L257D/L302E dropped by 1.5 units and enzyme specific-activity was improved.（2）Integration of the gene encoding GDH in C.glutamicum enhances the accumulation of glutamic acid.The above optimal mutant enzyme Ax GDHS150D/T188D/L257D/L302E and wild enzyme encoding genes were inserted in plasmid p K18mobsac B.After electric shock conversion and sucrose screening of transformant,recombinant C.glutamicum E01::axgdhS150D/T188D/L257D/L302E and C.glutamicum E01::axgdh were obtained.With glucose as a carbon source,the maximum yield of L-glutamic acid was 115.0 g·L-1 of C.glutamicum E01::axgdhS150D/T188D/L257D/L302E after 96 h of fermentation in a 5 L fermenter.（3）Enzymatic properties determination and rational design of GAD.Clone GAD encoding gene from Lactiplantibacillus plantarum HG-06 strain that has a high production of GABA,construct recombinant plasmid p ET-28a-lpgad for expression of Lp GAD in E.coli BL21（DE3）.The enzymatic properties of Lp GAD were determined using its pure enzyme solution and the results showed that the optimal p H of Lp GAD is 4.8 and the catalytic p H range is narrow.The Lp GAD was taken as the object,and the above-mentioned Rosetta software was used for rational design.Nine mutation points were designed:S24R,D88R,L135K,E170R,H196R,A225K,Y309K,A359R,and E417K.Construct the corresponding recombinant E.coli,purify the expressed enzyme of each mutant strain,and determine each mutant enzyme’s relative activity under specific p H,the final optimal mutant enzyme Lp GADS24R/D88R/Y309K was obtained.Then the optimum catalytic conditions,stability,and substrate bonding of Lp GADS24R/D88R/Y309K were compared with the wild enzyme by enzymatic properties determination and molecular dynamics simulation.The results showed that the activity of Lp GADS24R/D88R/Y309K was improved overall in the p H range of action,the enzyme activity was 1.68 times that of the wild enzyme at p H 6.0,and the p H range of enzyme action was widened by 0.5 units.（4）Fermentation and whole-cell transformation production of GABA using recombinant C.glutamicum.Taking the C.glutamicum E01::Ax GDHS150D/T188D/L257D/L302E as a host,Lp GAD and Lp GADS24R/D88R/Y309K were heterologously expressed through the shuttle plasmid p XMJ19.The maximum yield of GABA was 32.14 g·L-1 of recombinant strain and the conversion rate from glucose to GABA was 11.94%after 96 h of fermentation in a 5 L fermenter using a two-stage p H control strategy.Due to the low yield of GABA prepared by fermentation,the recombinant C.glutamicum was constructed to produce GABA by whole-cell transformation.The shuttle plasmid p XMJ19 was used to express Lp GAD and Lp GADS24R/D88R/Y309K in the C.glutamicum E01,and the transformation optimization results were as follows:the temperature was 40℃,the dosage of pyridinal-5’-phosphoric acid（PLP）was 100μmol·L-1,the substrate L-glutamic acid was 100.0 g·L-1,and the bacterial volume OD600 was 20.At last,the GABA titer of the C.glutamicum E01/p XMJ19-lpgad and C.glutamicum E01/p XMJ19-lpgadS24R/D88R/Y309K reached 247.1 g·L-1 and 402.8 g·L-1 separately in a fed-batch reaction carried out in a 5 L fermenter under the optimized conditions. |
