Production Of Gamma-aminobutyric Acid By Microbial Fermentation

Gamma-aminobutyric acid(GABA),the main inhibitory neurotransmitter in human cortex,has great potential uses as pharmaceuticals and dietary supplements in functional foods and fodders.Biosynthesis of GABA has been intensively studied over the past decade.The mostly used bacteria for GABA production are lactic acid bacteria and Escherichia coli.In the current study,lactic acid bacteria and E.coli were employed to biosynthesize GABA.Their GABA productions and productivities were enhanced by optimizations of fermentation conditions and strategies,especially genetic and metabolic engineering modifications.The main work was described as follows:(1)Identification and fermentation optimization of a GABA-producing lactic acid bacterium.Firstly,WPZ001,a GABA-producing bacterium isolated from fermented sausages in previous research,was identified as Lactobacillus buchneri species according to the sequence similarity of 16 S rDNA.Then the GABA production of WPZ001 was enhanced by the optimizing the media and fermentation conditions.After optimization,GABA production of WPZ001 dramatically increased from 6.2 to 75.5 g/L.Glucose is used as carbon source for most lactic acid bacteria,while WPZ001 could utilize both glucose and xylose,and the cell growth and GABA production were better when using xylose as the sole carbon source.At 1-L scale fermentation which combined static cultivation and resting-cell biotransformation,cells grown in media with xylose as sole carbon source produced 129 g GABA from monosodium glutamate in total;when corncob hydrolysate replaced xylose as the carbon source,total GABA production reached 117 g.(2)Sequence identification and activity analysis of Glutamate decarboxylase(GadB)from the WPZ001.In this study,gene gadB from WPZ001 was PCR amplified by primes designed according to the genome of L.buchneri NRRL B-30929.By sequence alignment,GadB from WPZ001 had more than 99% homology with NRRL B-30929 Gad B,but only about 35% homology with E.coli W3110 Gad B.GadB form WPZ001 and W3110 were overexpressed in E.coli BL21(DE3),the GadB activity of recombinant BL21(DE3)expressing Gad B from WPZ001 was much lower than that expressing GadB from W3110.Then GadB form WPZ001 and W3110 were purificated by Ni2+ affinity column chromatography and characterized.The two GadB were of highest activities and low stabilities at 50oC;and the optimum pH for Gad B form WPZ001 and W3110 were 5.0 and 4.6,respectively.(3)Extracellular expression of Gad B to improve GABA production in E.coli.GadB from E.coli W3110 was respectively fused to Sec pathway signal peptide gene pelB and Tat pathway signal peptide gene torA,and overexpressed in E.coli BL21(DE3).It was found that only TorA could facilitate GadB efficient secretion.After optimization of the media and fermentation conditions,at flask shaking scale,extracellular Gad B activity reached 5.11 U/mL,2.7-fold higher than that under non-optimized conditions.In 3-L bioreactor,adopting the two-stage glycerol strategy,extracellular GadB activity reached 13.12 U/mL.BL21(DE3)/pET20b-tor A-gadB could efficiently accumulate GABA from monosodium glutamate.Extracellular GadB played a major role in the increase of the production and productivity of GABA.At flask shaking scale,228.9 g/L GABA was produced after 76 h fermentation.At 3-L bioreactor scale,264.4 and 313.1 g/L GABA were obtained at 36 and 72 h,respectively.(4)Direct production of GABA from xylose in recombinant E.coli.In this study,recombinant plasmid pWZtac-g2 was constructed,which co-expressed gadB and gdhA under tac promoters;and recombinant plasmid pWZt7-g2 was constructed,which co-expressed gadB and gdhA under T7 promoters as well as T7 RNA polymerase under ftsZ promoter.The plasmids were transformed into E.coli JM109 and W3110 which accumulate no GABA.In the two-stage pH control fermentations,E.coli W3110/pWZtac-g2,W3110/pWZt7-g2,JM109/pWZtac-g2 and JM109/pWZt7-g2 produced 0.35-0.52 g/L GABA.The xylose metabolic pathway in E.coli is complex,while Caulobacter crescentus provides a simpler pathway named by Weimberg pathway,which is encoded by five genes.By co-expressing the gadB and gdhA,as well as genes encoding Weimberg pathway CcxylA,CcxylB,CcxylC,CcxylD and CcxylX,the metabolic pathway of xylose to GABA could be simplified to 7 steps.Recombinant plasmids pWZtac-xyl and pWZt7-xyl were constructed,which co-expressed the five Weimberg pathway genes under the control of tac promoter and T7 promoter,respectively.The plasmids were transformed into recombinant strains,results in four recombinants harboring two plasmids E.coli W3110/pWZtac-g2/pWZtac-xyl,W3110/pWZt7-g2/pWZt7-xyl,JM109/pWZtac-g2/pWZtac-xyl and JM109/pWZt7-g2/pWZt7-xyl,which produced more GABA from xylose.And the GABA production of E.coli W3110/pWZtac-g2/pWZtac-xyl reached 0.83 g/L.(5)Further increase of the GABA production from xylose in E.coli by metabolic engineering.In order to enhance the carbon flux of Weimberg pathway,decrease the further metabolism of GABA and increase the membrane permeability,genes xylA,xylB,sucA,gabT,puuE,gabP,waaF and waaC were deleted step by step in E.coli W3110 and JM109,resulting in a series of mutants.Plamid pairs pWZt7-g2/pWZt7-xyl and pWZtac-g2/pWZtac-xyl,which co-expressed the genes of the seven-step pathway for GABA production from xylose under the control of tac promoter and T7 promoter,were transformed into the mutants,constructing 16 recombinants harboring two plasmids.Among the recombinants,GABA production of JWZ08/pWZt7-g2/pWZt7-xyl reached 2.47 g/L,higher than other recombinants.In order to enhance the Gad B activity,plasmid pWZt7-g3 was constructed by fusing signal peptide gene torA to gadB in pWZt7-g2.pWZt7-g3 was transformed into E.coli mutants and the GABA production of E.coli JWZ08/pWZt7-g3/pWZt7-xyl was further increased to 3.95 g/L,which was 3-fold of the highest reported production of GABA from glucose in E.coli.